diagnostic microbiologique de la tuberculose: … · diagnostic microbiologique de la tuberculose:...

Diagnostic microbiologique

de la tuberculose:

nouveaux tests, nouveaux

algorithmes et pièges à

éviter

Dr Onya Opota, PhD, FAMH

Dre Jaton Katia, PhD, FAMH, Cheffe de Laboratoire

Laboratoire de Diagnostic Moléculaire et Mycobactéries

Institut de Microbiologie, CHUV

ARL-Yverdon le 23.03.2017

No conflict of interest

Onya Opota & Jaton Katia 23.03.2017

Transmissible disease: Infection due to the inhalation of droplet

nuclei (airborne particles 1 to 5 microns in diameter) which production is

facilitated by coughing and singing

infectious dose <10 cells

TB Disease

Adapted from Small PM, Fujiwara PI. Management of tuberculosis in the united states. The New England journal of medicine. 2001; 345: 189-200.

Immunité cellulaire et hypersensibilité tissulaire

BK+++Age < 4 years oldImmunodepressionPoor sanitary conditions Malnutrition

Transmission of tuberculosis and progression from latent infection to reactivated disease

TB Disease

Pulmonary and extra-pulmonary tuberculosis

Meningitis

lymph node

Bone and joint

TB Disease

http://coursewareobjects.elsevier.com/objects/elr/ExpertConsult/Mandell/infectiousdiseases7e/IC/images/0250009.jpg






Plan

I- Issue and challenge of TB diagnosis

II- Microbiology: New tests-New paradigms

III- Avoiding pitfalls

IV- Future of TB diagnosis

Suspicion of pulmonary TB

PREVENT THE SPREAD OF THE DISEASE

SEVERE (Lethal) DISEASE

LONG, SIDE-EFFECTS,

RESISTANCE

Healthcare workersLaboratory workers

PatientsVisitorsFamilly

Etc...

1. Patient management

2. Transmission control

DIAGNOSTIC:Rapid

Sensitive & SpecificAntibiotic

susceptibility

H


II- Microbiology: New tests-New

paradigms


IV- Vision on the direction of TB

diagnosis

Issue and challenge of TB diagnosis

Based on:Clinical presentation and epidemiologyImagery ImmunologyMicrobiology

Microscopy (on respiratory sample)

CulturePCR

5’000-10’000 CFU/ml, Same day result

10-100 CFU/ml, 6-8 weeks

100-1’000 CFU/ml, Same day result



paradigms



diagnosis

Issue and challenge of TB diagnosis

Historical approach

Microscopy Culture

SensitivityTime to result- +

5’000-10’000

CFU/ml

10-100

CFU/ml

GOLD STANDARD

<1hour 6-8 weeks

Hazardous (BSL3)Risk of contamination

Initiate TB diagnosis

Address patients’

infectiousness



paradigms



diagnosis

Sputum smear examination

AFB* negative AFB positive

tuberculosis suspicion tuberculosis suspicion

low infective potential high infective potential

2 x AFB negative3 x AFB negative

*AFB=Acid Fast Bacilli

Patients suspected for tuberculosis are placed in negative pressure isolation room until 3 consecutive sputum smear examinations are negative



paradigms



diagnosis

Limits of smear examination

M. tuberculosis complex (MTBC):M. tuberculosisM. africanumM. bovisBCG (Bacillus Calmette–Guérin)

M. microtiM. canettiiM. capraeM. pinnipediiM. suricattaeM. mungi

Non tuberculosis mycobacteria (NTM):M. abscessusM. chelonaeM. fortuitumM. aviumM. intracellulareM. kansasiiM. marinumM. szulgaiM. genavenseM. haemophilumM. ulceransM. scrofulaceumM. xenopi

Aerobic ActinomycetesNocardia sp.Rhodococcus sp…Endospores

Non bacterialNuclear inclusion bodies Some parasites- Cryptosporidium parvum-Taenia eggs- Echinococcus spFungal (Some yeast forms…)

Possible other acid-fast structures

Peptidoglycan Peptidoglycan

Peptidoglycan

Peptidoglycan

Mycolic acid

Principle:Persistence of staining after acid-alcooldecolorization

Limited sensitivity and specificity

Limits of smear examinationI- Issue and challenge of TB diagnosis


paradigms



diagnosis

So far the gold standard

Solide medium (Löwenstein)12 weeks, rough colonies

Liquid medium in automated

systems (Mgit)6-8 weeks

Limit of detection 10-100

cfu/ml



paradigms



diagnosis

Culture: Gold standard

Microscopy PCR Culture


GOLD STANDARD

5’000-10’000

CFU/ml

100-1’000

CFU/ml

10-100

CFU/ml

<1hour 2-24hours 6-8 weeks

Hazardous (BSL3)Risk of contamination


Address patients’

infectiousness



paradigms



diagnosis

PCR

2 mains NAAT (Nucleic Acid Amplification Technologies) approaches

Homemade platformSpecific detection of Mtbc

Sensitivity +++

Batches, High throughput

+/- Coast effective

Specialized lab

TAT 4-24h, opening hours

Greub G, Sahli R, Brouillet R, Jaton K. Ten years of r&dand full automation in molecular diagnosis. Future microbiology. 2016; 11: 403-425.



paradigms



diagnosis

PCR

Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, KrappF, et al. Rapid molecular detection of tuberculosis and rifampinresistance. The New England journal of medicine. 2010; 363: 1005-1015.

Drancourt M, Michel-Lepage A, Boyer S, Raoult D. The point-of-care laboratory in clinical microbiology. Clinical microbiology reviews. 2016; 29: 429-447.

POCT (Point of Care Test)Xpert MTB/RIF (Cepheid)

Specific detection of Mtbc

Sensitivity +++

Detection of resistance marker (rpoB)

TAT 2h, emergency, WE

Limit of detection 100-1000 DNA

copies/ml

Microbiology: New tests-New paradigms???

IDSA :

- recommends that 3 AFB smear microscopy be performed, rather than no AFB smear microscopy, in all patients suspected of having pulmonary TB (strong recommendation, moderate-quality evidence)

- suggests that both liquid and solid mycobacterial cultures be performed, rather than either culture method alone, for every specimen obtained from an individual with suspected TB disease (conditional recommendation, low-quality evidence).

- suggests performing a diagnostic NAAT, rather than not performing a NAAT, on the initial respiratory specimen from patients suspected of having pulmonary TB (conditional recommendation, low-quality evidence).

Lewinsohn DM, Leonard MK, LoBue PA, Cohn DL, Daley CL, Desmond E, et al. Official american thoracic society/infectious diseases society of america/centers for disease control and prevention clinical practice guidelines: Diagnosis of tuberculosis in adults and children. Clinical infectious diseases.2017; 64: 111-115.



paradigms



diagnosis



GOLD STANDARD

5’000-10’000

CFU/ml

100-1’000

CFU/ml

10-100

CFU/ml

<1hour 2hours 6-8 weeks


Address patients’

infectiousness

Xpert MTB/RIF (Cepheid)

all inclusive<2 hours

131* CFU/ml

Microbiology: New tests-New paradigms???I- Issue and challenge of TB diagnosis


paradigms



diagnosis

1954: Infectivity of pulmonary tuberculosis in relation to sputum status. Shaw, J. B. and Wynn-Williams, N., Am Rev Tuberc. May;69(5):724-32.

1957: The infectiousness of human tuberculosis; an epidemiological investigation. Hertzberg G., Actatuberculosea Scandinavica. Supplementum38: pg 1-146.

1960: The influence of the number of bacilli on the development of tuberculous disease in children.Van Zwanenberg D., The American review of respiratory disease July 1st,.

Smear status (positive/negative) is historically used to determine the infectivity potential of TB patients

International guidelines: Patients with positive smear examination are the most infectious and require airborne isolation

Smear negative patients can also transmit TB!

% of people infected by smear-negative patients

Methods:1574 TB patients (culture-positive) from San Francisco.Strains clustering determined by DNA fingerprints (RFLP).Results: The AFB smear identifies the most infectious patients~17% of tuberculosis transmission due to AFB negative sputum smears patients.

Behr MA, Warren SA, Salamon H, Hopewell PC, Ponce de Leon A, Daley CL, et al. Transmission of mycobacterium tuberculosis from patients smear-negative for acid-fast bacilli. Lancet. 1999; 353: 444-449.

Infective potential of smear negative patients

Methods:

Guinea pigs exposed to air from a tuberculosis

ward, Lima, Peru 2007

Results:

transmission from AFB positive patients > AFB

negative patients

... not statistically significant

Microscopy PCR

PCR rather than microscopy to address

patients infective potential

?

CULTURE


GOLD STANDARD

5’000-10’000

CFU/ml

100-1’000

CFU/ml

10-100

CFU/ml



Address patients’

infectiousness

Xpert MTB/RIF (Cepheid)

all inclusive<2 hours

131* CFU/ml



paradigms



diagnosis

1) Determine the performances of microscopy and Xpert MTB/Rif for TB diagnosis, culture as gold standard.

2) Compare the semi-quantitative results of microscopy and Xpert MTB/Rif in order to find a correlation between the two tests.



paradigms



diagnosis

Study IMU-CHUV:

Opota O, Senn L, Prod'hom G, Mazza-Stalder J, Tissot F, Greub G, and Jaton K. Added value of molecular assay xpertmtb/rif compared to sputum smear microscopy to assess the risk of tuberculosis transmission in a low-prevalence country. Clinical microbiology and infection . 2016; 22: 613-619.



paradigms



diagnosis

1. Negative GX 100% Negative smear: corresponds to a limited risk of

transmission.

2. Smear positive samples are all GX positive (100%).

3. GX positive medium/high corresponds to a high risk of transmission

(~100% smear positive).

4. For patients with positive GX low/very-low an evaluation of the clinical

chart is necessary to decide whether airborne isolation is needed.

Distribution of Xpert MTB/RIF semi-quantitative results according to smear microscopy results

Smear microscopy result

negative scanty 1+ 2+ 3+

Total

smear

positive(a)

Total

Prediction of smear

positivity (95% CI)

Xpert

MTB/Rif

Positive high 0 1 1 1 3 6 6PPV 100%

(54.1-100)

Positive

medium1* 6 14 5 2 27 28

PPV 95.4%

(81.6-99.9)

Positive low 11 7 3 2 0 12 23PPV 52.2%

(30.6-73.2)

Positive very

low8 - 1 - - 1 9

PPV 11.1%

(0.3-48.2)

Negative 166 8§ 2§ 0 0 0 176NPV(a) = 100%

(97.8-100)

+

-

Transmission potential

Opota O, Senn L, Prod'hom G, Mazza-Stalder J, Tissot F, Greub G, and Jaton K. Added value of molecular assay xpertmtb/rif compared to sputum smear microscopy to assess the risk of tuberculosis transmission in a low-prevalence country. Clinical microbiology and infection . 2016; 22: 613-619.



paradigms



diagnosis

Opota O, Senn L, Prod'hom G, Mazza-Stalder J, Tissot F, Greub G, and Jaton K. Added value of molecular assay xpert mtb/rif compared to sputum smear microscopy to assess the risk of tuberculosis transmission in a low-prevalence country. Clinical microbiology and infection . 2016; 22: 613-619.



paradigms



diagnosis

New algorithm: smear microscopy independentalgorithm of TB microbial diagnosis



paradigms



diagnosis

Opota O, Senn L, Prod'hom G, Mazza-Stalder J, Tissot F, Greub G, and Jaton K. Added value of molecular assay xpert mtb/rif compared to sputum smear microscopy to assess the risk of tuberculosis transmission in a low-prevalence country. Clinical microbiology and infection . 2016; 22: 613-619.

Not anymore in emergency!

Emergency (24/7h)

Microbiology: New tests-New paradigmsI- Issue and challenge of TB diagnosis


paradigms



diagnosis



III- Avoiding pitfallsFalse negativesFalse positives


False Negative



5’000-10’000

CFU/ml

100-1’000

CFU/ml

10-100

CFU/ml


A negative PCR does not exclude a TB

Decrease sensitivity of Xpert for pleural fluid!

Note recommended for extra pulmonary TB

1st analysis

2nd analysis

3rd analysis

53.8%

64.1%

66-70%

92.2%

96.0%

97.6%

Opota O, Senn L, Prod'hom G, Mazza-Stalder J, Tissot F, Greub G and Jaton K. Added value of molecular assay xpert mtb/rifcompared to sputum smear microscopy to assess the risk of tuberculosis transmission in a low-prevalence country. Clinicalmicrobiology and infection . 2016; 22: 613-619.

Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, Krapp F, et al. Rapid molecular detection of tuberculosis andrifampin resistance. The New England journal of medicine. 2010; 363: 1005-1015.

Lewinsohn DM, Leonard MK, LoBue PA, Cohn DL, Daley CL, Desmond E, et al. Official american thoracic society/infectiousdiseases society of america/centers for disease control and prevention clinical practice guidelines: Diagnosis of tuberculosisin adults and children. Clinical infectious diseases.2017; 64: 111-115.



paradigms



diagnosis

Homemade platformsLaboratory practices compatible with molecular diagnosis

Order of sample processing (for NAATs & Culture)

NAATs analysis performed in triplicate

Investigation of weak positive (low copy number, 1/3 reaction)

Clinic and pre-test probability

POCT (Point of Care Test)Molecular diagnostic test!

(Sample preparation in BSL2)

Greub G, Sahli R, Brouillet R, Jaton K. Ten years of r&d and full automation in molecular diagnosis. Future microbiology. 2016; 11: 403-425.

False Positive (TB detection)I- Issue and challenge of TB diagnosis


paradigms



diagnosis

Xpert MTB/RIF

Low prevalence of MDR TB (low pre-test probability)

Technological issue

Increased risk of false positive resistance detection

Proven for Xpert “positive very-low”

Need confirmation by sequencing

Ocheretina O, Byrt E, Mabou MM, Royal-Mardi G, Merveille YM, Rouzier V, et al. False-positive rifampicin resistant results with Xpert MTB/RIF version 4 assay in clinical samples with a low bacterial load. Diagnostic microbiology and infectious disease. 2016; 85: 53-55.

False Positive (Resistance detection)I- Issue and challenge of TB diagnosis


paradigms



diagnosis

False Positive (Resistance detection)I- Issue and challenge of TB diagnosis


paradigms



diagnosis

Conclusions (1)

NAATs (PCR) improve and accelerate the diagnosis of active TB

New paradigms are emerging:

Smear independent diagnosis

Definition of transmission potential

Xpert MTB/RIF design for high prevalence regions Caution when the pre-test probability is low

Added value for patient management

Cost effectiveness of POCT (community, hospital, patient)





Will we still need culture?I- Issue and challenge of TB diagnosis


paradigms



diagnosis

1. Issue of the limit of detection of PCR

Microscopy PCR CULTURE


5’000-10’000

CFU/ml

100-1’000

CFU/ml

10-100

CFU/ml



paradigms



diagnosis

Can be improved by targeting multiple

copy genes or mRNA

2. Antibiotic susceptibility

Whole genome sequencingAlready reliable on positive culture

Walker TM, Merker M, Kohl TA, Crook DW, Niemann S, Peto TE. Whole genome sequencing for m/xdr tuberculosis surveillance and for resistance testing. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 2017; 23: 161-166.

Precise correlation to the MICs for a defined strain (personalized medicine)

Walker TM, Kohl TA, Omar SV, Hedge J, Del Ojo Elias C, Bradley P, et al. Whole-genome sequencing for prediction of mycobacterium tuberculosis drug susceptibility and resistance: A retrospective cohort study. The Lancet Infectious diseases. 2015; 15: 1193-1202.



paradigms



diagnosis

3. Treatment follow-up? patients with previous TB?

DNA can be detected several months after a successfultreatmentNo clear cutoff of DNA quantity to distinguish re-infection/re-activation versus DNA detection in patients withprevious tuberculosis



paradigms



diagnosis

Theron G, Venter R, Calligaro G, Smith L, Limberis J, Meldau R, et al. Xpert mtb/rif results in patients withprevious tuberculosis: Can we distinguish true from false positive results? Clinical infectious diseases. 2016;62: 995-1001.

3. Treatment follow-up? patients with previous TB?

Proof of concept of the utility of PET-CT andmRNA detection for the follow up of TBtreatment



paradigms



diagnosis

Malherbe ST, Shenai S, Ronacher K, Loxton AG,Dolganov G, Kriel M, et al. Persisting positron emissiontomography lesion activity and mycobacteriumtuberculosis mRNA after tuberculosis cure. Nat Med.2016; 22: 1094-1100.



paradigms



diagnosis

Walker TM, Merker M, Kohl TA, Crook DW, Niemann S, Peto TE. Whole genome sequencing for m/xdr tuberculosis surveillance and for resistance testing. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 2017; 23: 161-166.

Conclusions (2/2)

TB is still there...

Importance of the pre-test probability

rather than prevalence

Immune status

Age

Travel

Toward culture independent diagnostic?

Acknowledgements

&

The Laboratory of Tuberculosis Diagnosis

The Diagnostic Microbiology Laboratory of the

Lausanne University Hospital.

Dre Katia Jaton

René Brouillet

Prof. Gilbert Greub

Dr Guy Prod’hom

Dre Laurence Senn

Dre Jessica Stalder

Dr Frédéric Tissot

Thank you

diagnostic microbiologique de la tuberculose: … · diagnostic microbiologique de la tuberculose:...

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