‘’ THE WHITE PLAGUE OF EUROPE’’17 & 18 CENTURY

23/04/2013Updated may 2015

HISTORYNEOLITH AGESEGYPTIAN MUMMIESROBERT KOCH ISOLATED IN 1882SENITORIASTREPTOMYCIN 1946ISONIAZID 1952

Member of Mycobacterium tuberculosis Complex

1.M tuberculosis2.M bovis3.M BCG4.M ulcerans5.M africanum

MORPHOLOGYBACILLUS : rod shaped, non- spore bearing 2-4um *0.2-0.5 umObligate Aerobe, growth enhanced by CO2Slow growingCarbon compds; oxidation & energyHigh lipid content of wall hydrophobicResistant to oral fluids

MAJOR HEALTH PROBLEMWHO 2008:1/3 population effected8.8 million new infected cases1.6 million died in 2000Every second a case infectedBy end of 2014; 12.3 million smear positive cases & 150,000 MDR treated

WHO 2014 global reportt9 million developed tb;;6.1 million cases ; 5.7 new & 0.4 million already on TM

2014 report: 122 countries 56% SEA & pacific; ¼ Africa

India 24% & China 11%1.5 million died3,60000 had HIV tooON DECLINE:Diagnosis n therapy from 2000-2013MDR 2013; 3.5 %

PAKISTAN: GFATMFifth highest burden country with TBFourth highest burden with MDR tbWHO’s National Professional Officer for Tuberculosis Control Dr Ghulam Nabi Kazi pointed out that

2012 over 284,000 cases of Tb detected & put on tm

this year the figure would touch around 300,000.

around 420,000 new cases appeared every year.12 tertiary care hospitals involved in Pakistan in diagnosis & t/m

CELL STRUCTURE• Slow growing: generation time 15-20hrs• Intracellular parasite• CELL WALL: 60% lipids• 3 components: 1. MYCOLIC ACIDS: long chain FA C78- C90

Bound to proteins, polysaccharides• Alpha branched structure• 50% dry weight of cell envelope• Form hydrophobic shell around MTB

LIPID BENEFITSPERMEABILTY BARRIER FOR1. Stains & dyes2. resist antibiotics3. resist killing by acids & alkalis

4. resist osmotic lysis by complement

5. resist oxygen and survive intracellular killing by macrophages

VIRULENCENO ATTACK BY:• CATIONIC PROTEINS• LYSOZYME• OXYGEN IN PHAGOCYTIC GRANULES

EXTRACELLULAR: Complement not deposited

2.CORD FACTOR: abundant in virulent strains serpentine cords. parallel chains;

toxic to mammalian cells

inhibit PMN migraton

3 Wax D : Freund’s adjuvant

STAINING CHARACTER’SBASIC STAINS

Gram’s stain: not taken/weakly taken Ziehl Neelsen stain;Carbol fuchsin: not readily taken, pushed by heat

ACID FAST: once taken cannot be decolorized by alcohol. 95% alcohol + 3%HCL(ACID-ALCOHOL) decolourize all but mycobacteria

HOT ACIDS & Sonication: destroy acid fastness

Lipid effects on tissueMuramyl dipeptide + mycolic acid complex Granuloma formation

Phospholipds: Caseous necrosis:CORD FACTOR: trehalose 6,6’-dimycolate1. Inhibit migration of leukocytes 2. Chronic granulomas3. Immunologic adjuvantProteins: tuberculin reaction; elicit Anti bodies

CULTURE MEDIA(non/selective)Semisynthetic Agar medium.Middlebrook 7H10 & 11Contents: salts, cofactors + malachite green/ABglycerol albuminoleic acid catalase

Mjddlebrook 7H11; caesin hydrolysate as well

Large inocula needed in several weeksColonial morphology, sensitivity testing

INSPISSATED EGG MEDIALOWENSTEIN JENSEN MEDIA:Complex organic substances;

fresh egg, yolks. potato flour

SaltsGlycerolMalachite green; selectiveSmall innocula yield growth in 3-6 weeks

BROTH MEDIAMiddlebrook 7H9, 7H12Tweens: water -soluble esters of fatty acids

wet the surface, dispersed growth in liquid

Rapid growth PHYSICAL & CHEMICAL AGENTS;Resistant bacterium; hydrophobic nature, clumping

Pencillin, malachite green, acids, alkalis kill others

PATHOGENICITYM tuberculosisSusceptible ; Humans & guinae pigsResistant: fowl, cattle

M bovisSusceptible; cattle, humans

M KansasiiLesion like MTB

PATHOGENESISDroplet infection; 1-5um nuclei

family, hospital personnel. < 25umSpeak, cough, sneeze settle in alveoliCytokines, lymphokines stimulate monocytes, Primary infection of ALVEOLAR MACROPHAGES. Unrestrained intracellular proliferation in non immune alveolar macrophages

Lesions; 1-2 months in lungsHypersensitivity: polysaccharides

Disseminated infection via macrophages to LN& blood

In 3-8 weeks cell mediated immunity develops

Activated macrophages at infection sites

Granuloma formation

Ag-specific activation of CD4+ T cells, clonal expansion

IL2 secretion increased & IF -gamma

IL2 receptors expression increased

Activation of gamma interferon to kill M tuberculosis by macrophages

Population exposure; Asia, Africa, latin America

overcrowding, socioeconomic conditions medical care provisionGenetic predisposition HLA Bw15AgAge ; extremesMalnutrition, alcoholics, destituteCoexisting disease : diabetes, silicosis, AIDS

PATHOLOGYHOST FACTORSMTB factors….no; multiplication LESIONSExudative; like pneumonia Oedema, polys, mononuclears surround Mtb

Fate; 1 resolution LN; enlarge/ Gohn’s complex

2 necrosis TT; positive 3 .productive

GOHN’S COMPLEX L NODE; enlarged, Caseation, calcificationChildrenAdultsREACTIVATION; surviving Mtb multiply; APEX

chronic tissue lesion; tubercle, caseation. Fibrosis

LN: Less effect

REACTIVATIONLatent infection reactivatedPartial immunityMycobacterial Ag increased in tissues intense inflammatory response by monocytesType 4 hypersensitivity responseDense mononuclear infilterates cause tissue damage due to release of O2 radicles and neutral proteases

Tissue damage: Caseous necrosis followed by liquifactive necrosis in absence of treatment

PRODUCTIVE TYPEChronic Granuloma/tubercle; 3 zones1 central area: large multinucleate giant cells with Mtb; later caseous necrosis

2. Mid zone; Pale epithelliod cells; radially set

3 Peripheral zone; fibroblasts, lymphocytes, monoytes cells; later fibrosis

Tubercle; empty in bronchus; form cavity

heal by fibrosis & calcification

SPREADBlood; LN.tubercle erosion in vein

Lymphatics; LN; miliary tbBronchi… aspirated to lungsGIT; swallowed food, lung

INTRACELLULAR GROWTH. Resist chemothera1.MONOCYTES2.RES3.GIANT CELL

SYMPTOMSCOUGHSPUTUM… early tissue necrosisDysponea; late tissue necrosis ;

parenchymal damage of lung

Fever; IL1 systemic effects

Weight loss; TNF alpha; cachectin

X-ray: Apical cavitary lesion of lung

PPD positiveHITOPATHOLOGYGRANULOMASEpitheloid cells; activated macrophages

Giant cells; most successful host tissue response

Activated lympocytesTubercle/granulation tissue

TUBERCULIN TESTMATERIAL;OLDTUBERCULIN; 6 week growth in broth

Conc.filterate has tuberculoprotein

PPD; chemical fractionation of OT PPD-S; Siebert’s Lot no 49608 standardTU; Biological activity of sp.wt of PPD S

Dose; 1 TU, 5 TU, 250TUstandardize by comparative activity in humans

TUBERCULIN TEST5 TU O.I ml injected intracutaneousRead in 48-72 hrs10 mm or > Positive: 4-6 weeks of infectionOedema. induration, erythemaPersist many days; weak disappear Central necrosis may be seen

DIAGNOSISClinicalSpecimenSmearCultureMolecular probe Gene sequencing

Tuberculin testPositive: infection (4-6 weeks)/ or past BCG vaccinationNEGATIVE:No exposureAnergy;, AIDS, Hodgkins measles, immunosuppression,

sarcoidosis overwhelming TB,

LAB DIAGNOSISSputum: 3-5 morning specimen CULTURE; decontaminate/centrifugeEgg based medium; LJAgar & Broth based media:Middlebrook AFB; Z-N stainAuramine-rhodaminePCR; respiratory smear positive samples

NEW DIAGNOSTIC TECHNOLOGIES BACTEC RAPID RADIOMETRIC CULTURE SYSTEMDeveloped by Becton DickinsonLiquid medium with C14 labelled palmetic acid

Automated early detection growing Mycobacteria use acid and release C14

Growth detected in 12 daysExpensiveFacility should handle radioactive material

HIGH PERFORMANCE LIQUID CHROMATOGRAPHYREF LAB: SPECIATION of mycobacteriaMycolic acid profiles vary in speciesDNA detection:PCR; 99% specific, 55-90% sensitive

STRAIN; clinical/epidemilogical typing

gene seq 6110 targeted:

DNA frag generated by restriction endonuclease digestion,

separated by electrophoreses.

Probe against 6110 used

ID ID OF M TUBERCULOSIS CULTURES

1.CONFIRM BY ZN STAIN; difficult to emulsify; others easily emulsify2.PIGMENT: leave in light for 2hrs re-incubate at 370 C overnight non- chromogen in light/dark3. Incubation of s/c at 25 0 C; no growth4.Growth on LJ Medium with p Nitrobenzoic acid 500 ug/ml No growth

PCRDNA/RNA probes and amplification usedDNA & RNA sequences extracted from mycobacteria in sputum

EpensiveSpecialist training neededMYCODOT ANTI-BODY

20 minute immuno-assay

Pureified lipoarabinomannan; highly immunogenic

Detects AB in active disease ie high levels; not screening

BACTERIOPHAGE TEST FOR SPUTUMFAST PLAQUE ASSAY:Mix sputum with a reagent with bacteriophageMycophages infect mycobacteriaUse virucide to kill bacteria outside mycobacteria

Mycobacteriophages replicate inside mycobacteria, cause their lysis and released out

Other rapid growing non pathogenic mycobacteria are addded,infected by these phages.

Sample is incorporated in agar mixture, plated and incubated overnight at 37 0 c

Phages lyse mycobacteria, leaving zones of clearing ie holes

Thi proves pt sample had M TBsensitivity like culture: can detect 100 tubercle bacilli/ml

GLOBAL TB REPORT 201415 vaccines under trial10anti tb drugs undergoing clinical trials

4 month versus 6 month therapy duration2 approved delamanidBedaquilineTARGETS: 203595% reduction in deaths90% reduction in incidence

GAMMA INTERFERON RELEASE ASSAYMTB specific Ag: (not BCG/non myco tb) ESAT 6 CFP 10CD4 cells release gamma interferon in whole blood

ELISA ASSAYQuantiferon GoldT spot TB ; mononuclear cells used

conversionTo negative:Isoniazid T/MBCG VACCINE; + 3-7 yrs - elimination of viable Mtb

HEALTHY; after yrs negative retest: 2 weeks positive by boost inj

Molecular probes1 day to detect/ 2hrsRapid, Sensitive, SpecificMycobacterium 10,000 rRNA copies /cell; natural amplification, easy detection

Hybridization of DNA with rRNA sequences single strands separated from hybrids

DNA probes attached to chemicals that chemiluminescence in hybrids & measure by lumino- meter & equivalent to amount of hybrid

Probes for M tub complex & MAC

TREATMENTFIRST LINE DRUGSPRIMARY: daily 1—2 months; 9 biweeklyIsoniazidRifampinOTHERS:PyrazinmideEthambutalStreptomycin6 months regime ; 4 drugs 2 months; iso/rif biweekly

SECOND LINE DRUGSTOXIC; Used in T/M failure with first-line

KENAMYCINCAPREOMYCINETHIONAMIDECYCLOSERINEOFLOXACINCIPROFLOXACIN

DRUG RESISTANCEOne in 106 or 108 mutate spontaneouslySingle drugFirst line drugsCombinations used: 4 drug regimeIsoniazid, rifampine, pyrazinamide, ethambutal

Second line; toxic, less effective, bothReserved for special circumstances; t/m failure, MDR

riskfactors: HIV, Asian, Latin American migrants, MDR area,

Mechanisms of drug resistanceIsoniazid resistance:katG; deletion, mutation in catalase-peroxidase gene

no or less catalase functionInhA gene: enzyme encoding mycolic acid synthesis

Streptomycin resistance: mutation in genes coding

Ribosomal S12 protein, 16SrRNA, rpsl, rrs

MDR: isoniazid & rifampin resistance problem in T/M & control hospitals, prisons, HIV t/m of contacts after sensitivity test , 3 or > drugs

XDR: extensive drug resistance WHO defination R : INZ & PYR flouroquinolone

3 second line injectables

CAUSES 1. Poor infection control2. Ineffective T/M3. Diagnostics poorFATE:PoorMortality; 64%

FUNDS8 billion US dollars/ year needed for global full response to TB epidemic

2/3 for detection & tm of drug susceptible tb

20% MDR T/M10% rapid tests & diagnostics5% HIV associated tb activity

PREVENTION & CONTROL1.Public health control: t/m cases: prompt & effective with follow up

Contacts: diagnose{ tt, x ray} & treat 2.Asymptomatic cases: tt + Treat with drugs: children, immunosuppressed3. Host resistance of person: reactivate or convert asymptomatic to disease

Malnutrition, gastrectomy, infection, HIV, steroids4. BCG 5. Cattle eradication 6. milk pasteurization

Atypical mycobacteria

ANONYMOUS MYCOBACTERIAENVIRONMENTAL MYCOBACTERIAMYCOBACTERIA OTHER THAN TUBERCLE; MOTTFound in soil, waterCause opportunistic infection in HIV/Immunosuppressed

DIAGNOSTIC FEATURESCHROMOGENSSCOTOCHROMOGENS; Pigment in darkPHOTOCHROMOGENS; Pigment in lightNON-CHROMOGENSTEMPERATURE:Grow at 25 0 CGrow in presence of P nitrobenzoic acidGROWTHSlow growers; 2 rapid growers

Pul inf S/R 25 oC Pigm PNBMAC /MAI S + N/S +

M kansasi S + N/S +

M xenopi S + N/S +

M malmoense S + N +

M scrofulaceum S + S +

M chelonae R + N +

M fortuitum R + N +

TRADITIONAL RUNYON Classification of MYCOBACTETB COMPLEX M tuberculosis, M africanum, M bovis PHOTOCH; M kansassi,marinum, simiae,asiaticumSCOTOC; M gordonae,scrofulaceum,szulgi, flavescens

NON CHROM; MAC, Ulcerans, xenopi, trivale, terrae, malmoense,gastri, genavense, hemophilium, celatum

RAPID GROWERSM abscessus M phlei M immunogenum

M fortuitum M smegmatis M vaccae

M chelonae M mucogenicum

MAC MAIGrow optimally at 41 CSmooth, soft non pigmented colonies,Ubiqutous; water, soil, food, animals, birdsImmuno-competent humans affectedAIDS: Opportunistic esp <100/ul25-50% pts develop bacteremia, disseminated infection. Decrease with HAART and azithromycin

Tissue infilteration…organ dysfunction eg lung,skin, soft tissue, LN, bone, CNS

C/O fever,night sweat,abd pain, diarrhea, wt loss

M kansasiiPhotochromogen, complex media at 37CPulmonary and systemic disease like TB esp ICompro

t/m; Rifampin, ethambutol,isoniazidM scrofolacium; scotochromogen, waterAdults with chronic lung disease lymphadenitis in childrenM marinum/ulcerans: water at 31 C, fish infected; swimming pool granulomas

M chelonae-abscessus; rapid grower; skin, bone. soft tissue infection,rep tract, cystic fibrosis