esm files/esm09.pdf · 2 esm 2009 welcome message dear colleagues, it is with great pleasure that...

ESM

European Societyof Mycobacteriology

In co-organization with the Instituto Nacional de Saúde Dr. Ricardo Jorge

1European Society of Mycobacteriology | 30th Annual Congress | July 2009 | Porto - Portugal

CONTENTS

Welcome Message•

Introduction to the European Society of Mycobacteriology•

Congress Organization•

Program at a glance•

Programme of Guest Lectures, Oral Presentations and Symposia•

Programme of Poster Presentations•

Abstracts of Guest Lectures (GL)•

Abstracts of Oral Presentations (OP)•

Abstracts of Poster Presentations (PP)•

Author Index•

2 ESM 2009

WElCOmE mESSagE

Dear Colleagues,

It is with great pleasure that we warmly welcome you to the 30th Annual Congress of the

European Society of Mycobacteriology, held in the city of Porto in Portugal from July 5-8, 2009.

The aim of ESM2009 is to promote the exchange of distinguished experts from all around

theworldwhowillhavetheopportunitytoupdate information inthe front-lineofscientific

achievement,shareexperienceandideas,andactivelyparticipateincontributiontothefieldof

mycobacteriology.

We have worked hard to make your stay in Porto a pleasant, useful and memorable occasion.

On behalf of the ESM and the Local Organisers,

Suzana David


EurOpEaN SOCiETy Of myCObaCTEriOlOgy 30Th aNNual CONgrESS July 5-8, 2009 pOrTO, pOrTugal

TheEuropeanSocietyofMycobacteriology(ESM,http://www.esmycobacteriology.eu),foundedin1980,isanon-profitinternationalscientificsocietydealingwithdifferentaspectsofmycobacteriologyandrelateddiseases.Itisconsideredoneofthemostactiveinternationalscientificsocietiesinthisarea,beingcommittedto:

- Encouraging the highest standards for research to facilitate the discovery of new knowledge;

- Coordinating and providing information and expertise to other organizations worldwide;

-Disseminatingknowledgeonallaspectsofmycobacteriologyandrelateddiseases,throughscientific meetings and publications,

- Encouraging and providing the highest standards of training to interested health care providers;

- Establishing, reviewing and revising guidelines;

- Promoting high quality and cost effective diagnostic procedures;

- Advising, cooperating and participating with government and non-government agencies in matters of common interest;

- Participating in activities whose aim is to prevent mycobacterial diseases worldwide.

The ESM meetings are held each year in a different country of Europe. The ESM2009 congress is hosted in the city of Porto, inPortugal. International specialistswill treat themesat the front-lineof scientificachievement in thefieldofmycobacteriologyandrelateddiseases.Asinpreviousmeetings,thescientificprogramoftheconferencescoversawiderange of topics in both applied and fundamental research in areas of priority such as:

- Diagnostics of active and latent tuberculosis

- Diagnostics of atypical mycobacteria

- Molecular epidemiology of tuberculosis

- Antibiotic resistance, MDR, XDR

- Tuberculosis drug development

- Immunology of mycobacterial infections

- Molecular Biology of mycobacteria

- Taxonomy of the genus Mycobacterium

- Veterinarian and environmental Mycobacteriology

- Methods for diagnostics adapted to economically disfavoured settings

- Laboratory safety

- Short and long term programs and recommendations

4 ESM 2009

CONgrESS OrgaNizaTiON

EurOpEaN SOCiETy Of myCObaCTEriOlOgy http://www.esmycobacteriology.eu

Steering Committee

hONOrS COmmiTTEE ESm 2009

General Secretary

President

Treasurer

Members

Enrico Tortoli (Firenze, Italy)

TodorKantardjiev(Sofia,Bulgaria)

Malcolm Yates (East Dulwich Grove, U.K.)

Maria-Jesus Garcia (Madrid, Spain)

Sven Hoffner (Solna, Sweden)

Stefan Niemann (Borstel, Germany)

Gabriela Pfyffer (Luzern, Switzerland)

Nalin Rastogi (Guadeloupe, France)

Veronique Vincent (Geneva, Switzerland)

Ana Jorge

Mariano Gago

Manuel Pizarro

Francisco Ramos

José Pereira Miguel

Jorge Sampaio

Mario Raviglione

Maria do Céu Machado

Francisco George

Jorge Soares

Jorge Torgal

Rui Rio

Fernando Augusto Fiuza de Melo

Minister of Health

Minister of Science, Technology and Higher Education

Secretary of State for Health

Secretary of State for Health

President of the National Health Institute Dr. Ricardo Jorge

UN Secretary General’s Special Envoy to Stop TB

Director of the Stop TB Department, World Health Organization

High Comissioner for Health

Director-General of Health

Director of the Health and Human de-velopment Service Calouste Gulbenkian Foundation

Director do Institute of Hygiene and Tropical Medicine

Mayor of the city of Porto

Director of the Clemente Ferreira Institute, São Paulo, Brasil

Chairman of the ESM 2009 congress Suzana David (Lisbon, Portugal)


COOrgaNizErS

European Society of mycobacteriology http://www.esmycobacteriology.eu

instituto Nacional de Saúde Dr. ricardo Jorge, iNSa http://www.insa.pt

parTNErS

foundation for Science and Technology (fCT) http://www.fct.mctes.pt

fundação Calouste gulbenkian http://www.gulbenkian.pt luso-american foundation (flaD) http://www.flad.pt

STOp-Tb Working group on New Diagnostics, point of Care sub group http://www.stoptb.org

SpONSOrS

The Organization expresses its thanks and appreciation to all those who gener-ously contributed to the success of the 30th Annual Congress of ESM.

haiN lifeSciences http://www.hain-lifescience.com

Platinum Medal Sponsor

beckton Dickinson & Quilaban http://www.bd.com http://www.quilaban.pt

Gold Medal Sponsor

biomerieux http://www.biomerieux.com

Gold Medal Sponsor

microsens (logo) http://www.microsens.com

Cepheid http://www.cepheid.com

genoscreen http://www.genoscreen.com

6 ESM 2009

Sunday, July 5th 09h00 REGISTRATION

11h00 – 12h00 SYMPOSIUM SPONSORED BY HAIN LIFESCIENCE GMBH: RAPID MOLECULAR GENETIC DETECTION OF MDR- AND XDR-TB Chair: Michael Weizenegger Speakers: Doris Hillemann and Christopher M Gilpin

12h00 – 13h30 SYMPOSIUM SPONSORED BY STOP-TB WORKING GROUP ON NEW DIAGNOSTICS, POINT OF CARE SUB GROUP: A SYMPOSIUM ON POINT-OF-CARE TESTS FOR TUBERCULOSIS Speakers: Catharina Boehme, Carol Nawina Nyirenda, Rosanna Peeling, Gerd Michel, Ruth McNerney and Amy P Wong

13h30 – 15h30 Break for Lunch

15h30 – 16h30 SYMPOSIUM CO-SPONSORED BY BECTON, DICKINSON AND COMPANY AND QUILABAN - QUÍMICA LABORATORIAL ANALÍTICA LDA: SUCCEPTIBILITYTESTING AND TREATMENT OF TB IN THE ERA OF MDR-TB AND XDR-TB. DO WE USE THE RIGHT DRUGS AND TOOLS? Chair: Francoise Portaels and Salman Siddiqi Speakers: Stefan Winkler, Andre De Bock, and Virginia Crews

17h00 – 19h00 OPENING SESSION Welcome Address

TB CONTROL PROGRAMS Chair: Jaime Nina and Cristina Furtado Guest Lecture-1: Miguel Villar Guest Lecture-2: Fernando Fiuza de Melo

19h15 Welcome Reception

monday, July 6th 09h00 – 13h15 SCIENTIFIC SESSION ON MOLECULAR EPIDEMIOLOGY AND DRUG RESISTANCE SURVEILLANCE Chair: Stefan Niemann and Cristina Gutierez

9h00 – 9h45 Guest Lecture-3: Sebastien Gagneux

9h45 – 11h00 Oral Presentations

11h00 – 11h30 Coffee and tea

Chair: Nalin Rastogi and Dr. Philip Supply

11h30 – 12h15 Guest Lecture-4: Dick van Soolingen


13h15 – 14h00 Lunch

prOgram aT a glaNCE


14h00 – 15h00 POSTER SESSION

15h00 – 16h45 SCIENTIFIC SESSION ON NON TUBERCULOUS MYCOBACTERIA Chair: Enrico Tortoli

15h00 – 15h45 Guest Lecture-5: Joseph O. Falkinham, III



17h15 – 19h15 SCIENTIFIC SESSION ON ISSUES IN THE MODERN TB LABORATORY Chair: Gabriela Pfyffer and Thomas Shinnick

17h15 – 18h00 Guest Lecture-6 : Dr. Jean-Pierre Zellweger

18h00 – 18h30 Guest Lecture-7 : Lee W. Riley


19h30 Visit and Dinner at the Port Wine Cellars

Tuesday, July 7th 09h00 – 12h30 SCIENTIFIC SESSION ON VACCIN DEVELOPMENT AND PATHOGENESIS

Chair: David Minnikin

09h00 – 09h45 Guest Lecture-8: Carlos Martin

09h45 – 10h30 Guest Lecture-9 : Lee W. Riley



12h30 – 13h15 Lunch

13h15 – 14h15 BUSINESS MEETING

14h15 – 18h15 SCIENTIFIC SESSION ON LABORATORY STRENGTHENING Chair: Dr. Sven Hoffner and Dr. Mark Perkins

14h15 – 15h00 Guest Lecture-10: Thomas Shinnick

15h00 – 15h45 Guest Lecture-11: Afrânio Kritski

15h45 – 16h30 Guest Lecture-12 : Moisés Palaci



17h45 – 18h15 BEST POSTER

18h30 Cultural Evening Dinner

8 ESM 2009

Wednes-day, July 8th

09h00 – 11h00 SCIENTIFIC SESSION ON DRUG DEVELOPMENT

Chair: Dr. Nalin Rastogi

09h00-09h45 Guest Lecture-13: Clarice Queico Leite

09h45 – 10h30 Guest Lecture-14 : Moisés Palaci



11h30 – 12h20 SESSION ON PRACTICAL ASPECTS AND QUALITY ASSURANCE IN MOLECULAR EPIDEMIOLOGY Chair: Dr. Elvira Richter and Prof. Christophe Sola

11h30 – 12h00 Guest Lecture-15 : Philip Supply

12h00 – 12h20 Oral Presentation

12h20 – 12h30 Discussion

12h30 – 13h30 SYMPOSIUM SPONSORED BY INSTAND, SOCIETY FOR RESEARCH PROMOTION OF QUALITY ASSURANCE IN MEDICAL LABORATORIES, WHO COLLABORATING CENTRE, DUESSELDORF, GERMANY: EXTERNAL QUALITY ASSURANCE Speakers: Elvira Richter, Girts Skenders, Akos Somoskövy

13h:30 – 13h45 Closing Remarks

13h45 – 14h15 CLOSING OF CONGRESS

14h15 Conference Closes


Sunday, July 5th

9h00 – 11h00 REGISTRATION

11h00 – 12h00 SYMPOSIUM SPONSORED BY HAIN LIFESCIENCE GMBH RAPID MOLECULAR GENETIC DETECTION OF MDR- AND XDR-TB Chair: Dr. Michael Weizenegger

Dr. Doris Hillemann, National Reference Center for Mycobacteria (Borstel, Germany)

rapid detection of XDr-Tb with the genotype mTbDrsl assay

Dr. Christopher M Gilpin, PhD MPH, International Organization for Migration (Geneva, Switzerland)

implementation of new diagnostic tools and algorithms for enhanced case detection of drug resistant forms of tuberculosis

12h00-13h30 SYMPOSIUM SPONSORED BY STOP-TB WORKING GROUP ON NEW DIAGNOSTICS, POINT OF CARE SUB GROUP A SYMPOSIUM ON POINT-OF-CARE TESTS FOR TUBERCULOSIS

Dr. Catharina Boehme, M.D., Foundation for Innovative Diagnostics (FIND) (Geneva, Switzerland)

What is a pOC test?

Carol Nawina Nyirenda (Zambia)

Why do we need rapid tests: a patient’s perspective?

Prof. Rosanna Peeling, Ph.D., London School of Hygiene & Tropical Medicine (London, UK)

rapid tests for Tb: what is wrong with them?

Dr. Gerd Michel, Ph.D., Foundation for Innovative Diagnostics (FIND) (Geneva, Switzerland)

biomarker discovery: are we making progress

Dr. Ruth McNerney, Ph.D., London School of Hygiene & Tropical Medicine (London, UK)

Volatile markers for Tb: myth or reality?

Dr. Amy P Wong, Ph.D., X PRIZE Foundation (California, U.S.A.)barriers to Tb test development

Discussion: The way forward Platformandfloor

13h30 – 15h30 Break for Lunch

15h30 – 16h30 SYMPOSIUM CO-SPONSORED BY BECTON, DICKINSON AND COMPANY AND QUILABAN - QUÍMICA LABORATORIAL ANALÍTICA LDA

SuCCEpTibiliTyTESTiNg aND TrEaTmENT Of Tb iN ThE Era Of mDr-Tb aND XDr-Tb. DO WE uSE ThE righT DrugS aND TOOlS? Chair: Prof. Francoise Portaels and Dr. Salman Siddiqi

Prof. Stefan Winkler, University of Vienna (Vienna, Austria)

antibiotic therapy for Tb and new treatment options

prOgrammE Of guEST lECTurES, Oral prESENTaTiONS aND SympOSia

10 ESM 2009

Dr. Andre De Bock (BD)

Extended Drug Susceptibility Testing of m. tuberculosis

Virginia Crews (BD)

The new BD MGIT TBc Identification Test

16h30 – 17h00 OPENING SESSION

WELCOME

MinisterofHealth(Portugal)(toconfirm)

MinisterofScience,TechnologyandHigherEducation(Portugal)(toconfirm)

17h00 – 17h45 Welcome from the President of the Instituto Nacional de Saúde Dr. Ricardo Jorge, Prof. José Pereira Miguel (Portugal)

Welcome from the General Secretary, Dr. Enrico Tortoli (Italy)

Welcome address, Dr. Suzana David (Portugal)

Tb CONTrOl prOgramS

Chair: Prof. Jaime and Prof Cristina Furtado

17h45 – 18h20 Guest lecture: Dr. Miguel Villar, Consultant or Thuberculosis, Directorate-General of Health (Lisboa, Portugal)

Tuberculosis in portugal

18h20 – 19h00 Guest lecture: Dr. Fernando Fiuza de Mello, Director of the Clemente Ferreira Institute (São Paulo, Brasil)

The brazilian experience in the control of multi-drug resistant tuberculosis

Welcome reception Welcome Reception

monday, July 6th SCIENTIFIC SESSION ON MOLECULAR EPIDEMIOLOGY AND DRUG RESISTANCE SURVEILLANCE

9h00 – 9h45 Guest lecture: Dr. Sebastien Gagneux, Ph.D., National Institute for Medical Research (London, UK)

gl-3

Evolutionary forces in mycobacterium tuberculosis

Chair: Dr. Stefan Niemann and Dr. Cristina Gutierez

9h45 – 10h00 Honisch C, Mosko M, Arnold C, Gharbia S, Feuerriegel S, Niemann S mass Spectrometry for molecular typing of the mycobacterium tuberculosis complex: One platform and multiple assay formats

Op-1

10h00 – 10h15 Borile C, Refrégier G, Labarre M, Franz S, Mézard M, Sola C Clustering of spoligo-patterns: towards an automation of Mycobacterium tuberculosis complex classification

Op-2

10h15 – 10h30 Sandoval A, Cubillos A, Reyes A, Correa N, Robledo J, Zambrano MM, Del Portillo P Identification of the insertion element IS6110 in phop promoter in a high transmission mycobacterium tuberculosis strain: a clue to phenotypic variation

Op-3

10h30 – 10h45 Oelemann MC, Gomes HM, Willery E, Lima KVB, Possuelo L, Locht C, Goguet de la Salmonière YOL, Gutierrez MC, Supply P, Suffys PN

Op-4


12h15 – 12h30 Millet J, Miyagi-Shiohira C, Yamane N, Mokrousov I, Rastogi N The unique endemic nature of beijing genotype strains in Okinawa, ryukyu islands of Japan as revealed by newly de-scribed 15 and 24-loci miru-VNTr typing schemes

Op-6

12h30 – 12h45 Shamputa IC, Lee J, Allix-Béguec C, Cho E-J, Lee J-I, Min JH, Goldfeder LC, Kim JH, Kang HS, Hwang SH, Eum SY , Lee H, Park SK, Supply P, Cho SN, Via LE, Barry III CE Mycobacterium tuberculosis genetic diversity in South Korea

Op-7

12h45 – 13h00 Feuerriegel S, Homolka S, Post E, Oberhauser B, George AG, Westman L, Dafae F, Rüsch-Gerdes S, Niemann S Correlation of molecular resistance mechanisms and phenotypic resistance to first-line drugs in Mycobacterium tuberculosis strains from Sierra leone

Op-8

13h00 – 13h15 McNerney R, Mallard K lam and hiV: correlation or co-incidence?

Op-9

13h15 – 14h00 Lunch

14h00 – 15h00 POSTER SESSION

SCIENTIFIC SESSION ON NON TUBERCULOUS MYCOBACTERIA

Chair: Dr. Enrico Tortoli

15h00 – 15h45 Guest lecture: Prof. Joseph O. Falkinham, III, Ph.D., Virginia Tech (Virginia, U.S.A.)

gl-5

Surrounded by mycobacteria

15h45 – 16h00 Lyberopoulos P, Frangopoulos F, Kontos F, Zerva L, Malagari Ai, Papiris S Mycobacterium celatum: an emerging pathogen in the immuno-competent. a case report

Op-10

16h00 – 16h15 Radomski N, Thibault V, Karoui C, De Cruz K, Cochard T, Gutiérrez C, Supply P, Biet F, Boschiroli ML Mycobacterium avium subspecies strains from human and animal origin

Op-11

16h15 – 16h30 Leão SC, Tortoli E, Viana-Niero C, Ueki SYM, Lima KVB, Lopes ML, Yubero J, Menendez MC, Garcia MJ The characterization of mycobacteria from an outbreak sug-gests a revision of the taxonomic status of members of the Mycobacterium chelonae-abscessus group

Op-12

genomic interrogation of mycobacterium tuberculosis isolates from brazil

10h45 – 11h00 Mokrousov I, Valcheva V, Sovhozova N, Aldashev A, Rastogi N, Isakova J penitentiary population of mycobacterium tuberculosis in Kyrgyzstan: Exceptionally high prevalence of the beijing geno-type and its Russia-specific subtype

Op-5

11h00 – 11h30 Coffee and Tea

11h30 – 12h15 Guest lecture: Dr. Dick van Soolingen, Ph.D., National Institute for Public Health and the Environment (Bilthoven, Netherlands)

gl-4

advances in the molecular epidemiology of tuberculosis

Chair: Dr. Nalin Rastogi and Dr. Philip Supply

12 ESM 2009

18h30 – 18h45 Kaal E, Kolk A, Kuijper S, Janssen HG Fast identification of Mycobacterium tuberculosis in sputum and cultures based on thermally-assisted hydrolysis and methylation by gas chromatography-mass spectrometry

Op-14

18h45 – 19h00 Ängeby K, Juréen P, Giske C, Chryssanthou E, Werngren J, Hoffner S, Kahlmeter G, Sturegård E, Schön T how Wild-type miC distributions can be useful to determine clinical breakpoints in Mycobacterium tuberculosis

Op-15

19h00 –19h15 Miotto P, Cirillo DM molecular techniques to monitor Tb patients’ treatment: selec-tive removal of DNa from dead bacteria in mixed populations by use of ethidum monoazide

Op-16

Visit and Dinner at the Port Wine Cellars

Tuesday, July 7th SCIENTIFIC SESSION ON VACCIN DEVELOPMENT AND PATHOGENESIS Chair: Dr. David Minnikin

9h00 – 9h45 Guest lecture: Prof. Carlos Martin, M.D., Ph.D., University of Zaragoza (Zaragoza, Spain)

gl-8

New live tuberculosis vaccines strategies

09h45 – 10h30 Guest lecture: Prof. Lee W. Riley, M.D., Ph.D., University of California at Berkeley (California, U.S.A.)

gl-9

regulation of Mycobacterium tuberculosis cell wall lipid composi-tion and its effect on in vivo bacterial persistence


11h00 – 11h15 Brzostek A, Pawelczyk J, Rumijowska-Galewicz A, Dziadek B, Dziadek J Mycobacterium tuberculosis is able to accumulate and utilize cholesterol

Op-17

11h15 – 11h30 Rodríguez-Güell E, Alonso C, del Val-Romero B, Clivillé R, Secanella SP, Roura-Mir C, Cañete C, Navarro A, de Gispert FX , Luquin M, Julián E mycolic acid-induced ifN-g production by CD1-restricted T cells from tuberculous patients

Op-18

11h30 – 11h45 Farnia P, Ali Veleyati A, Masjedi MR, Ibrahim TA, Tabarsei P, Haroun RZ, Kuan HO, Omar AR a report on new adapted forms of extensively drug resistance tubercle bacilli : Transmission Electron microscopy analysis

Op-19

16h30 – 16h45 Santos R, Marques M, Oliveira P, Carvalho F, Carvalho C, Monteiro G, Cabral J, Frade R, Silva M, Fernandes P The sunny side of mycobacteria

Op-13


SCIENTIFIC SESSION ON ISSUES IN THE MODERN TB LABORATORY

Chair: Dr. Gabriela Pfyffer and Dr. Thomas Shinnick

Guest lecture: Dr. Jean-Pierre Zellweger, M.D., Swiss Lung Association (Bern, Switzerland)

17h15 – 18h00 The use of interferon gamma release assays as an aid in the con-trol of tuberculosis

gl-6

18h00 – 18h30 Guest lecture: Prof. Lee W. Riley, M.D., Ph.D., University of California at Berkeley (California, U.S.A.)

gl-7

a novel diagnostic test to differentiate latent Tb infection and active disease


14h15 – 15h00 guest lecture: Dr. Thomas m. Shinnick, ph.D., Centers for Disease Control and prevention (georgia, u.S.a.)

gl-10

CDC’s global Tb laboratory activities

15h00 – 15h45 Guest lecture: Prof. Afranio Kritski, M.D., Ph.D., Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil)

gl-11

Development and validation of new Tb diagnostic tests in brazil: experience of rede-Tb

15h45 – 16h30 Guest lecture: Prof. Moisés Palaci, Ph.D., Universidade Federal do Espírito Santo (Vitória, Brazil)

gl-12

Experience of a successful mycobacteriology laboratory network in Espirito Santo- brazil


17h00 – 17h15 Portugal C, Cardoso N, Sancho L, Sousa G Tuberculosis software in a general hospital, working instrument

Op-23

17h15 – 17h30 den Hertog A, Koeleman M, Ingham C, Fey F, Langerak E, Klatser P, Anthony R Development of an automated culture system for m. tuberculo-sis with autofluorescence detection

Op-24

17h30 – 17h45 Morcillo N, Imperiale B, Di Giulio B Second-line drug susceptibility testing of mycobacterium tuber-culosis by mgiT 960 system, the microplate colorimetric-based method and the proportion method

Op-25

17h45 – 18h15 BEST POSTER

Cultural Evening Dinner

Wednesday, July 8th

SCIENTIFIC SESSION ON DRUG DEVELOPMENT

Chair:Dr. Nalin Rastogi

9h00 – 9h45 Guest lecture: Prof. Clarice Queico Fujimaro Leite, M.Sc., Ph.D.; Universidade Estadual Paulista (Araraquara, Brazil)

gl-13

11h45 – 12h00 Homolka S, Niemann S, Russell DG, Rohde KH Growth profile of clinical isolates of Mycobacterium tuberculosis complex in murine macropghages

Op-20

12h00 – 12h15 van Ingen J, van der Wel N, Dekhuijzen R, Boeree M, van Soolingen D presence of esat-6 and cfp-10 genes does not lead to phagolyso-some translocation of mycobacterium szulgai

Op-21

12h00 – 12h30 Fraga AG, Braga JE, Cruz A, Martins TG, Pereira DR, Meyers WM, Portaels F, Castro AG, Pedrosa J Development of an adaptive immune response in the draining lymph node during mycobacterium ulcerans infection

Op-22

12h30 – 13h15 Lunch

13h15 – 14h15 BUSINESS MEETING

SCIENTIFIC SESSION ON LABORATORY STRENGTHENING

Chair: Dr. Sven Hoffner and Dr. Mark Perkins

14 ESM 2009

11h30 – 12h00 Allix-Béguec C, HubansC, Ferreira S, Supply P New, easy-to-use tools for quality-controlled genotyping of m. tuberculosis complex strains

gl-15

12h00 – 12h20 Abadia E, Zhang J, Refrégier G, Sola C membrane- based versus microbead- based spoligotyping: preliminary results on a quality- insurance study on 10 sites worlwide

Op-28

12h20 – 12h30 Discussion

SYMPOSIUM SPONSORED BY INSTAND, SOCIETY FOR RESEARCH PROMOTION OF QUALITY ASSURANCE IN MEDICAL LABORATORIES, WHO COLLABORATING CENTRE, DUESSELDORF, GERMANY

12h30 – 13h30 EXTERNAL QUALITY ASSURANCE

PD Dr. Elvira Richter, NRL (Borstel, Germany)

EQa in a low incidence, high income country

Dr. Girts Skenders, State Agency of TB and Lung Disease (Latvia)

Organization and EQa of latvian Tb laboratory network

Dr. Akos Somoskövy, M.D., Ph.D., D.Sc., Foundation for Innovative Diagnostics (FIND) (Geneva, Switzerland)

Quality assurance for new techniques – what is necessary, what is possible?

13h30 -13h45 Closing remarks

13h45 – 14h15 CLOSING OF CONGRESS

14h15 Conference closes

Screening of molecules with anti-Tb activity, from the brazilian cerrado plants, and synthetic metallo- organic compounds

09h45 – 10h30 Guest lecture: Prof. Moisés Palaci, Ph.D., Universidade Federal do Espírito Santo (Vitória, Brazil)

gl-14

Clinical trials of drugs and diagnostic tests: the challenges in mycobacteriology

10h30 – 10h45 van Ingen J, Boeree M, Amaral L, Pando RH, van Soolingen D Thioridazine shows promising activity in a murine model of multidrug-resistant tuberculosis

Op-26

10h45 – 11h00 Rodrigues L, Sampaio D, Couto I, Machado D, Kern WV, Amaral L, Viveiros M Contribution of efflux pump activity for macrolide resistance in m. avium complex

Op-27


SESSION ON PRACTICAL ASPECTS AND QUALITY ASSURANCE IN MOLECULAR EPIDEMIOLOGY Chair: Dr. Elvira Richter and Prof. Christophe Sola


Miotto P, Baldan R, Cirillo DMEvaluation of the high-throughput repetitive-sequence-based pCr diversilab system in M. tuberculosis molecular epidemiology studies

pp-1

Zhang J, Abadia E, Refrégier G, Ruimy R, Boschiroli ML, Guillard B, Sola C68 spacers Mycobacterium tuberculosis complex spoligotyping : a study using a microbead-based high throughput format

pp-2

Niemann S, Khechinashvili G, Gegia M, Mdivani N, Tang YWassociation between beijing genotype and drug resistance among Mycobacterium tuberculosis isolates circulating in the republic of georgia

pp-3

Baboolal S, Millet J, Akpaka PE, Ramoutar D, Rastogi NMycobacterium tuberculosis epidemiology and genetic diversity in the Twin island republic of Trinidad and Tobago

pp-4

Mestre O, Luo T, Rauzier J, Golec M, Rastogi N, Rasolofo V, Tonjum T, Sola C, Matic I, Mei J, Gao Q, Vultos TD, Gicquel BDiversity and evolution of M. tuberculosis

pp-5

Sharaf-Eldin GS, Elmoula IF, Ali MS, Saaed NS, Ali AB, Mallard K, McNerney R, Algamdi SSpoligotype patterns and drug resistant profile of Mycobacterium tuberculosis in Sudan

pp-6

Valcheva V, Mokrousov I, Panaiotov S, Bachiiska E, Zozio T, Sola C, Markova N, Rastogi NControversial dissemination pattern of the Bulgaria-specific M. tuberculosis spoligotype ST125_bgr

pp-7

Panaiotov S, Bachiyska E, Brankova N, Levterova VMycobacterium tuberculosis beijing genotype and origins of the bulgarians

pp-8

Al-Maniri AA, Singh JPN, Al-Rawas O, Al Busaidi S, Al Balushi L, Ahmed I, Al- Mahruqi S, Haile M, Diwan V, Hoffner Sa Snapshot on biodiversity and clustering of Mycobacterium tuberculosis among nationals and immigrants in Oman using spoligotyping

pp-9

David S, Ribeiro JN, Maio JN, João I, Amorim A, Pereira EThe extent of the latin american-mediterranean Mycobacterium tuberculosis spoligotype family in portugal

pp-10

Von Groll A, Martin A, Felix C, Prata P, Honscha G, Portaels F, Almeida da Silva P, Palomino JCfitness Study of the rDrio lineage and lam family of Mycobacterium tuberculosis in a study population in rio grande, brazil

pp-11

Perdigão J, Silva C, Portugal Igenomic characterization of lisboa family strains by deletion analysis

pp-12

Obrovac M, Katalinic-Jankovic V, Grce M, Zmak Limportance of molecular typing in suspected intra-familial transmission of tuberculosis

pp-13

Oral Zeytinli U, Kayar Mb, Karacali A, Sahan Kipalev A, Yula E, Köksal FDetection of clonal complexity in clinical M. tuberculosis isolates by miru-VNTr in Cukurova region, Turkey

pp-14

Leite CQF,SantosACB,PandolfiJRC,MalaspinaAC,PavanFR,MendesNH,VianaBHJmolecular epidemiology study of tuberculosis patients in a small city of São paulo – brazil, from 2002 to 2006

pp-15

Leite CQF, Nogutia EN, Malaspina AC, Santos ACB, Hirata RDC, Hirata MH, Cardoso RFgenotyping of Mycobacterium tuberculosis in northwest of paraná State of brazil

pp-16

prOgrammE Of pOSTEr prESENTaTiONS

16 ESM 2009

Mello FAF,AlbarralMIP,MendesNH,PandolfiJRC,SantosACB,AlmeidaEA,CardosoRFSpoligotyping of Mycobacterium tuberculosis isolated from patients of Clemente ferreira ambulatory in São paulo, Sp – brazil

pp-17

Tajeddin E, Farnia P, Kargar M, Noroozi J, Ahmadi M, Kazempour M, Hadadi M, Masjedi M, Velayati A Comparison Of Mycobacterium beijing genotype with VNTr, Spoligotyping and rflp-iS6110

pp-18

Ritacco V, Reniero A, Beltrán M, López B, Kantor I, Barrera Lmultiply recurrent tuberculosis in a pacient living with hiV: reinfection or reactivation?

pp-19

Tavares Magalhães A, Alves A, BragaR, Valente I, DuarteR, Miranda Amolecular epidemiology of tuberculosis in Vila Nova de gaia, portugal

pp-20

Alves A, Miranda Amolecular study of recurrent tuberculosis cases

pp-21

Ehricht R, Slickers P, Monecke Sgenotyping of drug resistance in Mycobacterium tuberculosis using diagnostic microarrays

pp-22

Al-Hajoj S, Varghese B, Herbawi M, Al-Omari R, Allix-Béguec Cgenotyping of mono and multi-drug resistance Tb in Saudi arabia

pp-23

Machado D, Viveiros M, Rodrigues L, Couto I , Amaral LEarly detection of mDrTb by molecular tools in the control of drug resistant tuberculosis in portugal: a case of success

pp-24

Vladimirov K, Zaitseva E, Ivanov ADrug-resistance of Mycobacterium tuberculosis at penitentiary institutions of St. petersburg, russian federation

pp-25

Stoffels K, Fauville-Dufaux Man increase of drug resistance since 2001 in multidrugresistant M. tuberculosis isolates from belgium

pp-26

Perdigão J, Ferreira A, Malaquias A, Macedo R, Brum L, Portugal Imutational analysis of genes associated with resistance to injectable second-line drugs in Mycobacterium tuberculosis clinical isolates from lisbon, portugal

pp-27

Nuak J, Ferreira D, Carvalho T, Gomes MH, Sarmento Amultidrug-resistant tuberculosis

pp-28

Tudó G, Rey E, Alcaide F, Coll P, Codina G, Martín-Casabona N, Montemayor M, Moure R, Salvadó M, González-Martín JCharacterisation of streptomycin mutations in Mycobacterium tuberculosis clinical isolates in the area of barcelona

pp-29

Fattorini L, Pardini M, Cirillo D, Borroni E, Miotto P, Filippini P, Cassone ASurveillance of Drug-resistant Tuberculosis in italy

pp-30

Sancho L; Portugal C; Tancredo L; Silva M; Dias A; Silva F, Sousa GTuberculosis resistance in a general hospital in portugal – 9 years surveillance

pp-31

Yates M, Brown T, Drobniewski FDoes a mutation in the rpob mean that the M. tuberculosis is resistant to rifampicin?

pp-32

Zaldumbide MA, Mazarrasa CF, Martinez-Martinez L, Balbin JAgenotypic detection of isoniazid and rifampin resistance in Mycobacterium tuberculosis clinical isolates

pp-33

Chan CYR, Chan WCE, Au TKM, Lai WMR, Yew WW, Yip CW, Kam KMPhysiological fitness and transmission potential of multi-drug resistant Mycobacterium tuberculosis clinical isolates in hong Kong

pp-34


Sousa AS, Pinheiro MD, Carvalho T, Gonçalves HMycobacterium tuberculosis: 1999-2008 antituberculosis drugs surveillance in clinical isolates from patients in the largest hospital in the North of portugal

pp-35

Von Groll A, Martin A, Jureen P, Hoffner S, Portaels F, Palomino JC, Almeida da Silva Pfitness cost of Mycobacterium tuberculosis clinical isolates resistant to fluoroquinolones

pp-36

Von Groll A, Martin A, Jureen P, Hoffner S, Portaels F, Almeida DA Silva P, Palomino JCIn vitro activity of ofloxacin, moxifloxacin and gatifloxacin against Mycobacterium tuberculosis by the resazurin colorimetric method

pp-37

Paasch F, Martin A, Docx S, Fissette K, Portaels F, Palomino JCrapid detection of extensively drug-resistant Mycobacterium tuberculosis by the resazurin microtiter assay plate

pp-38

Montoro E, Yzquierdo S, Lemus D, Echemendia M, Takiff HDetection of embb gene codon 306 mutations in ethambutol susceptible and resistant Mycobacterium tuberculosis strains

pp-39

Yew WW, Yan SW, Fung SL, Chau CH, Chan Chiu YTolerance of moxifloxacin in routine clinical treatment of tuberculosis

pp-40

Perdigão J, Sabino A, Milho C, Macedo R, Brum L, Portugal ICharacterization of gidB gene in Mycobacterium tuberculosis isolates in lisbon health region: role in streptomycin resistance and epidemiological markers

pp-41

Gaile I, Skenders G, Leimane V, Jansone I, Bauskenieks M, Pole I, Baumanis Vfluorquinolone resistant Mycobacterium tuberculosis isolates and their molecular characteristics

pp-42

Samper S, Millan I, Lopez-Calleja AI, Gavin P, Lezcano MADesign of a rapid method of identification of a highly transmitted strain based on the localization of iS6110

pp-43

Gutierrez MC, Brosch R, Marceau M, Tap J, Bourdon E, Brisse Smangenot S, Salvignol G, Barbe V, Médigue C, Supply PDriving forces on the evolution of the progenitor of m. tuberculosis

pp-44

Ruiz P, Causse M, Zerolo FJ, Gutierrez J, Casal Mresistance, mDr and XDr of M. tuberculosis in Spain in the last years

pp-45

Radomski N, Lucas F, Cambau E, Moulin L, Haenn S, Régis MDetection of non tuberculous mycobacteria in surface waters: comparison of culture methods

pp-46

Spicic S, Cvetnic Z, Pate M, Duvnjak S, Zdelar-Tuk M, Racic ITyping of Mycobacterium avium subsp. avium from different sources using pvuii–psti–iS901 restriction fragment length polymorphism (rflp) in Croatia

pp-47

Spicic S, Duvnjak S, Obrovac M, Zdelar-Tuk M, Katalinic-Jankovic V, Racic I, Cvetnic ZTuberculosis in pets and wild animals living in urban environment

pp-48

Spicic S, Cvetnic Z, Pate M, Katalinic-Jankovic V, Duvnjak S, Ocepek M, Zdelar-Tuk M, Krt BiS1245-rflp based genetic relatedness of the Mycobacterium avium subsp. hominissuis strains isolated from humans, animals and environment in Croatia

pp-49

Lucas F, Radomski N, Cambau E, Moulin L, Haenn S, Moilleron RDevelopment of real-time PCR assay for quantification of mycobacteria in surface waters

pp-50

Amorim A, Macedo R, Pereira ENontuberculous mycobacteria, isolated from patients with lung disease, from lisboa e Vale do Tejo region, during 2008

pp-51

18 ESM 2009

Lima KVB, Lopes ML, Furlaneto IP, Lima EJC, Conceição EC, de Sousa MS, Costa ARFNontuberculous mycobacteria infections in the State of pará, amazon region, brazil

pp-52

Jahromi NS, Seif S, Farnia P, Kazempour M, Kargar M, Nowroozi J, Kazempour M, Masjedi M, Velayati AEvaluation of hsp65, Tb ,Sp regions in identifying Mycobacterium Other Than Tuberculosis (mOTT); using pCr-rflp

pp-53

Svensson E, Ridell M, Åkerström M, Andersson EMycobacterium avium alveolitis after cleansing hotel spa whirlpools

pp-54

Couto I, Machado D, Viveiros M, Rodrigues L, Amaral LIdentification of nontuberculous mycobacteria in clinical samples using molecular methods: a three-year study

pp-55

PateM,FermeD,ŽolnirDovcM,Ocepek Mmycobacteria in animals in Slovenia – an overview of the last decade

pp-56

Leite SRA, Silva P, Sato DN, Santos ACB, Miyata M, Leite CQFIsolation and identification of Rhodococcus and Nocardia genders in sputum samples with tuberculosis suspect

pp-57

Neonakis IK, Kontos F, Gitti Z, Baritaki S, Bazigos S, Mihailelis E, Zerva L, Spandidos DApCr-rflp of hsp65 for identification of Mycobacterium leprae directly from a clinical sample

pp-58

Neonakis IK, Kontos F, Gitti Z, Baritaki S, Kosmadakis G, Baritaki M, Zerva L, Spandidos DAa case-report of Mycobacterium thermoresistibile from greece

pp-59

Portugal C, Sancho L, Dias A, Tancredo L, Silva M; Sardinha T, Sousa JGisolation and frequency of Mycobacterium sp in a general hospital during a 9-year period

pp-60

Diogo J, Rodrigues A, Nascimento I, Sardinha E, Raposo A, Figueira R, Monge I, Silva K, Gil MJ, Rodrigues Slaboratory microbiology contribution to Mycobacterium spp. Diagnosis in three district councils of Setubal (portugal), an area with high mycobacterial infection prevalence

pp-61

Santos C, Mendes AC, Fernandes SJ, Ramos MHMycobacterium lentiflavum as a causative agent of adenopathy

pp-62

Watson C, Lockwood DTeaching old bones new tricks; single nucleotide polymorphism analysis of european archaeological m. leprae DNa

pp-63

Greib C, Lazaro E, Viallard JF, Pellegrin JL, Maugein JInterpretation of positive M. tuberculosis antigen specificifnγ release assays in tuberculosis diagnosis

pp-64

Wang S, Neo ZY, Mak KX, Quieng MD, Sing LHDirect Identification of Mycobacterium tuberculosis Complex, Mycobacterium avium Complex and Mycobacterium kansasii in Smear-positive Clinical Specimens

pp-65

Müllerova Mrapid diagnosis and drug susceptibility testing of tuberculosis infection: mTD-Test2 and bactec mgiT 960 system

pp-66

Levina K, Dementieva A, Saluotsa Mfirst experience with genotype mTbDr assai for rapid evaluation of mDr cases

pp-67

Fajfar N, Zolnir - Dovc MDrug resistant tuberculosis in Slovenia and evaluation of genotype mTbDrpluS test in clinical laboratory

pp-68

^


Causse M, Gutierrez-Aroca JB, Casal MEvaluation of a new real-time pCr kit for the diagnosis of tuberculosis inrespiratory specimens

pp-69

Karabela S, Papaventsis D, Nikolaou S, Konstantinidou E, Sainti A, Ioannidis P, Kanavaki SQuantiferon-Tb gold assay (QfT) and tuberculine skin test (TST) clinical performance for the diagnosis of active tuberculosis

pp-70

Karabela S, Papaventsis D, Nikolaou S, Konstantinidou E, Sainti A, Ioannidis P, Kanavaki SClinical performance of Quantiferon-Tb gold assay (QfT) for the diagnosis of latent tuberculosis in different patient groups

pp-71

Nikolaou S, Karabela S, Papaventsis D, Sainti A, Konstantinidou E, Ioannidis P, Kanavaki STuberculosis diagnosis by Quantiferon Tb gold assay in areas with differences in Tb incidence

pp-72

Havelkova M, Bartu V, Kubin MQuantiferon -Tb gold in-Tube test used in prague patients listed in the National Tuberculosis register

pp-73

Cacho J, García-Cañas A, González Torralba A, Cano I, Pérez Meixeira A, Ramos Martos, Sánchez-Concheiro MPractical experience of using a DNA amplification assay for rapid detection of Mycobacterium tuberculosis complex in respiratory specimens

pp-74

Morgan Kreal-time polymerase chain reaction for the direct detection of Mycobacterium tuberculosis in clinical specimens

pp-75

Kontos F, Zerva LThe utility of molecular testing in routine mycobacteriology diagnosis

pp-76

Salas S, Hernández J, Ojeda P, Awad C, de la Hoz F, Murcia MDetection of Mycobacterium tuberculosis DNA in formalin-fixed, paraffin-embedded tissue specimens by spoligotyping: application to histopathological diagnosis

pp-77

Cardoso S, Coelho R, Paulo C, Abreu C, Silva S, Gomes H, Sarmento Apott´s Disease: an ancient disease?

pp-78

Loureiro C, Matos G, Balacó I, Mota M, Nogueira C, Lemos S, Rocha GOsseous tuberculosis at age of 9 months

pp-79

Secanella SP, Luquin M, Julián EDifferences in direct antitumoral capacity among the various Mycobacterium bovis bCg substrains

pp-80

Anoosheh S, Farnia P, Noruzi J, Kargar M, Kazempour M, Seif S, Masjedi MR, Velayati AArole of TNf-a gene polymorphisms in host genetic susceptibility to pulmonary tuberculosis

pp-81

Torrado E, Fraga AG, Logarinho E, Martins TG, Carmona JA, Gama JB, Carvalho MA, Proença F, Castro AG, Pedrosa Jmycolactone interferes with the protective ifN-γ-dependent activation of macrophages during infection with Mycobacterium ulcerans

pp-82

Montoro E, Valdés I, Aguilar D, Orozco H, Hernández-Pando RVirulence, immunogenicity and protection induced by ´Mycobacterium habana´ strains in a murine model of pulmonary tuberculosis

pp-83

Saraiva M, Sousa C, Carmona JA, Cruz A, Pedrosa J, Castro AGDendritic cells differentially express il12-family cytokines after infection with Mycobacterium tuberculosis or M. bovis bCg

pp-84

Simões MF, Jordão L, Teles JMM, Couto S, Moniz-Pereira J, Pimentel Manalysis of M. smegmatis mutants resistant to ms6 infection

pp-85

20 ESM 2009

Julián EG, Rodríguez-Güell E, del Val-Romero B, Clivillé R, Cañete C, Navarro A, de Gispert FX, Luquin M, Alonso Chumoral response in tuberculous patients against the mycolic acids of Mycobacterium tuberculosis

pp-86

López AGStructural, functional and bioinformatic characterization of Tlya protein from Mycobacterium tuberculosis

pp-87

Ferreira C, Afonso A, Duarte R, Lyashchenko K, Silva A, Rodrigues F, Miranda A, Tavares M, Caldas C, Valente F, Valente A, Vasconcelos O, Amado J, Correia-Neves MEvaluation of the applicability of serodiagnosis for tuberculosis in portugal

pp-88

Martin A, Munga Waweru P, Babu Okatch F, Amondi Ouma N, Bonte L, Palomino JC, Varaine F, Portaels Fimplementation of the thin layer agar (Tla) for the diagnosis of smear negative pulmonary tuberculosis in a high hiV prevalence setting

pp-89

Ferro RS, Shikama M-L, Villela G, Sato DN, Giampaglia CS, Martins MC, Martin A, Palomino JCDirect detection of rifampin resistance in Mycobacterium tuberculosis by the nitrate reductase assay applied directly in sputum samples

pp-90

Ferro RS, Shikama M-L, Villela G, Sato DN, Giampaglia CS, Martins MC, Martin A, Palomino JC, Telles MASDirect detection of rifampin resistance in Mycobacterium tuberculosis by the nitrate reductase assay applied directly in sputum samples

pp-91

De Haas P, Zenhorst R, Mwamba P, Muvwimi M, Mwanza W, Mbulo G, Kapata N, Ayles HmTbDrpluS assay is a useful tool to screen for multi-drug resistant tuberculosis in a national survey

pp-92

de Haas P, Moyoyeta M, Samutela M, Mwanza W, Musunsa A, Mbulo G, Muvwimi M, Ayles HContribution of laboratory factors to high mgiT culture contamination rate in zambia

pp-93

Ahmed A, Qazi F, Khan AJprogrammatic Community-based management of mDr-Tb: Experience in Karachi, pakistan

pp-94

Muchwa C, Akol J, Mumbowa F, Orikiriza P, Morgan K, Eisenach K, Joloba M, Etwom A, Mugyenyi P, Mugerwa REvaluation of Capilia (TAUNS) for rapid identification of Mycobacterium tuberculosis complex from cultures

pp-95

Muchwa C, Akol J, Orikiriza P, Morgan K, Mumbowa F, Eisenach K, Etwom A, Joloba MComparison of capilia (TAUNS) and IS6110 PCR for rapid identification of Mycobacterium tuberculosis complex from cultures in Kampala, uganda

pp-96

Bwanga F, Hoffner S, Haile M, Joloba MLDirect testing for multi drug resistant tuberculosis with four assays evaluated at Kampala, uganda

pp-97

McNerney R, Turner C, Mallard K, O’Sullivan DpEa production by mycobacteria and its application in a rapid drug susceptibility test

pp-98

Balmoi FVarious strategies to decontaminate acid fast bacilli positive liquid cultures from bactec mgiT 960

pp-99

Orikiriza Plow cost isolation of Mycobacterium tuberculosis (mTb) from blood

pp-100

Kayar B, Oral Zeytinli U, Karacali A, Soyal A, Nagiyev T, Köksal FComparison of rapid Colorimetric method, proportion method and baCTEC460 Tb System for testing susceptibility of M. tuberculosis to rifampine and isoniaside

pp-101


Crews V, Warns M, Pfeltz R, Beaty PS, Rosales J, Kopher K, Joshi S, Hoosen A, Said HEvaluation of the mgiT Tbc iD test vs two commercially available rapid immunoassays for M. tuberculosis complex organism detection from liquid and solid culture

pp-102

Montoro E, Milián Y, Lemus D, Echemendía M, Yzquierdo S, Martin A, Van der Stuyft P, Palomino JCNitrate reductase assay applied to direct detection of drug resistance in Mycobacterium tuberculosis

pp-103

Montoro E, Lemus D, Madruga M, Mirabal N, Milián Y, Yzquierdo S, Echemendía M, Martín A, Van der Stuyft P, Palomino JCuse of nicotinamide in colorimetric methods for rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis

pp-104

Hepple P, Novoa-Cain J, Cheruiyot C, Richter E, Ritmeijer Kimplementation of liquid culture for tuberculosis diagnosis in a remote setting: lessons learned

pp-105

Ichijo T, Izumi Y, Yamaguchi N, Nasu Mrapid detection of respiratory active mycobacteria by auramine O-CTC double staining

pp-106

Rey E, Tudó G, González-Martín JSynergistic activity of two antituberculous drug combinations against clinical isolates of Mycobacterium tuberculosis resistant to isoniazid

pp-107

Stoffels K, Traore H, Van Hoof R, Fauville-Dufaux MTobramycin-clarithromycin combination on Mycobacterium tuberculosis clinical isolates

pp-108

Au-Yeang CKW, Au TK, Chan EWC, Chan RCYPrevalence of Efflux-Mediated Rifampicin Resistance in Mycobacterium tuberculosis Clinical isolates

pp-109

Leite CQF,PavanFR,MaiaPIS,DeflonVM,SatoDN,AzevedoAA,PoelhsitzGV,LeiteSRA,FranzblauSGintra and extracellular activity of ruthenium complexes against Mycobacterium tuberculosis and their cytotoxicity

pp-110

Leite S,PavanF,MaiaP,DeflonV,BatistaA,SatoD,FranzblauS,LeiteCanti-Mycobacterium tuberculosis activity of thiosemicarbazones, semicarbazones and hydrazones

pp-111

Ramos J, Rodrigues L, Couto I, Amaral L, Viveiros Mmethods for assessment of ethidium bromide transport across Mycobacterium smegmatis cell wall

pp-112

Martins M, Viveiros M, Couto I, Amaral LThe human macrophage as a model to select compounds active against mDr/XDr-Tb

pp-113

Cynamon M, Mookherjee S, Shoen Cin vitro activities of JpC 2067 alone and in combination with SmX against nocardia species

pp-114

Nina JNosocomial Tb in a laboratory setting

pp-115


abSTraCTS Of guEST lECTurES (gl)


gl-1

TubErCulOSiS iN pOrTugal

Miguel VillarConsultant on Tuberculosis, Directorate-General of Health, Lisbon, Portugal

Tuberculosis is a global problem with an estimated number of 9 million cases per year, 83% of which are in Sub-Saharian AfricaandSouth-EastAsia,wherewefindmanyofthehighburdencountries.

Concerning multidrug-resistance tuberculosis (MDR-TB), WHO estimates about half a million cases globally, per year, including 50.000 extensively drug-resistant tuberculosis (XDR-TB).

In 2007, European Union (EU) had an incidence rate of 17/105, having Portugal one of the highest rates in the EU (27/105). Inthelast20years,theincidenceinPortugalhasdecreasedconsistentlymorethan7%peryearinthelastfiveyears.

This reduction is mainly in the age group between 25 and 44 years old, leading to a shift to the right of the median age both in the nationals and in the immigrants.

The foreign born cases have represented about 12% of the cases and the prevalence of HIV has been around 14%.

Most of the cases are pulmonary forms (74.1%) between 2003-2007, 67.5% of which are SS+ and 75.3% are culture positive.

Mixed multidrug-resistance tuberculosis, including XDR-TB, during the same period, represents 1.9% (154 cases) of the TB cases at the start of treatment, varying from 1.3% (22 cases) in 2006 to 2.4% (38 cases) in 2004, with an average of 31 cases per year (1.9%). These proportions are representative as the coverage of drug sensibility tests (DST) is over 80%.

WewilladdresstheimportanceoftheMicobacteriologyLaboratorynetwork,concerningcasedetection,definitionofconfirmedcases,earlydiagnosisofMDR-TBand1stand2ndlineDST.

As an important complement of the DOTS Strategy, the analysis of the outcomes will be discussed, concerning not only the general population but also the different risc groups, having as a goal the 85% cure rate proposed by WHO.

WefinishourpresentationaddressingthestrategyforMDR/XDR-TBcontrolinPortugal,namelytheimportanceofthereference network for MDR-TB with its national coordination.

26 ESM 2009

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ThE braziliaN EXpEriENCE CONTrOliNg Tb mulTi-Drug rESiSTaNCE

Fernando Augusto Fiuza de Melo

Médico, Diretor do Instituto Clemente Ferreira – Coordenadoria de Controle de Doenças da Secretaria de Estado • da Saúde de São Paulo – ICF/CCD/SP

Doutorado em Medicina, área de pneumologia, pela Escola Paulista de Medicina da Universidade Federal de São • Paulo - EPM/UNIFESP

Membro do Comitê deAssessoriaTécnico-Científico do ProgramaNacional de Controle daTuberculose do • Ministério da Saúde - PNCT/MS

Correspondence:

Rua Santo Estácio, 248 – Cidade Vargas, CEP. 04319-010 - Brasil - São Paulo,SPTele-fax:00551132188653-Telemóvel00551184694330-e-mail:[email protected]

Brazilwasthefirstdevelopingcountryusingtheshortdurationregimenofrifampin(R)andisoniazid(H)combinedinonecapsuleforsixmonths,pluspyrazinamide(Z)duringthefirsttwomonthsafterreorganizingtheBrazilianNationalTuberculosis Control Program of (BNTBCP, Programa Nacional de Controle da Tuberculose, PNCT) in 1980. At that moment Brazil adopted the anti-TB regimen named E-1 (2RHZ/4RH) for all forms of TB with no known previous treat-ment. A similar regimen was adopted for Meningitis TB, named E-2, adding corticoids in the intensive phase with the recommendation to lengthen the continuation phase of treatment to 7 months (2RHZCort/7RH). For those TB cases of relapsing (RC) or re-treatment after defaulting (RA) the anti-TB regimen adopted was E-1R with ethambutol (E) during the intensive phase (2RHZE/4RH). The recommended regimen for failure (F-1) cases was named E-3 and included Z and E, associated with streptomycin (S) and the ethionamida (Et) during at least 12 months (3SZEEt/9EEt)¹

The E-1 was evaluated under pragmatic clinical “non-study” conditions during decades 1980/90 and also during half of the firstdecadeofthenewmillenniumandshownanefficacyof94.6and93.9%,aneffectivenessof77.8and77.1%,adefaultrate of 13.7 and 13.1%13%, a failure rate of 1.5 and 1.7%%, a serious adverse events rate of 3.1 and 3.3% and a mortality rate of 3.9 and 4.8%, respectively. (²,³). The small worsening on mortality rate might be related to high rates of defaulting and the HIV co-infection. The good quality of the National Aids Program Control and the increasing rate of TB treatment under direct supervision in Brazil are possible reasons for Brazil rates of resistance were not too high. On the other hand, results ofE-3werenotnicewithefficacyandeffectivenessvaryingbetween57,5%-85,2and66,7-84.7%,respectively(4).

4%of80,000casesofTBnotifiedinBrazilwereresistanttoR+HorwereunabletobetreatedwiththesedrugsforanyotherreasonandweredefinedasF-1case.ThesepatientsweretreatedwithE-3(3SZEEt/9EEt)andareconsideredinBrazil as a case of MDR-TB. Brazil estimates a rate between 0,3 and 0,4% of cases of F-1 not responding to E-3. These casesaredefinedinBrazilasaMultiresistantTB(MRTB)andthereisnowellestablishedanti-TBregimenforthesepa-tients in the BNTBCP5.

In1995ananti-TBregimenincludingamicacin(AM),ofloxacin(OFX),terizidona(TRZ),clofazimine(CFZ)andEwasevaluated in some TB Centers in Brazil with reasonable results (6).

In2000theMonitoringProgramforMRTBwascreated,centralizingthenotificationofallcasesofTBMRinBrazilandcreatingaworkgroupincludinghealthprofessionalsinvolvedwithMRTBinordertodefineandorganizeawaytocontrolMRTB in Brazil. In 2007, Guidelines for MRTB were published presenting the knowledge about TBMR and establishing rules for diagnosis, treatment, prevention and biosafety; providing orientation on epidemiologic surveillance, building hu-manandmaterialresources,andimplementingaspecificNationalNotificationSystemforthesepatients.ThecurrentalternativeregimenforMRTBcasesisdefinedbythesensitivitytestsandadministeredundersupervision,including:AM(or S if sensible) for 12 months, OFX, TRZ and E for 18 months and Z (if sensible) for 6 months7. Metronidazole (MTZ) replacing Z, especially in cases of intolerance or resistance, is used in some clinics.

TBMR rates were evaluated in Brazil between 2000 and 2005, showing the following results:

Between323and334caseshadbeenannuallynotifiedfrom2000to2004,withanincreasein2005to383casesnotifiedamong a total of 80.000 TB cases.

Increasing cure rates over time (40 to 62%), with some organized units showing better rates (75 to 85%).


Default rates between 5 and 7%; failure rates between 10 e 15%; mortality rates had decreased from 33% to 11% over time.

Most of MRTB were post-primary cases (74 to 80%); 6 to 8% were the primary cases, especially contacts and risk groups; 11 to 20% were indeterminate.

TBMR rate among HIV co-infected patients was low, between 1.6 e 3%7,8.

Extensivelymulti-drugresistant(X-MDR)TBcases,presentingresistanceto2firstlinedrugsand3secondlinedrugs,have been observed since 20009, however for more accurate estimates, a national survey is necessary. In recent survey attheClementeFerreiraInstitute,34casesresistanttofluoroquinolonewerenotified,and16caseswerealsoresistantto AM and 18 to S. The patients were treated with an alternative regimen indicated for MRTB, with 9 cure cases and 25 failurecases,including17deaths.Animportantfindingwastheoccurrenceof3primaryX-MDRcases10.

references

MinistériodaSaúde/FundaçãoNacionaldeSaúde/ComitêTécnico-CientíficodeAssessoramentoàTuberculose/ComitêAssessor para Co-infecção HIV-Tuberculose. Tuberculose: guia de vigilância epidemiológica, Brasília, 2002.

Ministério da Saúde/Fundação Nacional de Saúde/Centro de Referência Prof. Hélio Fraga-Rio de Janeiro, Documento Básico da Reunião de Avaliação operacional e epidemiológica do PNCT na década de 80. Bol Pneumol Sanit 1993, Numero Especial.

Ministério da Saúde/Secretaria de Vigilância em Saúde/Centro de Referência Prof. Hélio Fraga-Rio de Janeiro. Análise da situação da tuberculose no Brasil nos anos 90 e início da década atual. Bol Pneumol Sanit 2005;13:133-179.

Campos HS, Melo FAF. Efetividade do esquema 3 (3sSZEEt/9EEt) no retratamento da tuberculose na rotina das unidades de saúde. Bol Pneum Sanit 2000;8:7-14.

MeloFAF,IdeNetoJ,SeiscentoM,PintoJA,AfiuneJB:TuberculoseMultirresistente.JPneumol1993;19:73-82.

Dalcolmo MP, Fortes A, Melo FAF, Motta R, Ide Neto J, Cardoso N, Andrade M, Barreto AW, Gerhardt G. Estudo de efetivi-dade de esquemas alternativos para o tratamento da tuberculose multirresistente no Brasil. J Pneumol 1999;25:70-77.

Ministério da Saúde/Secretaria de Vigilância em Saúde/Centro de Referência Prof. Hélio Fraga/Projeto MSH. Tuberculose multirresistente: guia de vigilância epidemiológica;2005:89pg

MeloFAF,AfiuneJB,IdeNetoJ,AlmeidaEA,SpadaDTA,AntelANL,CruzML.Aspectosepidemiológicosdatuberculosemultirresistente em serviço de referência na cidade de São Paulo Rev da Soc Brasil Med Trop 2003;36:733-40.

Emergence of Mycobacterium tuberculosis with extensive resistance to second line drugs worldwide 2000 – 2004. MMWR 2006;55(11)

Savioli MTG, Melo FAF, Morrone N e Rodrigues DS. Tuberculosis with extensive resistance to drugs in a TB reference center in Sao Paulo, Brazil. Poster accepted for UICTER 2009;Cancum, Mexico.

28 ESM 2009

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EVOluTiONary fOrCES iN MyCoBaCTeriuM TuBerCuloSiS

Sebastien Gagneux

Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, NW7 1AA, London,UnitedKingdom;[email protected],phone:+44208816-2399,fax:+44208816-2564

The Mycobacterium tuberculosis complex (MTBC) consists of genetically monomorphic organisms. Studying the genetic population structure and evolution of monomorphic bacteria is hindered by the lack of DNA sequence variation; methods such as multilocus sequence typing (MLST), which have been well established in other bacteria, are not applicable. Because of this limitation, most current genotyping methods for MTBC are based on mobile or repetitive DNA elements (e.g. IS6110 RFLP, spoligotyping, MIRU-VNTR). Mobile and repetitive DNA regions change relatively quickly, which makes them ideal markers for molecular epidemiological analyses. However, because these markers can exhibit convergent evolution leading to homoplasy (similar patterns emerging in unrelated strains), they are less robust to infer phylogenetic relation-ships. Furthermore, actual DNA sequence data is preferred for population genetic analyses. To get around this problem, we sequenced 89 genes in 108 MTBC strains. We used these DNA sequence data to explore the evolutionary forces that haveshapedthegeneticdiversityinMTBC.OurfindingsshowthatMTBCisundergreatlyreducedselectiveconstraint(i.e.purifying selection is reduced in MTBC), and as a result, much of the genetic diversity in MTBC is likely to have functional consequences.Thesefindingshaveimportantimplicationsforthedevelopmentofnewtoolstocontroltuberculosis.


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aDVaNCES iN ThE mOlECular EpiDEmiOlOgy Of TubErCulOSiS

Dick van Soolingen1, Jakko van Ingen1, Philip Supply2, Anita Schürch1, Ida Parwati3, Reinout van Crevel4, Nguyen Van Hung5, 1- Frank Cobelens6, and Kristin Kremer1 National Tuberculosis Reference Laboratory, Nat. Inst. for Public Health and the Environment(RIVM),Bilthoven,theNetherlands;[email protected],InstitutPasteur,Lille,France3 - Dept. of Clin. Path. Hasan Sadikin Hosp., Med. Fac. Padjadjaran Univ.,Bandung, Indonesia4 - Dept. Int. Med., Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands5 - Nat. Hosp. of Tuberculosis and Respiratory Diseases. Hanoi, Vietnam6 - Center for Poverty-related Comm. Dis., Acad. Medical Center, Amsterdam, the Netherlands

Although elimination of tuberculosis in Europe is not yet in sight, the ECDC held a meeting in Stockholm in April 2009 tore-definetheindicatorsofasuccessfulTBcontrolonthiscontinent.Molecularepidemiologywasidentifiedasakeycomponent to detect the level of active transmission. In 2009, a new ECDC project has been initiated to re-activate the molecular surveillance of (MDR/XDR) TB in Europe, with strong focus on a high coverage of MDR/XDR cases in Central and Eastern Europe. In the previous project transmission of MDR/XDR-TB in Europe was largely caused by Mycobacterium tuberculosis Beijing genotype strains.

Inarecent(2009)resistancesurveyinVietnam,asignificantcorrelationbetweenresistanceandBeijingstrainswasob-served. Moreover, in previous studies in Ho Chi Minh City treatment failures and relapses were more frequently found in patients infected by Beijing strains. However, in a recent, larger study in Indonesia patients infected with Beijing geno-type strains also more often had a positive sputum culture after six months treatment (RR:1.95; CI 95%:1.25-3.02), but this was not correlated with differences in drug resistance. Therefore, this suggests that M. tuberculosis Beijing genotype strains have a higher capacity to withstand tuberculosis treatment, even in the absence of drug-resistance.

The new European network on molecular epidemiology will implement 24-loci VNTR typing as a standard. Although the utility of VNTR typing has been shown in multiple studies, a broad and nation wide comparison of IS6110 RFLP typing and VNTR typing is still missing. In the Netherlands 4400 M. tuberculosis isolates from the period of 2004-2008 have been subjected to IS6110 RFLP as well as VNTR typing and a concordance of 81% has been observed. Moreover, VNTR typing showedahigherdegreeofconcordancewithfindingsintheconventionalcontacttracingthanRFLPtyping.

To come to the highest resolution of DNA typing, two isolates from the Harlingen tuberculosis outbreak, that have been isolated with an interval of 12.5 years and which were separated by four person-to-person transmissions were subjected to whole genome sequencing. Four single nucleotide polymorphisms (SNPs) and one tandem repeat polymorphism (TRP) and a IS6110 transpositionwereidentified.Typingofall104isolatesintheIS6110 RFLP cluster with the six DNA polymorphismsendorsedtheseparatelineoftransmissionestablishedbycontacttracing.Thesefindingssuggestthatthemicroevolution of M. tuberculosis can be used to resolve separate transmission chains in large outbreak clusters.

30 ESM 2009

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SurrOuNDED by myCObaCTEria

Joseph O. Falkinham, IIIDepartment of Biological SciencesVirginia TechBlacksburg, Virginia 24061-0406Phone 1-540-231-5931FAX 1-540-231-9307E-mail [email protected]

Humans, animals, and plants are surrounded by mycobacteria. The environmental opportunistic mycobacteria (also called nontuberculous or atypical mycobacteria) include over 100 species; many of which cause disease. Infections include cer-vical lymphadenitis in children and pulmonary disease and skin infections in adults. Evidence that the environment was thesourceofhumandiseasewasgainedfromtheidentityofDNAfingerprintsofmycobacterialisolatesfrompatientsand either their household water or potting soils. Recently, a number of reports have documented a dramatic increase in pulmonary disease caused by these mycobacteria amongst elderly and slender men and women who lack all of the classic predisposing risk factors (e.g., smoking, exposure to dusts). Although slowly growing with generation times of one-half to one day, the environmental opportunistic mycobacteria survive, grow, and persist in a number of habitats that are shared with humans and animals. The environmental opportunistic mycobacteria are oligotrophs; able to grow in water containing greater than 50 µg AOC/L. Survival and persistence in the environment is due, in part, to the thick, impermeable, hydrophobic, lipid-rich envelope of mycobacterial cells. Although the hydrophobic wall reduces the rate of transferorhydrophilicnutrients,itpromotesattachmenttosurfaceswheremycobacteriaformbiofilms.Hydrophobicityalso contributes to disinfectant- (e.g., chlorine and biocides) and antibiotic-resistance. For example, mycobacteria enter-ing a water treatment system on particulates survive disinfection and grow during travel in the distribution system in the absence of competitors. Hydrophobicity also promotes the aerosolization of mycobacteria from water to air in environ-ments such as showers and hot tubs in the home and occupations where aerosols are generated.


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ThE uSE Of iNTErfErON gamma rElEaSE aSSayS aS aN aiD iN ThE CONTrOl Of TubErCulOSiS

Jean-Pierre ZellwegerSwiss Lung Association, Berne, Switzerland

Interferon Gamma Release Assays (IGRAs) are in vitro tests detecting the presence of latent tuberculosis infection (LTBI) in asymptomatic persons who may have been infected by M. tbc inarecentorremotepastandwhomaybenefitfromapreventive treatment to decrease the risk of later reactivation of tuberculosis.

Basically, the IGRA tests rely on the same immunological phenomenon as the tuberculin skin tests, but they do it in a muchmorespecificway,becausethetestsarenotinfluencedbyapriorvaccinationwithBGCorbyaninfectionwithmost of the non-tuberculous mycobacteria present in the environment. Therefore, the indications and the use of the IGRA tests are fundamentally the same as for the tuberculin skin tests :

Detection of LTBI in persons in contact with an index case of tuberculosis•

Detection of LTBI in persons with a high risk of tuberculsois, if infected (immunosuppressed patients, patients • receiveing or due to receive immunosuppressive therapy, small children)

Surveillance of exposed health care workers (as the test can be repeated without risk of inducing a booster effect)•

Aid to the diagnosis of tuberculosis in cases where a bacteriological examination is not feasible or not reliable • (severe extrapulmonary TB, TB in children)

In spite of their superiority, the IGRAs are not totally devoid of problems in practice and the best use of them is still amatterofdebate.SomeGuidelinesrecommendtheiruseonlyfortheconfirmationofpositiveTSTamongcontacts(the so-called two-step testing procedure) whereas others recommend the routine replacement of the TST by IGRAs. Performingonlyonetestiseasier,andavoidsapossibleinfluenceofapriorTSTontheIGRAresponse.

The predictive value of the new IGRAs seems to be superior to the predictive value of TST, reinforcing their usefulness, if the preventive treatment is corectly precribed and followed. One intriguing phenomenon is the possible reversion of a positive IGRA after conversion, possibly indicating that some infected contacts may be able to eradicate the mycobac-teria without treatment.

32 ESM 2009

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a NOVEl DiagNOSTiC TEST TO DiffErENTiaTE laTENT Tb iNfECTiON aND aCTiVE DiSEaSE

Lee W. RileyMD, School of Public Health, University of California, Berkeley

It is well recognized that the treatment of latent TB infection (LTBI) is a highly effective TB prevention strategy, which is still not widely practiced in most parts of the world. Most TB-endemic countries rely on BCG vaccine to prevent TB. LTBI treatment requires contact investigation, which is not done in most “BCG countries”. One reason for this reluc-tance to practice contact investigation is the lack of a reliable test that can distinguish LTBI from TB. Thus, a test that can unequivocally distinguish LTBI from TB could alter the current national prevention programs in TB-endemic countries. WehaveidentifiedasetofM. tuberculosis cell wall proteins that are expressed when the bacilli replicate in vivo, but not when they are in a nonreplicative state. Their continued expression is associated with disease progression in infected mice, and mouse T cells are sensitized as these proteins are continually expressed in vivo. Exploiting this observation, we developed a bioassay that is able to distinguish LTBI from active disease in a mouse model. The assay is based on IFNγ induction by T cells exposed to a set of synthetic peptides based on the cell wall protein called Mcep1A. The Cornell mouse model was used to study the response of spleen cells exposed ex vivo to these peptides. Cells from untreated mice expressed 7-9-fold higher levels of IFNγ than those from treated mice at 24 and 32 weeks of infection, as mea-sured by ELISA. Blood cells from healthy tuberculin skin-test positive, QuantiFERON-negative (n=3) and TST-negative, QuantiFERON-negative (n=3) human volunteers showed no response to the peptides. These peptides are currently under evaluation in newly diagnosed TB patients. If the assay can show a response in these TB patients at levels similar to those observed in diseased mice, this assay can be converted into an immunochromatographic (“dip stick”) format. Such a test then can be used to readily differentiate those with LTBI and active disease, and could then be incorporated as part of National TB Control Programs.


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NEW liVE TubErCulOSiS VaCCiNE STraTEgiES

Jesus Gonzalo Asensio, Ainhoa Arbues and Carlos MartínDepartment of Microbiology, University of Zaragoza. Spainhttp://genmico.unizar.es

BCG, the current vaccine against tuberculosis (TB), has been used for more than 80 years but is ineffective at providing protection against adult pulmonary TB. New tuberculosis vaccine candidates and TB vaccination strategies, conferring better protection against pulmonary tuberculosis than the current vaccine BCG, are needed.

In the recent decade, a global pipeline of novel TB candidates has emerged. Pioneering strategies for the development of more effective vaccines today have lead to the discovery of subunit vaccines, which have proved ineffective at provid-ing better protection that BCG in various animal models. Different heterologous prime BCG and boost with subunit strategiesareinclinicaltrialswiththeaimtoimproveefficacyofBCG.Morerecently,clinicaltrialswithrecombinantBCGvaccineshavestartedwiththeaimtofindcandidatestobeusedasprime,preventivevaccines.Anotherinnovativestrategy, live attenuated Mycbacterium tuberculosis vaccines, in late preclinical investigation, are promising new preventive vaccine candidates to replace BCG.

Based upon the observation that phoP is an essential gene for M. tuberculosis virulence, we rationally attenuated the tubercle bacillus by inactivating phoP (Perez et al, Mol Micro 2001). The mutant was shown to be strongly attenuated in cellular and animal models. Moreover, the phoP mutant resulted more attenuated than BCG Pasteur in immunocompro-mised SCID mice and this vaccine candidate protected guinea pigs and non human primates against tuberculosis infec-tion (Martin et al Vaccine 2006, Verreck et al PLoS ONE 2009).

Both, the attenuated phenotype and the protective immunity conferred against tuberculosis infection can be accounted for by the mechanism of action of PhoP, which has been recently shown to be crucial for intricate virulence network of M. tuberculosis (Gonzalo Asensio et al PLoS ONE 2008). This observation was used to construct a new generation of live vaccines based on phoP inactivation carrying a second additional mutation which affects the synthesis of a new family of lipids associated to M. tuberculosis virulence.

It is estimated that at least 20 vaccine candidates should enter phase I safety trials with around half going forward for immunologicalevaluationinphaseIItrialsandleadingtofourphaseIIIefficacytrialswiththegoaltoreachaneffectivelicensed vaccine in 5-7 years (Young and Dye, Cell 2006). The discovery and use of a new TB vaccine better than BCG is key to reach the 2050 objective of TB eradication.

34 ESM 2009

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rEgulaTiON Of MyCoBaCTeriuM TuBerCuloSiS CEll Wall lipiD COmpOSiTiON aND iTS EffECT ON iN ViVO baCTErial pErSiSTENCE

Lee W. RileySchool of Public Health, University of California, Berkeley

The hallmark of M. tuberculosis is its ability to survive for many years in an infected host to establish latent tuberculosis infection (LTBI). We propose a new model of latent infection that is based on the idea that this organism may simply have readapted its “housekeeping” metabolic function to a new environment for its long-term survival. We propose that M. tuberculosis, which evolutionarily most likely originated in soil, has readapted a soil-survival strategy its ancestral species possessed to the granuloma environment in the human host. Granuloma cells constantly turn over every few days to weeks, and after they die, they undergo replacement by new cells that migrate into the granuloma. Hence, M. tuberculosis needs to readapt to this constantly changing environment, and we provide evidence that this adaptation is mediated by M. tuberculosis remodeling its cell envelope in response to signals produced by dead granuloma cells. This remodeling is mediated by a family of operons called mce (mce1,2,3,4). Disruption of the operons results in profound changes in lipidprofileofthecellwall.Themce1 operon mutant causes free mycolic acids (MA) to accumulate on its surface, and other operon mutants (mce2,3,4)showevidenceoflipidprofilechangesinthecellwall.Theseoperonproductsserveasenergy-dependent lipid importers. Lipid products released from dead host granuloma cells that turnover may be used as carbon sources for the resident M. tuberculosis. Thus, the “housekeeping” lipid metabolic function of M. tuberculosis may have been readapted in the granuloma environment as a way for this organism to survive, similar to the way its ancestral saprophytic organism survived in soil by scavenging dead organic materials as carbon sources. Further elucidation of the interaction between M. tuberculosis cell wall and granuloma cell turnover may contribute to a new understanding of the mechanism of LTBI.


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CDC’S glObal Tb labOraTOry aCTiViTiES

Thomas M. Shinnick,Associate Director of Global Laboratory Activities, Division of TB Elimination, Centers for DiseaseControlandPrevention,1600CliftonRoad,MS-G35,AtlantaGeorgia30333USA.email:[email protected];FAX:1-404-639-1287; Tel: 1-404-639-1474

A key bottleneck in health service delivery is weak laboratory capacity. This is particularly true for drug-resistant TB — less than 5% of MDR TB cases are currently being detected globally. To meet the 2015 targets of the Stop TB Global Plan, 60 million culture tests and 5 million drug susceptibility tests will be needed annually. This will require estab-lishing at least 2,000 new culture laboratories and training of more than 20,000 laboratorians. At least US$ 1 billion will beneededannuallyforbuildingTBlaboratoryinfrastructureandrecurringcosts.However,thebenefittocostratioofsuch investments is estimated to be 9:1 in populations with a high prevalence of HIV infection. Meeting the 2015 goals could save countries in sub-Saharan Africa alone as much as US$ 52 billion annually.

While trainingofbench-level technicians reliesonTB-specificexpertise, laboratorycapacitybuilding reliesmoreoncross-cutting expertise in infrastructure, biosafety, human resource development, supply chain management, logistics, quality assurance programs, management principles, information systems, data management, and accreditation processes. As such, TB laboratory strengthening efforts can build on lessons-learned from the building of laboratory networks for polio,measles,SARS,influenza,HIV/AIDS,andotherdiseases.

The goal of our TB laboratory strengthening efforts is the creation of a network of laboratories that can provide reli-able, high quality testing and which is based on quality laboratory management principles and integrated across disease programs, especially HIV and TB. A systems approach is used to optimize laboratory testing and information exchange. The approach involves understanding the structure, performance, and cost of the network; developing referral processes toensurepromptflowofspecimensandinformation;andusingquality-improvementprinciplestocontinuallyevaluateand improve the performance of the network. While TB laboratory strengthening plays the central role in our efforts, the overriding goal is to ensure that a broader health systems approach is used to maximize the impact and sustainability of the investments.

36 ESM 2009

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DEVElOpmENT aND ValiDaTiON Of NEW Tb DiagNOSTiC TESTS iN brazil: EXpEriENCE Of rEDE-Tb

Brazilian Tuberculosis Research Network: A Multi Disciplinary Collaborative Research Project

introduction

InBrazil,whichhasanestimated124,000casesofTBperyear,therehasbeenasignificantgapincommunicationandunderstanding between TB programmatic experts, academics, the community, and nongovernmental organizations. In rec-ognition of this gap, a National TB Research Network was established in 2002 to bring these constituencies together to promote an integrated, multi-disciplinary and multi-institutional strategy for TB control in Brazil. In the last years, Rede-TB has established a solid relationship among the National Tuberculosis Control Program and Oswaldo Cruz Foundation thathashelpedtofosterBrazilianleadershipandcompetencyinthedevelopmentandevaluationunderfieldconditionsof new diagnostics for TB.

Objectives

To develop new diagnostics, vaccines and drugs for the prevention and cure of TB, including MDR-TB, and develop improved, therapeutic alternatives with the renewal of drugs, new formulations, drug associations, use of drugs already available in the market, and immunotherapy.

To perform pre-clinical and clinical studies of new diagnostic tests against TB using adequate ethical standards and to capacitate clinical sites for explanatory and pragmatic trials.

To carried out pragmatic clinical trials and cost-effective analysis of alternative interventions for TB control that include diagnostic methods and health service strategies

To improve case detection by changing health behaviour and mobilizing communities.

To build a successful research partnership model that has every potential to stimulate changes in national standards and practice in ways that serve country needs.

Conclusions

Through this approach, it is expected that locally algorithms based on clinical features, antibiotic response, and chest ra-diographywillbevalidated,andclearguidelinesissuedaboutwhichpatientsmightalsobenefitfrommycobacteriacultureor new phenotypic / molecular diagnostic techniques. Additionally, the incorporation of new tests into clinical practice will better planned and regulated, and national policy makers with better decision-making models will be provided.


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EXpEriENCE Of a SuCCESSful myCObaCTEriOlOgy labOraTOry NETWOrK iN ESpiriTO SaNTO- brazil

Moisés PalaciUniversidade Federal do Espírito Santo, Vitória, Brazil

The State of Espírito Santo occupies an area of approximately 6,750 square miles on the coast of Brazil. The economy is mainly based on the production of steel, harborage activity, agriculture, a large number of small industries, and tour-ism. The population of the State is approximately 3.2 million, with the majority living in metropolitan Vitória, the capital, which is located on an island and connected to the mainland by several bridges. The annual incidence of TB on the island of Vitória is approximately 70 cases per 100,000 inhabitants. Each year approximately 1,500 new cases of TB (65% smear positive) are reported for the State of Espírito Santo with 60% occurring in the City of Vitória and its 5 neighboring cities.The Núcleo de Doenças Infecciosas has organized a local network of mycobacteriology laboratories in the Epírito State, Brazil, Five local laboratories have been integrated into this network. This network was established by NDI researchers and is committed to keep a partnership with the City Department of Health of each location and their laboratories. The firstphaseofthesepartnershipsconsistedinreformingandrestructuringthelaboratoriestoenablethemtoaccomplishthe technical requirements, data processing, and biosafety regulations involved in these projects. To this extent, basic equipment and computers were installed to allow for the maintenance of mycobacterial culture and sharing of data over an Internet database. The second phase consisted of training laboratory staff to properly and safely complete the neces-sary bench work and data processing procedures. Therefore, a considerable efforts and time has been spent for setting up and to keep this system working. With the establishment of this network, NDI has gained earlier access to TB patients and better conditions to conduct several clinical trials, including IND trials. In addition, capacitating local TB laboratories in the metropolitan region of Vitória to perform mycobacterial cultures, allowed us to increase the detection rate of TB cases at about 24%.

38 ESM 2009

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SCrEENiNg Of mOlECulES WiTh aNTi-Tb aCTiViTy, frOm ThE braziliaN CErraDO plaNTS, aND SyNThETiC mETallO-OrgaNiC COmpOuNDS

Clarice LeiteUniversidade Estadual Paulista, Faculdade de Ciências Farmacêuticas

Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death. Among all countries in the Americas, Brazil reports the second-highest TB mortality and morbidity, comprising a preva-lence of 62/100.000. The global resurgence of TB and the rapid emergence of MDR-TB, underscore the importance of the development of new antituberculous drugs.

Plants have provided many drugs in the past, and they remain a rich source of novel compounds. Plant extracts are among the most attractive sources for developing new drugs and have been shown to produce promising results in the treat-ment of several diseases. Our research group deals in projects that integrate the chemical and anti-TB activity of plants that compose the bioma of the Brazilian Cerrado, a savannah like vegetation. Many of those plants are commonly used as natural remedies by people living in these areas to treat many illnesses. To perform the phytochemical step we used chromatographic techniques, and to determine the structure of the isolated compounds we used mainly spectrometric methods. To evaluate the activity of the extracts, enriched fractions and pure substances against M. tuberculosis we use the resazurin microtiter assay (REMA) and M. tuberculosis H37Rv ATCC 27294 strain. In total were studied 77 extracts from 39 plants, distributed into 20 families. From all extracts assayed 23% showed promising activity, bellow or equal to 125 µg/mL. The triterpene bassic acid from B. fagifolia showed strong antitubercular activity with MIC values of 2.5 µg/mL comparabletoMICsofsomefirst-linetuberculosisdrugs.Theresultsindicatedthatplantsof"cerrado"presentfractionsand compounds with promising anti tuberculosis activity.

Bytheway,theuseofnaturalcompoundsfromplantsisproblematic,duetodifficultyinobtainingpuresubstancesandtheir low availability.

Within the pipeline of new synthetic compounds with potential effectiveness in the treatment of TB, there are 7 novel compounds, which are in various stages of clinical development. Inside this group however, there are complexes that associate metals to organic compounds. Using the thiosemicarbazones, semicarbazones and hidrazones derivates as li-gands, we proposed the complexation with Vanadium, to obtain organo-metallic compounds. We determined the anti-M. tuberculosis activity of these compounds using REMA and the study of the citotoxicity of the ligands and complexes was performed using murine macrophage cell line J774. We analyzed 37 compounds (14 free ligands and 23 vanadium com-plexes) and from of this, 17 (46%) presented promising MIC values varying between 0.97 and 7.80 µg/mL. The vanadium complexes of hydrazones, semicarbazones and tiossemicarbazones derivates showed high antiTB activity, most of the time this activity was increased from 2 to 10 times when compared with the free ligands. However due to high citotoxic-ity of hydrazones, semicarbazones and tiossemicarbazones derivates, the increase in the activity of the complexes didn’t compensate the citotoxicity of the ligands.


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CliNiCal TrialS Of DrugS aND DiagNOSTiC TESTS: ThE ChallENgES iN myCObaCTEriOlOgy

Moisés PalaciUniversidade Federal do Espírito Santo, Vitória, Brazil

Tuberculosis (TB) therapy has three major microbiologic goals: (1) initial killing of actively multiplying organisms in order to achieve early control of the disease and reduce infectivity (early bactericidal activity [EBA]); (2) eliminating slowly growing mycobacteria in order to minimize relapses (sterilizing activity); and (3) preventing the emergence of drug resistance (1). Currently, two month sputum culture conversion on solid medium is the best established predictor of treatment outcome. Early bactericidal activity (EBA), determined by the serial decline in sputum M. tuberculosis colony counts(CFU),isacommonlyusedtoolforcomparingnewdrugstocurrentanti-TBdrugsanddosefinding.EBAhasbeenmeasured as the rate of decrease in colony counts of mycobacteria in quantitative sputum cultures obtained during the firstdaysoftherapy.MeasurementofEBAisintendedtobearapidmeansofassessingtherelativepotencyofnewdrugsduring early treatment. The development of new drugs for TB treatment has been hampered by the lack of an early sur-rogatemarkerthatreflectslongtermnon-relapsingcure.MeasurementofEBAbyquantitativeculturehoweveristimeconsuming and labor intensive. The ideal marker would measure events early during treatment and be accurate regard-less of the drug action or regimen being tested. In this lecture we will explore the potential role and the main limitation of surrogate markers for TB treatment.

40 ESM 2009

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NEW, EaSy-TO-uSE TOOlS fOr QualiTy-CONTrOllED gENOTypiNg Of M. TuBerCuloSiS COmplEX STraiNS

Caroline Allix-Béguec1,2,3, Christine Hubans3, Stéphanie Ferreira3, and Philip Supply1,2,3

1 - INSERM U6292 - Institut Pasteur de Lille, Lille3 - Genoscreen, Lille, France, France

Mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing has become a major method for fast and high-resolution genotyping of Mycobacterium tuberculosis complex isolates. A system based on 24 loci has been proposed for international standardization. Several population-based studies have been published, showing similar predictive value of this method compared to the previous gold standard IS6110 RFLP for studying tuberculosis transmission in Western European settings. As a result, this method is being internationally adopted, often in combina-tion with spoligotyping, as the new standard method for TB molecular epidemiology. New, easy-to-use tools and options have recently become available, which facilitate quality-controlled use of this technique and interpretation of the results obtained. MIRU-VNTR typing services are already used by international Reference Centers and laboratories, for out-sourcing their genotyping (including of M. bovis strains) and/or for QA/QC evaluation. Quality-controlled MIRU-VNTR calibration,validationandtypingkits,aswellason-sitetrainingsgreatlyfacilitatestandardizedsetupandefficientuseofMIRU-VNTR typing in user’s laboratory. Bioinformatic tools, including MIRU-VNTR Data Manager, have been developed for further automating and streamlining the genotyping process, as well as ensuring direct compatibility with MIRU-VNTRPlusDatabasefordatainterpretation.Wehopethattheavailabilityofthesetoolsforeasierandmoreefficientreal-time genotyping will contribute to improve molecular-guided TB control and surveillance.


abSTraCTS Of Oral prESENTaTiONS (Op)


Op-1

maSS SpECTrOmETry fOr mOlECular TypiNg Of ThE myCoBaCTeriuM TuBerCuloSiS COmplEX: ONE plaTfOrm aND mulTiplE aSSay fOrmaTS

C. Honisch1, M. Mosko1, C. Arnold2, S. Gharbia2, S. Feuerriegel3, S. Niemann3

1 - SEQUENOM, Inc., San Diego2 - Health Protection Agency, London3 - Molecular Mycobacteriology, NRC for Mycobacteria, Forschungszentrum Borstel, Borstel

Objectives

The analysis of nucleic acids by mass spectrometry has evolved to a user friendly technology for characterizing DNA, and RNA via SNP genotyping and comparative sequencing in clinical research, agricultural applications, molecular medi-cine and non-invasive prenatal diagnostics research. Recently, the technology has become a versatile tool for microbial detectionandidentificationutilizingcomparativesequenceanalysis.Anexampleisthesuccessfulapplicationto16Sbasedtyping of mycobacteria. Here, we adopted the technology to perform high throughput spoligotyping and detection of resistance conferring SNPs.

methods

Assays were designed in silico for spoligotyping analysis and detection of key resistance mutations. Both assays were evalu-ated by using well characterized reference collections.

results

For MassARRAY 43 spacer oligonucleotide probes were designed and grouped into two multiplexed assays (TypePLEXTM). Over 200 characterized strains from different reference centers representing the major M. tuberculosis complex lineages were analyzed by the MassARRAY spoligotyping assays. Results were in concordance with classical spoligotyping data. For detection of resistance mutations, assay were developed based on the MassCLEAVETM system. Resistance regions areamplifiedbyPCRwithataggedprimersystemfollowedbyin vitro transcription of both DNA strands. Subsequent endonuclease digests of the RNA transcripts at the bases cytosine and uracil result in four mixtures of RNA cleavage products.Resistanceis identifiedbycorrelatingacquiredspectrawiththeoreticalpeakpatternspredictedfor in silico cleavagesofsequencescontainedinareferencedatabase.Thefirstassayshavebeensuccessfullyevaluatedinasetofreference strains, further analyses are in progress.

Conclusion

Massspectrometryspecificassayformatsforgenotypingandcomparativesequenceanalysisgeneratehighlyaccuratequalitative and quantitative data and provide a toolbox for molecular typing of microbes and viruses. Existing typing schemes can be translated onto the mass spectrometry platform and new typing schemes can easily be developed.

44 ESM 2009

Op-2

CluSTEriNg Of SpOligO-paTTErNS: TOWarDS aN auTOmaTiON Of MyCoBaCTeriuM TuBerCuloSiS COmplEX ClaSSifiCaTiON

Borile C 1, Refrégier G 2, Labarre M 1, Franz S 1, Mézard M 1, Sola C 2

1-LPTMS,bât.100,UniversitéParis-Sud,Centrescientifiqued’Orsay,15rueGeorgesClémenceau,91405Orsaycedex2-IGEPEbât.400,UniversitéParis-Sud,Centrescientifiqued’Orsay,rueGregorMendel,91405Orsaycedex

Spoligotyping isatypingmethoddetectingthepresenceorabsenceofspecificregionscalledspacers.Thesespacersare grouped on what is called the DR locus (Direct Repeat locus) that belongs to the CRISPR locus family (Clustered Regularly Interspaced Palindromic Repeats). The DR locus in Mycobacterium tuberculosis complex is believed to evolve solely by deletion.

Usingthespoligotypingtechnique,specificfamiliesofMycobacterium tuberculosis complex strains have been recognized, showingthattheDRlocusisphylogenetically informative.Specificsignaturesarerecognizedbyexpertssothateachspoligo-patterniseasilyassignedtoaspecificfamily.Amazinglyhowever,whenusingallavailablesoftwaresforclusteringthedata,thestrainsofaspecificfamilyarenotalwaysclusteredinthesamegroupeusingvariousmethods.

We implemented a new algorithm to cluster these data. Until now, the single publicly available software to achieve this task, SpotClust, provided only sub-optimal results. Our system is based on an evolutionary model taking into account that spacers can be deleted as a large group. This is not the case when using the Jaccard distance in the commonly used Bionumerics software, that mimicks a one-by-one spacer deletion process.

Wewillpresentdatashowingunderwhatconditionsthisalgorithmgivessignificantlybetterresultsthanthecommonlyused one. This studies leads toward an automation of Mycobacterium tuberculosiscomplexclassification,anotherwisetimeconsuming and sometimes debatable topic.


Op-3

iDENTifiCaTiON Of ThE iNSErTiON ElEmENT iS6110 iN PHoP prOmOTEr iN a high TraNSmiSSiON MyCoBaCTeriuM TuBerCuloSiS STraiN: a CluE TO phENOTypiC VariaTiON

Andrea Sandoval1,2, Andrés Cubillos1,2, Alejandro Reyes3, Nidia Correa2,4, Jaime Robledo2,4, Maria Mercedes Zambrano1, and Patricia Del Portillo1,2

1 - Corporación CorpoGen, Bogotá, Colombia. 2 - Centro Colombiano de Investigación en Tuberculosis CCITB, Bogotá, Colombia.3 - Center for Genome Sciences, Washington University School of Medicine, St. Louis, Missouri, USA4 - Corporación para Investigaciones Biológicas CIB, Medellín, Colombia

aim

The insertion element IS6110 can mediate genetic diversity in Mycobacterium tuberculosis (MTB) strains due to its capac-ity to move and cause rearrangements, deletions and insertions. Transposition of these elements can therefore affect gene expression and alter the phenotype of MTB. In this study we analyzed the insertion sites of IS6110 in two clinical MTB Haarlem genotype strains that present differences in transmissibility as demonstrated in a cohort of patients and household contacts from Colombia.

methods

Restriction Fragment Length Polymorphism (IS6110-RFLP) was performed and revealed genomic differences between the two strains. DNA was isolated, digested with XmaIandligatedtospecificadaptersdesignedforthispurpose.LigationMediatedPCR(LM-PCR)wascarriedouttoamplifytheregionsflankingIS6110 insertion sites. A library containing the amplifiedproductswasconstructedandsequencedclonesweremappedagainsttheannotatedsequencedgenomes.PCRwasusedtoconfirmthesitesofinsertion.

results

Twelvedifferent insertionswereidentified;ninewerecommontobothstrains(Rv2336,Rv1754c,Rv0963c,Rv0403c,Rv1358,Rv2813/DR,Rv2254c,Rv0795andPPE34),twowerespecificforthehightransmissionstrain(DRregionandtranscriptional regulator phoP), and one was found just in the low transmission strain (PPE46).

Conclusions

LM-PCR allowed us to identify IS6110flankingregionsandlocalizedthegenomicdifferencesbetweentwostrainswithcontrasting transmission pattern. Most of the insertions occur in conserved hypothetical proteins, followed by proteins involved in cell wall synthesis and cell processes, regulatory proteins and members of the PE/PPE family. The DR region, considered a hotspot for insertions, had three insertions, two common to both strains and one in the high transmission strain. Since the phoP gene regulates several functions implicated in virulence, it is possible that the IS6110 insertion in the phoP promoter could enhance transmission by acting as a portable promoter and inducing gene expression, as has been reported before in M. bovis.

acknowledgment

Consorcio Colombiano de Investigación en Tuberculosis, CCITB. 4312004

46 ESM 2009

Op-4

gENOmiC iNTErrOgaTiON Of MyCoBaCTeriuM TuBerCuloSiS iSOlaTES frOm brazil

Oelemann, Maranibia C 1, Gomes, Harrison M 1, Willery, Eve 2, Lima, Karla Valéria B 3, Possuelo, Lia 4, Locht, Camille 5, Goguet de la Salmonière, Yves-Olivier L 6, Gutierrez, Maria Cristina 6, Supply, Philip 7, Suffys, Philip N 1

1 - Laboratory of Molecular Biology Applied to Mycobacteria, Oswaldo Cruz Institute, Rio de Janeiro, Brazil2 - Laboratory of Molecular Mechanisms of Pathogenesis of Respiratory Pathogens, INSERM U629 and Institut Pasteur de Lille, France3 - Evandro Chagas Institute, Belém, Brazil4-CenterofScientificandTechnologicalDevelopment,PortoAlegre,Brazil5 - Laboratory of Molecular Mechanisms of Pathogenesis of Respiratory Pathogens, INSERM U629 and Institut Pasteur de Lille, France6 - Department of Infection and Epidemiology, Institut Pasteur, Paris, France7 - Laboratory of Molecular Mechanisms of Pathogenesis of Respiratory Pathogens, INSERM U629 and Institut Pasteur de Lille, France1 - Laboratory of Molecular Biology Applied to Mycobacteria, Oswaldo Cruz Institute, Rio de Janeiro, Brazil

background

The Latin-American Mediterranean (LAM) spoligotype “clade” is among the six major M. tuberculosis spoligotype families and is particularlyprevalentinSouthAmerica.Incertainregions,thereisadominanceofgeographicallyspecificandgeneticallyhomo-geneous strain lineages. Here, we have studied the genetic diversity and the consistency of the LAM and other families, by analyzing Mtb isolates from three Brazilian regions including Rio de Janeiro (South East), Belém (North), and Rio Grande do Sul (South).

methods and findings

A PCR-based standardized genotyping system, based on amplification of 15 to 24 mycobacterial interspersed re-petitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to beproficient formolecular-guidedevaluationofTB transmission.We tested the applicabilityof this system formo-lecular epidemiological analysis of 369 M. tuberculosis isolates from three regions of Brazil. Deligotyping, target-ing multiple large sequence polymorphisms (LSPs), and MIRU-VNTRplus identification database were additionallyused to confirm phylogenetic identification.The high congruence between the different typing results showed thecountrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. Nevertheless, by distinguishing 321 genotypes among the 369 isolates, combined MIRU-VNTR typing and spoligo-typingdemonstratedthepresenceofmultipledistinctclones.Noteworthy,27of the32clusters identifiedwereex-clusively composed of patient isolates from a same city, consistent with expected patterns of local TB transmission.

Conclusions

Notwithstanding the challenges, high-capacity mycobacterial genotyping may now become a usable tool to guide TB control efforts, at least on sentinel sites or targeted risk-populations in high TB burden countries. The interro-gation of large sequence polymorphisms (LSPs) revealed that certain lineages or clones present genomic fea-tures that could change the phenotype and play a role in the clinical properties of specificMtb strains.This is thefirst countrywide report of an in-depth analysis on the genomic diversity of M. tuberculosis isolates from Brazil.

acknowledgments

Fiocruz, CAPES, CNPq, FAPERJ, ICOHRTA, NIH, INSERM and Institut Pasteur.


Op-5

pENiTENTiary pOpulaTiON Of MyCoBaCTeriuM TuBerCuloSiS iN KyrgyzSTaN: EXCEpTiONally high prEValENCE Of ThE bEiJiNg gENOTypE aND iTS ruSSia-SpECifiC SubTypE

Igor Mokrousov 1, 2, Violeta Valcheva 1, 3, Nurmira Sovhozova 4, Almaz Aldashev 4, Nalin Rastogi 1, Jainagul Isakova 4

1 - Institut Pasteur de Guadeloupe, France2 - St. Petersburg Pasteur Institute, St. Petersburg, Russia3-InstituteofMicrobiology,Sofia,Bulgaria4 - Institute of Molecular Biology and Medicine, Bishkek, Kyrgyz Republic

Objective

To identify genotypes and drug resistance properties of M. tuberculosis isolates from Kyrgyzstan’s prison inmates, a population with high risk for TB; to compare in regional and global context.

methods

56 M. tuberculosis DNA samples from sputum of HIV-negative Kyrgyz prison inmates, 2008, were typed by spoligotyping, VNTR (12 MIRU and 3 hypervariable [HV] loci), IS6110-inverse-PCR, LAM-PCR. rpoB and katG mutations were detected using TB-Biochip kit.

results

Beijing genotype was detected in 42 of 56 samples. 12-locus MIRU-VNTR typing showed 8 of 56 samples to be mixed cases; 7 of them contained a Beijing strain. MIRU analysis demonstrated a high homogeneity of the studied collection (HGI=0.66)while28of56strainshadaprofile223325153533correspondingtoBeijing/M2subtypehighlyprevalentindifferentRussiansettings(Mokrousov,2004,2008).FourBeijingstrainsbelongedtotypesM33andM70specificforEastAsia.Regardingnon-Beijingvariants,acomparisonoftheirspoligoprofileswithSITVIT2database(InstitutPasteurdeGuadeloupe)revealedapresenceofminorglobalandEurasia(Europe/Russia)specifictypesSIT262/Haarlem,SIT73,SIT254/LAM found in ex-USSR and Europe but very rare in East Asia and global type SIT42/LAM that is also prevalent in different parts in Eurasia. Three hypervariable loci, QUB-3232, VNTR-3820 and VNTR-4120, permitted to subdivide 28 BeijingstrainswithMIRU12profile223325153533into11subtypessharedby1to9strains.RIFandINHresistancewasdetected in 28% and 55% samples. 13 of 15 MDR strains belonged to Beijing genotype. Comparison of the rate of drug resistancemutationsindifferentBeijingsubclustersrevealednostatisticallysignificantdifference.

Conclusions

The penitentiary population of M. tuberculosisinKyrgyzstanshowsastrongaffinitytothenorth-westEurasia,especially,Russia, and a weak relatedness to East Asia. Beijing genotype constituted 75% of the entire collection while half of the studiedstrainsbelongedtotheBeijing/M2MIRU-definedsubtypethatisthemajorBeijingvariantinRussia.MDR-TBwasdetected in 27% samples that is similar to the Kyrgyzstan’s civilian population. IS6110-inverse PCR and HV-VNTR loci were shown to be useful for detection of and subtyping within the Beijing genotype, directly in sputum-extracted DNA.

48 ESM 2009

Op-6

ThE uNiQuE ENDEmiC NaTurE Of bEiJiNg gENOTypE STraiNS iN OKiNaWa, ryuKyu iSlaNDS Of JapaN aS rEVEalED by NEWly DESCribED 15 aND 24-lOCi miru-VNTr TypiNg SChEmES

Julie Millet, 1 Chika Miyagi-Shiohira, 2 Nobuhisa Yamane, 2 Igor Mokrousov, 1,3 Nalin Rastogi 1

1 - Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, Abymes, Guadeloupe2 - Department of Laboratory Medicine, Graduate School and Faculty of Medicine, University of the Ryukyus, Okinawa, Japan3 - St. Petersburg Pasteur Institute, St. Petersburg, Russia

Tuberculosis (TB) in the eastern Asiatic countries is mainly caused by strains of Mycobacterium tuberculosis belonging to the Beijing lineage which was identified back in early nineties using IS6110-RFLP and spoligotyping. Associated with mul-tiple drug-resistance (MDRTB), this highly homogeneous genogroup is characterized by little molecular diversity. Several studies have recently emphasized the utility of using minisatellites in conjunction with IS6110-RFLP or spoligotyping for a better discrimination of Beijing strains. In a recent study, we genotyped Beijing TB strains collected in Okinawa (Ryukyu Islands, Japan), by using a discriminative selection of 8 MIRU loci (chosen from the classical 12-loci MIRUs), and 7 QUB markers. This typing scheme excluded 4 MIRU loci (MIRU2, 4, 20, and 24), that were found to have a too low discrimina-tory power within an in-house Beijing database containing 694 strains (Millet et al., J. Clin. Microbiol. 2007, 45:3606–3615). In the present study we evaluated the full “classical” 12-loci typing as compared to the newly described 15-loci and 24-loci MIRU-VNTR typing schemes, which include 9 and 12 new MIRU loci respectively. We compared the results obtained in Okinawa (an insular setting; n=72) with those recently published for Osaka (n=174 strains) and Kobe (n=175) (Wada et al., FEMS Microbiol Lett. 2009, 291:35-43). A higher discriminatory power of 15-loci versus 12-loci format was seen through percentage of clustered isolates; clustering for 12-loci format in Osaka, Kobe, and Okinawa was 78.3, 81.7, and 68.1% respectively, as compared to 55.7, 48.0, and 37.1% using 15-loci format. Corresponding discriminatory index (HGI) in Osaka, Kobe, Okinawa were 0.901, 0.936, and 0.944 respectively for 12-loci format, as compared to 0.989, 0.989, and 0.992for15-lociformat.Afinercomparisonof15-locipatternsinthe3settingsrevealedthatcontrarytoKobeandOsaka which shared together a high number of similar patterns (25/114 and 25/107 respectively), Okinawa shared a single pattern out of 55 with Kobe, and none with Osaka. The full 24-loci format results further reduced the clustering observed (from 37.1% to 20%, HGI 0.996). The results analyzed by drawing a minimum spanning tree underlined the unique endemic nature of the Beijing genotype strains in the insular setting of Okinawa, and suggest a local evolution of M. tuberculosis Beijing genotype in this island starting from a common pool in mainland Japan.


Op-7

MyCoBaCTeriuM TuBerCuloSiS gENETiC DiVErSiTy iN SOuTh KOrEa

Isdore Chola Shamputa1, Jongseok Lee2, Caroline Allix-Béguec3, Eun-Jin Cho2, Ji-im Lee2, Jin Hong Min4, Lisa C. Goldfeder1, Jin Hee Kim4, Hyung Seok Kang4, Soo Hee Hwang4, Seok Yong Eum2 ,Hyeyoung Lee5, Seung Kyu Park2,4, Philip Supply3,6, Sang Nae Cho7, Laura E. Via1, Clifton E. Barry III1

1 - Tuberculosis Research Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland2 - International Tuberculosis Research Center, Masan South Korea3 - Genoscreen, Lille, France4 - Masan National Tuberculosis Hospital, Masan, South Korea5 - Department of Biomedical Laboratory Sciences, Yonsei University, Wonju, South Korea6-CentreNationaldelaRechercheScientifique,Institut Pasteur de Lille/Institut de Biologie de Lille, Lille France7 - Department of Microbiology, Yonsei University College of Medicine, South Korea

South Korea has recorded a nine fold decrease in the incidence of TB in the last four decades, however challenges of TB controlremainsignificantastheRepublicofKoreaisamongthe30countrieswiththehighestnumbersofestimatedMDR-TB cases. Genotypic analysis of M. tuberculosis has greatly contributed to the control of TB by providing information on transmission dynamics, assessing clonal distribution and expansion of the tubercle bacilli, in investigating of outbreaks and pseudo-outbreaks, and in identifying laboratory cross contamination. However, there is limited information on the molecular epidemiology of TB in South Korea.

Genetic diversity of 208 M. tuberculosis isolates from subjects enrolled in a prospective observational cohort study at National Masan Tuberculosis Hospital in South Korea was determined using spoligotyping, IS6110-RFLP and standardised MIRU-VNTR typing based on 24 loci. MIRU-VNTR analysis was performed independently and blindly from spoligotyping and IS6110-RFLP results.

Analysis of MIRU-VNTR typing results use in conjunction with MIRU-VNTRplus database predicted that 202 (97.1%) isolatesbelongedtotheBeijinggenotype.Thispredictionwasfullyconfirmedbyspoligotyping.Congruenceanalysisindi-cated the prevalence of 3 branches among Beijing strains respectively named Korea, Masan and China. Preliminary analy-sis did not show differential distribution of resistant, MDR or XDR strains among the 3 branches. MDR or XDR isolates weredetectedinatleast4clustersconcordantlyidentifiedbythe3genotypingmethods.UsingMIRU-VNTR typing and spoligotyping, 23 clusters of 66 isolates were detected indicating a relatively large diversity of circulating strains despite the prevalence of the Beijing lineage.

StandardisedMIRU-VNTR typing appearedefficient as a first linediscriminatorymethod for this countrywithhighprevalence of Beijing strains. However, preliminary analyses suggest that clustered cases may not be epidemiologically linked and rather correspond to endemic strains circulating in South Korea.

50 ESM 2009

Op-8

COrrElaTiON Of mOlECular rESiSTaNCE mEChaNiSmS aND phENOTypiC rESiSTaNCE TO firST-liNE DrugS iN MyCoBaCTeriuM TuBerCuloSiS STraiNS frOm SiErra lEONE

Silke Feuerriegel1, Susanne Homolka1, Erik Post2, Barbara Oberhauser2, Abu Garawani George3, Lars Westman3, Foday Dafae4, Sabine Rüsch-Gerdes1, Stefan Niemann1

1 - Research Center Borstel, National Reference Center for Mycobacteria, Parkallee 18, 23845 Borstel, Germany2 - German Leprosy and TB Relief Association, Würzburg, Germany3 - National Leprosy/TB Reference Laboratory, Freetown, Sierra Leone4 - National Program Manager for Tuberculosis and Leprosy, Freetown, Sierra Leone

background

Resistancetofirst-linedrugs(INH,RMP,SM,EMBandPZA)displaysaseriousproblemforthetreatmentofMycobacterium tuberculosis infections. Resulting MDR-tuberculosis (resistance to at least INH and RMP) implies an enormous threat for tuberculosis control worldwide. It is therefore of great importance to analyze the genetic basis of clinical resistance, especially in high incidence settings and to correlate molecular resistance data with phenotypic resistance data.

methods

A total of 97 M. tuberculosis strains from previously treated patients in Sierra Leone which displayed resistance to INH (n=32), RMP (n=16), SM (n=39), EMB (n=15) and PZA (n=10), respectively, were sequenced concerning the predomi-nant resistance determining regions (katG, rpoB, rrs, rpsL embB and pncA). Strains resistant to INH with no mutation in katG were also sequenced in the promoter regions of inhA and ahpC. From all strains analyzed 11 showed resistance to INH and RMP and were therefore MDR-TB. Drug susceptibility testing was done by using the proportion method on Löwenstein-Jensen medium.

results

Among INH resistant strains the most common mutation detected is katG315 (65.6%). From all 32 resistant strains 3 had mutations in the promoter regions of inhA and ahpC. Among RMP resistant strains 50% displayed mutations at rpoB531.SensitivityandspecificityoftheDNAsequencingofkatG and rpoB for detection of INH and RMP resistance were about 90%. Concerning SM resistance none of the resistant strains showed any mutation in rrs, but 46.2 % had rpsL mutations, either at codon 43 or 88. Among EMB resistant strains 46.7% showed mutations at embB306 and 13.3% at codon 332 and 497 respectively. Strains resistant to PZA displayed a number of different mutations throughout the pncA gene.SpecificitiesofsequencingofrpsL, embB and pncA for detection of the respective resistance phenotypes were high (96-100%), whereas sensitivities were lower (46% for sequencing of rpsL, 60% for embB, 70% for pncA).

Conclusion

There is a close correlation between data from molecular and phenotypic resistance testing for the determination of INH and RMP resistance in strains from Sierra Leone. Sensitivities of sequencing of resistance determining genes for SM, EMB and PZA resistance were low due to so far unknown resistance determining regions. Thus it is of great importance to gather information on further mechanisms leading to drug resistant MTB strains in different settings.


Op-9

lam aND hiV: COrrElaTiON Or CO-iNCiDENCE?

McNerney, Ruth, Mallard, KimLondon School of Hygiene & Tropical Medicine

The deadly synergy between TB and HIV presents a serious challenge to heath and development. Three decades after the onset of the AIDS pandemic the region with the highest prevalence of HIV infection is sub-Saharan Africa, followed by the Caribbean and Latin America. Molecular typing methods permit differentiation of M. tuberculosis into strain families or genotypes. We have undertaken analysis of genotyping data to compare the prevalence of spoligotype lineage with that of HIV infection. Data was taken from the peer reviewed literature, surveys testing only drug resistant strains or those not fully describing the genotype population were excluded. The Latin-American-Mediterranean (LAM) lineage are identifiedastypicallylackingspoligotypespacerscorrespondingtooligonucleotides21to24and33to36.Theyhavebeen observed in many geographic locations and include the F11 family reported in South Africa. Our analysis suggests that LAM strains are more frequently found in regions with a high prevalence of HIV. Conversely, they are rare in set-tings where HIV has yet to emerge as a major threat to public health. Interestingly LAM genotypes appear less frequent in African countries such as Uganda, Tanzania and Cameroon which have lower HIV prevalence’s than countries such as Malawi, Zimbabwe and South Africa where HIV rates are in excess of 10%. Why tuberculosis of this genotype should be linkedtotheHIVepidemicremainsamatterofspeculation.Althoughsocioeconomicandpoliticalfactorsplayasignifi-cant role they do not explain the uneven distribution of HIV in sub Saharan Africa. It is now evident that there is diversity intheinteractionofM.tuberculosiswithitshostandstrainsofdifferinggenotypeappeartoelicitsubtlebutsignificantdifferences in immune response. We present the hypothesis that strains of tuberculosis belonging to the LAM genotype are associated with enhanced transmission of TB in immunosuppressed populations.

52 ESM 2009

Op-10

MyCoBaCTeriuM CelaTuM: aN EmErgiNg paThOgEN iN ThE immuNOCOmpETENT. a CaSE rEpOrT

Lyberopoulos Panagiotis 1, Frangopoulos, Fragiskos1, Kontos, Fanourios 2, Zerva Loukia 2, Malagari Aikaterini 3, Papiris Spyridon 1 1 - 2nd Department of Pulmonary Medicine, Attikon University Hospital, Athens, Greece2 - Department of Clinical Microbiology, “Attikon” University Hospital, Athens, Greece3 - 2nd Department of Radiology, Attikon University Hospital, Athens, Greece

Mycobacterium celatum is a pathogen for immunocompromised patients but there is little evidence of its pathogenicity among immunocompetent individuals. We report the isolation of M. celatum from a middle-aged immunocompetent wom-anwithbronchiectasisthatwasinitiallyconsideredanon-significantfinding,buteventuallyprovedextremelydangerous.

A 55-year-old Caucasian female never smoker, was referred to our hospital complaining of productive cough over the preceding two weeks accompanied by general malaise. She lived in an urban area and had no history of alcoholism, use of steroids or immunosuppressive drugs. A high resolution computed tomography (HRCT) of the chest showed ill-definedperibronchialdensitiescontainedwithintherightupperlobewithair-bronchogramsandbranchingpatternwitha few opacities of tree-in bud formation. There was no lymphadenopathy or pleural effusion.

Smearsoffivesputumspecimens(beforeanytreatment)werenegativeforacid-fastbacilli.Thesespecimenswerepro-cessed with the NALC/NaOH method and cultured in the BACTEC MGIT 960 system (Becton Dickinson, USA) and on Löwenstein-Jensen medium (BioMerieux, France). Two mycobacterial strains were recovered from these cultures andbothstrainswereidentifiedasM. celatum with the use of the Genotype Mycobacterium CM and AS assays (Hain-Lifescience). PCR restriction analysis (PRA) of a 439-bp fragment of the hsp65 gene and sequencing of the 16S rRNA geneconfirmedthattheywereM. celatum type 1.

Fromthefiveaforementionedsputum specimens Pseudomonas aeruginosa and Serratia liquefaciens were also isolated and werebothsensitivetociprofloxacin.Ciprofloxacin(1gr/day,per-os)pluslowdoseazithromycin(500mgtwiceaweek)were administered. Two months later, a control chest HRCT scan showed a considerable improvement; no pathogens were isolated from consecutive sputum samples.

Eight months later the patient complained for general malaise, night sweats and cough with blood tinged sputum. Imaging studies revealed a lung apical cavity. Two bronchial washing and two sputum cultures were smear positives and M. celatum was recovered from all.

In conclusion, when the American Thoracic Society criteria for the diagnosis of NTM infection are met and M. celatum isidentified,itshould be considered as the pathogen causing the pulmonary infection even in patients with apparently normal cellular immunity.


Op-11

MyCoBaCTeriuM aviuM SubSpECiES STraiNS frOm humaN aND aNimal OrigiN

Radomski, Nicolas 1, Thibault, Virginie 2, Karoui, Claudine 1, De Cruz, Krystel 1, Cochard, Thierry 1, Gutiérrez, Cristina 3, Supply, Philip 3, Biet, Frank 2, Boschiroli, María Laura 1

1 - AFSSA-LERPAZ, Maisons Alfort2 - INRA-UR1282, Nouzilly3 - INSERM U629-Institut Pasteur, Lille

The Mycobacterium avium sbsp. avium and Mycobacterium avium sbsp. hominissuis are pathogenic emergent bacterial spe-cies belonging to the Mycobacterium avium complex (MAC). These two subspecies can infect and lead to disease to nu-merous animal species: birds, pigs, cattle, deer, sheep, goats, horses, cats, dogs, etc. Moreover, Mycobacterium avium subspe-cies have been isolated in HIV infected patients and in immuno-competent patients with pulmonary pathologies. MAC is an ubiquitous bacterial group that can be found in water, in the environment, or even in food. A molecular typing study was undertaken with the primary goal of improving the taxonomic and epidemiological knowledge of MAC. Different strains of Mycobacterium avium sbsp. avium, Mycobacterium avium sbsp. hominissuis, and also of Mycobacterium avium sbsp. silvaticum, isolated from animals, humans, and the environment, were typed by two methods: the Restriction Fragments Length Polymorphism on insertion sequences IS1311 (RFLP1311), which is a standard method to characterise MAC, and the Variable Number of Tandem Repeats-Mycobacterial Interspersed Repetitive Units (VNTR-MIRUs) characterisation, which has been recently developed on Mycobacterium avium sbsp. paratuberculosis. Our results demonstrate that the discrimination power of both methods is comparable (DI of more than 0.92). Therefore, VNTR-MIRUs seems a much better typing method since it is a PCR based method that requires little genetic material for being performed, which is easy to standardize in any laboratory and because the deduced numerical patterns do not require special softwares for being compared in an inter-laboratories fashion.

54 ESM 2009

Op-12

ThE CharaCTErizaTiON Of myCObaCTEria frOm aN OuTbrEaK SuggESTS a rEViSiON Of ThE TaXONOmiC STaTuS Of mEmbErS Of ThE MyCoBaCTeriuM CHelonae-aBSCeSSuS grOup

Sylvia Cardoso Leão1, Enrico Tortoli2, Cristina Viana-Niero1, Suely Yoko Mizuka Ueki3, Karla Valeria Batista Lima4, Maria Luiza Lopes4, Jesus Yubero5 María Carmen Menendez5, Maria Jesus Garcia5

1 - Universidade Federal de São Paulo, São Paulo, Brazil2 - Centro Regionale di Riferimento per la Diagnostica dei Micobatteri, Ospedale di Careggi, Firenze, Italy3 - Instituto Adolfo Lutz, São Paulo, Brazil4 - Instituto Evandro Chagas, Belém, Brazil5 - Universidad Autonoma de Madrid, Madrid, Spain

An outbreak of infections by rapidly growing mycobacteria (RGM) related to invasive procedures has been ongoing in Brazil since 2004. Isolates from patients submitted to laparoscopic or plastic surgery and to mesotherapy, a cosmetic procedure,werepreviouslyidentifiedbymolecularmethodsasMycobacterium massiliense and M. bolletii, respectively. The similarity of rpoB and hsp65 sequences from the clinical isolates and the corresponding sequences from both M. massil-iense and M. bolletiitypestrainswereabovetheacceptedlimitforinterspeciesvariability,leadingtoconflictingresults.Therefore, an extensive characterization was carried out with six Brazilian clinical isolates from this outbreak study and type strains from the members of the M. abscessus-M. chelonae group – M. abscessus, M. chelonae, M. immunogenum, M. massiliense and M. bolletii.Phenotypicidentificationwasperformedbybiochemicaltests,highperformanceliquidchro-matography(HPLC)anddrugsusceptibilitytesting.Molecular identification includedPCR-restrictionenzymepatternanalysis (PRA) of the hsp65 gene, as well as rpoB and hsp65 gene sequencing and analysis of the corresponding phylo-genetic trees. DNA-DNA hybridization (DDH) and restriction fragment length polymorphism (RFLP) of the 16S rRNA gene were used as the gold standards for RGM speciation. The clinical isolates and the M. abscessus, M. massiliense and M. bolletii type strains could not be separated by phenotypic tests and were grouped in the phylogenetic trees obtained. Also,DDHresultsconfirmed>70%relatedness,andindistinguishableRFLP16SrRNApatternswereobtained.Onthecontrary, separation from M. chelonae and M. immunogenum was supported by results from PRA-hsp65, rpoB and hsp65 phylogenetic trees, DDH and RFLP-16S. Taken together, these results led to the proposition that M. abscesus, M. massil-iense and M. bolletii represent a single species, that of M. abscessus. Two subspecies are also proposed, M. abscessus subsp. abscessus and M. abscessus subsp. massiliense which can be distinguished by two different PRA-hsp65 patterns, differing in a single Hae III band, and by differences in rpoB (3.4%) and hsp65 (1.3%) sequences.


Op-13

ThE SuNNy SiDE Of myCObaCTEria

Santos, Ricardo 1, Marques, Marco 2, Oliveira, Pedro 2, Carvalho, Filipe 2, Carvalho, Carla 2, Monteiro, Gabriel 2, Cabral, Joaquim 2, Frade, Raquel 2, Silva, Maria 3, Fernandes, Pedro 4

1 - Laboratório de Análises2 - IBB-CEBQ-IST3 - Faculdade de Farmacia, Univ. Coimbra4 - IBB-CEBQ-IST

Mycobacteria are typically seen as cause of morbidity and mortality worldwide. There is however a Jekyll side to these acid-fast bugs. Taking advantage of some key metabolic pathways, as well as of the particular nature of the hydrophobic cell wall, that enables operation in aggressive, non-conventional environments, non-pathogenic mycobacteria can be used for the production of compounds with application in the pharmaceutical, food and environmental areas. The pres-ent work aims to illustrate this concept, by providing examples of the application of non-pathogenic mycobacteria for theproductionofsteroidandsiderophoremolecules,andforthepinpointmodificationofcarbocycles.Theassessmentof the required biocatalytic activity and the early stages of process characterization have been carried out in miniatur-ized bioreactors, allowing for a high level of parallelization, hence providing a high throughput platform for bioprocess development. Going into detail, Mycobacterium sp. NRRL B-3805 was shown to effectively yield androstenedione (AD), a key intermediate in the production of therapeutic steroids, from phytosterols, recovered from residues of the paper industry, while operating in phthalate or liquid silicone environments, an approach that enhances process productivity. Furthermore, this particular strain was shown to also yield the AD out of several polyhydroxylated steroids. Relying on the high-throughput methodology, a library of non-pathogenic Mycobacterium spp. siderophore producers was devel-oped in-house. Scaling-up the production process to bench bioreactor using the most promising strains is envisaged. The samehigh-throughputmethodologyhasbeenrecentlyusedtoscreennon-pathogenicmycobacteriaforspecificcatalyticactivity, namely hydroxylation, on carbocyclic molecules, with promising results.

56 ESM 2009

Op-14

faST iDENTifiCaTiON Of MyCoBaCTeriuM TuBerCuloSiS iN SpuTum aND CulTurES baSED ON ThErmally-aSSiSTED hyDrOlySiS aND mEThylaTiON by gaS ChrOmaTOgraphy-maSS SpECTrOmETry

Erwin Kaal 1,2*, Arend Kolk 3, Sjoukje Kuijper 3, Hans-Gerd Janssen 1,4

1 - Polymer-Analysis group, Van ’t Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands.2 - ATAS GL International, P.O. Box 17, 5500 AA Veldhoven, The Netherlands.3 - KIT Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands. 4 - Unilever Research and Development, Advanced Measurement and Imaging, P.O. Box 114, 3130 AC Vlaardingen, The Netherlands.

A fast gas chromatography-mass spectrometry (GC-MS) method with minimum sample preparation is described for early diagnosis of tuberculosis (TB). The automated procedure is based on the injection of sputum samples which are then methylated inside the GC-injector using thermally assisted hydrolysis and methylation (THM). The THM-GC-MS procedurewasoptimizedfortheinjectionofsputumsamples.FortheidentificationofMycobacterium tuberculosis the known marker tuberculostearic acid (TBSA) and other potentional markers were evaluated. Hexacosanoic acid in com-binationwithTBSAwasfoundtobespecificforthepresenceofM. tuberculosis. For validation of the method several sputum samples with different viscosities spiked with bacterial cultures were analyzed. The detection limit was better than 1 x 104 bacteria/ml. 17 methyl octadecanoic acid was used as standard. The detection limit for this compound was 20 pg/ml. Finally, 18 stored sputum samples collected in Vietnam from patients suspected to suffer from TB were re-analyzed in Amsterdam by microscopy after decontamination/concentration and using the new THM-GC-MS method. No false positives were found by THM-GC-MS and all patients who were diagnosed with TB were also found positive using our newly developed THM-GC-MS method. These results show that the new fast and sensitive THM-GC-MS method holds great potential for the diagnosis of TB.

This work was partially supported by the Foundation of New Diagnostics (FIND) and the Optimus Foundation.


Op-15

hOW WilD-TypE miC DiSTribuTiONS CaN bE uSEful TO DETErmiNE CliNiCal brEaKpOiNTS iN MyCoBaCTeriuM TuBerCuloSiS

Ängeby, Kristian 1, Juréen, Pontus 2, Giske, Christian 1, Chryssanthou, Erja 1, Werngren, Jim 2, Hoffner, Sven 2, Kahlmeter, Gunnar 3, Sturegård, Erik 4, Schön, Thomas 5

1 - Department of Clinical Microbiology, Karolinska University Laboratory and Karolinska Institute, Sweden2 - Department of Bacteriology, Swedish Institute for Infectious Disease Control, Sweden3 - Department of Clinical Microbiology, Växjö Hospital, Sweden4 - Department of Clinical Microbiology, Malmö University Hospital, Sweden5 - Department of Clinical Microbiology, Kalmar County Hospital, Sweden

The increasing prevalence of multidrug resistant (MDR) and extensively drug resistant (XDR) tuberculosis (TB) un-derscores the need for accurate drug susceptibility testing (DST) .It is unfortunate that the current critical antibiotic concentrations(breakpoints)arebasedmostlyonempiryandonlytoa limitedextentonscientificevidence.This isespecially true for second line drugs.

For most other bacterial pathogens, wild-type minimal inhibitory concentration (MIC) distributions has been successfully applied as one of other tools (such as pharmacokinetic and pharmacodynamic data) to determine clinical breakpoints for DST.Amicroorganismisdefinedaswild-typebytheabsenceofacquiredandmutationalresistancemechanismstothedrug in question. In modern breakpoint determination, breakpoints that divide divide wild type distributions of MICs are avoided. An isolate with a MIC above the wild-type distribution is highly likely to harbor resistance mechanisms and is considered clinically resistant until there is evidence to the contrary.

At present, wild-type MIC-distribution data for M. tuberculosis is scarce, presumably in part because of the complexity of DST. In order to establish wild-type MIC distributions for M. tuberculosis we used a 96-stick replicator method, which allowedustoefficientlydeterminetheMICsof95clinical isolatessimultaneouslyforfourfirst linedrugs(isoniazid,rifampicin, ethambutol and streptomycin) and 15 second line drugs (including four quinolones, four injectables, ethiona-mide,prothionamide,cykloserine,PAS,thioacetazone).Ourfindingsclearlydemonstratehowwild-typeMICdistributionsamongothertoolscanbeusedtodefineclinicalbreakpointsalsoinM.tuberculosis.

58 ESM 2009

Op-16

mOlECular TEChNiQuES TO mONiTOr Tb paTiENTS’ TrEaTmENT: SElECTiVE rEmOVal Of DNa frOm DEaD baCTEria iN miXED pOpulaTiONS by uSE Of EThiDum mONOaziDE

Miotto Paolo, Cirillo Daniela M.EmergingBacterialPathogensUnit,SanRaffaeleScientificInstitute,Milan–ITALY

Drug-resistant tuberculosis (TB) represents a public health problem worldwide and is considered a real threat for TB controlprograms.Modernnucleicacidamplificationtechniques(NATs)thatexploitnucleicacidssignalsfromclinicalsamples represent useful tools to allow rapid detection of resistant M. tuberculosisstrains.Identificationofresistantbac-teria in clinical samples is fundamental in order to prevent inadequate treatment and further development of resistant strains. However, NATs can not be used for patients’ follow-up because DNA-derived signals can originate from nonviable bacterial cells and, therefore, generate data that could be misinterpreted. A method developed for microbial food patho-gens and already tested on environmental samples is here tested to distinguish between live and dead mycobacteria.

Ethidium monoazide bromide (EMA) is membrane impermeant that intercalates into both extracellular DNA and DNA in nonviable cells and is excluded from viable bacteria. Exposure to a light source renders EMA-DNA incapable of con-tributing to PCR.

Liquid culture of M. tuberculosis H37Rv was heat inactivated by incubation at 95 °C for 30 min and then treated with 6 µM, 9 µM, 12 µM, respectively, EMA concentrations. EMA treatment consists of 20 min of incubation at +4 °C in the dark followed by 30 min of exposure to a 500 Watt light. Controls used in the study were: inactivated bacteria untreated with EMA, live bacteria untreated with EMA, and live bacteria treated with the same 3 EMA concentrations. After EMA treat-ment, samples and controls were processed for DNA extraction by thermal lysis (95 °C for 30 min) and directly used in PCR. Amplicons were analyzed by capillary electrophoresis (Agilent Technologies).

EMAtreatmentatallthreeconcentrationstestedsuppressedPCRamplificationfromheatinactivatedcultureswithoutaffectingamplificationfromlivebacteria,provingthatEMAcouldbeusedalsoformycobacterialsamples.

Preliminary studies were also conducted on mixed live/dead populations using two strains with a different MIRU-VNTR genotype. Performing MIRU typing on mixed samples after EMA treatment, only the live strain could be detected. Comparisonoftreatedanduntreatedsampleshighlightedthesignificantcontributionthatnonviablebacteriacanmaketo DNA-based diagnostic analysis. EMA approach could be a simple and effective means to monitor patients’ treatment allowing the use of NATs during follow-up.


Op-17

MyCoBaCTeriuM TuBerCuloSiS iS ablE TO aCCumulaTE aND uTilizE ChOlESTErOl

Brzostek Anna 1,2, Pawelczyk, Jakub 2, Rumijowska-Galewicz, Anna 2, Dziadek, Bozena 3, Dziadek, Jaroslaw 2

1 - Laboratory of Mycobacterium Genetics and Physiology PAS, Institute for Medical Biology2 - PAS, Institute for Medical Biology3 - University of Lodz, Department of Immunoparasitology

Mycobacterium tuberculosis, is the causative agent of tuberculosis that infects one third of the human population. Tubercle bacilli are able to persist in a dormant state, from which they may reactivate disease state. The presence of lipid me-tabolism genes in the genome of M. tuberculosis suggests that lipids, including steroids, are important carbon and energy sources for this pathogen. One potential nutrient that is available in the mammalian host is cholesterol, a major sterol of the plasma membrane. Cholesterol is essential for the uptake of mycobacteria by macrophages, and it has been found to accumulate at the site of M. tuberculosis entry. Moreover cholesterol can be utilized by fast-growing, non-pathogenic mycobacteria,butpathogenicmycobacteriamightnotbeabletousecholesterol.Here,weshowforthefirsttimethatM. tuberculosis grown in media containing carbon source other than cholesterol is able to accumulate cholesterol in the free lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary anti-tuberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore, M. tuberculosis was able to grow on mineral media supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537 (kstD) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of theintermediate,9-hydroxy-4-androstene-3,17-dione.OurfindingsthatM. tuberculosis is able to accumulate cholesterol in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future studies into the pathophysiology of this pathogen.

TheworkwassupportedpartiallybygrantsICGEB(CRP/POL07-01)andStateCommitteeforScientificResearch(N302035 31/3172).

60 ESM 2009

Op-18

myCOliC aCiD-iNDuCED ifN-γ prODuCTiON by CD1-rESTriCTED T CEllS frOm TubErCulOuS paTiENTS

Rodríguez-Güell, E 1, Alonso, C 2, del Val-Romero, B 2, Clivillé, R 2, Secanella,SP 1, Roura-Mir,C 3, Cañete, C 4, Navarro, A 4, de Gispert, FX 4, Luquin, M 1, Julián,E 1

1. Dept. Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona)2. Dept. Microbiologia, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona)3. Dept. Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona)4. Unitat de Respiratori, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona)

introduction

The cell wall of Mycobacterium tuberculosis (MTB) has a large number of structurally diverse lipids. Mycolic acids (MAs) merit specialinterestbecausetheirimmunogenicityhasbeendemonstratedinCD1-restrictedTcellclones;specifically,theyarepresented by CD1b molecules. To date, the recognition of MAs in tuberculous patients (TB) has not been studied.

The purpose of the study was to determine whether MAs are recognized by the immune system of TB patients and, in the event of this being so, to study the evolution of this response throughout anti-TB treatment.

methods

Immature dendritic cells (iDCs) isolated from 31 TB patients at the time of diagnosis and throughout anti-TB treatment, 20 PPD-positive and 20 PPD-negative donors were analysed by cytometry for CD1b surface expression. Subsequently, the samples were irradiated and cultured with autologous lymphocytes in the presence of phytohemaglutinin, MTB and purifiedMAsasstimuli.After48hours,IL-10andIFN-γwerequantifiedbyenzyme-linkedimmunosorbentassay.

results

NosignificantdifferencesamongthesurfaceexpressionofCD1biniDCsfromhealthydonorsorTBpatientswereob-served at any time during the disease. iDCs from all the individuals were therefore able to present MAs.

Median levels of stimuli-induced IL-10 in samples obtained at the outset of anti-TB treatment were lower than those obtainedattheend;however,nosignificantdifferenceswereobserved.NorwereanydifferencesobservedamongtheIL-10 levels obtained from the different groups of individuals.

In TB patients, MA-induced IFN-γmedianvaluesobtainedattheendofprophylaxisweresignificantlyhigher,statistically,than those elicited at the beginning. Grouping the samples of the 31 TB patients in terms of collection time, IFN-γ median levels followed an upward trend throughout anti-TB treatment, reaching maximum levels at the point of disease cure.

Furthermore, in PPD-negative donors IFN-γmedianvalueswerelowerthanthosefromPPD-positivedonors,significantdifferences only being established when MTB was used as antigen.

Conclusions

ThespecificcellularimmuneresponseagainstMAsinTBpatientsdescribedhereforthefirsttimesuggestsapotentialimmunoprotective role for MAs.


Op-19

a rEpOrT ON NEW aDapTED fOrmS Of EXTENSiVEly Drug rESiSTaNCE TubErClE baCilli : TraNSmiSSiON ElECTrON miCrOSCOpy aNalySiS

Parissa Farnia(PhD)1. Ali Akbar Veleyati (MD)1, Mohammal Reza Masjedi (MD)1, 2 Tengku Azmi Ibrahim (PhD)2, Payam Tabarsei(MD),RafiuzZamanHaroun(MSC)2,HoOiKuan(MSC))2,AbdulRahmanOmar(PhD)11 - Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Disease (NRITLD), WHO Collaborating Centre, Shahid Beheshti University (Medical Campus), Darabad, Tehran ,19556, P.O: 19575/154, Iran. E-mail:[email protected] - Microscopic unit, Institute of Bioscience , University Putra Malaysia ,43400 UPM ,Serdang, Selangor Darul Ehsan , Malaysia

background

Extensively drug resistance tuberculosis bacilli (XDR-TB), is caused by a strain of M. tuberculosis(MTB) that are resistant to isoniazidandrifampin(whichdefinesMDRtuberculosis)inadditiontoanyfluroquinoloneandatleastoneofthethreefollowing injectable drugs: caperomycin, kanamycin and amikacin. Viewed under transmission electron microscopy (TEM), spore like structure was observed inside the XDR-TB bacilli.

methods

ThesusceptibilitytestingagainstfirstandsecondlinedrugswasperformedonisolatedM. tuberculosis strains. Subsequently, a homogenous thick suspension of 107 to 108 cells at exponential phase was prepared and observed under Transmission Electron Microscopy (TEM). Five isolates from each group (susceptible, MDR, and XDR TB) were used in this study.

results

Viewed under the TEM, the XDRTB bacilli at exponential phase had three types of cell division 1) 80-70% of bacilli were looks as norm one with symmetrical or asymmetrical cell division 2) 5-7% with extra ordinary thick cell wall ( 21 to 26 nm ) which was similar to stationary or dormant phase bacilli. But surprisingly the stationary phase XDR-TB bacilli were at dividing process 3) 15-20% bacilli had spore formation inside them These spores were different from buds or a polar division that formed during cell branching of MTB. No spore’s and stationary phase bacilli were detected in susceptible or multidrug resistant strains of MTB.

Discussion

Dividing phenomena in XDR-TB bacilli with stationary phase appearance will generate new adapted types of M. tubercu-losis which can resist the effect of all available anti tuberculosis drugs. At the same time, it is possible that under certain conditions, the XDR-TB bacilli produce spore to overcome the hostile environment. Spore formation in XDR-TB bacilli should take very seriously from epidemiological point of view. With these new forms of adaptation that occurs in XDRTB bacilli , how should we treat and control the diseases.

62 ESM 2009

Op-20

grOWTh prOfilE Of CliNiCal iSOlaTES Of MyCoBaCTeriuM TuBerCuloSiS COmplEX iN muriNE maCrOpghagES

Susanne Homolka1, Stefan Niemann1, David G. Russell2 and Kyle H. Rohde2

1 - Molecular Mycobacteriology, National Reference Center of Mycobacteria, Borstel Germany2 - Department of Microbiology/Immunology, Cornell University, Vet. Med. Center, Ithaca, USA

background

Mycobacterium tuberculosis and other members of the Mycobacterium tuberculosis complex (MTC) remain a major cause of morbidity and mortality worldwide. Several studies based on spoligotyping, IS6110 fingerprintingandMIRU-VNTRtyping demonstratedthattheglobalpopulationstructureofMTCisdefinedbyphylogeographicallineagesandgenotypesthatarealsoassociatedwithpathogenicdifferences.However,theinfluenceofstraingenomicvariationontheoutcomeofinfectionand the clinical presentation are not completely understood. In our study, we investigated growth differences of 15 strains offivedifferentgenotypesincomparisontotheCDC1551referencestrainsinliquidcultureandinmacrophages.

methods

Murine bone marrow derived macrophages were infected with liquid culture of different clinical isolates (MOI 3:1; 2x106CFU/ml). Survival of strains in resting and activated macrophages was determined at different time points up to 11 days post-infection. Additionally, growth kinetics of all strains in 7H9 liquid media were analyzed.

results

GrowthanalysesofclinicalisolatesinliquidculturebasedonODmeasurementsshowednosignificantdifferencesincomparisontoCDC1551.However,weobservedbothstrain-andgenotype-specificsurvivalandgrowthprofileswithinresting and activated macrophages.

Conclusions

The genomic diversity of clinical isolates influences the complex interactionof the pathogenwith host phagocytes.Furtheranalysestocorrelateintracellulargeneexpressionprofileswiththeoutcomeofinfectionareinprogress.


Op-21

prESENCE Of eSaT-6 aND CFP-10 gENES DOES NOT lEaD TO phagOlySOSOmE TraNSlOCaTiON Of MyCoBaCTeriuM Szulgai

Jakko van Ingen1,2, Nicole van der Wel3, Richard Dekhuijzen2, Martin Boeree2, Dick van Soolingen1

1 - National Mycobacteria Reference Laboratory, National Institute for Public Health and the Environment, Bilthoven, the Netherlands2 - Department of Pulmonary diseases, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands3 - Department of Cell Biology, Netherlands Cancer Institute, Amsterdam, the Netherlands

background

Bacterial virulence factors in nontuberculous mycobacteria are mostly unknown. In Mycobacterium tuberculosis complex bacteria, the esat-6 and cfp-10 genes are important virulence factors, which facilitate translocation from the phagolyso-some to the cytosol of macrophages. Their presence and role among nontuberculous mycobacteria is largely unknown.

methods

We assessed the presence of esat-6 and cfp-10 genes in all 5 M. kansasii subtypes based on 16S-23S internal transcribed spacer sequencing (n=15), M. szulgai (4), M. marinum (4), M. avium (2), M. conspicuum (4), M. genavense (1), M. bohemicum (2), M. interjectum (2), M. flavescens (5), M. xenopi (2), M. malmoense (2), “M. riyadhense” (1) and M. tuberculosis H37Rv by PCR. We used Esa-12 CATGACAGAGCAGCAGTG and Esa-303 5’-GCCCTATGCGAACATCCC-3’ primers for esat-6 and opBR78 5’-GTAGCCCGGGATGGCAGAGATGAAGACCGATGCC-3’ and opBR103 5’-TCAGAAGCCCATTT-GCGAGGACAGC-3’ primers for cfp-10. For PCR negatives, we performed Southern blotting with probes based on the esat-6 gene of M. tuberculosis H37Rv.

One NTM species with esat-6 and cfp-10 genes was selected for macrophage infection. By cryo-immunogold electron mi-croscopy we tested whether these genes effect translocation from the phagolysosome to the cytosol of macrophages.

results

We were able to amplify esat-6 and cfp-10 genes in M. tuberculosis H37Rv, all M. kansasii subtypes, M. szulgai, M. marinum and “M. riyadhense”. All esat-6 and cfp-10sequencesamongthesenontuberculousmycobacteriawerespecies-specific;multisequence alignment revealed an average of 90% homology with the M. tuberculosis sequences. The remaining spe-cies were repeatedly negative by both PCR and Soutern blotting. Mycobacterium szulgai was subsequently used for a macrophage (THP-1) infection experiment, with an M. tuberculosis positive control. Seventy-two hours after infection, no cytosolic M. szulgai bacteria were found, while 53% of the M. tuberculosis bacteria had translocated to the cytosol.

Conclusions

While some nontuberculous mycobacteria harbor esat-6 and cfp-10 genes, their products, at least for M. szulgai, do not effect translocation of the bacteria from the phagolysosome to the cytosol in macrophages. ESAT-6 and CFP-10 protein structure or secretion could be critical factors. While the presence of esat-6 and cfp-10 genes adds an interesting phylo-genetic marker, the pathogenesis of NTM infections remains unexplained.

64 ESM 2009

Op-22

DEVElOpmENT Of aN aDapTiVE immuNE rESpONSE iN ThE DraiNNg lymph NODE DuriNg MyCoBaCTeriuM ulCeranS iNfECTiON

Alexandra G. Fraga; Joana E. Braga; Andrea Cruz; Teresa G. Martins; Daniela R. Pereira; Wayne M. Meyers; Françoise Portaels; António G. Castro; Jorge Pedrosa1 - Life and Health Sciences Research Institute (ICVS). School of Health Sciences, University of Minho. Braga, Portugal2 - Mycobacteriology Unit, Department of Microbiology, Institute of Tropical Medicine, Antwerp, Belgium3 - Armed Forces Institute of Pathology, Washington, D. C., USA

Buruli ulcer is a neglected tropical disease caused by infection with Mycobacterium ulcerans. The necrotic cutaneous le-sions of BU patients are associated with the cytotoxic properties of the exotoxin mycolactone. Previous results from our group show that the protective role of IFN-γ is impaired during infection with the highly virulent strain 98-912. To further characterize the development of an adaptive immune response during progressive infection with a highly virulent M. ulcerans strain, we decided to study the T cell dynamics in the draining lymph node (DLN) of mice infected with M. ulcerans 98-912, as compared tothelowvirulent,mycolactone-deficientstrain5114.

Subcutaneous infection of mouse footpads with M. ulcerans 5114 induced a modest increase in the number of cells in the DLN, prevalent throughout the experimental infection. Surprisingly, infection with strain 98-912 led to a 26-fold increase inthenumberofcellsintheDLN,followedbyasignificantdroptobasallevels.However,thisincreaseinthenumberofcells did not correlate with a protective response in the infected footpad.

Following this observation, we explored whether the induction of an adaptive immune response occurs during infection with strain 98-912. M. ulceransinfectionelicitedantigen-specificTcellsproducingIFN-γ in the DLN, with the response being more prominent in M. ulcerans 98-912 infected mice. Additionally, infection with M. ulcerans followed by adoptive transferofOVA-transgenicTcellsdidnotimpairtheaccumulation,proliferationoractivationoftheOVA-specificTcellsintheDLNuponchallengewithOVA.Moreover,thisTcellaccumulationandactivationwassignificantlyincreasedinmice infected with strain 98-912. Interestingly, analysis of the DLN at later time points revealed the presence of viable bacilli that correlated with the dramatic decrease in the number of cells. This decrease was due to apoptosis, as dem-onstrated by the higher number of activated-caspase 3 positive cells. Supporting our observations in the mouse model, presence of bacilli and tissue destruction were also observed in DLN of BU patients.

In summary, the failure of the host to generate a protective response against infection with this highly virulent strain of M. ulceransisnotassociatedwithanimpaireddevelopmentofspecificTcellsintheDLN,butinsteadtotheirdestructionin the infection foci and, later, to the destruction of the DLN itself.


Op-23

TubErCulOSiS SOfTWarE iN a gENEral hOSpiTal, WOrKiNg iNSTrumENT

Portugal Clara 1; Nuno Cardoso 2, Luísa Sancho 1; Germano Sousa 1

1 - Laboratory of Microbiology, Department of Clinical Pathology2 - Planning and Management Control DirectorHospital Fernando Fonseca (HFF) - Amadora, [email protected]

background

Our Hospital is located in a Lisbon’s surroundings, its construction was completed in 1995 and, in this same year, it started the operation.

The HFF was designed to house a population much smaller than that now covers, 750 000.

Our population has a low socioeconomic conditions and a high level of immigrants from Africa and in Eastern Europe, which may be the main reason for concentrating a large number of tuberculosis’s cases. Portuguese Health Authorities reported that 66% of TB cases in Portugal were concentrated in Lisbon’s surroundings.

In a few years after the opening of the Hospital, all laboratory’s data relating to tuberculosis became too numerous to be recorded and cross in the general program.

purpose

Given the need to gather and cross all the tuberculosis’s data, in 2000 was developed a software (MYCOHFF) that we are going to present.

methods

MYCOHFFresultsfromtheinterconnectionof3Excelfiles;MYCOYEARrelatedtotheyearinquestion,MYCOZIEHLthat brings only patient’s data with positive Ziehl-Nielseen (since 1997) and MYCOCULT that joins only the patient’s data with positive cultural examinations (liquid and/or solid) (since 2000).

For each specimen submitted for mycobacterial culture, we record the name of the patient, process number, analysis number, product’s type, the origin patient’s Service and product inoculation’s date. Thus takes place an observation’s weekly list. For each week, we note the medium (liquid and Lowenstein-Jensen) growth or not, the contamination and/or degradation, during 6 or 8 weeks (sometimes more).

The year’s records are placed in alphabetical order (patient’s name).

The patient’s analysis with positive culture (liquid and/or solid) automatically appears in red.

If the patient had already a positive culture, all the negative analysis appears in purple. If the patient had a positive Ziehl-Nielseen, then all the analysis will appear in yellow, becomes purple if it has other positive culture or red if this analysis is positive.

Onpatient’sfirstisolation,weperformtheIdentificationandSusceptibilityTest.Thisdateisnotedaswellasthefinaldatewiththedefinitiveresults,whicharepresentedtotheclinician.

results

Warning Signs: When the patient’s registration happens, we know immediately, with different colors (program • alerts), if the patient has Ziehl-Nielseen positive, culture positive, and when.

Search: Any search is fast because we are talking about an application that is made in Excel software.•

Weeklyobservation’slist:Theweeklysheetsareeasilycreatedbyfilteringtheinoculation’sdatefield.•

Statistical indicators and graphics: With only a few records, statistical indicators are automatically calculated and • graphical analyses are available at any time.

66 ESM 2009

Statistical indicators for each year are concerning to different parameters. 1- Concerning the analysis/patient relation-ship: Nº of patients, nº of analysis, total positive patients, total positive analysis, analysis/patient ratio, patients positive rate, analysis positive rate; 2- Concerning the Ziehl-Nielseen (Z) and liquid/solid medium relationship: Nº of positive direct tests (Z+), Nº of positive cultural tests (C+), true Z positive’s (Z+/C+) rate, false Z negative’s (Z-/C+) rate, false Z positive’s (Z+/C-) rate, real Z negative’s (Z-/C-) rate, Ziehl-Nielseen’s positivity rate per analysis and per patient; 3-ConcerningtheidentificationandTSA:NºofMycobacterium sp.isolated,totalidentifiedstrains,totalstrainswiththeTSAandidentificationstillinstudy,NºandrateofMycobacterium tuberculosis complex (CMT), CMT without resis-tance, with one resistance, MDR (Multi Drug Resistant - TB), XDR (Extensively Drug Resistant – TB), Nontuberculous mycobacteria,averageresponsetimebeginningwiththeisolationculture(identificationplusTSA).

Thestatisticsgraphsshow:1-Analysisrequested/positiveanalysisbyorganicproduct;pleuralbiopsy,pleuralfluid,sputum,bronchialsecretions,bronchoalveolarlavage,gastricfluid(respiratoryspecimens),urine,pus,mieloculture,cerebrospinalfluid,biopsy,ascitesfluid,fluids,blood;andtheirpositivity,2-TheisolatedMycobacterium sp.’s strains, including the differentiation in CMT, zero resistance, one resistance, MDR, XDR, 3 - Compare the tuberculosis’s inci-dence rate in two populations, MDR and non-MDR, for the age range of patients, sex and HIV.

Conclusions

For us, MYCOHFF software is an essential tool for the process of gathering and managing tuberculosis data.

Quick reports of any patient are possible. In a few seconds, we can make the patient laboratory story on tuberculosis.

Each year a statistical report is communicated to all our hospital’s departments. Monthly, or when requested, for the CDP (centre that follows the patients in the community), is sent a list identifying new positive cases and results (preliminary and/orfinal).


Op-24

DEVElOpmENT Of aN auTOmaTED CulTurE SySTEm fOr M. TuBerCuloSiS WiTh auTOfluOrESCENCE DETECTiON

den Hertog, Alice 1, Koeleman, Marc 1, Ingham, Colin 2, Fey, Frank 3, Langerak, Edwin 3, Klatser, Paul 1, Anthony, Richard 1

KIT Biomedical Research, Amsterdam, The NetherlandsMicrodish BV, Wageningen, The NetherlandsCCM, Nuenen, The Netherlands

Due to the unavailability of rapid sensitive diagnostic methods for tuberculosis (TB), culture remains one of the most important diagnostic tools - even though it is technically demanding and slow. The time required for culture leads to a delay in TB diagnosis and treatment with effective drugs. A standardized method for more rapid drug sensitivity testing (DST) of TB would be valuable.

Wearedevelopingasysteminwhichmycobacterial(micro-)coloniesaredetectedbytheirautofluorescencebasedonthe MODS (microscopic observation of drug susceptibility) method, which is much more rapid than the traditional cul-ture methods, but is laborious.

By using microscopy with low-power magnification, colony growth could be determined much earlier thanwhen viewed by eye. Studying Mycobacterium smegmatis initially as a model organism, we have made se-quential brightfield and fluorescence microscopy photographs of colonies growing on movable solid sup-ports on media. These images were used to develop an image analysis protocol to determine the growth rate. We found that the time to detection of microcolonies was less than half that required for visual detection. Furthermore, as soon as the colonies could be visualized, further growth of colonies could be detected by the image analysis protocol within 1-2 division times, from which the growth rate could be determined. As the bacteria were inoculated on movable solidsupports,exposingthemicrocoloniestoselectivemediaaftertheirfirstdetectioncouldmakeDSTofmanyindi-vidual colonies possible after only a few additional division times, allowing the rapid determination of the proportion of antibiotic resistant and sensitive mycobacterial colonies present in a sample.

Afterconfirmationoftherecentlydescribedautofluorescenceofmycobacteria,thismethodwasusedforthedetectionandenumerationofviablemycobacteria.Thespecificityoftheautofluorescencemaybeusedtoconfirmthepresenceof mycobacteria.

Theuseof(semispecific-)autofluorescenceincombinationwithgrowthratewillincreasethefeasibilityofdevelopingan automated detection system based on this method, which is very challenging for light microscopy methods (such as MODS). Additionally, the possibility of performing quick DST without re-inoculation may lead to a reduce time to cor-rect treatment.

68 ESM 2009

Op-25

SECOND-liNE Drug SuSCEpTibiliTy TESTiNg Of MyCoBaCTeriuM TuBerCuloSiS by mgiT 960 SySTEm, ThE miCrOplaTE COlOrimETriC-baSED mEThOD aND ThE prOpOrTiON mEThOD

Nora Morcillo1, Belén Imperiale,1, 2, Beatriz Di Giulio3

1 - Reference Laboratory of Tuberculosis Control Program of Buenos Aires Province, Dr. Cetrángolo Hospital Buenos Aires, Argentina.2-NationalCouncilofScientificandTechnologicalResearch,BuenosAiresCity.3 - P. V. de Cordero Hospital, San Fernando, Buenos Aires, Argentina.

The accurate treatment of tuberculosis (TB) cases due to multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis emphasizes the necessity of new tools for rapid detection of these strains in clinical laboratories. Minimal inhibitory concentrations (MICs) by MGIT960 and the colorimetric microplate method using dyes as MTT or resarzurin (CMM) were determined for the following drugs (µg/ml): amikacin (AMK): 2.0, 4.0, 8.0; kanamycin (KM), capreomycin (CPM),ethionamide(ETH):2.5,5.0,10.0;cycloserine(CS):15.0;ofloxacin(OFX)andlinezolide(LZ):0.5,1.0,2.0;andmoxifloxacin(MOX)0.25,0.5,1.0.MICswereperformedon94clinicalisolates.TheproportionmethodonMiddlebrook7H11 (PM) was used as gold standard. Inoculated MGITs were incubated in the instrument for no longer than 21 days. AstraintestedbyMGIT960wasconsideredresistantifapositivesignalflaggedfromthedrug-containingtubewithin5days of the positive control tube. Microplates of the CMM were incubated for an average of 8 days. Statistical methods wereappliedtodefinedrug-resistantstrainsonthebasisofthecomparisonbetweenresultsobtainedbyMGIT960andCMMwiththePM.Thefollowingcriticalconcentrationswereidentified(µg/ml):AMK:4.0;CPM,ETHandKM:5.0;CS:30.0; LZ: 1.0; MOX: 0.5; OFX: 2.0. Accuracy of MGIT960 and M-MTT was 100% for AMK, CPM, OFX, MOX and LZ. In this study tubes incubation and positivity detection was manually obtained from the MGIT960 instrument which actually can be adapted to automatically detect both susceptible and resistant strains to each one of the second-line drugs. Results were obtained in less than 10 days for both MGIT960 and CMM. On the other hand CMM, as a complete homemade method, was cheaper but more laborious than MGIT960. Our results showed that both methods could be promissory implemented as a rapid diagnosis tools to detect MDR and XDR-TB cases in clinical practice.


Op-26

ThiOriDaziNE ShOWS prOmiSiNg aCTiViTy iN a muriNE mODEl Of mulTiDrug-rESiSTaNT TubErCulOSiS

Jakko van Ingen1,2, Martin Boeree1, Leonard Amaral3, Rogelio Hernandez Pando4, Dick van Soolingen2

1 - Department of Pulmonary Diseases, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands2 - National Mycobacteria Reference Laboratory, National Institute for Public Health and the Environment, Bilthoven, the Netherlands3 - Mycobacteriology Unit, Institute of Hygiene and Tropical Medicine, Universidade Nova de Lisboa, Lisbon, Portugal4 - Experimental Pathology Section, Department of Pathology, National Institute of Medical Sciences and Nutrition SalvadorZubiràn,MexicoCity,Mexico

background

Multidrug-resistant tuberculosis (MDR-TB) is a threat to TB control efforts worldwide for which very few active drugs are currently available. The phenothiazines are antipsychotic agents that have potential as anti-tuberculosis drugs, at least in vitro.Withinthispharmacologicalclassthioridazineisthemostefficaciousandcausesthemildestside-effectswhenused as an antipsychotic agent. Thioridazine is active against mycobacteria by targeting the type II NADH dehydrogenase, succinate dehydrogenase, the binding of calcium to proteins and disruption of aerobic respiration under micro-aerobic conditions. We tested its in vivo activity in a murine model.

methods

We infected 3 groups of 40 Balb/c mice with pansusceptibe M. tuberculosis H37Rv. After 60 days, 20 mice in each group started 2 months of thioridazine monotherapy at 16, 32 and 70 mg/kg dosages; the remaining 20 served as controls. This experiment was repeated using a clinical multidrug-resistant M. tuberculosis (MDR-TB) isolate and two months daily oral administration of 32 and 70 mg/kg. In a third experiment, 3 groups of 20 mice were infected with M. tuberculosis H37Rv; one group received rifampicin, isoniazid and pyrazinamide, another received these 3 drugs and thioridazine, one un-treatedgroupservedascontrols.Inallexperiments,groupsoffivemicepergroupweresacrificedafter2,4and8weeksof treatment; lung tissue was homogenized for quantitative cultures. The bacillary load of the lungs was determined by colonyformingunits(CFU)quantification;histologicaldamagewasobservedbymicroscopicexamination.

results

In both the M. tuberculosisH37RvandMDR-TBinfection,monotherapywith32and70mg/kgthioridazineleadtosignifi-H37RvandMDR-TBinfection,monotherapywith32and70mg/kgthioridazineleadtosignifi-cant reductions in CFU counts from the lung tissue homogenates and in the extent of histological damage, at all time points. Moreover, when 32 mg/kg of thioridazine was added to a regimen containing rifampicin, isoniazid and pyrazin-Moreover, when 32 mg/kg of thioridazine was added to a regimen containing rifampicin, isoniazid and pyrazin-amideforsusceptibletuberculosis,asignificantsynergisticeffectwasachieved.

Conclusions

Thioridazine shows promising activity in our murine model. It has a potential effect in the treatment of susceptible and MDR-TB, where few active drugs are available. The low price of this out of patent drug enables extended use in resource–poor settings where the burden of multidrug-resistance is most grave.

70 ESM 2009

Op-27

CONTribuTiON Of EffluX pump aCTiViTy fOr maCrOliDE rESiSTaNCE iN M. aviuM COmplEX

Liliana Rodrigues1,2*, Daniela Sampaio1, Isabel Couto1,3, Diana Machado1, Winfried V. Kern4,5, Leonard Amaral1,2,5 and Miguel Viveiros1,5

1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal2 - UPMM, IHMT/UNL, Lisbon, Portugal3 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal4 - Center for Infectious Diseases and Travel Medicine, University Hospital, Freiburg, Germany5 - COST ACTION BM0701 (ATENS)

Mycobacterium avium complex (MAC), comprising M. avium and M. intracellulare, is clinically important since it can cause severe infections in AIDS patients and other immunocompromised individuals. Therapy of MAC infections is problematic due to the intrinsic resistance of these bacteria to many of the available antimicrobial drugs. The use of the macrolides clarithromycin and azithromycin has improved the outcome of MAC infections, but therapeutic failure is still a major problem.WehaverecentlyshownthateffluxpumpsofMACplayanimportantroleonthisresistancephenotype.Infact,increasedactivityofeffluxpumpsisknowntocontributetoamultidrugresistancephenotypebyextrudingawidevarietyof chemically and structurally unrelated compounds from the cell, preventing them from reaching their cellular targets. Thus,thecharacterizationofsucheffluxpumpsiscrucialforthedesignofnewantimycobacterialtherapeuticstrategies.

Inthiswork,wehavestudiedtheeffluxpumpactivityinMACclinicalstrainsbyafluorometricmethodthatdetectseffluxactivityonareal-timebasis,andevaluatedthecontributionofactiveeffluxtotheresistancetomacrolides.

Theresultstobepresentedshowthatresistancetoclarithromycinwassignificantlyreducedinthepresenceofeffluxpumpinhibitors(EPIs)suchasthecalcium-channelinhibitorsthioridazineorchlorpromazineandthecalciumioninfluxinhibitor verapamil.ThesameEPIswereeffectiveindecreasingtheeffluxofethidiumbromide(acommoneffluxpumpsubstrate)fromMACcells,asshownbyfluorometricanalysis.Moreover,theretentionof[14C]-Erythromycin by the same inhibitorsdemonstratedthatactiveeffluxcontributestoMACresistancetomacrolides.

Inconclusion,thisstudydemonstratesthateffluxpumpsplayanimportantroleinMACresistancetoantibiotics,par-ticularly to macrolides, and opens the possibility to explore the usefulness of these EPIs, already used in clinical practice for other purposes, such as thioridazine, as adjuvants to enhance the effectiveness of the therapeutic regimens against MAC infections.


Op-28

mEmbraNE- baSED VErSuS miCrObEaD- baSED SpOligOTypiNg: prElimiNary rESulTS ON a QualiTy- iNSuraNCE STuDy ON 10 SiTES WOrlWiDE

Abadia E1, Zhang J1, Refregier G1, Sola C1.1 - Institut de Génétique et Microbiologie, UMR8621, CNRS-Université Paris-Sud (Universud), Equipe IGEPE, bât. 400. France.

Spoligotyping have been developed 12 years ago and have been used worlwide for molecular epidemiological or mo-lecular evolutionary studies. This technique is based on a reverse line- blot hibridization method. Due to it’s wide accep-tance (458 references in Pubmed on 2009 April 27th) it can be considered as a basic technique in genotyping strains of Mycobacterium tuberculosis complex together with MLVA (Multi locus variable analysis) typing. However, spoligotyping has not been the focus of standarization nor of quality insurance studies. The transfer from the membrane- based to a microbead- based format, through the development of the multiplex Luminex plataform was done in 2004 in the CDC in Atlanta. We implemented this technique in our team in 2008.

We wanted to evaluate membrane- based spoligotyping results obtained on 10 sites (around 1000 DNA samples from clinical isolates) comparing them with those from the Luminex microbead- based results obtained on the same samples. TheDNAsampleswereselected to fulfill the followinggoals: (1)Retrospectivelyassess thequalityof spoligotypingresults, that have been produced in various laboratories worlwide during a decade (2) Solve inconsistencies, resolve dis-crepancies, improve quality of hard- to- interprete membrane- based spoligotyping results (3) Study if DNA extraction procedure (quality, quantity) may have produced errors in spoligotyping patterns production (4) assess the economical interest of the new Luminex- based technique and it’s contribution (or not) to and increased quality of services in mo-lecular epidemiological studies.

We will present all results available until today. Our current experience and preliminary results suggest that the Luminex platform it’s very versatile to produce high quality spoligotypnig results with both, a higher throughput and sensitivity than the membrane- based spoligotyping and at affordable costs for developed countries.


abSTraCTS Of pOSTEr prESENTaTiONS (pp)


pp-1

EValuaTiON Of ThE high-ThrOughpuT rEpETiTiVE-SEQuENCE-baSED pCr DiVErSilab SySTEm iN M. TuBerCuloSiS mOlECular EpiDEmiOlOgy STuDiES

Miotto Paolo, Baldan Rossella, Cirillo Daniela M.EmergingBacterialPathogensUnit,SanRaffaeleScientificInstitute,Milan–ITALY

PCR-based methods have been developed to simplify and reduce the time required for genotyping M. tuberculosis by stan-dard approaches based on IS6110-Restriction Fragment Length Polymorphism (RFLP). Of these, MIRU-VNTR comple-mented with spoligotyping has been proposed as an alternative. Repetitive-sequence-based PCR (rep-PCR) is useful for generatingDNAfingerprintsofdiversebacterialspecies.Rep-PCRampliconfingerprintsrepresentgenomicsegmentslying between non-coding repetitive sequences. A commercial system (Diversilab, Biomerieux) that electrophoretically separates rep-PCR amplicons onmicrofluidic chips, and provides computer-generated readouts of results has beenadapted for use with Mycobacterium species. The ability of this system to type M. tuberculosis was evaluated in comparison with spoligotyping and MIRU-VNTR in two different panels.

First, we evaluated a 35 strains panel by MIRU-15 complemented with spoligotyping and Diversilab rep-PCR. Results were analyzed with MIRU-VNTRplus database for spoligo-MIRU, and with Diversilab Software for rep-PCR using two differentalgorithms(PearsonCorrelation[PC]andKullback-Leibler[KL]).Thresholdforclusterswasfixedat98%ofsimilarity for rep-PCR. MIRU-15 showed a clustering rate of 11.4% whereas the rep-PCR reported a clustering rate of 28.6% (PC) and 17.1% (KL). The discriminatory power (Hunter-Gaston discriminatory index [HGDI]) for spoligo-MIRU-15 resulted 0.983 whereas for rep-PCR was 0.978 (PC) and 0.983 (KL).

We also compared the two techniques on a panel composed by 8 closely related strains from a probable outbreak. In this case the rep-PCR showed a discriminatory power of 0.571 (PC) and 0.679 (KL), compared to a HGDI of 0.643 for spoligo-MIRU-15. Nevertheless, clustering rate for MIRU-15 was 50.0% whereas rep-PCR algorithms showed a cluster-ing rate of 100.0% (PC) and 62.5% (KL), respectively. To understand the meaning of the discrepancies still found between spoligo-MIRU-15 and rep-PCR, analysis of epidemiological data for the clustered patients will be taken in consideration.

Preliminary data obtained by Diversilab suggest that the KL algorithm is more appropriate for M. tuberculosis typing analy-sis. Nevertheless, even if rep-PCR results analyzed by KL algorithm showed a discriminatory power similar to MIRU-15, clustering rate remains higher. This new technique could be useful for a routine use in clinical laboratories for real-time genotyping and laboratory contaminations control.

76 ESM 2009

pp-2

68 SpaCErS MyCoBaCTeriuM TuBerCuloSiS COmplEX SpOligOTypiNg : a STuDy uSiNg a miCrObEaD-baSED high ThrOughpuT fOrmaT.

Zhang J1, Abadia E1, Refrégier G1 , Ruimy R2, Boschiroli ML3, Guillard B4 and C. Sola1.1 - Institut de Génétique et Microbiologie, UMR8621, CNRS-Université Paris-Sud (Universud), Equipe IGEPE, bât. 400, Centrescientifiqued’Orsay,rueGregorMendel,91405Orsay-Cedex2-APHP,HôpitalBichat-ClaudeBernard,Paris3 - Agence française de sécurité sanitaire des aliments AFSSA, Maisons-Alfort4 - Institut Pasteur du Cambodge

New captures probes for the microbead-based spoligotyping assay (Luminex) were designed to test for the presence/absence of 25 spacers that are not used in routine spoligotyping. These spacers are expected to provide an improved discriminatory power for Major Genetic Group I, including the « ancestral » TbD1+ MTC (Mycobacterium tuberculosis complex) clinical isolates.

The new 68 spacers spoligotyping format developed on a Luminex platform was shown to give excellent results. Around 400 strains of MTC from 3 different centers (Bichat Hospital, AFSSA, Institut Pasteur of Cambodia) were studied. We show that the 68 spacers format is more discriminant to study the strains of the East African Indian family (EAI) : among 86 strains of the EAI family, it could distinguish 44 clusters compared to 27 clusters by routine 43 spacers spoligotyping. Whereas for a total of 210 clinical isolates of Mycobacterium bovis, we reported 31 types instead of 25, and for a total of 30 “Beijing” clinical isolates, 4 clusters instead of 3 clusters were found. For Mycobacterium africanum, a total of 17 strains were assayed and 11 instead of 9 clusters were found. High-thr(East-African Indian - Indo-Oceanic clade) and otherMajorGeneticGroupIstill-undefinedspoligotypingsignatures.Thisobservationoftenconcernsthefirstspacerof a string of several missing spacers (border domains). These observations are currently under further investigation by sequencing. Extended High-throughput spoligotyping is also a new mean to detect mutational events that could be diag-nosticsofclade-specificMTCevolution.


pp-3

aSSOCiaTiON bETWEEN bEiJiNg gENOTypE aND Drug rESiSTaNCE amONg MyCoBaCTeriuM TuBerCuloSiS iSOlaTES CirCulaTiNg iN ThE rEpubliC Of gEOrgia

Stefan Niemann1, G. Khechinashvili2, M. Gegia2, N. Mdivani2, and Y. W. Tang3

Molecular Mycobacteriology, National Reference Center for Mycobacteria, Research Center Borstel, Borstel, GermanyGeorgian Foundation against Tuberculosis and Lung Diseases, Tbilisi, GeorgiaVanderbilt University Medical Center, Nashville, TN, USA

Rising tuberculosis (TB) rates and high levels of multidrug-resistant TB (MDR-TB) have become a major public health problem in several parts of the former Soviet Union. High rates and transmission of MDR-TB have been noticed to be as-sociated with the presence of Mycobacterium tuberculosis Beijing genotype strains pointing to the importance of pathogen geneticfactorsforthemodulationofinfectionoutcomeandepidemiology.Herewepresentthefirstdataonthepopula-tion structure of M. tuberculosis strains from the Republic of Georgia, a high incidence setting at the Black Sea Coast.

All strains were analysed by spoligotyping and 24-loci MIRU-VNTRgenotyping. IdentificationofmajorM. tuberculosis genotypes was carried out by using the MIRU-VNTRPlus database (www.miru-vntrplus.org) and a similarity analysis performedto identifystrainswith identicalgenotypingprofiles(clusters),whichare indicative fortherateofrecenttransmission. Anti-tuberculosis drug resistance was determined by in vitro antimicrobial susceptibility testing.

Genotypingprofilesweresuccessfullygeneratedfor187M. tuberculosis isolates which were further investigated. The most prominent genotype found, was Beijing (29%), followed by LAM (18%), Ural (12%), and Haarlem (n=10) strains. Approx 50% of the isolates were grouped in clusters. When the distribution of drug resistance is considered, it was noticed that MDR-TB was nearly completely restricted to Beijing strains. Further detailed analyses are currently in progress.

Our data underline the importance of Beijing genotype strains for the TB epidemiology in former Soviet Union countries. However,Ural,Haarlemgenotypeandalargevarietyofstrainsofsofarundefinedlineagesrepresentnearlytwothirdofthe strains found in Georgia. Although Beijing strains are not as dominant as in others Eastern European countries such asKazakhstan,weconfirmaclearassociationbetweenMDR-TBandtheBeijinggenotypeinRepublicofGeorgia.

78 ESM 2009

pp-4

MyCoBaCTeriuM TuBerCuloSiS EpiDEmiOlOgy aND gENETiC DiVErSiTy iN ThE TWiN iSlaND rEpubliC Of TriNiDaD aND TObagO

Shirematee Baboolal, 1, 2 Julie Millet,3 Patrick Eberechi Akpaka, 1 Dottin Ramoutar, 4 Nalin Rastogi 3

1 - Department of Para-Clinical Sciences, Faculty of Medical Sciences, The University of the West Indies, St. Augustine, Trinidad & Tobago2 - Caribbean Epidemiology Centre, Jamaica Boulevard, Port of Spain, Trinidad & Tobago3 - Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, Abymes, Guadeloupe4 - Caura Chest Hospital, Caura, Trinidad & Tobago

Thisworkdescribesthefirstapplicationofmoleculartoolsforstudyingtuberculosis(TB)epidemiology,geneticdiversity,and transmission in the twin island Republic of Trinidad and Tobago (T&T). The study population (n=132) represented one year recruitment of all culture positive TB cases from T&T, and was characterized by a high male to female sex-ratio of 4 (mean age 42.8 years, range 17 to 78 years), and a HIV/TB coinfection rate of nearly 30%. It mainly occurred among African descendants who represented 37.5% of total population but 69.7% of all TB cases (p<0.001). Spoligotyping resulted in 25 different patterns and 12 clusters (2 to 74 strains per cluster). In total, 81.3% of the isolates in our studywasdefinedasmoderntuberclebacillibelongingtothePrincipalGeneticGroups2/3.Fivemajorlineageswereobserved: East-African Indian (EAI), Latin-American and Mediterranean (LAM), X, Beijing, and Haarlem. A comparison with international SITVIT2 database showed that the overall lineage distribution in T&T was completely different from other Caribbean neighbors (n=2653 isolates). A high clustering rate of 90% was observed essentially due to a single large cluster of 74 strains designated as Spoligotype International Type - SIT566 (T&T clone). Patients harboring this genotype were overrepresented in St George Central, the capital city of Port-of-Spain, younger (mean 39.1 years vs. 47.7 years for other genotypes, p<0.0005), and more frequently prison inmates and drug users, while those harboring “other geno-types” were older and showed diabetes as an associated factor. A study of the evolutionary relationships suggested prob-able relatedness of SIT566 with X1 prototype SIT119. A database search localized most of the SIT566 related patterns in USA. Second-line typing using Mycobacterial Interspersed Repetitive Units (MIRUs) suggested the highly conserved natureofSIT566,itsphylogeographicallyspecificitytoT&T.

acknowledgements

We thank the Caribbean Epidemiology Center, Trinidad & Tobago for support, and the staff of the Chest Clinics at the Eric Williams Medical Sciences Complex, the San Fernando General Hospital, and Trinidad Public Health Laboratory for assistancewithdatacollection.SBisgratefultotheUniversityofWestIndiesforfinancialsupport,andJMtotheRegionalCouncil of Guadeloupe and European Social Funds for a Ph.D. fellowship. NR and JM thank J. Driscoll for providing infor-mation regarding the SIT566 clone in USA.


pp-6

SpOligOTypE paTTErNS aND Drug rESiSTaNT prOfilE Of myCObaCTErium TubErCulOSiS iN SuDaN

Ghada Suliman Sharaf-Eldin 1, Imad F.Elmoula 1, Mohammed S. Ali 1, Nageeb S. Saaed 2, Ahammed B Ali 1, Kim Mallard 3, Ruth McNerney 3, Saad Algamdi 3

1 - Al Neelain University-Sudan2 - National Health Laboratory-Sudan3 - London School of Hygiene & Tropical Medicine.

Sudan has a high burden of tuberculosis with an estimated 93,000 new cases each year. The purpose of this study was to investigate the genotypic patterns of M. tuberculosis strains circulating in Sudan and to assess their susceptibly to anti-tuberculosis drugs. Isolates from 237 smear positive tuberculosis patients were col-lected from different geographic regions of the country. Spoligotyping was performed by the Kamerbeek method and results were compared with the international SpolDB4 database (Institut Pasteur, Guadeloupe). Results revealed 28 clusters ranging in size from 12 to 57 isolates. Seventy unique (unclustered) strains were observed, representing 30% of the strains examined. The most frequently observed spoligotype patterns belonged to the CAS fam-ily which represented 115 (48.5%) of isolates studied. T1, H3, U and Beijing strains were found in 12 (5.1%), 11 (4.6%), 7 (3%) and 6 (2.5%) patients respectively. Strains belonging to the Beijing family were found mainly in Western Sudan. Resistance to isoniazid, rifampicin, ethambutol and streptomycin was observed in 18.1, 22.4, 22.2 and 32% of strains re-spectively. Twenty patients (8.4%) had MDR-TB of which 10 were new cases. Seventeen patients with rifampicin resistant tuberculosis were infected with CAS1-DELHI strains matching SIT 25 of the SpolDB4 database and 3 were of the SIT 1 Beijing family. 15 loci MIRU-VNTR typing subdivided the 17 CAS strains into one cluster of 5, two clusters of 2 and 8 individual MIRU types. Similarly the 3 Beijing spoligotypes were differentiated into a cluster of 2 and a single strain. The use of molecular strain typing provides a proactive approach that may be used to initiate, and not just augment, traditional surveillance outbreak investigation in Sudan. However, caution must be used when interpreting clustered spoligotype patterns in this region.

80 ESM 2009

pp-7

CONTrOVErSial DiSSEmiNaTiON paTTErN Of ThE bulgaria -SpECifiC M. TuBerCuloSiS SpOligOTypE ST125_bgr

Violeta Valcheva 1, 2, Igor Mokrousov 1, 3, Stefan Panaiotov 4, Elizabeta Bachiiska 4, Thierry Zozio 1, Christophe Sola 1, Nadya Markova 2, Nalin Rastogi 1

1 - Institut Pasteur de Guadeloupe, France2-InstituteofMicrobiology,Sofia,Bulgaria3 - St. Petersburg Pasteur Institute, St. Petersburg, Russia4-NationalCenterofInfectiousandParasiticDiseases,Sofia,Bulgaria

Objective

To investigate phylogenetic position and geographic genetic diversity of spoligotype ST125 in Bulgaria.

material and methods

Study sample included all available 47 DNA samples belonging to spoligotype ST125 that were taken from two previously published M. tuberculosis collections from Bulgaria (Valcheva et al., 2005, 2007, 2008abc; Panaiotov et al., 2005, 2006). These DNA were additionally typed using new 24-loci MIRU format, IS6110-RFLP typing and LAM-PCR. Phylogenetic analysis was done using PAUP and PHYLIP packages.

results

Comparison with SITVIT2 database (Institut Pasteur de Guadeloupe) revealed a high gradient of ST125 in Bulgaria (14.3%) compared to its negligible presence in the world. Typing of Bulgarian ST125 strains revealed that they: (i) did notharboraLAM-specificIS6110 insertion (ii) formed a monophyletic cluster in 24-MIRU tree of Bulgarian strains (iii) grouped closely with ST34 that is a prototype of the S family. A similarity of the IS6110-RFLPprofilesconfirmedatruerelatedness of these ST125 strains whereas a diversity of the MIRU loci suggested a long-term evolution of this spoligo-type in Bulgaria. Minimum spanning tree of the 24-MIRU-based subtypes of ST125, and comparison with their geographic distribution revealed an enigmatic and complex dissemination pattern of this spoligotype across Bulgaria.

Conclusion

T125 likely belongs to S family and may have originated from spoligotype ST4 by a deletion of a single spacer #40 in the DRlocus.ST125isphylogeographicallyspecificforBulgaria;weproposeitsrenamingasST125_BGR.Ahighdiversityofthe MIRU loci suggests a long-term evolution of this spoligotype in Bulgaria.

acknowledgments.

This work was supported by NATO grant SFP-982319 “Detect drug-resistant TB”.


pp-8

myCObaCTErium TubErCulOSiS bEiJiNg gENOTypE aND OrigiNS Of ThE bulgariaNS

Panaiotov, Stefan; Bachiyska, Elizabeta; Brankova, Nadia; Levterova, VictoriaNationalCenterofInfectiousandParasiticDiseases,Sofia1504,Bulgaria

RecentstudiesdemonstratedthatexistgenotypesofM.tuberculosislocallydistributedtospecificgeographicregion.Other genotypes are distributed globally or on vast geographic areas. These facts led to other recent fundamental stud-iesandconclusionsthatexistscertaingeneticpredispositionoftheethnicgroupstospecificM.tuberculosisgenotypes,hencegeneticpredispositiontotuberculosisspecificgenotypesissuspected.Inthepastandnowadaysthewavesofhu-man migration coincide with expansion of tuberculosis genotypes. In our study we associated the global distribution of MTB Beijing genotype, its’ distribution in Bulgaria, the geographic regions of historical origin of the Bulgarian tribes, and theethnicaffiliationoftheBulgarians.

For our analysis we used data published in the fourth international spoligotyping database (SpolDB4), publications, per-sonalcommunicationsandofficialhistoricaltheoriesregardingoriginsoftheBulgarians.

From the literature and in SpolDB4 exists about 330 spoligotypes of Bulgarian M. tuberculosis clinical strains collected fromalloverthecountry.Beijingspoligotypewasnotidentifiedamongthem.WeconcludedthatthisMTBgenotypeisnot(orveryrare)distributedinBulgaria.InRomaniaBeijinggenotypewasnotidentifiedtoo(personalcommunication).OthercountriesassociatedwiththeoriginsandmigrationoftheBulgarianswhereBeijinggenotypeisnotidentifiedisIran.SignificantpartoftheBulgarianspoligotypesarephylogeneticallyrelatedtoMANUfamily,widelydistributedinIndia.These facts correlate with the widely accepted theory that the origins of the Bulgarian tribes are of Indo-Iranian lineage. In contrary, this fact does not support the theory for the Turkik lineage or more precisely the Turano-Hunnic origins of the Bulgarian ethnos.

Beiging genotype is widely distributed in Central Asian countries, Kazahstan, Turkmenistan, Russia, Turkey, China etc. Bulgaria has very active tourist, cultural, political, trade and immigration links with all these countries. Based on these empiric facts we indirectly conclude that there is genetic predisposed resistance of the Bulgarian and Iranian ethnos to MTB Beijing genotype.

82 ESM 2009

pp-10

ThE EXTENT Of ThE laTiN amEriCaN-mEDiTErraNEaN MyCoBaCTeriuM TuBerCuloSiS SpOligOTypE family iN pOrTugal

Suzana David 1, João Nuno Ribeiro 1,2, José-Nuno Maio 1,2, Inês João 1, António Amorim 3, Edna Pereira 3

1 - Unidade de Referência e Vigilância Epidemiológica, Laboratório de Infecções Respiratórias – Micobactérias, Instituto Nacional de Saúde Dr. Ricardo Jorge, Portugal2 - Grupo de Microbiologia e Imunologia da Infecção, Instituto de Biologia Molecular e Celular, Porto, Portugal3 - Laboratório de Saúde Pública, Micobacteriologia / Tuberculose, Administração Regional de Lisboa e Vale do Tejo, Lisboa, Portugal

Portugal isclassifiedat the intermediate levelwithrespect to the incidence for tuberculosis.Geographical spread isheterogeneous with the majority of cases concentrated in the large urban areas mainly in the Lisbon and Porto districts. Previous efforts in the characterization of the population structure of Mycobacterium tuberculosis isolates stressed the importance of this approach to tuberculosis control. Spoligotyping data restricted to the metropolitan area of Lisbon, revealed a 51% prevalence of the Latin American-Mediterranean (LAM) spoligotype family, and a high proportion of SIT20 (LAM1) and SIT42 (LAM9) sub-families. In the present study this characterization has been extended to encompass both the Lisbon and Porto areas. With the use of complementary data from SpotClust and Ag85C103 RFLP, the proportion of the LAM genotype was shown to be as high as 62% of the total number of isolates. The LAM genotypes were further characterized for the RDRio deletion detected in up to 60% of the LAM isolates. This study suggests that Portugal may have one of the highest global proportions of the LAM family. Monitoring and further characterizing of this relevant spo-ligotype family is considered of major importance for the understanding of the dynamics of tuberculosis in Portugal.


pp-11

fiTNESS STuDy Of ThE rDriO liNEagE aND lam family Of MyCoBaCTeriuM TuBerCuloSiS iN a STuDy pOpulaTiON iN riO graNDE, brazil

Von Groll, Andrea 1;Martin, Anandi 1; Felix, Carolina 2; Prata, Pedro 2; Honscha, Günther 3; Portaels, Françoise 1; Almeida da Silva, Pedro 2; Palomino, Juan Carlos 1

1 - Institute of Tropical Medicine, Antwerp, Belgium2 - Universidade Federal do Rio Grande, Rio Grande, Brazil3 - Laboratório Municipal de Tisiologia, Rio Grande, Brazil

RDRio is a novel Mycobacterium tuberculosis lineage member of the Latin American-Mediterranean (LAM) family. LAM has been found worldwide but it is more predominant in South America. The aim of this study was to assess the presence of the RDRiolineageandLAMfamilyinthecityofRioGrande,Brazil,andtoinvestigatethefitnessofthesestrainsbasedon the determination of their rate of growth. Fifty clinical isolates of M. tuberculosis were genotyped and 43 different pat-terns were found by spoligotyping and MIRU-VNTR. The predominant genotypes belonged to the LAM family (54% of the strains) followed by clade T (22%) and Haarlem (16%). The RDRio lineage represented 38% of the total strains and 70.4% oftheLAMstrainsfoundinthisstudy.StrainsbelongingtotheLAMfamilyshowedafitnessadvantagecomparingtheirrate of growth with that of non-LAM. RDRiostrainswerepredominantwithintheLAMfamily,butasignificantdifferenceinfitnessbetweenRDRio and the non-RDRiostrainswasnotconfirmed.

84 ESM 2009

pp-12

gENOmiC CharaCTErizaTiON Of liSbOa family STraiNS by DElETiON aNalySiS

João Perdigão, Carla Silva and Isabel PortugalCentro de Patogénese Molecular, URIA, Faculdade de Farmácia da Universidade de Lisboa

Multidrug and extensive drug resistant tuberculosis poses a very serious threat for public health. Lisbon Health Region has one of the world’s most serious situations regarding this problem. Such, is the result of a continued circulation of an endemic and predominant strains of a particular genetic family – Lisboa family. Little is known regarding the phylogeny, relativevirulenceandgeneticbackgroundofthesestrains.Thelossordeletionofspecificgenomicregionsconstitutesthe most important way by which Mycobacterium tuberculosis diverges and adapts. Several deletions, named Regions of Difference (RD), have already been described and associated with phylogeographic lineages. The characterization of Lisboa family in this manner may elucidate its origin and perhaps be helpful in explaining its high prevalence.

Three representative clinical isolates of different genetic clusters of strains circulating in Lisbon Health Region were screenedforthepresenceof16distinctRDs.DeletiondetectionwasperformedbyPCRcarriedoutusingprimersflank-ingeachRD.Confirmationwasperformedbysequencinganalysis.

All three isolates were found to possess four of the tested deletions: TbD1, pks15/1, RD174 and RDRIO. It was not pos-sible to discriminate between the strains using this deletion typing approach. However, it was possible to infer on the phylogeography of these strains. The presence of TbD1 and pks15/1 deletion positions the strains in the modern and Euro-American lineage, respectively. On the other hand, RD174 suggests that the analyzed strains are related to the West-African sub-lineage.

The present study point toward the fact that Lisboa strains and others circulating in Lisbon belong to Mycobacterium tuberculosis modern lineages of the Euro-American lineage, West-African sub-lineage, therefore revealing more on Lisboa family’s origin.


pp-13

impOrTaNCE Of mOlECular TypiNg iN SuSpECTED iNTra-familial TraNSmiSSiON Of TubErCulOSiS

Obrovac, Mihaela 1, Katalinic-Jankovic, Vera 1, Grce, Magdalena 2, Zmak, Ljiljana 1

1 - Croatian National Institute of Public Health2 - Rudjer Boskovic Institute

Tuberculosis (TB) is most commonly transmitted from a person suffering from infectious pulmonary TB to another person by infected droplet nuclei. The chance that a person will be infected with TB depends on the intensity, frequency and dura-tion of the exposure to tubercle bacilli. Also, numerous studies emphasize the importance of host resistance and hereditary susceptibility, indicating that the development of TB is a result of a complex interaction between the host and the pathogen influencedbyenvironmentalfactors.Assumingthatthereisprolongeddurationandfrequencyofcontactbetweenfamilymembers and among household contacts, it is estimated that the risk of TB transmission will be high. There are several studiesconfirmingintra-familialtransmissionusinggenotypingofisolatedM.tuberculosisstrains.However,thepossibilityofunsuspected transmission should not be disregarded. We report here of two cases of suspected TB transmission in families causedbyM.tuberculosisstrainsthatwerefoundnottobeidenticalaccordingtogenotypingprofilesobtainedbydetermin-ing variabile number of tandem repeats (VNTR) of mycobacterial repetitive interspersed units (MIRU). Genotyping was per-formed using complete set of 24 VNTR loci. Epidemiological data were collected by contact tracing and interviewing patients. Our results show that TB cases in family do not necessarily have to be caused by the same M. tuberculosis strain. Epidemiologic investigations need to be combined with genotyping data for better understanding of transmission dynamics. Transmission of TBcannotbeconfirmedbycontactinvestigationonly,evenwhenintra-familialtransmissionissuspected.

86 ESM 2009

pp-14

DETECTiON Of ClONal COmplEXiTy iN CliNiCal M. TuBerCuloSiS iSOlaTES by miru-VNTr iN CuKurOVa rEgiON, TurKEy

Ülkü ORAL ZEYTINLI, M. Begüm KAYAR, Ayse KARACALI, Arzu SAHAN KIPALEVErkan YULA, Fatih KÖKSAL University of Cukurova

Purpose of the study: Tuberculosis remains one of the most prevalent infectious disease in the world . The applica-tion of molecular typing methods to the analysis of clinical Mycobacterium tuberculosis (MTB) complex isolates has greatly facilitated the understanding of epidemiology of tuberculosis (TB) and revealed that the infection by this patho-gen can be clonally complex and reinfection, coinfection. Genotyping using RFLP-IS6110 (Restriction Fragment Length Polymorphism) is based on transposable element IS6110 and mycobacterial interspersed repetitive unit-variable number of tandem repeat typing (MIRU-VNTR) has become a major method for epidemiological tracking of Mycobacterium tuberculosis complex clones. Our aim was to establish the range of applicability of 12 loci MIRU–VNTR genotyping in epidemiology of TB and evaluate the discriminatory power obtained with RFLP-IS6110 and MIRU-VNTR used alone or in combination.

methods

In this study, we analyzed 94 clinical MTB complex isolates from sputum in patients with pulmonary TB between February 2008- February 2009 in Cukurova region, Turkey.

results

MIRU–VNTR typing detected 45 different patterns, 61 strains were grouped into 12 clusters and 33 strains had uniqe patterns. The largest cluster comprised 9 strains. In addition, 2 clusters contained 5 strains, 6 clusters contained 3 strains and 1 cluster contained 2 strains. It is also determined that the loci including MIRU 04, MIRU 10, MIRU 26 and MIRU 40 have the highest allelic diversities and discriminatory power. On the other hand with IS6110-RFLP typing of same clini-cal MTB strains, 33 genotypes were founded. 76 strains of which were gruoped into15 clusters. 18 of the 94 strains had unigue RFLP patterns.

Conclusion

MIRU-VNTR is more discriminative methods for phylogenetic studies than IS6110-RFLP and could define from reinfections to reactivations important to treatment of MTB complex organisms.


pp-15

mOlECular EpiDEmiOlOgy STuDy Of TubErCulOSiS paTiENTS iN a Small CiTy Of SÃO paulO – brazil, frOm 2002 TO 2006

Leite, Clarice;Santos,Adolfo;Pandolfi,JoséRodrigo;Malaspina,AnaC;Pavan,Fernando;Mendes,NatáliaUniversidade Estadual Paulista, Faculdade de Ciências Farmacêuticas

The molecular epidemiology study using different techniques revolutionized the understanding of the epidemiology of tuberculosis allowing comparison between strains of Mycobacterium tuberculosis and tracking the movements of individual lines. This project aimed to use the techniques of molecular epidemiology (MIRU) trying to understand more about the phenomenon of transmission of tuberculosis among patients with pulmonary tuberculosis in a small city of São Paulo - Brazil, attended the Special Health Service of Araraquara (SESA - clinic of reference in the diagnosis and treatment of tuberculosis) in the city of Araraquara. From total 163 positive cultures received, the MIRU technique was performed in 74,2% (121/163) of the isolates from patients attended by SESA in the period from 2002 to 2006. 5 isolates were also identifiedasenvironmentalmycobacteriaand4unidentifiedmycobacteria.Fromthe121isolatessubmittedtogenotyp-ing, six did not present all alleles among the 12 loci of MIRU. From the 115 isolates submitted to the dendrogram, 29 (25,2%) are grouped into 13 clonal groups with similarity of 100%. 10 groups with two isolates each and 3 groups con-taining3isolateseach.Theothers86isolates(74,8%)hadasinglegeneticprofile.Aboutthegroupof29patients,only10were female and 19 males. Except for one patient, the other 28 were treated with the schedule I having cure in 82,8% of the cases. Whereas the similarity of 83% or greater, highlighted 3 major clonal groups called A, B and C, involving 86 of all isolates analyzed. In these large groups were included all 13 clonal groups with 100% similarity. These data suggest the possibility the tuberculosis in Araraquara is because of the presence of persistent endemic strains responsable for 74,8% of cases, besides the existence of recent transmission in this work was 25,2%.

Keywords: Epidemiology, Tuberculosis, MIRU

Financial support: FUNDUNESP.

88 ESM 2009

pp-16

gENOTypiNg Of myCObaCTErium TubErCulOSiS iN NOrThWEST Of paraNÁ STaTE Of brazil

Leite, Clarice Queico Fujimura 1, Nogutia, Erika N 2, Malaspina, Ana Carolina 1, Santos,Adolfo Carlos Barreto 1, Hirata, Rosáro DC 3, Hirata, Mário H 3, Cardoso, Rosilene Fressatti 2

1 - São Paulo State University2 - Maringá State University3 - University of São Paulo

introduction

Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death. The molecular epidemiology study using different techniques revolutionized the understanding of the epidemiology of tuberculosis allowing comparison between strains of Mycobacterium tuberculosis and tracking the movements of indi-viduallines.WereportedherethefirstinsightaboutthegeneticdiversityofM.tuberculosisinnorthwestofParanáState,south of Brazil. This knowledge encourages additional prospective epidemiological study for evaluation of the Regional Tuberculosis Control Plan in this setting.

Objectives

Provide information about the genetic diversity and prevalent genotype of Mycobacterium tuberculosis and compare the usefully of two methodologies in epidemiological study of tuberculosis in low endemic area in south of Brazil. Material and Methods: We used spoligotyping and MIRU-VNTR typing to genotype M. tuberculosis isolates.

results

The 93 isolates analyzed by spoligotyping were divided into 36 different patterns and 25 were described in the SpolDB4.0 database. Latin American and Mediterranean, Haarlem and T family were responsible for 26.9%, 17.2% and 11.8%, of tuberculosis cases respectively. From the 84 isolates analyzed by MIRU-VNTR typing, 58 showed unique pattern and 26 belonged to 9 clusters. The MIRU loci 40, 23, 10 and 16 were the most discriminatory. MIRU-VNTR and spoligotyping combined showed 85.7% of discriminatory power (HGI=0.995).

Conclusions

Spoligotyping and MIRU-VNTR typing combined are useful tool for epidemiological study in this low endemic setting in south of Brazil and tuberculosis predominantly develops through reactivation of latent infection.


pp-17

SpOligOTypiNg Of myCObaCTErium TubErCulOSiS iSOlaTED frOm paTiENTS Of ClEmENTE fErrEira ambulaTOry iN SÃO paulO, Sp – brazil

Mello, Fernado Augusto Fiuza 1, Albarral, Maria Idemar Pedrosa 1, Mendes, Natália Helena 2,Pandolfi,JoséRodrigoCláudio 2, Santos, Adolfo Carlos Barreto 2, Almeida, Elisabete Aparecida 1, Cardoso, Rosilene Fressatti 3, Leite, Clarice Queico Fujimura 2 1 - Clemente Ferreira Institute2 - São Paulo State University3 - Maringá State University

The molecular epidemiology study using different techniques revolutionized the understanding of the epidemiology of tuberculosis allowing comparison between strains of Mycobacterium tuberculosis and tracking the movement of individual strains. This project aims to use the technique of molecular epidemiology, Spoligotyping, trying to understand more about the phenomenon of transmission in patients with pulmonary tuberculosis treated at Clemente Ferreira Ambulatory (ambulatory of reference for the treatment of tuberculosis) in São Paulo city, from August 2006 to July 2008. Theclinicalisolateswerere-identifiedbymoleculartechnique(PCRandPRA),andthestrainsidentifiedasM.tuberculo-sisconductedbythegenotypingtechniqueofSpoligotyping.From102isolates,thetechniqueofIS6110-PCRconfirmedtheidentificationofM.tuberculosisin96clinicalisolatesandthePRAin99,theremaining3,2isolatesidentifiedasM.aviumsubtype2and1unidentifiedmycobacteria.Theresultsshowedthat3clinicalisolatesofM.tuberculosishadnottheIS6110insertionsequencespecificofM.tuberculosis,aswellas3isolatesidentifiedintheclinicasM.tuberculosisbymoleculartechniqueswereatypicalmycobacteria.Of96isolatesconfirmedasM.tuberculosis,wereanalyzedbythetechnique of Spoligotyping, a total of 89 isolates, which revealed the presence of 21 strains (23.6%) with spoligotipes not yet described in the data base world (spolDB4) and 68 (76.4%) of isolates involved in 7 different families, containing 2 to 30 isolates. The most frequent was T family with 30 isolates), followed by LAM (with 20 isolates) and Haarlem (with 10 isolates),whichtogetheraccountedabout67.4%ofallisolates.Werealsoidentified4genotypesoftheBeijingfamily,allsimultaneously resistant to isoniazid and rifampicin and / or more drugs.

90 ESM 2009

pp-18

COmpariSON Of myCObaCTErium bEiJiNg gENOTypE WiTh VNTr, SpOligOTypiNg aND rflp-iS6110

Elahe Tajeddin , Parissa Farnia, Mohammad Kargar,Jamileh Noroozi, Mojtaba ahmadi, Mehdi kazempour, Maryam Hadadi,Mohammadreza Masjedi, Aliakbar VelayatiMycobacteriology Research Center (MRC) National Research Institute Of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University Medical Campus.Tehran,Iran.

background

Beijing strains constitute more than 1/4 of Mycobacterium tuberculosis (MTB) genotypes. Beijing genotype is considered an important genotype because of its reasonable characteristics such as: association with multi-drugs resistance TB. Accordingly these strains are reluctant to conventional TB drugs. Therefore, it is necessary to investigate the transmission rate among Beijing strains within the studied communities. In this study, three molecular methods (Spoligotyping, VNTR, and RFLP-IS6110) were used to identify transmission among patients infected with Beijing strains.

materials and methods

The susceptibility tests were performed on 238 M. tuberculosis culture positive specimens. Thereafter, the isolated Beijing genotype was subjected to VNTR and RFLP. The results of Spoligotyping were analysed by using SPOLDB4 database. VNTR typing was used to identify alleles diversity in 9 locus (MPTR-A, ETR-A, ETR-B, ETR-C, ETR-D, ETR-E, ETR-F, QUB11B, QUB3232) of isolated Beijing strains.The allelic diversity of VNTR was measured by using Hunter Gaston Index (HGI).

results

The spoligotyping of M. tuberculosis isolates revealed the following 8 patterns: Haarlem (27.7%), CAS1 (25.2%), EAI3 (21.8%), CAS2 (6.7%), T1 (6.3%), Beijing(5.5%) U(5%), T(0.4), EAI2 (1.2%).

ThefollowingVNTRloci(QUB3232),(QUB11b,ETR-EandETR-F)and(otherloci)wereidentifiedasmost(HGI≥ 0.6), median (HGI≥0.4-0.6) and weakest (HGI=0) distinctive loci for Beijing families respectively. Whereas the Beijing strains demonstrated diverse patterns in RFLP,13/13(100%) and VNTR 10/13(77%).

Conclusions

Beijing is one of the dominated circulating strains in Iran and interestingly majority of infected cases were due to reactiva-tionratherthanrecenttransmission.TheVNTRandspoligotipingmethodsweremoreefficienttodetectBeijingstrainsthan by use VNTR and RFLP allow.

Keywords

Spoligotyping / VNTR / RFLP / Mycobacterium tuberculosis Beijing genotype.


pp-19

mulTiply rECurrENT TubErCulOSiS iN a paCiENT liViNg WiTh hiV: rEiNfECTiON Or rEaCTiVaTiON?

Ritacco, Viviana 1, Reniero, Ana 2, Beltrán, Marcelo 2, López, Beatriz 1, Kantor, Isabel 3, Barrera, Lucía 1

1 - ANLIS, CONICET, Argentina2 - Hospital Municipal de San Isidro3 - PAHO/WHO consultant

purpose

To determine the cause of recurrent clinical episodes of tuberculosis in a patient living with HIV. Patient: Male, 24 year-old andillegaldrugintravenoususerfor10yearsatthetimeoffirstconsultation,assistedbetween1995and2009inouroutpatient clinic, San Isidro Municipal Hospital, Argentina.

methods

Clinical-epidemiological follow up.Mycobacterial culture, identification and drug susceptibility testing.Mycobacterium tuberculosis IS6110 RFLP and spoligo genotyping within the frame of a population-based study performed in San Isidro, an outskirt of Buenos Aires City.

findings

Ourpatient’sfirstdiagnosisoftuberculosiswasconfirmedbyculturein1995,almostsimultaneouslywithHIVserologicalconversion. At that time, the isolate was found only resistant to isoniazid and its genotype matched that of a isoniazid-resistantoutbreakstrainpreviously identified intwoofourpatient’sprison inmates,whowerealsomembersofhisintravenous-drug-user gang. One year after cure, our patient suffered from a relapse of his tuberculosis due to the same strain,whichnowhadaddedresistancetorifampicinandlostabandintheIS6110fingerprint.In2002,thepatientsuf-feredfromathirdepisodeoftuberculosis,thistimeduetoafullydrugsusceptiblestrain,identifiedpreviouslyinotherof our patient’s intravenous drug user friends. At that time, given his poor clinical condition, our patient underwent a prolonged hospitalization in the Muñiz Hospital, the epicentre of a large tuberculosis outbreak caused by the notorious multidrug-resistant strain “M”. After successful treatment and cure, our patient, who was never compliant with antiretro-viral treatment, was discharged from the Muñiz Hospital. A few months later, he sought again assistance at our clinic with active tuberculosis due to the multidrug resistant strain “M”. He is currently struggling with a relapse of disease caused by this outbreak strain.

Conclusion

Whether recurrent tuberculosis is due to a newly acquired infection or to reactivation of a previous one is a century-long controversial question. In our case, both conditions alternated throughout the 14 years of the patient living with HIV. Work partially funded by FP7 grants 201690 and 223373 of the EC.

92 ESM 2009

pp-20

mOlECular EpiDEmiOlOgy Of TubErCulOSiS iN Vila NOVa DE gaia, pOrTugal

Tavares Magalhães, Ana1, Alves, Adriana1, Braga, Rosário2, Valente, Isabel2, Duarte, Raquel3 and Miranda, Anabela1

1 - Tuberculosis Reference Laboratory, Department of Infectious Diseases, National Institute of Health, Porto, Portugal2 - Central Hospital, Vila Nova de Gaia, Portugal3 - Chest Clinic, Vila Nova de Gaia, Portugal

DNAfingerprintingofMycobacterium tuberculosis has provided a better understanding of the epidemiology of tubercu-has provided a better understanding of the epidemiology of tubercu-losis. Restriction fragment length polymorphism based on IS6110 (RFLP-IS6110) has been the gold standard for typing M. tuberculosis since 1993. In recent years, mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR)hasbeenproposedasafirst-linetypingmethod for M. tuberculosis. This technique generates easily ana-lyzed and portable data, has a good discrimination power and has been proven useful for studying the epidemiology of tuberculosis and the phylogeography of tuberculosis bacilli.

In the present study we evaluated the genetic diversity of M. tuberculosis clinical strains, isolated from 115 patients from Vila Nova de Gaia, Portugal, during the period of 2004 to 2005, using the standardized MIRU-VNTR typing method based on 15 loci, proposed by Supply and collaborators in 2006. Strain lineage designation, allelic diversity and clustering rate were determined using the MIRU-VNTRplusidentificationdatabase.Thediscriminativepowerofthemethodwasana-lysed using the Hunter and Gaston diversity index.

Based on MIRU-VNTR typing, the 115 strains were divided into 62 types as follows: 69 strains were distributed into 16 clusterscontainingtwotoeightisolates,and46strainshaduniqueprofiles.MIRU-VNTRrevealedaclusteringrateof46% in the sample under study. The Hunter-Gaston index was 0.976. The most discriminatory loci were Mtub04, MIRU 40, MIRU10, QUB11b, Mtub30, Mtub39 and QUB26 showing an allelic diversity higher than 0,6.

The phylogenetic analysis revealed the presence of two major lineages, LAM and Haarlem, representing 60% and 27% of the total isolates, respectively. This fact is in agreement with the M. tuberculosis phylogeography for the South of Europe.

Fourteenstrainsshoweddrugresistancebutnoassociationwasestablishedbetweendrugresistanceprofileandphylo-genetic family.

MIRU-VNTR showed a good discriminatory power for typing of M. tuberculosis strains in this setting and the MIRU-VNTRplus databaseisanimportanttoolforlineageidentification.Thistypingapproachoffers,simultaneously,epidemio-isanimportanttoolforlineageidentification.Thistypingapproachoffers,simultaneously,epidemio-lineageidentification.Thistypingapproachoffers,simultaneously,epidemio-logic and phylogenetic information, as demonstrated. Overall, this study reveals that recent transmission of tuberculosis is high in Vila Nova de Gaia, and that import of strains in this region is not a problem.


pp-21

mOlECular STuDy Of rECurrENT TubErCulOSiS CaSES

Alves, Adriana; Miranda, Anabela; Tuberculosis Reference Laboratory GroupTuberculosis Reference Laboratory, Department of Infectious Diseases, National Institute of Health, Porto, Portugal

Tuberculosisrecurrenceisfrequentlyattributedtoreactivationoftheisolateresponsibleforthefirstepisodeofthedisease.Nevertheless,thiscanalsobeduetoaninfectionwithanotherisolate,ortomixedinfections.Clarificationofthe cause of recurrence is very important and can be achieved by molecular typing of serial isolates of M. tuberculosis. The most appropriate methods to do so are IS6110 RFLP and MIRU-VNTR.

In this work, 39 clinical isolates of M. tuberculosis belonging to nine individuals with recurrent disease were studied throughout time. Seven of these patients were resistant to isoniazid and rifampicin as well as to most other 1st and 2nd line drugs. Another patient was resistant to isoniazid and streptomycin, and the last one was resistant to rifampicin only. For some of these individuals, resistance to drugs worsened during the cause of the disease, which in some cases has lastedformorethanadecade.All39strainswereanalyzedbyIS6110RFLP.MIRU-VNTRwasusedtotype:(i)thefirststrainofeachpatientwhenserialisolatesshowednochangeintheIS6110RFLPprofile;or(ii)eachstrainwithapatternchange in relation to the previous one.

RFLP results showed that only one out of nine patients displayed a change in the pattern of serial isolates with gain of one IS6110 element. Analysis of the results using Bionumerics grouped these patients in nine clusters, being that: (i) strains from two patients belong to the same cluster; and (ii) strains of one patient are divided in two clusters. Results of 15 MIRU-VNTR locitypingcorroboratetheIS6110RFLPfindings.Inotherwords,theserialisolatethatgainedoneIS6110element also shows a change in MIRU-VNTR results. In this case, we observed a reduction of one repeat in loci Mtub21 and QUB11b. Finally, 40% of these strains belong to the LAM family, 10% to the Haarlem family, and the remaining 50% didnotfindamatchinthedatabase.

This work shows that the cause of recurrent tuberculosis of the nine patients included in this study is due to persistence of initial M. tuberculosis strain or to reactivation when there is more than one episode of disease. Antibiotic resistance is the most important cause of chronic tuberculosis in these patients, since all analyzed strains are resistant to the majority of the 1st and 2nd line drugs.

94 ESM 2009

pp-22

gENOTypiNg Of Drug rESiSTaNCE iN MyCoBaCTeriuM TuBerCuloSiS uSiNg DiagNOSTiC miCrOarrayS

Ehricht, Ralf 1, Slickers, Peter 1, Monecke, Stefan 2

1 - CLONDIAG, Jena, Germany2 - University Hospital Dresden

Tuberculosis is a disease of worldwide concern. Antimicrobial resistance in Mycobacterium tuberculosis is an increasing challenge and, in contrast to other bacteria, not caused by the acquisition of certain genes, but by acquisition of single point mutations in genes which are present in all strains. Unfortunately, culturing and subsequent growth inhibition assays are still time demanding preventing fast detection and treatment. Genotyping methods as PCR followed by sequencing are an alternative. Here the bottlenecks are processing time, overall costs and lack of parallelisation. Hybridisation of such PCR products against highly discriminatory oligonucleotide probes is an alternative approach which could solve these problems. We developed an assay using a diagnostic oligonucleotide microarray and covering probes for relevant mutations in genes rpoB, katG, embA, and embB, for the embC/embA-intergenic region, and the mabA/inhA promoter. PCR is employed to amplify these genes and to incorporate biotin 16-dUTP during elongation. These labeled amplicons are hybridised to the array which are inserted into ArrayStrips, and hybridisation is visualised using dye precipitation trig-gered by streptavidin-peroxidase complexes bound to the biotin labels and the ArrayMate reading device. The procedure is currently tested using DNA from previously characterized strains for which conventional susceptibility test results and relevant sequences are available.


pp-23

gENOTypiNg Of mONO aND mulTi-Drug rESiSTaNCE Tb iN SauDi arabia

Sahal Al-Hajoj1, Bright Varghese1, Mais Herbawi1 , Ruba Al-Omari1 and Caroline Allix-Béguec2

1 - King Faisal Specialist Hospital and Research Centre, Comparative Medicine Tuberculosis Research Unit Saudia Arabia2 - Genoscreen

A total of 150 isolates collected from different regions in Saudia Arabia were the subjectoffingerprintingusingGenoScreenMIRU-VNTR kit (do you know the timeframe?). Upon genotype the data were compared to the international MIRU-VNTRplus database (www. Miru-vntrplus.org). The data showed that Saudia Arabia harbors the following major clades EAI 13.07%, Haarlem 12.42%, TUR 13.07%, Beijing 12.42%, unknown12.42%, Dehli/CAS 7.84%, LAM 7.19%, Cameroon 6.54%, UgandaI 5.23%, S 3.92%, multiple matches1.96%, NEW-1 1.31%, URAL 0.65%, Ghana 0.65%, X, 0.65%, UgandaII, 0.65%. Indicate the clustering rate for this panel of 150 isolates. Ongoing transmission may be an indication to the need of improving TB control program. The unknown clades represent a considerable % (12.42%). These could represent unique endogenous clades. However, further analysis is needed to gain insight into the nature of the unknown clades.

96 ESM 2009

pp-24

Early DETECTiON Of mDrTb by mOlECular TOOlS iN ThE CONTrOl Of Drug rESiSTaNT TubErCulOSiS iN pOrTugal: a CaSE Of SuCCESS

Diana Machado1, Miguel Viveiros1,2, Liliana Rodrigues1,3, Isabel Couto1,4, Leonard Amaral1,2,3 1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal2 - COST ACTION BM0701 (ATENS)3 - UPMM, IHMT/UNL, Lisbon, Portugal4 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal

Multidrug resistant tuberculosis (MDRTB) represents a threat to public health and a challenge to tuberculosis (TB) con-trol programs. From 1994 to 1997, Portugal had an incidence of 48.3 new cases of TB per 100 000 inhabitants and an average of 22.7% of these cases were MDRTB, the highest in Western Europe. In an attempt to assist the National Health Authorities in the control of TB, we implemented in 2002 with the support of Fundação Calouste Gulbenkian, the “TB Fast-Track” program as part of the TB Task Force of Greater Lisbon, involving 12 Lisbon hospitals and based on the use of the BACTECTMMGIT960system,coupledwiththedirectidentificationofM. tuberculosis complex (MTBC) and the de-tection of mutations in the rpoB gene, using the INNOLiPA Rif.TB Assay (LiPA) (Innogenetics). Because mutations in the rpoB result in resistance to rifampicin (RIF), and resistance to RIF is almost always accompanied by resistance to isoniazid (INH),thisapproachallowedustoidentifytheMDRTBpatientwithin24-48hours.Afullreportconfirmingidentificationand antibiotic susceptibility (AST) of MTBC by conventional methods (BACTEC culture plus AST and Accuprobe ID) was issued within additional 12 days.

From September 2002 to January 2006, the LiPA assay was directly applied to 630 acid fast positive respiratory speci-mens. The comparison between data from this assay and conventional methods revealed 84 discrepancies. The 11 false positive results corresponded to patients with therapy already established by the time of specimen sampling, whereas the73falsenegativeresultedfrominhibitionofamplification.Atotalof487ofthe600MTBCpositiveisolatesweresusceptibletoall5first-lineanti-TBdrugs.ThefrequencyofMDRTB(resistanttoatleastINHplusRIF)was10%.Fromthe 63 MTBC resistant to RIF, 62 were detected by the LiPA as carrying mutations S531L (60 isolates), H526Y and D516V (1isolateeach).NomutationwasdetectedbyLiPAforonesample,repeatedlyidentifiedasresistantbyAST.Detectionof rpoB mutations proved to be a good surrogate marker for MDRTB, since only 2 out of 600 MTBC isolates were monoresistant to RIF.

The early detection of active TB, particularly the detection of MDRTB, is essential for the success of any TB control program.Theapplicationofmoleculartechniquesfortheearly identificationofMDRTBassistedtheNationalHealthAuthorities in the reduction of MDRTB rates in Lisbon to less than 8% (average 2003 to 2007).


pp-25

Drug-rESiSTaNCE Of MyCoBaCTeriuM TuBerCuloSiS aT pENiTENTiary iNSTiTuTiONS Of ST. pETErSburg, ruSSiaN fEDEraTiON.

Vladimirov, Kirill; Zaitseva, Elena; Ivanov, AleksandrInstitution: State Medical Academy named after I.I. Mechnikov, St. Petersburg, Russia.

background

Morbidity with tuberculosis (TB) in Russian Federation and the whole world remains high. This index is up to 40 times above the average level among prison population, with high prevalence of multi-drug resistant (MDR) TB.

Setting. Central hospital for prisoners in St. Petersburg.

Study design. We retrospectively reviewed data of the patients, who were admitted to the hospital for active culture-positive TB between 2005 and 2008. Between 2005 and 2007, new and re-treatment cases were admitted. In 2008, only new TB cases were admitted. We studied the results of drug-susceptibility of Mycobacterium to Isoniazid (H), Rifampicin (R),Ethambutol(E),Streptomycin(S),Kanamycin(K),Ofloxacin(O)insolidLowenstein-Jensenmedium.Casesofpan-sensitivity,drugresistance(DR),includingMDRandextradrugresistance(XDR)weredefined.

results

As much as 163 cultures were studied. From 52 cases in 2005, 36.5% were pan-sensitive, 25.0% were DR and 38.5% were MDR. In 2006, 36.0% of 50 cultures were pan-sensitive, while casualty of MDR increased to 48.0%. In 2007, number of pan-sensitive cultures decreased to 21.4%, while 35.7% were MDR and 7.2% were XDR. In 2008, among 47 cultures nearly half were MDR (48.9%) and 12.8% were XDR, only 27.7% cultures were pan-sensitive.

Conclusions

Among prisoners in St. Petersburg, the value of the cases of primary MDR and XDR TB increased dramatically. Introduction of the fast methods of drug resistance detection is required.

98 ESM 2009

pp-26

aN iNCrEaSE Of Drug rESiSTaNCE SiNCE 2001 iN mulTiDrugrESiSTaNT M. TuBerCuloSiS iSOlaTES frOm bElgium

Karolien Stoffels, Maryse Fauville-DufauxReferenceLaboratoryofTuberculosisandMycobacteria,ScientificInstituteofPublicHealth,642RueEngeland,1180Brussels, Belgium. Tel. +32-23733210 | Fax. +32-23733281. [email protected],[email protected]

Between January 1994 and December 2008, MDR clinical isolates of 174 patients were analyzed in our National Reference Laboratory.Theyrepresent90%ofalltheMDR-TBpatientsidentifiedinBelgiumduringthis15-yearsperiod.Since2000,thenumberofMDRpatientsidentifiedinourcountryisstable(inaverage16peryear,i.e.anaverageof1,3%ofthepatients tested for susceptibility to drugs) but the isolates are resistant to more and more second line drugs. We observe adramaticallyincreaseinresistancetoethambutol,rifabutin,amikacinandofloxacin,aswellastopyrazinamid.

Wedividedthestudiedperiodin2ranges,1994to2000and2001to2008.OnlythefirstMDRclinicalisolateofeachpa-tient was taken into account. So these results do not consider the evolution of the isolates during treatment in Belgium, butonlytheinitialMDRresistanceprofile.

In the second period (2001 to 2008) 75,6% of the MDR clinical isolates showed resistance to ethambutol versus 45,5%inthefirstperiod(increaseofresistanceof30,1%);75,4%wereresistanttorifabutinversus70,4%inthefirstperiod (increase of 5%). Resistance to pyrazinamid increased from 39,6% to 55,6% (difference of 16%). Resistance level to amikacin showed an increase of 12,2% (3,6% to 15,8%) and resistance level to ofloxacin showed an increase of 8,6% (3,6% to 12,2%).

NoprimaryXDRisolatewasobservedduringthefirstperiod,but5weredetectedsince2001.ThreeMDRisolatesdevelopedintoXDRwhatthetotalamountofXDRstrainsbroughtto6andonly2forrespectivelythesecondandfirstperiod.

Concerningthegeneticfamiliesidentified,14,7%moreBeijingstrainswereregisteredinthesecondperiodcomparedtothefirstperiod.AdecreaseofthemembersoftheLAMandHaarlemfamilywasnoted(16,4%and17,3%).

Inconclusion,animportantincreaseofresistancetoethambutol,pyrazinamid,amikacinandofloxacinisobservedinMDRclinicalisolatesdetectedinBelgium.Thisconfirmstheurgentneedfornewanti-tuberculosisdrugs.


pp-27

muTaTiONal aNalySiS Of gENES aSSOCiaTED WiTh rESiSTaNCE TO iNJECTablE SECOND-liNE DrugS iN MyCoBaCTeriuM TuBerCuloSiS CliNiCal iSOlaTES frOm liSbON, pOrTugal

João Perdigão1, Ana Ferreira1, Ana Malaquias1, Rita Macedo2, Laura Brum2 and Isabel Portugal.1,2

1 - Centro de Patogénese Molecular, URIA, Faculdade de Farmácia da Universidade de Lisboa2 - Laboratório de Micobactérias, Centro de Bacteriologia, Instituto Nacional de Saúde Dr. Ricardo Jorge

Objectives

Multidrug resistance (MDR) constitutes a serious problem to tuberculosis (TB) control program in Portugal. An even more serious threat is the one posed by the high rate of extensive drug-resistant TB (XDR-TB). Our laboratory has already shown that high rates of this form of TB exist in Lisbon. Given the fact that MDR-TB and XDR-TB are currently associated with a limited number of genetic clusters, mainly Lisboa family clusters, the diversity of genetic polymorphisms conferring resistance to second-line drugs is also probably limited. In this study we intended to characterize the genetic polymorphisms associated with resistanace to second-line injectable drugs and to assess the clinical isolates clonality.

methods

We have analyzed 19 MDR-TB strains resistant to one or more second-line injectable drugs, collected from several hos-19 MDR-TB strains resistant to one or more second-line injectable drugs, collected from several hos-pital units across Lisbon Health Region during the year of 2005. All isolates were typed by Mycobacterial Interspersed Repetitive Units (MIRU-VNTR) and, screened for mutations in tlyA and rrs genes.

results

ThreedifferentmutationswereidentifiedontlyA gene and another three at rrs gene. Overall, 9 isolates had mutations in tlyA gene and 8 isolates had mutations in rrs gene; two isolates didn’t have any mutation in either gene. The most frequent mutations found were A1401G in rrs gene (6/19) and 755InsGT in tlyAgene(6/19).Wealsoverifiedthattherewasnooverlapping of mutations from different genes. The genotyping analysis revealed that the isolates could be distributed through two different MIRU-VNTR genetic clusters: Lisboa3 and Q1. Cluster Q1 contained all clinical isolates bearing the A1401G mutation in rrs gene, while Lisboa 3 cluster contained all isolates that had the 755InsGT mutation in tlyA gene.

Conclusion

Wehaveidentifiedseveralmutationsthatmightbeassociatedwithresistancetodifferentbutrelatedsecond-linedrugs:kanamycin, amikacin and capreomycin. The two most prevalent mutations were associated with different genetic clus-ters, which suggests recent transmission and, ultimately, that XDR-TB transmission is taking place. The most prevalent mutationsassociatedwithinjectablesecond-linedrugshavethereforebeendefined,whichopensthewayformoleculardetection of resistance to second-line drugs in the region.

100 ESM 2009

pp-28

mulTiDrug-rESiSTaNT TubErCulOSiS

Nuak, Joao; Ferreira, Danina; Carvalho, Teresa; Gomes, Maria Helena; Sarmento, AntonioHospital S. Joao, Porto - Portugal

introduction

Multidrug-Resistant Tuberculosis (MDRTB) is caused by M.tuberculosis (MT) resistant to at least Isoniazid (INZ) and Rifampin (RIF), and can be due to unsuitable or irregular treatment.

purpose

To know the epidemiological and clinical characteristics of the disease in patients (pts) hospitalized with MDRTB in an Infectious Diseases Service.

patients and methods

Review of clinical records of pts with MDRTB. Diagnosis was based on drug susceptibility testing.

results

Between 1996 and 2007, 16 pts had MDRTB. Eleven were male. Ages ranged between 27-40y(X=32.2±4.33). Fifteen (93.8%) had HIV infection (14 drug addicts; 1 sexual risk). One pt had professional contact with MDRTB. The disease was only pulmonary in 7(44%) pt, disseminated in 5(31%), pulmonary and extra-pulmonary in 3(19%)-meningeal, lymph nodes (LN) and urine in one each pt; another pt had only meningeal disease. Eleven (69%) pts had previous irregular antituber-culosis treatment. MT was isolated in sputum in 12/16 pt (75%); CSF in 5/6 pt (83%), bronchoalveolar lavage in 2/6(33%), blood in 3/6(50%), urine in 2/6(33%), LN in 2/6(33%), gastric aspirate and feces in one each. Most pts had MT isolated in more than one sample. All MT were resistant to INZ and RIF. Thirteen out of 16 pt (81%) were also resistant to strep-tomycine, 6/12(50%) to pyrazinamide, 6/16(37%) to ethambutol, 5/12(42%) to rifabutine, 10/15(67%) to ethionamide, 4/14(28%)tokanamycin,4/13(31%)toofloxacilline,3/7(43%)toPAS,2/5(40%)tokapriomycinee1/5(20%)tocyclocerin.In 4 pts MT was simultaneously resistant to at least three 2nd line drugs (XDRTB). Fifteen pts had medical treatment ac-cording to the drug susceptibilities testing. In two pts pneumectomy was performed. Eight pts died: One before diagnosis and 7 between 30-720 days of diagnosis. One pt survived for 6y. maintaining positive cultures of sputum despite medical and surgical therapy. Two pts were treated for 18 months with clinical and radiological improvement, without evidence of disease 1 and 5y. later. Five pts were lost to follow-up. One is on treatment with clinical and radiological improvement.

Conclusion

MDRTB is a serious public health problem. HIV, drug addiction and irregular treatment were important factors for its development. Prognosis depends on early detection of drug resistance and institution of appropriate therapy. We empha-size the very high mortality.


pp-29

CharaCTEriSaTiON Of STrEpTOmyCiN muTaTiONS iN MyCoBaCTeriuM TuBerCuloSiS CliNiCal iSOlaTES iN ThE arEa Of barCElONa

Griselda Tudó1, Emma Rey1, Fernando Alcaide2, Pere Coll3, Gemma Codina4, Núria Martín-Casabona4, Michel Montemayor3, Raquel Moure2, Margarita Salvadó5, JuliàGonzález-Martín1

1 - Servei de Microbiologia, CDB. Hospital Clínic de Barcelona-IDIBAPS, Universitat de Barcelona2 - Servei de Microbiologia, Hospital Universitari de Bellvitge-IDIBELL, Universitat de Barcelona3 - Servei de Microbiologia, Hospital de la Santa Creu i Sant Pau de Barcelona, Universitat Autònoma de Barcelona4 - Servei de Microbiologia, Hospital Universitari Vall d’Hebron. Universitat Autònoma de Barcelona5 - Laboratori de Referència de Catalunya, Barcelona. All the authors are members of Spanish Network for the Research in Infectious Diseases (REIPI, RD06/0008).

Objective

To determine the proportion and type of mutations in Mycobacterium tuberculosis (Mtb) isolates resistant to streptomycin (SM) and their relationship with the level of resistance and their genotype.

methods

SM resistant isolates from an Mtb strain collection (1995-2007) were studied. Minimum inhibitory concentrations (MIC) of SM for each isolate were determined using the proportion method with Middlebrook 7H10 medium. The entire rpsL geneand2specificfragments(loop530andregion912)oftherrs gene were sequenced. IS6110-RFLP and spoligotyping techniques were used to type Mtb isolates.

results

Of 69 SM resistant isolates, 36 (52.17%) presented a mutation in either the rpsL gene and/or the rrs530 gene, with no mu-tation in the rrs912 region. No mutations were found in 33/69 (47.8%) SM resistant isolates (all of them with MIC≤16µg/ml). Seventeen (24.63%) isolates showed rpsL mutations: 9 (13.04%) at position 88 (7: AAG→AGG, 1: AAG→ACG and 1: AAG→CAG) and 8 (11.59%) in codon 43 (AAG→AGG). Isolates with mutations in the rpsL gene (94.1%) had a MIC≥512 µg/ml. Among isolates with alterations in the rrs gene (27.53%):10 (14.49%) had a 491 C→T change and low MIC level; 7 (10.1%) had a mutation at position 513 A→T or A→C and 2 (2.89%) had a 516 C→T substitution. These mutation points were related to intermediate and high MIC levels. One isolate with a codon 88 mutation had a second mutation in the rrs530 gene at position 491. IS6110-RFLPtypingidentified4clusters(11/69,13%).ClustersIandIIweremonoresistantto SM, with a low MIC level and a mutation at position 491 in the rrs gene. Cluster III was multidrug-resistant with a high MIC level and a mutation in codon 88 in the rpsL gene. Cluster IV and V was monoresistant to SM with a low MIC level and no mutation. Interestingly, all the isolates with a mutation at position 491 in the rrs530genewereidentifiedasLAM3 lineage. All the Beijing family presented mutations in the rpsL gene (2 and 1 at codons 88 and 43, respectively).The spoligotyping lineageT5-MAD2 was detected in non-mutated isolates.

Conclusions

Mutations in the rpsL and rrs genes were detected in at least 50% of SM resistant isolates. Mutations in the rpsL gene were associated with high-level resistance while mutations in the rrs530 gene were associated with different MIC levels. The isolates with no mutations had low-level resistance. Mutations in the rrs530 gene at position 491 were associated with LAM3 lineage

102 ESM 2009

pp-30

SurVEillaNCE Of Drug-rESiSTaNT TubErCulOSiS iN iTaly

Fattorini Lanfranco 1, Pardini Manuela 1, Cirillo Daniela 2, Borroni Emanuele 2, Miotto Paolo 2, Filippini Perla 3, Cassone Antonio 3, TB-CCM Study Group 4

1-IstitutoSuperiorediSanità,Rome,Italy2-IstitutoScientificoSanRaffaele,Milan,Italy3-IstitutoSuperiorediSanità,Rome,Italy4 - Italian network of mycobacteriology laboratories

introduction

Drug-resistant TB is an increasing problem worldwide. In order to update the national data on drug resistance, we start-ed a surveillance program including the most representative diagnostic centres in Italy. Purpose of the study. The study was designed to determine: 1) Accuracy of drug susceptibility testing (DST) for streptomycin (S), isoniazid (I), rifampicin (R), ethambutol (E). 2) Drug resistance in new cases (NC), previously treated cases (PTC), patients born in Italy (PBI), patients born abroad (PBA). 3) Molecular typing.

methods

1)Thirty laboratorieswereenrolledin2006-7in18/20regionsforproficiencytesting(PT),basedontheamountofDST performed every year and geographic location. The laboratories received 20 strains each, and sent DST results to theWHOSupranationalReferenceLaboratory(SRL)ofIstitutoSuperiorediSanità(ISS)whichcomparedthemwiththe judicial results of the Global Network of SRLs. 2) A questionnaire was returned to ISS with DST results of TB cases diagnosedin2007;strainsresistantto>1drugsand10%ofthesusceptibleswerecollected.3)moleculartypingwasperformed at the SRL of San Raffaele Hospital (Milan) by spoligotyping and MIRU-VNTR. Results. 1) Twenty-nine labora-toriescompletedthePT.Theaverageefficiency(correctresults/totalresults)washigh(95.5±2.8%forS,97.6±2.9%forI,95.7±2.9% for R, 96.2±4.4% for E). 2) In 2007, a total of 1,698 antibiograms for SIRE were examined. As to NC and PTC, total monoresistances were 10.6 and 16.5%, respectively; MDR cases were 2.5 and 26.6%, respectively. As to PBI and PBA, total monoresistances were 10.9 and 11.2%, respectively; MDR cases were 2.6 and 5%, respectively. 3) In 257 strains collected in 2006-07 and examined for molecular typing, 146 spoligotypes and 29 clusters were found. Beijing genotype was detected in 5% of cases. In 44 MDR strains typed by the MIRU-12 technique the clustering rate was 0.023 showing low transmission rate.

Conclusions

1) PT indicated that the DST in this network of laboratories was accurate. 2) MDR rate in Italy consistently increased in NC from 1998-2001 (1.1%) to 2007 (2.5%), a phenomenon likely related to immigration. 3) No MDR-TB outbreak was detected. Noteworthy, unlike reported from other European countries, MDR strains were not associated with the Beijing lineage (This work was supported by the Italian Ministry of Health, CCM Project Surveillance of resistance to anti-TB drugs, Grant N92)


pp-31

TubErCulOSiS rESiSTaNCE iN a gENEral hOSpiTal iN pOrTugal – 9 yEarS SurVEillaNCE

Sancho L.; Portugal C.; Tancredo L.; Silva M.; Dias A.; Silva F.; Sousa GermanoLaboratory of Microbiology, Department of Clinical PathologyHospital Fernando Fonseca – Amadora, [email protected]

Tuberculosis remains a serious public health problem in Portugal. In 2008, the Portuguese Health Authorities reported a TB incidence of 25,3/100.000 inhbitants (13,6% immigrants). TB Multi Drug Resistant (MDR) were 2,5%, 34% of which were Extensively Drug Resistant (XDR).Resistance to any of the primary drugs makes the disease more difficult and expensive to treat. Our Hospital is located in Lisbon’s surroundings and covers a population of 750.000 inhabitants most of them with poor socioeconomic level and immigrants from Africa and East Countries. In the Great Lisbon are located 66% of TB cases of Portugal.

purposeThe aim of this study was to investigate the frequency of drug resistance of Mycobacterium tuberculosis Complex in a general Hospital in Amadora, Portugal, during a 9-year period (2000-2008).

methodsA total of 19.417 clinical specimens (15.159 pulmonary and 4.261 extra pulmonary), collected from 9.525 patients, were cultured for mycobateria.MoleculargeneticidentificationofM.tuberculosis Complex and its resistance to Isoniazid and/or Rifampicin was made with the technology Genotype MTBDR plus (HAIN-Lifecience-Germany).Antimycobacterial susceptibility test to the primary drugs, Streptomycin (STR), Isoniazid (INH), Rifampicin (RIF), Ethambutol (ETB) was performed in BACTEC MGIT 960 System

resultsIn 19.417 cultured clinical specimens for mycobateria, 1094 (14,2%) were positive by cultural methods.1029wereidentifiedasM.tuberculosis Complex; 783 (76,1%) were strains without resistance, 150 (14,6%) with one re-sistance, 73 (7.1%) were MDR, being more than 25% XDR.The proportion of M.tuberculosis strains resistance rate to antituberculosis drugs during the 9-year period was: Isoniazid 11,1% (114), Streptomycin 20.2% (208)), Rifampicin 7.2% (74), and Ethambutol 4.7% (48); but in 2008 was: Isoniazid 4,8%, Streptomicin 17%, Rifampicin 4,1% and Etambutol 2%.On our tuberculosis population (661), in the last 6-years (2003-2008), we have compared the resistance rate related to 3 parameters: sex, age and HIV.The tuberculosis (661) and MDR (38) populations have the same incidence rate: in male (67%) and in females (33%).The age distribution in the MDR population (38) was 0% [0-15], 5% [16-25], 29% [26-35], 39% [36-45], 13% [46-55], 11% [56-65], 0% [66-75], 3% [76-100]; and in patients without resistance (623) was 3% [0-15], 13% [16-25], 30% [26-35], 20% [36-45], 14% [46-55], 8% [56-65], 7% [66-75], 5% [76-100].The HIV parameter results were analysed on a 554 tuberculosis population. The MDR incidence rate for the HIV group (213) was 7%, and for the no HIV group (341) was 4%.

ConclusionThelevelofresistanceinourpopulation(MDR7%)issignificantlyhigherthanPortugal’saverage(2,5%)The Multi Drug Resistance tends to be lower in the last years. The same can be observed in each of the tested drugs.HIV infection, age and sex patient are factors that contributed to the variation of tuberculosis/MDR incidence rate.Comparing the resistance rate by sex parameter, we didn’t found differences for tuberculosis or MDR populations; they both have a bigger incidence in the male sex (67%).The major incidence of tuberculosis is among active population, between 26 and 45 years old, but, it is between 36 and 45 that we found most of the MDR strains (39% of all), mostly because of drug abuse and HIV infection in this age group.The incidence of MDR tuberculosis is clearly bigger in HIV positive (7%) than in HIV negative (4%).

DiscussionIn spite of our population have a level of resistance above the Portuguese average, we noted that the number of tuber-culosis cases, including MDR, decreased 7,8% during this 9-year period analysed, which is comparable to 2008 national data (-7.2% in the last decade).

104 ESM 2009

pp-32

DOES a muTaTiON iN ThE rpOb mEaN ThaT ThE M.TuBerCuloSiS iS rESiSTaNT TO rifampiCiN?

Yates, Malcolm; Brown, Tim; Drobniewski, FrancisHPA National Mycobacterium Reference Unit

Testing for mutations in the “hot spot” region of the rpoB gene is gaining momentum for the diagnosis of rifampicin resistant strains of M.tuberculosis and as a surrogate marker for MDRTB.

The use of PCR combined with hybridisation to commercial strips (Hain Lifesciences, Innolipa)has decreased time and increased ease of use so that these investigations are being performed by more and more laboratories.

The strips consist of a series of overlapping probes that cover the whole region (S bands) and are all present in Wild Type isolates. There are also a series of probes (R bands) covering the commonest mutations which are linked with rifampicin resistance and with the deletion of the corresponding S band.

OccasionallyanSbandisdeletedwithnoRbandappearing.Wereporttheseas“unidentifiedmutations”.Thequestionis whether these isolates are resistant or sensitive to rifampicin.

Recently we identified three patients from SaoTome, an island off theWest Coast ofAfrica (population 55000),with a strains of M.tuberculosis that had the same S band deleted. Sequencing data showed that the strains had the samesynonymousmutation,VNTR/MIRUprofileswereidentical,andallstrainswerefullysensitivetofirstlinedrugs. Onanalysisofourdatabase,466strainswerefoundtohaverpoBmutations,103(22%)ofthesewereunidentifiedmuta-tions of which 13% were sensitive to rifampicin. A further 15 isolates gave a WT result but were rifampicin resistant.

All strains were sequenced to identify the mutation.

Before rpoB mutations can be used as a surrogate for MDRTB the rate of mono-resistance to rifampicin in the area must be determined: e.g. in the London area 30% of rifampicin resistance is mono.

Care must, therefore, be taken when reporting these rpoB mutation results to Clinicians as

1)unidentifiedmutationsdonotalwayscorrespondtorifampicinresistanceand2)rpoBmutationsmaynotalwayscor-respond to MDRTB


pp-33

gENOTypiC DETECTiON Of iSONiaziD aND rifampiN rESiSTaNCE iN MyCoBaCTeriuM TuBerCuloSiS CliNiCal iSOlaTES

Maitane Aranzamendi Zaldumbide, Carlos Fernandez Mazarrasa , Luis Martinez-Martinez, Jesus Agüero Balbin.Hospital Universitario Marques de Valdecilla, Servicio de Microbiologia, Santander, Spain.

background

The emergence of Mycobacterium tuberculosisresistanttofirst-linedrugsunderlinestheurgentneedfornewresistance-profilingmethodsthatwouldallowfortimelydeterminationofpropertreatment.Theaimofthisstudywastoevaluatethedevelopmentoftargetedandfastmoleculardiagnosticmethodsuitableforspecificgenomeregionsresponsibleforisoniazid (INH) and rifampin (RAMP) resistance in M. tuberculosis clinical isolates.

methods

79 strains known to be resistant to INH (n=71) or INH+RAMP (n=8) by the automated system BacT ALERT (bioMérieux) were selected from a stock collection of clinical isolates (January 2000- March 2009). The genome regions associated with INH-R (including the codon 315 of the katG gene and the fabG1(mabA)-inhA regulatory region) and RAMP-R (81-bp hot spot region of the rpoB genecalledRRDR)wereamplifiedbyPCRandtheDNAsequenceswerestudied.

results

Of the 79 isolates, 22 (27.84%) had the mutation S315T in the katG gene, 5 (6.32%) showed changes at -15 nucleotide of the fabG-inhA regulatory region and 2 (2.53%) presented both mutations. Asignificantproportionofstrains, 50 (63.29%), had no detectable alterations at the studied loci. INH + RAMP-R strains were associated with mutations in the RRDR of the rpoB gene in all cases. From these, majority (5 of 8, 62.5%) presented the mutation S531L, whereas the others involved changes atthecodon516:mutationsD516VandD516F,whichwereidentifiedin1(12.5%)andin2(25%)strainsrespectively.

Conclusions

Our results demonstrated a low sensitivity of this method to detect INH-R strains, andpointstotheneedoffindingout other mutant regions. On the other side, confirmtheusefulnessofthisstrategyforfastassessmentofresistancetoRAMP, which in turn is a marker for multiresistance. This analysis can also be done with assays based on reverse line blot hybridization detecting the same mutations, except D516F, which is not targeted.

106 ESM 2009

pp-34

phySiOlOgiCal fiTNESS aND TraNSmiSSiON pOTENTial Of mulTi-Drug rESiSTaNT MyCoBaCTeriuM TuBerCuloSiS CliNiCal iSOlaTES iN hONg KONg

CHAN Chiu Yeung Raphael 1, CHAN Wai Chi Edward 1, AU Tai Kong Mike 1, LAI Wai Man Raymond 2, YEW Wing Wai 3, YIP Chi Wai 4, KAM Kai Man 4.1 - Department of Microbiology, the Chinese University of Hong Kong;2 - Department of Microbiology, Prince of Wales Hospital3 - Tuberculosis & Chest Unit, Grantham Hospital,4 - Tuberculosis Reference Laboratory, Department of Health, HKSAR, Hong Kong.

This study evaluated the infectivity and transmission potential of multi-drug resistant Mycobacterium tuberculosis (MDR-MTB) strains by determining (i) whether resistance developed at a physiological cost which rendered them less capable of surviving environmental stress and infecting human host, and (ii) the degree of genetic relatedness shared by resistant organisms, which indicated the extent by which they spread among humans. The relative growth rates of selected isolates were measured using the MGIT system and compared to that of drug-sensitive strains. Our data showed that their aver-age initial growth rate, measured within 7 days of inoculation, was inversely proportional to the number of mutations they harbored in key resistance genes, with strains carrying 5 mutations growing at a rate 46% slower than that of the wildtype.ThesefindingssuggestedthatresistancegenemutationsinMTBimposedarangeofphysiologicalcostchar-acterizedbyreducedgrowthfitness.However,resultsofepidemiologicaltypingshowedthat34%ofthe402MDR-MTBisolatesanalyzedexhibitedgeneticrelationshiptootherstrains,indicatingthatsuchfitnesscostdidnotsignificantlyaffecttheabilityofMDR-MTBtotransmitbetweenhumanhostsandcauseinfection.Importantly,theidentificationoffivema-jor clusters of 50 strains strongly suggests that infection due to dissemination of ‘parental’ MDR-MTB clones is common. Ontheotherhand,themajorityofteststrainsdisplayedauniquegeneticprofile,indicatingthatMDR-MTBmightalsoemergeindependentlythroughdrugselectionwithinindividualpatient.TheDNAfingerprintsandgrowthfitnessdataoflocal resistant isolates may be used for matching the genetic identities, predicting transmission potential, and tracing the routes of dissemination of future MDR-MTB isolates in the community.

This work was supported by the Research Fund for the Control of Infectious Diseases (Project code 6902191 and 6902200)


pp-35

myCObaCTErium TubErCulOSiS: 1999-2008 aNTiTubErCulOSiS DrugS SurVEillaNCE iN CliNiCal iSOlaTES frOm paTiENTS iN ThE largEST hOSpiTal iN ThE NOrTh Of pOrTugal

Sousa,AnaSofia; Pinheiro, Maria Dolores; Carvalho, Teresa; Gonçalves, HelenaLaboratório de Microbiologia do Serviço de Patologia Clínica. Hospital de S. João, Porto, Portugal

introduction

Tuberculosis, whose causal agent is Mycobacterium tuberculosis (MT), is still an important cause of morbidity and a leading cause of death by infection worldwide. Multidrug resistant (MDR) and extreme resistant drug (XRD) strains of MT are fre-quently isolated. They have become an emerging global public health problem and an obstacle for tuberculosis (TB) control. Insights on prevention of disease dissemination and its empirical treatment may be obtained from resistance surveil-lance and monitorization of its trends. Therefore, in our study we present the susceptibility data of the strains isolated in Hospital de S. João over the past ten years.


A prospective ten year study was done using susceptibility data of MT strains isolated in the hospital. Susceptibility testing tofirstlinedrugs(streptomycin,isoniazid,rifampinandethambutol)wasmadeonthefirstisolateofeachpatient,usingthe proportion method (in Lowenstein-Jensen medium until 2000, from 2001 to 2003 in Bactec 460TB and in Bactec MGIT thereafter). When MDR strains were present, second line drugs (ethionamide, cycloserine, capreomycin, kanamy-cin,amikacin,ciprofloxacinandrifabutin)weretested.

results

Inthese10years,1493MTstrainsisolatedforthefirsttimeinthesamenumberofpatients,including1103males,weretested for in vitro susceptibility. Streptomycin was the drug with high index of resistance (8,8%); isoniazid (6,0%) was the second one; rifampin (2,4%) the third and ethambutol (1,3%) was the less resistant. We found no resistance in 1328 (88,9%) of our patients, but in 29 (1,9%) we isolated MDR strains and 13 (0,8%) patients had XDR ones. From 1999 to 2005, the number of MDR strains isolated was relatively stable (an average of 3 patients/year); in 2006, 2007 and 2008 the number of patients with MDR was 8, 3 and 1 respectively. In what XDR strains are concerned, we had no isolates from 1999 to 2002; in the following years, 2003-2008, XDR strains were isolated in 1,0,2,7,2 and 1 patients.

Conclusion

The results obtained in our patients are similar to previous reports in Portuguese and international literature, support-ing the view that tuberculosis is currently a serious public health problem. The knowledge of susceptibility results will provide evidence in support of preventive health policies. It also emphasizes the role of Laboratory as a cornerstone in diagnosis, management of individual patients and effective TB control.

108 ESM 2009

pp-36

fiTNESS COST Of MyCoBaCTeriuM TuBerCuloSiS CliNiCal iSOlaTES rESiSTaNT TO fluOrOQuiNOlONES

VON GROLL, Andrea 1; MARTIN, Anandi 1; JUREEN, Pontus 2; HOFFNER, Sven 2; PORTAELS, Françoise 1; PALOMINO, Juan Carlos 1; ALMEIDA DA SILVA, Pedro 3 1 - Institute of Tropical Medicine, Antwerp, Belgium2 - Swedish Institute for Infectious Disease Control, Solna, Sweden3 - Universidade Federal do Rio Grande, Rio Grande, Brazil

Fluoroquinolones (FQs) have been used as effective second-line drugs in the treatment of the tuberculosis. However, the emergence of M. tuberculosis resistant to FQs has contributed for the occurrence of XDR-TB. This study investigated the fitnesscostrelatedtothemechanismofresistancetoFQsinM. tuberculosis clinical isolates. A total of 37 isolates had theofloxacin,moxifloxacinandgatifloxacinsusceptibilitydeterminedbytheproportionmethodandweresequencedto look for mutations in gyrA and gyrB.Theroleofeffluxpumpswasevaluatedbydeterminingtheminimalinhibitoryconcentration of the FQs in the presence and absence of verapamil by resazurin microtiter assay. Growth curves of the isolates were obtained using the MGIT960 automated system and the lag phase time and rate of growth were established tocomparethefitness. On the 25 FQ resistant isolates (FQR), the most frequent mutation was at Ala-90→Val, followed bymutationsatAsp-94toGly,Tyr,AlaandAsn.SomeunusualmutationswereidentifiedatAsp-89→Asn, Asn-533→Thr (gyrB) and deletion of the codon 678 and 679 in gyrB. The isolate with mutation at Asn-533→Thr was the only case of no whole cross resistance among the three FQs tested. One FQR isolate was wild type for the region investigated. The effluxmechanismwasindentifiedin36%oftheFQRisolates,beingmorefrequentlyfoundinmoxifloxacinandgatifloxacin.Inregardtothefitnessparameters,themutationatAsp-94showedalongerlagphasewhilethemutationatAsn-90hadnotanysignificantdifferencerelatedtothewildtypeFQS. The establishment of FQRisolateswithoutfitnesscostwarnsfor the possibility of a continue emergence of XDR-TB and highlights for a more rational use of FQs, not only for the treatment of TB, but also, for other bacteria.


pp-37

iN ViTrO aCTiViTy Of OflOXaCiN, mOXiflOXaCiN aND gaTiflOXaCiN agaiNST MyCoBaCTeriuM TuBerCuloSiS by ThE rESazuriN COlOrimETriC mEThOD

VON GROLL, Andrea 1; MARTIN, Anandi 1; JUREEN, Pontus 2; HOFFNER, Sven 2; PORTAELS, Françoise 1; ALMEIDA DA SILVA, Pedro 3; PALOMINO, Juan Carlos 1

1 - Institute of Tropical Medicine, Antwerp, Belgium2 - Swedish Institute for Infectious Disease Control, Solna, Sweden3 - Universidade Federal do Rio Grande, Rio Grande, Brazil

Theinvitroactivityofofloxacin,moxifloxacinandgatifloxacinwastestedagainst41strainsofMycobacteriumtubercu-losis by the resazurin microtiter assay (REMA) plate and the proportion method on 7H11 agar. A critical concentration of2.0µg/mlforofloxacinand0.5µg/mlformoxifloxacinandgatifloxacinwasobtainedbytheproportionmethodon7H11agar.ForREMAweproposeacriticalconcentrationof2.0µg/mlforofloxacinand0.25µg/mlformoxifloxacinandgatifloxacin.Fullcross-resistanceamongthethreefluoroquinolonescouldnotbeconfirmedsinceonestrainresistanttomoxifloxacinandgatifloxacinwasstillsusceptibletoofloxacin.Thisfindingcouldhaveimportantimplicationsforthetreatment of tuberculosis patients.

110 ESM 2009

pp-38

rapiD DETECTiON Of EXTENSiVEly Drug-rESiSTaNT myCObaCTErium TubErCulOSiS by ThE rESazuriN miCrOTiTEr aSSay plaTE Paasch, Fabienne; Martin, Anandi; Docx, Sven; Fissette, Kristina; Portaels, Françoise; Palomino, Juan Carlos Institute of Tropical Medicine, Antwerp, Belgium

introduction

A major concern for tuberculosis control programs is the emergence of multidrug-resistant (MDR) tuberculosis and especially extensively drug-resistant (XDR) tuberculosis. Conventional drug susceptibility testing (DST) requires 3 to 6 weekstoyieldresults.Therefore,thereisanurgentneedofnewtimelyandaccuratedetectionoffirstandsecondlineanti-tuberculosis drug resistance.

purpose of the study

The aim of this study was to evaluate the first and second line drugs rifampicin (RIF), isoniazid (INH), ofloxa-cin (OFX), kanamycin (KAN), amikacin (AMK) and capreomycin (CAP) with clinical isolates of M. tuberculosis by the colorimetric resazurin microtiter assay (REMA) plate in comparison to the indirect proportion method (PM).

method

A total of 150 clinical isolates were studied. DST by PM was performed on Löwenstein Jensen for RIF and INH and on 7H11 agar for the other drugs. The minimal inhibitory concentration obtained by REMA was compared with the PM.

results

REMA results were easily determined visually after 8 days compared to 21 to 42 days by the PM. Out of 150 isolates 92 wereMDRand20wereXDR.Afterdefiningthecriticalconcentrationforeachdrugbythecolorimetricassay,excellentresultswereobtainedforfirstandsecondlinedrugswithlevelsofspecificityandsensitivitybetween93%and100%.

Conclusion

In this study drug resistance detection by REMA has shown high level of agreement with the conventional PM. Therefore, REMA could be a reliable alternative method for rapid detection of MDR and XDR M. tuberculosis.


pp-39

DETECTiON Of Embb gENE CODON 306 muTaTiONS iN EThambuTOl SuSCEpTiblE aND rESiSTaNT myCObaCTErium TubErCulOSiS STraiNS.

Montoro, Ernesto 1; Yzquierdo, Sergio 1; Lemus, Dihadenys 1; Echemendia, Miguel 1; Takiff, Howard 2

1 - Institute of Tropical Medicine Pedro Kourí (IPK), Havana, Cuba2-VenezuelanInstituteforScientificResearch(IVIC),Caracas,Venezuela

Ethambutol(EMB)isoneofthefirstlinedrugsinthetreatmentoftuberculosis.The major mechanism of acquisition of resistance to EMB in Mycobacterium tuberculosis seems to be associated with points mutations in the embCAB operon en-coding different arabinosyl transferases. In particular, amino acid replacements at embB gene codon 306 occur frequently in EMB-resistant M. tuberculosis strains. However, this alteration has been also reported in multidrug-resistant (MDR) strains susceptible to EMB. The aim of this work was to detect the most frequently mutation at codon 306 in EMB-resistant strains as well as MDR strains. For this purpose, the indirect Proportional Method (PM) on Löwenstein-Jensen was carried out as susceptibility test to isoniazid (0,2 µg/mL), streptomycin (4 µg/mL), EMB (2 µg/mL) and rifampicin (40 µg/mL) on 86 M. tuberculosis strains from the collection at the National Reference Tuberculosis Laboratory. All strains that showed resistance to EMB by PM, all MDR strains and a selection of EMB-susceptible strains were extracted the DNA to obtain a fragment of 803 bp by PCR, corresponding to embB gene codon 306. All this fragments were sequenced by automatic form using appropriate primers and they data were assembled, edited electronically, and compared with wildtype gene sequences. From 34 MDR strains, only 18 showed mutations (53%) being 8 resistant and 10 susceptible strains to EMB. The 61,5% of EMB-resistant strains showed mutations at codon 306 whereas EMB-susceptible strains no hadalterationsinthisfragment.Inconclusion,theresultsconfirmthatembB gene codon 306 is responsible of majority EMB-resistant in M. tuberculosis. All mutations were founded in MDR strains and because of this, the simple detection of changes in this fragment could be considered as multidrug-resistance marker.

112 ESM 2009

pp-40

TOlEraNCE Of mOXiflOXaCiN iN rOuTiNE CliNiCal TrEaTmENT Of TubErCulOSiS

Yew, Wing Wai 1, YAN, See-Wan 1, FUNG, Siu-Leung 1, CHAU ,Chi-Hung 1, CHAN, Chiu-Yeung 2

1 - Tuberculosis and Chest Unit,Grantham Hospital, Hong Kong, CHINA2 - Department of Microbiology. The Chinese University of Hong Koong, Hong Kong, CHINA

From Sep 07 through Feb 09, in a tertiary centre of pulmonary diseases, 65 patients [all male, age 70.0;16.6 yrs (mean;SD)] withtuberculosis(TB)werecommencedonmoxifloxacintreatmentaspartoftheirroutinedrugregimens,duetoin-tolerance/contraindicationtostandardfirst-lineanti-TBagentsordrug-resistantdisease.96.9%ofpatientsreceived400mgmoxifloxacinoncedaily.Thedurationofmoxifloxacintreatmentforallpatientswas51.2;37.3days,exceptonewhoreceivedthedrugfor330days.28patients(43.1%)hadcompletedtheirdesignateddurationofmoxifloxacintreat-ment. 9 patients had nausea, thrush and headache not necessitating drug withdrawal. 15 patients (23.1%) developed probablymoxifloxacin-relatedadverseeventsrequiringdrugcessation:QTcprolongation(5),skinrash(3),arthralgia(2),neutropenia /thrombocytopenia (2), vomiting (1), angioedema (1) and ECG T-wave inversion (1). Time to occurrence of these events was 43.7;42.0 days. A 93-yr old patient died of stroke and myocardial infarct after 3 days of treatment. 2 otherpatientsdiedofpneumoniaduringmoxifloxacintherapy.A82-yroldpatientwhodevelopedECGT-waveinversionhad sudden death 9 days after drug cessation. Another 6 patients died of serious comorbidities, such as lung carcinoma, 1–80daysaftermoxifloxacincessation.Otherwiseallpatientsrespondedfavourablytotheiranti-TBtherapy,withclini-cal,radiographicandbacteriologicimprovement.Univariatebutnotmultivariateanalysisrevealsthatolderage(>65yrs)might increasetheriskofmoxifloxacinassociatedadverseeffects(P=0.07).Similarly,mortality isassociatedwiththenumberofcomorbidities(P=0.025).Thus,patienttoleranceto“long-term”moxifloxacinuseintreatmentofTBappearsreasonable.However,inolderindividualswithsignificantcomorbidities,vigilanceshouldbeexercisedregardingputativedrug-associated events of potential severity, especially those of cardiovascular nature.


pp-41

CharaCTErizaTiON Of gidB gENE iN MyCoBaCTeriuM TuBerCuloSiS iSOlaTES iN liSbON hEalTh rEgiON: rOlE iN STrEpTOmyCiN rESiSTaNCE aND EpiDEmiOlOgiCal marKErS

João Perdigão 1, Ana Sabino 1, Catarina Milho 1, Rita Macedo 2, Laura Brum 2, Isabel Portugal 1,2

1 - Centro de Patogénese Molecular, URIA, Faculdade de Farmácia da Universidade de Lisboa, Portugal 2 - Departamento de Doenças Infecciosas, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal

Objectives

Streptomycin(STP)wasthefirstantibacillarydrugintroducedinthetreatmentoftuberculosisin1944.Withthede-velopment of further antibacillary drugs, streptomycin has become less used. Development of STP-resistance is usually explained by the acquisition of mutations in rpsL gene or in the rrs gene. Our laboratory regularly isolates STP-resistant strains without any mutation in the referred genes. Recently, mutations occurring in a rRNA methyltransferase (encoded by gidB gene) were shown to be involved in the acquisition and resistance to STP. In this study, we examined the gidB gene of STP-resistant isolates in search of mutations that may explain the acquisition and STP low-level resistance on these strains.

methods

We have analyzed by sequencing and/or endonuclease analysis the gidB gene of 57 STP-resistant clinical isolates and 30 STP-susceptible clinical isolates, recovered in 2005 and 2006 from different hospital units. The entire rpsL ORF of all isolateswasamplifiedandscreenedformutationsbyendonucleaseandsequencinganalysis.Allclinicalisolateswerealsogenotyped by MIRU-VNTR.

results

The gidB gene of 19 STP-resistant isolates was sequenced and two missense mutations, A80P and F12L, were found in 5 and 1 out of 19 isolates, respectively. We have found that these gidB mutations were only present in isolates without rpsL mutations. The remaining isolates were screened by endonuclease analysis for mutations A80P and K43R in gidB and rpsL genes, respectively. Overall, mutation A80P in gidB gene was found in 10/57 STP-resistant isolates; 11/14 STP-susceptible multidrug resistant isolates; and, none of 16 pansusceptible isolates. GidB mutation A80P was also associated with MIRU-VNTRgeneticclusterQ1,althoughanindependentoccurrencehasbeenidentified.

Conclusion

We conclude that gidB mutations may in fact explain the high number of STP-resistant strains with no mutation in rpsL or rrs, isolated in our laboratory. These mutations probably confer STP low-level resistance that may pass undetected in regular drug susceptibity testing. The independent occurrrence suggests however, that the acquisition of such mutations present an adaptative advantage.

114 ESM 2009

pp-42

fluOrQuiNOlONE rESiSTaNT MyCoBaCTeriuM TuBerCuloSiS iSOlaTES aND ThEir mOlECular CharaCTEriSTiCS

I Gaile 2, G Skenders 1, V Leimane 1, I Jansone 2, M Bauskenieks 2, I Pole 1, V Baumanis 2

1 - State Agency of Tuberculosis and Lung Diseases, Stopini , Riga District, Latvia, LV 2118 2 - Latvian Biomedical Research and Study Centre, University of Latvia , Ratsupites 1, Riga LV 1067, Latvia

Tuberculosis incidence decreased in Latvia from 74 per 100.000 populations in 1998 till 40.3 in 2008. However, multi drug resistance (MDR) is still high and ~10% of it is extensive drug resistance (XDR). The goal of present study is analysis of fluoroquinolone(Q)resistanceandgeneticcharacteristicsofanappropriateisolates.

60 MDR cultivated and resistant to Q isolates from 2001-2007 were analysed for mutations in the gyrA gene, 25 of themusingprimaryclinicalmaterialalso.Mutationswereevaluatedbysequencingorusinginhousedevelopedmodified(Giannoni et.al.2005) reverse hybridisation method, but kanamycine (K) resistance by sequencing of rrs gene fragment. 48isolateswereconfirmedastypicalXDRthen.GenotypingbyPvuII restriction and spoligotyping was performed as well retrospective case control studies.

35 isolates (58%) contained mutations in the codone 94 (D94 to G, A or H), 14 (23%) in the 90 and 3 (5%) in the codone 91. In 8 cases (13%) mutations were not found. 8 samples (14%) contained whether two mutations or wild type sequenc-es also. 15 K resistant isolates (of 48) contained mutation in the codone A1400G, the rest were wild type. Genotyping revealed 10 clusters with 2-6 isolates in each. 62% of isolates were of Beijing genotype, but 33% to other typical in Latvia MDR genotype – C (related to LAM). The clustering rate (47%) indicates on transmission, however, small cluster size shows, that it is more among hospitalised persons or imprisoned ones. 23 patients suffered from primary XDR TB. Treatment outcome of XDR patients is 28% cured, failures 55%, the rest defaulted or died.

More profound biochemical and genetic properties of these MDR and XDR isolates ought to be studied in order to improve the cure rate. Typical genotypes mutations in the gyr gene in Q resistant isolates indicate on the necessity to search among Q line new drugs affecting other metabolic processes also.


pp-43

DESigN Of a rapiD mEThOD Of iDENTifiCaTiON Of a highly TraNSmiTTED STraiN baSED ON ThE lOCalizaTiON Of iS6110

SofiaSamper1, Isabel Millan 2, Ana I. Lopez-Calleja 3, Patricia Gavin 1, M. Antonia. Lezcano 4

1 - Hospital Univesitario Miguel servet / I+CS / CIBER enfermedades respiratorias2 - Universidad de Zaragoza / Hospital Universitario Miguel Servet / I+CS / CIBER enfermedades respiratorias3 - Hospital Universitario Miguel Servet / I+CS4 - Hospital Universitario Miguel Servet / CIBER enfermedades respiratorias

Efficientmolecularmethodsallowedthedetectionofalargeandunsuspectedtuberculosisoutbreak,involving85pa-tients in Zaragoza (Spain), caused by a strain named Mycobacterium tuberculosis Zaragoza “MTZ”, representing nearly 20% of the isolates in 2001 and being still present among the isolates from our tuberculosis population.

We mapped and localized 8 of the insertion sites of IS6110 in its genome observed by RFLP. The insertion sequence 6110 besides being a very useful tool in molecular epidemiology, induces loss of gene activity either by mediating deletion events or disrupting coding sequences and regulatory domains. It also could modulate expression of neighboring genes by acting as a promoter sequence, driving or enhancing their expression.

In the present work, we designed a rapid method for identifying this particular M. tuberculosis MTZ. For this purpose, differentpairofprimerswhichtargetedtheflankingsitesofIS6110 in MTZ were designed. One hundred isolates among clinical isolates already molecular typed were chosen randomly. and were tested in these isolates. Separate PCR reactions were performed for these isolates. Among the 8 sites localized, 4 of them were previously described as preferential locus for IS6110 transposition.

AftertestingwithdifferentlocationsfinallyoneintragenicinsertioninRv2823c was selected for rapid diagnosis. Only 4 of the 100 hundred samples tested were positives, being the four positives isolates MTZ. None more of the isolates werepositive,theother96showedthesamesizeoftheamplifiedproductthanthepositivecontrolusedH37Rv,indi-cating that IS6110 was not present.

Conclusion

the mapping of the IS6110 insertion sites in the genome of “MTZ” strain resulted in both, offers clues for better understanding of the adaptability and virulence of M. tuberculosis, and the design of a rapid method for identifying this particular M. tuberculosis MTZ.

Presentations Type: Poster

116 ESM 2009

pp-44

DriViNg fOrCES ON ThE EVOluTiON Of ThE prOgENiTOr Of M. TuBerCuloSiS

M. C. Gutierrez 1,2, R. Brosch2, M. Marceau1, J. Tap2, E. Bourdon2, S. Brisse2, S. Mangenot3, G. Salvignol3, V. Barbe3, C. Médigue3, and P. Supply1

1 - Institut Pasteur de Lille –INSERM2 - Institut Pasteur, Paris3 - CEA/DSV/IG/Génoscope, Evry, FRANCE

Genomic and functional plasticity of agents of tuberculosis (TB) suggest that they are the legacy of extremely long evolutionaryfightforsurvivalwhichstartedwiththeirenvironmentalancestorsthatevolvedtotheexclusivelyhumanintracellular parasite of our days. Almost certainly, TB has impacted on humankind through pre-history. Mycobacterium tuberculosis and its earlier relatives have probably been co-evolving with Homo sapiens and its earlier relatives for hundred of thousand of years. Despite extensive research, the cause of M. tuberculosis speciation and the factors that have led to its predominance as a human pathogen are still unknown.

Previous studies of rare human TB clinical isolates from East-Africa (Van Soolingen et al., Int J Syst Bacteriol 1997; Fabre et al., J Clin Microbiol, 2004; Gutierrez et al., PLoS Pathogens 2005) showed that they share many properties of other agents of TB,butradicallydifferintermsofamorediversifiedpopulationstructureandobvioustracesofintra-specieshorizontal gene transfer (HGT). These features suggest that these smooth TB bacilli are extant representatives of a much broader and older progenitor species, named “M. prototuberculosis”.

We performed comparative genomics on a comprehensive collection of 56 strains of “M. prototuberculosis” to under-omparative genomics on a comprehensive collection of 56 strains of “M. prototuberculosis” to under-stand the roles played by various evolutionary processes in shaping the structure of M. tuberculosis genome and of its population.Multi-locussequencetypingof16house-keepinggenesandthreeintein-encodingsequencesconfirmedthelarge genetic diversity of the TB bacilli and suggests that the M. tuberculosis complex (MTBC) is just a particularly suc-cesful clonal lineage that has emerged from the M. prototuberculosis progenitor pool, probably involving multiple HGT episodes (clonal epidemic structure). Preliminary analysis of whole-genome sequences from the four most genetically distant strains of smooth TB bacilli indicate extensive chromosomal rearrangements and the existence of multiple ge-nomic islands compared to MTBC genomes. Genome downsizing, mutations, HGT of genomic islands, and intra-species recombination appear as major driving forces on the evolution of the ancestor of extant M. tuberculosis.


pp-45

rESiSTaNCE, mDr aND XDr Of M. TuBerCuloSiS iN SpaiN iN ThE laST yEarS.

P. Ruiz, M. Causse, F.J. Zerolo, J. Gutierrez, M. CasalMycobacteria Reference Center. Department of Microbiology. Faculty of Medicine. HospitL “Reina Sofía” . Córdoba. Spain.

Tuberculosis is among the leading causes of death worldwide. The World Health Organization (WHO) estimates that 32% of the world population is infected with Mycobacterium tuberculosis . The resistance to antituberculous drugs is a big problem to the control of the illness . The emergence of multidrug-resistant strains (MDR), extensively drug-resistant (XDR) and extreme drug-resistant (XXDR) strains, is a global problem that has made a considerable alarm. There is an increasing demand to determinate in vitro susceptibilities of clinical isolates to antimicrobial agents other than those considered primary drugs.

The purpose of this study, was determinate the resistance, MDR and XDR strains in our Reference Center in the four last years.

material and method

We are studied 624 samples from patients suspects of tuberculosis. 355 strains of M. tuberculosis were isolates , in BACTECMGIT960andLowenstein-JensenmediumandidentifiedusingAccuprobeandGENOTYPEMYCOBACTERIA.The susceptibility testing was made for primary drugs and: Amikacin (AK) 1.0 µg/ml; Kanamycin (K) 1 µg/ml; Capreomycin (CM)2.5µg/ml;Ethionamyde(ETH)5.0µg/ml;Ofloxacin(OF)2.0µg/ml;Ciprofloxacin(CI)2µg/ml;Moxifloxacin(MX)2µg/ml;Levofloxacin(LE)4µg/ml;Rifabutin(Rb)0.5µg/ml;Rifapentine5µg/mlandLinezolid(Lz)1.0µg/ml.TheBactecMGIT technique with standart protocol was strictly followed as recommended,

results

From 624 samples, in 355 were isolated M. tuberculosis and 55 (15,49 %) of theme were resistant to some of the antimi-crobial agents studied. The resistance to streptomycin was 3´38 %, to rifampin 7,88%. ethambutol 1,12 %, isoniazid 11.26 % and pyrazinamide 1,97%. A 7,6 % of strains were resistant to some of the second line drugs.The MDR was 5,6 % and the XDR 1,4 %. No XXDR were isolated.

Conclusion

The resistance to secondary drugs made necessary the in vitro studies. This method, BACTEC MGIT 960 is a reliable and rapid method to determinate the secondary drugs.

118 ESM 2009

pp-46

DETECTiON Of NON TubErCulOuS myCObaCTEria iN SurfaCE WaTErS: COmpariSON Of CulTurE mEThODS

Radomski, Nicolas 1, Lucas, Francoise 1, Cambau, Emmanuelle 2, Moulin, Laurent 3, Haenn, Sophie 3, Régis, Moilleron 4 1 - Leesu, Universite Paris-Est AgroParisTech 2 - CNRMYC, CHU Saint-Louis de Paris 3 - Crecep, Etude biologie 4 - Leesu, Universite Paris-Est AgroParisTech

Since there is no evidence for person-to-person transmission, environment is considered a likely source of non tubercu-lous mycobacteria (NTM) infections. Particularly, environmental water, from river, lake, pond or hot spring seems being a major source of NTM. NTM has been also isolated from wastewater, from sources of drinking water, from drinking water distribution system, from tap water, and even from bottled mineral water. Among human infections caused by NTM from water origin, pulmonary infections and cutaneous infections are often described. These NTM human infections linked to water could stem from changes in water use, from human vulnerability or from increase of virulence level among environmental strains. Also, it seems necessary to determine the origin of these NTM in the environment, in order to evaluate the importance of non-clinical habitats and to detect the emergence of virulence. Lack of knowledge about life cycle of harmful bacteria from surface water, particularly NTM, require more analytical tools which are not currently standardized or adapted to environmental samples. The aim of this study is to propose an improved culture method that could be used for counting and isolating NTM from surface water and wastewater. Based on literature, we selected different bacteriological methods from medical applications that had previously been applied to water samples. Samples from the river Seine (Paris, France) were used to select a culture method that will prevent the growth of interfering microbiota, with minimal inhibition of mycobacteria. The effect of antibiotics (polymyxin B, amphotericin B, nalidixic acid, triméthoprime, azlocillin, vancomycin) and chemical decontamination process (methods using acids, bases, detergent or cetylpyridininium chloride) on have been compared. Results will be discussed and a method adapted to water with high densities of interfering bacteria will be proposed.


pp-47

TypiNg Of MyCoBaCTeriuM aviuM SubSp. aviuM frOm DiffErENT SOurCES uSiNg pVuii–pSTi–iS901 rESTriCTiON fragmENT lENgTh pOlymOrphiSm (rflp) iN CrOaTia

Spicic, Silvio 1, Cvetnic, Zeljko 1, Pate, Mateja 2, Duvnjak, Sanja 1, Zdelar-Tuk, Maja 1, Racic, Ivana 1

1 - Croatian Veterinary Institut, Zagreb2 - Veterinary Faculty Ljubljana, Ljubljana, Slovenia

A total of 9 M. avium subsp. avium strains isolated from bovines (N=1), pigs (N=2), wild boars (N=2) and poultry (N=4) were genotyped by PvuII–PstI–IS901 restriction fragment length polymorphism (RFLP) analysis. The isolates were col-lectedintheperiodfrom2001to2006.Revealedprofilesweredesignatedaccordingtothenomenclatureestablishedand used in the OIE reference laboratory for avian tuberculosis in Brno, Czech Republic. Digestion with restriction en-donucleasePvuIIresultedin3PvuIIRFLPprofiles(F,QandM),comprising8-9bands.Digestionwithrestrictionendonu-cleasePstIwassuccessfullyaccomplishedin8isolatesdemonstrating4differentprofilesof11-13bands.Amongthem,3werefoundtobenewinthedatabase(A31,A32andA33).CombinationofPvuII–PstIdigestionrevealed4RFLPprofiles(A29/F, A31/F, A32/F, A33/M). No epizootiological connection was found between the isolates expressing the predominant profile(A29/F),foundinpigs,wildboarsandpoultry.ThisisthefirstresearchinthefieldofgenotypingM. avium subsp. avium strains isolated from different animal species in Croatia.

120 ESM 2009

pp-48

TubErCulOSiS iN pETS aND WilD aNimalS liViNg iN urbaN ENVirONmENT Spicic, Silvio; Duvnjak, Sanja; Obrovac, Mihaela; Zdelar-Tuk, Maja; Katalinic-Jankovic, Vera; Racic, Ivana; Cvetnic, ZeljkoCroatian Veterinary Institut Zagreb Apart from pets and domestic animals humans are often in direct or indirect contact with wild animals, especially those living in Zoos. Because of their origin these animals are a possible source of infection with mycobacteria ati-pical for certain regions. In that manner, in 2004. M. africanum type I was isolated from organs of a hyrax (Procavia capensis) that died in zoological garden in Zagreb. The hyrax had been imported from United Arab Emirates (UAE). Also, in the same year, Mycobacterium tuberculosis infection was diagnosed in a dog. This was a pet who lived it’s intire life in a city (Zagreb, Croatia) and whose owner was negative on tuberculosis. Both isolates were MIRU typed on 12 locuses (2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39 i 40). Sinse no extensive researsch was done in the past with regards to molecular typisation of M. africanum with MIRU typing, we compared our results to the ones available online (data base www. miru-vntrplus. org )(ALLIX-BÉGUEC et. al., 2008). M. africanum type I isolated from the hy-rax had a unique code (235424253422). MIRU type of M. tuberculosis isolated from the dog was identical to 8 hu-man isolates originating from all over Croatia in the period from 2004.-2006. Out of these 8 cases, 3 originated from ZagrebandCountyofZagreb;3fromneighbouringcounties(Sisako–moslavaka,KarlovakaandVaraždinska)and2 from more distant counties (Me imurska and Primorsko – Goranska). Considering that the highest tuberculosis in-cidence of the same MIRU genotype was localised in a 50 km radius in as many as 6 human cases we deducted that it’s very likely that these people were source of infection for this dog. According to these examples, pets and zoo ani-mals are important links in the chain of import and spread of pre-existing mycobacteria species in urban environment.

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pp-49

iS1245-rflp baSED gENETiC rElaTEDNESS Of ThE MyCoBaCTeriuM aviuM SuBSP. HoMiniSSuiS STraiNS iSOlaTED frOm humaNS, aNimalS aND ENVirONmENT iN CrOaTia

Spicic, Silvio 1, Cvetnic, Zeljko 1, Pate, Mateja 2, Katalinic-Jankovic, Vera 1, Duvnjak, Sanja 1, Ocepek, Matjaz 2, Zdelar-Tuk, Maja 1, Krt, Brane 2

1 - Croatian Veterinary Institut, Zagreb, Croatia3 - Veterinary Faculty Ljubljana, Ljubljana, Slovenia

SignificanceofinfectionswithMycobacteriumavium,inparticularsubspeciesM.a.hominissuis,inanimalsandhumansis constantly increasing. Susceptibility of humans, cattle and swine to various mycobacteria and the prevalence of these bacteria in the environment, although very important from the aspect of epizootiology and epidemiology, have not been sufficientlyinvestigatedinCroatia.

This study is based on massive tuberculin skin tests of cattle and swine from large farms, bacteriology and molecular identificationofM.a.hominissuisisolatedfromdomesticandwildanimals,humansandenvironment.Genotypingofthe23 isolates was conducted by IS1245 RFLP method. The selected cut-off value for cluster designation was similarity level of75%.Atotalof5clusterscontaining86.9%ofallgenotypedstrainswereidentified.Highsimilarityleveloftheprofileswithin a cluster was found for the isolates from man, swine from intensive breeding, deer, wild boar and sawdust from variouscounties.Asnoepizootiologicallinksbetweenanimalsandhumanswereidentified,humansandanimalscouldbeconsidered as dead-end hosts. According to our results, the source of infection for humans and animals is most probably the environment.

122 ESM 2009

pp-50

DEVElOpmENT Of rEal-TimE pCr aSSay fOr QuaNTifiCaTiON Of myCObaCTEria iN SurfaCE WaTErS Lucas, Francoise 1, Radomski, Nicolas 1, Cambau, Emmanuelle 2, Moulin, Laurent 3, Haenn, Sophie 3, Moilleron, Regis 11 - Leesu, University Paris-Est2 - CNRMYC, CHU Saint-Louis de Paris3 - Etude Biologie, Crecep

Most non-tuberculous mycobacteria (NTM) are saprophytes living in natural environments. However, some species are opportunistic pathogens involved in various human diseases. An increase in incidence of mycobacteriosis has been recognized worldwide, probably linked to changes in water use, population vulnerability. One important question aris-ing from the increasing occurrence of NTM diseases is the origin of these pathogens. Several cases showed that water playasignificantroleinthetransmissionofNTM.IndeedNTMcanbefoundinvariousaquaticecosystems,andalsoinwater distribution systems. However their number and diversity in environmental water bodies and in wastewaters are often underscored by the actual detection methods and no standard protocol exist. Cultural studies are time consuming andpoorlyspecificforNTMgrowth.FewquantitativePCRhavebeendevelopedforNTMspecies.However,thesePCRmethods have only been applied to clinical samples and may not be adapted for environmental samples. The aim of this studyistodevelopareal-timePCRassaytoquantifyNTMinsurfacewatersamples.Firstthespecificityandsensitiv-ity towards NTM of several published target genes, such as 16S rRNA, rpoB, hsp65, ITS and gyrA and gyrB have been evaluatedusingthealgorithmBLAST.Thisfirstscreeningresultedintheselectionof8primerpairs,whichspecificityandsensitivitywereempiricallyevaluatedbyDNAamplificationof50microbialisolatesfromtheriverSeine(France),whichbelong to Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes and Mycetes. This strain library was completed with 25 NTM and other 8 Actinobacteria strains from national collections. This second step of screening resulted in the selectionoftwohighlyspecificprimerpairstargetinggyrBandrrsgenes,showingrespectively94,83%et93,10%ofspecificity.TheefficiencyoftherealtimePCRwasevaluatedforbothprimerpairsusingthestrainlibraries.


pp-51

NONTubErCulOuS myCObaCTEria, iSOlaTED frOm paTiENTS WiTh luNg DiSEaSE, frOm liSbOa E ValE DO TEJO rEgiON, DuriNg 2008

António Amorim1*, Rita Macedo, Edna PereiraLaboratório de Saúde Pública - Micobacteriologia/Tuberculose, Administração Regional de Saúde de Lisboa e Vale do Tejo, I.P., Lisboa, Portugal*Presentingauthor.Phone:00351213602520.E-mailaddress:[email protected]

Despite the clinical relevance of most nontuberculous mycobacteria (NMT) in pulmonary infection in immunocompetent patientsisstillunclear,thismycobacteriaplayanincreasingsignificantpathogenicroleinHIV-positive,andotherimmuno-compromised patients. Nevertheless, no data about NTM species isolated from patients with lung disease, from Lisboa e Vale do Tejo region, are published or available until now.

We carried out a study during the entire year of 2008 with the purpose of identify, determine the incidence of each specie, and correlate this data with some epidemiological data in patients with lung disease from Lisboa e Vale do Tejo region.

A total of 46 patients with lung disease were detected with NTM infection. Among this patients the isolated nontuber-Among this patients the isolated nontuber-culous mycobacteria were M. intracellulare (n=7, 15,2%), M. fortuitum (n=6, 13%), M. kansassi (n=6, 13%), M. chelonae (n=4, 8,7%), M. gordonae (n=4, 8,7%), M. avium (n=3, 6,6%), M. peregrinum (n=3, 6,6%), M. spp (n=3, 6,6%), M. abscessus (n=2, 4,3%), M. mucogenicum (n=2, 4,3%), M. szulgai (n=2, 4,3%), M. triplex (n=2, 4,3%), M. lentiflavum (n=1, 2,2%) and M. simiae (n=1, 2,2%). ThepatientswithlungdiseasethatweidentifiedasinfectedwithNMThaveamedianageof51,6years,45,7%(21/46) were male and 54,3% (25/46)were female. Despite 43,5% (20/46) of our patients were HIV-state unknown, in the group of known HIV-state, 88,5% (23/26) were HIV-negative, and only 11,5% (3/26) were HIV-positive.

OurresultssuggestthatthepatternofNTMpulmonaryinfection,inLisboaeValedoTejo,hasnosignificantdifferencesfromthosereportedintherestofEuropeandUSA,andalso,nosignificantcorrelationwithHIVstatus.Wecarriedoutthe study during only one year, so, further studies are needed to better clarify the NMT infection in patients with lung disease, in Portugal, in both immunocompromised and immunocompetent patients.

124 ESM 2009

pp-52

NONTubErCulOuS myCObaCTEria iNfECTiONS iN ThE STaTE Of parÁ, amazON rEgiON, brazil Lima, Karla Valéria Batista 1, Lopes, Maria Luíza 1, Furlaneto, Ismari Perini 2, Lima, Elys JulianeCardoso 2, Conceição, Emilyn Costa 2, Sousa, Maísa Silva de 2, Costa, Ana Roberta Fusco 1

1 - Instituto Evandro Chagas, Belém, Pará, Brazil2 - Universidade Federal do Pará, Belém, Pará, Brazil

introduction

The genus Mycobacterium currently has more than 130 species. This genus includes M. tuberculosis complex and M. le-prae and other organisms referred to as nontuberculous mycobacteria (NTM). In recent years, there has been a marked increase in the number of cases of human disease due NTM, and the NTM diseases seems to be related to the geo-graphic distribution of these species in the environment.

purpose of the study

The aim of the present study was to describe the diversity of NTM from clinical isolates received at the Instituto Evandro Chagas, Pará, Amazon Region of Brazil, between 2004 and 2008.

methods

NTM included in this study were isolated from clinical specimens of 95 patients, whom 84 had pulmonary in-fection, and 11 infections cases related to other sites (lymphonod, biopsy and abscess fluid). Löwenstein-Jensenmedium was used for the recovery of mycobacteria from clinical specimens. Genetic characterization to spe-cies level was determined by PCR-RFLP analysis of hsp65 gene (PRA), and 16S rDNA and hsp65 sequencing.

results

NinetyfivepatientspresentedNTMinfections,ofwhom88.4%(84/95)manifestedpulmonarysymptoms,1.1%(1/95)presented lymphadenopathy and 10.5% (10/95) represented cases of healthcare-associated infections. A total of 13 spe-cieswere identifiedand included:M.abscessus;M.bolletii;M.massiliense;M. fortuitum;M.avium;M. intracellulare;M.scrofulaceum; M. colombiense; M. kansasii; M. simiae; M. interjectum;M. smegmatis and M. szulgai. M. chelonae-M. abscessus (26), M. avium-M. intracelullare-M. scrofulaceum (27) and M. simiae (23) complexes were the most frequent in pulmonary infections. Lymphadenopathy case was caused by M. fortuitum infection. We encountered M. chelonae-M. abscessus com-plex (6), M. smegmatis (2) and M. fortuitum (2) in cases of healthcare-associated infections.

Conclusion

We showed the diversity of species associates the NTM infections isolated at the Instituto Evandro Chagas and we en-counteredhighvarietyofspeciesinisolatedfrompulmonarysamples.AfindingwasthepresenceofM.simiaecomplexspecies in human infections, which is not common.


pp-53

EValuaTiON Of hSp65, Tb ,Sp rEgiONS iN iDENTifyiNg myCObaCTErium OThEr ThaNTubErCulOSiS( mOTT);uSiNg pCr-rflp

Noorolhoda Saadaee Jahromi ,Shima Seif, Parissa Farnia, Mehdi Kazempour,Mohammad Kargar, JamilehNowroozi, Mehdi Kazempour, Mohammadreza Masjedi,Aliakbar Velayati Mycobacteriology Research Center (MRC),National Research Institute Of Tuberculosis and Lung Disease(NRITLD),Shahid Beheshti University Medical Campus.Tehran,Iran.

background

Mycobacteria Other Than Tuberculosis (MOTT) are frequent causes of pulmonary infections resembling TB, but differ from MTB complex by being opportunistic pathogens and are acquired mainly from the environment. Recent investiga-torsreportedanincreasingcauseofpulmonaryinfectionbyMOTTspecies,inIran.IdentificationofMOTTbyconven-tional biochemical methods is cumbersome and time-consuming. Therefore in the present study,the capabilities of 3 different regions (Tb,Sp,Hsp

65) were examined using three primers. We demonstrated that Hsp

65 genotyping would be

very helpful to identify mycobacteria at the species level.

material & method

DNAwereextractedfrom121culturepositivespecimensduringtheyear2007-2009.Theamplificationcarriedoutusing following primers ( Tb ; Tb 115’-ACCAACGATGGTGTGTCCAT-3’ ,Tb 12 5’-CTTGTCGAACCGCATACCCT) ,(Sp ; Sp 15’-ACCTCCTTTCTAAGGAGCACC-3’ ,Sp 2 5’- GATGCTCGCAACCACTATCCA-3’), ( Hsp ; HSP F3 5’-ATCGC-CAAGGAGATCGAGCT-3’ , HSP R4 5’-AAGGTGCCGCGGATCTTGTT-3’)

The PCR products (Tb: 439bp ,Sp: 250-330 bp ,HSP: 644 bp) were digested using restriction enzymes with BsteII and HaeIII for Tb ,HaeIII for Sp and AvaII ,HphI,HpaII for HSP regions.The digested patterns were analyzed on 2% agarose gel.

result

The results demonstrated different sensitivity ratio for various Mycobacterium species by Tb ,Sp and Hsp regions. For RGM , Rapid Growing Mycobacteria , group(e.g., M. furtitium and M. chelonei ) the sensitivity of Tb primer was the highest among the other two regions(92%). Although, for slow growing mycobacterium(nonphotochromgen & phtochromgen) the combination of two primers(i.e., Tb+Sp)wasrequired.Thereafter,thesensitivityforidentificationofsuchspeciesreached upto 94%. Incontrast , scotochromoghen(e.g M. gordonae and M. scrofalceum) were differentiated more precisely using Sp primer(74.3% ).

Conclusion

The recent increase in MOTT species within the country , underline the need to rapidly distinguish such Mycobacteria fromtuberculosiscomplex.Wedemonstratetheuseofcombinedprimersforaccurateidentificationofmycobacteriumup to species level.

KeyWord:Identification,AtypicMycobacteria,PCR-RFLP,RGM

126 ESM 2009

pp-54

myCObaCTErium aVium alVEOliTiS afTEr ClEaNSiNg hOTEl Spa WhirlpOOlS

Svensson; Erik 1; Ridell; Malin 1; Åkerström; Magnus 2; Andersson; Eva 2

1 - Institute for Biomedicine, University of Gothenburg2 - Department of Occupational and Environmental Medicine, University of Gothenburg

Hotelstaffcleaningspawhirlpoolsandfiltersbecameillinadiseasesuspectedtobetheso-calledhottublung,whichisanallergic alveolitis-like granulomatous lung disease. In total seven employees at three hotels were involved. Mycobacterium sp.wassuspectedtobethecause.Culturesfrompatientsandfromwaterandoutletfiltersweredone.Aquantitativeculture method was developed and water from different parts of the equipment was analysed.

OnepatienthaddefiniteallergicalveolitisandM. avium was isolated. Two other employees from the same hotel had suspectedalveolitis,butnocultivationformycobacteriawasdone.Inthishotelthecleansingofthespafilterswasdonewith high-pressure washers.

Two employees at another hotel had fever, chills and dyspnea related to cleansing the equipment. Their disease was not regarded as allergic alveolitis, but both of them were colonized with M. avium

In the third hotel, two employees were colonized with M. avium, but no one hade symptoms related to work. One patient, though,hadflulikesymptomsshortlyafterbathinginthepool.

InrespiratorysamplesfromfiveofthesevenpatientsM. aviumwasisolated.ThepoolwaterandthesurfacefilmsofthewaterfilterscontainedM. avium, often mixed with other, rapidly growing mycobacteria, however a pure culture was never obtained. In the quantitative water culture 0 - 16000 CFU/mL of M. avium was isolated.

Thesearethefirstreportedcasesofhottub lungalveolitis(hypersensitivitypneumonitis) inSweden.Cultures frompatients,filtersandwaterhavecontainedM. avium. The symptoms of the patients have presently ceased. The cleaning practiceshavebeenchangedandthepoolfilterequipmenthasbeenrebuilt.


pp-55

iDENTifiCaTiON Of NONTubErCulOuS myCObaCTEria iN CliNiCal SamplES uSiNg mOlECular mEThODS: a ThrEE-yEar STuDy

Isabel Couto1,2*, Diana Machado1, Miguel Viveiros1,3, Liliana Rodrigues1,4 and Leonard Amaral1,3,4

1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal2 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal3 - COST ACTION BM0701 (ATENS)4 - UPMM, IHMT/UNL, Lisbon, Portugal

Although Mycobacterium tuberculosis, the etiologic agent of human tuberculosis is the main cause of mycobacteriosis in Man, other species of mycobacteria may also cause infection in humans. The increasing importance of nontuberculous mycobacteria(NTM)isnowconsensuallyrecognizedanddemandsforfastermethodsfortheiridentificationandse-lectionofappropriatetherapy.InthisworkwereportourexperienceontheidentificationofNTMreceivedfrom12hospitals of the Lisbon Health Region (Portugal) over a three-year period using the GenoType Mycobacterium (CM/AS) assays (HAIN Lifescience). From 1 January 2005 to 31 December 2007, our laboratory received a total of 1192 acid-fast bacilli (AFB) positive isolates from 1174 patients presenting with presumptive active mycobacteriosis. All isolates were processed for Ziehl-Neelsen staining and inoculated into MGIT tubes of the BACTEC MGIT 960 system. M. tuberculosis isolatedfromculturewereidentifiedbytheAccuprobesystem(Gen-Probe).Full-grownAFBcultures,negativeforM. tuberculosis,wereidentifiedbyGenoTypeMycobacterium(CM/AS)kits.Outofthe1192specimenreceived,1181wereidentifiedasmembersoftheMycobacterium genus. From these, 1032 cultures (87.4%) were positive for M. tuberculosis complex. The remaining 149 cultures were NTM, corresponding to 12.6% of the total number of cultures from which mycobacteria were isolated. During the study period, NTM prevalence increased steadily, starting with 8.7% in 2005 and risingto15.2%in2007.ThejointuseoftheCMandASkitsidentified96.6%ofallNTMisolatestested.Amongthe18NTMspeciesidentified,M. avium complex was the most frequent, although it accounted for only 34% of all NTM. In countries with high incidence of tuberculosis and, particularly, multidrug resistant tuberculosis (MDRTB) such as Portugal, therapeutic failure with isoniazid and rifampicin is anticipated to be due to an MDRTB strain. Since many NTM species areresistanttothesedrugs,theidentificationofthemycobacteriacausingtherapeuticfailure(MDRTBversusNTM)isofmajorimportance.TheintroductionofmolecularmethodsfortheidentificationofNTMinourlaboratoryhasresultedin an increased awareness of the importance of being able to rapidly identify NTM as potential pathogens and the key role played by the laboratory in assisting the selection of therapeutic modality.

128 ESM 2009

pp-56

myCObaCTEria iN aNimalS iN SlOVENia – aN OVErViEW Of ThE laST DECaDE

Mateja PATE1, Darja FERME1,MancaŽOLNIRDOVC2,MatjažOCEPEK1

1 - Veterinary Faculty Ljubljana, Gerbiceva 60, SI-1115 Ljubljana, Slovenia; e-mail:[email protected];fax:+386147793522 - University Clinic of Respiratory and Allergic Diseases Golnik, Golnik 36, SI-4204 Golnik, Slovenia

Successful national control program carried out between 1962 and 1973 contributed to eradication of bovine tubercu-losis (BTB) in Slovenia. Since then, infections with the causative agents of BTB were seldom detected. The main role of veterinarymycobacteriologistshasthereforebecomethedetectionandidentificationofthecausativeagentsofaviantuberculosis and opportunistic mycobacterial pathogens. The aim of this study was to summarize the work done in the pasttenyears(1999-2008)inthefieldofveterinarymycobacteriologyinSlovenia.

Identificationoftheisolateswasbasedonthefollowingfeaturesandtests:colonymorphologyandgrowthcharacteris-tics,biochemistry,PCRandcommercialidentificationkitsAccuProbe(Gen-Probe)andGenoType(HainLifescience).

From 1999 to 2008, a total of 292 mycobacteria were isolated from domestic, pet and wild animals in captivity. The vast majority of isolates were represented by M. avium(80.1%),identifiedtosubspecieslevelin92.7%(M. a. subsp. avium – 36.3%, M. a. subsp. hominissuis–56.4%),while7.3%isolatesremainedidentifiedasM. avium. These mycobacteria were found predominantly in pigs, followed by cattle, poultry and exotic birds. M. caprae was found in 1.4% cases and was related to an outbreak in a zoo, affecting bisons and camels, and to a single culture-positive case of BTB in cattle in the last 15 years. M. tuberculosis was found in one cow (0.3%) as a consequence of human-to-animal transmission proven by means of molecular epidemiology. Other species detected included M. terrae (0.7%, pigs), M. fortuitum (0.7%, pig & cattle), M. scrofulaceum (0.3%, bison) and M. celatum (0.3%, pig). A relatively large proportion of isolates (16.1%) originating from pigs,cattle,sheep,goat,mouflon,bisonandmonitorlizardwereidentifiedtothegenuslevelonly.Thiscouldbepartlyattributedtothelackofreliableidentificationmethodsinthepast.

Development of diagnostic kits based on molecular tests led to improved and easier diagnostics of mycobacteria, espe-ciallyofthespeciescommonlyfoundintheenvironment,thereforereducingtheburdenofunidentifiedmycobacteriaina routine laboratory.

c^ C

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pp-57

iSOlaTiON aND iDENTifiCaTiON Of rhODOCOCCuS aND NOCarDia gENDErS iN SpuTum SamplES WiTh TubErCulOSiS SuSpECT Leite, Sérgio Roberto A; Silva, Paulo; Sato, Daisy Nakamura; Santos, Adolfo; Carlos Barreto; Miyata, Marcelo; Leite, Clarice Queico Fujimura São Paulo State University

Species from Rhodococcus and Nocardia genders are partially alcohol acid resistant and could be isolated from clini-cal samples (sputum, bronchial-alveolar rinsing and lung biopsy). These genders grow successfully in Löwenstein-Jensen medium and can cause pulmonary infections similar to tuberculosis cases. Our purpose was to evaluate the presence of these bacteria from 1636 sputum samples of patients with clinic suspect of pulmonary tuberculosis at Ribeirão Preto city, São Paulo state, Brazil, between the years 2000 and 2002. The samples were analyzed in the laboratory of Instituto Adolfo Lutz, Ribeirão Preto unit. From 426 acid-fast bacilli positive sputum samples, applying phenotypic methods and chemotax-onomy,weisolated296mycobacteria(identifiedas224M.tuberculosisand72NTM),29Nocardiasp.,51Rhodococcusequiand09non-identifiedpartiallyacid-fastbacilli(PAFB).Thesuccessofthetreatmentdependsonearlydiagnosis,thusthe differential diagnostic between Mycobacterium, Nocardia and Rhodococcus genders deserves special importance. Due to the high incidence of tuberculosis and the similarity in symptoms, probably the nocardiosis and rodococosis are under-notifiedinBrazil.ThereforehumanhealthprofessionalsneedtoobservecarefullytheimportanceofRhodococcusequi and/or Nocardia sp., that were found on 12% and 6.8% respectively of patients suspected with tuberculosis, attended at Ribeirão Preto city between 2000 to 2002.

130 ESM 2009

pp-58

pCr-rflp Of HSP65 fOr iDENTifiCaTiON Of MyCoBaCTeriuM lePrae DirECTly frOm a CliNiCal SamplE

Neonakis Ioannis K1, Kontos Fanourios2, Gitti Zoe1, Baritaki Stavroula1, Bazigos Stavros1, Mihailelis Efstratios1, Zerva Loukia2, Spandidos Demetrios A1

1 - Microbiology Labolatory, University Hospital of Heraklion, Heraklion, Greece.2 - Clinical Microbiology Laboratory, Medical School of Athens, “Attikon” University Hospital, Athens, Greece.

introduction

Mycobacterium leprae cannot be cultured in vitro. The application of PCR-RFLP analysis of hsp65foridentificationofM. leprae had been previously proposed (Rastogi et al., J Clin Microbiol, 1999, 37, 2016-19) and, to our knowledge, the method has been used only once.

Objective

The identification ofM. leprae directly from a clinical sample by application of PCR- Restriction Fragment Length Polymorphism analysis (PCR-RFLP) of hsp65 gene.


A sample, taken with a swab from open lesions with exudates from a 51-y old patient suspected of suffering from lep-rosy, was suspended in sterile water. Mycobacteria were heat-inactivated at 80o C for 1 h and the DNA was extracted usingtheguanidiniumthiocyanatelysisbuffer(Casasetal.,JMedVirol,1995,47,378-385).PCRamplificationofa439-bpfragment of hsp65 was performed using the protocol and primers Tb11 and Tb12 as previously described (Telenti et al., JClinMicrobiol,1993,31,175-178).ThePCRproductwasfurtheranalyzedbysequencingandRFLP.TheamplifiedPCRproduct was digested using the Hae III and BsteII (New England Biolabs) restriction enzymes and the mixtures were electrophoresed on a 3% Metaphor agarose.

results

The BstEII digestion produced two fragments of 315 and 135 bp and the HaeIII digestion produced two fragments of 265 and130bp.ThisprofilematchedtheonepreviouslyreportedforM. leprae. Sequencing of the PCR product (GenBank accessionnumber:FJ497239)verifiedtheidentityofM. leprae.

Conclusions

PCR-RFLP could be a useful molecular tool as an adjunct to careful clinical and pathological assessment of patients sus-pected of suffering from leprosy.


pp-59

a CaSE-rEpOrT Of MyCoBaCTeriuM THerMoreSiSTiBile frOm grEECE.

Neonakis Ioannis K1, Kontos Fanourios2, Gitti Zoe1, Baritaki Stavroula1, Kosmadakis Georgios1, Baritaki Maria1, Zerva Loukia2, Spandidos Demetrios A1

1 - Microbiology Laboratory, University Hospital of Heraklion, Heraklion, Greece.2 - Clinical Microbiology Laboratory, Medical School of Athens, “Attikon” University Hospital, Athens, Greece.

Mycobacterium thermoresistibileisanon-tuberculousmycobacteriumthatwasfirstrecoveredinJapanbyTsukamurain1966. Although it is strongly associated with pulmonary and dermal diseases, there have only been six reports of its isola-tionfromclinicalsamples.HerewereportonthefirstcasefromGreece.

The mycobacterium was isolated from the solid culture (Lowenstein-Jensen slant at 37oC) of a sputum sample after 14 days of incubation. The sample was taken from a 67-year-old male, who was a heavy smoker (1pack/day for 30 years) and had a history of COPD, type II respiratory distress syndrome, diverticulosis and diabetes, and was presented to our hospital with fever (38o C), productive cough, dyspnea, weakness, and acute purpura. The chest radiograph revealed an elevatedcardiothoracicratio,aswellaschronicobstructivelungdisease,peribronchialinfiltrations,consolidationsintheright middle and lower lung zones and a small blunt at the left pneumodiaphragmatic angle.

The Accuprobe Mycobacterium tuberculosis complex assay (Gen-Probe, San Diego, CA) was negative. Accuprobe also yielded negative results for the Mycobacterium avium complex and Mycobacterium gordonae.Biochemicalprofilecouldnot distinguish it from other mycobacteria. The banding patterns obtained with GenoType CM and GenoType AS (Hain, Lifescience,Nehren,Germany)werenotspecies-specific[GenoTypeCM:1,2,3and10,andGenoTypeAS:1,2,3and12].TheidentificationofM. thermoresistibilewasachievedwiththeamplificationandsequencingofthe16SrRNAgeneandthe 16S-23S internal transcribed region (GenBank accession: FJ236481).

Moreover, a 439-bp fragment of the 65-kDa heat shock protein (hsp65) gene (GenBank accession: FJ236482) was further used for restriction fragment length polymorphism analysis with Hae III (New England Biolabs) and BsteII (New England Biolabs). The BstEII digestion produced two fragments of 235 and 210 bp and the HaeIII digestion produced four frag-ments of 180, 135, 70 and 50 bp.

Although M. thermoresistibile is considered pathogenic, it is rarely isolated from clinical samples. Molecular techniques are essentialforitsidentification.

132 ESM 2009

pp-60

iSOlaTiON aND frEQuENCy Of MyCoBaCTeriuM Sp iN a gENEral hOSpiTal DuriNg a 9-yEar pEriOD

Portugal C.; Sancho L.; Dias A.; Tancredo L.; Silva M.; Sardinha T.; Sousa GermanoLaboratory of Microbiology, Department of Clinical PathologyHospital Fernando Fonseca – Amadora, [email protected]

Tuberculosis remains a major public heath problem in Portugal with an incidence rate of 25,3/100.000 inhabitants in 2008, being the majority of the cases in the surroundings of the two major cities (Lisbon and Oporto).

Our Hospital is located in the Lisbon’s surroundings and covers a population of 750.000 inhabitants most of them with poor socioeconomic level and immigrants from Africa and East Countries.

purpose

The aim of this study was to investigate the isolation frequency of Mycobacterium sp. in a general Hospital in Amadora, Portugal, during a 9-year period (2000-2008).

methods

A total of 19.417 clinical specimens (15.159 pulmonary and 4.261 extra pulmonary), collected from 9.525 patients, were cultured for mycobateria. All specimens were processed by the NaCl-NaOH method as recommended by CDC, stained by Ziehl-Neelsen, cultured on Lowenstein-Jensen and liquid medium MGIT. Identificationwasmadewiththetechnology Genotype MTBDR plus (HAIN-Lifecience- Germany).

results

Of the 19.417 cultured specimens for mycobateria, 2751 (14,2%) were positive by cultural methods.

The positive rates by clinical specimen were: 17% (279/1684) in respiratory specimens and 6% (26/476) in extra pulmo-nary tuberculosis; 21% (5/23) in pus, 11% (2/14) in biological liquids, 8% (5/66) in biopsy, 6% (7/112) in blood, 5% (1/22) inasciticsfluid,4%(1/22)inmieloculture,and3%inurine(4/133)andcerebrospinalfluid(2/84).

In 9525 suspected TB patients, 1094 were effective tuberculosis cases (122 TB cases/year average, 97 cases in 2008). The incidence rate by patient was 11,5%.

In comparison with the positive results of the culture, Ziehl-Neelsen stain was positive in 39% of the samples (47% of the patients).

96% (1029) of mycobateria isolated were Mycobacterium tuberculosis Complex, 2% (20) M.avium, 2% (20) other species (M.chelonae, M.intracellulare, M.alsiensis / malmoense / szulgai, M.scrofulaceum, M.lentiflavum).

Conclusion

In spite of the increase of TB suspected patients (+11,3%), the number of effective tuberculosis cases have decreased (-7,8%) for a 9-year period, which is comparable to 2008 national data (-7,2%) in the last decade.

Tuberculosis is a major problem in this area with an recovery rate of 14,2% in the clinical specimens that we receive and in 11,5% of the patients, most of them with Mycobacterium tuberculosis Complex (96%).

We have one positive patient every 3 days.

In attempt to minimize the impact of this disease, that depend on a large number of factors, we know that the laboratory playsanimportantroleinmakingadefinitivediagnosisinashorttimeperiod(ASAP).Wealsoknowthatisimportantaclose relation between the microbiology laboratory and the hospital doctors and also with the centre that follows the patients in the community (CDP). This is our policy.


pp-61

labOraTOry miCrObiOlOgy CONTribuTiON TO MyCoBaCTeriuM Spp. DiagNOSiS iN ThrEE DiSTriCT COuNCilS Of SETúbal (pOrTugal), aN arEa WiTh high myCObaCTErial iNfECTiON prEValENCE.

José Diogo, Ana Rodrigues, Isabel Nascimento, Elisabete Sardinha, Ana Raposo, Rita Figueira, Isabel Monge, Kátia Silva, Maria José Gil, Susana Rodrigues.Laboratory Microbiology, Hospital Garcia de Orta (Almada)

The purpose of this work is to evaluate the incidence of Mycobacterium spp. infection in three district councils of Setubal (Almada, Seixal and Sesimbra) with a high prevalence of this disease, superior to the average in Portugal. The number of patients with mycobacterial infection and positive mycobacteriological study in Laboratório de Microbiologia (LM), Serviço de Patologia Clínica, Hospital Garcia de Orta, E. P. E. (HGO) between 1993 and 2008 were evaluated.

Each biological sample was processed as follows: 1) for acid-fast stain with Kinyoun’s carbolfuchsin and microscopic ob-servation with a 100x immersion oil objective; 2) inoculation in Lowenstein-Jensen medium with aerobic incubation at 37ºCand/orinoculationinMiddlebrock7H9andincubationinMGITSystem(Bactec®);and3)speciesidentificationandfirstlineantimycobacterialsusceptibilitytesting.

In these sixteen years, LM processed more than 25000 biological products, 3813 were positive and isolated from biologi-cal products of 1704 patients.

The age, gender, positive samples for BAAR and number of cases of tuberculosis (including variation per year) in Almada, Seixal and Sesimbra was determinated. A relation was established between the disease localization (pleuro-pulmonar, extra-pulmonar and disseminated) by clinical and microbiological criteria. The mean age of the patients was 40,6 years, 1199 (70.3%) were male and 505 (29,7%) were female.

Speciesidentification(since2004)was:664(84,1%)M. tuberculosis, 67 (8,5%) M. gordonae, 20 (2,5%) M. avium-intracelular and 39 (4,9%) other mycobacteria.

Thefirst lineantimycobacterialresistancewasdeterminatedin649M. tuberculosis strains. The resistance level was: 116 (17,9%) to streptomycin, 76 (11,7%) to isoniazid, 28 (4,3 %) to rifampicin, 17 (2,6%) to ethambutol and 14 (2,2%) to pyrazi-namide. The multiresistance (simultaneous resistance to isoniazid and rifampicin) was detected in 27 (4,1 %) strains. Six pa-tients had extensively drug-resistance tuberculosis (XDR-TB). Susceptibility pattern variation per year was also evaluated.

134 ESM 2009

pp-62

MyCoBaCTeriuM lenTiFlavuM aS a CauSaTiVE agENT Of aDENOpaThy

Santos, Claudia, Mendes, Ana Constança, Fernandes, Sandra João, Ramos, Maria HelenaCentro Hospitalar do Porto - Hospital Santo António

We report the case of a 23 year old female patient, HIV positive, presenting with mesenteric adenopathies of unknown origin. A biopsy was performed and sent for microbiological study. Results turned out negative for aerobic and anaerobic bacterial culture, but with positive AFB examination. Following this information, patient initiated anti-tubercular therapy – Isoniazid, rifabutin, pyrazinamide and ethambutol. Molecular detection of Mycobacterium tuberculosis complex, directly from the clinical sample, was negative.

We then performed an in house universal real time PCR targeting a 370 bp region of 16S rDNA bacterial gene, witch gaveapositivesignal.Inordertoidentifytheamplifiedproduct,sequencingwasperformedusingtheBigDye®Terminatorv3.1 Cycle Sequencing Kit (Applied Biosystems), in ABI PRISM 310 Genetic Analyser (Applied Biosystems). The obtained sequenceswereanalyzed,andidentifiedasMicobacteriumlentiflavum.

Thisinformationledtotreatmentalteration–(ciprofloxacin,clarothromycin,rifabutinandethambutol).

Conventional mycobacterial cultures (MGIT™ and Lowenstein-Jensen medium) turned out negative after incubation time.

Three months later a new biopsy was sent for microbiological study, with positive AFB examination, but negative cultures. Samplewasinsufficientformolecularstudy.

Sequencebasedbacterialidentificationhasbeenwidelyusedtoidentifymicroorganismsisolatedinclinicalsamples,par-ticularly when applied to poorly described, rarely isolated or phenotypically aberrant species. We report sequence based bacterialidentificationofMycobacteriumlentiflavumdirectlyfromaclinicalsample.Lackofculturalgrowthdidnotallowsusceptibilitytesting,andclinicalspecimenwasinsufficientforfurthermoleculartests, includingtestingformutationsassociated with antibacterial resistance. However resistance to rifampin has been reported, as has been susceptibility to isoniazid,ethambutol,clarithromycin,amikacinandciprofloxacin,invitrosusceptibilitypatternswhoseclinicalrelevanceremains uncertain.


pp-63

TEaChiNg OlD bONES NEW TriCKS; SiNglE NuClEOTiDE pOlymOrphiSm aNalySiS Of EurOpEaN arChaEOlOgiCal m. lEpraE DNa

Watson, Claire, Lockwood. DianaLSHTM - London School of Hygiene and Tropical Medicine, Keppel Street, London, UK

background

LeprosywascommoninEuropeeighttotwelvecenturiesagobutmolecularconfirmationofthishasbeenlacking.Wehave extracted M. leprae DNA from medieval bones and SNP typed the DNA, this provides insight into the pattern of leprosy transmission in Europe and may assist in the understanding of M. leprae evolution.

Methods and findings

Skeletons have been exhumed from 4 European countries (the United Kingdom, France, Denmark and Croatia) and aredatedaroundthemedievalperiod(476to1350A.D.).wetestedforthepresenceof5previouslyidentifiedsinglenucleotide polymorphisms (SNPs) in 50 aDNA extractions chosen at random from the collection. M. leprae aDNA was extracted from 20 of the 50 bone samples. SNP analysis of these 20 extractions were compared to previously analysed European SNP data using the same PCR assays. Testing for the presence of SNPs in M. leprae DNA extracted from an-cientbonesamplesisanovelapproachtoanalysingEuropeanM.lepraeDNAandthefindingsconcurwiththepreviouslypublished data that European M. leprae strains fall in to one group (SNP group 3).

Conclusions

ThesefindingssupportthesuggestionthattheM.lepraegenomeisextremelystableandshowthatarchaeologicalM.leprae DNA can be analysed to gain detailed information about the genotypic make-up of European leprosy, which may assist in the understanding of leprosy transmission worldwide.

136 ESM 2009

pp-64

iNTErprETaTiON Of pOSiTiVE M. TuBerCuloSiS aNTigEN SpECifiC ifNΓ rElEaSE aSSayS iN TubErCulOSiS DiagNOSiS Greib Carine 1, Lazaro, Estibaliz 1, Viallard, Jean-François 1, Pellegrin, Jean-Luc 1, Maugein, Jeanne 2

1 - Medecine Interne et Maladies Infectieuses CHU Haut-Leveque Bordeaux2 - Laboratoire de bactériologie, CHU Haut-Leveque Bordeaux

QuantiFERON-TB Gold in tube test is an accurate test to detect immune responses against active Mycobacterium tuberculosis infection (TB) and has the advantage to eliminate false positive outcomes due to BCG vaccina-tion and non-TB mycobacteria. Howevever there are still some false positive tests which remain unexplained. In this study we aimed to focus on the prevalence and the potential explanation of false positive tests among a cohort of patients accurately screened without TB.

A total of 250 adults patients with suspicion of active tuberculosis were enrolled in this study between January 2007 and December 2008.

Among them, 88 had positive result in accordance with manufactured interpretation (Nil ≤ 8I/mL, TB antigen ≥ 0.35 IU/mL and ≥25%ofNilvalue).For28patients,tuberculosiswasconfirmedbycultureofMycobacteriumtuberculosisin26cases and Mycobacterium bovis in 2 cases. For 9 patients, diagnosis of active tuberculosis was made according to a clinical and paraclinical body of arguments. For 10 patients without active tuberculosis, we could suspect a technical mistake to explain the positive result of the test (TB antigen near 0.35IU/mL for 4 patients and mitogen < 0.5IU/mL for the 6 others).

Among the other 41 patients out of 88 with a positive test without any argument for TB, we found that 12 had previous diagnosis of active tuberculosis in the past and 13 came from tuberculosis endemic areas. Finally, 16 positives results out of 88 (18 %) remain without explanation. Errors of diagnosis, bias in collection of medical information (previous or latent tuberculosis infection), or technical mistakes could be possible reasons.

In conclusion, quantiFERON-TB is a useful tool for diagnosis of active TB. However the results have to be carefully analy-sed according to the high rate of false positive diagnosed in this study.


pp-65

DirECT iDENTifiCaTiON Of MyCoBaCTeriuM TuBerCuloSiS COmplEX, MyCoBaCTeriuM aviuM COmplEX aND MyCoBaCTeriuM KanSaSii iN SmEar-pOSiTiVE CliNiCal SpECimENS

SUXING WANG, ZHI YU NEO, KE XIN MAK, MARIA DOLORES QUIENG, LI HWEI SNGCentral Tuberculosis Laboratory, Department of Pathology, Singapore General Hospital

GenoType@MycobacteriaDirect(GTMD)wasusedfordirectidentificationMycobacterium tuberculosis complex (MTBC), M. avium, M. intracellulare, M. kansasii in 136 specimens from 122 patients. All 136 specimens were AFB smear positive with rangesfrom1+to4+.TheGTMDcorrectlydetectedandidentified134of136ofthemycobacteriapresent.Comparedtoresultsfromculture,thisindicatesasensitivityandspecificityofGTMDassayof98.5and100%,respectively.Thesevalues increased to 100% when specimens with only MTBC isolation were considered. There were two discrepant re-sultsduringthestudy.ThefirstwassputumfromwhichM. avium complex was isolated which had been kept at –70oC for nearly 3 years. The test was negative for inhibitors and another sputum collected from same patient at same period was detected as being positive for M. avium by GTMD. This may have been accounted for by degradation of the RNA or a sampling issue. The second was a stool specimen from a HIV-positive patient who had M. avium complex isolated from hisstool.BothRNAisolationandamplificationwererepeatedforthesamespecimen,andthepresenceofinhibitorwasconfirmed.TwostoolspecimensfromoneHIV-positivepatientwereinitiallyidentifiedascontainingM. avium complex by AccuProbe. GTMD results showed bands matching M. intracellulae and M. kansasii, indicating possible co-infections in thisHIV-positivepatient.ThiswasconfirmedwhenDNAprobewasperformedongrowthfromtheLJslanteventhoughthere were no pigmented colonies and both specimens turned out to be positive for M. kansasii. It was most likely that the mixed infection was not detected by routine methods as the M. intracellulare in this case, had outgrown M. kansasii as the predominant organism in broth and solid cultures.

DirectidentificationbyGTMDtakesabout5workinghourscomparedto3to5weeksrequiredforcultureisolationandspeciesidentificationbyconventionalmethods.

138 ESM 2009

pp-66

rapiD DiagNOSiS aND Drug SuSCEpTibiliTy TESTiNg Of TubErCulOSiS iNfECTiON: mTD-TEST2 aND baCTEC mgiT 960 SySTEm

Müllerova, MariaKlinlabLtd,Uvojenskenemocnice1200,16900Prague6,CzechRepublic,[email protected]

The Czech Republic is a country situated in the heart of the Europe. It has a low incidence of tuberculosis in the last 10 years. However, the increasing number of migrants from countries with high incidence of TB is changing the situation.

Samples were collected from patients in the Central Bohemian Region and in a part of Prague (2 millions inhabitants).

All samples were tested by MTD-Test2 and conventional methods, partly also by BACTEC MGIT 960, for the detection of M.TB complex.

Drug susceptibility tests were performed by conventional methods and BACTEC MGIT 960 (S.I.R.E.+PZA).

For the differentiations of separate species of M.TB complex, the GenoType Mycobacteria MTBC test was used.

MTD-Test2fordetectingtheM.TBcomplextakesonly3.5hrsanditsperformanceishighlysensitiveandspecific.

From December 16, 1999 to December 31, 2008 a total of 39,592 different samples were collected for detecting the M.TB complex, comprising of 23,582 sputa, 10,910 bronchoalveolar lavages, 1,492 laryngeal swabs and 3,662 non-pulmo-nary samples.

From 35,930 pulmonary samples were MTD-T2 pos., cult. neg. 416 (1.16%), MTD-T2 neg., cult. pos. 116 (0.32%), MTD-T2 pos., cult. pos. 1,108 (3.08%), and MTD-T2 neg., cult. neg. 34,290 (95.44%).

Of the 1,296 isolated strains of M.TB tested for susceptibility to basic AT (S.I.R.E.+PZA), 1,109 (85.57%) strains were susceptible and 187 (14.43%) were resistant for varying combinations of AT; however, further 78 strains (6.02%) were resistant to INH+RIF, i.e. MDR TB.

The number of multiresistant patients is rising, especially in the last 3 years: in 2006 10.17% patients were resistant, and from them 4.81% MDR TB, in 2007 8.78% res., 5.36% MDR TB and in 2008 35.51% res., 13.76% MDR TB. These patients are mostly young males, 25-35 years old, from foreign countries.

The increase in the number of MDR TB is becoming a problem in our country in view of the need of using alternate AT.


pp-67

firST EXpEriENCE WiTh gENOTypE mTbDr aSSai fOr rapiD EValuaTiON Of mDr CaSES

Klavdia Levina, Anna Dementieva, Maret SaluotsaNorth Estonia Medical Centre, Tallin

Tuberculosis (TB) incidence in Estonia decreased from 56.6 per 100.000 population in 1998 till 30.7 in 2008. However, multidrug resistance (MDR) is still very high ~ 12%. Therefore rapid determination of Rifampin (RMP) and Isoniazid (INH) resistance in M.tuberculosis (MTB) isolates remains actual for the initiation of effective chemotherapy to break the transmission of the MDR strains.

Drug susceptibility testing (DST) by conventional methods is time-consuming. More rapid results could be achieved by using molecular methods.

The goal of our study was to investigate the possibility of application of the Hain Lifescience GenoType MBTDRplus assay as a rapid diagnostic tool for detection of RMP and INH resistance in MTB isolates by comparing the results with those obtained by conventional phenotypic resistance studies.

61 smear positive clinical material and 97 MTB strains recovered from smear negative patients’ specimens by culture have been studied by MBTDRplus assay.

Additionally DR was analyzed by standardized DST method on BACTEC MGIT 960 system when culture was later available. Among tested MTB strains, 108 were phenotypically sensitive to INH and RMP, 39 were resistant to both drugs, 9 and 2 have got mono resistance to INH and to RMB correspondently. Wild type patterns were found using the MTBDRplus assay in 107 samples. SpecificmutationsassociatedwithresistantpatternsMTBDRplusassayweredisplayedin48samples.

MTBDRplus assay and the DST prepared from strains isolated by cultural methods from smear negative specimens showed 100% agreement between the two methods.

Concordant results between MTBDRplus test directly from smear positive samples and conventional DST from later obtained culture strains was found in 95.7 % of cases tested, agreement of the results regarding RMPwas100%.

In two (3.4%) smear positive samples resistance-linked mutations were found, but no phenotypically resistance was de-tected. One strain recovered from AFB positive specimens was INH resistant, but had not displayed mutations conferring INH resistance by MTBDRplus assay directly from the same smear positive specimens.

Our results suggest that GenoType MTBDRplus assay is a valuable alternative for rapid detection of resistance and in agreement with the classical methods gives opportunity to break the transmission of the MDR strains.

140 ESM 2009

pp-68

Drug rESiSTaNT TubErCulOSiS iN SlOVENia aND EValuaTiON Of gENOTypE mTbDrpluS TEST iN CliNiCal labOraTOry Natasa Fajfar, Manca Zolnir - DovcUniversity Clinic of Pulmonary and Allergic Diseases Golnik, Golnik 36, SI-4204 GOLNIK, SLOVENIA

Slovenia is one of the countries with relative low rate of drug resistant tuberculosis (TB). In the period 1995-2008 the rate was 4.2% (1.6-6.5) and only 0.70 % (0.0-1.8) of Slovenian TB patients had MDR TB in the same period. The aim of our retrospective study was to evaluate the performance of MTBDRplus assay (Hain Lifescinece GmbH, Nehren, Germany) for the detection of rifampicin (RMP) and isoniazid (INH) resistance in comparison to phenotypic drug susceptibility testing method.

A total of 48 drug-resistant Mycobacterium (M.) tuberculosis isolates were included in the study. M. tuberculosis drug-resis-tant strains were obtained from patients living in Slovenia between 1995 and 2008. In total, 20 RMPr/INHr, 27 RMPs/INHr, 1 RMPr/INHs strains were analysed with MTBDRplus assay. The assay was performed according to the manufacturer’s instructions.

In comparison to conventional drug susceptibility testing MTBDRplus was able to identify RMP resistance in 21 of the 21 strains (100%), but for INH the accordance of both methods was lower - only in 37 of 47 strains (79%). The most com-mon mutation carried in RMP-resistant isolates was rpoB MUT3-S531L (48%) followed by mutation rpoB MUT1-D516V (33%). In INH-resistant strains dominating mutation was in the gene katG in 60% (MUT1-S315T1 in 68%, MUT2-S315T2 in 29%) and the mutations in the inhA promotor gene in 19%.

Comparative analysis demonstrated very good agreement between molecular and phenotypic drug susceptibility results for RMP resistance, but not so for INH. Thus MTBDRplus assay is useful method for the rapid detection of drug resis-tance,buttheresultsshouldalwaysbeconfirmedbythephenotypicmethodsaswell.


pp-69

EValuaTiON Of a NEW rEal-TimE pCr KiT fOr ThE DiagNOSiS Of TubErCulOSiS iNrESpiraTOry SpECimENS

Causse, M; Gutierrez-Aroca, JB; Casal, MMycobacteriaReferenceCenter.MicrobiologyDepartment.ReinaSofiaUniversityHospital,Cordoba(Spain)

introduction

Early diagnosis of tuberculosis is one of the major objectives of the World Health Organization. In its latest update, the Center for Disease Control and Prevention (CDC) recommends the use of a rapid molecular diagnostic technique on at least one sample per patient.

Objectives

To evaluate a new kit (COBAS Taqman MTB®, Roche) for the diagnosis of tuberculosis in respiratory samples, and com-pare results with those obtained by the COBAS Amplicor MTB kit. Culturing (Lowenstein-Jensen or BACTEC MGIT 960) was used for reference purposes.


A total of 170 respiratory and 19 non-respiratory samples were processed. Specimens decontaminated were stained with auramine and cultured. A single manual extraction was performed using the AMPLICOR Respiratory Specimen Preparation Kit; eluate aliquots were thenamplifiedusingtheCOBASAmplicorMTBandtheCOBASTaqManMTBkit. Automatic sample extraction was also performed, using the Ampliprep TNAI kit, 200-µl aliquots being previously incu- TNAI kit, 200-µl aliquots being previously incu-200-µl aliquots being previously incu-bated at 95ºC for 15 minutes.

results

All77smear-positivesampleswereclassifiedaspositivebybothkits.

Of the 170 respiratory samples tested using the TaqMan, 2 false positives (FP) and one false negative (FN) were recorded. Of the 169 tested using the Amplicor kit, there were 2 FP (different samples than TaqMan) and 2 FN.

TaqManamplificationwithautomaticextractionyieldedonefalsenegativeandnofalsepositives.

Real-timePCRsensitivityandspecificitywere98.7%and97.7%respectively.UseofTaqMan with automatic extraction yielded98.8%sensitivityand100%specificity.Kappaindiceswere0.95and0.97withrespecttothecomparator.

Forsmear-negativesamples,sensitivitydeclinedto80%andspecificityremainedat97%fortheTaqMankit.

For the 19 non-respiratory specimens, all three techniques tested recorded one false negative.

Conclusions

TaqMan MTB kit is a real-time PCR method offering the same reliability as the Amplicor MTB kit. TaqMan MTB kit ap-. TaqMan MTB kit ap-TaqMan MTB kit ap-peared to display good sensitivity using non-respiratory specimens.

It cuts down time-to-results from 6 to 2.5 hours

Automatic extraction reduced the number of false positives and considerably shortened handling time.

142 ESM 2009

pp-70

QuaNTifErON-Tb gOlD aSSay (QfT) aND TubErCuliNE SKiN TEST (TST) CliNiCal pErfOrmaNCE fOr ThE DiagNOSiS Of aCTiVE TubErCulOSiS

Karabela Simona, Papaventsis Dimitrios, Nikolaou Stavroula, Konstantinidou Efthimia, Sainti Asimina, Ioannidis Panayotis, KanavakiSofiaNational Reference Center for Mycobacteria, “Sotiria” Hospital, Athens, Greece

Objective

The purpose of this study was to evaluate and compare Quantiferon-TB Gold In-Tube (QFT, Cellestis, Australia) and the tuberculine skin test (TST) in patients with active TB, with and without previous BCG vaccination.

methods

Patients with symptoms compatible with active TB were included. The TST was performed according to the Mantoux method and the QFT assay according to the manufacturer’s instructions. The cut-off value for a positive result was ≥0.35 IU/ml interferon-gamma (IFN-γ).Sensitivity,specificity,positiveandnegativepredictivevalueswerecalculatedandcom-pared for QFT and TST tests. Agreement between QFT and TST was assessed by the kappa (κ) coefficient.

results

A total of 296 patients were enrolled in the study. One hundred eighty-nine had a record regarding BCG vaccination. Forty-four(23%)ofthe189patientshadbeenvaccinated.Intotal,thesensitivityandspecificityofQFT,excludingthosewith indeterminate results, was 79% (52/66; 95% CI: 66-88%) and 66% (153/167; 95% CI: 58-73%), respectively. The sensi-58-73%), respectively. The sensi--73%), respectively. The sensi-73%), respectively. The sensi-%), respectively. The sensi-tivityandspecificityofTSTwas72%(46/64;95%CI:58-83%) and 67% (156/232; 95% CI: 59-74%), respectively. The overall concordance between the QFT and TST tests was 70.2%, with a kappa value of 0.46 (95% CI: 0.289-0.524). In the BCG-vaccinated subgroup, agreement between the two assays was 66%, with a kappa value of 0.350 (95% CI: 0.103-0.597). The difference with the non-vaccinated subgroup (κ=0.463; 95% CI: 0.303-0.623) was considered to be not quite statistically significant(p>0.05).InitialTSTpositivescreeningfollowedbyaQFTpositiveresultwasfoundtohavegreatersensitivityandspecificityinthenon-vaccinated[sensitivity=79%(95%CI: 59-92%);specificity=81%(95%CI:71-88%)] compared to the BCC-vaccinated subgroup [sensitivity=67% (95% CI: 30-92%);specificity=75%(95%CI:57-89%)].

Conclusion

ThisstudyconfirmedpreviousreportsthatQFTassayhashighersensitivityfordetectingactiveTBcomparedtoTST.An overall moderate agreement between TST and QFT was found. The difference in agreement between non-vaccinated andBCG-vaccinatedsubgroupscouldbeattributedtoTSTinfluencebyvaccination.InpatientswithactiveTBandnoBCG-vaccination history, TST screening followed by subsequent QFT testing proved to present the highest sensitivity andspecificityforTBdiagnosis.Largerprospectivestudiesareneededtoconfirmourresults.


pp-71

CliNiCal pErfOrmaNCE Of QuaNTifErON-Tb gOlD aSSay (QfT) fOr ThE DiagNOSiS Of laTENT TubErCulOSiS iN DiffErENT paTiENT grOupS

Karabela Simona, Papaventsis Dimitrios, Nikolaou Stavroula, Konstantinidou Efthimia, Sainti Asimina, Ioannidis Panayotis, KanavakiSofiaNational Reference Center for Mycobacteria, “Sotiria” Hospital, Athens, Greece

Objective

The purpose of this study was to evaluate the performance and usefulness of Quantiferon-TB Gold In-Tube (QFT, Cellestis, Australia) in the diagnosis of latent tuberculosis and to compare it with the tuberculin skin test (TST).

methods

A cohort of 395 high risk adults was prospectively evaluated. Study groups consisted of 139 neoplastic patients, 98 with autoimmune diseases (SLE, rheumatoid and psoriatic arthritis), 20 immunossupressed (e.g. HIV, hemodialysis) and 26 Intensive Care Unit (ICU) patients, and 112 patients with no underlying disease. The TST was performed according to the Mantoux method and the QFT assay according to the manufacturer’s instructions. The cut-off value for a positive result was ≥0.35 IU/ml interferon-gamma (IFN-γ). QFT and TST were simultaneously performed in 297 patients.

results

Overall, QFT gave a positive result in 72/395 (18.22%) patients. Among the different risk groups, ICU and immuno-suppressed patients represented the highest positive rates (19% and 20%, respectively). Indeterminate results (7% on the whole) were more often seen in ICU (34.6%), neoplastic (7.2%) and in patients with autoimmune disease (7.2%). Indeterminate results represented <1% in patients with no underlying disease. Strength of agreement between GFT and TST results was poor (agreement=55.44%, kappa=0.171; 95% CI: [0.064-0.278]).

Conclusion

QFT is a very usefully method in TB diagnosis because in contrast to TST, it reduces over diagnosis of latent TB in previ-ously BCG vaccinated, distinguishing truly tuberculosis cases from BCG vaccinated individuals and/or non-tuberculous infections. As a consequence, QFT provides valuable information for therapeutical decision-making.

144 ESM 2009

pp-72

TubErCulOSiS DiagNOSiS by QuaNTifErON Tb gOlD aSSay iN arEaS WiTh DiffErENCES iN Tb iNCiDENCE

Nikolaou Stavroula, Karabela Simona, Papaventsis Dimitrios, Sainti Asimina, Konstantinidou Efthimia, Ioannidis Panayotis, KanavakiSofiaNational Reference Center for Mycobacteria, “Sotiria” Hospital, Athens, Greece

Objective

Evaluation of Quantiferon TB Gold in Tube method (QFT) for the latent TB diagnosis in patients originated from countries with high (group A) and low (group B) incidence of TB.

material

948 whole blood samples from 153 (16,15) individuals belonging to group A and 795 (83,9%) to group B.

method

Performance of Quantiferon TB Gold in Tube (Cellestis, Australia) method according to the manufacturers’ instructions.

results

QFT positive results was detected in 93/153(60.8%) individuals of group A and 273/795 (29,35) of group B (p<0,0001). Clinical information for previous BCG vaccination was available in 351 cases: 65 of group A and 286 of group B, where QFTconfirmedlatentTBin36/65(50,8%)and46/286(16,1%)ofgroupB(p<0,0001).Asitwasexpected,TuberculinSkinTest (TST) was positive in all 351 cases with previous BCG vaccination.

Conclusion

QFT is a highly diagnostic and useful method, especially in patients originated from areas with high incidence of TB. In contrast to widely used TST, it reduces overdiagnosis of latent TB in previously BCG vaccinated.


pp-73

QuaNTifErON® -Tb gOlD iN-TubE TEST uSED iN praguE paTiENTS liSTED iN ThE NaTiONal TubErCulOSiS rEgiSTEr

Havelkova, Marta 1, Bartu, Vaclava 2, Kubin, Milan 3

1 - National Institute of Public Health – NRL for mycobacteria2 - Charles University , Faculty of Medicine, Faculty Thomayer Hospital3 - Prague Hygiene Institute

In 2006-2007, 65 (29.0%) of 224 patients registered in the A15, A16 and A18/19 categories (58.5%, 32.3% and 9.2% of all patients, respectively) were assessed using both the QuantiFERON – TB Gold In-Tube (QFT) method and the tuberculin skin test (TST) with 2 TU PPD.

Afterstimulationwithtuberculosis-specificantigens,alowlevelofinterferon-gamma(IFG)production(<0.35IU)wasfoundin18(27.7%)patientsandmoderateorhighlevels(>0.35IU)intheremaining47(72.3%)patients.Afterincuba-tionwithanon-specificmitogen,alowlevelofIFGproductionwasrecoveredin13(20%)individuals,withmoderateorhigh levels being found in the remaining 52 (80%) patients.

TheTSTrevealedlowlevelsofskininfiltratereaction(0-5mm)in25patients(39.1%)andmoderateorhighlevelsofskinreaction(range6->30mm)intheremaining39individuals(60.9%).

The high proportion of low reagent levels in both the QFT and tuberculin skin tests can be explained by a high number of older patients, comorbidity with malignant tumours, diabetes or general fatigue in pre-terminal patients, and the immune systéminsufficiencyrelatedtothesefactors.

This presentation was fully supported by project KAN 200520702 of the Grant Agency of Czech Academy of Sciences (GAAV qR).C^

146 ESM 2009

pp-74

praCTiCal EXpEriENCE Of uSiNg a DNa amplifiCaTiON aSSay fOr rapiD DETECTiON Of MyCoBaCTeriuM TuBerCuloSiS COmplEX iN rESpiraTOry SpECimENS

J. Cacho1, A. García-Cañas1, A. González Torralba1, I. Cano2, A. Pérez Meixeira3, A. Ramos Martos2, and M. Sánchez-Concheiro1

1 - Servicio de Microbiología, Hospital Universitario de Getafe, Madrid2 - Servicio de Neumología, Hospital Universitario de Getafe, Madrid3 - Servicio de Salud Pública, Comunidad de Madrid, Madrid.

Objectives

ToevaluateexperienceinaclinicalmicrobiologylaboratoryofusingaDNAamplificationassayforroutinedetectionof Mycobacterium tuberculosis complex (MTC) performed once weekly, and to compare this method with microscopy and culture.

methods

A total of 507 respiratory specimens from 419 patients were screened for tuberculosis (TB). Smear examinations, culture and polymerase chain reaction (PCR) were performed on each sample. Specimens were processed according to standard laboratory protocols. All samples were processed exactly as described in Cobas Amplicor MTB Methods Manual (Roche Molecular Systems, USA). This method was performed once per week.

The following two groups of samples were considered to be true positive: (1) samples which were culture positive for MTC; and (2) all samples which were culture negative for MTC but positive to PCR, provided that one or more of the following criteria were met: (i) the samples originated from a patient whose other samples were culture positive; (ii) the patient’sclinicalhistoryprovidedevidenceofTBsufficienttowarrantinitiatingtreatmentforTB.

results

Inhibition of PCR was seen in 15 (2.9%) specimens. A total of 37 (7.3%) samples were considered to be true positive: 33 samples grew MTC in culture and 4 were culture negative for MTC but positive for PCR and met the criteria as de-scribed previously. TheoverallsensitivityandspecificityofPCRascomparedtotruepositivesampleswas83.8%(31/37)and 99.1% (466/470), respectively; that of culture 89.2% (33/37) and 100% (470/470), respectively; and that of direct microscopy 64.9% (24/37) and 99.8% (469/470), respectively.

The average time to reporting for true positive samples was 6 days for positive PCR and 11.4 days for positive culture. Of the smear-positive, true positive samples, 91.7% (22/24) were PCR positive. Of the smear-negative, true positive samples, 69.2%(9/13)werePCRpositive.The9samplesclassifiedastruepositiveandsmearnegativewerefrom9differentpa-tients.Treatmentwasstartedearlierin44.5%ofthesepatientsbecausepositivePCRfindingswereobtained.Theaveragetimetoreportingwas5.4daysforpositivePCRfindingsand17.4daysforpositiveculturefindings.

Conclusion

MTCinfectionwasconfirmedforPCRin91.7%ofsmear-positivespecimens. Although PCR was performed once weekly, treatment in 44.5% of patients was initiated earlier because of positive PCR results from smear-negative samples.


pp-75

rEal-TimE pOlymEraSE ChaiN rEaCTiON fOr ThE DirECT DETECTiON Of myCObaCTErium TubErCulOSiS iN CliNiCal SpECimENS Karen, MorganJoint Clinical Research Centre, Kampala, Uganda

introduction

The resurgence of tuberculosis is a leading cause of death worldwide. Since M. tuberculosis, is slow growing, methods of diagnostic testing based on culture are delayed. This study describes the development of a real-time polymerase chain reaction (RT-PCR) assay and subsequent clinical testing.

methods

Patients were recruited from routine TB suspects in the Monterey County Public Health laboratory, CA, USA. Initially, using Bactec MGIT 960 cultures as the gold standard, 231 respiratory specimens composed of 76 specimens from cul-ture-confirmedtuberculosiscasesand155culturenegativespecimenswereanalyzedbyRT-PCR.Overthenext2years206respiratorypatientspecimensweretestedfrom81flurochromesmearAFBpositiveand125negativesputa.DNAextractionwasperformeddirectlyfrompatientspecimensbythemodifiedQiagenmini-Ampkit(Valencia,CA).TheRT-PCR was performed on the Roche LightCycler instrument (Mannheim, Germany). The assay was designed to target the ITS region of the 16S rRNA gene of M. tuberculosis using Taqman hybridization probes for detection of amplicons.

results

ThedevelopedRT-PCRassayyieldedresultsinonedayandachievedasensitivityof85.5%andspecificityof100%.InsubsequentclinicaluseoverthetwoyearperiodtheRT-PCRassayachievedasensitivityof92.3%andspecificityof100%indirectdetectionofMTBfrom206respiratorypatientspecimens,while incontrastfluorochromesmearsachievedonlyasensitivityof60.5%%fornon-specificAFBdetectioninthesamepopulation.TheRT-PCRassaydetectedMTBin64.7%ofthesmearnegativebutcultureconfirmedpatientspecimens.TheRT-PCRassaycosts$14pertest,inexpensivecompared to commercial MTB assays ($60).

Conclusion

RT-PCR methodolgy can be used to detect MTB directly in patient specimens within eight hours, without waiting for culturegrowth.ThisrapidlyincreasesandenhancesspecificdetectionandtreatmentofMTB.

148 ESM 2009

pp-76

ThE uTiliTy Of mOlECular TESTiNg iN rOuTiNE myCObaCTEriOlOgy DiagNOSiS

Fanourios Kontos, Loukia Zerva. Clinical Microbiology Laboratory, Medical School of Athens, “Attikon” University Hospital, Athens, Greece.

Objective

Molecular methods increasingly replace phenotypic tests in Mycobacteriology. This study describes a “molecular” strat-egy that was developed for routine testing of clinical samples and isolates with the goal of shortening turnaround time (TAT) and providing accurate results.

methods

All samples submitted for mycobacterial cultures (from 12/2006 to 3/2009) were processed and cultured by standard methodology.Thefirstsmear(+)specimenofanypatientandeveryfirst(+)cultureweretestedbyanin-houseIS6110-PCR in order to differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) (TAT 4 hours). IS6110-PCR (+) specimens or cultures were tested by Genotype MTBDRplus (Hain-LIFESCIENCE) forMTBCspeciesconfirmationanddetectionofIsoniazidandRifampicinresistance(TAT2days).Subsequentculture(+) specimens from the same patient were tested by Accuprobe MTBC (Biomerieux) (TAT 2 hours). The susceptibility testing of MTBC isolates was performed by MGIT960 (Becton Dickinson). If a (-) result was obtained by the IS6110-PCR,identificationofisolatesproceededusingbothGenotypeΜycobacterium CM and AS (Hain-LIFESCIENCE) (TAT twodays).ForNTMspeciesconfirmationaPCR-RFLPanalysisofthehsp65genewasapplied(TAT3days);additionally,sequencingofthe16SrRNAgene(TAT5days)representedthereferenceidentificationmethodforselectedisolates.

results

Out of 4.000 specimens, 160 were culture positive (4%) including 85 MTBC positives (63.5% acid fast stain [AFS] posi-tive) and 75 NTM positives (26.7% AFS positive). Five samples contained more than one NTM species, which were iden-tifiedonlybymoleculartesting.Therewascompleteagreementbetweenallmolecularidentificationmethods;however16S rRNA sequencing was more informative (examples: M. fortuitum and M. lentiflavum, M. celatum). All MTBC isolates were Rifampicin susceptible, two were high- and one low-level Isoniazid resistant by MGIT960; only the latter was not recognized by Genotype MTBDRplus.

Conclusions

Focusingonmolecularmethodologyresultedinfastandaccuratefinalreporting.ThelowincidenceofMTBCpositivityamongtestedspecimens(2,1%)justifiedthedecisionnottouseindiscriminatelyadirectmoleculartestfortuberculosisdiagnosis. The frequent occurrence of NTM (48,5% of all culture positive samples) necessitates direct molecular testing of all AFS (+) specimens.


pp-77

DETECTiON Of MyCoBaCTeriuM TuBerCuloSiS DNa iN fOrmaliN-fiXED, paraffiN-EmbEDDED TiSSuE SpECimENS by SpOligOTypiNg: appliCaTiON TO hiSTOpaThOlOgiCal DiagNOSiS.

Salas S1, Hernández J1, Ojeda P2, Awad C2, de la Hoz F3, Murcia MI1.1 - Departamento de Microbiología, Facultad de Medicina. Universidad Nacional de Colombia. Bogotá, Colombia2 - Hospital Santa Clara E.S.E., Bogotá, Colombia3 - Departamento de Salud Pública, Facultad de Medicina. Universidad Nacional de Colombia. Bogotá, Colombia

In Colombia, Tuberculosis (TB) remains as a public health problem with an incidence of 25 per 100 000 population while extra-pulmonary TB has increased. The Spoligotyping method has demonstrated to be a good tool to improve the TB extra-pulmonary diagnosis.

purpose of study

To identify DNA of Mycobacterium tuberculosis fromFormalin-fixedparaffinembeddedTissuesspecimens(FFPET).

methods

We examined 160 FFPET storaged for 13 years and with a suspected diagnosis of Tuberculosis infection according to histopatologicalanalysis.Spoligotypingwascarriedoutaspreviouslydescribedwithsomemodifications.DNAextractionwithCHELEXwasusedandhumanDNAwasnegativecontrol.TheSpoligotypefilmswerescannedandclassifiedbyusingGeneToolssoftwarefollowedbymanualeditingandconfirmation.StatisticalanalysiswasperformedusingSPSSV.15.0.

results

Of the 160 samples analyzed by Spoligotyping, 78 (48.8%) cases showed absence of signal at the spot 33 to 36 compat-ible with M. tuberculosis. Nevertheless SPOL-DB4 was made with DNA obtained from cultures, we in tented to compare our patterns obtained and we founded only 4 samples compatible with pattern previously described LAM (2), U (1) and Haarlem lineage (1). Incomplete patterns were observed in 34 (21.2%) and no patterns were obtained in 48 (30.0%). Negativecontrolsdidnotyieldanyspoligopatterns.ExactFisher’sstatisticshowedasignificantdifferencebetweenthepositivityofSpoligotypingandstoragetime(P<0.05)whileforthekindoftissuefoundnodifferences(P>0.05).

Conclusions

In48.8%casesweconfirmedthediagnosisofM. tuberculosis. The spoligotyping method is a tool that provides some information about FFPET, the results of this can be affected by storage time. In order to obtain better results is necessary to examine the samples immediately after her obtention.

150 ESM 2009

pp-78

pOTT´S DiSEaSE: aN aNCiENT DiSEaSE? Cardoso, Sara, Coelho, Rui, Paulo, Cristiana, Abreu, Candida, Silva, Susana, Gomes, Helena, Sarmento, AntónioHospital S.João

introduction

Skeletaltuberculosisisararediseaseindevelopedcountriesalthoughit´sstillasignificantcauseofdiseaseinPortugalduetothehighprevalenceoftuberculosisandaninsidiousandnon-specificpresentationofthisformofdisease.Clinicalreport: We report 3 cases of Pott´s disease.

Case 1: Male, 62y. History of multiple sclerosis under imunomodulator and leg trauma with disability. He went several timestotheemergencyroomduetointenselombalgywithouttraumaanddischargedwithnon-steroidalanti-inflam-matory (NSAIs) medication, without relief. D12-L1 fracture was diagnosed and the patient was discharged under con-servative treatment. Fifteen days later, he appeared with paraparesia, fever, asthenia and weight loss and was submitted to surgery. Histology of the bone fragments revealed acid fast bacilli. The direct and cultural exam of gastric lavage (GL) were positive for M.tuberculosis complex (MTC). Thorax X-ray (XR) was normal.

Case 2: Female, 88y. History of previous ribs and femur trauma fractures. She had complaints of lombalgy and progressive paraparesia during the last year and was chronically medicated with NSAIs. Three months before admission anorexy and weight loss appeared. She had no fever. The magnetic ressonance revealed D6-D7 vertebral body fracture with cavitation and medular compression. She was submitted to surgery. The bone cultural exam was positive to MTC. The thorax XR was suggestive of pulmonary involvement but the GL mycobacteriology exam was negative.

Case 3: A 77y woman with past history of pulmonary tuberculosis and recent history of dorsal trauma with D12 frac-ture, treated conservatively. Later she was admitted with paraplegia and submitted to surgery. The bone culture, broncho-alveolarlavagecultureandDNA(polymerasechainreaction)werepositiveforMTC.Thecerebrospinalfluidhad24cells/uL, high ADA (123U/L), proteins (10,20g/l) and low glucose (0,14g/l), but DNA for MTC and culture were negative. All three patients were treated surgically and medically with rifampin, isoniazid, ethambutol and pyrazinamide. All of them had come with dorsal or lombar pain and progressed to paraparesia and plegia, which showed some improvement with antibiotics and physiotherapy.

Conclusion

Skeletal tuberculosis is a silent re-emergent disease, for which our doctors are not aware, leading to delayed diagnosis and causing severe neurological damage like Pott´s paraplegia as it was observed in our patients.


pp-79

OSSEOuS TubErCulOSiS aT agE Of 9 mONThS Loureiro, Carla 1, Matos, Gabriel 1, Balacó, Inês 1, Mota, Marta 2, Nogueira, Célia 2, Lemos, Sónia 1, Rocha, Graça 1

1. Pediatric Department/H. Pediatrico, CHC2. Mycrobiology Laboratory, Coimbra Medicine Faculty

background and aims

Osseous tuberculosis is rare, mainly as a primary disease in a previously healthy infant. Several mycobacteria may be involved such as M. tuberculosis, M. bovis and M. avium. When there is no association with a primary pulmonary disease in a child vaccinated with Calmet-Guérin Bacillus, M. bovis may be the causal agent.

Case report

A previously healthy 11 month-old infant BCG vaccinated at birth was admitted with a 2 month evolution of a right elbow tumefaction. The X-ray presented destruction of the trochea, severe periostic reaction and soft tissue tumefaction, and theecographyafluid-filledcavity.MRIsuggestedneuroblastomaorEwingsarcoma.Sedimentationrateandspecificeno-lasewereelevated.Onsurgeryawhitishsoftmassassociatedwithnecroticfluidcompressingthecubitalnervewasfound. The histological examination revealed characteristic features of caseum and allowed the diagnosis of osseous tubercu-losis.MycobacteriumtuberculosiscomplexwasidentifiedbyReal-TimePCRinosseousbiopsy.Gastricfluidandurinewere negative. Our patient recovered after surgical debridement and combination drug therapy. He developed a local cutaneousfistula(noagentidentifiedonfluidculture)andposteriorsofttissuecalcification.At22monthsageheremainswith no other relevant infections.

Conclusions

Skeletal tuberculosis with extravertebral location is rare. Tuberculous osteomyelites of the limb bones requires a high index of clinical suspicion along with radiological and histopathological investigation in order to establish the diagnosis. Tuberculosis is still an important differential diagnosis in unusual bone conditions

152 ESM 2009

pp-80

DiffErENCES iN DirECT aNTiTumOral CapaCiTy amONg ThE VariOuS MyCoBaCTeriuM BoviS bCg SubSTraiNS

SP Secanella, M Luquin, E JuliánDept. Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona)

The administration of Mycobacterium bovisBacillusCalmette-Guérin(BCG)isthefirsttreatmentoptionforavoidingtherecurrence of bladder cancer. Various BCG substrains are used. These substrains differ genetically and display differential antigenicdeterminants,whichithasbeensuggestedmayinfluencetheefficacyofBCGasvaccineintuberculosisstudies.Intuberculosis,evolutionaryearlystrainsaremoreefficaciousthanarethemoreattenuatedevolutionarylatestrains.Theimpact of these differences on BCG antitumoral capacity in bladder-cancer cell lines has not been addressed.

We aimed to compare the direct antitumoral activity of different BCG substrains by inhibiting cell proliferation and by triggering the production of cytokines in bladder-cancer cell lines.

T24, J82 and RT4 human bladder-cancer cell lines were cultured with different doses of BCGs. We tested three evolutio-human bladder-cancer cell lines were cultured with different doses of BCGs. We tested three evolutio-uman bladder-cancer cell lines were cultured with different doses of BCGs. We tested three evolutio-naryearlyBCGsubstrains,Japan,MoreauandRussia,andfiveevolutionarylatestrains,Connaught,Danish,Glaxo,Phipps,and Tice. Inhibition of cell proliferation was assessed by using a colorimetric assay at different time points; the production of interleukin (IL)-6 and IL-8 was measured in cell culture supernatants using enzyme-linked immunosorbent assay.

Among the different BCG substrains, in the case of T24 and J82 cell lines, Connaught and Russia induced both the high-est inhibition of proliferation and cytokine production. In contrast, Glaxo and Phipps (for the T24 cell line) and Glaxo andTice(fortheJ82)weretheleastefficaciousbothinreducingcellviabilityandininducingcytokineproduction.Theremaining BCGs behaved differently depending on the cell line.

Finally, for the RT4 cell line, all BCG strains inhibit cell proliferation at the same level, except for Danish and Glaxo, whichwereseentobelessefficacious.Asregardscytokineproduction,IL-6productionwasnotdetectedinanyculture,whereas low levels of IL-8 production were observed, the lowest being for Danish and Glaxo cultures.

TheresultsshowedthatConnaughtandRussiaarethemostefficientBCGsandthatGlaxoistheleasteffective,bothin the inhibition of tumoral-cell proliferation and the induction of cytokine production. No correlation was observed betweenBCGantitumoralefficacyandgenotypicevolutionaryclassification.


pp-81

rOlE Of TNf-a gENE pOlymOrphiSmS iN hOST gENETiC SuSCEpTibiliTy TO pulmONary TubErCulOSiS

Anoosheh, Saber 1, Farnia, Parissa 1, Noruzi, Jamileh 2, Kargar, Mohammad 3, Kazempour, Mehdi 1, Seif, Shima 1, Masjedi, Mohammad Reza 4, Velayati, Ali Akbar 4

1 - Mycobacteriology Research Center, NRITLD, Shahid Beheshti University (M.C)2 - Microbiology Department, Iran University of Medical Science3 - Microbiology Department, Jahrom Azad University4 - National Research Institute of Tuberculosis and Lung Disease, Shahid Beheshti University (M.C)

preface and goal

Tuberculosis is one of most common infectious diseases and it causes death of more than 3 million people a year, world-wide. It caused by Mycobacterium tuberculosis and approximately one - third of world population are infected with this bacteria, but only 5 - 10 % of them develop active TB. Therefore, individual differences in susceptibility to TB are expected. These differences might be due to host factors especially genetic diversity between populations. TNF-aasapro-inflam-matorycytokine,playakeyroleinhostdefenseagainsttuberculosis.Presenceofmutationinthisgenecaninfluencetheeffectiveness, performance and capability of immune Responses against infection. The Aim of this study was to investigate the frequency of TNF-a alleles and relationship between susceptibility to TB and TNF-a gene variations.


A case-control study was conducted and 65 healthy controls and 65 TB patients were enrolled. Genotype of TNF-238, TNF -244, TNF-308, TNF -857 and TNF-863 were distinguished using by PCR-RFLP method. The results were analyzed by SPSS v.16, Fisher exact and Hardy-Weinberg tests.

results

Obtained results showed that, TNF-308, TNF -857 and TNF-863 were as a high frequency mutation regions in population levels, andalsowefoundasignificantdifferencesatTNF-308 and TNF -857 between two group of controls and patients ( P-value < 0.05 ).

Conclusion

presence of mutation in TNF-308 and TNF -857 regions probably increases host susceptibility to mycobacterial infection and Genotyping of these regions can be used for screening of high risk persons. Also according to high frequency distribution of mutations in TNF -857 and TNF-863 regions, further studies on association of these regions is suggested.

Key words

Tuberculosis, Genetic Susceptibility, Cytokines, Gene Polymorphism

154 ESM 2009

pp-82

myCOlaCTONE iNTErfErES WiTh ThE prOTECTiVE ifN-γ-DEpENDENT aCTiVaTiON Of maCrOphagES DuriNg iNfECTiON WiTh MyCoBaCTeriuM ulCeranS

Egídio Torrado1; Alexandra G. Fraga1; Elsa Logarinho1; Teresa G. Martins1; Jenny A. Carmona1; José B. Gama1; Maria A. Carvalho2; Fernanda Proença2; António G. Castro1; Jorge Pedrosa1

1 - Life and Health Sciences Research Institute (ICVS). School of Health Sciences, University of Minho. Braga, Portugal2 - Chemistry Research Center. School of Sciences. University of Minho. Braga, Portugal

Mycobacterium ulcerans is the etiological agent of a necrotizing cutaneous disease, known as Buruli ulcer. The pathology caused by this pathogen is associated with the production of the lipidic exotoxin mycolactone. Following our recent demonstration of an intramacrophage growth phase for M. ulcerans, we investigated the biological relevance of interfer-on-gamma (IFN-γ), as well as the mechanisms activated by this cytokine in M. ulcerans-infected macrophages.

We used three different M. ulcerans strains selected based on their virulence for mice and the type of mycolactone produced: the low virulent mycolactone-negative strain 5114; the intermediate virulent, mycolactone C-producing strain 94-1327; and the highly virulent, mycolactone D-producing strain 98-912.

IFN-γ-deficientmiceshowedanincreasedsusceptibilitytoinfectiononlywithstrains5114and94-1327,suggestingthatthis cytokine plays a protective role in infections with the low and intermediate virulent strains of M. ulcerans, but not with the highly virulent strain. In line with this, IFN-γ-activated mouse primary bone marrow-derived macrophages con-trolled the proliferation of the low virulent and the intermediate virulent strains, the latter only at low multiplicities of infection. The effector mechanisms induced by IFN-γ in infected macrophages leading to M. ulcerans growth restriction involvedbothphagosomematurationandacidification,aswellasincreasednitricoxideproduction.Inagreement,theadditionofmycolactoneD,purifiedfromculturesofthehighlyvirulentstrain,ledtoadose-dependentinhibitionofpha-gosome maturation and nitric oxide production in IFN-γ-activated cultured-macrophages infected with the mycolactone-negative strain, resulting in an increased bacterial burden.

Our results suggest that the protection mediated by IFN-γ during the intramacrophage phase of M. ulcerans infection depends on the type and amount of mycolactone produced.


pp-83

VirulENCE, immuNOgENiCiTy aND prOTECTiON iNDuCED by ´MyCoBaCTeriuM HaBana´ STraiNS iN a muriNE mODEl Of pulmONary TubErCulOSiS. Montoro, Ernesto 1; Valdés, Iliana 1; Aguilar, Diana 2; Orozco, Hector 2; Hernández-Pando, Rogelio 2

1. Institute of Tropical Medicine “Pedro Kourí”(IPK), Havana, Cuba2. National Institute of Medical Science and Nutrition ¨Salvador Zubirán¨. Mexico DF, Mexico

Mycobacterium habana’wasfirstisolatedinCubabyValdivia, in1971.Later,wasdemonstrateditsprotectioncapacityagainst M. tuberculosis and other mycobacteria. We studied the virulence, immunogenicity and protection of 3 strains of ‘M. habana’ usingBalb/cmice.Thefirstexperimentwasdonetoknowthevirulencepotentialof‘M. habana’ using a progressive pulmonary TB model. In the 2nd assay mice were vaccinated with 3 doses of bacilli. The grade of immunoge-nicity was related with the induction of IFNγ by the stimulation of the main organs with antigens of M. tuberculosis. The best doses that induced immunogenicity were used in the 3rd experiment for animal vaccination. Two months later mice were challenged with M. tuberculosis H37Rv and Beijing genotype. All the animals infected with M. habana TMC-5135 and IPK-337 were alive until the end of the experiment. IPK-220 strain showed about 20% of death to the seven week post-infection.AllthestrainshadsignificativedifferenceswhenwecomparedwiththecontrolgroupinfectedwithH37Rvstrain. The values of the colony forming units (CFU) were in correspondence with the survival rate. The percentage of pneumonia was higher for ‘M. habana’IPK-220,showingfinalvaluessimilartomiceinfectedwithH37Rvstrain.Duetothe virulent behaviour of ’M. habana’ IPK-220 we discharged it for the coming assays. The most important results of the 2nd experiment were the statistical differences in the IFNγ production founded in groups vaccinated of ‘M. habana’ IPK-337 and TMC-5135 strains, respectively, with the BCG group. The lung CFU for these doses showed a decreasing tendencywithatotalsterilizationtothefinaloftheexperiment.Animalvaccinatedwith‘M.habana’strainsandchal-lenged with M. tuberculosis H37Rv had the highest survival. These results are in accordance with the low percentage of pneumoniawithbothstains.NeverthelessthisdifferenceswasnotstatisticalsignificativeincomparisonwithBCGgroup.With the Beijing challenge we observed differences between vaccination with TMC-5135 and BCG group. The lungs of the animals that received BCG and challenged with Beijing showed more than 70% of pneumonia and a lower granuloma area.TheCFUrevealslowerlungbacilliloadinmicevaccinatedwith‘M.habana’strains.Thefinalresultsdemonstratethepotential of ‘M. habana’ to protect against TB infection.

156 ESM 2009

pp-84

DENDriTiC CEllS DiffErENTially EXprESS il12-family CyTOKiNES afTEr iNfECTiON WiTh MyCoBaCTeriuM TuBerCuloSiS Or M. BoviS bCg

Margarida Saraiva, Carole Sousa, Jenny A. Carmona, Andrea Cruz, Jorge Pedrosa, A. Gil CastroLife and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal

IL-12 family is a group of heterodimeric cytokines, composed of 4 related cytokine members: IL-12, IL-23, IL-27 and IL-35. These cytokines, that share some functions and receptor components of IL-12, act on CD4+ T cells modulating the type of T helper and T regulatory responses, and initiating the development of the acquired T cell response to intracellular pathogens, such as Mycobacterium tuberculosis (MTb) or M. bovis BCG. Bone marrow derived dendritic cells (BMDC) originating from wild-type mice were exposed to live MTb strain H37Rv or to BCG for different periods of time. The kinetics of mRNA production of each monomer that form the cytokines of the IL-12 family (p19, p28, p35, p40, Ebstein-Barr-Virus-induced gene 3 (Ebi-3)) was determined. We show that MTb-stimulated BMDC were strong producers of p40, p35 and p19, whereas BMDC exposed to BCG expressed much lower levels of these cytokines. The main TLR involved in the recognition of MTb and BCG and activation of BMDC is TLR2, since in its absence the expression of the various monomerswasnearlyabrogated.AconsequenceofthisdifferentialactivationofBMDCwasreflectedonthedistincttype of T helper responses developed when MTb- or BCG-infected BMDC presented OVA peptide to TCR-transgenic CD4 T cells. MTb-infected BMDC were able to induce the development of both Th1 and Th17 responses, whereas BCG-infected BMDC induced Th17 responses. We are currently addressing the molecular mechanisms that differentiate the response of BMDC in the context of an MTb or a BCG infection. Understanding the details of DC activation by MTb or BCG and its consequences on the CD4+ T cell arm of the immune response will help to reveal important aspects to be improved for a better vaccination strategy.


pp-85

aNalySiS Of M. SMegMaTiS muTaNTS rESiSTaNT TO mS6 iNfECTiON

Simões, Marta Filipa; Jordão, Luísa; Teles, JMM; Couto, S; Moniz-Pereira, José; Pimentel, MadalenaCentro de Patogénese Molecular-Unidade dos Retrovirus e Infecções Associadas, Faculty of Pharmacy, University of Lisbon, Portugal

Mycobacteriophages represent excellent model systems for studying mycobacterial hosts. Ms6 is a temperate myco-bacteriophage that infects Mycobacterium smegmatis.ThefirststepofadsDNAphageinfectionisadsorptiontoahostreceptor followed by injection of DNA into the cytoplasm. Characterization of the phage resistant mutants represents acommongeneticstrategyfortheidentificationofmycobacterialgenesinvolvedinthesynthesisofparietalphagere-ceptors. The study of these genes is of highly importance, as phage receptors are also involved in the entry of others molecules, such as antibiotics, into the cell. In order to identify the Ms6 receptor we started to select from a mutant library obtained after a transposition event using the pCG79 system (Guilhot et al, 1994), M. smegmatis mutants to a Ms6 infection. DNA obtained from these mutants was submitted to an enzymatic restriction analysis, which allowed the selectionoffourmutantswithdifferentprofiles.

In order to understand which step of the phage infection was affected, adsorption and infection assays with DAPI stained phages were performed.

Wefounddifferentprofilesamongtheselectedmutantssuggestingthatdifferentstepsoftheinfectionareaffected.Withthese results, our goal is to identify the mycobacterial genes disrupted by the transposition event.

Identificationoftheaffectedgenesisanimportantachievementwhichmaycontributetothecharacterizationofthephage receptor.

Guilhot,C.,I.Otal,I.VanRompaey,C.Martín,andB.Gicquel.1994.Efficienttranspositioninmycobacteria:constructionof Mycobacterium smegmatis insertional mutant libraries. J. Bacteriol. 176: 535-539.

158 ESM 2009

pp-86

humOral rESpONSE iN TubErCulOuS paTiENTS agaiNST ThE myCOliC aCiDS Of MyCoBaCTeriuM TuBerCuloSiS

Julián, Esther Gómez 1; Rodríguez-Güell, E 1; del Val-Romero, B 2; Clivillé, R 2; Cañete, C 3; Navarro, A 3; de Gispert, FX 3; Luquin, M 1; Alonso, C 2

1 - Dept. Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona)2 - Dept. Microbiologia, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona)3 - Unitat de Respiratori, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona)

Although numerous serological tests have been developed for TB serodiagnosis, none of these have shown adequate levels of accuracy; in consequence, they have not been widely implemented.

Diverse mycobacterial antigens have been evaluated in these tests, among which are cell-wall glycolipidic antigens such ascordfactor(CF).Thismoleculeismadeupofatrehaloseresiduesterifiedtotwomycolicacids(MAs);ithasbeenestablished that MAs are the epitopes for the anti-CF antibodies recognition. Furthermore, a possible cross-reaction between anti-MAs antibodies and cholesterol (COL) in TB patients has recently been published.

The purpose of this study was to detect and compare the presence of IgG, IgM and IgA antibodies against MAs with regard to the presence of anti-CF and anti-COL antibodies in TB patients.

Toaddressthisquestion,MAsandCFwerepurifiedfromM. tuberculosis H37Rv (ATCC 27294T), and commercially avail-able COL was used. An ELISA was developed to detect IgG, IgM and IgA antibodies to MAs and COL, and an ELISA previously developed for CF was performed.

The presence of IgG, IgM and IgA anti-MAs, CF and COL in the sera of 31 HIV-negative TB patients at the time of TB diagnosis and throughout anti-TB prophylaxis, 20 PPD-positive donors, 20 PPD-negative donors and 20 patients affected by other pneumonias were determined.

No antibodies to MAs were determined in any sera of the groups studied, whether for TB patients at the onset or during prophylaxis, or in control groups.

Anti-CF IgG and IgA antibodies in sera from TB patients were detected, following a downward kinetic from the beginning totheendoftheprophylaxis.Testsensitivityandtestspecificitywere41%and78%,respectively,forIgGdetection,and19% and 95%, respectively, for IgA detection. Additionally, anti-CF IgM antibodies were detected in all the groups.

In contrast, neither IgG nor IgA anti-COL antibodies were detected in any of the groups; however, IgM anti-COL antibod-ies were also detected in sera from all the groups studied.

Each serum showing anti-CF and COL antibodies was individually analysed, and their titres against each antigen were compared.Analysisshowedthatresponseprofilesweredifferentagainstthetwoantigens.Moreover,giventhattheserareacting against COL or/and CF did not react against MAs, it is therefore possible to rule out a cross-reaction between MAs and COL.

The overall results invalidate MAs as possible antigens for the serodiagnosis of TB.


pp-87

MyCoBaCTeriuM TuBerCuloSiS

Arley Gómez LópezInfectious Diseases and Tropical Medicine Unit; Universidad del Rosario

TlyA protein has a controversial function as a virulence factor on Mycobacterium tuberculosis (Mtb). Updated studies have not demonstrated a possible hemolytic activity conferred by TlyA and contrary to recent evidence have suggested a function enzymatically similar to RNA methyltransferase at ribosomal level which confers antibiotic susceptibility to capreomycin and viomycin.

Our aim was to determine the In vitro hemolytic activity of MtbTlyAoverexpressedandpurifiedfromE. coli BL21-AI and to carry out an in silico phylogenetic and structural analysis.

Based on tlyA gene sequence (Rv1694) from MtbH37Rvspecificprimersweredesigned.Theamplificationproductwasligated on pEXP5-CT/TOPO vector (Invitrogen), transformed and overexpressed on E. coliBL-21-AI.Afterpurificationbyaffinitychromatography,TlyA-His6recombinantproteinwasanalyzedbySDS-PAGEunderdenaturingconditionswhichwas detected as 28 KDa single band. Recombinant protein as recognized by anti-His monoclonal antibody.

Protein structural characterization by circular dichroism was carried out. On the other hand, hemolytic activity assays usingTlyA-His6purifiedwerenegativeaswellasonbacteriallysates.

HemolyticassayswithTlyA-His6supplementedwithcalciumandmagnesiumwerenegativesuggestinglackofspecificrequirements for this activity.

By using bioinformatics tools, a ribosomal binding called S4 located between 5 and 68 residues and FtsJ among 62 and 247residueswereidentifiedonTlyA.Thesedomainshavebeendescribedonproteinsassociatedwithribosomaltrans-lationalmachinery.TheHidrophobicityprofilewasindisagreementwithapossibletransmembranehelixalthoughnonpolar amino acid composition suggesting that TlyA might not be membrane attachment. Nevertheless, three-dimensional model (Structural homology with 1L9K model) reveals a consensus structure with a common core, comprising a parallel β-sheet of six strands, sandwiched between two layers of a-helices corresponding to a RNA methyltransferase structure. Phylogenetic analyses showed that TlyA is highly conserved among mycobacteria species and it does not exhibit changes among Mtb complex strains. tlyA gene evolution might operate under purifying selection model. Additionally differences were observed among TlyA and bacterial pore forming proteins.

Thisevidencesupportsalinkbetweenribosomalmodificationstoposttranslationallevelandsuggestsafunctionalan-notation error of this family protein at GENBANK as well as missannotation on several genomes as Mtb genome. Studies on resistance mechanisms of Mtb mediated by ribosomal proteins might be useful for understanding new alternative therapeutic approaches for tuberculosis control applied to knowledge of second line antibiotics such as capreomycin.

Key words

TlyA, Mycobacterium tuberculosis, hemolysin, Structural modeling, RNA methyltransferase.

160 ESM 2009

pp-88

EValuaTiON Of ThE appliCabiliTy Of SErODiagNOSiS fOr TubErCulOSiS iN pOrTugal Carina Ferreira 1, Andrea Afonso 2, Raquel Duarte 3, Konstantin Lyashchenko 4, AnabelaSilva 5, Filomena Rodrigues 5, Anabela Miranda 5, Margarida Tavares 6, Cátia Caldas 6, FátimaValente 2, António Valente 2, Olga Vasconcelos 7, Joana Amado 7, Margarida Correia-Neves 1

1 - Life and Health Sciences Research Institute (ICVS), University of Minho, Braga, Portugal2 - Centro de Saúde de Bragança, Bragança, Portugal3 - Centro de Diagnóstico Pneumológico de Vila Nova de Gaia, Portugal4 - Chembio Diagnostic Systems, New York, USA5 - Centro de Tuberculose e Micobactérias, Instituto Nacional de Saúde Dr Ricardo Jorge, Delegação do Porto, Portugal6 - Serviço de Doenças Infecciosas, Hospital de São João, Porto, Portugal7 - Hospital Joaquim Urbano, Porto, Portugal

Diagnosis of tuberculosis requires laboratory techniques to complement clinical information. Among the different tech-niques in use, the only accurate diagnostic method is based on the search for Mycobacterium tuberculosis growth in cultures of human biological samples, usually sputum. However, this approach is long, lacks sensitivity for samples with low bacterial load, and is invasive in the case of non-pulmonary tuberculosis. In order to overcome these constraints, severalnovelmethodologicalapproachesareunderevaluation.Amongtheseisthedeterminationoftuberculosis-specificantibodies in the sera - serodiagnostic methods. Validation of serodiagnosis tests in any given country is mandatory, since sensitivityandspecificityvarydependingonfactorssuchasthecompositionandfrequencyofenvironmentalmycobac-teria, previous vaccination with BCG, prevalence of tuberculosis and other diseases, and host genetic background. We evaluatedthespecificityandsensitivityoftwoserodiagnosistests:TBSTAT-PAKII-animmunochromatographictestforthe detection of antibodies to Mycobacterium tuberculosis; and MAPIA - Multi-Antigen Print Immunoassay (both devel-opedatChembioDiagnostics,NY,USA).Thespecificityofthetestswasveryhigh,rangingfrom94to100%dependingon the test and control population analysed. The sensitivity was 37 and 54% for TB STAT-PAK II and MAPIA, respectively, anditincreasedduringthefirst3monthsoftreatment,forTBSTAT-PAKII.Interestingly,whenthesmearresultswerecombined with the ones from the TB STAT-PAK II, the sensitivity increased from 71 (just smear) to 79%. Thus, taking into considerationthegreatspecificityoftheTBSTAT-PAKII,itsuseforsmearnegativepatientscouldincreasetherateofdetection in early TB diagnostic.


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implEmENTaTiON Of ThE ThiN layEr agar (Tla) fOr ThE DiagNOSiS Of SmEar NEgaTiVE pulmONary TubErCulOSiS iN a high hiV prEValENCE SETTiNg Martin, Anandi 1, Munga Waweru, Peter 2, Babu Okatch, Fred 2, Amondi Ouma, Naureen 2, Bonte, Laurence 2, Palomino, Juan Carlos 1, Varaine, Francis 2, Portaels, Françoise 1

1 - Institute of Tropical Medicine, Antwerp, Belgium2 - Médecins Sans Frontières, Paris, France

Early diagnosis of smear-negative pulmonary tuberculosis in countries with high incidence of TB/HIV co-infection is crucial to limit the mortality and control the disease. The objective of this study was to evaluate the performance of a low-cost method, the Thin Layer Agar (TLA), for the diagnosis of smear-negative patients compared to the gold standard Löwenstein-Jensen method. TLA relies on microscopic detection of cording growth that is characteristic of M. tuber-culosis and is able to differentiate between M. tuberculosis and non-tuberculous mycobacteria. This prospective study was performed in Homa Bay district Hospital in Kenya. Out of 1584 smear-negative sputum samples, 212 were positive by LJ (13.5%) and 220 positive by TLA (14%). The sensitivity of LJ and TLA was 71% and 74 % respectively. With further improvement for decreasing the contamination rate in both methods, TLA could become an affordable method for the diagnosisofsmear-negativetuberculosisinresource-limitedsettingsallowingthesimultaneousdetectionandidentifica-tion of M. tuberculosis, within 2 weeks.

162 ESM 2009

pp-90

DirECT DETECTiON Of rifampiN rESiSTaNCE iN MyCoBaCTeriuM TuBerCuloSiS by ThE NiTraTE rEDuCTaSE aSSay appliED DirECTly iN SpuTum SamplES Regina Ferro e Silva 1, Maria de Lourdes Shikama 1, Gleize Villela 1, Daisy Nakamura Sato 1, CarmenMaria Saraiva Giampaglia 1, Maria Conceição Martins 1, Anandi Martin 2, Juan Carlos Palomino 2

1 - Instituto Adolfo Lutz (IAL), São Paulo, Brazil2 - Institut of Tropical Medicine, Antwerpen, Belgium

Resistance to rifampin (RIF) is an important predictor for the early diagnosis of MDRTB. Conventional methods for DST of M. tuberculosis require several weeks to give results.Thus, an alternative in vitro method which can detect drug susceptibility directly from sputum and presents quick results and low cost, will be very useful for tuberculosis diagnosis. The nitrate reductase assay uses the detection of nitrite as an indication of growth when used as a drug susceptibility test. Objective:to compare the nitrate reductase assay (NRA) with the proportion method (PM), considered as gold standard, to detect RIF resistance in M. tuberculosis directly from sputum samples. Method: the study was carried out by 4 regional laboratories from the state of São Paulo, Brazil. A total of 206 sputum samples tested smear positive from patients with pulmonary tuberculosis. The sputum was decontaminated by Petroff method and DST to RIF was carried out using the PM and the NRA. Nitrate Reductase Assay: Sputum samples, after decontamination were inoculated in one tube control (without drug) and one tube containing 40 µg/ml of rifampicin. Findings: the results of the DST obtained directly from sputum were: 6 samples resistant to RIF and 200 samples susceptible. The comparison between NRA and the traditional goldstandardmethodshowedagreementof100%.ThesensitivityandspecificityoftheNRAforrifampicinwas100%.Results were available in 10 days for 66 (34%) samples, 15 days for 102 (53%) samples and 20 days for 24 (13%) samples while the results of PM took 30 days to be available. Conclusions: the NRA proved to be a promising method for the screening of suspect MDRTB cases directly from sputum samples. The simplicity of the method, its low cost and celerity to give the results make it a good alternative method for laboratories in resource-poor settings.


pp-91

DirECT DETECTiON Of rifampiN rESiSTaNCE iN MyCoBaCTeriuM TuBerCuloSiS by ThE NiTraTE rEDuCTaSE aSSay appliED DirECTly iN SpuTum SamplES

Ferro Regina Silva 1, Shikama Maria de Lourdes 1, Villela Gleize 1, Sato Daisy Nakamura 1, Giampaglia Carmen Saraiva 1, Martins Maria Conceição 1, Martin Anandi 2,3, Palomino Juan Carlos 2, Telles Maria Alice da Silva 1

1 - Instituto Adolfo Lutz, São Paulo, Brazil2 - Institute of Tropical Medicine, Antwerp, Belgium3 - Médecins Sans Frontières, Paris, France

A cost-effective and rapid drug susceptibility testing (DST) method is required to guide TB treatment. Commercially available systems such as BACTEC MGIT 960 and MB/BacT are faster but demand costly equipment and supplies, there-fore, are not feasible in most resource-poor settings. Resistance to rifampin (RIF) is an important predictor for the early diagnosis of MDRTB. To compare the nitrate reductase assay (NRA) with the proportion method (PM) to detect RIF resistance in M. tuberculosis directly from sputum samples. The study was carried out by 4 regional laboratories from the state of São Paulo, Brazil. A total of 210 sputum samples tested smear positive from patients with pulmonary tubercu-losis. The sputum was decontaminated by Petroff method and DST to RIF was carried out using the PM and the NRA. The results of the DST obtained directly from sputum were: 6 samples resistant to RIF and 204 samples susceptible. No discordancewasobservedbetweenthetwomethods.ThesensitivityandspecificityoftheNRAwas100%.Resultswereavailable in 10 days for 75 (36%) samples, 15 days for 107 (51%) samples and 20 days for 28 (13%) samples. The results of PM took 30 days to be available. The NRA proved to be a promising method for the screening of suspect MDRTB cases directly from sputum samples. The simplicity of the method, its low cost and celerity to give the results make it a good alternative method for laboratories in resource-poor settings.

acknowledgements

This study was partially funded by INCO-Dev ICA4-CT-2001-10087.

164 ESM 2009

pp-92

mTbDrpluS aSSay iS a uSEful TOOl TO SCrEEN fOr mulTi-Drug rESiSTaNT TubErCulOSiS iN a NaTiONal SurVEy De Haas, Petra 1, Ramona Zenhorst 2, Pike Mwamba 2, Mweemba Muvwimi 3,Winnie Mwanza 2, Grace Mbulo 2, Nathan Kapata 2, Helen Ayles 1

1 - Zambart project, Lusaka, Zambia and LSHTM London2 - Zambart project, Lusaka, Zambia3 - National Reference laboratory, Lusaka, Zambia

background

TheWorldHealthOrganizationrequeststhatcountriesconducttuberculosisdrugresistancesurveys(DRS)everyfiveyears to monitor trends of drug resistance and to determine rates of multi-drug resistant tuberculosis (MDR-TB). Zambia conducted its second nation-wide DRS in 2008. The objective of this study is to investigate whether the MTBDRplus assay (HAIN), a new molecular assay performed directly on sputum, is a useful tool in conducting a DRS.

method

Throughout Zambia approximately 900 sputum specimens were collected from consecutive smear-posi-tive TB patients and transported to the TB Reference Laboratory in Lusaka. Specimens were decontaminat-ed and concentrated smears were prepared. MGIT and LJ cultures were inoculated. Drug susceptibility test-ing (DST) was performed on positive cultures. Remaining decontaminated sputum was heat-killed, sonicated and stored at -80C. The MTBDRplus assay was performed using a 1:5 or 1:10 dilution of decontaminated sputum. Results:Ofthefirst340specimenstestedusingtheMTBDRplusassay,307(90.3%)showednoevidenceofresistance,while thirty-three (9.7%) showed mutations consistent with resistance: 10 were MDR-TB, 20 were isoniazid (INH) mono-resistant and 3 were rifampicin (RIF) mono-resistant. MGIT DST results were obtained from 271 (79.7%) of 340 specimens with an MTBDRplus result. We were unable to obtain MGIT DST results from the remaining 69 (20.3%) specimens due to contamination or lack of growth. Thirteen (39.4%) of 33 specimens that showed mutations consistent with resistance in the MTBDRplus assay failed to yield a DST result on MGIT. Six out of these 13 samples are according to the MTBDRplus assay MDR, 5 are INH mono-resistant and 2 RIF mono-resistant . One sample that showed RIF mono-resistance using the MTBDRplus assay, showed both isoniazid and rifampicin resistance uses the MGIT DST.

Conclusion

In our study, the MTBDRplus assay performed directly on sputum was more rapid and cost-effective than culture to screen for MDR-TB.


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CONTribuTiON Of labOraTOry faCTOrS TO high mgiT CulTurE CONTamiNaTiON raTE iN zambia de Haas, Petra 1, Moyoyeta, Monde 2, Samutela, Mulemba 2, Mwanza, Winnie 2, Musunsa, Alan 3, Mbulo, Grace 2, Muvwimi, Mweemba 3, Ayles, Helen 1

1. Zambart project, Lusaka, Zambia and LSHTM, London2. Zambart project, Lusaka, Zambia3. National reference laboratory, Lusaka, Zambia

background:

An evaluation study of the MGIT liquid culture system in Zambia found a high contamination rate. It was suggested that fac-tors such as delayed sample processing due to long distances, inadequate storage of samples once submitted, poor labora-tory infrastructure and inexperienced staff may have contributed to the high contamination. The objective of this study was to investigate the contribution of laboratory factors to the higher than expected rate of MGIT contamination.

method:

As part of the National drug resistance survey, 917 sputum specimens were collected from smear-positive TB patients andtransportedtotheTBReferenceLaboratoryinLusaka.AfterdecontaminationusingNALC-NAOH(1,5%finalcon-centration) MGIT and LJ cultures were inoculated. In addition 584 of the decontaminated samples were inoculated onto blood agar plates (BA). When the MGIT culture showed growth Ziehl Nielsen staining was performed. If micro-organisms otherthenacidfastbacilliwereseen,aBAwasinoculatedandsubsequentcoloniesidentifiedusingbiochemicaltools.

results

Time between sample submission and processing varied from 1-50 days with a median of 9 days. Increased contamina-tion in MGIT was found when the time between sample submission and processing was extended; 18.6% for 1-7 days, 27.4% for 8-14 days and 37.5% for more than 15 days. The overall contamination rate was 27.6% (95% CI 24.0-31.4). Of 584 decontaminated sputum 197 (33.7%) showed growth on BA. Out of the 197 samples showing growth 86 (43.6%) were also contaminated in MGIT whereas for samples that showed no growth on BA, 70 (18%) out of 387 were con-taminated.ContaminatedorganismfromthesputumandfromthecontaminatedMGITculturewereidentifiedfor35outofthe86.Identicalspecieswereidentifiedinonlysixteen(45.7%)ofthese35samples,whereasin19(54.3%)casesa different contaminanting species was found in the MGIT culture compared to the decontaminated sample.

Conclusion

High contamination on MGIT is a problem in our setting. Delayed processing of samples increases the chance of samples being contaminated. The contamination of samples that did not show any growth on BA from decontaminated sputum as well as different species isolated from the contaminated MGIT compared to the decontaminated sputum suggest that a major part of the contamination may be due to laboratory factors.

Oral Communication

166 ESM 2009

pp-94

prOgrammaTiC COmmuNiTy-baSED maNagEmENT Of mDr-Tb: EXpEriENCE iN KaraChi, paKiSTaN Ahmed, Altaf; Qazi, Fahad; Khan, Amir JavedThe Indus Hospital, Karachi, Pakistan

introduction

MultidrugResistantTuberculosis(MDR-TB)isdefinedasTBresistanttothetwomostpowerfulanti-TBdrugs,Isoniazid(H)and Rifampicin (R). Pakistan is ranked 8th among the high TB burden countries.In November 2007, the Karachi DOTS-Plus program was established with an objective to provide free, comprehensive, community-based management of MDR-TB pa-tients in Karachi and Hyderabad based on Partners in Health (PIH) and World Health Organization (WHO) guidelines. The purpose of this paper is to share our initial experience at programmatic management of MDR-TB in a low income setting.

methodology

At baseline registration, smears, cultures, DST, radiology, and ancillary tests were performed on each patient and each patient house was mapped using GPS devices.

Inclusion criteria were as follows:

1) Acid Fast Bacilli Culture and Sensitivity (AFBCS) showing MDR-TB or PDR-TB

2) Clinical, Radiological or Bacteriological (smear and culture) evidence of active disease

Enrollment was based on availability of funds, clinical judgment and proximity to center. All patients were managed in the community. Each enrolled patient received monthly consultation, uninterrupted and monitored second line drug supply, laboratory tests as per program guidelines. Social support was also provided to all patients in the form of:

1) Monthly Food Baskets

2) Professional Counseling

3) Treatment Supporters (TS)

results

As of May 2009, 106 MDR-TB patients were registered in the program (49 males, 57 females). The mean age was 29.58 (+/-12.8).73patientswereputontreatment.Theirclassificationaccordingtoprevioustreatmentswasasfollows:

1) After failure of retreatment ……30 (41%)

2) Relapse ………………………...14 (19%)

3)Afterfailureoffirsttreatment…12(16%)

4) New …………………………….6 (8%)

5) Registered after default…………5 (7%)

6) Other…………………………….1 (1%)

7) Transfer in ………………………1 (1%)

68(90%)patientshadreceivedfirstlinetreatmentpreviously,1(2%)hadreceivedsecondlinetreatmentand6(8%)werenew patients. HIV testing on 68 of 73 enrolled patients came negative for all. Adverse events recorded were recorded. Comorbid conditions included DM, Tobacco use, substance abuse, chronic lung disease, hepatitis, respiratory failure, preg-nancy,hypertension,gastritis,renalfailureandneuropathy.Althoughtreatmentdurationforthefirstpoolofp


pp-95

EValuaTiON Of Capilia (TauNS) fOr rapiD iDENTifiCaTiON Of myCObaCTErium TubErCulOSiS COmplEX frOm CulTurES

C Muchwa2,3, J Akol2,3, F Mumbowa5, P Orikiriza2,3, K Morgan2,3, K Eisenach4 , M Joloba1,3, A Etwom2,3, P Mugyenyi2, R Mugerwa3

1 - Department of Medical Microbiology, Makerere University Medical School, Kampala, Uganda2 - Joint Clinical Research Centre, Kampala, Uganda3 - Uganda-CASE Research Collaboration, Kampala, Uganda4 - University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, USA;. 5Infectious Diseases Institute (IDI), Kampala, Uganda

introduction

The Capilia TB assay is a simple immunochromatographic assay which uses anti-MPB64 monoclonal antibodies to dis-criminate MTB from non-tuberculosis mycobacteria. Evaluation of Capilia to determine its costs, performance and turn around time (TAT) was done using PCR IS6110 assay (PCR) as a gold standard.

methods

Respiratory and blood samples specimens were digested and decontaminated using 1.5% NAOH/NALC, concentrated by centrifugation and inoculated into BACTEC MGIT 960 culture tubes for incubation. Blood was aseptically inoculated and incubated in the BACTEC 9120 instrument. All BACTEC positive cultures were screened for acid fast bacilli by the Ziehl-Neelsen method before testing for MTB. Blood cultures were then inoculated on 7H10 agar and incubated for MTB isolation. The Capilia test was performed according to the manufacturer’s instructions while PCR was done accord-ing to laboratory protocol. The test included 155 respiratory and 70 blood samples were tested.

results

Overall agreement between Capilia and PCR IS6110 methods was 98.2%. Capilia achieved a sensitivity of 98.4% and specificityof97.9%.InitialPCRcomparisonforrespiratoryculturesresultedinsensitivityandspecificityof97.4%and98.7%respectively.Bloodachievedspecificityof27.4%only;thismaybeduetofalsenegativePCRresultscausedbyPCRinhibitors in blood cultures. PCR testing was performed from colonial growth on 7H10 accuracy and reliability of PCR asagoldstandardandtheresultingCapiliatestsensitivityincreasedto100%and94.4%specificity.

Conclusion

TheCapiliahasanoverallsensitivityof98.4%andspecificityof97.9%andisfarmoreaccuratemethodofidentifyingMTB directly in blood cultures. It is less expensive (≈ $5) compared to PCR (≈ $45). It is easier to perform with TAT of 20 minutes while PCR has TAT of 8 hours for respiratory cultures.

168 ESM 2009

pp-96

COmpariSON Of Capilia (TauNS) aND iS6110 pCr fOr rapiD iDENTifiCaTiON Of MyCoBaCTeriuM TuBerCuloSiS COmplEX frOm CulTurES iN Kampala, ugaNDa.

C Muchwa2,3, J Akol2,3, P Orikiriza2,3, K Morgan2,3, F Mumbowa5, KEisenach4, A Etwom2,3, M Joloba13

1 - Department of Medical Microbiology, Makerere University Medical School, Kampala, Uganda2 - Joint Clinical Research Centre, Kampala, Uganda3 - Uganda-CASE Research Collaboration, Kampala, Uganda4 - University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, USA5 - Infectious Diseases Institute (IDI), Kampala, Uganda

introduction

The Capilia TB assay, a simple immunochromatographic method which uses anti-MPB64 monoclonal antibodies to dis-criminate MTB from non-tuberculous mycobacteria has been successfully evaluated in other settings. Before adopting capilia in our laboratory, an evaluation to determine its costs, performance and turn around time (TAT) compared to the existingmolecularidentificationmethodusingPCRIS6110assay(PCR)wasdone.

methods

Sputum/Gastric samples were processed using the 1.5%NAOH/NALC decontamination method and the sediment culturedusingtheBACTECMGIT960system..Blood/PleuralfluidswereasepticallyinoculatedintheBACTEC9120instrument.AllBACTECpositivecultures, (sputumandbodyfluids)werescreened foracid fastbacilliby theZiehl-Neelsen method before testing for MTB. The Capilia test was performed on all ZN positive MGIT 960 tubes according to the manufacturer’s (TAUNS ) instructions while the PCR was done according to laboratory protocol. The ZN positive bloodandotherbodyfluidcultureswereinoculatedon7H10agar for MTB isolation. Capillia was performed on ZN positiveisolates.Thetestincluded155sputum/gastricsand70bodyfluidsamples.TheCapiliaandPCRassaysweredoneby separate technicians who were blinded to the other test results.

results

There was an overall agreement of 98.2% between the Capilia and PCR IS6110 methods. Capilia achieved a sensitivity of98.4%andspecificityof97.9%.TheseresultsconcurredwiththefindingofJannWangetalpublicationof98.6%&97.9% respectively. The initial PCR comparison for sputum/gastric cultures resulted in a 97.4% and 98.7% sensitivity and specificityrespectively.WhenthePCRtestwasperformedfromcolonialgrowthon7H10,theaccuracyandreliabilityofPCRasagoldstandardincreasedandtheresultingCapiliatestsensitivitywas100%and94.4%specificity.ThesensitivityandspecificityofCapiliaoncontaminatedcultureswas97.0%and98.7%respectivelywhilethatonpurecultureswas99.0%and94.4%.Therewasnosignificantdifferenceinthetestperformancebetweencontaminatedandpurecultures(p values of 0.12405 and 0.786 respectively). The recurrent cost for capilia was ~$5 and that of PCR ~$45. The average TAT for Capilia was 20 minutes while that of PCR 8 hours for sputum/gastric cultures and 22 days for blood cultures. Overall Capilia was much easier to learn, had fewer steps and requires no instruments

Conclusion/Discussion

ThesensitivityandspecificityofCapiliaiscomparabletoPCRforbothpureandcontaminatedMTBcultures.•

Capilia is less expensive, faster and easier to perform than PCR. •

Capilia is capable of identifying MTB directly in blood cultures.•


pp-97

DirECT TESTiNg fOr mulTi Drug rESiSTaNT TubErCulOSiS WiTh fOur aSSayS EValuaTED aT Kampala, ugaNDa

Freddie Bwanga1,2,3, Sven Hoffner2,3, Melles Haile2,3, Moses L Joloba1

1 - Department of Medical Microbiology Makerere University College of Health Sciences Kampala, Uganda2 - Department of Bacteriology, Swedish Institute for Infectious Diseases Control, Solna Sweden3 - Department of Microbiology, Tumour and Cell Biology (MTC), Karolinska Institute, Stockholm, Sweden

background

Multi drug resistant tuberculosis (MDR TB) is on the rise worldwide. Early detection of MDR TB is important for effective control of MDR TB transmission. In most TB high burden countries this remains a challenge due to lack of rapid tests. This study evaluated four rapid tests for MDR TB detection.

methods

Smear positive re-treatment TB patients were consecutively recruited at Mulago – Uganda’s National referral Hospital. Sampleswereprocessedusingafinalconcentrationof1.5%NAOH-NALCmethod.Sedimentswereuseddirectlytoset susceptibility to isoniazid and rifampicin with four rapid tests at the National TB Reference Laboratory Kampala. The four tests included the Nitrate Reductase Assay (NRA), Microscopic Observation Drug Susceptibility (MODS), Mycobacterium Growth Indicator Tube (MGIT 960) and Genotype® MTBDRplus (Hain Life Sciences, Nehren, Germany). Results of the four tests were compared to those of the conventional indirect Lowenstein-Jensen proportion method (L-J PM).

results

A total of 66 patients were recruited. Interpretable results were obtained for all the samples with the LJ PM and MODS assay, 64 (97%) with the NRA and MGIT 960, and 62 (94%) with the Genotype® MTBDRplus. Interpretable results across allthefivetestswereavailablefor56samplesandresultsobtainedoninitialtestingwere100%,98%,91%,82%and68%with the Genotype® MTBDRplus, MODS, LJ PM, NRA, and MGIT 960, respectively. Repeat testing with the MGIT 960 was due to power failure -13 samples, contamination - 4 samples and undergrowth - one sample.

Sensitivityandspecificityfordetectionofresistancetoisoniazidwas100%and100%forNRA,100%and95%forMODS,93% and 98% with the Genotype® MTBDRplus, and 93% and 100% with the MGIT 960, respectively. For rifampicin it was 100% and 100% with NRA, 91% and 93% with MODS, 100% and 96% with Genotype MTBDR®plus, and 73% and 100% with the MGIT 960, respectively.

The average time to results was 2 days (range 1-3 days) for Genotype® MTBDRplus, 8 days for MGIT 960 (range 5-13 days), 8 days for MODS (range 7-18 days) and 11 days for NRA (range 10-21 days). Results obtained within 10 days were 91%, 88%, and 75% for the MODS, MGIT 960, and NRA, respectively.

Conclusion

Findings show excellent performance of the direct NRA, MODS, and Genotype® MTBDRplus for MDR TB detection, with most of the interpretable results obtained on initial testing in <14 day.

170 ESM 2009

pp-98

pEa prODuCTiON by myCObaCTEria aND iTS appliCaTiON iN a rapiD Drug SuSCEpTibiliTy TEST. McNerney, Ruth 1, Turner, Claire 2, Mallard, Kim 1, O’Sullivan, Denise 1

1 - London School of Hygiene & Tropical Medicine2-CranfieldUniversity

Metabolites produced during bacterial growth may be used to monitor the impact of drugs on bacteria. We have inves-tigated volatile organic compounds emitted by mycobacteria growing on Lowenstein Jensen (LJ). Mass spectrometry determined one of the major volatile compounds from M. bovis BCG to be phenylethyl alcohol (PEA), a bacteriostatic compound that is a reversible inhibitor of DNA synthesis. PEA production was also observed in mycobacteria other than tuberculosis (MOTT). That such a compound is produced in quantity by mycobacteria growing on LJ is surprising and may explain the limited growth of mycobacteria on this media. PEA is only produced during bacterial growth and monitoring production during exposure may provide a means of determining susceptibility to antimicrobial compounds. To test this hypothesis, bacteria were placed on LJ slopes containing drug and incubated at 37C. Headspace vapors from the culture tubes were analysed using the zNose, an ultra-rapid capillary gas chromatograph coupled to a SAW detector. The test required no sample preparation and each slope was tested in less than 2 minutes. To minimize the risk of creating infected aerosols culture tube caps incorporated a PTFE/silicone septum enabling air to be drawn from the tube without remov-ing the cap. Samples collected were heat sterilized during testing. Headspace samples from drug containing slopes were compared to control slopes without drug. The incubation time required for differentiation between positive and negative samples or ‘growth’ and ‘no growth’ depended on the size of the inoculum and the species of mycobacteria and ranged from hours to a few days. MOTT could be differentiated from M. tuberculosis complex bacilli by continued production of PEA in the presence of 500ug/ml para-nitrobenozic acid. We conclude that detection of volatile metabolites emitted by mycobacteria growing on Lowenstein Jensen offers a simple, rapid alternative to assess their drug susceptibility.


pp-99

VariOuS STraTEgiES TO DECONTamiNaTE aCiD faST baCilli pOSiTiVE liQuiD CulTurES frOm baCTEC mgiT 960 Balmoi, FaithJoint Clinical Research Centre, Kampala, Uganda

introduction

Though liquid culture is enriched and more sensitive in the recovery of MTB, it yields a greater percent-age of contaminants as well. It is essential that MTB cultures be isolated pure to permit drug susceptibil-ity testing which is vital for prognosis of patients besides determining the presence of MDR-TB and XDR-TB. In this study we evaluated various approaches to reduce or eliminate contamination in Bactec MGIT 960 cultures.

method

Contaminated AFB PCR positive MGIT cultures were subjected to decontamination methods of:

i) Double PANTA Polymyxin B (750µg/ml); Amphotericin (75µg/ml); Nalidixixic (300µg/ml); Trimethoprim (75µg/ml) and Azlocillin (75µg/ml)

ii) VAN vancomycin (30.5µg/ml), Amphotericin (106µg/ml) and Nalidixic acid (226.7µg/ml)

iii) 2% NaOH each contaminated culture was divided and subjected to all three decontaminat-ing procedures. The decontaminated cultures were then inoculated in MGIT tubes and 7H10 se-lective whole plates and incubated in Bactec MGIT 960 and a 5%-10% CO2 at 37°C respectively. Theplateswerereadweeklyuntilasufficientcolonialgrowthwasachieved.ThepositiveMGITwassubjectedtoZiehl-Neelsen (ZN) smear and blood agar culture for purity check.

results

A total of 50 specimens had analyzable results for each method. Double PANTA was able to recover 62.0%, VAN 60.0% and 2% NaOH 24.4% while 16.0%, 14.0% and 6.7% respectively remained contaminated. However, 22.0%, 26.0% and 68.9% were non-viable after decontamination with double PANTA, VAN and 2% NaOH respectively. Further testing is being done so as to generate more reliable results.

Conclusion

VAN and double PANTA were comparable as far as giving more specimens (p value = 0.614). With pure cultures •when they are used for decontamination.

More specimens were unrecoverable when 2% NaOH was used than the other two methods. •

The cost of decontaminating a sample using VAN was ~$1 while double PANTA was ~$5.70 •

172 ESM 2009

pp-100

lOW COST iSOlaTiON Of myCObaCTErium TubErCulOSiS (mTb) frOm blOOD Orikiriza, PatrickUganda-CASE Research Collaboration, Kampala

introduction

Routinely, identificationofMTBfrombodyfluidsrequiresaPCRbasedtechniquewhichhasbeenadoptedasagoldstandard. This technique however has been unsuccessful against blood cultures which are often affected by inhibitors. In this study, we evaluated a simple but effective way of isolating and identifying MTB in blood cultures.

method

A total of 91 blood cultures positive for acid fast bacilli were sub-cultured onto 7H10 medium and Lowenstein Jensen slants and incubated at 37ºc in a 5% carbon dioxide incubator. The isolates that grew on solid media were further tested byPCRtoconfirmMTB.TheoriginalbloodculturesweretestedbydilutinganaliquotofbloodcultureinPCRwatersandrepeatingtheIS6110PCRandalsobyCapiliaforidentificationofMTB.

results

The study found 15 cultures PCR positive when diluted in PCR water and 56 positive with Capilia. On the other hand, cultures grown on the solid media before performing PCR yielded 53 PCR positives from 7H10, 44 PCR positives from LJ and 28 did not grow on either medium.

The time it took for the cultures to turn positive on the different media was also noted

Conclusion

IdentificationofMTBbyPCRwasmoreefficientbloodculturesweresubculturedtosolidmediaforidentificationassay Capiliatestsperformeddirectlyfrombloodculturemediagavecomparableresults(95%)withMTBidentificationbyPCR from solid growth.

There was an overall better turn around time using the 7H10 media compared to LJ medium


pp-101

COmpariSON Of rapiD COlOrimETriC mEThOD, prOpOrTiON mEThOD aND baCTEC460 Tb SySTEm fOr TESTiNg SuSCEpTibiliTy oF M.TuBerCuloSiS TO rifampiNE aND iSONiaSiDE Begum KAYAR, Ülkü ORAL ZEYTINLI, Ayse KARACALI, Ayben SOYAL, Togrul Nagiyev, Fatih KÖKSALCukurova University, Medical Faculity

introduction

Multidrug-resistant (MDR) M. tuberculosis (MTB) strains founded resistant to at least isoniaside [INH] and rifampin [RIF], thetwomostimportantfirst-linedrugsposeaseriousthreattothecontroloftuberculosis(TB)andthespreadofthesestrains has become a major public health problem. In this study, the performance of antimycobacterial susceptibility test-ingforthefirstlinedrugs(RIFandINH)withcolorimetricnitratereductase-basedantibioticsusceptibility(CONRAS)and conventional proportion method depend on solid agar culture (LJ) were compared to BACTEC 460 TB system as the gold standard.

methods

Of the total of 187 MTB strains, when 24 isolates were found as resistant to RIF and INH by Bactec 460 TB other 163 strains were susceptible for these antibiotics. All of strains were isolated in sputum from patient with pulmonary dis-easeandidentifiedasMTBbyNAPtestinBactec460system.TheCONRAStestwasperformedwith0.1mg/mLofINHand1mg/mLofRIFonLJasmodificationofstandartmethodsdescribedbyH.Syreetal.Antibioticsusceptibilitytesting with proportion method was performed on LJ medium according to the standard protocol laid down by WHO. Results:Thesensitivity,specificityandoverallagreementoftheCONRAStestwere95.83%(23/24),99.38%(162/163)and97.60% for RIF and 83.33% (20/24), 99.38% (162/163) and 91.35% for INH, respectively. Results for proportion tests were found as; 91.66% (22/24), 93.25% (152/163) and 92.45% for RIF and 75% (18/24), 97.54% (159/163) and 86.27% for INH, respectively by Bactec 460TB system as the gold standart.

interpretation & conclusion

CONRAS test showed good agreement with Bactec 460 (the overall agreement of this test was 94.47%) for each of the antimicrobial tested. CONRAS test is simple, easy to perform, less expensive, reliable and may be used as a preliminary screen for susceptibility testing of MTB in particularly for resource-poor countries.

.. o,

.

174 ESM 2009

pp-102

EValuaTiON Of ThE mgiT TbC iD TEST VS TWO COmmErCially aVailablE rapiD immuNOaSSayS fOr M. TuBerCuloSiS COmplEX OrgaNiSm DETECTiON frOm liQuiD aND SOliD CulTurE

Crews, Virginia 1, Warns, Matthew 1, Pfeltz, Richard 1, Beaty, P. Shawn 1, Rosales, Julie 1, Kopher, Ken 1, Joshi, Sudhaunshu 1, Hoosen, Anwar 2, Said, Halima 2

1 - BD Diagnostic Systems, Sparks, Maryland, United States of America2 - Department of Medical Microbiology, Faculty of Health Sciences, University of Pretoria and National Health Laboratory Service, Tshwane Academic Division, Pretoria, South Africa

introduction

ChromatographiclateralflowimmunoassaysfortheMPT64proteinantigencanprovidearapidandcost-effectivemeth-od to qualitatively detect Mycobacterium tuberculosis complex (Mtbc) organisms from acid-fast bacillus (AFB) positive cultures.Thepurposeofthisstudywastocomparethesensitivityandspecificityofthreesuchdeviceswithmycobacteriafrom liquid and solid media cultures.

methods

Positive liquid (BACTEC MGIT 960 7 mL tubes) and solid (BBL Löwenstein-Jensen slants) culture media seed-ed with mycobacteria provided inocula for side-by-side evaluation of two commercially available tests, Capilia TB (“Capilia”, Tauns Laboratories, Numazu, Japan) and SD Bioline TB Ag MPT64 Rapid Test (“Bioline”, Standard Diagnostics, Inc, Kyonggi-do, Korea), and the BD MGITTBc IdentificationTest (“TBc ID”, BD Diagnostic Systems,Sparks, MD, USA). The MGIT TBc ID test is currently in clinical trials. Devices were inoculated per manufacturer’s in-structions and visually assessed as positive for detection (visible test line) and reagent function (visible control line). RESULTS. All three devices detected each of the 20 Mtbc organisms tested (100% sensitivity), which included 15 M. tuberculosisstrains.Specificitiesforthe18non-tuberculousmycobacteria(NTM)testedwereasfollows:100%fortheTBc ID device, 94% for the Capilia device (due to cross-reactivity with M. marinum), and 94% for the Bioline device (due tocross-reactivitywithM.gastri).Additionaltestingwiththetwocross-reactiveNTMspeciesconfirmedtheseresults.Sensitivityandspecificityresultsforagivendevicewereidenticalbetweensolidandliquidmedia.However,flowissuesafter inoculation from solid medium as well as background discoloration after inoculation from liquid medium occurred regularly with the Bioline device.

Conclusions

ThethreerapidMtbcorganismidentificationimmunoassayproductsexhibitedverygoodsensitivity.Thespecificitydem-onstratedbytheTBcIDdevicewasalsoverygood,butspecificitywaslowerfortheCapiliaandBiolinedevicesduetocross-reactivitywithspecificNTMs.


pp-103

NiTraTE rEDuCTaSE aSSay appliED TO DirECT DETECTiON Of Drug rESiSTaNCE iN myCObaCTErium TubErCulOSiS. Montoro, Ernesto 1, Milián, Yoslaine 1, Lemus, Dihadenys 1, Echemendía, Miguel 1,Yzquierdo, Sergio 1, Martin, Anandi 2, Van der Stuyft, Patrick 2, Palomino, Juan Carlos 2

1 - Institute of Tropical Medicine Pedro Kourí (IPK), Havana, Cuba2 - Institute of Tropical Medicine, Antwerp, Belgium

Tuberculosis still represents a major public health problem; especially in low-resource countries were the burden of the disease is more important. Standard methods for drug susceptibility testing of Mycobacterium tuberculosis, such as the proportional method (PM), the absolute concentration method, and the resistance ratio method, are used globally but dependoncultureonsolidmediaandarethereforetime-consuming.Thetimelagisasignificantthreattothepatient,the community, and health care workers. To reduce this period, we have evaluated the nitrate reductase assay (NRA) on smear-positive sputa for the direct detection of drugs resistance in Mycobacterium tuberculosis. A total of 63 smear-positivesputumwereusedinthisstudy.ThesamplesweredecontaminatedusingthemodifiedPetroffmethod,apor-tion was used to carried out the NRA as susceptibility test to isoniazid (INH, 0,2 µg/mL), streptomycin (SM, 4 µg/mL), ethambutol (EMB, 2 µg/mL) and rifampicin (RIF, 40 µg/mL). The resulting sample was used to perform the indirect PM on Löwenstein-Jensen which was used as gold standard method. The NRA results were obtained between 14 and 28 days. However, were necessary 28 or 42 days to obtain PM results. The sensitivity of MNR to INH, SM, EMB and RIF was 88,9%, 75%, 0% and 71,4% respectively. The low sensitivity obtained for all drug evaluated was due to a small amount of resistancestrainsfounded.Ontheotherhand,thespecificityofMNRtoeachdrugswas96,3%(INH),96,1%(SM),95,2%(EMB) and 98,2% (RIF). In general, the concordance between MNR and PM was 95,2% to INH, 92,1% to SM, 93,7 to EMB and 95,2% to RMP. In conclusion, the MNR applied in sputum samples is more rapid than any conventional method. Two drugsthatdefinemultidrug-resistanceandarethemostpotentinthetreatmentoftuberculosis,INHandRIF,showedthe higher values of concordance wit PM. The MNR results are easy to performance, use each drug with identical critical concentration in the same solid media than PM and could be a useful tool for detection of tuberculosis drug resistance in low-resource countries with limited laboratory facilities.

176 ESM 2009

pp-104

uSE Of NiCOTiNamiDE iN COlOrimETriC mEThODS fOr rapiD DETECTiON Of pyraziNamiDE rESiSTaNCE iN myCObaCTErium TubErCulOSiS Montoro, Ernesto 1, Lemus, Dihadenys 1, Madruga, Mariela 1, Mirabal, Niuris 1, Milián Yoslaine 1, Yzquierdo, Sergio 1, Echemendía, Miguel 1, Martín, Anandi 2, Van der Stuyft, Patrick 2, Palomino, Juan Carlos 2 1 - Institute of Tropical Medicine Pedro Kourí (IPK), Havana, Cuba2 - Institute of Tropical Medicine, Antwerp, Belgium

Pyrazinamide (PZA) is one of the most effective anti-tuberculosis drugs. It is also bactericidal to semidormant Mycobacterium tuberculosis and it reduces the total treatment time. The current susceptibility testing methods for this drugaredifficultdue to the poor growth of the bacteria in acid medium which is required for drug activity. One alterna-tive has been the use of nicotinamide (NIC), an analogue of PZA that can be converted by PZAase into active form in a physiological pH that does not hinder bacterial growth. Recently, the NIC has been applied successfully in inexpensive susceptibility testing such as nitrate reductase assay (NRA) for rapid detection of PZA resistance. The purpose of this study was to develop the NRA and malachite green indicator (MGI), both with NIC, as rapid susceptibility testing to PZA in M. tuberculosis. The NRA and MGI were carried out on 120 M. tuberculosis strains from the collection at Tuberculosis National Reference Laboratory. The concentration of NIC applied in both methods was 1 000 µg/mL and all results were compared with Wayne method which was used as gold standard, employing 100 µg/mL of PZA. The Wayne method results were obtained in 4-7 days whereas MGI and NRA results required 7-14 days. A total of 17 and 85 strains were reported as resistant and susceptible respectively by the three methods but MGI had 16 discordant results and NRA only 4 discrepancies. The MGM showed asensitivityandspecificityof80,95%and87,88%respectivelywhereasNRAprovidedasensitivityof90,48%andspecificityof97,98%.Ingeneral,theconcordanceofMGIandNRAwere86,67%and96,67%respectively. The MGI employing NIC showed a low sensitivity to detect resistance to PZA. In contrast, the NRA showed a high concordance with Wayne method. This assay is rapid, accurate and could be an attractive option for rapid detection of PZA resistance, especially in limited-resource countries with high levels of resistance.


pp-105

implEmENTaTiON Of liQuiD CulTurE fOr TubErCulOSiS DiagNOSiS iN a rEmOTE SETTiNg: lESSONS lEarNED

Pamela Hepple1, Jonathan Novoa-Cain2, Chris Cheruiyot2, Elvira Richter3, Koert Ritmeijer4 1 - Manson Unit, Médecins sans Frontières, London, UK2 - Médecins sans Frontières OCA South Sudan, Lokichoggio, Kenya3 - Forschungszentrum Borstel, Nationales Referenzzentrum für Mykobakterien, D-23845 Borstel, Germany4 - Médecins sans Frontières OCA, Amsterdam, the Netherlands

issues

The diagnosis of tuberculosis (TB) in Médecins sans Frontières (MSF) projects is based on sputum smear micros-copy, which has low sensitivity. Following WHO recommendations, MSF established a TB liquid culture laboratory in Lokichoggio, Kenya, processing samples from 4 South Sudan projects, for diagnosis of smear-negative and extra-pulmo-nary (EP) TB and follow-up of patients.

Description

The manual MGIT (mycobacterial growth indicator tube, Becton Dickinson) system was used with Lowenstein-Jensen media. One positive culture per patient was sent to Borstel Supranational Reference Laboratory, Germany for speciation using the Hain Genotype Mycobacteria series and sequencing techniques.

From March 2007 to December 2008, sputum culture was performed for 64 diagnostic and 24 follow-up patients. Ten EP samples were also cultured.

For diagnostic patients, of two smear-positives, one was culture-positive for both Mycobacterium tuberculosis (MTB) and M. fortuitum, and one for M. fortuitum.

Eight of 62 (13%) smear-negatives were culture-positive for MTB complex (3 for MTB and 5 for MTBC) with nine (14%) culture-positiveforotheridentifiedmycobacteria;six(9%)grewunknown,notvalidly-describedmycobacteria,and39(61%) were culture negative.

For follow-up patients, of seven smear-positives, one was culture-positive for MTB, one for M. intracellulare and one for M. fortuitum complex, and four (57%) were culture-negative. Among the smear-negatives, eight of 17 (47%) were culture-positive, four of which were unknown mycobacterial species, and four non-tuberculous mycobacteria (NTM). Nine (53%) were culture-negative.

SamplesfromthreeEPpatientsof10grewmycobacteria,speciesunidentifiable.

Intotal,only10of36(28%)culture-positivepatientsgrewmycobacteriafromsputumwhichcouldbeidentifiedasMTBorMTBC.

lessons learned

Duetothelongturn-aroundtimebetweensampleproductionandspeciesidentificationduetoshipmentissues(ap-proximately 4-6 weeks), clinicians often did not wait for results before initiating or adjusting therapy. The high proportion ofNTMwasdifficulttointerpret.Thecultureresultshadlittleclinicalimpact,andculturelabactivitiesweresuspendedin February 2009.

recommendations

Future TB culture programmes require facilities for on-site speciation of mycobacteria or, if working with reference labs, should minimize turn-around time to results by ensuring timely shipment of samples.

178 ESM 2009

pp-106

rapiD DETECTiON Of rESpiraTOry aCTiVE myCObaCTEria by auramiNE O-CTC DOublE STaiNiNg Ichijo, Tomoaki; Izumi, Yoko; Yamaguchi, Nobuyasu; Nasu, MasaoOsaka University

introduction

Clarifying the dynamics of nontuberculosis mycobacterial (NTM) and determining their hot spots in aquatic environ-ments are important to prevent their infection to human beings. For this purpose, methods for the detection of active mycobacterial cells are required. Culture methods are widely used for detection of mycobacterial cells, but these meth-odsareratherdifficulttodetectmycobacteriaquantitativelyandrapidly.Mycobacterialcellsarequantifiableunderafluorescentmicroscopewithinseveralhourswithacid-faststaining.Inaddition,severalfluorochromesareavailabletodetermine the bacterial activities. In this study, we attempted to detect mycobacteria with physiological activity rapidly by combining Auramine O acid-fast staining with 5-cyano-2, 3-ditoryl tetrazolium (CTC) as an indicator of respiratory activity (Auramine O-CTC).

methods

MycobacteriumsmegmatiswasstainedwithCTCandfixedwithformaldehyde.FixedcellswerestainedwithAuramineOandobservedunderanepifluorescentmicroscopewithblueexcitation.

results and Discussion

WefirstlyoptimizedtheconditionoftheAuramineO-CTCdoublestainingmethodtoaddressthefollowingproblems;(i)CTCformazandissolvedinthedecolorizationstepand(ii)fluorescentintensityofAuramineOwasdecreasedbyfluorescentresonantenergytransfer(FRET).Specificityofthismethodwasconfirmedbystainingnon-targetedbacteria.Results can be obtained within 90 min by the optimized procedure, while more than one week is required for the cul-ture-dependent approach. In conclusion, the Auramine O-CTC double staining method was useful for rapid detection of respiratory active mycobacteria. This method may contribute to identifying dynamics of NTM in aquatic environments.

acknowledgement

ThisworkwassupportedbytheJSPSGrant-in-AidforScientificResearch(A)(20249007).


pp-107

SyNErgiSTiC aCTiViTy Of TWO aNTiTubErCulOuS Drug COmbiNaTiONS agaiNST CliNiCal iSOlaTES Of MyCoBaCTeriuM TuBerCuloSiS rESiSTaNT TO iSONiaziD.

Emma Rey,GriseldaTudóandJuliàGonzález-MartínServei de Microbiologia. Hospital Clínic-IDIBAPS. Dept. Microbiologia i Parasitologia Sanitaria, Universitat de Barcelona. Spanish Network for the Research in Infectius Diseases (REIPI, RD06/0008).

Objective

Todeterminethesynergisticactivityof2drugcombinations:isoniazid(H)+rifampicin(R)+ethambutol(E)andofloxacin(O)+R against M. tuberculosis clinical isolates resistant to H versus drug susceptible isolates.

methods

Individual MICs of the strains were studied. Both combinations were studied crossing 7 concentrations of each antibiotic (including their MIC). The inoculum was of 104CFU/ml. All cultures were performed with the proportional method in Middlebrook 7H11 medium and incubated 4 weeks. On analysing the results of the combinations, the fractional inhibitory concentration (FIC) was interpreted: FIC≤0.5,synergisticactivity;FICfrom1-4indifferenceandFIC>4antagonisticactivity.

results

H+R+E Combination: 11 H resistant isolates were studied: 4 (36%) isolates had MIC=52µg/ml and 7 (64%) had MIC=0.8µg/ml. 9 drug-susceptible isolates were also studied. Among the 20 isolates the individual MIC for R ranged from 1-2 µg/ml, from 2.5-5 µg/ml for E, being 0.05µg/ml for H in susceptible isolates. In H-resistant isolates, the MIC in combination for H and R decreased up to 3 dilutions (average) respect to their individual MIC. A lesser decrease (2-3 dilutions) was ob-served for E. In H-resistant isolates, 9/11 (81.1%) showed synergistic activity while 2/11 (18.18%) of the resistant isolates indicated indifference (FIC index =0.7). MICs in combination of susceptible strains decreased an average of 2 dilutions compared to their individual MIC. The susceptible strains had FIC indices ≥ 0.748. O+R Combination: 21 isolates were studied:11 had MIC≤1µg/ml,4MIC=1-6.4µg/mland6MIC>6.4.9drug-susceptibleisolateswerestudied.IndividualMICsfor R ranged from 1-2, with 1 for O. MICs in combination of all strains were the same or decreased up to 1 dilution with regard to their individual MIC. The combination of OR in 21 strains did not show synergism or antagonism in either the resistant or the susceptible strains being the FIC values mainly 1.5.

Conclusions

1)These results suggest that in strains resistant to H with an MIC of 0.8µg/ml in vitro, the standard antibiotic regimen, in-cluding H, would be effective due to the possible compensatory effects of R and E. 2) Although strains resistant to H with an MIC of 51.2 µg/ml showed synergism, these strains would be within the range of resistance. 3)The combination of HRE against susceptible strains and that of OR did not show synergism probably because of high individual bactericide action.

180 ESM 2009

pp-108

TObramyCiN-ClariThrOmyCiN COmbiNaTiON ON MyCoBaCTeriuM TuBerCuloSiS CliNiCal iSOlaTES

Karolien Stoffels1, Hamidou Traore2, Raymond Van Hoof3 and Maryse Fauville-Dufaux1

1-NationalReferenceLaboratoryofTuberculosisandMycobacteria,ScientificInstituteofPublicHealth,Department Institut Pasteur, Rue Engeland 642, 1180 Brussels, Belgium2 - Laboratoires SMB, Rue de La Pastorale 26-28, 1080 Brussels, Belgium3-NationalReferenceLaboratoryofResistancetoAminoglycosides,ScientificInstituteofPublicHealth,Department Institut Pasteur, Rue Engeland 642, 1180 Brussels, Belgium

Objectives

This study investigated the in vitro susceptibility of 25 Mycobacterium tuberculosis clinical isolates to two well-known drugs, tobramycin (TM) and clarithromycin (CL). The effect of both drugs administered together was also investigated to detect a possible synergistic effect.

methods

MICofisolateswithvariableresistanceprofiles wasdeterminedbytheradiometricBACTEC460TBandwasdefinedasthe minimal antibiotic concentration for which at least 99% of the mycobacterial population growth was inhibited. The influenceofTMontheMICofCLwasinterpretedbyFractionalInhibitoryConcentration(FIC).

results

The median MIC for both TM and CL was 8 µg/ml (range: 2 to 8 µg/ml and ≤2to>16µg/mlrespectively).For36%(9/25)of the tested isolates a decrease of the MIC of CL by a single or twofold dilution was observed (FIC ≤ 0,5) when a sub-inhibitoryconcentrationofTMwasadded.Noantagonisticeffect(FIC>4)wasobserved.Similarresultswereobservedforisolatessusceptibleorresistanttofirst-lineantituberculosisdrugs.

Conclusion

Although the MICs for CL and TM seem high compared to conventional anti-tuberculosis drugs, these antibiotics should notimmediatelyberuledoutasclinicallyinsignificant.Indeed,afteradministrationof300mgaerosolisedTM,antibioticcon-centrationsintheepithelialliningfluidarehigherthan10timesthemedianMICobservedfortheTBisolatestestedinthisstudy (8µg/ml). Oral administration of 500 mg CL twice daily leads to a peak concentration of 13,50 +/- 3,3 µg/g in the lungs, 505±293µg/mlinalveolarcellsand34±5µg/mlinepithelialliningfluid,thusallexceedingtheMICof8µg/ml.

Promising new drug delivery systems such as the Dry Powder Inhaler allow achieving very high local antibiotic concen-tration, e.g. 7 times higher for TM compared to aerosol administration and thus far beyond the MIC of resistant isolates. Theseresultssuggestthatbothdrugsshouldbeinvestigatedfurtheraspotentialadjunctstothedifficulttreatmentofmulti-drug resistant tuberculosis where other alternatives have failed.


pp-109

prEValENCE Of EffluX-mEDiaTED rifampiCiN rESiSTaNCE iN MyCoBaCTeriuM TuBerCuloSiS CliNiCal iSOlaTES

Carrie K. W. Au-Yeang, T. K. Au, Edward W. C. Chan, Raphael C. Y. ChanDepartment of Microbiology, The Chinese University of Hong Kong, The Prince of Wales Hospital, Shatin, New Territories, Hong Kong

Rifampicin is one major ingredient in the cocktail-regimen used in treatment of Mycobacterium tuberculosis (MTB) infec-tion. Although mutations in the rpoBgeneareconsideredthebasisofrifampicinresistance,wenotedthatasignificantproportionoflocalresistantcasescouldnotbeattributedtomutations.Alternativemechanismssuchasdrugeffluxhave been proposed.InordertoevaluatetheroleofdrugeffluxmechanismsinmediatingrifampicinresistanceinMTB,weexaminedtheprevalenceofeffluxactivities inrifampicinresistant isolatesusingthreeefflux inhibitors:reserpine,carbonyl cyanide chlorophenylhydrazone and verapamil. Forty-two rifampicin resistant and nine drug susceptible MTB clinical isolates were studied. The minimum inhibitory concentration (MIC) values for rifampicin were determined in the presenceandabsenceofeffluxinhibitors.ThemagnitudeofMICreductionforeacheffluxinhibitorandtheprevalenceofefflux-mediatedresistancewereexamined.Wefoundthatamongthethreeeffluxinhibitorstested,significantMICreduction, ≥ 2-fold MIC decrease, was observed only for verapamil. 61% (31/51) of the test isolates had an MIC reduc-tionbetween2to8-foldinthepresenceofverapamil.Thisverapamil-sensitiveefflux-mediatedMICreductioneffectwas much more apparent in rifampicin resistant isolates than the drug susceptible controls: 71% (30/42) versus 11% (1/9). Likewise, this phenomenon was more prevalent in isolates resistant to 3 - 5 anti-tuberculosis drugs than isolates resistantto1-2drugs:77%(24/31)versus55%(6/11).ThisdatasupportthenotionthatdrugeffluxsystemscontributesignificantlytorifampicinresistanceinMTBclinicalisolatesandhighlighttheneedfordeterminingtheextentbywhichthese systems contribute to resistance to other anti-tuberculosis drugs.

This study is supported by a grant (08070292) from Research Fund for the Control of Infectious Diseases.

182 ESM 2009

pp-110

iNTra aND EXTraCEllular aCTiViTy Of ruThENium COmplEXES agaiNST MyCoBaCTeriuM TuBerCuloSiS aND ThEir CyTOTOXiCiTy

Leite, Clarice Queico Fujimura 1, Pavan, Fernando Rogério 1, Maia, Pedro I da S 2,Deflon,VictorM2, Sato, Daisy Nakamura 3, Azevedo, Alzir A 4, Poelhsitz, Gustavo V 4, Leite, Sérgio Roberto Andrade 1, Franzblau, Scott G 5

1 - São Paulo State University2 - University of São Paulo3 - Adolfo Lutz Institute4 - Federal University of São Carlos5 - University of Illinois

introduction

Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death. One-third of the world’s population is infected with Mycobacterium tuberculosis (MTB), the etiologic agent of TB. The World Health Organization estimates that about eight million new TB cases occur annually. The pulmonary TB (most common form of TB), is highly contagious and has been increasing in incidence in many areas, not only in developing coun-tries but also in industrialized countries. The global resurgence of TB and the rapid emergence of MDR-TB, underscore the importance of the development of new antituberculous drugs.

Objectives

ObjectingtofindnewcompoundswithhighactivityagainstTB,wedeterminedthecytotoxicity,andintra/extracellularanti-M. tuberculosis activity of 29 new compounds involving different class of ligands such as diimines, phosphines and Schiff bases with ruthenium.


As analytical methods, three standardized techniques were used: 1- in vitro Microplate Rezarurin Assay (REMA) for de-tection of minimal concentration of compounds necessary to inhibit 90% of bacillary growth (PALOMINO et al., 2002), 2- Cytotoxicity (IC50) of compounds against mamarian cells (AHMED et al., 1994) and 3- Determination of compounds activity against M. tuberculosis internalized in a macrophage cells (SNEWIN et al., 1999).

results and Conclusion

Fromthe29compoundsanalyzed,7ofthemcontainingtheruthenium,werequalifiedaspromisinganti-TBagents.Thesecomplexes presented inhibitory activity ranging of 0.25 – 3.9 µg/mL, whose values are better than some drugs commonly usedintheTBtreatment,lowcytotoxicity(IS>10)andintracellularinhibitoryactivityhighthan70%.


pp-111

aNTi-myCObaCTErium TubErCulOSiS aCTiViTy Of ThiOSEmiCarbazONES, SEmiCarbazONES aND hyDrazONES Leite; Sergio 1, Pavan; Fernando 2, Maia; Pedro 3,Deflon;Victor3, Batista; Alzir 4, Sato; Daisy 5, Franzblau; Scott 6, Leite; Clarice 2

1 - Universidade Estadual Paulista, Instituto de Química2 - Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas3 - Universidade de São Paulo, Instituto de Química de São Carlos4 - Universidade Federal de São Carlos, Departamento de Química5 - Instituto Adolfo Lutz, Unidade de Ribeirão Preto6 - University of Illinois at Chicago, College of Pharmacy

The aim of this study was to identify a candidate drug for anti-tuberculosis therapy development from previously synthe-sized thiosemicarbazones, semicarbazones and hydrazones, comprising a total of 17 compounds. The Minimal Inhibitory Concentration (MIC) of these compounds, determined by the resazurin reduction method, was investigated in order to determine their in vitro antimycobacterial activity against Mycobacterium tuberculosis. In vitro cytotoxicity values (IC50) of the same compounds were determined on J774 cells to establish a selectivity index (SI = IC50/MIC). Lower values of MIC were found for four thiosemicarbazones and four hydrazones, namely: 2-acetylpyridine N4 (etil) thiosemicarbazone; 2-acetylpyridine N4 (cyclohexyl) thiosemicarbazone; di-2-pyridyl ketone N4 (phenyl) thiosemicarbazone; 2-acetylpyridine morpholyl thiosemicarbazone; mono benzoylacetone isonicotinoyl hydrazone; mono-acetylacetone isonicotinoyl hydra-zone; di-2-pyridyl ketone isonicotinoyl hidrazone; di-2-pyridyl ketone thiophene hidrazone (MIC values ranging from 0.78 to 6.25 µg/mL). All the compounds presented very low cytotoxicity, with the exception of 2-acetylpyridine morpholyl thiosemicarbazone (IC50 ≤ 3.9 µg/mL and SI ≤ 5). The results obtained with the other 7 compounds (SI ranging from 100 to800)qualifythemascandidatesforanti-TBdrugs,oncetheirinvitroresultsarecomparabletosomeof“firstline”and“second line” drugs commonly used in the TB treatment.

184 ESM 2009

pp-112

mEThODS fOr aSSESSmENT Of EThiDium brOmiDE TraNSpOrT aCrOSS MyCoBaCTeriuM SMegMaTiS CEll Wall

Jorge Ramos1*, Liliana Rodrigues1,2, Isabel Couto1,3, Leonard Amaral1,2 and Miguel Viveiros1

1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisboa, Portugal2 - UPMM, IHMT/UNL, Lisboa, Portugal3 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal*Correspondig author: Jorge Ramos, Unit of Mycobacteriology, Instituto Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Rua da Junqueira 96, 1349-008 Lisboa, Portugal. Tel:+351213652600;Fax:+351213632105;E-mail:[email protected]

Activeeffluxsystemsandreducedcellwallpermeabilityareconsideredtobethemaincausesofmycobacterialintrinsicresistancetomanyantimicrobials.Althoughseveralmycobacterialeffluxpumpshavealreadybeendescribed,theirroleindrugresistanceisnotyetfullyunderstood.RecentstudiesshowedthatbothLfrAandMspA,themaineffluxpumpandporin in M. smegmatis, respectively, are involved in reduced susceptibility to several antimicrobials.

We have compared the M. smegmatis wild-type strain mc2155 with LfrA and MspA M. smegmatis deleted mutants, for their ability to extrude ethidium bromide (EtBr), a knowneffluxpump substrate, underdifferent energy conditionsandinthepresenceorabsenceofeffluxpumpsinhibitors(EPIs),by(i)a96wellmicroplatescreeningassaywiththemycobacterial cells grown in Middlebrook 7H9 with 10% of OADC in presence of increasing concentrations of EtBr and different concentrations of the EPIs and the plates examined with a UV transilluminator and photographed after 24 hours of incubation; and (ii)asemi-automatedfluorimetricmethodthatdetectseffluxonarealtimebasisduringaperiod of 30 minutes. The EPIs employed were chlorpromazine, thioridazine, carbonyl cyanide m-chlorophenylhydrazone and verapamil.

Theeffluxactivitydetectedforeachstrainbythesetwomethodswasthencorrelatedwithresistancetoseveralanti-biotics (ATBs), by the determination of their minimal inhibitory concentrations in the presence or absence of the EPIs. TheATBstestedwerestreptomycin, isoniazid(INH),rifampicin(RIF),ethambutol(ETB),amikacin,ciprofloxacin(CIP)and clarithromycin (CLR).

In the absence of the major porin of M. smegmatis, MspA, it was observed that accumulation of EtBr decreased and the cellsbecamemoreresistanttoseveralATBs.Ontheotherhand,themutantforthemajoreffluxpumpLfrAshowedincreased accumulation of EtBr. This strain also presented increased susceptibility to EtBr, INH, RIF, ETB, CIP and CLR.

These results show that MspA is an important channel for entrance of quaternary ammonium compounds and ATBs and that the pump LfrA is involved in low-level resistance to several ATBs and quaternary ammonium compounds in M. smegmatis.


pp-113

ThE humaN maCrOphagE aS a mODEl TO SElECT COmpOuNDS aCTiVE agaiNST mDr/XDr-Tb

Marta Martins1,2,*, Miguel Viveiros1,3, Isabel Couto1,4 and Leonard Amaral1,2,3.1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal2 - UPMM, IHMT/UNL, Lisbon, Portugal3 - COST ACTION BM0701 (ATENS)4 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal* Corresponding author: Unit of Mycobacteriology and UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Rua da Junqueira, 96, 1349-008, Lisbon, Portugal; Telf: +351213652600; Fax: +351213632105; e-mail:[email protected]

The emergence of Multi- and Extensively-Drug Resistant Mycobacterium tuberculosis (MDR/XDR-TB) represents a major threat to public health worldwide. Both infections result in high mortality, especially if the patient is co-infected with HIV. The selection of therapy for these multi-drug resistant infections is limited, and for most situations, ineffective as many of these strains are untreatable with the available drugs. Thus, there is an urgent need to design and develop new compounds against drug resistant M. tuberculosis that are effective within the main target of this infection, the human macrophage.Wehaverecentlydemonstratedthateffluxpumpsinhibitorsareactive against mycobacteria, by enhancing the killing activity of the human macrophage and may represent an alternative to the conventional antibiotherapy for the treatment of the MDR/XDR-TB infections.

From previous studies we have demonstrated that thioridazine (TZ) enhances the killing of MDR-TB phagocytosed by human macrophages. However, the mechanism of action of TZ on these cells is not fully understood. We have studied the activity of TZ, several of its derivatives, organosilicon (SILA) compounds and other known inhibitors of K+ and Ca2+

transport (ouabain, reserpine and verapamil) on macrophages infected with MDR-TB and XDR-TB. After phagocytosis, the compounds were added to the macrophage cultures. Following incubation cells were lysed and the intracellular bac-terial concentration determined. Our results demonstrate that TZ, three of its derivatives and one SILA compound (SILA 421) enhanced substantially the macrophage killing activity.

The killing activity of neutrophils is correlated with the K+ availability, which is dependent upon transport processes affected by agents that inhibit Ca2+-activated K+ pumps. Based on this and on our results, we postulate that the enhance-ment of the macrophage killing activity by these compounds could be due to the inhibition of Ca2+ and K+ transport that promotes the activation of hydrolases and the killing of intracellular bacteria. A model describing the sequence of events that lead to the killing of intracellular bacteria will be presented. Moreover, the ex-vivo testing of compounds using pa-the ex-vivo testing of compounds using pa-tient’s own macrophages in the clinical TB laboratory might allow screening the most effective compounds against MDR/XDR-TB providing the basis for the intelligent selection of drugs to be used in the therapy of these infections.

186 ESM 2009

pp-114

in viTro aCTiViTiES Of JpC 2067 alONE aND iN COmbiNaTiON WiTh SmX agaiNST NOCarDia SpECiES

Michael Cynamon, Swagatam Mookherjee, Carolyn ShoenVeterans Affairs Medical Center, Syracuse, New York, USA E-mail:[email protected]:315-425-4871

background

JPC 2056, a biguanide prodrug of JPC 2067, a dihydrotriazine DHFR inhibitor, is being developed as an antimalarial thera-peutic. Previously we demonstrated that earlier compounds related to JPC 2067 had promising activities in vitro alone and in combination with sulfamethoxazole (SMX) against nocardia species. JPC 2067 is active against M. tuberculosis, M. kansasii, and M. marinum at ≤ 1µg/ml. The purpose of the present study was to evaluate the in vitro activities of JPC 2067 alone and in combination SMX against a group of clinical nocardia isolates.

methods

JPC 2067 was provided by Jacobus Pharmaceutical Co., Princeton, NJ. SMX was purchased from Sigma Chemical Co, St. Louis,MO.EachdrugwasdissolvedinDMSOatafinalconcentrationof1mg/ml.Aliquotswerefrozenat-200C. Drugs werethawedpriortotestinganddilutedinmodified7H10broth(pH6.6;7H10agarformulationwithagarandmalachitegreen omitted) with 10% OADC enrichment and 0.05% Tween 80. JPC 2067 and SMX were tested alone from 64µg/ml –0.06µg/ml.WhentestedtogetherSMXwasevaluatedatfixedconcentrationsof1µg/mlandJPCat16µg/ml–0.015µg/ml. Twenty eight nocardia isolates (from the ATCC and clinical isolates provided by B. Body and B. Forbes) were used in the study. An in vitrobrothdilutionmethodsimilartothatdefinedbyCLSIwasutilized.

results:

The MIC50 and MIC90forJPC2067,SMXandthecombination(SMXfixedat1µg/ml)were0.125µg/mland4µg/ml,16µg/ml and 32 µg/ml, and 0.03 µg/ml and 2 µg/ml respectively.

Conclusions

JPC 2067 was more active than the previously tested dihydrotriazine analogs against nocardia. The addition of SMX had a modest but consistent effect on lowering the JPC 2067 MIC. It is likely that this effect would be more pronounced if the concentration of SMX was increased to perhaps10 µg/ml (a readily achieved serum level for this agent). JPC 2067 alone and in combination should be evaluated in animal models of both nocardial and mycobacterial infection to understand the clinical potential of these agents.


pp-115

NOSOCOmial Tb iN a labOraTOry SETTiNg

Jaime M S Nina1,2,3

1 - Instituto Nacional de Saúde Doutor Ricardo Jorge2 - Universidade Nova de Lisboa3 - Hospital Egas Moniz

Abstract

TB is recognized as a major cause of morbidity and mortality worldwide. Its easily transmissibility is also generally rec-ognized, both at the family level, in the household, at the place of work and inside health care facilities. This last way of transmission, properly called nosocomial transmission, has been suspected for long time, and was formally demonstrated in the ward, both among patients, and health care workers. Several professional bodies and other institutions, both at the national and international level, produced guidelines trying to minimize TB transmission to health care workers in-side wards and emergency services. Furthermore several countries produced legislation to protect health care workers against nosocomial TB, and/or included TB in the list of professional diseases or hazards to health care workers.

However, much less attention has been given to the TB transmission potential to laboratory workers. Even if several countries moved laboratory work with live TB samples to LSB-3 facilities, the evidence on which to base this decision is thin, and no systematic study has been published.

Herein are presented three cases of nosocomial transmission inside laboratory settings, in Lisbon. These cases cover all spectra of professional differentiation, from basic level auxiliary personnel, to a laboratory technician, and to a micro-biologist physician. In one case the way of transmission was a very common kind of laboratory accident, in another a commoninattention,andinthelastonenospecificwayoftransmissionwasfound.OneofthecaseswasfoundtobeaMDX TB.

In conclusion, the trend to carry out all routine work with live TB samples only inside a BSL-3 facility seems a right one, andsoitisfullyjustified.Alsojustifiedwouldbetheinclusionofhealthlaboratoryworkersinthelegislationthatprovidesafety measures and insurance cover to health care workers.


auThOr iNDEX

Abadia EAbreu CAfonso AAguilar DAhmadi MAhmed AAhmed IÅkerström MAkol JAkpaka PE Al Balushi LAl Busaidi SAl- Mahruqi SAlbarral MIPAlcaide FAldashev AAlgamdi SAl-Hajoj SAli ABAli MSAli Veleyati AAllix-Béguec CAl-Maniri AAAlmeida da Silva PAlmeida EAAl-Omari RAlonso CAl-Rawas OAlves AAmado J,Amaral LAmondi Ouma NAmorim AAndersson EÄngeby KAnoosheh SAnthony RArnold CAu TKAu-Yeang CKWAwad CAyles HAzevedo AABaboolal SBabu Okatch FBachiiska EBalacó IBalbin JABaldan RBalmoi FBarbe VBaritaki MBaritaki SBarrera LBarry III CEBartu VBatista ABaumanis VBauskenieks MBazigos SBeaty PS

Beltrán MBiet FBoehme CBoeree MBonte LBorile CBorroni EBoschiroli MLBourdon EBraga JEBragaRBrankova NBrisse Smangenot SBrosch RBrown TBrum LBrzostek ABwanga FCabral JCacho JCaldas CCambau ECañete CCano ICardoso NCardoso RFCardoso SCarmona JACarvalho CCarvalho FCarvalho MACarvalho TCasal MCassone ACastro AGCausse MChan Chiu YChan CYRChan EWCChan RCYChan WCEChau CHCheruiyot CCho E-JCho SNChryssanthou ECirillo DCirillo DMClivillé RCochard TCodina GCoelho RColl PConceição ECCorrea NCorreia-Neves MCosta ARFCouto ICouto SCrews VCruz A

Cubillos ACvetnic ZCynamon MDafae FDavid SDe Bock ADe Cruz KDe Gispert FXDe Haas PDe la Hoz FDe Sousa MSDeflonVDeflonVMDekhuijzen RDel Portillo PDel Val-Romero BDementieva ADen Hertog ADi Giulio BDias ADiogo JDiwan VDocx SDrobniewski FDuarteRDuvnjak SDziadek BDziadek JEchemendía MEhricht REisenach KElmoula IFEtwom AEum SY Fajfar NFalkinham, III, JOFarnia PFattorini LFauville-Dufaux MFelix CFerme DFernandes PFernandes SJFerreira AFerreira CFerreira DFerreira SFerro RSFeuerriegel SFey FFigueira RFilippini PFissette KFiuza de Melo FFrade RFraga AGFrangopoulos FFranz SFranzblau SFranzblau SGFung SL

Furlaneto IPFurtado CGagneux SGaile IGama JBGao QGarcia MJGarcía-Cañas AGavin PGegia MGeorge AGGharbia SGiampaglia CSGicquel BGil MJGilpin CMGiske CGitti ZGoguet de la Salmonière YOLGoldfeder LCGolec MGomes HGomes HMGomes MHGonçalves HGonzález Torralba AGonzález-Martín JGrce MGreib CGuillard BGutiérrez CGutierrez JGutierrez MCGutierrez-Aroca JBHadadi MHaenn SHaile MHaroun RZHavelkova MHepple PHerbawi MHernández JHernández-Pando RHillemann DHirata MHHirata RDCHoffner SHomolka SHonisch CHonscha GHoosen AHubansCHwang SHIbrahim TAIchijo TImperiale BIngham CIoannidis PIsakova JIvanov AIzumi Y

Jahromi NS Jansone IJanssen HGJoão IJoloba MJoloba MLJordão LJoshi SJulián EJulián EGJureen PKaal EKahlmeter GKam KMKanavaki SKang HSKantor IKapata NKarabela SKaracali AKargar MKaroui CKatalinic-Jankovic VKatalinic-Jankovic VKayar BKayar MbKazempour MKern WVKhan AJKhechinashvili GKim JHKlatser PKoeleman MKöksal FKolk AKonstantinidou EKontos FKopher KKosmadakis GKritski AKrt BKuan HOKubin MKuijper SLabarre MLai WMRLangerak ELazaro ELeão SCLee HLee JLee J-ILeimane VLeite CLeite CQFLeite SLeite SRALeite SRALemos SLemus DLevina K

190 ESM 2009

Levterova VLezcano MALima EJCLima KVBLocht CLockwood DLogarinho ELopes MLLópez AGLópez BLopez-Calleja AILoureiro CLucas FLuo TLuquin MLyashchenko KLyberopoulos PMacedo RMachado DMadruga MMaia PMaia PISMaio JNMak KXMalagari AIMalaquias AMalaspina ACMallard KMarceau MMarkova NMarques MMartín AMartin CMartín-Casabona NMartinez-Martinez LMartins MMartins MCMartins TGMasjedi MMasjedi MRMatic IMatos GMaugein JMazarrasa CFMbulo GMcNerney RMdivani NMédigue CMei JMello FAFMendes ACMendes NHMendes NHMenendez MCMestre OMeyers WMMézard MMichel GMihailelis EMilho CMilián YMillan IMillet JMin JHMiotto PMirabal NMiranda AMiyagi-Shiohira CMiyata MMoilleron RMokrousov I

Monecke SMonge IMoniz-Pereira JMonteiro GMontemayor MMontoro EMookherjee SMorcillo NMorgan KMosko MMota MMoulin LMoure RMoyoyeta MMuchwa CMugerwa RMugyenyi PMüllerova MMumbowa FMunga Waweru PMurcia MMusunsa AMuvwimi MMwamba PMwanza WNagiyev TNascimento INasu MNavarro ANeo ZYNeonakis IKNiemann SNikolaou SNina JNogueira CNogutia ENNoroozi JNoruzi JNovoa-Cain JNowroozi JNuak JNyirenda CNOberhauser BObrovac MObrovac MOcepek MOelemann MCOjeda POliveira POmar AROral Zeytinli UOrikiriza POrozco HO’Sullivan DPaasch FPalaci MPalomino JCPanaiotov SPando RHPandolfiJRCPapaventsis DPapiris SPardini MPark SK Pate MPaulo CPavan FPavan FRPawelczyk JPedrosa JPeeling R

Pellegrin JLPerdigão JPereira DRPereira EPereira Miguel JPérez Meixeira APerkins MPfeltz RPfyffer GPimentel MPinheiro MDPoelhsitz GVPole IPortaels FPortugal CPortugal IPossuelo LPost EPrata PProença FQazi FQuieng MDRacic IRadomski NRamos JRamos MartosRamos MHRamoutar DRaposo ARasolofo VRastogi NRauzier JRefrégier GRégis MReniero ARey EReyes ARibeiro JNRichter ERidell MRiley LWRitacco VRitmeijer KRobledo JRocha GRodrigues ARodrigues FRodrigues LRodrigues SRodríguez-Güell ERohde KHRosales JRoura-Mir CRuimy RRuiz PRumijowska-Galewicz ARüsch-Gerdes SRussell DGSaaed NS Sabino AŞahanKipalevASaid HSainti ASalas SSaluotsa MSalvadó MSalvignol GSampaio DSamper SSamutela MSánchez-Concheiro M

Sancho LSandoval ASantos ACBSantos CSantos RSaraiva MSardinha ESardinha TSarmento ASato DSato DNSchön TSecanella SPSeif SShamputa ICSharaf-Eldin GSShikama M-LShinnick TShinnick TMShoen CSiddiqi SSilva ASilva CSilva FSilva KSilva MSilva PSilva SSimões MFSing LHSingh JPNSkenders GSlickers PSola CSomoskövy ASousa ASSousa CSousa GSousa JGSovhozova NSoyal ASpandidos DASpicic SStoffels KSturegård ESuffys PNSupply PSvensson ETabarsei PTajeddin ETakiff HTancredo LTang YWTap JTavares MTavares Magalhães ATeles JMMTelles MASThibault VTonjum TTorrado ETortoli ETraore HTudó GTurner CUeki SYMValcheva VValdés IValente AValente FValente I

Van der Stuyft Pvan der Wel NVan Hoof Rvan Ingen JVan Soolingen DVaraine FVarghese BVasconcelos OVelayati A Velayati AAVia LEViallard JFViana BHJViana-Niero CVillar MVillela GViveiros MVladimirov KVon Groll AVultos TDWang SWarns MWatson CWeizenegger M Werngren JWestman LWillery EWinkler SWong APYamaguchi NYamane NYan SWYates MYew WWYip CWYubero JYula EYzquierdo SZaitseva EZaldumbide MAZambrano MMZdelar-Tuk MZellweger J-PZenhorst RZerolo FJZerva LZhang JZmak LZolnir - Dovc MŽolnirDov- MZozio T


Levterova VLezcano MALima EJCLima KVBLocht CLockwood DLogarinho ELopes MLLópez AGLópez BLopez-Calleja AILoureiro CLucas FLuo TLuquin MLyashchenko KLyberopoulos PMacedo RMachado DMadruga MMaia PMaia PISMaio JNMak KXMalagari AIMalaquias AMalaspina ACMallard KMarceau MMarkova NMarques MMartín AMartin CMartín-Casabona NMartinez-Martinez LMartins MMartins MCMartins TGMasjedi MMasjedi MRMatic IMatos GMaugein JMazarrasa CFMbulo GMcNerney RMdivani NMédigue CMei JMello FAFMendes ACMendes NHMendes NHMenendez MCMestre OMeyers WMMézard MMichel GMihailelis EMilho CMilián YMillan IMillet JMin JHMiotto PMirabal NMiranda AMiyagi-Shiohira CMiyata MMoilleron RMokrousov I

Monecke SMonge IMoniz-Pereira JMonteiro GMontemayor MMontoro EMookherjee SMorcillo NMorgan KMosko MMota MMoulin LMoure RMoyoyeta MMuchwa CMugerwa RMugyenyi PMüllerova MMumbowa FMunga Waweru PMurcia MMusunsa AMuvwimi MMwamba PMwanza WNagiyev TNascimento INasu MNavarro ANeo ZYNeonakis IKNiemann SNikolaou SNina JNogueira CNogutia ENNoroozi JNoruzi JNovoa-Cain JNowroozi JNuak JNyirenda CNOberhauser BObrovac MObrovac MOcepek MOelemann MCOjeda POliveira POmar AROral Zeytinli UOrikiriza POrozco HO’Sullivan DPaasch FPalaci MPalomino JCPanaiotov SPando RHPandolfiJRCPapaventsis DPapiris SPardini MPark SK Pate MPaulo CPavan FPavan FRPawelczyk JPedrosa JPeeling R

Pellegrin JLPerdigão JPereira DRPereira EPereira Miguel JPérez Meixeira APerkins MPfeltz RPfyffer GPimentel MPinheiro MDPoelhsitz GVPole IPortaels FPortugal CPortugal IPossuelo LPost EPrata PProença FQazi FQuieng MDRacic IRadomski NRamos JRamos MartosRamos MHRamoutar DRaposo ARasolofo VRastogi NRauzier JRefrégier GRégis MReniero ARey EReyes ARibeiro JNRichter ERidell MRiley LWRitacco VRitmeijer KRobledo JRocha GRodrigues ARodrigues FRodrigues LRodrigues SRodríguez-Güell ERohde KHRosales JRoura-Mir CRuimy RRuiz PRumijowska-Galewicz ARüsch-Gerdes SRussell DGSaaed NS Sabino AŞahanKipalevASaid HSainti ASalas SSaluotsa MSalvadó MSalvignol GSampaio DSamper SSamutela MSánchez-Concheiro M

Sancho LSandoval ASantos ACBSantos CSantos RSaraiva MSardinha ESardinha TSarmento ASato DSato DNSchön TSecanella SPSeif SShamputa ICSharaf-Eldin GSShikama M-LShinnick TShinnick TMShoen CSiddiqi SSilva ASilva CSilva FSilva KSilva MSilva PSilva SSimões MFSing LHSingh JPNSkenders GSlickers PSola CSomoskövy ASousa ASSousa CSousa GSousa JGSovhozova NSoyal ASpandidos DASpicic SStoffels KSturegård ESuffys PNSupply PSvensson ETabarsei PTajeddin ETakiff HTancredo LTang YWTap JTavares MTavares Magalhães ATeles JMMTelles MASThibault VTonjum TTorrado ETortoli ETraore HTudó GTurner CUeki SYMValcheva VValdés IValente AValente FValente I

Van der Stuyft Pvan der Wel NVan Hoof Rvan Ingen JVan Soolingen DVaraine FVarghese BVasconcelos OVelayati A Velayati AAVia LEViallard JFViana BHJViana-Niero CVillar MVillela GViveiros MVladimirov KVon Groll AVultos TDWang SWarns MWatson CWeizenegger M Werngren JWestman LWillery EWinkler SWong APYamaguchi NYamane NYan SWYates MYew WWYip CWYubero JYula EYzquierdo SZaitseva EZaldumbide MAZambrano MMZdelar-Tuk MZellweger J-PZenhorst RZerolo FJZerva LZhang JZmak LZolnir - Dovc MŽolnirDov- MZozio T

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ÓRGÃO OFICIAL DA SOCIEDADE PORTUGUESA DE PNEUMOLOGIA

30.º CONGRESSO ANUAL DA SOCIEDADE EUROPEIA DE MICOBACTERIOLOGIA

30th ANNUAL CONGRESS OF THE EUROPEAN SOCIETY OF MYCOBACTERIOLOGY

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REVISTA PORTUGUESA DEPNEUMOLOGIAPORTUGUESE JOURNAL OF PULMONOLOGY

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Dr.ª Lurdes CarvalhoDr. J. Rosal Gonçalves

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Patologia do SonoCoordenador: Dra. Marta Drummond Secretário: Dra. Paula Pinto


R e v i s t a P o r t u g u e s a d e P n e u m o l o g i a

Vol XVI Suplemento 1 A Janeiro 2010

ÍNDICEINDEX

30.º CONGRESSO ANUAL DA SOCIEDADE EUROPEIA DE MICOBACTERIOLOGIA30th ANNUAL CONGRESS OF THE EUROPEAN SOCIETY OF MYCOBACTERIOLOGY

Introdução 5Introduction

A tuberculose em Portugal 7Tuberculosis in Portugal

A experiência brasileira de controlo da multidroga-resistência 11Brazilian experience in the management of multidrug-resistance

Forças evolutivas do Mycobacterium tuberculosis 21Evolutionary forces in Mycobacterium tuberculosis

Epidemiologia e ecologia de micobactérias não tuberculosas 27Epidemiology and ecology of nontuberculous mycobacteria

O uso da análise de libertação de gama interferão como auxiliar no controlo da tuberculose 31The use of interferon gamma release assays as an aid in the control of tuberculosis

Regulação da composição lipídica da parede celular do Mycobacterium tuberculosis e o seu efeito na persistência bacteriana in vitro 37Regulation of Mycobacterium tuberculosis cell wall lipid composition and its effects on in vitro bacterial persistence

Uma nova vacina viva contra a tuberculose com base na inativação do phoP 43A new live tuberculosis vaccine based on phoP inactivation

Simpósio: Testes rápidos para o rastreio preliminar da tuberculose 49Symposium: Point-of-care tests for tuberculosis

Criar capacidade laboratorial para a tuberculose: Necessidades e estratégias 57Building tuberculosis laboratory capacity: needs and strategies

Susana David

Miguel Villar

Fernando AF de Melo

Sebastien Gagneux

Joseph O Falkinham III

Jean-Pierre Zellweger

Lee W Riley

Jesus Gonzalo AsensioAinhoa Arbues

Dessi MarinovaCarlos Martín

Ruth NcNerney

Thomas M Shinnick


Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento e avaliação de novos métodos de diagnóstico da tuberculose 67The experience of the Brazilian Tuberculosis Research Network in the development and evaluation of new methods of diagnosing tuberculosis

Ensaios clínicos de novas drogas e testes diagnósticos em tuberculose: Desafi os micobacteriológicos 77Clinical trials of new tuberculosis drugs and diagnostic tests: mycobacteriological challenges

Avaliação das moléculas com atividade antiTB das plantas do cerrado brasileiro 83Screening of molecules with anti-TB activity from the brazilian cerrado plants

Novas ferramentas de fácil utilização para genotipagem padronizada e com qualidade controlada de estirpes do complexo Mycobacterium tuberculosis 89New, easy-to-use tools for standardised and quality-controlled genotyping of Mycobacterium tuberculosis complex strains

Simpósio: Avaliação externa da qualidade 95Symposium: External quality assurance

Editora convidada/Guest editor: Susana David

Afranio Kritski

Moisés Palaci

Karina de PrinceFernando R Pavan

Daisy N SatoWagner VillegasSergio RA LeiteClarice QF Leite

Caroline Allix-BéguecChristine Hubans

Stéphanie FerreiraPhilip Supply

Elvira Richter

R e v i s t a P o r t u g u e s a d e P n e u m o l o g i a4


A edição deste suplemento foi patrocinada com fi ns educacionais pelo Instituto Nacional de Saúde Doutor Ricardo Jorge e pela Fundação Luso-Americana para o Desenvolvimento/

This supplement is made possíble thanks to an unrestricted educational grant from Instituto Nacional de Saúde Doutor Ricardo Jorge and Luso-American Development Foundation

Este suplemento foi escrito ao abrigo do Novo Acordo Ortográfi co


R e v i s t a P o r t u g u e s a d e P n e u m o l o g i a S 5


IntroduçãoIntroduction

Susana David

A Sociedade Europeia de Micobacteriologia (ESM, http://www.esmycobacteriology.eu) é considerada uma das sociedades científicas internacionais mais ativas na área da mico-bacteriologia e doenças relacionadas. As reuniões da ESM, que têm lugar cada ano num país europeu diferente, promovem a comunicação de especialistas internacionais de renome, criando oportunidades para atua-lização do conhecimento dos últimos avan-ços científicos, compartilhar experiências e ideias e participar ativamente no desenvol-vimento da micobacteriologia.Este número especial da Revista Portuguesa de Pneumologia é dedicado ao 30.º Con-gresso Anual da Sociedade Europeia de Mi-cobacteriologia (ESM2009), que se realizou no Porto, Portugal, de 5 a 8 de julho de 2009. A organização deste congresso deveu--se à ESM e ao Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), o braço la-boratorial do sistema português de saúde (http://www.insa.pt).O Congresso ESM2009 deu particular aten-ção ao esforço global e multidisciplinar em micobacteriologia necessário na luta contra a tuberculose. As sessões científicas e minis-simpósios versaram os seguintes temas:

Programas de controlo da tuberculose;• Epidemiologia molecular e vigilância da • resistência a fármacos;Deteção da resistência a fármacos por • genética molecular;

The European Society of Mycobacteriology (ESM, http://www.esmycobacteriology.eu) is considered one of the most active interna-tional scientific societies in the area of my-cobacteriology and related diseases. The ESM meetings, held each year in a different country of Europe, promote the exchange of distinguished experts from all around the world creating the opportunity to update information in the front-line of scientific achievement, share experience and ideas, and actively participate in contribution to the field of mycobacteriology.This special issue of the Revista Portuguesa de Pneumologia (RPP) is dedicated to the 30th Annual Congress of the European Society of Mycobacteriology (ESM2009) hosted in Por-to, Portugal, from July 5-8, 2009. This con-gress was co-organized by the ESM and the National Health Institute Doutor Ricardo Jorge (INSA), the laboratory arm of the Por-tuguese health system (http://www.insa.pt).The ESM2009 congress gave particular at-tention to the multi-disciplinary effort needed, from mycobacteriologists on a world wide basis, in the fight against tuber-culosis. Scientific sessions and mini-sympo-sia covered the following themes:

Tuberculosis control programs;• Molecular epidemiology and drug resis-• tance surveillance;Molecular genetic detection of drug re-• sistance;

1 Laboratório Nacional de Referência das Infecções Respiratórias para as Micobactérias da Unidade de Referência e Vigilância Epidemiológica do Departamento de Doenças Infecciosas do Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal

E-mail: [email protected]

Pneumologia 16 Supl 1A - Miolo - 4ª PROVA.indd Sec1:5Pneumologia 16 Supl 1A - Miolo - 4ª PROVA.indd Sec1:5 11-05-2010 14:35:2211-05-2010 14:35:22

R e v i s t a P o r t u g u e s a d e P n e u m o l o g i aS 6


introduçãoSusana David

Testes de susceptibilidade e tratamento • da tuberculose;Micobactérias não tuberculosas;• Novas questões no papel do laboratório • de tuberculose;Desenvolvimento de vacinas e patogenia;• Testes • point-of-care para tuberculose;Fortalecimento do laboratório;• Desenvolvimento farmacológico;• Aspetos práticos e controlo de qualida-• de na epidemiologia molecular;Controlo de qualidade externo.•

Este número é constituído por uma com-pilação de breves comunicações baseadas nos temas de algumas das principais confe-rências. O programa completo pode ser consultado nos websites da ESM e do INSA ou em http://www.esm2009.org.Gostaríamos de manifestar o nosso especial agradecimento ao Dr. Renato Sotto-Mayor e à Revista Portuguesa de Pneumologia pelo seu interesse na publicação deste número e a to-dos os que amavelmente deram a sua contri-buição científica. A realização do Congresso ESM 2009 não teria sido possível sem o apoio das instituições: Fundação Calouste Gulbenkian (http://www.gulbenkian.pt); Fundação Luso--Americana de Desenvolvimento (http://www.flad.pt); STOP-TB Working Group on New Diagnostics, Point of Care sub group (www.stop tb.org) e patrocinadores: HAIN LifeScien-ces (http://www.hain-lifescience.com); Becton Dickinson (http://www.bd.com); Quilaban (http://www.quilaban.pt); BioMerieux (http://www.biomerieux.com); Cepheid (http://www.cepheid.com); Microsens (http://www.micro-sens.co.uk); Genoscreen (http://www.genoscre-en.com).

Susana DavidPresidente do Congresso ESM2009

Susceptibility testing and treatment of • tuberculosis;Non tuberculous mycobacteria;• Issues in the modern tuberculosis labo-• ratory;Vaccine development and pathogenesis;• Point-of-care tests for tuberculosis;• Laboratory strengthening;• Drug development;• Practical aspects and quality assurance • in molecular epidemiology;External quality assurance.•

This issue is comprised of a compilation of short communications based on the themes of some of the main conferences. The full program may be consulted on the ESM and INSA websites or at http://www.esm2009.org.We would like to give special thanks to Dr. Renato Sotto-Mayor and to the RPP for their interest in putting together this issue and to all those who kindly accepted to give their scien-tific contribution. The ESM2009 congress would not have been possible without the support of the partners: Fundação Calouste Gulbenkian (http://www.gulbenkian.pt); Lu-so-American Foundation (http://www.flad.pt); STOP-TB Working Group on New Di-agnostics, Point of Care sub group (www.stoptb.org) and sponsors: HAIN LifeSciences (http://www.hain-lifescience.com); Becton Dickinson (http://www.bd.com); Quilaban (http://www.quilaban.pt); BioMerieux (http://www.biomerieux.com); Cepheid (http://www.cepheid.com); Microsens (http://www.microsens.co.uk); Genoscreen (http://www.genoscreen.com).

Susana DavidChairman of the ESM2009 congress




30.º Congresso Anual da Sociedade Europeia de Micobacteriologia30th Annual Congress of the European Society of Mycobacteriology

Miguel Villar1

A tuberculose (TB) é um problema glo-bal com um número estimado de 9 mi-lhões de novos casos por ano, 83% dos quais se situam na África subsariana e no Sudeste Asiático, onde se encontram mui-tos dos países com a maior carga de TB (Fig. 1). A Organização Mundial de Saú-de (OMS) estima a emergência global de cerca de meio milhão de novos casos de tuberculose multirresistente (MDR -TB), por ano, incluindo 50 000 casos de tu-berculose extensamente resistente a fárma-cos (XDR -TB).Em 2007, a União Europeia (UE) teve uma incidência média de 17/100 000, com Por-tugal a registar um dos mais elevados índices na UE (27/100 000). Nas últimas duas dé-cadas, a incidência em Portugal tem vindo a diminuir consistentemente, e em mais de 7% anualmente nos últimos 5 anos (Fig. 2). Esta diminuição é mais notória no grupo etário dos 25 aos 44 anos, o que resulta numa alteração da média de idades, tanto nos do-entes nacionais como nos imigrantes (Fig. 3).

Tuberculosis is a global problem with an es-timated 9 million new cases per year, 83% of which occur in Sub-Saharan Africa and South-East Asia, where many of the high burden countries are found (Fig. 1). The World Health Organisation (WHO) esti-mates an emergence of about half a million new cases of multidrug-resistant tuberculo-sis (MDR-TB), globally, each year, includ-ing 50 000 of extensively drug-resistant tu-berculosis (XDR-TB).In 2007, the European Union (EU) had an incidence rate of 17/100 000, with Portugal registering one of the highest rates in the EU (27/100 000).Over the last two decades, the incidence in Portugal has decreased consistently, and more than 7% per year, in the last five years (Fig. 2). This reduction is mainly in the age group between 25 and 44 years old, leading to a shift to the right of the median age, both in national citizens and in immigrants (Fig. 3). The foreign-born have represented about 12% of the TB cases, and the preva-

A tuberculose em Portugal

Tuberculosis in Portugal

1 Em representação do Director-Geral de Saúde/On behalf of the General-Director of Health




a tuberculose em portugalMiguel Villar

Os estrangeiros têm representado cerca de 12% dos casos, e a prevalência de VIH tem sido de aproximadamente de 14%, com uma redução de 34% nos últimos cinco anos.Entre 2003 e 2007, a maioria dos casos de TB foram das formas pulmonares (74,1%), dos quais 67,5% SS+ e 75,3% confirmados

lence of HIV has been around 14%, with a 34% reduction in the last five years. Between 2003 and 2007, most TB cases were pulmonary forms (74.1%), 67.5% of which were SS+ and 75.3% were confirmed by culture. MDR-TB and XDR-TB, during the same period, represents 1.9% (154) of

Fig. 1 – Taxas de incidência da tuberculose por país, 2006. Fonte: OMS

Fig. 1 – Tuberculosis incidence rates, by country, 2006. Source: WHO

Casos de tuberculose notifi cados (novos e relapsos) por cada 100 000 habitantes/Notifi ed TB cases (new and relapse) per 100 000 population

0-2425-4950-99100 ou mais/100 or moreNão reportado/No report

Fig. 2 – Evolução das taxas de incidência de tuberculose notifi cada no continente e regiões autónomas (todas as formas, 10–5 habitantes). Fonte: DGS

Fig. 2 – Evolution of the tuberculosis incidence rates notifi ed in Mainland Portugal and Autonomous Regions (all forms/10-5

inhabitants). Source: DGS





por cultura. Neste mesmo período, os casos de MDR -TB e XDR -TB representam 1,9% (154) de todos os casos de tuberculose no início do tratamento, variando de 1,3% (22), em 2006, e de 2,4% (38), em 2004, com uma média de 31 casos por ano (1,9%). Estes números são representativos, já que a cobertura dos testes de sensibilidade a fár-macos (TSF) é superior a 80%.No que respeita ao tema deste congresso, a Rede de Laboratórios de Micobacteriologia é uma componente importante do Progra-ma Nacional para a Tuberculose (PNT), com colaboração na deteção e definição de casos, diagnóstico precoce de MDR -TB, identificação de Mycobacterium tuberculosis e realização de TSF de 1.ª e 2.ª linha. Para além da confirmação dos casos de TB, permite -nos, também, acompanhar o índice de negatividade, especialmente até aos dois meses de tratamento, o que é importante para o controlo da transmissão e da eficácia do tratamento. No mesmo período, verificou -se um índice de negatividade do-cumentada de 34% aos dois meses.

the TB cases at the beginning of treatment, varying from 1.3% (22), in 2006, and 2.4% (38), in 2004, with an average of 31 cases per year (1.9%). These proportions are re-presentative, since the coverage of drug sen-sibility tests (DST) is over 80%.As regards the theme of this Congress, the Mycobacteriology Laboratory Network is an important component in the National Tuberculosis Programme (NTP), with col-laboration in case detection, case defini-tion, early diagnosis of MDR-TB, identifi-cation of Mycobacterium tuberculosis and performance of 1st and 2nd line DST. Be-sides confirmation of TB cases, it also al-lows us to accompany the negativity rate, especially up to two months of treatment. This is important for the control of trans-mission and treatment efficacy. In the same period mentioned above, we had 34% doc-umented negativity at two months.The Laboratory is also very useful in the screening strategy of the tuberculosis sur-veys, mainly through the use of nuclei c-acid amplification tests (NAAT). Analysing the

Fig. 3 – Evolução da taxa de incidência de tuberculose notifi cada, por gru-pos etários (105 habitantes). Fonte: DGS

Fig. 3 – Evolution of the tuberculosis incidence rates, according to age groups (105 inhabitants). Source: DGS




O laboratório é também muito útil na estra-tégia de monitorização dos rastreios da tuber-culose, principalmente pelos testes de am-pliação do ácido nucleico (TAAN). Ao analisar os resultados, como um complemen-to importante da estratégia DOTS, obtive-mos um índice de sucesso de 85,6% na po-pulação em geral, de acordo com os objetivos da OMS. No que se refere aos grupos de ris-co, o índice de sucesso no grupo sem risco foi de 90%, mas, nos outros grupos, e de acordo com os objetivos da OMS, foi nomeadamen-te de 69% no de TB/VIH, de 53% no de MDR -TB e de 82% no de imigrantes.Finalmente, terminámos a nossa apresenta-ção abordando a estratégia para controlo de MDR/XDR -TB em Portugal, através da criação, em junho de 2007, do Centro Na-cional de Referência da Tuberculose Multi--Resistente (Circular Informativa 14 -DT, da Direcção -Geral de Saúde). Os seus prin-cipais objetivos são os de reduzir a prevalên-cia e evitar a transmissão da MDR -TB, apoiar os médicos na escolha dos regimes terapêuticos, colaborar com eles no ajuste desses regimes por efeitos adversos e, se ne-cessário, na determinação do internamento dos doentes e nas condições de isolamento.Em 2009, uma das grandes prioridades do PNT foi a implementação de uma rede de centros regionais de referência para trata-mento de casos de MDR/XDR -TB.

outcomes is an important complement of the DOTS Strategy; we had an 85.6% suc-cess rate in the general population, in ac-cordance to the WHO goals. As to risk groups, the success rate in the risk-free group was of 90%, but under WHO goals, in the other risk groups, it was namely 69% in TB/HIV, 53% in MDR TB and 82% in immigrants.Finally, we finished our presentation ad-dressing the strategy for MDR/XDR-TB control in Portugal, through the creation of the National Reference Centre for Multi-drug-resistant Tuberculosis, in June 2007 (policy document 14-DT, General-Direc-torate of Health). Its main objectives are to reduce prevalence and prevent transmission of MDR-TB, support clinicians in choosing the therapeutic regimens, collaborate with them in the management of adverse effects and, if needed, in the hospitalization and isolation of patients in the best possible conditions.In 2009, one of the high priorities of NTP was the implementation of a network of re-gional reference centres for the treatment of MDR/XDR TB cases.





Fernando Augusto Fiuza de Melo1 A experiência brasileira de controlo da multi droga--resistência

Brazilian experience in the management of multidrug-resistance

1 Médico, Diretor do Instituto Clemente Ferreira – Coordenadoria de Controle de Doenças da Secretaria de Estado da Saúde de São Paulo – ICF/CCD/SES -SP/Physician, Director, Instituto Clemente Ferreira, Coordinator of Disease Control of the Secretary of State for Health of São Paulo – ICF/CCD/SES -SP

Doutorado em Medicina, área de pneumologia, pela Escola Paulista de Medicina da Universidade Federal de São Paulo – EPM/UNIFESP/PhD, Pulmonology, Escola Paulista de Medicina da Universidade Federal de São Paulo – EPM/UNIFESP

Membro do Comitê de Assessoria Técnico -Científico do Programa Nacional de Controle da Tuberculose do Ministério da Saúde (PNCT/MS)/Member of the Technical--Scientific Committee of the Ministry of Health’s National Tuberculosis Control Programme (PNCT/MS)

Correspondência/Correspondence to:Rua Santo Estácio, 248Cidade Vargas, CEP 04319 -010 – São Paulo, SP, BrasilTele -fax: 0055 11 3218 8653Telemóvel: 0055 11 8469 4330e -mail: [email protected]

ResumoNeste artigo de revisão, o autor faz uma revisão de como evoluiu a abordagem da tuberculose multirresis-tente (MDR) no Brasil, desde a introdução da rifampi-cina associada a isoniazida e a pirazinamida (RHZ). Mostra que o país foi um dos primeiros no mundo a aplicar o esquema RHZ dentro de um sistema de tra-tamento com um esquema de primeira linha, outro específico para as formas meningoencefálicas, para re-tratamento para recidivas ou retorno com tuberculose ativa após abandono, e um esquema de reserva. O sis-tema era de aplicação nacional com garantia de forne-cimento gratuito das drogas e autoadministrado. Avalia a evolução da resistência aos medicamentos, a emer-gência da resistência múltipla e como foi organizado o controlo desta forma da doença.

Palavras -chave: Tuberculose multirresistente a múl-tiplos medicamentos (TB -MDR), tuberculose super-resistente (TB -XDR).

AbstractIn this article the author reviews the evolution of the approach to multidrug-resistant tuberculosis (MDR-TB) in Brazil following the introduction of rifampicin associated to isoniazid and pyrazinamide (RHZ). It shows Brazil was one of the world’s first countries to use the RHZ regimen within a treatment system, with a first line regimen, another one specific for meningo-encephalic forms, for re-treatment of recurrences or of patients who returned with active tuberculosis after abandoning treatment, and a reserve regimen. The sys-tem was applied nationwide with guaranteed cost-free provision of medication, and self-administered. The author evaluates the growth of drug resistance, the emergence of multidrug-resistance and how manage-ment of this form of the disease has been organised.

Key-words: Multidrug-resistant tuberculosis (MDR-TB), extensively drug-resistant tuberculosis (XDR-TB).




IntroduçãoEmbora seja um fenômeno mundial, a tu-berculose (TB) apresenta características va-riáveis de acordo com a região do mundo, dependente de fatores raciais, ecológicos, socioeconômicos, interrelações com outras endemias, como a da VIH/SIDA, perfil de resistência às drogas em uso e do desenvolvi-mento do controlo da doença.Assim como a TB, também a tuberculose multi droga -resistente (MDR -TB), que es-pantou o mundo, apresenta um desenvolvi-mento próprio no Brasil1,2,3.

O sistema brasileiro de tratamento da tuberculoseO Brasil foi o primeiro país não desenvolvi-do a usar esquema de curta duração com participação da rifampicina (R) associada a hidrazida (H), além da pirazinamida (Z), ao reorganizar em todo o país o Programa Na-cional de Controle da Tuberculose, do Mi-nistério da Saúde (PNCT -MS), em 1979.Essa mudança estabelecida, após um ensaio dirigido comparando o esquema RHZ com esquema de longa duração (12 meses) com a H associada à estreptomicina (S) e ao etam-butol (E)4, estabelece, não somente altera-ções de medicamentos, mas normatiza um sistema de tratamento.Esse sistema (Fig. 1) conta com um esque-ma de primeira linha, de curta duração, o E -1 (2RHZ/4RH), indicado para todas as formas de TB sem tratamento anterior, me-nos para a forma meningoencefálica, com uso de corticosteroides na fase de ataque e prolongando a medicação para nove meses, o E -2 (2RHZCort/7RH). Para os recidivan-tes após cura (RC) ou reingresso após aban-dono com doença ativa (RA), a indicação

IntroductionAlthough a worldwide phenomenon, the characteristics of tuberculosis (TB) vary in line with the world region, depending on racial, ecological and socioeconomic factors, inter-relationships with other endemics, such as HIV/AIDS, the resistance profile of the drugs in use and the development of management of the disease.Similarly to TB, multidrug-resistant tuber-culosis (MDR-TB), which has alarmed the world, has had its own development in Brazil1-3.

The Brazilian tuberculosis treatment system

Brazil was the first developing country to use a short-course regimen involving rifampicin (R) associated to isoniazid (H) and pyrazi-namide (Z), in reorganising a nationwide Tu-berculosis Control Programme of the Minis-try of Health (TBCP-MH), in 1979.After a trial comparing the RHZ regimen with a 12-month long combination regi-men with the association of H streptomycin (S) and ethambutol (E)4, this change, once instituted, established changes in medica-tion and harmonised a treatment system.This system (Fig. 1) includes a short-course first line regimen, the E-1 (2RHZ/4RH), suitable for all forms of TB without prior treatment, except the meningoencephalic form, using corticosteroids during the attack stage and prolonging the medication for nine months, the E-2 (2RHZCort/7RH). The same E-1 was prescribed for recurren ces after cure (RC), or for patients who relapsed with active disease (RA) after abandoning treatment, reinforced with ethambutol E

a experiência brasileira de controlo da multidroga-resistênciaFernando Augusto Fiuza de Melo




do mesmo E -1, reforçado com o etambutol (E) na fase de ataque, o E -1R. Para os que apresentassem falência com o E -1, foi pre-visto um esquema de segunda linha, com duração de 12 meses, onde além da S, Z e E, se associava a etionamida (Et), o E -3 (3SZEEt/9EEt)5 (Fig. 1).A dose média da H no país é de 10 mg/kg//dia, bem maior do que a usada internacio-nalmente. As características operacionais básicas do sistema eram a aplicação nacio-nal, a garantia de fármacos, gratuita, com R+H em um único comprimido e de uso autoadministrado.Para os fracassos com o E -3, a orientação era encaminhar o doente para referências, a fim de avaliar drogas alternativas, se acessíveis, ou condutas cirúrgicas, se possíveis5.

during the attack stage, the E-1R. In those for whom the E-1 failed, there was a 12-month long second line regimen, which associated ethionamide (Et) to S, Z and E; the E-3 (3SZEEt/9EEt)5 (Fig. 1).The mean dosage of H used in Brazil is 10 mg/kg/day, substantially higher than that used internationally. The system’s basic logis-tical characteristics were: cost-free application nationwide, guaranteed medication, R+H in a single tablet to be self-administered.Patients whom E-3 failed were referred to reference units for evaluation of alternative medication, if available, or surgical proce-dures, if possible5.

Evolution of the system’s resistance and successA reference centre cohort study evaluated the growth of resistance in three-yearly cohorts in the 1960s, 1970s and 1980s. It showed that


E-12RHZ/4RH

E-2(m)2RHZ*/7RH

*steroid

NT

AR

CR

DEATH

CURE

ABANDON

E-1R

2RHZE/4RH

E-3

3SZEEt/9EEt

F F

FAILUREF F

F F TBMR

Fig. 1 – O sistema de tratamento da tuberculose no Brasil, 1999

Fig. 1 – Tuberculosis treatment system in Brazil, 1999




Evolução da resistência e do rendimento do sistemaUm estudo de coortes, numa referência, avaliou evolutivamente a resistência em co-ortes trienais nas décadas de 1960, 1970 e 1980, mostrando que o PNCT organizado na década de 1960 e a introdução da R na sua reorganização, na década seguinte, pos-sibilitaram uma excepcional proteção das drogas clássicas com significativa redução da resistência à H e à S. A redução entre 1960 e 1970 (p=0,002) foi mais significativa que entre 1970 e 1980 (p=0,032), sugerindo que um bom programa protege melhor a re-sistência do que a introdução de uma droga potente, como a R (Fig. 2)6.A avaliação na rotina do rendimento do E -1 na década de 1980, excluindo as transferên-cias e considerando as mudanças de drogas por toxicidade nos estudos de coortes, apre-sentou uma taxa de eficácia/efetividade de 94,6/77,8; com taxa de abandono de 13,7%,

the TBCP organised in the 1960s and the in-troduction of R in its reorganisation in the 1970s provided exceptional protection of the classic drugs with significant reduction in resis-tance to H and S. The reduction between 1960 and 1970 (p = 0.002) was more significant than that between 1970 and 1980 (p = 0.032), suggesting that a good programme offers better protection against resistance than the introduc-tion of a potent drug such as R (Fig. 2)6.The routine evaluation of the success of E-1 in the 1980s, excluding transfers and taking into consideration medication changes due to toxicity in the cohort studies, showed a 94.6/77.8 rate of efficacy. It had a 13.7% rate of abandonment, a 1.5% rate of failure, a 3.1% rate of change due to adverse effects and a 3.9% mortality rate7. In the 1990s and early 2000s (1990-2002) these rates were 93.9, 77.1, 13.1, 1.7, 3.3 and 4.8%, respectively8. The success rate of the reserve E-3 was low, with a rate of efficacy ranging

Fig. 2 – Evolução da resistência primária durante três décadas: 1960, 1970 e 1980 – ICF/SP

Fig. 2 – Evolution of primary resistance over three decades: 1960’s, 1970’s and 1980’s – ICF/SP





de falência de 1,5%, troca por efeitos adversos de 3,1% e de mortalidade de 3,9%7. Na déca-da de 1990 e início do novo milênio (1990--2002), estas taxas foram de 93,9, de 77,1, de 13,1, de 1,7, de 3,3 e de 4,8%, respetivamen-te8. Quanto ao E -3 de reserva, o rendimento foi baixo, com a taxa de eficácia/efetividade variando entre 57,5/85,2 e 66,7/84,7, respeti-vamente9.A piora da efetividade do sistema foi rela-cionada com o abandono, certamente re-sultante do regime autoadministrado e a emergência da coinfecção TB -VIH/SIDA, apresentando uma tendência de melhoria pela introdução e expansão do tratamento supervisionado e a boa qualidade do pro-grama de controlo do VIH no país.

A evolução e a abordagem resistência múltipla no BrasilCom base nestes números, acrescentando as taxas de RC e RA, em 1993 estimou -se que cerca de 4 a 5% dos notificados acabariam por apresentar resistência associada a R+H ou impossibilidade de uso destas duas drogas por toxicidade, com indicação do E -3. Seriam portadores de MDR -TB, chamados entre nós resistentes ao E -1. Como o rendimento do E -3 é baixo, um número de doentes acaba por sair do sistema sem perspetivas de tratamento com as drogas programáticas (R, H, Z, E, S e Et), estimado entre 0,3 e 0,4% dos notifica-dos anuais, denominados portadores de tu-berculose multirresistente (TBMR)10.Para esses doentes, teoricamente sem perspecti-vas terapêuticas com as drogas programáticas, a orientação das normas ministeriais no passado era que fossem encaminhados para unidades de referências sem lhes oferecer recursos e con-dições para sua atenção e abordagem.

from 57.5/85.2 to 66.7/84.7, respectively9.The system’s worsening rate of efficacy was related to abandonment, almost certainly a result of the self-administration regimen and the emergence of the TB-HIV/AIDS co-infection, tending towards improvement with the introduction and expansion of su-pervised treatment and the good quality of Brazil’s HIV management programme.

The progress and management of multidrug-resistance in BrazilBased on those numbers, to which are ad-ded the rates of RC and RA, in 1993 it was estimated that around 4 – 5% of those noti-fied as having the disease presented resis-tance associated to R+H or the impossibility of the use of these drugs due to toxicity, with indication for E-3. These patients had MDR-TB, and were classified as resistant to E-1. As E-3 has a low success rate, a number of patients ended up leaving the system with no prospect of treatment with the pro-grammed drugs (R, H, Z, E, S and Et). These were estimated as ranging from 0.3 – 0.4% of the annual number of those noti-fied and known to carry multidrug-resistant tuberculosis (MDR-TB)10. For these pa-tients, in theory with no perspectives of treatment with the programmed drugs, ear-lier Ministry norms referred them to refe-rence units without offering the latter the resources and possibilities to treat and ma-nage them.The first attempts at treatment were put together by state programmes (provincial) with bigger and better conditions. In the later 1980s and early 1990s there were se-veral trials of alternative regimens. All the programmed drugs, lung resection surgery





As primeiras tentativas de tratamento acaba-ram sendo realizadas por programas estaduais (provinciais), com maiores e melhores con-dições. Na segunda metade dos anos de 1980 e início da década de 1990, surgiram diver-sas experiências de esquemas alternativos. Associava -se de uma só vez todas as drogas programáticas, cirurgias de ressecção pulmo-nares e uso de antigos fármacos não norma-tizados pelo PNCT, como a canamicina (KM), o ácido para -aminossalicílico (PAS), tiossemicarbazona (TSCZ) e outros. Foram testadas a clofazenina (CFZ) usada no trata-mento da hanseníase, a amicacina (AM) e, mais recentemente, as novas quinolonas. A efetividade dos esquemas tomava como base a cura ou mesmo a sobrevida maior encon-trada entre os que evoluíam sem tratamento alternativo11,12,13.Em 1995, o Centro de Referência Profes-sor Hélio Fraga, do MS (CRPHF/MS)/Rio de Janeiro, organiza um protocolo na-cional com esquema associando AM, levo-floxacino (LFX), terizidona (TRZ), CFZ e E (se ainda sensível), testado em três centros de referências entre 1995 a 1997, com taxas de eficácia/efetividade razoáveis (n=187 -56/48), quando confrontados com estudos internacionais14.Em 2000, inicia no CRPHF/MS o Progra-ma de Vigilância Epidemiológica de TBMR e em 2004 um convénio com a associação civil brasileira sem fins lucrativos, “Projeto MSH” (Management Sciences for Health), e com recursos financiados pela USAID (Uni-ted States Agency for International Develop-ment), garante o financiamento das drogas alternativas. Um guia de vigilância epide-miológica de TBMR foi elaborado conden-sando o conhecimento acumulado no país, estabelecendo normas para o diagnóstico,

and use of earlier drugs non-standardized by the TBCP, such as kanamycin (KM), p-aminosalicylic acid (PAS), thiosemicarba-zone (TSCZ) and others were simultane-ously contemplated. Clofazimine (CFZ) used to treat hanseniasis, amikacin (AM) and, more recently, the new quinolones were tested. Cure or even the greater sur-vival seen in those who progressed without alternative treatment was the basis for as-sessing the regimens’ efficacy11-13.In 1995, the Centro de Referência Professor Hélio Fraga do MS (CRPHF/MS)/Rio de Janeiro organised a national protocol with a regimen associating AM, levofloxacin (LFX), terizidone (TRZ), CFZ and E (if still sensitive), tested in three reference cen-tres, from 1995 to 1997, with fair efficacy rates (n = 187– 56/48) in comparison with international studies14.In 2000, the CRPHF/MS initiated an MDR-TB epidemiological surveillance pro-gramme and, in 2004, had an agreement with a non-profit making Brazilian civil as-sociation “Project MSH” (Management Sciences for Health) financed by the USAID (United States Agency for International De-velopment), guaranteeing the financing of the alternative drugs. A guide to MDR-TB epidemiological surveillance was drawn up, summarising the country’s collected know-ledge, establishing diagnostic, treatment, prevention and biosafety norms, epidemio-logical surveillance guidelines, human re-sources training and financial support for carrying out the programme. A nationwide notification system specifically for MDR-TB was instituted15.Subsequent studies and routine review of notifications showed much smaller numbers than predicted, with regional variations dif-





tratamento, prevenção e biossegurança, orientações para a vigilância epidemiológi-ca, formação de recursos humanos e provi-são de recursos materiais para execução do programa. Foi instituído um sistema de no-tificação específico para a TBMR, aplicado nacionalmente15.Estudos posteriores e revisão de notificados na rotina acabaram por apresentar números bem menores do que os estimados, com va-riações regionais diferenciadas pela situação sócioeconômica, pela qualidade do progra-ma local e por uma provável subnotificação devido à baixa oferta da cultura e testes de sensibilidade15 (Fig. 3).O esquema alternativo atual para os porta-dores de TBMR usado no país dentro desse programa é: AM (ou S se sensível) por 12 meses; OFX, TRZ e E (se sensível) por 18 meses e Z (se sensível) por seis meses. Al-guns serviços usam, ao invés da Z, o metro-nidazol (MTZ), por 18 meses. As taxas de cura variam entre 62 a 85%, com o abando-

ferentiated by local socioeconomic situa-tions, the quality of the local program and by a probable under-notification due to the meagre amount of culture and sensitivity tests available15 (Fig. 3).The current alternative regimen for MDR-TB patients used in Brazil within the pro-gramme is AM (or S if sensitive) for 12 months; OFX, TRZ and E (if sensitive) for 18 months and Z (if sensitive) for six months. Some Units use metronidazol (MTZ) instead of Z for 18 months. Cure rates range from 62% to 85%, the rate of abandonment from 5-7%, failure 10-15% and the mortality rate dropped from 33% to 11%, depending on the greater or lesser degree of organisation and quality of treat-ment15,16.The MDR-TB cases were mostly post-pri-mary (74-80%), with the primaries around 6-8%, especially between contacts and risk groups (conscripts, the homeless, health care professionals and others with high ex-

Fig. 3 – Casos de TBMR no Brasil (1994-2006) (n = 2632 casos – incidência anual média: 75 000 casos/ano)

Fig. 3 – MDR-TB cases in Brazil (1994 -2006) (n=2632 cases, mean annual rate 75 000 cases/year)





no entre 5 e 7%, a falência entre 10 e 15% e o óbito decresceu de 33 para 11%, depen-dendo do maior ou menor grau de organiza-ção e qualidade do atendimento15,16.Os casos de TBMR são maioritariamente pós--primários de 74 a 80%, sendo os primários em torno de 6 a 8%, especialmente entre con-tatos e grupos de risco (conscritos, sem-abrigo e profissionais de saúde e outros com alta ex-posição) e, 11 a 20%, indeterminados (ausên-cia de informações capazes de estabelecer o grupo). A ocorrência de TBMR entre co-infectados pelo VIH no país é baixa, entre 1,6 e 3%. Não se pode afirmar se há uma tendên-cia a manter esse número ou se deve aumentar a ocorrência, ou mesmo se não existe uma subnotificação15,16,17. A grande maioria dos ca-sos apresenta lesões pulmonares bilaterais, cer-ca de 80%, o que inviabiliza condutas cirúrgi-cas de rotina, seja associado ao tratamento ou higiénicas (?), para prevenir recidiva, como propõem alguns cirurgiões no país18.Casos de TB superresistentes (TB -XDR), com resistência a duas drogas usuais e três al-ternativas, observados a partir de 2000, vêm sendo encontrados no Brasil. As baixas ofer-tas de testes de sensibilidade, em especial para drogas alternativas, dificultam a seleção des-sas formas da doença. No Instituto Clemente Ferreira, que conta com TS automatizados e tem convénios com serviços que avaliam a resistência pelo MIC em meio líquido e lei-tura pela técnica de Alamar -Blue, um levan-tamento recente documentou 34 casos resis-tentes a quinolonas, sendo 16 resistentes a AM e 18 a S. Tratados com o esquema alter-nativo indicado para TBMR, o resultado foi nove curas e 25 falências, entre estes 17 óbi-tos. Um dado alarmante foi o encontro de três casos de TB -XDR primários, um indício de que bacilos superresistentes podem estar

posure) and 11-20% undetermined (lack of information capable of establishing a group). There is a low incidence of MDR-TB in patients co-infected with HIV in Brazil, between 1.6% and 3%. We cannot state if there is a trend to maintain this number or if it will increase, or even if there was under-notification15-17. The vast majority of cases, around 80%, present bi-lateral pulmonary lesions, which rules out routine surgical procedures, be it associat-ed to treatment or hygiene (?) to prevent recurrences, as some Brazilian surgeons propose18.Cases of extensively drug-resistant tubercu-losis (XDR-TB) with resistance to two usual and three alternative drugs, seen after 2000, have been observed in Brazil. The scarcity of drug sensitivity testing, in particular to al-ternative drugs, makes it hard to select for these forms of disease. In the Instituto Cle-mente Ferreira, which has automated sus-ceptibility testing and agreements with units which evaluate resistance using minimal in-hibitory concentration (MIC) in liquid me-dium and reading using the Alamar-Blue technique, 34 quinolone-resistant cases were recently documented with 16 resistant to AM and 18 to S. Treatment with the al-ternative regimen indicated for MDR-TB resulted in nine cures and 25 failures, with 17 deaths among the latter. A cause for alarm was finding three cases of primary XDR-TB, indicating that super-resistant bacilli could be in our midst and not only occasioned by treatment errors19,20.

Note: Brazil, based on a review of the prog-ress of primary and post-primary resistance, decided to change the initial E-1 with asso-ciation of E as the fourth drug in the attack





circulando no nosso meio, e não apenas pro-duzidos por erros terapêuticos19,20.

Nota: O Brasil, com base em revisão da evo-lução da resistência primária e pós -primária, decidiu e está alterando o E -1 inicial com associação do E como quarta droga na fase de ataque, com implantação gradativa no início de 2010. Foram considerados os au-mentos da taxa de resistência a H da TB--MDR em coinfetados TB -HIV/AIDS e da longevidade populacional com estoque de TB -latente selecionado nos 30 anos de uso de R+H. Essa mudança faz-se com o uso de associação dos fármacos na forma de com-primidos com dose fixa combinada (4 em 1), altera também o E -3 de reserva agora para doentes portadores de TB -MDR e pas-sa a considerar os que não conseguem a cura dentro do sistema como TB -XDR21.

Bibliografia/Bibliography1. Melo FAF. Evolução dos conhecimentos, o controle e algumas questões pendentes na tuberculose pulmonar multirresistente, Pneumologia: atualização e reciclagem. Sociedade Paulista de Pneumologia e Tisiologia 2003; 5:285 -292.2. CDC/USA. National action plan to combat of multidrug -resistant tuberculosis. Meting the challenge of multidrug -resistant tuberculosis: summary of a con-ference. Management of persons exposed to multidrug--resistant tuberculosis. MMWR 1992; 41(RR -11).3. Snider DE Jr, Roper WL. The news tuberculosis (Eds.). N Engl J Med 1992; 267:703 -705.4. Gerhardt G, Teixeira GM, Hijjar MA, Feitosa JVP, Penna MLF. Resultados iniciales del tratamiento de corta duración en condiciones de rutina en los servicios de salud del Brasil. Bol UICT 1982; 57:87 -92.5. Ministério da Saúde/Fundação Nacional de Saúde/Centro de Referência Prof. Hélio Fraga -Rio de Janeiro – Sociedade Brasileira de Pneumologia e Tisiologia. Controle da tuberculose: uma proposta de integração ensino -serviço. 5.ª ed. 2000.

6. Melo FAF, Afiune JB, Ribeiro LHG, Almeida, EA, Castelo A. Resistência primária do M. tuberculosis num serviço ambulatorial de referência em São Paulo: evolução por três décadas e comparação com outros es-tudos nacionais. J Pneumol 1996; 22:3 -8.7. Ministério da Saúde/Fundação Nacional de Saúde/Centro de Referência Prof. Hélio Fraga -Rio de Janeiro Documento Básico da Reunião de Avaliação operacional e epidemiológica do PNCT na década de 80. Bol Pneu-mol Sanit 1993, número especial.8. Ministério da Saúde/Secretaria de Vigilância em Saúde/Centro de Referência Prof. Hélio Fraga -Rio de Janeiro. Análise da situação da tuberculose no Brasil nos anos 90 e início da década atual. Bol Pneumol Sanit 2005; 13:133 -179.9. Campos HS, Melo FAF. Efetividade do esquema 3 (3sSZEEt/9EEt) no retratamento da tuberculose na rotina das unidades de saúde. Bol Pneumol Sanit 2000; 8:7 -14.10. Melo FAF, Ide Neto J, Seiscento M, Pinto JA, Afiune JB. Tuberculose multirresistente. J Pneumol 1993; 19:73--82.

stage, phased in from 2010 on. The in-creased rates of resistance to H, MDR-TB in patients co-infected with TB-HIV/AIDS and longevity of the population with a stock of latent TB selected in the 30 years of use of R+H were considered. This change is in the use of the association of drugs in tablet form with fixed combined dosage (4 in 1) and also changes the reserve E-3 now for MDR-TB patients and considers those who cannot find a cure within the system as XDR-TB cases21.





11. Picon PD, Pereira AN, Dias CA. Tuberculose pul-monar: análise terapêutica de 62 casos crônicos. Rev AMERIGS 1980; 24:36 -8.12 Comissão de terceira linha do Hospital Sanatório Partenon: Eficácia terapêutica do esquema de terceira linha ofloxacina–amicacina–tiacetazona-hidrazida para tuberculose multirresistente. J Pneumol 1995; 21:215--224.13. Seiscento M, Fiuza de Melo FA, Ide Neto J, No-ronha AML, Afiune JB, Inomata T, Cruz ML. Tubercu-lose multirresistente (TBMR): aspectos clínico -laborato-riais, epidemiológicos e terapêuticos. J Pneumol 1997; 23:237 -244.14. Dalcolmo MP, Fortes A, Melo FAF, Motta R, Ide Neto J, Cardoso N, Andrade M, Barreto AW, Gerhardt G. Estudo de efetividade de esquemas alternativos para o tratamento da tuberculose multirresistente no Brasil. J Pneumol 1999; 25:70 -77.15. Ministério da Saúde/ Secretaria de Vigilância em Saúde/Centro de Referência Prof. Hélio Fraga -Rio de Janeiro. Tuberculose multirresistente: guia de vigilância epidemiológica; Rio de Janeiro, 2007.

16. Ministério da Saúde/Secretaria de Vigilância em Saúde/Centro de Referência Prof. Hélio Fraga -Rio de Janeiro. Dados coletados no Sistema de Informação da Tuberculose Multirresistente (Sistema TBMR), me-diante uso de senha. 2008.17. Melo FAF, Afiune JB, Ide Neto J, Almeida EA, Spa-da DTA, Antel ANL, Cruz ML. Aspectos epidemiológi-cos da tuberculose multirresistente em serviço de refe-rência na cidade de São Paulo. Rev da Soc Brasil Med Trop 2003; 36:733 -740.18. Leite LPS, Costa ALP, Andrade RNS, Galvão T. Tratamento cirúrgico adjuvante de tuberculose pulmonar multirresistente. Jornal de Pneumologia 1997; 23:11 -14.19 Emergence of Mycobacterium tuberculosis with exten-sive resistance to second line drugs worldwide 2000 – 2004. MMWR 2006; 55(11).20. Savioli MTG, Melo FAF, Morrone N e Rodrigues DS. Tuberculosis with extensive resistance to drugs in a TB reference center in Sao Paulo, Brazil. Poster accepted CHEST, San Diego, California 2009.21. III Diretrizes para a tuberculose da Sociedade Bra-sileira de Pneumologia e Tisiologia. J Bras Pneumol 2009; 35:1018 -1048.

a experiência brasileira de controlo da multi-droga-resistênciaFernando Augusto Fiuza de Melo




Sebastien Gagneux1 Forças evolutivas do Mycobacterium tuberculosis

Evolutionary forces in Mycobacterium tuberculosis

A análise da diversidade genética de patogé-nios bacterianos ajuda a compreender me-lhor a epidemiologia e a evolução destes mi-cróbios. A compreensão da evolução de patogénios é particularmente importante nos dias de hoje, com a crescente resistência antimicrobiana1. Porém, o estudo da diversi-dade genética do complexo Mycobacterium tuberculosis (CMTB) é um desafio, porque a variação da sequência de ADN nestes orga-nismos é baixa2 e os métodos -padrão de ge-notipagem, como a tipagem de sequência multiloco (TSML), já bem estabelecida nou-tras bactérias3, proporcionam pouca infor-mação4. Além disso, deveriam usar -se dife-rentes métodos de genotipagem quando se investigam questões diferentes5. A investiga-ção epidemiológica molecular clássica da transmissão de doenças e a diferenciação en-tre recidiva e reinfeção exógena requer mar-cadores de genotipagem com grande força discriminatória6, enquanto as análises evolu-tivas deveriam apoiar -se em marcadores filo-geneticamente robustos7. Infelizmente, ocorre frequentemente trade-off entre essas

Analyzing the genetic diversity of bacterial pathogens helps to better understand the epidemiology and evolution of these mi-crobes. Understanding the evolution of pathogens is particularly important in to-day’s era of increasing antimicrobial resis-tance1. However, studying the genetic di-versity of the Mycobacterium tuberculosis complex (MTBC) is challenging because the DNA sequence variation in these or-ganisms is low2, and standard genotyping tools such as multilocus sequence typing (MLST), which have been well established in other bacteria3, provide little informa-tion4. Moreover, different genotyping tools should be used when addressing dif-ferent research questions5. Classical mo-lecular epidemiological investigation of disease transmission and differentiating between relapse and exogenous re-infec-tion requires genotyping markers with a high discriminatory power6, whereas evo-lutionary analyses should rely on phyloge-netically robust markers7. Unfortunately, there is often a trade-off between these

1 Division of Mycobacterial Research, MRC National Institute for Medical Research, London, UKe-mail: [email protected]




propriedades nos marcadores individuais usados na genotipagem, particularmente quando se estudam bactérias geneticamente monomórficas, como CMTB8,9. Com o CMTB, a spoligotipagem e a tipagem por MIRU -VNTR têm sido usadas com grande sucesso para tipagem e investigação epide-miológica molecular, mas essas técnicas são limitadas quando se inferem relações filoge-néticas entre estirpes10. Isto deve -se ao facto de a spoligotipagem e a tipagem por MIRU--VNTR dependerem de marcadores mole-culares repetitivos que se alteram rapidamen-te e, por consequência, esses marcadores têm um elevado índice de evolução convergen-te10. Em contraste, as deleções genómicas ou polimorfismos de sequência (LSP) e os poli-morfismos de um nucleótido único (SNP) aglomeram -se de modo relativamente lento no genoma de CMTB, podendo assim se-rem usados para definir linhagens filogeneti-camente robustas11.Já mostrámos que os LSP definem seis li-nhagens de estirpes principais no CMTB humano12, que incluem as duas linhagens geralmente referidas como M. africanum África Ocidental 1 e 2. Mostrámos também que estas linhagens eram correspondentes a agrupamentos observados na tipagem de SNP11. No entanto, devido a limitações ine-rentes a esses estudos anteriores, a real dis-tância genética entre linhagens e nelas pró-prias é ainda desconhecida. Para definir mais quantitativamente a diversidade gené-tica em CMTB, realizámos uma análise de sequências multilocos em larga escala (MLSA) de 108 estirpes globalmente repre-sentativas13, que incluíram membros de CMTB adaptado a animais, como M. bovis, M. microti, M. caprae e M. pinnipedii. Para todas estas 108 estirpes, gerámos a sequên-

properties in individual markers used for genotyping, and this is particularly true when studying genetically monomorphic bacteria such as MTBC8,9. In MTBC, spo-ligotyping and MIRU-VNTR typing have been used very successfully for fine typing and molecular epidemiological investiga-tions, but these techniques are limited when inferring phylogenetic relationships between strains10. This is because spoligo-typing and MIRU-VNTR typing rely on repetitive molecular markers that change rapidly, and as a consequence these mar-kers exhibit a high rate of convergent evo-lution10. By contrast, genomic deletions, also known as large sequence polymor-phisms (LSPs), and single nucleotide poly-morphisms (SNPs) accumulate relatively slowly in the MTBC genome and can thus be used to define phylogenetically robust strain lineages11.We have previously shown that LSPs de-fine six main strain lineages within the human MTBC12. These include the two lineages generally referred to as M. africa-num West-Africa 1 and 2. We have also shown that these lineages were congruent with groupings found based on SNP-typ-ing11. However, because of the inherent limitations of those previous studies, the actual genetic distance within and be-tween strain lineages remained unknown. To define more quantitatively the genetic diversity in MTBC, we performed a large-scale multilocus sequence analysis (MLSA) of 108 globally representative strains13. These included members of the animal-adapted MTBC like M. bovis, M. microti, M. caprae, and M. pinnipedii. For all of these 108 strains, we generated the com-plete DNA sequence of 89 genes, which

Forças evolutivas do Mycobacterium tuberculosisSebastien Gagneux




cia de ADN completa de 89 genes, que compreenderam genes de housekeeping, ge-nes de virulência e genes antigénicos. Usá-mos os genes concatenatos completos para definir uma nova filogenia do CMTB com base na sequência do ADN13. Esta é a mais abrangente e robusta filogenia do CMTB até à data14 e é também altamente congruen-te com a nossa anterior classificação do CMTB, baseada na análise de LSP, em seis linhagens principais15. A natureza quantita-tiva dos novos dados das sequências de ADN proporcionou um novo discernimento. Em particular, enquanto todas as formas de CMTB adaptadas de animais se agrupavam, representavam apenas um subgrupo da di-versidade genética observada em toda a filo-genia, sugerindo que a diversidade genética do CMTB humano é mais pronunciada do que anteriormente se pensava13. Além disso, as duas linhagens M. africanum, quase ex-clusivamente observadas na África Ociden-tal, são as mais ancestrais. Conjuntamente com o facto de ser a África o único conti-nente onde se encontram as seis mais im-portantes linhagens do CMTB humano, estes achados apoiam a teoria de que o CMTB terá sido originado em África12.Os dados do nosso novo estudo por análise de sequências multilocos permitiram -nos estudar a história evolutiva do CMTB humano em maior detalhe. Quando comparámos os dados da diversidade genética com as distâncias geo-gráficas que separam os locais de origem das estirpes incluídas no estudo, encontrámos cor-relações estatisticamente significativas entre estas duas medidas13. Os presentes resultados apoiaram um cenário ‘Fora -de -e -de -volta -a--África’ para a evolução do CMTB humano13. De acordo com este cenário, o CMTB origi-nou-se na África e espalhou -se originalmente

comprised housekeeping genes, virulence genes, and antigenic genes. We used the complete gene concatenates to define a new DNA sequence-based phylogeny of MTBC13. This phylogeny is the most comprehensive and most robust phyloge-ny of MTBC to date14, and is also highly congruent with our previous LSP-based classification of MTBC into six main li-neages15. The quantitative nature of the new DNA sequence data offered some new insights. In particular, while all ani-mal-adapted forms of MTBC clustered together, they represented only a sub-set of all the genetic diversity observed across the whole phylogeny, suggesting that the genetic diversity of human MTBC is more pronounced than previously thought13. Furthermore, the two M. africanum li-neages, which are almost exclusively ob-served in West Africa or the most ances-tral lineages. Together with the fact that Africa is the only continent that harbours all six main lineages of human MTBC, these findings support to view that MTBC originated in Africa12.Our new MLSA data allowed us to study the evolutionary history of human MTBC in more detail. When we compared the ge-netic diversity data to the geographic dis-tances separating the places of origin of the strains included in the study, we found sta-tistically significant correlations between these two measures13. Our results supported an ‘Out-of-and-back-to-Africa’ scenario for the evolution of human MTBC13. Accord-ing to this scenario, MTBC originated in Africa and spread originally out of Africa ac-companying ancient human migrations which occurred approximately 50 000 years ago. Through these ancient migrations,





para fora desse continente, acompanhando antigas migrações humanas que ocorreram há aproximadamente 50 000 anos. Através dessas antigas migrações, três ‘modernas’ linhagens evolutivas de CMTB espalharam -se por áreas da Europa, da Índia e da China, respectiva-mente. Essas regiões passaram por grande au-mento da população humana durante as últi-mas centenas de anos, o que levou a uma expansão destas linhagens de CMTB. Conco-mitantemente, estas linhagens modernas co-meçaram a disseminar -se globalmente e re-gressaram a África, através de recentes ondas de colonização, comércio e conquista.Outra observação interessante que decorreu da nossa análise de sequências multilocos foi que o rácio SNP não sinónimos: SNP sinóni-mos (uma medida conhecida como dN/dS) é muito maior no MTBC do que na maioria das outras bactérias13. Um dN/dS elevado em patogénios bacterianos tem normalmente sido associado à ancestralidade recente16. Por outras palavras, ainda não houve tempo sufi-ciente para a purificação da seleção eliminar SNP não sinónimos, a maioria dos quais ten-dem a ser ligeiramente nocivos à competência bacteriana. Porque os SNP sinónimos se acu-mulam ao longo do tempo sem serem elimi-nados pela seleção natural, um elevado dN/dS pode ser indicador de ocorrência recente. Po-rém, as nossas análises apoiam a noção de que a razão dN/dS é elevada no CMTB porque a purificação da seleção deste organismo é redu-zida, provavelmente pela consequência do au-mento aleatório da deslocação genética asso-ciada às acumulações de populações durante a transmissão doente a doente13. Isto sugere que o ‘acaso’, e não apenas a seleção natural, tem conduzido a evolução do CMTB.Em resumo, o presente trabalho de análise de sequências multilocos confirmou que a estru-

three evolutionarily “modern” lineages of MTBC seeded areas in Europe, India, and China, respectively. These regions expe-rienced strong human population increase during the last few hundred years, leading to an expansion of these MTBC lineages. Concomitantly, these modern lineages star-ted to spread globally and back to Africa, through recent waves of colonization, trade, and conquest.Another interesting observation coming out of our MLSA work was that the ratio of nonsynonymous SNPs to synonymous SNPs (a measure known as dN/dS) is much higher in MTBC than in most other bacteria13. A high dN/dS in bacterial pathogens has generally been associated with recent ancestry16. In other words, there has not been enough time of purify-ing selection to remove nonsynonymous SNPs, the majority of which tend to be slightly deleterious to bacterial fitness. Because synonymous SNPs accumulate over time without being removed by na-tural selection a high dN/dS can be in-dicative of recent emergence. However, our analyses supported the view that the reason dN/dS is high in MTBC is because purifying selection in this organism is re-duced, most likely as consequence of in-creased random genetic drift associated with the serial population bottlenecks during patient-to-patient transmission13. This suggest that ‘chance’, and not just natural selection has been driving the evo-lution of MTBC.In summary, our MLSA work confirmed that the genetic population structure of human MTBC consists of six main strain lineages. Our findings also show that the human MTBC lineages are more geneti-





tura genética da população humana de CMTB consiste em seis linhagens de estirpes principais. Os achados mostram também que as estirpes do CMTB humano são mais gene-ticamente diversas do que se pensava anterior-mente. Há evidência crescente de que a linha-gem de CMTB pode afetar a evolução da tuberculose infeção e doença17 -20. O presente trabalho de sequenciação identificou muitos SNPS filogeneticamente informativos, o que permitirá aos investigadores classificar rapida-mente estirpes em agrupamentos robustos10 e continuar a explorar diferenças específicas das linhagens nos contextos experimental e clíni-co. As nossas análises de genética de popula-ções revelaram que a diversidade genética do CMTB foi moldada por migrações humanas antigas e mais recentes e que a deslocação ge-nética aleatória pode ser uma importante for-ça condutora na evolução do CMTB. À me-dida que os custos da sequenciação de ADN continuam a baixar, a sequenciação do geno-ma completo tem potencial para se tornar o instrumento ‘final’ para genotipagem de bac-térias21. A sequenciação genómica comparati-va de grandes coleções de estirpes de CMTB melhorará a nossa compreensão das forças evolutivas que moldam a diversidade genética deste importante patogénio15.

Bibliografia/Bibliography1. Borrell S, Gagneux S. Infectiousness, reproductive fit-ness and evolution of drug -resistant Mycobacterium tu-berculosis. Int J Tuberc Lung Dis 2009; 13:1456 -1466.2. Sreevatsan S, Pan X, Stockbauer KE, Connell ND, Kreiswirth BN, et al. Restricted structural gene poly-morphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc Natl Acad Sci USA 1997; 94: 9869 -9874.3. Maiden MC. Multilocus sequence typing of bacteria. Annu Rev Microbiol 2006; 60: 561 -588.

cally diverse than previously thought. There is mounting evidence that MTBC lineage can affect the outcome of tuber-culosis infection and disease17,18,19,20. Our sequencing work identified many phylo-genetically informative SNPs, which will allow researchers to rapidly classify strains into robust groupings10, and to further explore lineage-specific diffe-rences in experimental and clinical con-texts. Our population genetic analyses revealed that the genetic diversity in MTBC has been shaped by both ancient and more recent human migrations, and that random genetic drift might be an important driving force in the evolution of MTBC. As DNA sequencing costs continue to decrease, full genome-se-quencing has the potential to become the “ultimate” genotyping tools for bac-teria21. Comparative genome sequencing of large strain collections of MTBC will improve our understanding of the evolu-tionary forces shaping the genetic diver-sity in this important pathogen15.


4. Baker L, Brown T, Maiden MC, Drobniewski F. Si-lent nucleotide polymorphisms and a phylogeny for My-cobacterium tuberculosis. Emerg Infect Dis 2004; 10: 1568 -1577.5. Feil EJ. Small change: keeping pace with microevolu-tion. Nat Rev Microbiol 2004; 2: 483 -495.6. Mathema B, Kurepina NE, Bifani PJ, Kreiswirth BN. Molecular epidemiology of tuberculosis: cur-rent insights. Clin Microbiol Rev 2006; 19: 658--685.




7. Achtman M, Wagner M. Microbial diversity and the genetic nature of microbial species. Nat Rev Microbiol 2008; 6: 431 -440.8. Achtman M. Evolution, population structure, and phylogeography of genetically monomorphic bacterial pathogens. Annu Rev Microbiol 2008; 62: 53 -70.9. Pearson T, Okinaka RT, Foster JT, Keim P. Phyloge-netic understanding of clonal populations in an era of whole genome sequencing. Infect Genet Evol 2009; 9: 1010 -1019.10. Comas I, Homolka S, Niemann S, Gagneux S. Geno- typing of genetically monomorphic bacteria: ADNse-quencing in Mycobacterium tuberculosis highlights the limitations of current methodologies. PLoS One 2009; 4: e7815.11. Gagneux S, Small PM. Global phylogeography of Mycobacterium tuberculosis and implications for tuber-culosis product development. Lancet Infect Dis 2007; 7: 328 -337.12. Gagneux S, Deriemer K, Van T, Kato -Maeda M, de Jong BC, et al. Variable host -pathogen compatibility in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 2006; 103: 2869 -2873.13. Hershberg R, Lipatov M, Small PM, Sheffer H, Nie-mann S, et al. High functional diversity in Mycobacte-rium tuberculosis driven by genetic drift and human de-mography. PLoS Biol 2008; 6:e311.14. Smith NH, Hewinson RG, Kremer K, Brosch R, Gordon SV. Myths and misconceptions: the origin and

evolution of Mycobacterium tuberculosis. Nat Rev Microbiol 2009; 7: 537 -544.15. Comas I, Gagneux S. The past and future of tuber-culosis research. PLoS Pathog 2009; 5: e1000600.16. Rocha EP, Smith JM, Hurst LD, Holden MT, Cooper JE, et al. Comparisons of dN/dS are time de-pendent for closely related bacterial genomes. J Theor Biol 2006; 239: 226 -235.17. Caws M, Thwaites G, Dunstan S, Hawn TR, Thi Ngoc Lan N, et al. The influence of host and bacterial genotype on the development of disseminated disease with Mycobac-terium tuberculosis. PLoS Pathog 2008; 4: e1000034.18. de Jong BC, Hill PC, Aiken A, Awine T, Antonio M, et al. Progression to active tuberculosis, but not trans-mission, varies by Mycobacterium tuberculosis lineage in the Gambia. J Infect Dis 2008; 198: 1037 -1043.19. de Jong BC, Hill PC, Brookes RH, Gagneux S, Jef-fries DJ, et al. Mycobacterium africanum elicits an attenua-ted T cell response to early secreted antigenic target, 6 kDa, in patients with tuberculosis and their household contacts. J Infect Dis 2006; 193: 1279 -1286.20. Thwaites G, Caws M, Chau TT, D’Sa A, Lan NT, et al. The relationship between Mycobacterium tuberculo-sis genotype and the clinical phenotype of pulmonary and meningeal tuberculosis. J Clin Microbiol 2008; 46: 1363 -1368.21. Medini D, Serruto D, Parkhill J, Relman DA, Do-nati C, et al. Microbiology in the post -genomic era. Nat Rev Microbiol 2008; 6: 419 -430.





Joseph O Falkinham III1 Epidemiologia e ecologia de micobactérias não tuberculosas

Epidemiology and ecology of nontuberculous mycobacteria

As micobactérias não tuberculosas (MNT) são patogénios oportunistas dos humanos e animais encontrados no meio ambiente1,2. Nos Estados Unidos, a prevalência de doenças por MNT vem aumentando 8% ao ano, atingindo atual-mente quase 35 casos por cada 100 0003. Em Ontário, Canadá, a prevalência de doença pul-monar por MNT aumentou de 1,5 para 9,0 por 100 000 (6 vezes) no período 1997 -20034.A evidência de que o ambiente é a fonte das doenças por MNT em seres humanos foi conseguida a partir do ADN de isolados de MNT de doentes com SIDA, de água de be-ber5 e de doentes imuno competentes e de isolados de MNT de terra de vasos6 ou de um chuveiro doméstico7. Os seres humanos estão continuamente expostos a MNT, uma vez que estes vivem e crescem normalmente nos sistemas de distribuição da água de beber8, e 20% das amostras recolhidas em biofilmes nas cabeças dos chuveiros domésticos nos Es-tados Unidos tinham M. avium9.

Nontuberculous mycobacteria (NTM) are opportunistic human and animal environ-mental pathogens1,2. In the United States, the prevalence of NTM disease is increasing by 8% per year and now stands at almost 35 cases per 100 0003. In Ontario, Canada the prevalence of NTM pulmonary disease in-creased from 1.5 to 9.0 per 100 000 (6-fold increase) over the period 1997-20034.Evidence that the environment was the source of NTM disease in humans was gained from the identity of DNA fingerprints of NTM isolates from AIDS patients and drink-ing water5 and from immunocompetent pa-tients and NTM isolates from potting soils6 or a home shower7. Humans are continually exposed to nontuberculous mycobacteria (NTM) as they are normal inhabitants and grow in drinking water distribution systems8 and 20 % of showerhead biofilms (swab sam-ples) collected from households in the United States had M. avium9.

1 Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia 24061 -0406 Phone 1 -540 -231 -5931 Fax 1 -540 -231 -9307 e -mail: [email protected]




Os fatores de risco para doenças por MNT incluem: imuno competência diminuída devi-do a infeção por VIH, cancro, quimioterapia, ou imunossupressão associada a transplanta-ção, doença pulmonar preexistente, como pneumoconiose, silicose e doença do pulmão negro, alterações na arquitetura normal do tronco, alcoolismo e hábitos tabágicos10. Re-centemente, mutações no regulador da con-dutância transmembranária na fibrose quísti-ca (RCTFQ) e genes de α -1 -antitripsina têm sido associados ao risco aumentado de doença pulmonar por MNT11. É preocupante o facto de a doença pulmonar por MNT ter aumen-tado drasticamente entre homens e mulheres idosos magros que, não tendo os fatores de risco clássicos para doença micobacteriana, parecem ser substancialmente mais suscetíveis a doenças por MNT12. Com o envelhecimen-to das populações, é expectável que o número de idosos com doença pulmonar por MNT aumente, com o consequente peso para os prestadores de cuidados de saúde. A terapêu-tica recomendada para infeções por MNT inclui múltiplos antibióticos (e.g., claritromi-cina, etambutol e rifampin) por períodos de 24 meses13.As micobactérias não tuberculosas sobrevi-vem, crescem e persistem em habitats parti-lhados por seres humanos e animais. São oli-gotróficos, podendo crescer em água com mais de 50 μg de carbono orgânico assimilá-vel (COA/)L14. A presença de grandes quan-tidades de MNT em pântanos de águas escu-ras e ácidas15 e no solo de pinhais16 deve -se, em parte, ao crescimento estimulado pela matéria orgânica do solo raramente metabo-lizada por outros microrganismos, nomeada-mente ácidos húmico e fúlvico17.As micobactérias têm uma verdadeira mem-brana externa18, cujos ácidos micólicos de

Risk factors for NTM disease include: re-duced immune competence as a result of HIV infection, cancer, chemotherapy, or immunosuppression associated with trans-plantation, preexisting lung disease such as pneumoconiosis, silicosis, and black lung disease, altered normal chest architecture, alcoholism, and smoking10. Recently, mu-tations in the cystic fibrosis transmem-brane conductance regulator (CFTR) and α-1-antitrypsin genes have been associated with increased risk of NTM pulmonary disease11. Quite alarming is the fact that pulmonary NTM disease has increased dramatically amongst elderly slender men and women, who lack the classic risk fac-tors for mycobacterial disease, yet appear to be substantially more susceptible to NTM disease12. As the human population ages, it would be expected that the number of elderly with NTM pulmonary disease will increase, placing further demands on healthcare providers. Recommended the-rapy for NTM infections include multiple antibiotics (e.g., clarithromycin, ethambu-tol, and rifampin) for periods as long as 24 months13.NTM survive, grow, and persist in a num-ber of habitats that are shared with humans and animals. NTM are oligotrophs; able to grow in water containing greater than 50 μg assimilable organic carbon (AOC/)L14. The presence of high numbers of NTM in coastal acidic, brown water swamps15 and pine forest soils16 is due, in part, to the growth stimulation by soil organic matter material rarely metabolized by other mi-croorganisms; namely humic and fulvic acids17.Mycobacteria have a true outer mem-brane18, whose long chain mycolic acids

Epidemiologia e ecologia de micobactérias não tuberculosas (MNT)Joseph O Falkinham III




cadeia longa contribuem para a hidrofobici-dade, impermeabilidade e crescimento lento das células de MNT19. A hidrofobicidade da superfície das células de MNT é a mais eleva-da de todas as bactérias e é uma importante determinante da fagocitose dos macrófagos20. Embora a parede hidrofóbica reduza o índice de transferência de nutrientes hidrofílicos, contribui para a resistência a desinfetantes (e.g., cloro e biócidos) e a antibióticos21,22.As MNT penetram nos sistemas de trata-mento de águas aderentes a partículas, sobre-vivem à desinfeção e crescem em biofilmes na ausência de competidores mortos pela disin-feção8. A formação de biofilme também au-menta a resistência das MNT a desinfetan-tes23 e a antibióticos24. A hidrofobicidade também promove a aerossolização de mico-bactérias da água para o ar em ambientes como chuveiros e banheiras domésticos e nas profissões onde se manuseiam aerossóis25.

Bibliografia/Bibliography1. Wallace RJ, Brown BA, Griffith DE. Nosocomial outbreaks pseudo -outbreaks caused by nontuberculous mycobacteria. Annu Rev Microbiol 1998; 52;453 -490.2. Falkinham JO III. Surrounded by mycobacteria: non-tuberculous mycobacteria in the human environment. J Appl Microbiol 2009; 107;356 -367.3. Iseman MD, Marras TK. The importance of nontu-berculous mycobacterial lung disease. Am J Respir Crit Care Med 2008; 178:999 -1000.4. Marras TK, et al. Isolation prevalence of pulmonary non -tuberculous mycobacteria in Ontario, 1997 -2003. Thorax 2007; 62;661 -666.5. von Reyn CF, et al. Persistent colonisation of potable water as a source of Mycobacterium avium infection in AIDS. Lancet 1994;343;1137 -1141.6. De Groote MA, et al. Relationships between Myco-bacterium isolates from patients with pulmonary myco-bacterial infection and potting soils. 2006; Appl Envi-ron Microbiol 2006;72;7602 -7606.

contribute to the hydrophobicity, imper-meability, and slow growth of NTM cells19. The surface hydrophobicity of NTM cells is the highest amongst bacteria and is a major determinant of phagocytosis by macrophages20. Although the hydrophobic wall reduces the rate of transfer of hydro-philic nutrients, it contributes to disinfec-tant- (e.g., chlorine and biocides) and anti-biotic-resistance21,22.NTM enter a water treatment system on particulates, survive disinfection, and grow in biofilms in the absence of com-petitors killed by disinfection8. Biofilm formation also increases NTM resistance to disinfectants23 and antibiotics24. Hydro-phobicity also promotes the aerosolization of mycobacteria from water to air in envi-ronments such as showers and hot tubs in the home and occupations where aerosols are generated25.

7. Falkinham JO III, et al. Mycobacterium avium in a shower linked to pulmonary disease. J Water Health 2008; 6;209 -213.8. Falkinham JO III, Norton CD, LeChevallier MW. Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare, and other Mycobacteria in drinking water distribution systems. Appl Environ Microbiol 2001; 67;1225 -1231.9. Feazel LM, et al. Opportunistic pathogens enriched in showerhead biofilms. Proc Natl Acad Sci USA 2009;106;16393 -16399.10. Marras TK, Daley CL. Epidemiology of human pul-monary infection with nontuberculous mycobacteria. Clin Chest Med 2002; 23;553 -567.11. Kim JS, et al. Nontuberculous mycobacterial infec-tion: CT scan findings, genotype, and treatment res-ponsiveness. Chest 2005; 128;3863 -3869.12. Kennedy TP, Weber DJ. Nontuberculous mycobac-teria – an underappreciated cause of geriatric lung di-





sease. Am Rev Respir Crit Care Med 1994;149;1654--1658.13. Griffith DE, et al. An official ATS/IDSA statement: Diagnosis, treatment, and prevention of nontubercu-lous mycobacterial diseases. Am J Respir Crit Care Med 2007; 175;367 -416.14. Norton CD, LeChevallier MW, Falkinham JO III. Survival of Mycobacterium avium in a model distribu-tion system. Water Res 2005; 38;1457 -1466.15. Kirschner RA Jr, Parker BC, Falkinham JO III. Epi-demiology of infection by nontuberculous mycobacteria. Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in acid, brown -water swamps of the southeastern United States and their association with environmental variables. Am Rev Respir Dis 1992; 145;271 -275.16. Iivanainen EK, et al. Mycobacteria in boreal coniferous forest soils. FEMS Microbiol Ecol 1997;23;325 -332.17. Kirschner RA, Parker BC, Falkinham JO III. Hu-mic and fulvic acids stimulate the growth of Mycobacte-rium avium. FEMS Microbiol Ecol 1995; 30;327 -332.18. Hoffmann C, et al. Disclosure of the mycobacterial outer membrane: Cryo -electron tomography and vitre-ous sections reveal the lipid bilayer structure. Proc Natl Acad Sci USA 2008; 105;3963 -3967.

19. Brennan PJ, Nikaido H. The envelope of mycobac-teria. Annu Rev Biochem 1995; 64;29 -63.20. van Oss CJ, Gillman CF, Neumann AW. Phagocytic engulfment and cell adhesiveness. 1975; Marcel Dekker, New York.21. Rastogi N, et al. Multiple drug resistance in Myco-bacterium avium – Is the wall architecture responsible for the exclusion of antimicrobial agents. Antimicrob Agents Chemother 1981; 20;666 -677.22. Taylor R, et al. Chlorine, chloramine, chlorine diox-ide, and ozone susceptibility of Mycobacterium avium. Appl Environ Microbiol 2000; 66;1702 -1705.23. Steed KA Falkinham JO III. Effect of growth in bio-films on chlorine susceptibility of Mycobacterium avium and Mycobacterium intracellulare. Appl Environ Micro-biol 2006; 72;4007 -4011.24. Falkinham, JO III. Growth in catheter biofilms and antibiotic resistance of Mycobacterium avium. J Med Microbiol 2007; 56;250 -254.25. Parker BC, George KL, Falkinham JO III. Epidemio-logy of infection by nontuberculous mycobacteria. IV. Preferential aerosolization of Mycobacterium intracellu-lare from natural waters. Am Rev Respir Dis 1984; 123;652 -656.





Jean-Pierre Zellweger1 O uso da análise de libertação de gama interferão como auxiliar no controlo da tuberculose

The use of interferon gamma release assays as an aid in the control of tuberculosis

As análises de libertação de gama interferão (IGRA) são testes in vitro que detetam a presença de infeção por tuberculose latente (ITBL) em pessoas assintomáticas que po-dem ter sido infetadas por Mycobacterium tuberculosis num passado recente ou remoto e que podem beneficiar dum tratamento preventivo para diminuir o risco de reativa-ção posterior da tuberculose1, 2.A investigação dos contactos é a busca de casos secundários de tuberculose entre os contactos dum caso primário, e a busca de contactos com uma infeção latente de tuberculose que possam beneficiar dum tratamento preventivo de modo a evitar a futura progressão para a doen-ça ativa. Esta é a segunda prioridade no contro-lo da tuberculose, depois da deteção e trata-mento dos casos ativos. Se feito de modo sistemático, e em países com entidades bem organizadas no controlo da TB, esta atividade pode ser eficaz em termos de custo.A deteção de casos secundários assenta essen-cialmente nos exames clínico, radiológico e bacteriológico dos contactos com queixas. A

Interferon Gamma Release Assays (IGRAs) are in vitro tests detecting the presence of latent tuberculosis infection (LTBI) in as-ymptomatic persons who may have been infected by Mycobacterium tuberculosis in a recent or remote past and who may benefit from a preventive treatment to decrease the risk of later reactivation of tuberculosis1, 2.Contact investigation is the search for se-condary cases of tuberculosis among the contacts of a primary (index) case and the search for contacts with a latent tuberculosis infection who may benefit from a preven-tive treatment in order to avoid the future progression to active disease. This is the se-cond priority for the control of tuberculosis, after the detection and treatment of active cases. If conducted in a systematic way and in countries with a well-organized TB ma-nagement team, this activity may be very cost-effective.The detection of secondary cases relies mainly on the clinical, radiological and bac-teriological examination of contacts with

1 Swiss Lung Association, Berne, Switzerlande-mail: [email protected]




deteção de contactos com infeção latente de tuberculose, por definição assintomática, apoia -se no uso de testes indiretos capazes de detetar a sensibilização anterior de linfócitos T por antigénios micobacterianos. O tradi-cional teste cutâneo de tuberculina (TCT), com que se faz a maioria dos estudos epide-miológicos, enferma de graves deficiências, entre as quais a baixa especificidade. As novas análises de libertação de gama interferão, com a sua alta especificidade, são muito mais promissoras e permitem uma melhor seleção dos contactos que podem beneficiar duma terapêutica preventiva, evitando a prescrição dum tratamento desnecessário para contac-tos não infetados que podem ter um TCT falso positivo, devido, por exemplo, ao BCG, ao efeito booster ou à sensibilização com mi-cobactérias não tuberculosas3.Basicamente, os testes IGRA assentam no mesmo fenómeno imunológico dos testes cutâneos de tuberculina, mas duma maneira muito mais específica, porque os testes não são influenciados por uma anterior vacina-ção com BGC ou por uma infeção com a maioria das micobactérias não tuberculosas presentes no ambiente. Assim, as indicações e o uso de testes IGRA são fundamental-mente as mesmas dos testes cutâneos de tu-berculina4:

Deteção de ITBL em pessoas em con-1. tacto com um caso primário de tuber-culose;Deteção de ITBL em pessoas com alto 2. risco de tuberculose, se infetadas (doen-tes imunossuprimidos, doentes a rece-ber ou programados para receber terapia imunossupressora, crianças);Vigilância de trabalhadores dos serviços 3. de saúde expostos (uma vez que o teste

complaints. The detection of contacts with a latent tuberculosis infection, by definition asymptomatic, relies on the use of indirect tests able to detect the prior sensitization of T lymphocytes by mycobacterial antigens. The time-honoured tuberculin skin test (TST), with which most of the epidemio-logical studies are performed, suffers from severe deficiencies, among which a low specificity. The new Interferon-Gamma Re-lease Assays, with their high specificity, are much more promizing and allow a better targeting of the contacts who may benefit from a preventive therapy, avoiding the pres cription of an unnecessary treatment to uninfected contacts who may have a false-positive TST, for instance due to BCG, booster effect or sensitization with non-tu-berculous mycobacteria3.Basically, the IGRA tests rely on the same immunological phenomenon as the tuber-culin skin tests, but they do it in a much more specific way, because the tests are not influenced by a prior vaccination with BGC or by an infection with most of the non-tu-berculous mycobacteria present in the envi-ronment. Therefore, the indications and the use of the IGRA tests are fundamentally the same as for the tuberculin skin tests4:

Detection of LTBI in persons in contact 1. with an index case of tuberculosis;Detection of LTBI in persons with a 2. high risk of tuberculosis, if infected (immunosuppressed patients, patients receiveing or due to receive immuno-suppressive therapy, small children);Surveillance of exposed health care 3. workers (as the test can be repeated without risk of inducing a booster ef-fect);

O uso da análise de libertação de gama interferão como auxiliar no controlo da tuberculose





pode ser repetido sem risco de indução do efeito booster);Auxiliar no diagnóstico de tuberculose 4. em casos em que o exame bacteriológi-co não é viável ou fiável (TB extra-pulmonar grave, TB em crianças).

O nível de resposta ao IGRA é proporcional à intensidade da exposição à tuberculose e pode mudar sob tratamento preventivo ou curativo, possivelmente indicando que o nú-mero de micobactérias vivas também dimi-nuiu. Assim, é possível que a alteração no nível de IGRA reflita o efeito do tratamento, mas infelizmente, até agora, não foi possível provar que a diminuição do nível esteja clara-mente relacionada com a erradicação de mi-cobactérias e possa ser usada para documen-tar a cura5. Além disso, não está provado que um teste positivo evidencie sempre a persis-tência de micobactérias vivas. É possível que o teste detete apenas uma resposta imune a um anterior contacto com micobactérias que, entretanto, tenham desaparecido6

Os testes IGRA podem contribuir para o diagnóstico de casos difíceis de TB. Na tuberculose pulmonar com um esfregaço negativo, e na tuberculose extrapulmonar pode ser muito difícil obter evidência bac-teriológica da presença de micobactérias. A libertação de gama interferão de linfó-citos isolados a partir de órgãos potencial-mente envolvidos em casos suspeitos de tuberculose (lavagem broncoalveolar na tuberculose pulmonar com um esfregaço negativo, líquido cerebrospinal na menin-gite tuberculosa, líquido peritoneal na tu-berculose abdominal) pode ser medida e está elevada em casos com um diagnóstico final de tuberculose. Nesses casos, a deter-minação do nível de libertação de gama

Aid to the diagnosis of tuberculosis in 4. cases where a bacteriological examina-tion is not feasible or not reliable (severe extrapulmonary TB, TB in children).

The level of IGRA response is proportional with the intensity of exposure to tuberculo-sis, and may change under preventive or cu-rative treatment, possibly indicating that the number of living mycobacteria has also decreased. It is therefore possible that the change in the level of IGRA reflects the ef-fect of treatment, but there is unfortunately up to now no solid proof that a decrease in the level is clearly correlated with the eradi-cation of mycobacteria and could be used as a documentation of cure5. Furthermore, there is no proof that a positive test result always documents the persistence of living mycobacteria. It may be that the test only detects a lasting immune response to a prior contact with mycobacteria that have disap-peared in between 6

IGRAs may contribute to the diagnosis of difficult TB cases. In smear-negative pulmonary tuberculosis and in extrapul-monary tuberculosis, it may be very dif-ficult to obtain the bacteriological docu-mentation of the presence of mycobacteria. The release of interferon-gamma from lymphocytes isolated from the organs po-tentially involved in cases of suspect tu-berculosis (BAL in smear-negative pul-monary tuberculosis, CSF in tuberculous meningitis, peritoneal fluid in abdominal tuberculosis) can be measured and is ele-vated in cases with a final diagnosis of tuberculosis. In such cases, the determi-nation of the level of Interferon-Gamma release can be used as an aid to the diag-nosis of tuberculosis7.

O uso da análise de libertação de gama interferão como auxiliar no controlo da tuberculoseJean-Pierre Zellweger




interferão pode ajudar no diagnóstico da tuberculose7.Os testes IGRA podem indicar o futuro de-senvolvimento da doença. Nem todas as pes-soas em contacto com um caso de tuberculo-se são infetadas e nem todos os contactos infetados virão a desenvolver tuberculose. Estima -se o risco de reativação da tuberculo-se como sendo de 10% para contactos com um TCT positivo. Estudos recentes demons-traram que o risco de futura reativação é maior em contactos com um IGRA positivo do que em contactos negativos, independen-temente do resultado do TCT8. Por isso, um IGRA positivo pode ter maior valor preditivo para risco de reativação futura do que o TCT. Como a proporção de contactos com um IGRA positivo é menor do que a dos contac-tos com um teste cutâneo de tuberculina po-sitivo, o número de contactos considerados infetados e que podem beneficiar de um tra-tamento preventivo é menor se os testes IGRA forem usados como definição. A utili-zação de testes IGRA na deteção de contactos infetados e na seleção de contactos que neces-sitam de tratamento preventivo tem, assim, um efeito moderador9.Apesar da sua superioridade, os testes IGRA não são totalmente isentos de problemas na prática e a sua melhor utilização ainda vem sen-do debatida. A variabilidade inter e intra-observador é baixa, mas o nível de resposta pode variar se o teste for repetido, verificando -se con-versões e reversões espontâneas na ausência de exposição ou de tratamento10 -12. Observam -se ainda mais alterações nos casos com respostas borderline. Assim, têm sido propostos aumentos no cut -off da positividade e na definição da con-versão na repetição dos testes13.Algumas linhas de orientação recomendam o seu uso apenas para confirmação dum

IGRAs may predict the future development of TB disease. Not all cases in contact with a case of tuberculosis will be infected and not all infected contacts will develop tuber-culosis. The risk of reactivation of tubercu-losis is estimated to be 10% for contacts with a positive tuberculin skin test. Recent studies have demonstrated that the risk of future reactivation is higher in contacts with a positive IGRA test than in negative contacts, independently from the result of the tuberculin skin test8. Therefore, a posi-tive IGRA test result may have a higher predictive value for the risk of future reacti-vation than the tuberculin skin test. As the proportion of contacts with a positive IGRA test is lower than the proportion of contacts with a positive tuberculin skin test, the number of contacts considered as in-fected and who may benefit from a preven-tive treatment is lower if IGRA are used as a definition. Using IGRA for the detection of infected contacts and selection of con-tacts in need of a preventive treatment has therefore a sparing effect9.In spite of their superiority, the IGRAs are not totally devoid of problems in practice and the best use of them is still a matter of debate. The intra-observer and inter-ob-server variability are low, but the level of re-sponse may vary if the test is repeated and there are spontaneous conversions and re-versions in the absence of exposure or treat-ment10-12. More of the changes are observed for cases with borderline responses. There-fore, proposals have been made for an in-crease in the cut-off for positivity and for the definition of conversion in repeated testing13.Some Guidelines recommend their use only for the confirmation of positive TST among

O uso da análise de libertação de gama interferão como auxiliar no controlo da tuberculose





TCT positivo entre contactos (o chamado teste de dois passos), enquanto outras reco-mendam a substituição do TCT de rotina por IGRA. Fazer um só teste é mais fácil e evita a possível influência de um TCT ante-rior na resposta do IGRA.

Bibliografia/Bibliography1. Menzies D, Pai M, Comstock G. Meta -analysis: new tests for the diagnosis of latent tuberculosis infection: areas of uncertainty and recommendations for research. Ann Intern Med 2007; 146(5):340 -354.2. Pai M, Zwerling A, Menzies D. Systematic review: T -cell -based assays for the diagnosis of latent tuberculo-sis infection: an update. Ann Intern Med 2008; 149(3):177 -184.3. Diel R, Loddenkemper R, Meywald -Walter K, Gottschalk R, Nienhaus A. Comparative performance of tuberculin skin test, QuantiFERON -TB -Gold in tube assay, and T -Spot.TB test in contact investigations for tuberculosis. Chest 2009; 135(4):1010 -1018.4. Zellweger JP. Latent tuberculosis: which test in which situation? Swiss Med Wkly 2008; 138(3 -4):31 -37.5. Pai M, Menzies D. Interferon -gamma release assays: what is their role in the diagnosis of active tuberculosis? Clin Infect Dis 2007; 44(1):74 -77.6. Mack U, Migliori GB, Sester M, et al. LTBI: latent tuberculosis infection or lasting immune responses to M. tuberculosis? A TBNET consensus statement. Eur Respir J 2009; 33(5):956 -973.7. Jafari C, Ernst M, Strassburg A, et al. Local immuno-diagnosis of pulmonary tuberculosis by enzyme -linked immunospot. Eur Respir J 2008; 31(2):261 -265.

contacts (the so-called two-step testing pro-cedure) whereas others recommend the rou-tine replacement of the TST by IGRAs. Per-forming only one test is easier, and avoids a possible influence of a prior TST on the IGRA response.

8. Diel R, Loddenkemper R, Meywald -Walter K, Nie-mann S, Nienhaus A. Predictive value of a whole -blood IFN -{gamma} assay for the development of active TB disease. Am J Respir Crit Care Med 2008; 177:1164--1170.9. Diel R, Nienhaus A, Lange C, Schaberg T. Cost opti-mization of screening for latent tuberculosis in close contacts. Eur Respir J 2006; 28:35 -44.10. Pai M, Joshi R, Dogra S, et al. T -cell assay conver-sions and reversions among household contacts of tu-berculosis patients in rural India. Int J Tuberc Lung Dis 2009; 13(1):84 -92.11. van Zyl -Smit RN, Pai M, Peprah K, et al. Within--subject variability and boosting of T -cell interferon--gamma responses after tuberculin skin testing. Am J Respir Crit Care Med 2009; 180(1):49 -58.12. Detjen AK, Loebenberg L, Grewal HM et al. Short--term reproducibility of a commercial interferon -gamma release assay. Clin Vaccine Immunol 2009; 16:1170--117513. Pai M, Dendukuri N, Wang L, Joshi R, Kalantri S, Rieder HL. Improving the estimation of tuberculosis infection prevalence using T -cell -based assay and mix-ture models. Int J Tuberc Lung Dis 2008;12(8):895--902.

O uso da análise de libertação de gama interferão como auxiliar no controlo da tuberculoseJean-Pierre Zellweger




Lee W Riley1 Regulação da composição lipídica da parede celular do Mycobacterium tuberculosis e o seu efeito na persistência bacteriana in vitro

Regulation of Mycobacterium tuberculosis cell wall lipid composition and its effects on in vitro bacterial persistence

IntroduçãoA marca da infeção humana por Mycobacte-rium tuberculosis é a sua capacidade de per-manecer latente durante vários anos, com posterior reativação para doença ativa (tu-berculose). Pouco se sabe sobre como este organismo permanece em latência e o que desencadeia a sua reativação. Frequente-mente atribui -se aos lípidos do M. tubercu-losis um papel na patogénese, porque grande parte do genoma do M. tuberculosis é dedi-cado à biossíntese e à degradação dos lípi-dos1. Só no metabolismo dos ácidos gordos quase 250 genes estão envolvidos, compara-tivamente com apenas cerca de 50 em orga-nismos como a Escherichia coli. Mais de 50% do peso seco da parede celular do M. tuberculosis é composto por lípidos1 -3 e, além disso, o organismo utiliza os lípidos como fonte de energia durante a sua persistência num mamífero hospedeiro4. Esta despro-porcionada atenção dispensada ao metabo-lismo lipídico é muito provavelmente uma estratégia adotada por este organismo como

IntroductionThe hallmark of human infection by My-cobacterium tuberculosis is its ability to re-main latent for many years, only to reacti-vate to cause active disease (tuberculosis). Very little is known about how this orga-nism remains latent and what triggers it to reactivate. Much attention is often paid to the lipids of M. tuberculosis as playing a role in pathogenesis. This is because a large proportionof the genome of M. tuberculo-sis is dedicated to the biosynthesis and degradation of lipids1. Nearly 250 genes are involved in metabolism of fatty acids alone, compared to only about 50 in or-ganisms such as Escherichia coli. More than 50% of the dry weight of the cell wall of M. tuberculosis is composed of lipids1-3, and furthermore, the organism utilizes lipids as energy source during its persis-tence in a mammalian host4. This dispro-portionate attention paid to lipid metabo-lism is very likely a major strategy this organism has adopted to defend against

1 MD, School of Public Health, University of California, Berkeleye-mail: [email protected]




defesa contra o ambiente hostil do seu ni-cho natural – os granulomas humanos.Os ácidos micólicos são componentes lipídi-cos importantes e os mais abundantes ácidos gordos na parede celular do M. tuberculosis5,6. O envelope celular do M. tuberculosis contém três classes de micolatos – ácidos alfa, ceto e metoximicólicos6. Os micolatos estão nor-malmente ligados à camada arabinogalactana e ao carboidrato de trealose6,7. As alterações estruturais dos ácidos micólicos afetam a viru-lência do M. tuberculosis. A ausência de ácido micólico oxigenado está associada à alteração do M. tuberculosis num modelo de rato8. A cis ciclopropanação de ácidos micólicos me-diados por ciclopropano sintetase é necessária à formação do fator corda e à virulência em ratos9,10. Por outro lado, a transciclopropana-ção de ácidos micólicos oxigenados é impor-tante para a virulência na direção oposta. Um mutante do M. tuberculosis sem anéis transci-clopropanos nestes ácidos micólicos é hipervi-rulenta no modelo de rato11.O conjunto destas observações sugere um papel importante dos lípidos e, em particu-lar, dos ácidos micólicos, na modificação da resposta imune do hospedeiro e no poste-rior resultado clínico. Os autores vêm estu-dando a regulação dos lípidos do M. tuber-culosis, com particular atenção a um conjunto de operões que parecem servir como sistemas transportadores de lípidos na parede celular – mce operões.

Papel dos mce operões na modificação da estrutura da parede celularO Mycobacterium tuberculosis contém qua-tro cópias homólogas de um operão desig-nado mce1 -41. Já demonstrámos que o M.

the hostile environment of its natural niche – the human granulomas.Mycolic acids are the major lipid consti-tutent and the most abundant fatty acid found in the cell wall of M. tuberculosis5,6. The M. tuberculosis cell envelope contains 3 classes of mycolates-alpha, keto- and me-thoxy-mycolic acid6. Mycolates are normally attached to the arabinogalactan layer and carbohydrate trehalose6,7. Structural altera-tions of mycolic acids affect the virulence property of M. tuberculosis. Absence of oxy-genated mycolic acid is associated with at-tenuation of M. tuberculosis in a mouse model8. Cis-cyclopropanation of mycolic acids mediated by cyclopropane synthase is needed for cord formation as well as for virulence in mice9,10. On the other hand, transcyclopropanation of oxygenated my-colic acids is important for virulence in the opposite direction. An M. tuberculosis mu-tant lacking transcyclopropane rings in these mycolic acids is hypervirulent in the mouse model11.Taken together, these observations suggest an important role for lipids, and in particu-lar, mycolic acids, in modifying host im-mune response and the subsequent clinical outcome. We have been studying the regu-lation of M. tuberculosis lipids focusing on a set of operons that appear to serve as lipid transporter systems in the cell wall – mce operons.

Role of the mce operons in cell wall structural modificationM. tuberculosis contains 4 homologous co-pies of an operon designated mce1-41. We showed that M. tuberculosis disrupted in the mce1 operon is hypervirulent in mice12. The

Regulação da composição lipídica da parede celular do Mycobacterium tuberculosis e o seu efeito na persistência bacteriana in vitro

Lee W Riley




tuberculosis danificado no operão mce1 é hi-pervirulento em ratos12. O mutante não provoca uma forte resposta imune do tipo Th1 e causa migração aberrante de células pró -inflamatórias, resultando em granulo-mas mal organizados nos pulmões dos ra-tos12. A falta de resposta imune do tipo Th1 adequada e de formação granulomatosa or-ganizada impede o controlo da proliferação bacteriana no rato, o que leva à morte pre-matura neste animal. Esta observação foi obtida em ratos BALB/c e C57BL/6 imu-nocompetentes, exibindo ambos resposta diferencial tipo Th1 à infeção por M. tuber-culosis do tipo selvagem12,13.O mce1 operão é regulado negativamente no meio intracelular por mce1R, localizado imediatamente acima14. O mce1R pertence à subfamília FadR de reguladores transcricio-nais GntR1,14. Um mutante do gene mce1R também é hipervirulento em ratos, mas pela razão oposta, pois causa uma resposta imu-nopatológica acelerada, com rápida progres-são para a morte do animal após maciça for-mação granulomatosa nos pulmões15. A morte do animal deve -se, não à proliferação bacteriana, mas à resposta hiperpró--inflamatória. Descobrimos, assim, que a ausência de expressão do operão mce1 (como com o operão mce1 mutante) está associada aos granulomas mal organizados e migração aberrante de células pró -inflamatórias, en-quanto a sua expressão constitutiva (como com o mutante mce1R) causa maciça forma-ção granulomatosa nos pulmões dos ratos. Ambos os efeitos resultam em consequên-cias clínicas adversas nestes animais, pelo que achámos que o mce1 operão serve para regular homeostaticamente a resposta imu-ne do hospedeiro para manter as estruturas granulomatosas que permitem, não só a so-

mutant cannot elicit a strong Th1-type im-mune response and causes aberrant migra-tion of pro-inflammatory cells, resulting in poorly organized granulomas in the mouse lungs12. The lack of adequate Th-1 type res-ponse and organized granuloma formation precludes control of bacterial proliferation in mice, which leads to early death in this animal. This observation was made in im-munocompetent BALB/c as well as in C57BL/6 mice, both of which exhibit dif-ferential Th1 type response to wild type M. tuberculosis infection12,13.The mce1 operon is negatively regulated intracellularly by mce1R, located imme-diately upstream14. Mce1R belongs to the FadR subfamily of GntR transcriptional regulators1,14. A mutant disrupted in mce1R gene is also hypervirulent in mice but for an opposite reason. It causes accelerated immunopathologic response with rapid progression to death of the animal follow-ing massive granuloma formation in their lungs15. The animal dies, not from bacte-rial proliferation but from the hyper-proin-flammatory response. Therefore, we dis-covered that the absence of the mce1 operon expression (as with the mce1 operon mu-tant) is associated with poorly organized granulomas and aberrant pro-inflammato-ry cell migration, while its constitutive ex-pression (as with the mce1R mutant) causes massive granuloma formation in mouse lungs. Both of these outcomes result in ad-verse clinical outcomes in mice. We there-fore reasoned that the mce1 operon serves to homeostatically regulate the host im-mune response to maintain granuloma structures that allow not only the host to survive but also for M. tuberculosis to re-main persistent.

Regulação da composição lipídica da parede celular do Mycobacterium tuberculosis e o seu efeito na persistência bacteriana in vitroLee W Riley




brevivência do hospedeiro, mas também a persistência do M. tuberculosis.A análise filogenómica dos operões mce do M. tuberculosis mostrou que operões relacionados se encontram entre a maioria dos membros de Actinomycetales e que estes codificam uma fa-mília de transportadores ABC de captação de lípidos16. Um dos produtos do operão mce1 é o FadD5, semelhante na sequência (43%) em E. coli da acil -CoA sintetase FadD17. Além disso, a proteína Mce1R do M. tuberculosis é 33% idêntica à proteína repressora transcri-cional FadR encontrada em E. coli, que se liga à acyl CoA -gorda e induz a expressão dos ge-nes envolvidos na degradação e transporte dos ácidos gordos18.O M. tuberculosis danificado em fadD5 é ate-nuado em ratos e tem crescimento reduzido in vitro em meio mínimo, com ácido micóli-co como única fonte de carbono19. Assim, FadD5 pode servir para reciclar ácidos micó-licos que possam ser libertados na morte do M. tuberculosis durante o desenvolvimento natural da infeção. Este mecanismo de reci-clagem pode contribuir para a sobrevivência a longo prazo da população viva de M. tuber-culosis num ambiente granulomatoso. Deste modo, o operão mce1 pode incluir um siste-ma de importação de ácidos micólicos.Curiosamente, outro grupo propôs que o operão mce4 pode ser um sistema de impor-tação de colesterol20. Atualmente, a função dos operões mce2 e mce3 como transporta-dores de lípidos ainda não está estabelecida.No entanto, estas observações sugerem que, durante diferentes estádios da infeção, o M. tuberculosis utiliza uma variedade de fontes de carbono para a sua sobrevivência a longo pra-zo, utilizando vários sistemas distintos de assi-milação de lípidos. Estas fontes de carbono podem tornar -se diferencialmente disponíveis

A phylogenomic analysis of the M. tubercu-losis mce operons showed that related ope-rons are found among most members of Actinomycetales and that they encode a fam-ily of ABC lipid uptake transporters16. One of the gene products of the mce1 operon is FadD5, which is similar in sequence (43%) to the E. coli fatty-acyl-CoA synthetase FadD17. In addition, the M. tuberculosis Mc-e1R protein is 33% identical to the FadR transcriptional repressor protein found in E. coli, which binds fatty-acyl CoA and indu-ces the expression of genes involved in fatty acid degradation and transport18.M. tuberculosis disrupted in fadD5 is atte-nuated in mice and diminished in growth in vitro in minimal medium supplied with my-colic acid as the sole carbon source19. Thus, FadD5 may serve to recycle mycolic acids that may be released from dying M. tubercu-losis during a natural course of infection. Such a recycling mechanism may contribute to the long-term survival of live population of M. tuberculosis in a granuloma environ-ment. Thus, the mce1 operon may comprise a mycolic acid import system.Interestingly, another group proposed that the mce4 operon may serve as a cholesterol import system20. At this time, the function of mce2 and mce3 operons as lipid trans-porters is not established.Nevertheless, these observations suggest that during different stages of infection, M. tuberculosis utilizes a variety of carbon sources for its long-term survival using several distinct lipid assimilation systems. These carbon sources may become diffe-rentially available to M. tuberculosis du-ring the turnover of granuloma cells. Some of the lipids may be released from these cells turning over, while others may

Regulação da composição lipídica da parede celular do Mycobacterium tuberculosis e o seu efeito na peresistência bacteriana in vitro

Lee W Riley




ao M. tuberculosis durante o turnover das célu-las granulomatosas, quando alguns dos lípidos podem ser libertados por estas células, enquan-to outros podem advir de um subgrupo de células de M. tuberculosis mortas pelas molécu-las efetoras produzidas por células que consti-tuem os granulomas. Em todas estas situações, o M. tuberculosis parece ter o fornecimento contínuo de energia assegurado por um longo período de tempo. Assim, qualquer interrup-ção deste equilíbrio entre a população bacte-riana e o turnover das células granulomatosas poderia levar à completa eliminação das bacté-rias (e.g., por tratamento com fármacos anti-tuberculose) ou à produção da doença ativa (e.g., por imunossupressão, idade avançada, inóculo infeccioso inicial elevado e certos fato-res relacionados com a estirpe).

ConclusõesOs transportadores ABC semelhantes ao operão mce conservados nos membros dos Actinomyce-tales, a maioria dos quais saprófitas de solo, apoiam a ideia de que o próprio M. tuberculosis descende de alguma micobactéria de solo. No reservatório do solo, as saprófitas obtêm os seus nutrientes e carbono como fonte de energia a partir de matéria orgânica morta. O M. tubercu-losis tem como seu reservatório natural o hospe-deiro humano, particularmente a estrutura gra-nulomatosa dos órgãos humanos. Parece que o M. tuberculosis simplesmente readaptou a fun-ção ancestral de sequestração de carbono usada pelos seus antepassados ao retirar o carbono li-bertado pelas células granulomatosas mortas e bactérias no reservatório humano. Infelizmente, para os humanos, esta readaptação metabólica levou a que o M. tuberculosis se tornasse uma das mais frequentes causas de morte por infeção no adulto em todo o mundo.

come from a subset of M. tuberculosis cells that are killed by effector molecules pro-duced by cells that comprise the granulo-mas. In all of these situations, M. tubercu-losis appears to be assured of continued supply of energy for a long period of time. Thus, any disruption of this balance be-tween bacterial population and granulo-ma cell turnover could lead to either com-plete elimination of the bacteria (e.g., by treatment with anti-tuberculosis drugs) or active disease production (e.g., from immunosuppression, old age, high initial infe ctious inoculum, and certain strain-related factors).

ConclusionsThe highly conserved mce-operon-like ABC transporters across members of the Actinomycetales, most of which are soil sa-prophytes, support the idea that M. tuber-culosis itself descended from some soil my-cobacteria. In the soil reservoir, saprophytes derive their carbon nutrients as energy source from dead organic matter. M. tuber-culosis has as its natural reservoir the human host, in particular, the granuloma structure in human organs. It appears that M. tuber-culosis has simply re-adapted its ancestral carbon-sequestration function its ancestors used in soil to derive carbon released from dead granuloma cells and bacteria in the human reservoir. Unfortunately, for hu-mans, this metabolic readaptation has led M. tuberculosis to become one of the most common infectious causes of death in adults worldwide.

Regulação da composição lipídica da parede celular do Mycobacterium tuberculosis e o seu efeito na persistência bacteriana in vitroLee W Riley




Bibliografia/Bibliography1. Cole ST BR, Parkhill J, Garnier T, Churcher C, Har-ris D, Gordon SV, Eiglmeier KGS, Barry CE 3rd, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, CR, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd SHT, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K,, Osborne JQM, Rajandream MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares RSS, Sulston JE, Taylor K, Whitehead S, Barrell BG. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998; 393:537 -544.2. Goren, M. Biosynthesis and stucture of phospholi-pids and sulfatides. In The Mycobacteria, part A. L.W. P Kubica, editor. New York: Marcel Dekker 1984; 379--415.3. Minnikin, DE. Chemical principles in the organiza-tion of lipid components in the mycobacterial cell enve-lope. Res Microbiol 1991; 142:423 -427.4. McKinney JDBK, Munoz -Elias EJ, Miczak A, Chen B, Chan WT, Swenson D, Sacchettini JC, Jacobs WR, Russell DG. Persistence of Mycobacte-rium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 2000; 406:735 -738.5. Brennan PJ, Nikaido H. The envelope of mycobacte-ria. Annu Rev Biochem 1995; 64:29 -63.6. Brennan PJ, Nikaido H. Structure of mycobacteria: recent developments in defining cell wall carbohydrates and proteins. Rev Infect Dis 1989; 11:S420 -430.7. Takayama KWC, Besra GS. Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculo-sis. Clin Microbiol 2005; 18:81 -101.8. Dubnau E, Chan J, Raynaud C, Mohan VP, Laneelle MA, Yu K, Quemard A, Smith I, Daffe M. Oxygenated mycolic acids are necessary for virulence of Mycobacterium tuberculosis in mice. Mol Microbiol 2000; 36:630 -637.9. Glickman MS, Cox JS, Jacobs WR. A novel mycolic acid cyclopropane synthase is required for cording, per-sistence, and virulence of Mycobacterium tuberculosis. Molec Cell 2000; 5:717 -727.

10. Glickman MS, Jacobs WR. Microbial pathogenesis of Mycobacterium tuberculosis: Dawn of a discipline. Cell 2001; 104:477 -485.11. Rao V, Gao F, Chen B, Jacobs WR Jr, Glickman, MS. Trans -cyclopropanation of mycolic acids on treha-lose dimycolate suppresses Mycobacterium tuberculosis -induced inflammation and virulence. J Clin Invest 2006; 116:1660 -1667.12. Shimono N, Morici L, Casali N, Cantrell S, Sidders B, Ehrt SLWR. Hypervirulent mutant of Mycobacterium tu-berculosis resulting from the disruption of the mce1 operon. PNAS 2003; 100:15918 -15923.13. Lima P, Sidders B, Morici L, Reader R, Senaratne R, Casali N, Riley LW. Enhanced mortality despite control of lung infection in mice aerogenically infected with a Mycobacterium tuberculosis mce1 operon mutant. Mi-crobes Infect 2007; 9:1285 -1290.14. Casali N, White AM, Riley LW. Regulation of the Mycobacterium tuberculosis mce1 operon. J Bacteriol 2006; 188:441 -449.15. Uchida Y, Casali N, White A, Morici L, Kendall LV, Riley LW. Accelerated immunopathological response of mice infected with Mycobacterium tuberculosis disrupted in the mce1 operon negative transcriptional regulator. Cell Microbiol 2007; 9:1275 -1283.16. Casali N, Riley LW. A phylogenomic analysis of the Ac-tinomycetales mce operons. BMC Genomics 2007; 8:60.17. Trivedi OA, Arora P, Sridharan V, Tickoo R, Mo-hanty D, Gokhale RS. Enzymic activation and transfer of fatty acids as acyl -adenylates in mycobacteria. Nature 2004; 428:441 -445.18. DiRusso CC, Black PN. Bacterial long chain fatty acid transport: gateway to a fatty acid -responsive signa-ling system. J Biol Chem 2004; 279:49563 -49566.19. Dunphy KYSR, Masuzawa M, Kendall LV, Riley LW. Journal of Infectious Disease (in press).20. Pandey AK, Sassetti CM. Mycobacterial persistence requires the utilization of host cholesterol. Proc Natl Acad Sci USA 2008; 105:4376 -4380.

Regulação da composição lipídica da parede celular do Mycobacterium tuberculosis e o seu efeito na persistência bacteriana in vitro

Lee W Riley




Jesus Gonzalo Asensio1

Ainhoa Arbues1

Dessi Marinova1

Carlos Martín1

Uma nova vacina viva contra a tuberculose com base na inativação do phoP

A new live tuberculosis vaccine based on phoP inactivation

A BCG viva atenuada é a atual vacina con-tra a tuberculose (TB). Embora venha sen-do usada há mais de 80 anos, esta vacina não é eficaz na proteção contra a TB pulmo-nar no adulto, sendo necessárias novas vaci-nas e novas estratégias de vacinação que ofe-reçam melhor proteção contra esta doença do que a vacina BCG existente. As vacinas vivas atenuadas estão entre as mais eficazes contra doenças infecciosas humanas devido à resposta imune alargada e de longa dura-ção por elas induzida.Na última década verificou -se um ressurgi-mento global de tuberculose, e as estratégias pioneiras para o desenvolvimento de vaci-nas mais eficazes levaram à descoberta de sub unidades de vacinas que provaram não oferecer melhor proteção do que a BCG em vários modelos animais, mas melhoravam a eficácia da BCG quando usadas numa estra-tégia de vacinação prime -boost. Formas dife-rentes de BCG prime e boost com estratégias de sub unidades estão a ser utilizadas em en-saios clínicos, com o objetivo de melhorar a

The attenuated live BCG is the current vac-cine against tuberculosis (TB). It has been used for more than 80 years but is ineffec-tive at providing protection against adult pulmonary TB. New tuberculosis vaccine candidates and TB vaccination strategies conferring better protection against pulmo-nary tuberculosis than the current vaccine BCG are needed. Live attenuated vaccines are among the most effective vaccines against human infectious disease due to the broad and long-lived immune response they induce.In the recent decade, a global pipeline of novel TB candidates has emerged. Pioneer-ing strategies for the development of more effective vaccines today have lead to the dis-covery of subunit vaccines, which have proved ineffective at providing better pro-tection than BCG in various animal mo dels, but could improve the efficacy of BCG used in a prime-boost strategy. Different heterologous prime BCG and boost with subunit strategies are in clinical trials with

1 Department of Microbiology, University of Zaragoza. Spain http://genmico.unizar.es




eficácia da BCG. Mais recentemente, iniciaram -se ensaios clínicos com vacinas BCG recombinantes, com o intuito de en-contrar candidatas para serem usadas como vacinas prime preventivas. Outra estratégia inovadora, as vacinas vivas baseadas no My-cobacterium tuberculosis racionalmente ate-nuado, são candidatas promissoras (algumas delas no fim da investigação pré -clínica), um novo instrumento substituto da BCG, com melhor eficácia protetora contra as for-mas pulmonares de TB.Com base na observação de que a codifica-ção pelo gene phoP do fator de transcrição PhoP é essencial para a virulência do M. tu-berculosis (Perez et al., 2001), atenuámos ra-cionalmente o bacilo da TB por inativação do phoP. O mutante demonstrou forte ate-nuação em modelos celular e animal e resul-tou mais atenuado do que a BCG em rati-nhos SCID imunocomprometidos (Martin et al., 2006). A vacina candidata protegeu porquinhos -da -índia e primatas não huma-nos contra a infeção tuberculosa (Martin et al., 2006, Verreck et al., 2009).Estudos moleculares recentes (Gonzalo Asen-sio et al., 2008) demonstraram que o fator de transcrição phoP controla a expressão de apro-ximadamente 80 genes (muitos dos quais im-plicados na virulência do M. tuberculosis), responsável por cerca de 2% da open reading frame (ORF) no genoma do M. tuberculosis. Com base nestes estudos, pode pensar -se que o fenótipo atenuado e a imunidade protetora conferida pelo mutante phoP podem ser ex-plicados pelo mecanismo de ação alterado do fator de transcrição phoP, conforme descrito e resumido na Fig. 1.O mutante phoP diminui a regulação da biossíntese dos complexos lípidos da parede celular micobacteriana, incluindo a diacil-

the aim to improve efficacy of BCG. More recently, clinical trials with recombinant BCG vaccines have started with the aim to find candidates to be used as prime, preven-tive vaccines. Another innovative strategy, live vaccines based on rationally attenuated Mycobacterium tuberculosis are promising candidates (some of which in late preclini-cal investigation), is a promising new BCG-replacement tool with better protective ef-ficacy against pulmonary forms of TB.Based upon the observation that the phoP gene coding for the transcription factor phoP is essential for M. tuberculosis virulence (Perez et al., 2001), we rationally attenuated the tubercle bacillus by inactivating phoP. The mutant demonstrated strong attenua-tion in cellular and animal models and re-sulted more attenuated than BCG in im-munocompromised SCID mice (Martin et al., 2006). The vaccine candidate protec ted guinea pigs and non-human primates against tuberculosis infection (Martin et al., 2006, Verreck et al., 2009).Recent molecular studies (Gonzalo Asensio et al., 2008) have demonstrated that phoP transcription factor controls the expression of approximately 80 genes (many of which implicated in M. tuberculosis virulence), ac-counting for about 2% of the open reading frame (ORF) in M. tuberculosis genome. Based on these studies it could be deduced that the attenuated phenotype and the pro-tective immunity conferred by the phoP mutant can be explained by the altered mechanism of action of the transcription factor phoP, as described below and summa-rized in Fig. 1.The phoP mutant decreases byosinthesis regulation of complex mycobacterial cell-wall lipids, including diacyltrehalose (DAT)

Uma nova vacina viva contra a tuberculose com base na inativação do phoPJesus Gonzalo Asensio, Ainhoa Arbues, Dessi Marinova, Carlos Martín




trealose (DAT) e poliaciltrealose (PAT) im-plicadas na imunomodulação do sistema imunológico hospedeiro (Saavedra R et al. 2005), não expressos pelo mutante phoP (Gonzalo Asensio et al. 2006).Também se observou desregulação dos genes na região da diferença 1 (RD1) (Gonzalo Asensio et al., 2008) necessária à virulência e à secreção de ESAT -6 no mutante phoP, quando comparado com a estirpe do tipo selvagem (Friguie et al., 2008). A RD1 restringe -se às estirpes virulentas do complexo M. tuberculosis (CMTB) e perde -se por todas as sub estirpes de BCG (Behr et al., 1999, Pym et al., 2003).

and polyacyltrehalose (PAT) implicated in immunomodulation of the host immune system (Saavedra R et al 2005), which are not expressed by the phoP mutant (Gonzalo Asensio et al 2006).Down regulation of genes within the region of difference 1 (RD1) (Gonzalo-Asensio et al., 2008) required for virulence and ESAT-6 secretion were also observed in the phoP mutant when compared to wild-type strain (Friguie et al 2008). RD1 is restricted to virulent M. tuberculosis complex (MTBC) strains and is lost by all BCG substrains (Behr et al., 1999, Pym et al., 2003).

Fig. 1 – Estudos moleculares de vacina baseada em phoP (Gonzalo-Asensio, et al., 2008)

Fig. 1 – Molecular studies of phoP-based vaccine (Gonzalo-Asensio et al., 2008)

~2% ORF no genoma do M. tuberculosis sob controlo do phoP / ~2% ORFs in the M. tuberculosis genome under phoP control

RESPIRAÇÃO AERÓBICA E ANAERÓBICA/AEROBIC & ANAEROBIC RESPIRATION

RESPOSTAS AO STRESS E AO CHOQUE TÉRMICO/STRESS

& HEAT-SHOCK RESPONSES

Mutante phoP/PhoP mutant

METABOLISMO LIPÍDICO SL, DAT, PAT.../

LIPID METABOLISM SL, DAT, PAT...

Membrana plasmática/Plasma membrane

RESPOSTAS HIPÓXICAS PRECOCES E DURADOURAS/EARLY & ENDURING

HYPOXIC RESPONSES PERSISTÊNCIA/PERSISTENCE

REGIÃO RD1 E SECREÇÃO DE ESAT-6/RD1 REGION & ESAT-6 SECRETION

Frigui et al. PLoS Path 2008

Gonzalo Asensio et al. JBC 2006Cell

lysate

Culturefi ltrate

SO2

H37R

vH3

7Ra

H37R

a::ph

oPH3

7Ra::

rpsL





The phoP mutant showed altered regula-tion and control of other key functions re-quired for successful survival of the micro-organism within the host cell. As illustrated in Figure 1, these functions include early and enduring hypoxic responses, aerobic and anaerobic respiration, stress and heat-shock responses, lipid metabolism (normal-ly down regulated in the avirulent strain H37Ra), and expression of the persistence factor ICL, important for intracellular per-sistence of M. tuberculosis during infection (Gonzalo-Asensio et al 2008). ICL is posi-tively regulated in the phoP mutant and this characteristic could explain the ade-quate persistence state important for im-proved antigen presentation by the mutant during vaccination.These observations were used to construct a new generation of a live vaccine based on phoP inactivation carrying a second addi-tional mutation which affects the synthesis of a new family of lipids associated with M. tuberculosis virulence following the Geneva consensus on essential steps towards clinical development of new live attenuated mycobacterial vaccines (Kamath et al., 2005). The final construct is the first live attenuated candidate vaccine developed according with and fulfilling the Geneva consensus requirements for live mycobacterial vaccines with the aim to deliver on the Millennium Development Goal to combat TB. Rigorous preclinical studies to date with the live phoP-based vaccine prototype have demonstrated proof of principle for adequate attenuation, safety, immunogenicity, and protective efficacy against respiratory forms of TB in stringent animal models (Perez et al., 2001, Williams et al., 2005, Martin et al., 2006, Aguilar et

O mutante phoP evidenciou regulação altera-da e controlo de outras funções -chave neces-sárias à sobrevivência dos microrganismos na célula hospedeira. Como se mostra na Fig. 1, estas funções incluem respostas hipóxicas precoces e duradouras, respiração aeróbica e anaeróbica, respostas ao stress e ao choque tér-mico, metabolismo lipídico (normalmente desregulado na estirpe avirulenta H37Ra) e expressão do fator de persistência ICL, im-portante para a persistência intracelular do M. tuberculosis durante a infeção (Gonzalo Asensio et al., 2008). O ICL é regulado posi-tivamente no mutante phoP e esta caracterís-tica poderia explicar o adequado estado de persistência, importante para a apresentação melhorada do antigénio pelo mutante duran-te a vacinação.Estas observações foram usadas para construir uma nova geração duma vacina viva baseada na inativação do phoP com uma segunda mutação adicional que afecta a síntese duma nova famí-lia de lípidos associada à virulência da M. tuber-culosis depois do consenso de Genebra sobre os passos essenciais para o desenvolvimento clíni-co de novas vacinas micobacterianas vivas ate-nuadas (Kamath et al., 2005). O elemento fi-nal é a primeira candidata a vacina viva atenuada desenvolvida de acordo com o con-senso e recomendações de Genebra para vaci-nas micobacterianas vivas, com o objectivo de a apresentar ao Millennium Development Goal no combate à TB. Ensaios pré -clínicos rigoro-sos feitos até à data com o protótipo da vacina viva baseada no phoP demonstraram a sua ade-quada atenuação, segurança, imunogenicidade e eficácia na proteção contra as formas respira-tórias de TB em modelos animais (Perez et al., 2001, Williams et al., 2005, Martin et al., 2006, Aguilar et al., 2007, Cardona et al., 2009, Verreck et al., 2009).





al., 2007, Cardona et al., 2009, Verreck et al., 2009).The molecular studies with the phoP mutant have helped begin to understand the intricate puzzle behind the mechanism of action of the PhoP transcription factor required for virulence, persistence and intracellular survival of the wild-type strain during infection and whose altered expression probably accounts for the adequate attenuation phenotype of the prototype vaccine. Moreover, elimination of the extractable immunomodulatory cell-wall lipids (DAT and PAT) as a result of the targeted inactivation of the phoP gene, and the impaired synthesis of the complex virulence determinant and cell-wall lipid PDIM (Camacho et al., 1999, Cox et al., 1999) due to the second genetically engineered mutation in the prototype phoP-based vaccine further contribute to the attenuated phenotype of this novel candidate.Strong to date research provides robust scientific support adducing this new generation vaccine well–advanced to progress from research to development and be clinically evaluated for vaccine safety and efficacy for the first time in human. Moreover, considering the lack of immunological correlates of protection, Phase 3 clinical trials are indispensible to tell us the real value of any new vaccine used as a prophylactic tool against TB.Stop TB partnership estimate that at least 20 vaccine candidates should enter phase I safety trials before 2015 with the goal to reach an effective licensed vaccine against TB (Young and Dye, 2006). The discovery and use of a new TB vaccine that confers improved protection against pulmonary forms of TB than the current BCG is key to reach the 2050 objective of TB eradication.

Os estudos moleculares com o mutante phoP ajudaram na compreensão do comple-xo puzzle por detrás do mecanismo de ação do fator de transcrição do phoP necessário à virulência, persistência e sobrevivência in-tracelular da estirpe do tipo selvagem du-rante a infeção, e cuja expressão alterada é provavelmente responsável pela adequada atenuação do fenótipo do protótipo da vaci-na. Além disso, a eliminação dos lípidos imunomoduladores extraíveis da parede ce-lular (DAT e PAT) resultante da inativação do gene phoP e a deficiente síntese da com-plexa determinante da virulência e dos lípi-dos da parede celular PDIM (Camacho et al., 1999, Cox et al., 1999), devido à segun-da mutação geneticamente conseguida no protótipo da vacina baseada no phoP, tam-bém contribuem para o fenótipo atenuado desta nova candidata.Investigação atual de qualidade proporciona forte apoio científico a esta vacina de nova geração em franco progresso e em vias de ser clinicamente avaliada quanto à segurança e eficácia em seres humanos. Tendo em conta a falta de correlativos imunológicos de pro-teção, é indispensável realizar ensaios clíni-cos de fase 3 para conhecermos o valor real de qualquer nova vacina usada profilatica-mente contra a TB.A parceria Stop TB estima que pelo menos 20 vacinas candidatas deveriam iniciar os ensaios de segurança de fase I antes de 2015, com o propósito de conseguir o licencia-mento para uma vacina eficaz contra a TB (Young, Dye, 2006). A descoberta e utiliza-ção de uma nova vacina contra a TB que ofereça melhor proteção contra as formas pulmonares da doença do que a atual BCG é crucial para alcançar o objetivo de erradi-car a tuberculose até 2050.





Bibliografia/BibliographyAguilar D, Infante E, Martin C, Gormley E, Gicquel B, Hernandez Pando R. Immunological responses and pro-tective immunity against tuberculosis conferred by vac-cination of Balb/C mice with the attenuated Mycobacte-rium tuberculosis (phoP) SO2 strain. Clin Exp Immunol 2007; 147: 330 -338.Asensio JA, Arbues A, Perez E, Gicquel B, Martin C. Live tuberculosis vaccines based on phoP mutants: a step towards clinical trials. Expert Opin Biol Ther 2008; 8:201 -211.Camacho LR, Ensergueix D, Perez E, Gicquel B, Guil-hot C. Identification of a virulence gene cluster of Myco-bacterium tuberculosis by signature -tagged transposon mutagenesis. Mol Microbiol 1999; 34: 257 -267.Cardona PJ, Asensio JG, Arbues A, Otal I, Lafoz C, Gil O, Caceres N, Ausina V, Gicquel B, Martin C. Exten-ded safety studies of the attenuated live tuberculosis vac-cine SO2 based on phoP mutant. Vaccine 2009; 27: 2499 -2505.Cox JS, Chen B, McNeil M, Jacobs WR Jr. Complex lipid determines tissue -specific replication of Mycobacte-rium tuberculosis in mice. Nature 1999; 402: 79 -83.Frigui W, Bottai D, Majlessi L, Monot M, Josselin E, Brodin P, Garnier T, Gicquel B, Martin C, Leclerc C, Cole ST, Brosch R. Control of M. tuberculosis ESAT -6 secretion and specific T cell recognition by PhoP. PLoS Pathog 2008; 4: e33.Gonzalo Asensio J, Maia C, Ferrer NL, Barilone N, Laval F, Soto CY, Winter N, Daffe M, Gicquel B, Mar-tin C, Jackson M. The virulence -associated two--component PhoP -PhoR system controls the biosynthe-sis of polyketide -derived lipids in Mycobacterium tuberculosis. J Biol Chem 2006; 281: 1313 -1316.Gonzalo -Asensio J, Mostowy S, Harders -Westerveen J, Huygen K, Hernandez -Pando R, Thole J, Behr M, Gic-quel B, Martin C. PhoP: a missing piece in the intricate puzzle of Mycobacterium tuberculosis virulence. PLoS ONE 2008; 3: e3496.

Kamath AT, Fruth U, Brennan MJ, Dobbelaer R, Hu-brechts P, Ho MM, Mayner RE, Thole J, Walker KB, Liu M, Lambert PH. New live mycobacterial vaccines: the Geneva consensus on essential steps towards clinical development. Vaccine 2005; 23: 3753 -3761.Martin C, Williams A, Hernandez -Pando R, Cardona PJ, Gormley E, Bordat Y, Soto CY, Clark SO, Hatch GJ, Agui-lar D, Ausina V, Gicquel B. The live Mycobacterium tuber-culosis phoP mutant strain is more attenuated than BCG and confers protective immunity against tuberculosis in mice and guinea pigs. Vaccine 2006; 24: 3408 -3419.Perez E, Samper S, Bordas Y, Guilhot C, Gicquel B, Mar-tin C. An essential role for phoP in Mycobacterium tuber-culosis virulence. Mol Microbiol 2001; 41: 179 -187.Saavedra R, Segura E, Tenorio EP, López -Marín LM. Mycobacterial trehalos -containing glycolipid with im-munomodulatory activity on human CD4+ and CD8+ T -cells. Micr Inf 2006; 8:533 -540.Verreck FA, Vervenne RA, Kondova I, van Kralingen KW, Remarque EJ, Braskamp G, van der Werff NM, Kersbergen A, Ottenhoff TH, Heidt PJ, Gilbert SC, Gicquel B, Hill AV, Martin C, McShane H, Thomas AW. MVA.85A boosting of BCG and an attenuated, phoP deficient M. tuberculosis vaccine both show pro-tective efficacy against tuberculosis in rhesus macaques. PLoS One 2009; 4: e5264.Williams A, Hatch GJ, Clark SO, Gooch KE, Hatch KA, Hall GA, Huygen K, Ottenhoff TH, Franken KL, Andersen P, Doherty TM, Kaufmann SH, Grode L, Seiler P, Martin C, Gicquel B, Cole ST, Brodin P, Pym AS, Dalemans W, Cohen J, Lobet Y, Goonetilleke N, McShane H, Hill A, Parish T, Smith D, Stoker NG, Lowrie DB, Kallenius G, Svenson S, Pawlowski A, Blake K, Marsh PD. Evaluation of vaccines in the EU TB vac-cine cluster using a guinea pig aerosol infection model of tuberculosis. Tuberculosis (Edinb) 2005; 85: 29 -38.Young D, Dye C. The development and impact of tu-berculosis vaccines. Cell 2006; 124: 683 -687.





Ruth McNerney1 Simpósio: Testes rápidos para o rastreio preliminar da tuberculose

Symposium: Point-of-care tests for tuberculosis

Pensa -se que há mais casos de tuberculose no mundo hoje do que em qualquer outra altura através da história. As estimativas da Organização Mundial de Saúde (OMS) para 2007 sugeriam uma prevalência de 13,7 mi-lhões, com 9,3 milhões de novos casos e 1,8 milhões de mortes. Esta é essencialmente uma doença de pobreza, contando a Ásia e a África juntas mais de noventa por cento dos casos mundiais. Na ausência duma vacina eficaz, o controlo depende da prontidão do tratamento para reduzir a carga bacilar viá-vel, diminuindo assim a contagiosidade de indivíduos com formas pulmonares da doen-ça. Através dos esforços de iniciativas de saúde internacionais, como as do Global Fund to fight HIV/AIDS, Tuberculosis and Malaria, o tratamento da TB está agora dis-ponível e de forma gratuita para a maioria das pessoas. Porém, o acesso ao tratamento está dependente do diagnóstico e os atrasos na deteção podem resultar na deterioração do doente e aumentar as oportunidades de

It is believed there are more cases of tu-berculosis in the world today than at any previous time in history. WHO estimates for 2007 suggested a prevalence of 13.7 million, with 9.3 million incident cases and 1.8 million deaths. It is primarily a disease of poverty, with Asia and Africa between them sharing over ninety percent of the global TB burden. In the absence of an effective vaccine control depends on prompt treatment to reduce the viable bacillary load, thus reducing the infec-tiousness of individuals with open pul-monary forms of the disease. Through the efforts of international health initiatives such as the Global Fund to fight HIV/AIDS, Tuberculosis and Malaria treat-ment for TB is now widely available and free for the majority patients. However, access to treatment is dependent on diag-nosis and delays in detection may result in deterioration of the patient and in-creased opportunity for transmission.

1 Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine

Keppel Street, London, WC1E 7HT, UK e-mail: [email protected]




transmissão. Infelizmente, o diagnóstico nem sempre é fácil, uma vez que os sinto-mas não são específicos, particularmente em pessoas com problemas de saúde concomi-tantes, como a imunossupressão relacionada com o VIH.A maioria dos casos de TB residem em paí-ses com sistemas de saúde fracos e com pou-cos recursos, onde os laboratórios que reali-zam exames de diagnóstico são limitados em número e distribuição geográfica, e cujo acesso pode envolver longas viagens e despe-sa considerável. Esta situação é agravada pela insensibilidade dos testes disponíveis e pela necessidade de múltiplas consultas. Os atrasos também se devem ao comportamen-to dos doentes, quando estes não procuram uma consulta médica. A relutância em obter um diagnóstico reflete a natureza crónica da doença, a sua não perceção e, nalguns casos, o receio de estigmatização e exclusão social. As estimativas da OMS sugerem que, du-rante 2006, quatro milhões de casos de TB não foram diagnosticados. Ao contrário do que acontece com patologias como a malá-ria ou o VIH, não há testes rápidos nem dis-positivos (POC) suficientemente fiáveis para uso de pessoal não especializado na comuni-dade.Um painel de especialistas internacionais foi convidado para um simpósio onde seria abordado o tópico dos testes rápidos para o diagnóstico da tuberculose. O evento foi organizado pelo subgrupo POC do grupo de trabalho STOP TB sobre novos diagnós-ticos, em colaboração com a Sociedade Eu-ropeia de Micobacteriologia. A intenção do simpósio foi aumentar a perceção da neces-sidade de melhorar o acesso ao diagnóstico da TB, atualizar os técnicos de diagnóstico e os investigadores sobre o desenvolvimen-

Unfortunately diagnosis is not straight-forward as symptoms are not specific, particularly in those with an underlying health problem such as HIV related im-munosupression. The majority of TB cases reside in countries with weak and poorly resourced health care systems where labo-ratory facilities for diagnosis are limited in number and distribution. Access may involve long journeys and considerable expense, a situation compounded by the insensitivity of the available tests and need for multiple visits. Delays also arise from health seeking behaviour, where indivi-duals do not seek medical assistance. Re-luctance to seek a diagnosis reflects the chronic nature of the disease, a lack of awareness and, in some settings a fear of stigmatization and social exclusion. WHO estimates suggest that during 2006 four million cases of TB remained undiag-nosed. Unlike conditions such as malaria or HIV there are no sensitive rapid tests and no reliable point-of-care (POC) de-vices that can be used by non specialist personnel within the community. To ad-dress these issues a panel of international experts were invited to speak in a sympo-sium on the topic of point-of-care tests for the diagnosis of tuberculosis disease. The event was organized by the POC sub-group of the STOP TB working group on New Diagnostics in collaboration with the European Society of Mycobacteriolo-gy. The intention of the symposium was to increase awareness of the need for im-proved access to TB diagnosis, to update diagnostic practitioners and the research community on the current status of POC test development and to debate research needs and priorities.

Testes rápidos para o rastreio preliminar da tuberculoseRuth McNerney




to e o estado atual dos testes POC, e ainda debater as necessidades e prioridades da in-vestigação. O potencial dos testes POC na ajuda ao controlo da TB foi discutido por Catharina Boehme (Foundation for Innova-tive New Diagnostics). O teste ideal não de-veria necessitar de técnicos especializados nem de infraestruturas laboratoriais, facul-taria o resultado no próprio dia da consul-ta, teria uma elevada especificidade e sensi-bilidade superior à da microscopia de esfregaço. Os indivíduos com um resultado positivo seriam imediatamente registados e referenciados para tratamento. Os testes com sensibilidade inferior à dos testes com base no laboratório teriam um papel se pu-dessem ser usados para reduzir os atrasos no diagnóstico. Alternativamente, um teste de avaliação com elevada sensibilidade e espe-cificidade razoável poderia ser usado no ras-treio de populações de alto risco, em que, com um resultado positivo, o doente seria referenciado para confirmação do diagnós-tico antes do início do tratamento.As atuais dificuldades no acesso ao trata-mento foram ilustradas pelo testemunho de uma doente com TB natural da Zâmbia, país severamente atingido por TB e VIH//SIDA. Em 2003, Carol Nyirenda (co-ordenadora nacional da Community Initiati-ve for Tuberculosis, HIV/AIDS and Malaria) dirigiu -se ao seu centro de saúde, queixando--se de tosse que durava havia mais de dois meses. Como a doente não conseguia pro-duzir expetoração para análise, foi enviada para casa com suspeita de bronquite cróni-ca. Durante os seis meses seguintes foi sub-metida a um total de quatro raios -X, mas não lhe foi proposto um teste VIH nesse pe-ríodo. Foi -lhe, finalmente, proporcionado o tratamento para a TB mais de seis meses de-

The potential of POC tests to aid TB control was discussed by Catharina Boe-hme (Foundation for Innovative New Dia-gnostics). The ideal test would not require specialist skills or a laboratory infrastruc-ture, would yield a result within one visit, would be highly specific and have a sensi-tivity superior to that of smear microsco-py. Anyone found positive would be im-mediately registered and referred for treatment. Tests that are not more sensi-tive than laboratory based tests would have a role if they could be used to reduce diagnostic delay. Alternatively, a screen-ing test that had high sensitivity and a reasonable specificity could be used to screen high risk populations, where a po-sitive result triggered referral for confir-mation prior to initiation of treatment. The current difficulties in accessing treat-ment were illustrated through the testa-ment of a former TB patient from Zam-bia, a country hard hit by both TB and HIV/AIDS. Carol Nyirenda (National Co-ordinator, Community Initiative for Tuberculosis, HIV/AIDS and Malaria) had presented at her local health clinic in 2003 with a cough of over two months duration. Unable to produce sputum for testing she was sent home with suspected chronic bronchitis. Over the following six months she had a total of four chest x-rays but was not offered an HIV test dur-ing that time. She was finally offered treatment for her TB more than six months after reporting symptoms. Suc-cessfully treated she now campaigns on behalf of people living with HIV and TB. She proposed that a test be developed that could be used by community volunteers, a test that could be used in rural settings





pois de ter referido os sintomas. Tratada com sucesso, Carol Nyirenda agora defende ativamente os interesses de doentes com VIH e TB, e propôs que seja desenvolvido um teste que possa ser usado por voluntá-rios comunitários, um teste que possa tam-bém ser usado em áreas rurais onde não há eletricidade e que possa ser disponibilizado a pessoas que residem em comunidades afe-tadas por TB e VIH.Os países onde a TB é endémica frequente-mente não dispõem da moldura legislativa para regular dispositivos e testes de diagnós-tico. Nessas regiões, os kits -teste podem ser comercializados sem evidência de benefício e, em algumas circunstâncias, foram acom-panhadas de alegações enganadoras quanto ao desempenho. Rosanna Peeling (London School of Hygiene & Tropical Medicine) apre-sentou dados dum estudo em que o desem-penho de 19 testes rápidos de diagnóstico da tuberculose disponíveis comercialmente foi avaliado usando um painel de 355 amos-tras séricas recolhidas em diversas regiões geográficas. Infelizmente, nenhum dos tes-tes demonstrou um desempenho suficiente-mente bom para substituir os atuais testes de microscopia de esfregaço. Quando com-parado com a combinação de referência--padrão (microscopia, cultura, radiografia e follow -up clínico), o teste com a maior sen-sibilidade (59,71%) tinha uma especificida-de de apenas 57,72%. Os dois testes com maior especificidade (98,66%) tinham sen-sibilidades de 0,97 e 2,43%, respectivamen-te. É possível obter uma cópia deste relató-rio no website do Special Programme for Research and Training in Tropical Diseases (TDR): http://apps.who.int/tdr/svc/publications/tdr -research -publicationsdiagnos-tics -evaluation -2.

where there is no electricity and a test that was affordable by people living in com-munities affected by TB and HIV.Countries where TB is endemic frequently lack the legistrative framework to regulate diagnostic test devices. In such an environ-ment test kits may be marketed without evidence of their benefit and in some cir-cumstances have been accompanied by misleading claims regarding their perfor-mance. Rosanna Peeling (London School of Hygiene & Tropical Medicine) presented data from a study where the performance of 19 commercially available rapid diag-nostic tests for tuberculosis was assessed using a panel of 355 sera collected from eight geographically diverse sites. Unfortu-nately none of the tests were found to per-form well enough to replace the current test of smear microscopy. When compared to a combined reference standard of mi-croscopy, culture, radiography and clinical follow-up the test with the highest sensi-tivity of 59.71% had a specificity of just 57.72%. The two tests with the highest specificity (98.66%) had sensitivities of 0.97 and 2.43% respectively. A full copy of the report may be downloaded from the Special Programme for Research and Train-ing in Tropical Diseases (TDR) website. http://apps.who.int/tdr/svc/publications/tdr-research-publications/diagnostics-eva-luation-2. The failure of current know-ledge to provide a rapid test for TB has led to recognition of the need for novel ap-proaches. Gerd Michel (Foundation for In-novative New Diagnostics) described the application of genome, proteome and me-tabolomic based technologies to biomarker discovery. The search for suitable detection targets has expanded from traditional pro-





O insucesso do conhecimento atual em for-necer um teste rápido para TB levou ao re-conhecimento da necessidade de novas abordagens. Gerd Michel (Foundation for Innovative New Diagnostics) descreveu téc-nicas baseadas na aplicação de tecnologias do conhecimento do genoma, proteoma e metabolómica para a descoberta de biomar-cadores. A busca de alvos adequados foi além da química de proteínas tradicional para um âmbito mais alargado de compo-nentes estruturais e metabólitos. Os testes baseados na deteção de lipoarabinomanano (LAM), um componente lipossacarídeo da parede celular, ainda não conseguiram sen-sibilidade suficiente, mas, curiosamente, num estudo, pareceram funcionar melhor em indivíduos positivos para VIH do que nos que não estavam coinfectados.Com o desenvolvimento de ensaios de ADN transrenal fragmentado na urina, novas estra-tégias estão também a ser investigadas para análises com base em ácidos nucleicos. Os testes POC precisam de ser rápidos e fáceis de fazer. Uma das maneiras mais simples e rápi-das de identificar substâncias é pela análise do seu aroma e a identificação de compostos orgânicos voláteis (COV), e vem emergindo como um campo de interesse. A prova de conceito do diagnóstico da TB foi consegui-da com o treino de ratos pouch africanos para cheirarem TB em amostras de expectoração. Embora não tão sensível como a microscopia de esfregaço, os animais conseguem avaliar uma amostra em apenas alguns segundos. Os utensílios para detectar baixas concentrações de COV estão a ser investigados, incluindo a adaptação de novos instrumentos desenvolvi-dos para uso militar e para deteção de agentes de bioterrorismo. No entanto, o optimismo precoce no que respeita à aplicação da tecno-

tein chemistry to a broader range of struc-tural components and metabolites. Tests based on detection of lipoarabinomannan (LAM) a lipopolysaccharide component of the cell wall have not so far achieved suffi-cient sensitivity but intriguingly in one study appeared to function better in HIV positive individuals than those that were not co infected. New strategies are also be-ing investigated for nucleic acid based analysis with the development of assays for fragmented transrenal DNA in urine. POC tests need to be both rapid and easy to per-form. One of the simplest and fastest ways to identify substances is by their aroma and volatile organic compound (VOC) analysis is emerging as field of interest. Proof of concept for TB diagnosis has been provi-ded through the training of African pouch rats to smell TB in sputum samples. Al-though not as sensitive as smear microsco-py the animals are able to screen a sample in just a few seconds. Tools for detecting VOC at low concentrations are being in-vestigated, including the adaptation of novel instrumentation developed for mili-tary use and bioterror agent detection. However, early optimism regarding the ap-plication of electronic nose technology has dissipated following the realisation that they lack the robustness required for a dia-gnostic test. A more analytical approach is now being pursued and Ruth McNerney (London School of Hygiene & Tropical Medi-cine) outlined some of the challenges fa-cing those searching for VOC biomarkers that are predictive of TB disease. Small volatile molecules lack the distinctive cha-racter of macromolecules traditionally used to predict infection and they are more of-ten found in nature. To complicate matters





logia do nariz electrónico dissipou -se depois da constatação de que lhe falta a robustez ne-cessária a um teste de diagnóstico. Uma abor-dagem mais analítica está agora em curso e Ruth McNerney (London School of Hygiene & Tropical Medicine) referiu alguns dos desa-fios que se apresentam a quem investiga bio-marcadores em COV preditivos de TB. As pequenas moléculas voláteis não têm o cará-ter distinto das macromoléculas tradicional-mente usadas para indicar a presença de in-fecção e encontram -se mais frequentemente na natureza. Para complicar, os metabólitos produzidos por Mycobacterium tuberculosis variam, dependendo da disponibilidade de nutrientes e outros fatores ambientais, pelo que os COV emitidos durante uma infeção podem diferir com o local da infecção e o es-tádio da doença. Do mesmo modo, os COV emitidos pelo hospedeiro em resposta à infe-ção podem sofrer alterações com a progressão da doença. A complexidade e a natureza di-nâmica da emissão de COV sugere que para atingir elevada sensibilidade e especificidade será necessária uma abordagem analítica mul-tivariada.Há muitos passos ao longo do caminho do desenvolvimento de testes e Amy P. Wong (X PRIZE Foundation) discutiu a rota e identificou as potenciais barreiras ao apare-cimento de um novo diagnóstico de suces-so. Quando os biomarcadores tiverem sido descobertos e testados com protótipos de dispositivos, a capacidade de fabrico será desenvolvida e estabelecer -se -á uma rota de mercado. As novas tecnologias podem re-querer métodos de fabrico inovadores que assegurem a robustez dos dispositivos a pre-ços competitivos. É necessária melhor orientação das especificações de novos tes-tes. A incerteza quanto ao mercado para

metabolites produced by Mycobacterium tuberculosis vary depending on the avai-lability of nutrients and other environmen-tal factors, thus VOC emitted during in-fection may differ with the site of infection and stage of disease. Similarly, VOC emit-ted by the host in response to infection may alter with disease progression. The complexity and dynamic nature of VOC emission suggest that to attain high sensi-tivity and specificity a multivariate analyti-cal approach will be required.There are many steps along the path of test development and Amy P Wong (X PRIZE Foundation) discussed the route and identified potential barriers to the de-livery of a successful new diagnostic. Once biomarkers have been discovered and tes-ted with prototype devices the capacity to manufacture must be developed and a route to market established. New tech-nologies may require innovative manufac-turing practices to ensure delivery of ro-bust devices at a competitive price. Improved guidance on optimum test specification is badly needed. Uncertainty regarding the market for POC TB tests also acts as a disincentive to potential in-vestors. Innovative mechanisms for re-warding test development are required to stimulate participation and facilitate the development of devices that can be used to detect TB wherever in the world there is a need. In the words of an ex TB patient “TB infection and disease is not just about numbers, for some of us it is a reality… we sit and watch TB reversing all the ef-forts and gains of the HIV fight, as we die one by one from TB. It is not about num-bers, there are real people at the end of the chain we have run out of time”.





testes POC de TB também funciona como desincentivo para potenciais investidores. São necessários mecanismos inovadores que compensem o desenvolvimento de testes, de modo a estimular a participação e a faci-litar o desenvolvimento de dispositivos que possam ser usados para detectar a TB em qualquer parte do mundo. Nas palavras de um ex -doente de TB, “a infeção por TB e a própria doença não são apenas números, para alguns de nós é uma realidade… va-mos assistindo à inversão, pela TB, de todos os esforços e ganhos na luta do VIH, con-forme vamos morrendo, um a um, de TB. Não se trata de números, somos pessoas no extremo da cadeia e o nosso tempo está a terminar.”





Thomas M Shinnick1 Criar capacidade laboratorial para a tuberculose: Necessidades e estratégias

Building tuberculosis laboratory capacity: Needs and strategies

Palavras -chavePlanos estratégicos nacionais, abordagem de sistemas, rede de laboratórios de saúde pública.

FundamentosNo relatório “TB Global 2009”1, a Organi-zação Mundial de Saúde (OMS) utilizou informação de modelos epidemiológicos e dados de programas para estimar que, em 2007, havia 9,27 milhões de novos casos de tuberculose (TB); 1,37 milhões (15%) de casos entre pessoas infetadas com VIH; 511 000 novos casos de TB multirresistente (MDR TB); e 50 000 novos casos de TB ex-tensamente resistente a fármacos (XDR TB). Estas são apenas estimativas, em parte devido a falhas na disponibilidade de servi-ços laboratoriais de TB em muitas regiões do mundo1.2. Apenas cerca de 60% dos no-vos casos de TB são confirmados pelo labo-ratório e só aproximadamente 5% dos casos

Key-wordsNational strategic plans, systems approach, public health laboratory networks.

BackgroundIn the 2009 Global TB report1, the World Health Organization used information from epidemiologic models and program data to estimate that in 2007 there were 9.27 mil-lion new cases on TB; 1.37 million (15%) cases among persons living with HIV; 511 000 new cases of multidrug-resistant TB (MDR TB); and 50 000 new cases of extensively drug-resistant TB (XDR TB). These are only estimates, in part, because there are large gaps in the availability of TB laboratory services in many regions of the world1,2. Only about 60% of new TB cases are laboratory confirmed and only about 5% of MDR TB cases are actually identified and reported.

1 PhD, Associate Director for Global Laboratory Activities, Division of TB Elimination,

Centers for Disease Control e Prevention1600 Clifton Road, MS -G35, Atlanta Georgia 30333 USAe-mail: [email protected]




de MDR TB são realmente identificados e reportados.A inadequada capacidade laboratorial preju-dica o diagnóstico, a gestão dos casos e a vi-gilância da doença. Isto é particularmente importante para os doentes com TB resis-tente a fármacos porque, frequentemente, os cuidados eficazes só se iniciam depois dos resultados dos testes de suscetibilidade a fár-macos. O aparecimento da MDR TB e da XDR TB levou ao reconhecimento de que a falta de capacidade laboratorial representa uma crise global. Os fatores que têm contri-buído para as falhas nos serviços laborato-riais de TB incluem: 1) falta de reconheci-mento da importância do laboratório no tratamento e no controlo da doença; 2) má comunicação entre os programas nacionais de TB e as entidades que fornecem os servi-ços laboratoriais de TB; 3) recursos humanos e financeiros inadequados nesses laborató-rios; 4) falta de infraestruturas e de instala-ções; e 5) preocupações com biossegurança4.Ao reconhecer o importante papel do labora-tório, a 2007 World Health Assembly5 endos-sou o apelo do Global Plan to Stop TB2 para acesso universal a testes de cultura e de susce-tibilidade a fármacos. O acesso universal re-presenta capacidade para fazer anualmente 120 milhões de exames microscópicos, 60 milhões de testes de cultura e 6 milhões de testes de suscetibilidade a fármacos3,6. Cum-prir com este objetivo representará criar 5000 novos centros de microscopia, treinar 9000 novos técnicos de microscopia e criar ainda 2000 novos laboratório para testes de cultura e testes de suscetibilidade a fármacos e treinar 23 000 novos técnicos. Serão necessários anualmente pelo menos mil milhões de dóla-res americanos para construir e manter infra-estruturas laboratoriais de TB. No entanto,

The inadequate laboratory capacity hinders diagnosis, case management, and disease surveillance. This is particularly important for patients with drug-resistant TB because effective care often does not begin until re-sults of drug-susceptibility tests are avai-lable. Indeed, the emergence of MDR TB and XDR TB has led to the recognition that the lack of TB laboratory capacity is a glo bal crisis. Factors that have contributed to the gaps in TB laboratory services include: 1) a lack of recognition of the importance of the laboratory in TB treatment and control; 2) poor communication between National TB Programs and those providing TB labora-tory services; 3) inadequate human and fi-nancial resources for TB laboratories; 4) lack of infrastructure and physical facilities; and 5) biosafety concerns4.Recognizing the important role of the labo-ratory, the 2007 World Health Assembly5 endorsed the call of the Global Plan to Stop TB2 for universal access to culture and drug-susceptibility testing. Universal access will require the capacity perform 120 million microscopy investigations, 60 million cul-ture investigations, and 6 million drug-sus-ceptibility investigations per year3,6. Meet-ing this goal will require establishing 5000 new microscopy centers and training 9000 new microscopy technicians as well as estab-lishing 2000 new culture and drug-suscep-tibility testing laboratories and training 23 000 new technicians. At least US$ 1 bil-lion will be needed annually for building TB laboratory infrastructure and recurring costs. However, the benefit to cost ratio of such investments is estimated to be 9:1 in populations with a high prevalence of HIV infection7. Meeting the universal access goals could save countries in sub-Saharan

Criar capacidade laboratorial para a tuberculose: necessidades e estratégiasThomas M Shinnick




estima -se que o rácio custo-benefício de tais investimentos seja de 9:1 em populações com elevada prevalência de infeção por VIH7. Se se conseguir o objetivo de acesso universal, os países na África subsariana poderão poupar cerca de 52 mil milhões de dólares america-nos anualmente.

Planeamento estratégico para laboratórios nacionaisPara criar e manter os serviços laboratoriais em falta serão necessários recursos conside-ráveis, dedicação e vontade política. A De-claração de Maputo de 2008 sobre Fortale-cimento dos Sistemas de Laboratórios8 apelou para os governos tomarem posse das redes de serviços laboratoriais e implemen-tarem estratégias nacionais de reforço e apoio a esses serviços e, consequentemente, às principais doenças que ameaçam a saúde pública. Os governos deveriam criar um de-partamento de sistemas laboratoriais, no âmbito do Ministério da Saúde, capaz de gerir essa rede de sistemas e assumir como prioridade elaborar uma política laborato-rial inserida no plano nacional de saúde.Os esforços de planeamento estratégico de-veriam incluir programas para todas as doen-ças, serviços clínicos e outras estruturas e a padronização dos serviços laboratoriais as-sente na qualidade. Para implementar e manter o planeamento estratégico, os gover-nos deveriam: 1) reconhecer a importância dos serviços laboratoriais no controlo das doenças; 2) assegurar que o fortalecimento dos planos do sector da saúde inclui compo-nentes adequadamente orçamentados para o desenvolvimento da capacidade laborato-rial; 3) coordenar os esforços de todos os departamentos, programas específicos para

Africa alone as much as US$ 52 billion an-nually.

National laboratory strategic planscreating and sustaining the needed labora-tory services will require considerable re-sources, efforts, and political will. The 2008 Maputo Declaration on Strengthening La-boratory Systems8 calls upon national go-vernments to take ownership of their labo-ratory systems and develop national strategic laboratory plans that integrate laboratory support for the major diseases of public health importance. National governments should establish a department of laboratory systems within the Ministry of Health to provide leadership and should set as a prio-rity developing a laboratory policy within the health development plan that will guide the implementation of a strategic laboratory plan.Strategic planning efforts should include all disease programs, clinical services, and other stakeholders and strive to create a laboratory system based on quality laboratory manage-ment principles. To implement and sustain the strategic plans, national governments should: 1) acknowledge the importance of laboratory services in disease control; 2) en-sure that health sector strengthening plans include adequately budgeted components for laboratory capacity development; 3) co-ordinate efforts of all departments, disease-specific programs, donors, and technical partners responsible for laboratory services; 4) commit adequate human and financial resources for laboratory services; 5) develop and implement a human resource policy to create a qualified laboratory workforce and





cada doença, dadores e parcerias técnicas responsáveis pelos serviços de laboratório; 4) consignar recursos humanos e financeiros adequados para esses serviços; 5) desenvol-ver e implementar uma política de recursos humanos, a fim de se obterem profissionais qualificados para os laboratórios, e identifi-car e afastar barreiras ao desenvolvimento das suas carreiras e remunerações, bem como a sustentação da sua competência técnica; e 6) contratar fornecedores de serviços de la-boratório (e.g., privados, académicos e labo-ratórios de saúde pública) para melhorar o acesso aos testes de qualidade controlada para o diagnósticos da TB.

Construção de redes de laboratóriosUma componente essencial no esforço de combate a doenças infeciosas é uma rede de laboratórios que forneça testes de diagnóstico fiáveis, tratamento e monitorização da tera-pêutica. Tal rede deveria ser posta ao serviço de patologias com particular importância na saúde pública, especialmente o VIH e a TB. Uma abordagem de sistemas na criação dessa rede é fundamental para otimizar os testes de laboratório e a troca de informações necessá-ria para assegurar que os serviços apropriados estarão disponíveis em cada programa. A abrangência desse esforço envolve avaliação e compreensão da estrutura, do desempenho e do custo da rede de laboratórios; desenvolvi-mento duma rede de referência e informação que assegure o rápido fluxo de amostras e de resultados; e adoção de princípios de melho-ramento da qualidade para avaliar e melhorar o desempenho da rede de laboratórios9.Um desafio na abordagem integrada é o facto do nível global de programas e de subsídios

identify and remove barriers to laboratory staff career development, remuneration, re-tention, and sustainability of technical com-petency, and 6) engage all laboratory service providers (e.g., private, academic, and pub-lic health laboratories) to improve access to quality-assured TB diagnostic testing.

Building laboratory networksAn essential component of efforts to com-bat infectious diseases is a network of labo-ratories that can provide reliable laboratory testing for diagnosis, treatment, and moni-toring of therapy. Such a network should strive to be integrated across the diseases of public health importance, especially HIV and TB. A systems approach to creating such a network is necessary to optimize la-boratory testing and information exchange and to ensure that appropriate services are available in every program. Comprehensive laboratory strengthening efforts involve as-sessment and understanding of the struc-ture, performance, and cost of the labora-tory network; development of a referral and information network to ensure prompt flow of specimens and information; and use of quality-improvement principles to evaluate and improve the performance of the labora-tory network9.A challenge to an integrated approach is that at the global level programs and fund-ing are directed to specific diseases. Such vertical global programs are often mirrored by vertical national programs and laborato-ries. For a variety of reasons including fund-ing sources, technical requirements, and biosafety concerns, the national reference laboratory for a disease in many countries is a specialized, free-standing institution sepa-





serem dirigidos a doenças específicas. Esses programas verticais globais refletem frequen-temente programas e laboratórios verticais nacionais. Por uma variedade de razões, in-cluindo origens de recursos, necessidades téc-nicas e preocupações com a biossegurança, o laboratório de referência nacional para uma doença em muitos países é uma instituição individual especializada, separada do sistema geral de laboratórios de saúde pública e de outros laboratórios nacionais de referência. Além disso, os laboratórios nacionais de refe-rência concentram -se frequentemente no apoio a actividades epidemiológicas, de vigi-lância ou de investigação, e podem ter um papel direto limitado no cuidado de doentes, o que pode ser uma limitação no que respeita ao valor da integração de serviços de labora-tórios nacionais de referência nos programas dirigidos a doenças específicas.Por outro lado, os laboratórios nacionais de referência têm frequentemente a responsabi-lidade de desenvolver, dirigir e monitorizar as redes de laboratórios de saúde pública que fornecem serviços de diagnóstico e de cuida-dos a doentes. Essas redes podem fornecer serviços para mais de um programa de doen-ças específicas, representando oportunidades de associação de programas e harmonização ou integração de serviços. Na verdade, ao ní-vel mais periférico do sistema de saúde, os serviços de laboratório estão muitas vezes to-talmente integrados, com um técnico que executa os testes para todas as doenças.Conceber a capacidade do laboratório é, por si só, uma oportunidade para construir liga-ções entre programas e para integrar servi-ços, isto é, embora o treinamento de técnicos assente na experiência específica de determi-nadas doenças, a capacidade de construir la-boratórios tem mais que ver com perícia

rate from the general public health labora-tory system and other national reference laboratories. Also, national reference labora-tories often concentrate on support for epi-demiologic, surveillance, or research activi-ties and may have a limited, direct role in patient care. Such an emphasis may limit the perceived value of integrating the ser-vices of national reference laboratories across disease programs.On the other hand, national reference la-boratories often have responsibility for de-veloping, leading, and monitoring a net-work of public health laboratories that provide services for diagnosis and patient care. Such network laboratories may pro-vide services for more than one disease-specific program and represent opportuni-ties to build linkages between programs and harmonize or integrate services. In-deed, at the most peripheral level of the health system, laboratory services are often fully integrated with one technician doing testing for all diseases.Building laboratory capacity is by itself an opportunity to build linkages between programs and integrate services. That is, although training of bench-level techni-cians relies on disease-specific expertise, laboratory capacity building relies more on cross-cutting expertise in infrastruc-ture, biosafety, facilities, human resource development, specimen referral, supply chain management, logistics, equipment, maintenance, quality assurance pro-grams, management principles, informa-tion systems, data management, and ac-creditation processes. A potential role for a department of laboratory systems within the Ministry of Health of a coun-try would be to oversee programs that





transversal em infraestruturas, biosseguran-ça, instalações, recursos humanos, referência de espécimes, gestão da cadeia logística, lo-gística, equipamento, manutenção, progra-mas de garantia de qualidade, princípios de boa gestão, sistemas de informação, gestão de dados e acreditação de processos. Um pa-pel potencial para um departamento de sis-temas de laboratórios, sob a alçada do minis-tério da saúde de um país, seria o de inspecionar os programas que tratam destes temas de modo coordenado e integrado.Geralmente, deveria ser responsabilidade do laboratório de TB nacional de referência, em colaboração e coordenação com o programa nacional de TB, programas de outras doenças e departamentos do ministério da saúde, para desenvolver, implementar e monitorizar uma rede de laboratórios que assegure o acesso a testes de TB com qualidade. Em muitos paí-ses, existem redes de laboratórios de micros-copia de esfregaços para bacilos ácido-álcool resistentes (BAAR) inspecionados pelo labo-ratório de TB de referência nacional que po-deriam servir de ponto de partida para o de-senvolvimento da capacidade laboratorial necessária ao processamento de testes de TB resistente a fármacos e TB associada a VIH. Porém, há que ter o cuidado de evitar criar uma rede de laboratórios de TB isolada e ver-tical. Sempre que possível, os serviços dos la-boratórios TB devem ser uma parte integral dos cuidados de saúde primários e duma rede nacional de laboratórios de saúde pública.

Recursos e assistênciaComeçam a surgir recursos de apoio a países com capacidade para construir laboratórios para testar TB. A Global Laboratory Initiative (GLI) da Stop TB Partnership (http://www.

address these issues in coordinated, inte-grated manner.Generally, it should be the responsibility of the national TB reference laboratory, in col-laboration and coordination with the na-tional TB program, other disease programs, and MOH departments, to develop, imple-ment, and monitor a laboratory network that assures access to quality TB testing and complete, timely reporting. In many coun-tries, there exists a network of AFB-smear microscopy laboratories overseen by the na-tional TB reference laboratory, which could serve as a starting point for developing the laboratory capacity needed to address drug-resistant TB and HIV-associated TB. How-ever, care must be taken to avoid creating an isolated, vertical TB laboratory network. Where possible, TB laboratory services should be an integral part of primary health care and a national public health laboratory network.

Resources and assistanceResources are becoming available to assist countries with TB laboratory capacity build-ing. The Global Laboratory Initiative (GLI) of the Stop TB Partnership (http://www.stoptb.org/wg/gli/; 6) is working to develop 1) a roadmap for ensuring quality TB diag-nostics services within national laboratory strategic plans, 2) guidance on norms and standards for TB laboratory testing, equip-ment, and biosafety, 3) global policy gui-dance, 4) training materials, and 5) human resource strategies.Laboratory consultants are needed to assist countries with assessment, strategic plan-ning, implementation, and evaluation. Cur-rently expert laboratory diagnostic advice





stoptb.org/wg/gli/6 está a trabalhar para de-senvolver: 1) um roteiro para assegurar servi-ços de diagnóstico de TB com qualidade, integrados no planeamento estratégico de laboratórios; 2) orientação nas normas e pa-drões para laboratórios de testes de TB, equi-pamento e biossegurança; 3) orientação na política global; 4) materiais de formação; e 5) estratégias de recursos humanos.São necessários consultores de serviços de la-boratório para ajudar os países na avaliação, planeamento estratégico e implementação. Presentemente, aconselhamento e experiên-cia têm sido fornecidos por peritos de labo-ratórios de países industrializados, frequen-temente numa base ad hoc, com pouca ou nenhuma coordenação com parceiros glo-bais. O Plano Global 2006 -2015 da OMS2 indicava que o actual sistema de curtas workshops e visitas de aconselhamento são insuficientes para suprir a falta de pes soal de laboratório em países com estas carências e para criar sistemas laboratoriais sustentáveis. A GLI está a trabalhar no desenvolvimento duma estratégia de consenso para criar capa-cidade laboratorial, cursos para consultores de implementação da estratégia de consenso e um mecanismo que assegure a coordena-ção de esforços em cada país e entre parcei-ros. Os aspetos críticos no processo de con-sultadoria são: desenvolver uma abordagem consistente para criar a capacidade laborato-rial; promover o uso de sistemas e testes de laboratório apropriados para países com re-cursos limitados; e assegurar que a experiên-cia necessária para desenvolver, implementar e avaliar o plan eamento estratégico referido seja disponibilizada. De notar que há uma maior necessidade de experiência para criar capacidade laboratorial do que para as técni-cas específicas de laboratório. Um dos obje-

and expertise has been provided by industria-lized-world laboratory experts, often on an ad hoc basis with little or no coordination with global partners. The WHO Global Plan 2006-20152 indicated that the current system of short workshops and consultancy visits are insufficient to bridge the training gap of laboratory personnel in high burden countries and to build sustainable laborato-ry systems. The GLI is working to develop a consensus strategy for laboratory capacity building, courses to train consultants in the implementation of the consensus strategy, and a mechanism to ensure coordination of capacity building efforts within a country and among partners. The critical aspects in the consulting process are to develop a con-sistent approach to laboratory capacity building; to promote the use of laboratory systems and tests appropriate for resource-limited, high-burden countries; and to en-sure that the expertise needed to develop, implement, and evaluate a strategic plan for laboratory capacity building will be avai-lable to countries. Note that there is greater need for expertise in laboratory capacity building than there is for expertise in di-sease-specific laboratory techniques. A goal is to have cadre of consultants who can be stationed in country for extended periods of time that would be able to assist countries in building an integrated laboratory net-work capable of providing the laboratory services needed to combat TB, HIV, and malaria.

ConclusionAn effective response to the call for univer-sal access to TB diagnostics requires a mas-sive scale-up of TB laboratory services and





tivos é conseguir quadros de consultores que possam ser colocados nos países durante lon-gos períodos de tempo, a fim de ajudarem na criação duma rede integrada de laboratórios capaz de fornecer os serviços necessários ao combate de tuberculose, VIH e malária.

ConclusãoUma resposta eficaz ao apelo para acesso universal ao diagnóstico da TB exige um au-mento maciço dos serviços laboratoriais e correspondente atribuição de recursos. Para maximizar o impacto e a sustentabilidade destes investimentos é imprescindível uma abordagem alargada dos sistemas de saúde. A harmonização de esforços na criação duma sólida rede de sistemas laboratoriais capaz de responder às necessidades de todas as patologias de importância para a saúde pública é um benefício para todos os pro-gramas de saúde e contribui para o fortaleci-mento do setor da saúde em general e para a qualidade dos cuidados aos doentes.

Bibliografia/Bibliography1. World Health Organization. Global tuberculosis control: epidemiology, strategy, financing: WHO re-port 2009 (WHO/HTM/TB/2009.411). Geneva: World Health Organization; 2009. Available online at http://www.who.int/tb/publications/global_report/en. Accessed Nov. 1, 2009.2. Stop TB Partnership e World Health Organiza-tion. Global Plan to Stop TB 2006–2015 (WHO/HTM/STB/2006.35). Geneva: World Health Or-ganization; 2006. Available online at http://www.stoptb.org/globalplan/assets/documents/Global-PlanFinal.pdf. Accessed Nov. 1, 2009.3. Report of a Ministerial Meeting of High M/XDR--TB Burden Countries, Beijing China, April 1 -3, 2009. Key bottlenecks in M/XDR -TB control and pa-tient care. 5. Responding to the laboratory bottleneck.

correspondingly large commitment of re-sources. To maximize the impact and sus-tainability of the investments in TB labora-tory capacity building, a broad health systems strengthening approach is needed. Harmonized efforts using a systems ap-proach to build to a strong laboratory net-work that addresses the needs of all diseases of public health importance is a benefit to all disease programs and contributes to ge-neral health sector strengthening and quali-ty patient care.

Geneva: World Health Organization; 2009. Available online at http://www.who.int/tb/challenges/mdr/bot-tlenecks/bottlenecks_chapter5.pdf. Accessed Nov. 1, 2009.4. Abdel Aziz M, Ryszewska K, Laszlo A, Blanc L. Stra-tegic approach for the strengthening of laboratory services for tuberculosis control, 2006 -2009 (WHO/HTM/TB/2006.364). Geneva: World Health Organi-zation; 2006. Available online at: http://whqlibdoc.who.int/hq/2006/WHO_HTM_TB_2006.364_eng.pdf. Accessed on Nov. 1, 2009.5. World Health Organization. 60th World Health As-sembly. Resolutions and Decisions. World Health As-sembly Resolution WHA 60.19: Tuberculosis control: progress and long -term planning. Geneva: World Health Organization; 2007. Available online at http://apps.





who.int/gb/ebwha/pdf_files/WHA60/A60_R19 -en.pdf. Accessed Nov. 1, 2009.6. Global Laboratory Initiative. Moving tuberculosis (TB) laboratory capacity strengthening forward: A Global La-boratory Initiative. Supporting document for 15th Annual Meeting of the Stop TB Partnership Coordinating Board, Bagamoyo Tanzania, November 28 -29, 2008. Geneva: Stop Tb Partnership; 2009. Available online at http://www.stoptb.org/cb/meetings/20081028_Bagamoyo_Tanzania/assets/documents/2.08 -11.1%20GLI%20Synopsis%20FINAL.pdf. Accessed Nov. 1, 2009.7. Laxminarayan R, Klein E, Dye C, Floyd K, Darley S, Adeyi O. Economic Benefit of Tuberculosis Control (August 1, 2007). World Bank Policy Research Working Paper Series, No. 4295, 2007. Available at http://www.wds.worldbank.org/servlet/WDSContentServer/

WDSP/IB/2007/08/01/000158349_20070801103922/Rendered/PDF/wps4295.pdf. Accessed Nov. 1, 2009.8. World Health Organization. Maputo Declaration on Strengthening Laboratory Sistems. Presented at: Con-sensus Meeting on Clinical Laboratory Testing Harmo-nization and Standardization; January 22 -24, 2008; Maputo, Mozambique. Available at: http://www.who.int/diagnostics_laboratory/Maputo -Declaration_2008.pdf. Accessed Nov. 1, 2009.9. Centers for Disease Control and Prevention (Shin-nick TM, Iademarco MF, Ridderhof JC). National plan for reliable tuberculosis laboratory services using a sis-tems approach: recommendations from CDC and the Association of Public Health Laboratories Task Force on Tuberculosis Laboratory Services. MMWR (RR -6) 2005; 54:1 -12.





Afranio Kritski1 Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento e avaliação de novos métodos de diagnóstico em tuberculose

The experience of the Brazilian Tuberculosis Research Network in the development and evaluation of new methods of diagnosing tuberculosis

IntroduçãoDe acordo com estimativa da Organização Mundial de Saúde (OMS) de 2009, o Brasil está em 18.º lugar entre os 22 países que cons-tituem 80% da carga global da tuberculose (TB). A TB continua a ser a principal causa de morte em indivíduos infetados com VIH, mesmo nos submetidos a HAART (terapia antirretroviral potente) oferecida gratuitamen-te a todas as pessoas infetadas com VIH, desde 1997. Tal como acontece noutros países em desenvolvimento, há alguma dificuldade em harmonizar a comunicação e o entendimento entre especialistas programáticos em TB, aca-démicos e comunidade, e as organizações não governamentais. Em 2001, a investigação da TB não estava sujeita a quaisquer linhas de orientação quanto a: a) desenvolvimento de novos produtos; b) inovação tecnológica; e c) incorporação de novos instrumentos nos seto-res público e privado. A Rede Nacional de In-vestigação da Tuberculose (REDE -TB) foi criada, em 2002, para aproximar as diferentes

IntroductionBrazil ranks 18th out of the 22 countries that bear 80% of the worldwide tuberculo-sis (TB) burden, according to the World Health Organization (WHO) in 2009. TB is the leading cause of mortality in HIV in-fected persons, despite all HIV infected per-sons having received highly active antiretro-viral therapy (HAART) free of charge since 1997.As in other developing nations, there is a significant gap in communication and un-derstanding between TB programme ex-perts, academics, the community, and non-governmental organisations. In 2001, there was no TB research policy in place regard-ing: a) the development of new products, b) technological innovation and c) the incor-poration of new tools in public and private sectors.In recognition of this gap, a National TB Research Network was established in 2002 to bring together different constituencies to

1 Coordinator, Diagnostic Area, Brazilian TB Research Network (Rede TB). Academic Tuberculosis Programme, Medical School of the Federal University of Rio de Janeiro, Brazil




sensibilidades e promover uma estratégia inte-grada, multi disciplinar e multi -institucional para controlo e investigação da TB no Brasil. (Rede -TB – www.redetb.org). Nesta apresen-tação, serão comentadas as atividades da área diagnóstica da Rede TB.

Desenvolvimento, avaliação e incorporação de testes diagnósticos no BrasilA avaliação da incorporação de novas tecno-logias diagnósticas em TB tem sido também priorizada pela Organização Mundial de Saúde após o lançamento do Plano Global de TB da OMS/STOP, em 2006 (http://www.who.int/tb/strategy/stop_tb_strategy/en/). A partir de 2002, apercebemo -nos de que o desenvolvimento diagnóstico e o pro-cesso de avaliação proposto pela OMS rece-beram das partes interessadas atenção e apoios diferentes. Verificou -se um enorme interesse e disponibilidade de fundos relati-vamente às seguintes fases: a) pré -clínica: descoberta e investigação, desenvolvimento; b) fase 1: avaliação – prova de princípio; c) fase 2: avaliações laboratoriais; e d) fase 3: avaliações de ensaios de campo para análise de desempenho. Mas a fase 4 despertou me-nos interesse e obteve menos fundos: custo, estudos de impacto e, para a transferência de políticas, pré -qualificação, aprovisiona-mento, preços negociados, análise de acesso. Em 2007, procedeu -se à avaliação das ten-dências dos artigos científicos sobre tuber-culose no Brasil publicados entre 1986 e 2006. Entre os 1054 trabalhos avaliados, que continham a palavra tuberculose e cujos autores estavam ligados a instituições brasi-leiras, 486 (46,1%) artigos estavam relacio-nados com descrição e revisão de casos e sé-ries. Apenas 70 (6,7%) artigos eram estudos

promote an integrated, multi-disciplinary and multi-institutional strategy to TB con-trol and research in Brazil (www.redetb.org). This article describes the Network’s work in the area of diagnosis.

The development, evaluation and incorporation of diagnostic tests in BrazilEvaluating the incorporation of new TB dia gnostic techniques was prioritised by the WHO following the 2006 launch of the Global Plan Against TB – WHO/STOP (http://www.who.int/tb/strategy/stop_tb_strategy/en/). Since 2002, we have learned that the diagnostics development and evalua tion process proposed by WHO has received different attention and funding by the stakeholders. A lot of interest and avai-lable funding has been allocated to the fol-lowing stages: a) pre-clinical: discovery and research, development, b) phase 1: evalua-tion – proof of principle; c) phase 2: labora-tory evaluations, and d) phase 3: evaluation of field trials for performance analysis. There has been a scarcity of interest and available funding allocated, however, to phase 4: cost, impact studies and policy transfer, prequali-fication, bulk procurement, negotiated pri-cing and access analysis.In 2007 there was an evaluation of the trends in scientific articles on TB in Brazil pub-lished between 1986 and 2006. Of 1054 TB publications assessed containing the word ‘tuberculosis’ with the authors affilia ted to Brazilian Institutions, 486 (46.1%) articles were related to descriptive, review and case series reports. Only 70 (6.7%) articles des-cribed operational/effectiveness studies or clinical trials (Kritski AL et al., 2007).

Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento e avaliação de novos métodos de diagnóstico em tuberculose

Afranio Kritski




operacionais/de eficácia ou ensaios clínicos. (Kritski AL, et al., 2007).Em 2002, fomos abordados por uma empre-sa que manifestou interesse em ter os seus testes serológicos registados numa agência reguladora brasileira (ANVISA) para futura comercialização. A avaliação desses testes se-rológicos demonstrou uma elevada especifi-cidade (95%) entre os controlos saudáveis, mas baixa especificidade (46%) entre os in-divíduos suspeitos de serem portadores de TB (Gounder C, et al., 2002). Felizmente, a empresa deixou o mercado brasileiro. Esses resultados são semelhantes aos descritos na revisão sistemática publicada em 2007 (Steingart et al., 2007). Os autores concluí-ram que no momento não há indicação de kits sorológicos comercializados ou testes in house no diagnóstico da TB no mundo. Os profissionais de saúde deveriam ser alertados para não utilizarem tais tecnologias na sua prática clínica. Lamentavelmente, esses tes-tes ainda estão à venda em países em desen-volvimento, não tendo sido publicitadas quaisquer recomendações sobre este facto pelas organizações internacionais. Em 2005, iniciou -se uma interação com a “Gerência de produtos para diagnóstico de uso in vitro” da Agência Reguladora Nacional (ANVISA) e efetuou -se uma avaliação dos testes diag-nósticos de tuberculose registados na ANVISA no período de 2000 a 2004: 48 foram regis-tados desde 2000, 33 não tinham registo vá-lido mas continuavam no mercado e 14 (30%) eram testes imunosserológicos. Estes testes foram registados em diferentes cená-rios clínicos no Brasil, sem validação prévia. Estes resultados acentuam a dificuldade de harmonização entre a agência reguladora, os académicos e os decisores que tratam da in-corporação de novas tecnologias no Brasil.

In 2002, we were approached by a company interested in having its serological tests regis-tered at the Brazilian regulatory agency (AN-VISA) for future commercial use. The evalua-tion of these serological tests showed a high specificity (95%) in healthy control subjects, but a low specificity in TB suspects (46%) (Gounder C et al., 2002). Fortunately, they withdrew from the Brazilian market. These results are similar to those described in the systematic review published in 2007 (Stein-gart et al., 2007). The authors concluded that there is currently no indication for com-mercial serologic testing kits or in-house tests in the diagnosis of TB in the world. Healthcare professionals should be warned not to use these techniques in clinical prac-tice. Unfortunately, these tests are still sold in developing nations and no strict recom-mendations on them has been issued by in-ternational organisations. In 2005 contact was made with the management section of the in vitro diagnostic products section of the National Regulatory Agency (ANVISA) and an evaluation of TB diagnostic products tested registered at ANVISA in the period 2000 to 2004 was carried out: 48 had been registered since 2000; 33 had no valid regis-tration, but still remained on the market and 14 (30%) were immunoserological tests. These tests had been registered without prior validation in different clinical settings in Brazil. These results highlighted the gap be-tween the regulatory agency, academics and the policy makers who deal with the incor-poration of new technologies in Brazil.Over the last few years, Brazil, along with other developing countries, has seen new molecular technique kits for diagnosing TB or resistant TB enter the market, both poly-merase reaction (PCR) and phenotype

Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento e avaliação de novos métodos de diagnóstico em tuberculoseAfranio Kritski




Nos últimos anos, como ocorre noutros paí-ses em desenvolvimento, no Brasil foram comercializadas novas tecnologias molecula-res (kits de PCR) ou fenotípicas (MGIT960) no diagnóstico de TB e TB resistente. Entre-tanto, tais tecnologias têm sido disponibili-zadas praticamente na rede privada de labo-ratórios. Eventualmente foram incluídos em unidades de saúde do sistema público de saúde, mas sem continuidade, por não terem sido incorporados na lista de prioridades do ministério.

Testes fenotípicos para o diagnóstico de TB resistentePor meio de estudo realizado em três labo-ratórios de micobacteriologia participantes da Rede -TB, observou -se elevada concor-dância entre a performance do MGIT960 e os três métodos considerados padrão-ouro para o diagnóstico de TB resistente: a) Mé-todo de proporções; b) Bactec 460; e c) Ra-zão da resistência (Giampaglia et al., 2007). Em outro estudo, em colaboração com pes-quisadores nas Honduras e na Universidade John Hokpins, avaliamos a performance do MODS em 854 doentes suspeitos de TB pulmonar. A sensibilidade e a especificida-de do MODS foi respectivamente de 96,5% e de 92,6%, o tempo do diagnóstico infe-rior para MODS (6 dias; interquartil de 5 a 7), em comparação com LJ (21 dias; inter-quartil 17 a 25 dias) (Arias M et al., 2007) Os estudos sobre a técnica MODS sugerem que ela pode ser útil no diagnóstico da TB sensível às drogas, pois, além de apresentar sensibilidade e especificidade similares aos meios de cultura padronizados, o diagnósti-co é bem mais rápido. Entretanto, requer técnicos de laboratório com elevado grau

(MGIT960) kits. These technologies were made available almost exclusively in pri-vately owned laboratories. They were even-tually incorporated into healthcare units of the National Health System, but were dis-continued as they were not considered Ministry of Health priorities.

Phenotype techniques for diagnosing resistant TBA study performed in three TB Research Network mycobacteriology laboratories showed a strong agreement between MGIT960 performance and the three gold standard methods for diagnosing resistant TB; the proportion method, Bactec 460 and the resistance ratio method (Giampa-glia et al., 2007). Another study, performed by researchers in the Honduras and at the John Hopkins University, evaluated the per-formance of the microscopic-observation drug-susceptibility (MODS) assay in 854 patients with suspected pulmonary TB. MODS sensitivity and specificity was 96.5% and 92.6%, respectively, and time to diagnosis was quicker for MODS (6 days; 5 – 7 interquartile), than Lowenstein Jensen (LJ) medium (21 days; 17 – 25 inter-quartile), (Arias M et al., 2007).Studies on the MODS assay suggest that MODS may be of use in diagnosing drug-susceptible TB as in addition to its sensiti-vity and specificity – similar to that of stan-dardised culture media – it offers a far quicker diagnosis. It does, however, require laboratory techniciens with a high degree of proficiency and biosafety as it uses a liquid medium in Petri dishes. Furthermore, we evaluated the MODS performance for drug resistant TB (DR-TB). In 351 DR-TB sus-pects, MODS had sensitivity and specificity


Afranio Kritski




for isoniazid and rifampicin respectively of 97.2%, 79%, and of 96.4%, 86.5% (Mello FCQ, et al., 2007). These results suggest that MODS might be used as screening technique for DR-TB diagnosis.

Molecular biology techniques for diagnosing TB and resistant TBThere is a great variation in the accuracy of commercial molecular tests for the diagno-sis of active TB, mainly in immunosup-pressed patients, with lesser sensitivity than specificity (Palomino, 2009, Ling 2008, Barnard, 2008). Further, performance re-sults from studies have led to some molecu-lar techniques being approved by the regu-latory bodies of industrialised countries and sold for use in respiratory samples, that is to investigate pulmonary TB in adult patients with no prior history of TB treatment. The molecular tests currently sold should not be used to diagnose other forms of TB or mo-nitor treatment and do not replace a myco-bacterial culture exam.In developing nations, positive direct mi-croscopy results have a high positive predic-tive value for tuberculosis diagnosis. In those regions, molecular tests may provide a higher impact for diagnosis of smear nega-tive TB cases (SNTB), especially among HIV seropositive cases. However, little data are available on the cost effectiveness and clinical utility of PCR in SNTB, in a setting with a high burden of TB/HIV co-infection (van Cleef, 2005). We evaluated the perfor-mance of the PCR dot-blot (developed by Brazilian scientists) in parallel with pretest probability (clinical suspicion) in patients suspected of having SNTB, in a prospective study of 213 individuals with clinical and

de proficiência e de biossegurança, em ra-zão do uso de meio líquido em placas de Petri. Avaliámos, ainda, o desempenho do MODS com a TB resistente (DR -TB). En-tre 351 indivíduos suspeitos de DR -TB, o MODS teve sensibilidade e especificidade para isoniazida e rifampicina de 97,2%, 79%, 96,4% e 86,5%, respetivamente (Mello FCQ, et al., 2007). Estes resultados sugerem que o MODS poderá ser usado para triagem no diagnóstico da DR -TB.

Tecnologias de biologia molecular para diagnóstico de TB e TB resistenteHá grande variabilidade da precisão dos testes moleculares comercializados no diag-nóstico da TB ativa, principalmente em doentes imunossuprimidos, com menores valores de sensibilidade em relação a espe-cificidade (Palomino, 2009, Ling 2008, Barnard, 2008). Além disso, em razão dos resultados obtidos em estudos de perfor-mance, alguns testes moleculares foram aprovados nos órgãos regulatórios de países industrializados e comercializados para uso em amostras respiratórias, ou seja, para a investigação de TB pulmonar, em doentes adultos, sem história prévia de tratamento anti TB. Portanto, os testes moleculares co-mercializados no momento não deveriam ser utilizados para o diagnóstico de outras formas de TB, monitoramento do trata-mento, e não substituem o exame de cultu-ra para micobactérias.Nos países desenvolvidos, os resultados po-sitivos da microscopia direta têm um eleva-do valor indicativo positivo no diagnóstico da tuberculose. Nestas regiões, os testes moleculares podem ter maior impacto no





diagnóstico de casos de TB com esfregaço negativo TB (ENTB), especialmente entre seropositivos para VIH. Porém, há poucos estudos sobre a relação custo -benefício e utilidade clínica da PCR na ENTB, em re-giões com elevada incidência de coinfeção TB/VIH (van Cleef, 2005). Avaliámos o desempenho da PCR dot -blot (desenvolvida por cientistas brasileiros) paralelamente à probabilidade pré -teste (suspeita clínica) em doentes suspeitos de ENTB, num estu-do prospectivo de 213 indivíduos com sus-peita clínica e radiológica de ENTB num hospital de referência de TB/VIH, no Sul do Brasil. Não se observou diferença na sensibilidade da PCR relativamente ao esta-tuto de VIH(Scherer LC, et al., 2007). No mesmo grupo de estudo, comparámos duas estratégias: o uso de microscopia de esfrega-ço com bacilos acid fast por coloração Ziehl--Neelsen (esfregaço AFB) e cultura; e esfre-gaço AFB e teste colorimétrico (PCR dot -blot), e efectuámos uma análise de cus-tos que incluiu serviços de saúde e custos com os doentes. Os custos totais de scree-ning foram 3,7 vezes para o esfregaço AFB e cultura versus os custos para esfregaço AFB e PCR dot -blot (US$ 5 651 560 versus US$ 1 513 ,760). O esfregaço AFB e PCR dot -blot mostrou melhor relação custo--benefício do que o esfregaço AFB e cultura quando se considerou o custo de tratar to-dos os casos corretamente diagnosticados (Scherer LC et al., 2009), Estes resultados mostraram que os testes moleculares, mes-mo nos países desenvolvidos, são bem acei-tes e desempenham um papel importante no diagnóstico da ENTB, diminuindo não só o tempo necessário para o diagnóstico, mas ainda a morbilidade/mortalidade e a transmissão do M. tuberculosis à comunida-

radiological suspicion of SNTB in a TB/HIV referral hospital in southern Brazil. There was no difference in the sensitivity of PCR in relation to HIV status (Scherer LC et al., 2007).In the same study group we compared two strategies: use of acid fast bacilli smear mi-croscopy by Ziehl-Neelsen staining (AFB smear) plus culture and AFB smear plus colorimetric test (PCR dot-blot) and per-formed a cost analysis that included health services and patient costs. The total screen-ing costs were 3.7 times higher for AFB smear plus culture than for AFB smear plus PCR dot-blot costs (USD 5,651.560 versus USD 1,513. 760). AFB smear plus PCR dot-blot was more cost-effective than AFB smear plus culture, when the cost of treating all correctly diagnosed cases was considered (Scherer LC et al., 2009). These results show that molecular tests, even in developing na-tions, are welcome and play an important role in SNTB diagnosis, cutting diagnostic delay, morbid-mortality and M. tuberculosis transmission to the community, even in re-gions with a high rate of HIV infection.

Molecular tests for diagnosing multi-drug resistant TB (MDR-TB)Of the molecular tests sold worldwide for a swift diagnosis of resistant TB, the following are accurate: INNO-LIPA Rif.TB kit (Inno-genetics, Zwijndrecht, Belgium), GenoType® MDRTB and GenoType®MDRTB plus as-says (Hain Lifescience, GMBH, Germany),TB Research Network researchers under-took a study into the accuracy of selected samples using the INNO-Lipa Rif.TB kit and found a high level of agreement with the reference tests in selected samples (Oli-


Afranio Kritski




de, mesmo em regiões com grande incidên-cia de infecção por VIH.

Testes moleculares para o diagnóstico de TB multirresistenteEntre os testes moleculares comercializados, a nível mundial, para o diagnóstico rápido de TB resistente que mostraram boa acurácia: o kit INNO -LIPA Rif.TB (Innogenetics, Zwi-jndrecht, Bélgica), o ensaio de GenoType® MDRTB e GenoType® MDRTBplus (Hain Lifescience, GMBH, Alemanha), pesquisa-dores da Rede TB realizaram estudo de acurá-cia em amostras selecionadas ao utilizarem o kit INNO -Lipa Rif.TB e observaram elevada concordância com testes de referência em amostras selecionadas. [Oliveira M, 2005). Entretanto, até ao momento, não há estudos publicados na literatura sobre a performance de tais testes moleculares em condições de ro-tina, em países em desenvolvimento. Recen-temente, avaliamos o desempenho da sequen-ciação de ADN em 38 (26%) doentes (99 amostras clínicas) com resultados discordan-tes entre MODS e métodos proporcionais. Surpreendentemente, verificámos um elevado índice de heterorresistência a RIF e/ou INH (21,7%) e de superinfeção (28,9%), usando a técnica spoligotyping, sendo a maioria Lam, Haarlem, e T1 [Andrade et al., 2009]. Resul-tados semelhantes foram observados na África do Sul e no Usbequistão. Van Rie et al. (2005) avaliaram 186 doentes com TB, encontrando superinfeção em 23% (14/62) de casos relata-dos e em 17% (21/1254) dos casos. Hofmann--Thiel et al. (Hofmann -Thiel S, et al., 2009) avaliaram 35 casos de TB, encontrando super-infeção em 8,6%. Em ambos estudos, a maio-ria das superinfeções esteve relacionada com a família M.tb Beijing. Mais recentemente,

veira M, 2005). That said, there are as yet no published studies in the literature into the performance of these molecular tests under routine conditions in developing countries.We recently evaluated the performance of DNA sequencing in 38 (26%) patients (99 clinical samples) with discordant results be-tween MODS and the proportion method. Surprisingly, we found high rate of hetero-resistance to RIF and/or INH (21.7%) and of superinfection (28.9%) using the spolig-otyping technique, with the majority Lam, Haarlem, and T1 (Andrade et al., 2009). Similar results were seen in South Africa and in Uzbekistan. Van Rie et al. (Van Rie 2005) evaluated 186 TB patients, finding superinfection in 23% (14/62) retreatment cases and in 17% (21/1254) of cases. Hof-mann-Thiel et al. (Hofmann-Thiel S et al. 2009), evaluating 35 TB cases, found super-infection in 8.6%.In both studies the majority of superinfec-tion was related to the M.tb Beijing family. More recently, Hillemann et al. (Hillemann D, 2009), compared the accuracy of the new MTBDRsl assay for extensively drug-resistant TB (XDR-TB) which included de-tection of fluoroquinolone, amikacin-capreomycin and ethambutol resistance testing. Among 106 selected clinical isolates, resis-tance to rifampicin and isoniazid was found in 63 (59.4%) cases. Ofloxacin resistance was found in 32 (30.2%) cases and hete-roresistance was observed in 21.9% (7/32). The high rates of heteroresistance and su-perinfection identified in those studies in clinical samples collected from DR-TB sus-pects highlight the need to evaluate the im-pact of the use of line probe assays in DR-TB suspects before its incorporation into





Hillemann et al. (Hillemann D, 2009), com-pararam a precisão do novo ensaio MTBDR-sl para XDR -TB, que incluiu a deteção da resistência aos testes com fluoroquinolona, amikacin -capreomicina e etambutol. Entre 106 isolados clínicos selecionados, observa-ram resistência a rifampicina e a isoniazida em 63 (59,4%) dos casos. Observaram resistência à ofloxacina em 32 (30,2%) e hetero r-resistência em 21,9% (7/32). Os elevados ín-dices de heterorresistência e de superinfeção identificados nesses estudos em amostras clí-nicas colhidas de indivíduos suspeitos de DR--TB acentuam a necessidade de avaliar o im-pacto do uso de ensaios line probe nestes suspeitos, antes da sua incorporação na práti-ca clínica, especialmente nos países em desen-volvimento.Mais recentemente, a Rede TB, juntamente com pesquisadores da Union International Contra Tuberculosis, do Management Science for Health, do Centro de Referência Prof. Hé-lio Fraga, da Fiocruz, e técnicos do Programa Nacional de TB e do Departamento de Ciên-cia e Tecnologia do Ministério da Saúde, ela-boraram uma plataforma de protocolos de pesquisa de viabilidade e impacto econômico a serem realizados em diferentes regiões do país, que inclui o uso do teste Xpert™ MTB/Rif (Cepheid, Sunnyvale, CA, EUA) para o diagnóstico de TB e TB resistente e o uso do teste GenoType® MDRTBplus e MTBDRsl assay (Hain Lifescience, GMBH, Alemanha) para o manuseio do doentes suspeitos de TB--MDR e TB -XDR, respetivamente

clinical practice, especially in developing nations.More recently, the TB Research Network along with researchers from the Interna-tional Union Against Tuberculosis of the Management Science for Health of the Centro de Referencia Prof Helio Fraga da Fiocruz and technicians from the National TB programme and the Ministry of Health’s Department of Science Technology drew up feasibility and economic impact research protocols agreements. These are to be car-ried out in different regions of Brazil and include use of the Xpert™ MTB / Rif (Cep-heid, Sunnyvale, CA, USA) test for diag-nosing TB and resistant TB and use of the GenoType® MDRTB plus test and MTB-DRsl assay (Hain Lifescience, GMBH, Germany) for the management of patients with suspected MDR-TB and XDR-TB, respectively.


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Bibliografia/BibliographyAndrade MK, Dalcolmo M, Marsico AG, Mello FCQ, Dorman S, Rossetti ML, Fonseca LS, de Oliveira MM, Kritski A. 40th World Conference on Lung Health. Cancun, México 3 -7 December, 2009 (www.worldlung-health.org).Arias M, Mello FC, Pavon A, Marsico AG, Alvarado--Galvez C, Rosales S, Pessoa CL, Perez, Andrade MK, Kritski AL, Fonseca LS, Chaisson RE, Kimerling ME, Dorman SE. The microscopic observation drug suscep-tibility (MODS) assay for detection of tuberculosis and tuberculosis drug resistance: results from a multi -center study. Clin Infect Dis 2007;44(5):674 -80. Epub 2007 Jan 22.Barnard M, Albert H, Coetzee G, O’Brien R, Bosman ME. Rapid molecular screening for multidrug -resistant tuberculosis in a high -volume public health laboratory in South Africa. Am J Resp Crit Care 2008, 177: 787--792.de Oliveira MM, da Silva Rocha A, Cardoso Oele-mann M, Gomes HM, Fonseca L, Werneck -Barreto AM, Valim AM, Rossetti ML, Rossau R, Mijs W, Van-derborght B, Suffys P. Rapid detection of resistance against rifampicin in isolates of Mycobacterium tuber-culosis from Brazilian patients using a reverse -phase hybridization assay. J Microbiol Methods 2003; 53(3):335 -342.Flores LL, PaiM, Colford JM, Riley LW. In -house nucleic acid amplification tests for the detection of Mycobacterium tuberculosis in sputum specimens: meta -analysis and me-taregression. BMC Microbiol 2005; 5: 55.Giampaglia MS, Martins MC, Vieira GBO, Vinhas SA, da Silva Telles MA, Palaci M, Marsico AG, Hadad DJ, Mello FCQ, Kritski A, Siddiqi S, Fonseca LS. Multi--center Evaluation of Automated Bactec MGIT 960 System for testing susceptibility of M. tuberculosis as compared with BACTEC 460TB, proportion and re-sistance ratio methods in Southeast of Brazil. Int J Tu-berc Lung Dis 2007; 11(9):986 -991.Global Plan against TB – WHO/STOP em 2006 (http://www.who.int/tb/strategy/stop_tb_strategy/en/).Gounder C, Mello FCQ, Conde MB; Kritski A, Chais-son RE, Dorman S. Field evaluation of a rapid immuno-chromatographic test for tuberculosis. J Clin Microbiol 2002; 40(6):1477 -1451.Hofmann -Thiel S, van Ingen J, Feldmann K, Turaev L, Uzakova GT, Murmusaeva G, van Soolingen D, Hoff-

mann H. Mechanisms of heteroresistance to isoniazid and rifampin of Mycobacterium tuberculosis in Tashkent, Uzbekistan. Eur Respir J 2009;33(2):368 -374. Epub 2008 Oct 1.Hillemann D, Rüsch -Gerdes S, Richter E. Feasibility of the GenoType MTBDRsl assay for fluoroquinolone, amikacin -capreomycin, and ethambutol resistance tes-ting of Mycobacterium tuberculosis strains and clinical specimens. J Clin Microbiol 2009; 47(6):1767 -1772. Epub 2009 Apr 22.Kritski AL, Villa TS, Trajman A, Lapa e Silva, JR Me-dronho RA, Ruffino -Netto A. Two decades of research on tuberculosis in Brazil: state of the art of scientific publications. Rev Saude Publica 2007;41(Supl):9 -14.Ling DI, Flores LL, Riley LW, Pai M. Commercial nucleic -acid amplification tests for diagnosis of pulmo-nary tuberculosis in respiratory specimens: meta -analysis and meta -regression. PLoS One 2008; 2:e1536.Mello FCQ, Arias M, Pavón A, Marsico AG, Alvarado--Gálvez C, Rosales |S, Pessoa CEC, Pérez M, Andrade MK, Kritski AL, Fonseca LS, Chaisson RE, Kimerling M, Dorman, SE. Clinical evaluation of the micros-copic observation drug susceptibility (MODS) assay for detection of Mycobacterium tuberculosis resis-tance to isoniazid or rifampin. J Clin Microbiol 2007; 45(10):3387 -3389.Palomino JC. Molecular detection, identification and drug resistance detection in Mycobacterium tuberculosis. Minireview. FEMS Immunol Med Microbiol 2009; 1 -9.Rede Brasileira de Pesquisa em Tuberculose – Rede Tb (www.redetb.org)Scherer LC, Sperhacke RD, Mello FCQ, C Jarczewski, Cafrune P, Minghelli S, Osorio M, Rossetti ML, Kritski AL. PCR colorimetric dot -blot assay and clinical pretest probability for diagnosis of pulmonary tuberculosis in smear -negative patients BMC Public Health 2007; 7(1):356.Scherer LC, Sperhacke RD, Ruffino -Netto A, Rossetti, MLR, Kritski AL. Cost -effectiveness analysis of the PCR associated with Ziehl Neelsen smear examination (ZN) for the rapid diagnosis of pulmonary tuberculosis in subjects with and without HIV in a hospital setting. BMC Public Health 2009 (in press).Steingart KR, Henry M, Laal S, Hopewell PC, Ramsay A, Menzies D, Cunningham J, Weldingh K, Pai M. Commercial serological antibody detection tests for the





diagnosis of pulmonary tuberculosis: a systematic re-view. PLoS Med 2007 Jun; 4(6):e202. Erratum in: PLoS Med 2007; 4(8):e254.Van Cleeff M, Kivihya -Ndugga L, Githui W, Ng’ang’a L, Kibuga D, Odhiambo J, Klatser P. Cost -effectiveness of polymerase chain reaction versus Ziehl -Neelsen smear microscopy for diagnosis of tuberculosis in Kenya. Int J Tuberc Lung Dis 2005; 9(8):877 -883.Van Rie A, Victor TC, Richardson M, Johnson R, van der Spuy GD, Murray EJ, Beyers N, Gey van Pittius NC, van Helden PD, Warren RM. Reinfection and mixed infection cause changing Mycobacterium tubercu-

losis drug -resistance patterns. Am J Respir Crit Care Med 2005; 172(5):636 -642. Epub 2005 Jun 9.WHO – 2007 recomendations – use of liquid médium for TB and drug resistant TB diagnosis http://www.who.int/tb/dots/laboratory/en/index.html.WHO – 2008 recommendations – use of line molecular probe assays for drug resistant TB diagnosis (http://www.who.int/tb/features_archive/mdrtb_rapid_tests/en/index.html\).[WHO] World Health Organization 2009. Global Tu-berculosis Control. WHO Report. Geneva, Switzer-land.


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Moisés Palaci1 Ensaios clínicos de novas drogas e testes diagnósticos em tuberculose: Desafios micobacteriológicos

Clinical trials of new tuberculosis drugs and diagnostic tests: Mycobacteriological challenges

Desde que a Organização Mundial de Saúde declarou a da tuberculose (TB) como emer-gência global em 1993, esta doença, histori-camente importante e igualmente negligen-ciada, vem recebendo mais atenção por parte das agências dedicadas ao seu controlo e ao financiamento de pesquisas. Como conse-quência deste facto e do desenvolvimento científico e tecnológico alcançado nos últi-mos anos, novos testes diagnósticos e com-postos com potencial terapêuticos têm surgi-do e obrigado os fabricantes e sites de pesquisa clínica a avaliá -los para serem regis-tados em agências regulatórias. Em ensaios clínicos para avaliação de novas drogas, a principal metodologia utilizada é a atividade bactericida precoce (early bactericidal activity – EBA) descrita por Mitchson1, que consiste em quantificar a carga bacilar (CFU) presen-te em amostras de escarro recolhidas por um período de 12 horas durante os primeiros 7 dias de tratamento. Tal metodologia é basea-da no facto de a redução do número de CFU

Since the World Health Organization’s 1993 declaration that tuberculosis (TB) was a worldwide emergency, this historically im-portant and equally neglected disease has received more attention from agencies dedi-cated to its controlling and financing re-search. As a consequence of this and of re-cent scientific and technological advances, new diagnostic tests and compounds with therapeutic potential have emerged. These have obliged manufacturers and clinical re-search sites to evaluate and register them in regulatory bodies. The main methodology used in clinical trials to evaluate new drugs is early bactericidal activity (EBA), described by Mitchison1, which consists of quantify-ing the colony forming unit (CFU) in spu-tum samples collected over a 12-h period for the first 7 days of treatment.This methodology is based on the reduction in the number of CFU over the first two days being statistically related to the efficacy of the treatment regimens instituted, making it a

1 Núcleo de Doenças Infecciosas da Universidade Federal do Espírito Santo, Brasil/Infectious Diseases Unit, Universidade Federal do Espírito Santo, Brazile-mail: [email protected]




durante os primeiros dois dias estar estatisti-camente relacionada com a eficácia dos es-quemas terapêuticos empregues e representa um parâmetro clínico de redução da infec-ciosidade2,3. Os estudos de EBA são realiza-dos para comparar a atividade de várias doses de uma droga, diferentes drogas dentro da mesma classe e diferentes classes de drogas4. A realização deste tipo de ensaio clínico re-quer elevado investimento financeiro, profis-sionais qualificados em boas práticas clínicas e laboratoriais e um sério comprometimento e compreensão dos doentes. Diante destes factos, o laboratório de micobacteriologia as-sume grande responsabilidade e enfrenta muitos desafios para cumprir com êxito o seu papel nos ensaios clínicos. A seguir são des-critos resumidamente alguns dos obstáculos e estudos realizados para superá -los.

Recolha de amostras 12 a 16 horasUm pool de escarro recolhido nestas condi-ções requer internação do doente, o seu dis-tanciamento da família, custo financeiro de internação, maior risco de contaminação da cultura e longo período de tempo para mo-nitoramento. Com o objetivo de verificar se o pool de escarro recolhido durante 5 horas pela manhã poderia conter uma carga bacilar semelhante ao pool recolhido num período de 12 horas, Nascimento et al (estudo em fase final), ao comparar a carga bacilar em amostras de escarro de doentes com tubercu-lose pulmonar, recolhidas por períodos de 5 e 12 horas, não observaram diferenças esta-tísticas significativas entre os dois grupos avaliados (6,88 log10 CFU/ml, e 6,95 log10 CFU/ml, respectivamente) e demonstraram assim que uma recolha monitorizada de es-

clinical parameter of reduced infectiousness2,3. EBA studies are performed to compare the activity of various doses of a drug, different drugs of the same class and different classes of drugs4. This type of clinical trial implies heavy financial investment, professionals qualified in good clinical and laboratory practices and a serious commitment to and understanding of patients. Accordingly, the mycobacteriology laboratory faces heavy responsibility and a se-ries of challenges to be able to play a success-ful role in clinical trials. Some of the obstacles encountered and studies undertaken to over-come them are summarised below.

Sample collection over 12 and 16 hrsCollecting a pool of sputum under these condi-tions requires the patient being admitted to the hospital, which involves separation of the pa-tient from his/her family, the costs of hospita-lization stay, an increased risk of culture con-tamination and increased length of patient monitoring. To see if a pool of sputum collected over 5-h in the morning contained similar CFU to a pool collected over a 12-h period, Nasci-mento et al. (study currently in final stage) when comparing CFU in the sputum of patients with pulmonary TB collected over 5-h and 12-h pe-riods, did not find any statistically significant differences in the two groups evaluated (6.88 log10 CFU/ml vs. 6.95 log10 CFU/ml, respec-tively) and, thus, showed that sputum collected over 5-h could be used in future clinical trials.

New markers of bacteriological clearance in response to anti-tuberculosis therapyMicrobiological parameters can easily be as-sessed in spontaneously expectorated spu-

Ensaios clínicos de novas drogas e testes diagnósticos em tuberculose: desafios micobacteriológicos

Moisés Palaci




carro durante 5 horas poderá ser utilizada em futuros ensaios clínicos.

Novos marcadores de depuração bacteriológica em resposta à terapia anti tuberculoseOs parâmetros microbiológicos podem ser facilmente avaliados na expectoração espon-tânea, mas a cultura quantitativa é demorada e trabalhosa. O marcador ideal mediria even-tos no decurso do início do tratamento e se-ria rigoroso, independentemente da ação ou do regime a ser testado. Recentemente, Liwen et al.5 avaliaram e reportaram níveis de mRNA por RT -PCR quantitativa em amostras recolhidas de doentes com TB sob monoterapia, num anterior estudo de ativi-dade bactericida em fluoroquinolonas, e nos que estavam sob um regime -padrão com base em rifampina num ensaio de IL -2. Os autores demonstraram que o mensageiro RNA para a isocitrato liase da enzima do ci-clo glioxilato teve taxas de declínio seme-lhantes em doentes a receberem monoterapia com isoniazida, gatifloxicina, levofloxacina e moxifloxacina. A isocitrato liase (icl) mRNA esteve altamente relacionada com as unida-des que formavam colónias na expectoração antes da terapia e durante 7 dias de monote-rapia em todos os grupos de tratamento. Detectou -se icl mRNA na expectoração de doentes com cultura positiva de TB em regi-me de tratamento com base em rifampina durante 2 meses. Além disso, também de-monstraram que a proteína de ligação à fi-bronectina (fbpB) mRNA diminuiu 0,47 log10 moléculas/ml/d desde o início até ao segundo dia, o único mRNA que se relacio-nou com a atividade bactericida precoce da monoterapia com isoniazida. Em conclusão,

tum, but quantitative culture is time con-suming and labour intensive. The ideal marker would measure events early during treatment and be accurate regardless of the drug action or regimen being tested. Re-cently, Liwen et al.5 assessed and reported mRNA levels by quantitative RT-PCR in sputum specimens from TB patients receiv-ing monotherapy in an early bactericidal activity study of fluoroquinolones and in those receiving a standard rifampin-based regimen in an IL-2 trial. These authors demonstrated that messenger RNA for the glyoxylate cycle enzyme isocitrate lyase de-clined at similar rates in patients receiving isoniazid, gatifloxacin, levofloxacin, and moxifloxacin monotherapy. Isocitrate lyase (icl) mRNA correlated highly with CFU in sputum prior to therapy and during 7 days of monotherapy in all treatment arms. icl mRNA was detectable in sputum of culture-positive TB patients receiving a rifampin-based regimen for 2 months. In addition, they also demonstrated that fibronectin-binding protein (fbpB) mRNA decreased 0.47 log10 molecules/ml/d from baseline to day 2, the only mRNA correlating with the early bactericidal activity of isoniazid mono-therapy. In conclusion, icl and fbpB mRNAs are reliable markers of M. tuberculosis viabil-ity, however, both of these uses will require larger longitudinal studies to validate the re-liability of icl mRNA as a surrogate marker of response to drug therapy for TB.

Lower rates of contamination of mycobacterial cultureIn clinical trials to evaluate treatment re-gimes that require patient follow-up for up to 2 years after cure, and in studies to eva-

Ensaios clínicos de novas drogas e testes diagnósticos em tuberculose: desafios micobacteriológicosMoisés Palaci




icl e fbpB mRNAs são marcadores fiáveis de viabilidade do M. tuberculosis; no entanto, estas duas utilizações carecem de estudos longitudinais mais extensos que validem a fiabilidade de icl mRNA como marcador substituto de resposta à terapia farmacológi-ca para TB.

Diminuição das taxas de contaminação de culturas de micobactériasEm ensaios clínicos para avaliação de regimes terapêuticos que exigem o monitoramento do doente em até dois anos após a sua cura, e em estudos para avaliação de novos testes diagnós-ticos, culturas de escarro contaminadas podem causar prejuízos económicos, eliminação de dados da análise estatística (time points onde ocorreram contaminação) ou até a exclusão do doente do estudo. Diante destas situações pro-blemáticas, Peres et al. (artigo submetido a pu-blicação) realizaram um ensaio clínico prag-mático para avaliar a eficácia de métodos de anti ssepsia intrabucal na redução da taxa de contaminação de culturas de doentes suspeitos de tuberculose. Os autores constataram que, dentre os três procedimentos de higienização bucais utilizados isoladamente (somente água, digluconato de clorexidina e cloreto de cetilpi-ridínio), apenas o realizado como digluconato de clorexidina permitiu uma redução signifi-cativa da taxa de contaminação das culturas, sobretudo no meio líquido MGIT (Becton Dickinson). Verificaram ainda que o uso de uma concentração maior do antimicrobiano PANTA (PANTA 2x) nas amostras -controlo dos doentes reduziu significativamente a po-pulação de organismos contaminantes no meio MGIT, sem, contudo, reduzir a taxa de deteção do M. tuberculosis.

luate new diagnostic tests, contaminated sputum cultures could result in high costs, elimination of data from the statistical ana-lysis (time points where contamination oc-curred) or even exclusion of the patient from the study. In the face of these difficul-ties, Peres et al. (article submitted for publi-cation) performed a pragmatic clinical trial to evaluate the efficacy of methods of intra-oral antisepsis in reducing the contamina-tion rate of cultures from patients suspected of TB. The authors found that of the three intra-oral hygiene procedures employed separately (water only; chlorhexidine diglu-conate and cetylpyridinium chloride), only the one using chlorhexidine digluconate re-sulted in a significant reduction of the cul-ture contamination rate, particularly in MGIT liquid medium (Becton Dickinson). They also found that using a higher concen-tration of PANTA antimicrobial solution (PANTA 2x) in patients’ control samples, significantly reduced the population of con-taminating organisms in the MGIT liquid medium, without, however, lowering the rate of M. tuberculosis detection.

Sputum sample divisionPerforming clinical trials to evaluate new di-agnostic methods and treatments often re-quires sputum sample division for compara-tive analysis. In this case, the samples must be divided equally, maintaining a similar CFU in each part. The use of a mucolitic agent is recommended for this, but there are no studies proving its efficacy or that of other procedures for sample division. Mo-rais et al. (unpublished data) used a quanti-tative culture technique to verify if CFU was similar in divided samples after the di-


Moisés Palaci




Divisão em amostras de escarroA realização de ensaios clínicos para avaliação de novos métodos diagnósticos e terapêuticos frequentemente requer a divisão da amostra de escarro para análise comparativa. Neste caso é necessário que a divisão da amostra ocorra de maneira equitativa, mantendo a carga bacilar semelhante entre as partes. Recomenda -se o uso de um agente mucolítico para esta finalida-de; contudo, por não se dispor de estudos que comprovem a eficiência deste ou de outros procedimentos de divisão de amostras, Morais et al (dados ainda não publicados), utilizando técnica de cultura quantitativa, propuseram-se verificar se após a digestão de amostras escarro por procedimento químico (N -acetil -L -cisteina 50 mg/ml - 10% do volume da amostra duran-te 15 minutos), ou mecânico (agitação com pérolas de vidro), a carga bacilar seria seme-lhante nas amostras divididas. Ao comparar os resultados dos grupos entre si não observaram diferenças estatísticas significativas. Para o pri-meiro grupo que fez uso imediato do NALC foi observada na aliquota I uma média de 5,66 (±0,92) log10, enquanto na aliquota II 5,65 (±0,94) log10 UFC/ml. Para o segundo grupo, processado com pérolas de vidro, foi observado uma média de 5,53 (±0,91) e 5,49 (±0,94) log10 UFC/ml em cada aliquota, respetivamen-te, demonstrando assim que tanto o procedi-mento químico quanto o físico podem ser uti-lizados com igual eficiência para divisão de amostras de escarro.Como destacado anteriormente, os ensaios clínicos terapêuticos em tuberculose são complexos e onerosos. Poucos sites no mun-do possuem os requisitos e equipas necessá-rias à sua realização. Se considerarmos o mo-desto investimento feito nesta área, importantes avanços ocorreram nos últimos anos. Entretanto, muitos outros desafios

gestion of sputum samples by chemical pro-cedure (N-Acetyl-L-Cysteine 50mg/ml- 10% of the sample volume for 15 minutes), or mechanically (agitation with glass beads). No statistically significant differences were seen when the results of the groups were compared. In the first group (NALC), ali-quot I had mean 5.66 (±0.92) log10 and ali-quot II mean 5.65 (±0.94) log10 CFU/ml. In the second group (glass beads) the means were 5.53 (±0.91) and 5.49 (±0.94) log10C-FU/ml for each aliquot, respectively, thus showing that both chemical and physical procedures can be used with equal efficacy for division of sputum samples.As mentioned above, clinical treatment trials for TB are complex and expensive. Few sites worldwide have the necessary requirements and teams to perform them. If we consider the modest investment made in this area, important advances have been seen in re-cent years. There are, however, many chal-lenges which have yet to be met. One such challenge is the validation of the results de-scribed above and the development of sim-ple tests able to determine sensitivity to drugs for latent bacteria and bacterial viabil-ity over the long course of TB treatment.

Ensaios clínicos de novas drogas e testes diagnósticos em tuberculose: desafios micobacteriológicosMoisés Palaci




ainda necessitam de ser superados. Dentre os quais a validação dos resultados descritos e o desenvolvimento de testes simples e capazes de determinar a sensibilidade a drogas de bactérias em fase de latência e a viabilidade bacteriana durante o longo período de trata-mento da tuberculose.

Bibliografia/Bibliography1. Mitchison DA. Assessment of new sterilizing drugs for treating pulmonary tuberculosis by culture at 2 months. Am Rev Respir Dis 1993; 147: 1062 -1063.2. Jindani A, Aber VR, Edwards EA, Mitchison DA. The early bactericidal activity of drugs in patients with pulmonary tuberculosis. Am Rev Respir Dis 1980; 121:939 -949.3. Kennedy N, Fox R, Kisyombe GM, Saruni AOS, Uiso LO, Ramsay ARC, Ngowi FI, Gillespie S. Early bactericidal and sterilizing activities of ciprofloxacin in pulmonary tuberculosis. Am Rev Respir Dis 1993; 148:1547 -1551.

4. Johnson JL, Hadad DJ, Boom WH, Daley CL, Pelo-quin CA, Eisenach KD, Jankus DD, Debanne SM, Charlebois ED, Maciel E, Palaci M, Dietze R. Early and extended early bactericidal activity of levofloxacin, gati-floxacin and moxifloxacin in pulmonary tuberculosis. Int J Tuberc Lung Dis 2006; 10:605 -612.5. Li L, Mahan CS, Palaci M, Horter L, Loeffelholz L, Johnson JL, Dietze R, Debanne SM, Joloba ML, Okw-era A, Boom WH, Eisenach KD. Sputum Mycobacte-rium tuberculosis mRNA as a marker of bacteriologic clearance in response to anti -tuberculosis therapy. J Clin Microbiol 2009; 18 [Epub ahead of print].


Moisés Palaci




Karina de Prince1

Fernando R Pavan1

Daisy N Sato2

Wagner Villegas5

Sergio RA Leite5

Clarice QF Leite1*

Avaliação das moléculas com atividade antiTB das plantas do cerrado brasileiro

Screening of molecules with anti-TB activity from the brazilian cerrado plants

IntroduçãoA tuberculose (TB) ainda é, em todo o mun-do, a doença infeciosa mais frequente e im-portante, causando morbilidade e morte. Um terço da população mundial está infeta-da com Mycobacterium tuberculosis (MTB) e aproximadamente dois milhões de mortes são atribuídas à TB anualmente1. Entre to-dos os países do continente americano, o Brasil tem o segundo maior índice de morbi-lidade e de morte por TB, com uma preva-lência de 62/100 0002. O ressurgimento glo-bal da TB e o rápido aparecimento da tuberculose multirresistente (MDR -TB) su-blinha a importância do desenvolvimento de novos fármacos antituberculose3.Na busca de novos compostos a partir de plantas, as árvores forneceram muitos fárma-cos no passado e permanecem uma importan-te fonte de novos compostos. Recentemente os produtos naturais têm sido alvo de muita atenção como potenciais agentes anti TB4 -6.

IntroductionAcross the world, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death. A third of the world’s population is infected with Mycobacterium tuberculosis (MTB), and approximately two million deaths are attributable to TB annually1. Among all the countries in the Americas, Brazil reports the second-highest TB mortality and morbidity, with a prevalence of 62 in /100 0002. The global resurgence of TB and the rapid emer-gence of MDR-TB, underscore the impor-tance of the development of new antituber-culous drugs3.Concerning the search for new compounds from Plants, trees have provided many drugs in the past, and remain a rich source of no-vel compounds. Natural products have re-cently received a lot of attention as potential anti-TB agents4-6. In Brazil, there is a tradi-tional knowledge of how to use native plants

1 Faculdade de Ciências Farmacêuticas, UNESP, CEP 14801 -902, Araraquara (SP), Brasil2 Instituto Adolfo Lutz, Ribeirão Preto, CEP 14085 -410, Ribeirão Preto (SP), Brasil3 Departamento de Química, Universidade Federal de São Carlos, CP 676, CEP 13565 -905, São Carlos (SP), Brasil4 Instituto de Química de São Carlos, Universidade de São Paulo, CP 780, 13560 -970, São Carlos – SP, Brasil5 Instituto de Química de Araraquara, UNESP, CEP 14801 -902, Araraquara – SP, Brasil.e-mail: [email protected]




No Brasil, existe um conhecimento tradicio-nal de como utilizar plantas nativas no trata-mento de várias doenças, porque muitas co-munidades não têm acesso a medicamentos e usam essas plantas para se tratarem7.Embora tenham sido encontradas várias centenas de produtos naturais de plantas com atividade antimicobacteriana, nenhu-ma delas atingiu a fase de desenvolvimento para fármaco devido a dificuldades, como a escassez de compostos, a elevada complexi-dade estrutural e a falta de estudos de acom-panhamento de indícios promissores.

ObjetivoNeste contexto, iniciámos o projeto inte-grando análises químicas e testes de activi-dade antiTB de plantas, especialmente de espécies que compõem o cerrado brasileiro, bioma do tipo savana que predomina no planalto central brasileiro e que inclui mi-lhares de espécies vasculares nativas agrupa-das em centenas de famílias. Muitas destas plantas são habitualmente usadas como re-médios naturais pela população local para tratar doenças várias.

Material e métodosOs especímenes de plantas do cerrado foram colhidas nos estados de Tocantins (aproxima-damente 11° S, 48° O) e Mato Grosso do Sul (aproximadamente 21° S, 56° O). Para a sepa-ração fitoquímica, usámos técnicas cromato-gráficas, essencialmente adequadas para sepa-ração de substâncias polares (GPC, XAD2, DCCC, HSCC, HPLC, etc.). Para determi-nar a estrutura dos compostos isolados usámos principalmente métodos espectrofotométri-cos, como NMR, IR, UV e MS. Avaliámos a atividade dos extratos, frações enriquecidas e

to treat several diseases because many com-munities don’t have access to synthetic me-dicines and use those plants in treatments7.Although several hundred natural plant products with antimycobacterial activity have been found, none of them have moved towards drug development, be-cause of difficulties, such as very low com-pound availability, high structural com-plexity and lack of follow-up studies of promising leads

ObjectiveIn this context, we started the project in-tegrating chemical analysis and anti-TB activity tests of plants, especially species that compose the “Brazilian Cerrado”, a savannah like biome that predominates in the center-west region of the country. It includes more than several thousands na-tive vascular plant species, grouped in hundreds of families. Many of these plants are commonly used as natural remedies by people living in this area to treat va-rious illnesses.

Material and methodsThe cerrado plant specimens were collected in the states of Tocantins (at nearly 11° S by 48° W) and Mato Grosso do Sul (approxi-mately 21° S by 56° W). To perform the phytochemical separation, we used chro-matographic techniques, mainly suitable for polar substances (GPC, XAD2, DCCC, HSCC, HPLC, etc). To determine the structure of the isolated compounds we used mainly spectrophotometric methods such as NMR, IR, UV and MS. To evaluate the activity of the extracts, enriched frac-

Avaliação das moléculas com atividade antiTB das plantas do cerrado brasileiroKarina de Prince, Fernando R Pavan, Daisy N Sato, Wagner Villegas, Sergio RA Leite, Clarice QF Leite





substâncias puras contra o M. tuberculosis por resazurin microtiter assay (REMA), de acordo com Palomino et al. (2002) e M. tuberculosis H37Rv ATCC 272948.

Resultados e discussãoSessenta e cinco extratos de 37 plantas distri-buídas por 18 famílias foram testadas contra M. tuberculosis. Vinte e seis por cento dos ex-tratos avaliados demonstraram actividade promissora, nomeadamente concentração inibitória mínima (CIM) ≤ 125 μg/m, 13 (76%) deles extratos de clorofórmio e 4 (24%) de metanol. Esses extratos foram sele-cionados para fracionamento guiado por ativi dade e avaliação detalhada das proprie-dades antituberculosas, sendo a sua compo-sição química analisada (Quadro I).Do extrato de clorofórmio de B. fagifolia, a mistura de lupeol, α -amirina e β -amirina re-velaram CIM mais baixa (31,25 μg/mL) do que lupeol, α -amirina ou β -amirina isola-dos, cujos valores CIM foram ≥ 62,5 μg/mL. A mistura de lupeol e acetatos de α -amirina e β -amirina mostraram o mesmo valor CIM (31,25 μg/mL), sugerindo que a acetilação de α -amirina e β -amirina não influencia a sua atividade9. Observou -se a mesma situa-ção nas frações enriquecidas de extrato de clorofórmio de B. crassa. As CIM de 31,25 para a mistura de α -e β -amirina e de 62,5 μg/mL, o dobro do valor para o acetato puro de α -amirina, confirma o efeito sinergístico entre os componentes destas misturas contra M. tuberculosi10.Para C. adamantium, a 5,7 -dihidroxi -6,8 -di -C -metilflavanona (A) isolada mostrou uma CIM superior a 250 e 2’, 4’ -dihidroxi -3’,5’ -dimetil -6’ -metoxicalcona (B) uma CIM de 62,5 μg/mL, enquanto as suas misturas

tions and pure substances against M. tuber-culosis we used the Resazurin Microtiter As-say (REMA) according Palomino et al. (2002) and the M. tuberculosis H37Rv ATCC 27294 strain8.

Results and discussionsixty five extracts from 37 plants, distribu-ted in 18 families have been tested against M. tuberculosis. Out of all the extracts as-sayed, 26% showed promising activity, namely MICs below or equal to 125 μg/mL, 13 (76%) of these coming from chloroform extract and 4 (24%) from methanol extract. These extracts were selected for activity guided-fractionation and detailed evalua-tion of the anti-tuberculosis properties, and their chemical composition was analysed and showed in Table I.From the B. fagifolia chloroform extract, the mixture of lupeol, α-amyrin and β-amyrin displayed a lower MIC (31.25 μg/mL) than isolated lupeol, α-amyrin or β-amyrin whose MIC value were higher than or equal to 62.5 μg/mL. The mixture containing lu-peol and α-amyrin and β-amyrin acetates showed the same MIC value of 31.25 μg/mL, suggesting that the acetylation of α-amyrin and β-amyrin does not influence their activity9. The same situation was found in the enriched fractions from the B. crassa chloroform extract. MICs of 31.25 for the mixture of α-and β-amyrin and 62.5 μg/mL, double the value for the pure acetate of α-amyrin, confirms the synergistic effect among the components of these mixture against M. tuberculosis10.For C. adamantium, the isolated 5,7-dihy-droxy-6,8-di-C-methylflavanone (A) showed higher MIC of 250 and 2’,4’-dihydroxy-3’,5’-





Quadro I – Valores CIM das frações enriquecidas e compostos testados contra M. tuberculosis

Compostos CIM (μg/mL)Fração enriquecida / compostos de clorofórmio extrato de B. fagifolia Mistura de lupeol, α -e β -amirina 31,25Mistura de lupeol, acetatos de α - e β -amirina 31,25α -amirina 62,5α -amirina acetato 62,5Dotriacontano 62,5Ácido básico 2,5Fração enriquecida / compostos de extrato de clorofórmio de B. crassaMistura de α -e β - amirina 31,25Mistura de acetato de α -e β - amirina 31,25Mistura de ácidos ursólico e oleanólico 62,5Compostos de extrato de clorofórmio de C. adamantium5,7 -dihidroxi -6,8 -di -C -metilfl avanona (A) 2502’,4’ -dihidroxi -3’,5’ -dimetil -6’ -metoxicalcona (B) 62,5Mistura A + B (2:8) 7,8Mistura A + B (3:7) 15,6Mistura A + B (7:3) 31,25Mistura A + B (8:2) 62,5Compostos de extrato de clorofórmio de Qualea parvifl oraLupeol 62,5Lupenona 125Ácido betulínico 31,25Ácido 3 epibetulínico 62,5Friedelina 125β sitosterol 125

Table I – MIC values of enriched fractions and compounds tested against M. tuberculosis

Compounds MIC (μg/mL)Enriched fraction/compounds from chloroform extract of B. fagifolia Mixture of lupeol, α-and β-amyrin 31.25Mixture of lupeol, acetates of α- and β-amyrin 31.25α-amyrin 62.5α-amyrin acetate 62.5Dotriacontane 62.5Bassic acid 2.5Enriched fraction/compounds from chloroform extract of B. crassaMixture of α-and β-amyrin 31.25Mixture of α-and β-amyrin acetate 31.25Mixture of ursolic and oleanolic acid 62.5Compounds from chloroform extract of C. adamantium5,7-dihydroxy-6,8-di-C-methylfl avanone (A) 2502’,4’-dihydroxy-3’,5’-dimethyl-6’-methoxychalcone (B) 62.5Mixture A + B (2:8) 7.8Mixture A + B (3:7) 15.6Mixture A + B (7:3) 31.25Mixture A + B (8:2) 62.5Compounds from chloroform extract of Qualea parvifl oraLupeol 62.5Lupenona 125Betulinic acid 31.253 epi betulinic acid 62.5Friedelin 125β sitosterol 125




(A+B), em vários rácios, mostrou vários CIM, entre 62,5 μg/mL para o rácio 8:2 (A+B) e a atividade muito melhor de 7,8 μg/mL para o rácio 2:8. Há aqui claro siner-gismo entre os compostos A e B quando misturados, e este sinergismo depende for-temente do seu rácio de concentração5. Nos compostos isolados de Qualea parviflora, apesar da grande semelhança estrutural de lupeol e lupenona, observou -se uma redu-ção duas vezes maior da atividade antiTB de lupeol, que se deveu à substituição de β -hidroxilato C3 no lupeol para cetona na lupenona. Do mesmo modo, também se observou uma redução duas vezes maior da atividade antiTB entre os ácidos betulínico (CIM 31,2 μg/mL) e epibetulínico (62,5μg/mL), devido à epimerização no ácido epibe-tulínico de β -hidroxil C3. Esses resultados corroboram os do trabalho de Cantrells et al. (2001), em que nos triterpenos, β -hidroxil em C3 é importante para a atividade anti-TB4. A melhor MIC observada foi no triter-peno do ácido básico de B. fagifolia, que mostrou forte atividade antitubercular, com valores CIM de 2,5 μg/mL9. Este valor de concentração inibitória é comparável aos dos fármacos antiTB de primeira linha, como etambutol (1 -5 μg/mL) e estreptomi-cina (2 -8 μg/mL), e melhor do que a pirazi-namida (20 -100μg/mL)11.

ConclusãoOs resultados indicam que as plantas do cerrado podem proporcionar frações e com-postos com atividade antituberculose pro-missora.

dimethyl-6’-methoxychalcone (B) a MIC of 62.5 μg/mL, while their mixtures (A+B), in several ratios, showed various MICs, ranging from 62.5 μg/mL for the ratio 8:2 (A+B) down to the much higher activity of 7.8 μg/mL, for the ratio 2:8. Here there is a clear syn-ergism between compounds A and B when mixed and this synergism depends strongly on their concentration ratio5. For compounds isolated from Qualea parviflora, despite the great structural similarity of lupeol and lupe-none a two-fold reduction of anti-TB activity was observed with lupeol. This was due to the substitution of β hydroxylat C3 in lupeol to the ketone on lupenone. Similarly a two fold reduction of anti-TB activity was observed between betulinic (MIC 31,2 μg/mL) and epi-betulinic acid (62,5μg/mL), due to the epimerization in epi-betulinic acid of the β hydroxyl C3. Those results corroborate the report of Cantrells et al. (2001) in that within triterpenes, the β hydroxyl on C3 is impor-tant for anti-TB activity4. The best MIC found was for the triterpene bassic acid from B. fagifolia, this showed strong antitubercular activity, with MIC values of 2.5 μg/mL9. This inhibitory concentration value is comparable to those of first-line tuberculosis drugs, such as ethambutol (1-5 μg/mL) and streptomycin (2-8 μg/mL), and better than pyrazinamide (20-100μg/mL)11.

ConclusionThe results indicated that plants of the “cer-rado” can provide fractions and compounds with promising anti – tuberculosis activity.





Bibliografia/Bibliography1. Global Alliance for TB Drug Development, www.tballiance.org, accessed June 1, 2009.2. Malaspina AC, Cavalcanti HR, Leite CQF, Machado SM, Viana BH, Silva RM, Hage EF, Figueiredo WM, Marques E, Ferrazoli L, Arbex M, Lessi M, Fonseca LS, Rigouts L, Saad MH. Jpn J Infect Dis (2008); 231--233.3. Lourenço MCS, Ferreira ML, Souza MV, Peralta MA, Vasconcelos TR, Henriques MGO. Eur J Med Chem 2008; 43:1344 -1347.4. Cantrell CL, Franzblau SG, Fischer NH. Planta Med 2001; 67:1 -10.5. Pavan FR, Leite CQF, Coelho RG, Coutinho ID, Honda NK, Cardoso CAL, Vilegas W, Leite SRA, Sato DN. Quim Nova 2009; 32:1222 -1226.

6. Honda NK, Pavan FR, Coelho RG, Leite SRA, Micheletti AC, Lopes TIB, Misutsi MY, Beatriz A, Brum RL, Leite CQF. Phytomedicine (2009) in press.7. Almeida SP, Proença CEB, Sano SM, Ribeiro Cerrado JF. Espécies vegetais úteis. In: Sano SM, Almeida SP (Eds.). Planaltina, Distrito Federal, Brazil, 38 -39.8. Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F. Antimicrob Agents Chemoter 2002; 2720 -2722.9. Higuchi CT, Sannomiya M, Pavan FR, Leite SRA, Sato DN, Franzblau SG, Sacramento LVS, Vilegas W, Leite CQF. eCAM (2008) doi:10.1093/ecam/nen077.10. Higuchi CT, Pavan FR, Leite CQF, Sannomiya M, Vilegas W, Leite SRA, Sacramento LVS, Sato DN. Quim Nova 2008; 31:1719 -1721.11. Collins L, Franzblau SG. Antimicrob Agent Che-mother 1997; 1004 -1009.





Caroline Allix-Béguec3

Christine Hubans3

Stéphanie Ferreira3

Philip Supply1,2,3

Novas ferramentas de fácil utilização para genotipagem padronizada e com qualidade controlada de estirpes do complexo Mycobacterium tuberculosis

New, easy-to-use tools for standardised and quality-controlled genotyping of Mycobacterium tuberculosis complex strains

Harmonized and reliable typing of patho-genic bacteria permits easy identification of locally or internationally circulating clones, which is essential for optimal epi-demiological surveillance and disease con-trol. This is especially true for diseases such as tuberculosis (TB), with worldwide dis-tribution and global emergence of drug-resistant strains. In addition, typing can guide therapeutic decisions, for instance in case of suspected laboratory errors or con-taminations. Mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing has become a major method for fast and high-resolu-tion genotyping of Mycobacterium tubercu-losis complex isolates. A system based on 24 loci has been proposed for international standardization by an international con-sortium including 10 European and Ame-rican laboratories1. In population-based studies, standard MIRU-VNTR typing was shown to have an equal to slightly bet-

A tipagem harmonizada e fiável de bactérias patogénicas permite a fácil identificação de clones em circulação local ou internacional-mente, o que é essencial para bons serviços de vigilância epidemiológica e de controlo de doenças. Isto aplica -se particularmente a doenças como a tuberculose (TB), com inci-dência mundial e emergência global de estir-pes resistentes a fármacos. Além do mais, a tipagem pode ajudar nas decisões terapêuti-cas, por exemplo, em casos de suspeição de erros ou contaminações laboratoriais. A tipa-gem pela metodologia MIRU -VNTR (myco-bacterial interspersed repetitive unit -variable number of tandem repeat) tornou -se impor-tante para a genotipagem rápida e de alta re-solução dos isolados do complexo Mycobacte-rium tuberculosis. Um consórcio internacional que inclui 10 laboratórios europeus e ameri-canos1 propôs um sistema baseado em 24 loci para padronização internacional. Estudos de população mostraram que a tipagem MIRU--VNTR padrão tem um valor de previsão

1 INSERM U6292 Institut Pasteur de Lille, Lille3 Genoscreen, Lille, Francee-mail: [email protected]




igual a ligeiramente melhor do que o anterior padrão -ouro, IS6110 RFLP, no estudo da transmissão de TB, em ambientes com carac-terísticas epidemiológicas representativas das de muitos países desenvolvidos. A interroga-ção baseada na PCR de até 24 marcadores independentes e bem calibrados facilita a rá-pida e fiável elucidação dos mecanismos mo-leculares de situações complexas que envol-vam potenciais surtos, infeções mistas ou reinfeções2 -6. Como resultado, este método vem sendo adoptado internacionalmente, frequentemente em combinação com spoligo-typing, como o novo método de referência para epidemiologia molecular da TB, por exemplo, pelo Center for Disease Control (CDC) nos Estados Unidos, grandes consór-cios europeus de investigação e de vigilância epidemiológica e por centros de referência nacionais ou regionais.Novos equipamentos e serviços de uso fácil que ficaram recentemente disponíveis vie-ram facilitar o controlo de qualidade da uti-lização desta técnica, bem como a interpreta-ção dos resultados obtidos. Os serviços de tipagem MIRU -VNTR são propostos e usa-dos já por centros de referência e laborató-rios internacionais para realização de genoti-pagem (incluindo de estirpes de M. bovis) e//ou para avaliação do sistema de garantia de qualidade QA/QC. Os kits de calibração, validação e tipagem MIRU -VNTR de qua-lidade controlada, bem como o software para gestão de dados e formação on -site, facilitam enormemente a padronização e a utilização eficiente da tipagem MIRU -VNTR no labo-ratório do utilizador.Com o kit de tipagem MIRU -VNTR, 24 marcadores são ampliados a partir de ADN purificado ou de extrato bruto de ADN de colónias micobacterianas ou de grânulos de

ter predictive value than the previous gold standard IS6110 RFLP for the study of TB transmission, in settings with epidemio-logical characteristics representative of those of many developed countries. PCR-based interrogation of up to 24 indepen-dent and well-calibrated markers facilitates prompt and reliable molecular-guided elu-cidation of complex situations involving potential outbreak cases, mixed infections or re-infections2-6. As a result, this method is being internationally adopted, often in combination with spoligotyping, as the new reference method for TB molecular epidemiology, e.g. by the US CDC, large European research and epidemiological surveillance consortiums and National or Regional reference Centers.New, easy-to-use tools and services have re-cently become available, which facilitate quality-controlled use of this technique, as well as interpretation of the results obtained. MIRU-VNTR typing services are proposed and already used by international Reference Centers and laboratories, for outsourcing their genotyping (including of M. bovis strains) and/or for QA/QC evaluation. Quality-controlled MIRU-VNTR Calibra-tion, Validation and Typing kits, as well as dedicated data management software and on-site trainings greatly facilitate standar-dized set up and efficient use of MIRU-VNTR typing in user’s laboratory.With the MIRU-VNTR Typing Kit, 24 markers are amplified from purified DNA or crude DNA extract from mycobacterial colonies or cell pellets from liquid cul-tures, using 8 triplex PCR and fluorescent primers specific for the flanking regions of the targeted loci. Amplified fragment are separated by capillary electrophoresis on


Caroline Allix-Béguec, Christine Hubans, Stéphanie Ferreira, Philip Supply




células de culturas líquidas, usando 8 triplex PCR e primers fluorescentes específicos para os flancos dos loci alvo. Os fragmentos am-pliados são separados por eletroforese capi-lar em plataformas ABI para determinação das dimensões dos produtos da PCR. Con-forme se vão sabendo os tamanhos das uni-dades repetidas, estes refletem os números das sequências dos loci ampliados. A análise é feita usando o software ABI GeneMapper®, equipado com módulos optimizados forne-cida no GENOSCREEN MIRU -VNTR ty-ping calibration kit. O resultado final é um genótipo numérico portátil, que correspon-de ao número repetido em cada locus. As estirpes dos genótipos podem então ser ana-lisadas e comparadas com as estirpes de ge-nótipos de referência, recorrendo a bancos de dados locais ou a www.miru -vntrplus.org, um banco de dados de identificação multifuncional de livre acesso na Internet.O kit de calibragem MIRU -VNTR foi de-senhado para implementação e validação padronizada da técnica MIRU -VNTR no laboratório do utilizador. A migração relati-va entre o padrão de tamanho e os produtos do PCR depende dos locus e alelos do MIRU -VNTR e difere segundo o polímero usado para eletroforese capilar e entre ins-trumentos4. Assim, os bin sets GeneMapper específicos dos instrumentos são criados com o fim de calibrar para estes efeitos. Cada bin define o âmbito de tamanhos ob-servados para um dado número repetido (alelo) determinado a partir de 4 sequências diferentes. Estes bin sets são criados usando o software Bin Set Creator e 24 marcado-res allelic ladders, de acordo com os tama-nhos dos allelic ladder obtidos no analisa-dor de ADN do utilizador. Para validar o processo, é fornecido um painel de 12

ABI platforms to determine the PCR product sizes. As the length of the repeat units is known, these sizes reflect the num-bers of repeated sequences in the ampli-fied loci. Analysis is done using ABI Gene-Mapper® software customized with optimized modules provided in GENO-SCREEN MIRU-VNTR Typing Calibra-tion Kit. The final result is a portable nu-merical genotype, corresponding to the repeat number in each locus. Strain geno-types can then be analyzed and compared to reference strain genotypes using local databases or www.miru-vntrplus.org, a multi-functional identification database freely accessible via the Internet.The MIRU-VNTR Calibration Kit is de-signed for standardised implementation and validation of MIRU-VNTR technique in user’s laboratory. Relative migration be-tween the size standard and the PCR pro-ducts depends on the MIRU-VNTR locus and alleles, and differs upon polymer used for capillary electrophoresis and between instruments4. Therefore, instrument-speci-fic GeneMapper bin sets are created in order to calibrate for these effects. Each bin de-fines the observed size range for a given re-peat number (allele) determined from 4 dif-ferent runs. These bin sets are specifically created using the Bin Set Creator software and 24 marker-specific Allelic Ladders, in accordance with the Allelic Ladder sizes obtained on user’s DNA Analyzer. To vali-date the process, a panel of 12 reference samples (including one negative control) with known allelic profiles is provided. Af-ter PCR amplification and electrophoretic separation, allelic profiles are determined using the created bin sets and checked for validation.

Novas ferramentas de fácil utilização para genotipagem padronizada e com qualidade controlada de estirpes do complexo Mycobacterium tuberculosisCaroline Allix-Béguec, Christine Hubans, Stéphanie Ferreira, Philip Supply




amostras de referência (incluindo um con-trolo negativo) com perfis alélicos conheci-dos. Após ampliação por PCR e separação eletroforética, determinam -se os perfis aléli-cos usando os bin sets criados e validados.O software do gestor de dados MIRU--VNTR foi desenhado para facilitar a gestão de dados após análise do GeneMapper. As características incluem operações de contro-lo de qualidade (verificação dos controlos, etc.), gestão dos dados (dados em falta e ale-los duplos, criação de novas folhas de cálcu-lo, intercalação e controlo cruzado dos re-sultados do projecto GeneMapper®) e formatação final para compatibilidade com o banco de dados MIRU -VNTRPlus. A for-

The MIRU-VNTR Data Manager software is designed to facilitate data management after GeneMapper analysis. Features include quality control operations (verification of controls, etc), data management (missing data and double alleles, creation of new run spreadsheets, merging and cross-control of GeneMapper® project results), and final for-matting for compatibility with MIRU-VN-TRPlus database. MIRU-VNTR training is proposed in user’s laboratory, and includes calibration and validation of user’s platform, as well as training on technical and scien-tific interpretation. Dedicated MIRU-VN-TR Support is provided to kit users via spe-cific e-mailbox and telephone.

Fig. 1 – Tipagem MIRU -VNTR. A curva, as caixas e as setas à direita representam um cromossoma da estirpe do complexo M. tuberculosis, unidades repetidas dos loci MIRU -VNTR e os primers usados para ampliação PCR, respectivamente. Vinte e quatro loci MIRU -VNTR são analisados via 8 PCR multiplex. O resultado fi nal é um genótipo numérico, que corres-ponde ao número de unidades repetidas de cada um dos 24 marcadores

Fig. 1 – MIRU-VNTR typing. Close curve, orange boxes and arrows on the right represent a M. tuberculosis complex strain chromosome, repeat units of MIRU-VNTR loci, and labeled primers used for PCR amplifi cation, respectively. Twenty-four MIRU-VNTR loci are analysed via 8 multiplex PCRs. The fi nal result is a numerical genotype, corresponding to the repeat unit number of each of the 24 markers


Caroline Allix-Béguec, Christine Hubans, Stéphanie Ferreira, Philip Supply




It is hoped that the availability of these tools for easier and more efficient real-time geno-typing will contribute to improved molecu-lar-guided TB control and surveillance.

mação MIRU -VNTR, que se propõe tenha lugar no laboratório do utilizador, inclui ca-libragem e validação da plataforma do utili-zador, bem como formação quanto à inter-pretação técnica e científica. Aos utilizadores dos kits MIRU -VNTR é oferecido apoio específico via e -mail e telefone.Espera -se que a disponibilidade destes apa-relhos para genotipagem em tempo real mais fácil e eficiente contribua para melho-rar o controlo e a vigilância molecular da tuberculose.

Bibliografia/Bibliography1. Supply P, et al. Proposal for standardization of opti-mized mycobacterial interspersed repetitive unit -variable--number tandem repeat typing of Mycobacterium tuber-culosis. J Clin Microbiol 2006 44(12):4498 -510.2. Alonso -Rodriguez N, et al. Evaluation of the new ad-vanced 15 -loci MIRU -VNTR genotyping tool in Myco-bacterium tuberculosis molecular epidemiology studies. BMC Microbiol 2008; 8:34.3. Oelemann MC, et al. Assessment of an optimized mycobacterial interspersed repetitive -unit -variable--number tandem -repeat typing system combined with spoligotyping for population -based molecular epidemi-ology studies of tuberculosis. J Clin Microbiol 2007; 45(3):691 -697.

4. Allix C, Supply P, Fauville -Dufaux M. Utility of fast mycobacterial interspersed repetitive unit -variable number tandem repeat genotyping in clinical mycobac-teriological analysis. Clin Infect Dis 2004; 39(6):783--789.5. Allix -Béguec C, Fauville -Dufaux M, Supply P. Three--year population -based evaluation of standardized my-cobacterial interspersed repetitive -unit -variable -number tandem -repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 2008; 46(4):1398 -1406.6. Shamputa IC, et al. Mixed infection and clonal repre-sentativeness of a single sputum sample in tuberculosis patients from a penitentiary hospital in Georgia. Respir Res 2006; 7:99.

Novas ferramentas de fácil utilização para genotipagem padronizada e com qualidade controlada de estirpes do complexo Mycobacterium tuberculosisCaroline Allix-Béguec, Christine Hubans, Stéphanie Ferreira, Philip Supply




Elvira Richter1 Simpósio: Avaliação externa da qualidade

Symposium: External quality assurance

Os objetivos deste simpósio foram:avaliar a situação actual• explorar os progressos na AEQ• desenvolver redes• promover melhor colaboração•

Três oradores informaram quanto aos dife-rentes aspectos destes objetivos.

A AEQ num país com baixa incidência e elevados rendimentos (Alemanha)Há anos que a situação da tuberculose (TB) na Alemanha se caracteriza por uma dimi-nuição do número de doentes, com uma incidência de 5,5 por 100 000 habitantes em 2008. Quarenta e três por cento dos doentes com TB pulmonar confirmada por cultura tiveram esfregaços negativos. Vinte por cento de todos os doentes apresenta-vam exclusivamente TB extrapulmonar. O índice de estirpes resistentes atinge 11% para qualquer resistência e 2% para a TB

The aims of this symposium were:to estimate the current situation• to explore advances in EQA• to develop networks• to promote better collaboration•

Three speakers provided information to dif-ferent aspects of these aims.

EQA in a low incidence, high income country (Germany)The TB situation in Germany is character-ized since years by a decrease of the number of patients with an incidence of 5.5 per 100,000 inhabitants in the year 2008. 43% of patients with culture confirmed pulmo-nary TB were smear negative. 20% of all patients present with exclusively extrapul-monary TB. The rate of resistant strains reaches 11% for any resistance and 2% for MDR. XDR strains are reported. Infections with non tuberculous mycobacteria (NTM) comprise lymphadenitis in children, adults

1 NRL, Borstel, Germany




multirresistente (MDR). Estão descritas es-tirpes extensivamente resistentes (XDR). As infecções com micobactérias não tuber-culosas (MNT) incluem a linfadenite em crianças, adultos com doenças subjacentes [e.g., doença pulmonar obstrutiva crónica (DPOC), bronquiectasia), ou outras (como o granuloma de piscina).Esta situação reflete -se no tipo específico de AEQ com o objetivo de avaliar:

1) A especificidade e sensibilidade da mi-croscopia (lâminas positivas e negativas preparadas para coloração com a técnica de rotina do laboratório);

2) A sensibilidade das técnicas de cultura [amostras com ou sem micobactérias (TB ou MNT), preparados para isola-mento primário e identificação de TB ou MNT];

3) A sensibilidade e especificidade das téc-nicas de ampliação de ácidos nucleicos (as amostras de expetoração são prepara-dos para deteção de ácidos nucleicos es-pecíficos de TB);

4) A fiabilidade e rapidez dos testes de sus-cetibilidade (INH, RMP, EMB, SM, e PZA);

5) O rigor da identificação das bactérias da TB e das MNT (diferentes espécies mi-cobacterianas são preparadas para identi-ficação a nível de espécie).

Antes do envio das amostras para AEQ nos laboratórios, é necessário esclarecer os regu-lamentos (e.g., transporte, ou autorização para o manuseamento de bactérias de TB).Algumas causas importantes para os falsos resultados podem ser detectadas através da análise dos dados e reanálise adicional desses dados com a colaboração do laboratório que

with underlying diseases (e.g. COPD, bron-chiectasis), or others (like swimming pool granuloma).This situation is reflected by the specific kind of EQA aiming to the evaluation of:

1) specificity and sensitivity of microscopy (positive and negative slides prepared for staining with the routine technique of the laboratory)

2) sensitivity of culture techniques (speci-mens with or without mycobacteria [TB or NTM] are prepared for primary isola-tion and identification of TB or NTM)

3) sensitivity and specificity of nucleic acid amplification techniques (sputum speci-mens are prepared for detection of spe-cific nucleic acids of TB)

4) reliability and rapidity of susceptibility testing (INH, RMP, EMB, SM, and PZA)

5) the accuracy of identification of TB bac-teria and NTM (different mycobacterial species are prepared for identification to species level)

Before sending EQA specimens to the labo-ratories, regulations (e.g transportation, or the permission for handling with TB bacte-ria) have to be clarified.Some important causes for false results can be found by analysis of the data and addi-tional reanalysis in cooperation with the laboratory that reported false results. Rea-sons for false positive microscopic results were found to follow from staining in cu-vettes, not as single slides, or misidentifica-tion of staining artefacts. False positive cul-ture results derive from laboratory cross contamination. Incorrect susceptibility re-sults are most often found when testing SM,

Simpósio: avaliação externa da qualidadeElvira Richter




forneceu os falsos resultados. Observaram--se falsos positivos de microscopia após co-loração em cuvettes, e não em lâminas indi-viduais, ou identificação errada dos produtos de coloração. Os resultados falsos positivos de culturas resultam de contaminação cru-zada no laboratório. Encontram -se com al-guma frequência resultados com suscetibili-dade incorreta ao testar SM e, em parte, PZA. Ao fazer a identificação de especis, ocorrem por vezes erros significativos, sendo a principal razão a insuficiência da utiliza-ção exclusiva de análises bioquímicas.Na preparação de amostras para AEQ há que abordar alguns desafios.

– A questão da verdadeira avaliação da sen-sibilidade (no que respeita a microscopia, ampliação dos ácidos nucleicos e isola-mento primário). A preparação de amos-tras com pequenas quantidades de bacté-rias é difícil, porque pode resultar em amostras individuais sem micobactérias.

– Para evitar o uso de estirpes MDR, são selecionados isolados com padrões de re-sistência pouco comuns. Isto não reflete uma “situação da vida real”.

– Há um contraste entre as necessidades científicas e as do diagnóstico, como se observa no espantoso número de espé-cies de MNT com sequências que cons-tam das bases de dados de nucleótidos, com apenas algumas espécies diferentes isoladas em laboratório.

Organização e AEQ na Rede de Laboratórios de TB na LetóniaA incidência de TB na Letónia foi caracteri-zada por uma contínua diminuição até aproximadamente 1990, quando se verifi-

and in part PZA. Performing species identi-fication, sometimes significant errors occur. The main reason for this is the insufficiency of biochemical analyses alone.Some challenges have to be addressed in the preparation of samples for EQA.

– The question of a real estimation of sen-sitivity (with regard to microscopy, nu-cleic acid amplification, and primary iso-lation). The preparation of samples with low amount of bacteria is difficult since it can result in single samples without my-cobacteria.

– To avoid the use of MDR strains, iso-lates with uncommon resistance patterns are selected. This does not reflect a ‚real life situation‘.

– There is a contrast between scientific vs. diagnostic needs as we see in the over-whelming number of NTM-species with sequences delivered in the Nucleotide Databases, but only few different species isolated in the routine laboratory.

Organization and EQA in the Latvian TB laboratory networkThe TB incidence in Latvia was characte-rized by a continuous decrease until approx. 1990, when a sharp increase was noted, which peaked around 2000. Since then, the incidence again decreased as to 40.3 per 100,000 inhabitants for the year 2008.The Latvian TB-Laboratory Network is composed of the National Reference Labo-ratory in Riga, of 5 regional culture and mi-croscopy laboratories, and of several labora-tories (at district hospitals, policlinics, private labs) facilitating AFB microscopy at PHC level in each of 26 districts.





cou um brusco aumento, com pico cerca do ano 2000. Desde então, a incidência voltou a descer, atingindo 40,3 por 100 000 habi-tantes em 2008.A Rede de Laboratórios de TB na Letónia é composta pelo Laboratório Nacional de Refe-rência (LNR), em Riga, 5 laboratórios regio-nais para trabalho de cultura e microscopia e ainda vários outros laboratórios (em hospitais distritais, policlínicas, laboratórios privados) que proporcionam microscopia de esfregaço para pesquisa de bacilos ácido-álcool resisten-tes (BAAR) a nível PHC em cada um dos 26 distritos.Todos os centros de microscopia são audita-dos e certificados com base em padrões inter-nacionais (ISO 17025, ISO 15189). O con-trolo de qualidade interno é feito por coloração e microscopia de esfregaços positivos e negati-vos conhecidos. Os laboratórios de cultura fazem descontaminação de amostras e de cul-turas. As culturas são enviadas para o LNR para identificação e testes de suscetibilidade a fármacos. O laboratório nacional de referên-cia realiza microscopia de esfregaços, cultura de amostras, testes de susceptibilidade para fármacos de 1.ª linha (+ Kanamicina) e TSF de 2ª linha para estirpes de MDR -TB.Além disso, o LNR efectua o controlo da qualidade externo de todos os laboratórios, duas vezes por ano, utilizando um painel de 5 esfregaços fixados. Com este fim, 5 lâmi-nas preparadas a partir de expetoração de doentes, contendo amostras negativas e po-sitivas, são fornecidas aos laboratórios, que processam e interpretam as lâminas, regis-tam os resultados e enviam -nos ao LNR juntamente com as respectivas lâminas. Fi-nalmente, o LNR reexamina as lâminas, avalia a coloração e os resultados e emite re-latórios sobre o desempenho. Este LNR

All microscopy centers are audited and cer-tified based on international standards for testing and medical laboratories (ISO 17025, ISO 15189). Internal Quality Con-trol is performed by staining and microsco-py of known positive and negative smears. The culture laboratories perform decon-tamination of specimens and culture. Cul-tures will be sent to NRL for identification and drug susceptibility tests (DST). The National Reference Laboratory performs smear microscopy, culture of specimens, DST for 1st line drugs (+ Kanamycin), and 2nd line DST for MDR-TB strains.Furthermore, the NRL provides the external quality assurance for all laboratories by a panel of 5 fixed smears twice a year. For this, 5 slides prepared from patient sputum, com-prising negative, scanty, and positive speci-mens, are provided to the laboratories. These stain and read the slides, record the results and send back results and slides to NRL. Fi-nally, the NRL rereads the slides, assesses staining and results, and issues reports on performance. Furthermore, the NRL moni-tors the quality of sputum smear microscopy procedure during supervisory visits.For EQA of the performance of the culture, 2 specimens are sent to each lab twice a year. In detail, each specimen is divided into two parts, one part is decontaminated and incu-bated at NRL, and the other is sent to the culture laboratory. The culture laboratory performs decontamination, inoculation, in-cubation, record of results and sends the re-sults back to the NRL. Beside this, the NRL monitors the quality of culture procedures during supervisory visits.The NRL itself yearly participated in an ex-ternal proficiency programme for 1st line DST provided by in Swedish Institute for





também controla a qualidade do procedi-mento da microscopia dos esfregaços de ex-petoração durante visitas de inspecção.Para a AEQ do desempenho da cultura, 2 amostras são enviadas a cada laboratório duas vezes por ano. Cada amostra é dividida em duas partes, sendo uma parte desconta-minada e incubada no LNR e a outra envia-da para o laboratório de cultura. Este proce-de à descontaminação, inoculação, incubação e registo dos resultados, que envia ao LNR. Além disto, o LNR também controla a qua-lidade dos procedimentos de cultura durante visitas de inspecção.O próprio LNR participou anualmente num programa externo de proficiência para TSF de 1.ª linha disponibilizado pelo Insti-tuto Sueco para o Controlo de Doenças In-fecciosas. A partir de 2008, o LNR passou a ser o Laboratório Supranacional de Referên-cia, tomando, assim, parte nas atividades AEQ anuais para TSF de 1.ª e 2.ª linha.

A implementação do controlo da qualidade como requisito para a introdução de um novo teste de diagnósticoA integração de laboratórios de saúde pública é um dos principais alvos na luta contra as do-enças infeciosas. O controlo da qualidade do procedimento dos testes é um requisito essen-cial para a obtenção de resultados úteis e fiá-veis. O controlo da qualidade compreende o controlo interno da qualidade e a avaliação ex-terna da qualidade. Para a integração de novas técnicas moleculares num laboratório de TB, o controlo interno da qualidade compreende vários aspectos, como uma infraestrutura ela-borada, utilização e manutenção adequadas do equipamento e orientação nos procedi-

Infectious Diseases Control. Since 2008 the NRL itself became Supranational Reference Laboratory and thus taking part in the year-ly EQA rounds for 1st and 2nd line DST.

The implementation a of quality assurance as prerequisite for the introduction of a new diagnostic test.The integration of public health laborato-ries is one of the main targets in fighting emerging infectious diseases. Quality assu-rance of the test procedures is a prerequisite to obtain reliable and useful results. Quality assurance is composed of internal quality control and external quality assessment. For the integration of new molecular techniques into a TB laboratory, internal quality con-trol comprises several aspects, like elaborate infrastructure, equipment that is well main-tained and operated and procedures guided by efficient SOPs. Test reagents need to be controlled with every LOT, its integrity needs to be checked for every shipment and proper transport and storage has to be con-trolled. The test procedures itself require several quality control steps, like contami-nation controls, negative controls at differ-ent levels of the test process, the correct in-terpretation of adequate and inadequate runs, and the implementation of certain quality indicators. Furthermore, personnel need to be trained and re-trained.External proficiency testing can be conduc-ted by analysis of duplicate specimens from one patient. As example, proficiency testing for line probe assays was performed in four laboratories in India as prerequisite for the implementation of the new test. The advan-tage of panel testing for external quality





mentos por SOP eficientes. Os reagentes têm de ser controlados com cada LOT, a sua inte-gridade verificada em cada remessa e o trans-porte e armazenagem devidamente fiscaliza-dos. O processamento dos testes envolve vários passos de controlo da qualidade, como con-trolo de contaminação, controlos negativos em diferentes níveis do processo, interpretação correta das sequências adequadas e inadequa-das e implementação de certos indicadores de qualidade. Há ainda que treinar o pessoal e proporcionar a sua atualização contínua.Os testes externos de proficiência podem ser efetuados pela análise de duplicados de amostras de um doente. Como exemplo, os testes de proficiência para ensaios line probe foram realizados em quatro laboratórios, na Índia, um requisito essencial para a imple-mentação dos novos testes. A vantagem dos testes de painel para controlo externo da qualidade consiste na revisão de todo o pro-cedimento, no uso direcionado de estirpes selecionadas com determinadas resistências, com mutações comuns e incomuns, bem como na utilização de espécies de MNT. Há, no entanto, que ter em conta o custo dos testes de painel (incluindo preparação e expedição). O envio de resultados digitali-zados para interpretação, bem como para reverificação, pode constituir uma alternati-va menos dispendiosa para AEQ dos ensaios line probe, devendo a melhor solução ser apreciada para cada situação.Foram oradores: Elvira Richter, NRL, Bors-tel, Alemanha; Girts Skenders, State Agency of TB and Lung Disease, Letónia; Akos So-moskövy, FIND, Geneva, Suissa.O simpósio foi apoiado por INSTAND e.V. Society for Research Promotion of Quality As-surance in Medical Laboratories, WHO Colla-borating Centre, Düsseldorf, Alemanha.

control is the revision of the entire proce-dure, the directed use of selected strains with certain resistances, with common and uncommon mutations, as well as the use of NTM species. However, costs of panel tests (including preparation and shipment) have to be taken into account. Cheaper alterna-tives for EQA of line probe assays may be achieved by sending of digitized results for interpretation as well as for re-checking. The best solution has to be found for diffe-rent situations.The speakers were: Elvira Richter, NRL, Borstel, Germany; Girts Skenders, State Agency of TB and Lung Disease, Latvia; Akos Somoskövy, FIND, Geneva, Switzer-land.The symposium was sponsored by IN-STAND e.V., Society for Research Promo-tion of Quality Assurance in Medical Labo-ratories, WHO Collaborating Centre, Düsseldorf, Germany.





Normas de PublicaçãoInstructions for Authors

Normas de publicação na Revista Portuguesa de Pneumologia

Portuguese Journal of PulmonologyInstructions for authors

A Revista Portuguesa de Pneumologia considera para publicação trabalhos (artigos originais, de revisão, de actualização, casos clínicos, cartas ao editor, resumos críticos a livros, etc.) relacionados directa ou indirec-tamente com o Aparelho Respiratório.As opiniões expressas são da exclusiva responsabilida-de dos autores.

Os artigos publicados ficarão propriedade da Re-vista Portuguesa de Pneumologia, não podendo ser reproduzidos, no todo ou em parte, sem autoriza-ção do editor.

A aceitação dos originais enviados para publicação é condicionada à avaliação pelo Conselho Científico da Revista. Nesta avaliação os artigos poderão ser:

a) aceites sem alterações;b) aceites após as modificações propostas e aceites pe-

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Apresentação dos trabalhos – Os textos devem ser escritos em português, dactilografados, com margens largas (25 mm), a dois espaços, numa só face do papel e em três exemplares com as páginas numeradas no canto superior direito.

Solicita-se a todos os autores que enviem artigos para publicação que o façam acompanhados do respecti-

Revista Portuguesa de Pneumologia / The Portuguese Journal of Pulmonology publishes papers (original ar-ticles, revised articles, updated articles, case reports, letters to the editor, book reviews, etc) which are di-rectly or indirectly related to the respiratory system.The opinions expressed are the exclusive responsibili-ty of the authors.

The articles published are the intellectual property of Revista Portuguesa de Pneumologia/ The Portu-guese Journal of Pulmonology and may not be re-produced, in part or in whole, without permission from the editor.

All submissions are subject to a screening process by the Journal’s Scientific Board. There are three asses-sments possible:

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All articles submitted for publication must be sent in addition in a computerised format. The compu-




vo suporte magnético, que indiquem o programa de computador em que foram executados e que te-nham em atenção à reprodução das imagens (que deverá ser feita, idealmente, em suporte JPG ou TIFF) de modo a que fiquem nítidas na sua impres-são tipográfica.

Chama-se a atenção que a transcrição de imagens, qua-dros ou gráficos de outras publicações deverá ter a pré-via autorização dos respectivos editores para dar cum-primento às normas que regem os direitos de autor.Poder-se-ão considerar para publicação artigos redigi-dos em inglês. Neste caso, deve incluir-se o resumo, o título e as palavras-chave, também em português.

Deverão ser referenciados, pelos próprios autores, como artigos originais, de revisão, cartas ao editor, ou outros.

Todos os artigos originais serão também publicados em inglês, após retroversão para esta língua, pela(s) tradutora(s) da Revista Portuguesa de Pneumologia. Caso os autores assim o entendam, poderão enviar os artigos já traduzidos.

Estrutura – Sempre que possível será adoptado o es-quema convencional em que se iniciará cada parte do trabalho numa nova página pela seguinte ordem:

a) Na primeira página: – título do trabalho em português e inglêsb) Na segunda página:

– o nome dos autores com os respectivos títulos académicos e/ou profissionais;

– os serviços onde foi realizado, nome dos seus di-rectores e os respectivos endereços.

c) Na(s) página(s) seguinte(s):– o resumo em português que não deverá ultrapas-

sar 250 palavras para os trabalhos originais e de 150 para os casos clínicos;

ter program used must be clearly stated, and particu-lar attention must be paid to the reproduction of images, which should ideally be in JPG or TIFF for-mat, to give a clear printed quality.

If an image, figure or graph has been previously pu-blished, written permission from the editor in ques-tion must be submitted to safeguard the author’s in-tellectual property rights.

Articles may also be submitted in English. In this case, the summary, the title and key words should also be submitted in Portuguese.

The authores should categorise their submissions as original articles, revised articles, case studies, letters to the editor, technical notes, etc.

All original articles shall be also printed in English, after being translated by the Revista Portuguesa de Pneumologia / The Portuguese Journal of Pulmonology´s translators. Authors may submit their articles already translated, if they so wish.

Form – As far as possible, the convention will be ob-served of beginning each new part of the paper on a new page and in the following order:

a) Title page: – the full title of the paper in Portuguese and

English;b) Second page:

– the full names of the authors and their academic and /or professional titles;

– the full name and address of the institution(s) at which the work was carried out and the full name of the institution(s) director(s).

c) Following page(s):– a summary in Portuguese, not exceeding 250

words for original articles and 150 for case reports;

NORMAS DE PUBLICAÇÃO/INSTRUCTIONS FOR AUTHORS




– os resumos em inglês com características idênti-cas ao do inicial em português;

– as palavras-chave, em português e inglês (3 a 10), que servirão de base à indexação do artigo, de acordo com a terminologia do Index Medi-cus “Medical Subject Headings”.

d) O texto que, no caso dos artigos originais, terá em geral: Introdução, Material e Métodos, Resulta-dos, Discussão e Conclusões

e) O texto, também em inglês, tratando-se de um artigo original, e caso o(s) autor(es) assim o entendam fazer

f ) Agradecimentosg) Bibliografiah) Quadros e Figuras.

Bibliografia – As referências bibliográficas devem ser numeradas por ordem consecutiva da sua primeira citação no texto. Devem ser identificadas no texto com números árabes. As referências devem conter, no caso das revistas, o nome do primeiro autor (apelido e nome), seguido dos restantes, do título do artigo, do nome da publicação e da sua identificação (ano, volume e páginas).

Quadros e figuras – Os quadros e figuras devem ser apresentados em páginas separadas, em condições de reprodução. Devem ser acompanhados da respectiva le-genda em página à parte, mencionando no verso a lápis o número de ordem. Todos os gráficos deverão ser apre-sentados através de fotografia do respectivo original.

Modificações e revisões – No caso da aceitação do ar-tigo ser condicionada a modificações, estas devem ser realizadas pelos autores no prazo máximo de vinte dias.

As provas tipográficas serão da responsabilidade da Redacção, se os autores não indicarem o contrário. Neste caso elas deverão ser feitas no prazo determina-do pela Redacção, em função das necessidades edito-riais da Revista.

– a summary in English, with identical characte-ristics to that in Portuguese;

– between three and 10 key words in Portuguese and English, which will be used to index the ar-ticle, using terms from the Medical Subject Hea-dings list of the Index Medicus.

d) Original articles shall include, as a general rule, the following: Introduction, Subjects and Metho-ds, Results, Discussion and Conclusions.

e) Original articles may be written in English if the authors so wish.

f ) Acknowledgments.g) References.h) Tables and Figures.

References – The bibliographical references should be numbered consecutively, in the order in which they are cited in the text and should be identified in the text with Arabic numerals. Each reference to a journal article should contain the surname and initial of the first author followed by the rest. These should be followed by the ti-tle of the article, the title of the publication and its iden-tifying years, volume number and the page numbers.

Tables and figures – Each table and figure should be on a separate page, and in such conditions as to be re-produced. Each table and figure should have its own brief description on a separate page and bear its sequen-ce number on its back, written in pencil. Each table and figure should be represented via a copy of the original.

Alterations and changes – Should an article be ac-cepted for publication subject to alteration, these must be made by the authors within a twenty day period.

Publishing proofs – These are the editorial board’s responsibility, unless the authors state otherwise. Should this latter be the case, the proofs should be concluded within a deadline set by the editorial bo-ard, in line with the editorial norms of the Journal.





Cartas ao editor – Devem constituir um comentário crítico a um artigo da Revista ou uma pequena nota sobre um tema ou caso clínico. Não devem exceder as 500 palavras, nem conter mais de um quadro ou figu-ra e um máximo de 6 referências bibliográficas. As respostas do(s) autor(es) devem obedecer às mesmas características.

Pedido de publicação – Os trabalhos deverão ser en-viados à Redacção, em nome do editor, para a sede da SPP, Rua Ivone Silva, n.º 6 – 6.º Esq., Edifício Arcis, 1069-130 Lisboa, Portugal, acompanhados de uma carta com pedido de publicação, subscrito por todos os autores, indicação da cedência do copyright e que não foram publicados ou enviados para publicação em outra revista nacional ou estrangeira. Não serão aceites trabalhos já publicados ou enviados simul-taneamente a outras revistas.

Deverão ser acompanhados pelo endereço electró-nico do autor principal para eventuais pedidos de esclarecimentos por parte da Redacção.

Os trabalhos também poderão ser enviados por via electrónica (e-mail: [email protected]).

Nota final – Para um mais completo esclarecimento sobre este assunto aconselha-se a leitura dos requisi-tos do International Commitee of Medical Journal Edi-tors, publicados na integra no N Engl J Med 1991; 324:424-428.

Letters to the editor – These should contain a criti-cal appraisal of a Journal article or a small comment on a theme or a case study. They should not exceed 500 words, contain more than one table or figure and have a maximum of 6 bibliographical references. Au-thors’ replies should observe these same norms.

Submissions for publication – Papers should be sent to the editorial board, addressed to the editor: Sociedade Portuguesa Pneumologia office: SPP – Rua Ivone Silva, n.º 6 – 6.º Esq., Edifício Arcis, 1069-130 Lisboa, Portu-gal. Submissions should be accompanied by a letter re-questing that the work be submitted for publication, signed by all of the authors, stating that they waive their intellectual property rights and that they work has not be published or submitted for publication in any other Por-tuguese of international journal. Work already publi-shed or already sent to other journals will be rejected.

Submissions must bear the e-mail address of the corresponding author in order to facilitate contact with the editorial team should any clarification be necessary.

Papers may should be sent via electronic mail to:[email protected]

Final note – For a fuller clarification of this matter, a reading of the requirements of the International Com-mittee of Medical Journal Editors, published in full in the N Engl J Med 1991; 324:424-428, is advised.



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