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Mycobacterium tuberculosis AND BASIC TO DRUG

RESISTANCE

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BIOLOGICAL CHARACTER

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CLASIFICATION

HUMAN PATHOGEN

M tuberculosis

M bovis

M lepraeM avium-intracellulare

M kansasii

M scrofu laceum

M xenopi

M szulgai

M marinum

M ulcerans

M haemophi lum

M afr icanumM malmoense

M simiae

M asiaticum

M fortui tum-chelonae complex

M thermoresistible

OPPORTUNIS-SAPROFIT

M gordonae

M terrae-tr iviae complexM gastr i

M nonchromogenicum

M paratuberculosis

M flavescensM smegmatis

M vaccae

M parafortuitum complex

M phlei

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CLASIFICATION AND MYCOBACTERIOSIS

HABITUS LESSION

Tuberculosis complex

M tuberculos is Human Bronchopulmonal

M bovis Human,livestock Soft tissue & GI tract

Photochromogen

M kansasi i water, livestock Skeletal

M marinum fish, water skin & Soft tissueM simiae Primate Bronchopulmonal

M asiaticum Primate Pulmonary

Scotochromogen

M scrofulaceum land,water,food LimfadenitisM szulgai unclear Bronchopulmonal

M gordonae water Pulmonary

M Flavescens land,water Pulmonary

M xenopi water Bronchopulmonal

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HABITUS LESSION

Nonphotochromogen

M avium complex land,water,pig Pulmonary, KGB,systemiclivestock,bird

M ulcerans unclear skin & soft tissue

M gastr i i land,water Pulmonary

M terrae land,water Pulmonary

Rapid grower

M for tu i tum land,water, animal, skin,soft tissue, sistemic

sea biotic

M abscessus the same above The same above, skeletal

M chelonae the same above The same above, skeletal

M semgmatis moisture surface Pulmonary

M leprae Human,armadillo skin,soft tissue, systemic

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Clinical Manifestation ofM. aviumcomplex

Pediatric Adult

Common limfadenitis superficial colonization

ulcus, skin abses pulmonary

Rare pulmonary skin lession

systemic limfadenitis

systemic

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Pediatric Adult

Common systemic colonization

pulmonary systemic

Rare single limfadenitis pulmonary

single skin lession osteomielitis

peritonitis

oral lesion

appendicitis

M. aviumcomplex in HIV cases

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The M. tuberculosis cell wall is composed of

- Peptidoglycan (PG)- Arabinogalactan (AG)

- Mycolic acids and lipoglycans such as lipoarabinomannan

(LAM).

Other cell wall associated lipids include

- Trehalose dimycolate (TDM)

- Trehalose monomycolate (TMM)

- Phthiocerol Dimycocerosate (PDIM) and

- di-acyl trehalose (DAT)

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Stucture ofM. tuberculosis cell wall

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TRANSMITION AND REPLICATION

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Spreading ofM. tuberculosis through microdroplet

Infectious dose 10 cells

Spreading ofM. bovis through contaminated milk

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INHALATION OF MTB

IMMEDIATE KILLING PRIMARY COMPLEX

STABILIZATION LOCAL DISEASE DISSEMINATION

(LATENCY) (PRIMARY TB)

STABILIZATION ACUTE DISEASE

(LATENCY) (MENINGITIS

MILIARY TB)

REACTIVATION

(POST PRIMARY)

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Organs most commonly affected are : Kidney,Suprarenal gland , Fallopian tube ,

Epididymis.,Brain and meninges, Bone and joints.

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MR ( Mannose receptor ) MR on macrophage surface bound with carbohydrat Mtb.

Macrophage inactivated by these binding and this activity increased by IL4, IL13, and

glucocorticoid, but is inhibited by IFNy.

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M. tubercu losis-derived glycolipids promote survival in the host M and

evasion of the host immune response. M. tubercu losis(MTB) resident in thehost M phagosome sheds PIM and LAM to exert effects on intracellular

vesicle trafficking. LAM prevents trafficking of late endosomes (LE) to the

phagosome, thus inhibiting delivery of Ag presentation machinery and

inhibiting the adaptive immune response . PIM enhances delivery of early

and recycling endosomes (EE) to the phagosome and is suggested to

increase levels of essential nutrients in the phagosome, thus contributingto M. tubercu losissurvival . Tf, transferrin.

Mtbdormancy

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RESISTANT MECHANISM TO DRUG

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- Anti tuberculosis drug is not resistant inducers

-No evidence that plasmid nor transposon play arole in drug resistance

-Gene mutation is major basis for drug

resistance-it can be substitution, insertion and deletion

-mutation is a random event and ussually

sequential

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FACTORS FAVOURING 0CCURENCE OF

RESISTANT M. tubercu losis

Poor compliance

Low quality drugs

Inappropiate drug regimens and treatment durationCo-morbid :

Cellular Immunodeficiency

Disorder that interfere with pharmacokinetic and

pharmacodynamic of drugs

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RELATED GENE FOR RESISTANCE TO ANTI TB

DRUG GENE FUNCTION ROLE

H katG

inhANadh

ahpC

Catalase-peroxidase

Enoyl ACP reductaseNADH dehydrogenase

Alkyl hydroperoxidase

Prodrug conversion

Drug targetModulator of INH activity

Marker of resistance

R rpoB RNA polymerase Drug target

Z pncA Nicotinamidase Prodrug conversion

E embCAB - Drug target

S rpsl,

rrs

S12 ribosomal protein

16s rRNA

Drug target

A/K rrs 16s rRNA Drug target

Q gyrAgyrB

lfrA

DNA gyrase ADNA gyrase B

Efflux protein

Drug targetParticipate in drug binding

Et etaA/ethA

inhA

Flavinmonooxidase

-

Prodrug conversion

Drug target

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MECHANISM OF RESISTANCE

Drug Genes Notes

Isoniazid katG, inhA,Nadh.ahpC 50-90% in katG, 20-35 in inhA gene,10-

15% in ahpC-oxyR gene

Rifampicin rpoB >95%) of R-resistant Mtb is due tomutation at 81 base pair region ( codon

507-533

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EMB interacts with the EmbCAB proteins encoded by the embC, embA, and embB

genes, leading to inactivation of arabinogalactan synthesis. Mutations in theembB locus cause alterations in EmbB, possibly leading to an

altered target for EMB. Alternatively, hyperexpression of the

EmbCAB proteins could lead to EMB resistance.Inlet box:Organization of the emb operon in Mycobacterium tuberculosis (MTB).

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Resistance to FLQs, AM-CM in M. tuberculosis is most frequently

attributed to mutations in the gyrA and rrs ( 16S rRNA ),

respectively.

- 70-90 % of FLQ-resistant strain is due to mutations in codons

90, 91, and 94 in the gyrA gene.

- Mutations at A1401G, C1402T, and G1484T in the rrs gene

confer resistance to CM, CM, and KAN , each of them beingresponsible for a specific resistance pattern. Mutations

G1484T and A1401G were found to cause high-level

resistance to all drugs, whereas C1402T causes resistance to

only CM and KAN.- Mutation of the tlyA gene, encoding a putative rRNA

methyltransferase, confers capreomycin and viomycin

resistance

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AMPLIFICATION OF DRTB

SPONTANEOUS

MUTATIONSELECTION BY

ANTI TB DRUGS

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MICROBIOLOGY DIAGNOSTIC OFM. tuberculos is

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Sample

-Immediately process , especially sample is contaminated bynormal flora

- Multiple sample irregular/intermiten bacterial release. 3-5

times in 24 hours

-High volume of sample

-For sputum and urine sample

morning sample

-Avoid swab

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MIKROSKOPIS

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MIKROSKOPIS

ZIEHL NEELSEN (HOT)

MODIFIED-ZIEHL NEELSEN ( HOT )

KINYOUN ( COLD )

KINYOUN-GABBETT ( TAN THIAM HOK ) ( COLD )

AURAMINE FLUOROCHROME ( COLD )

Konfirmasi diagnosis

Follow up pengobatan, NON MDRTB

Tak membedakan kuman mati dan hidup

Tak membedakan species Mycobacteria

Cut off value 10.000 kuman per ml

Sensitifitas 1x pemeriksaan 40-60%. 3x pemeriksaan 80%

Sensitifitas rendah pada HIV & pauci basiler Tb

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ISOLASI DAN IDENTIFIKASI

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ISOLASI DAN IDENTIFIKASI

NON SELECTIVE MEDIA :

LJ, ATS, MIDLDLEBROOK 7H10, MIDDLEBROOK

7H11,PETRAGANISELECTIVE MEDIA :

MODIFIED LJ, LJ PLUS, MIDDLEBROOK 7H10 PLUS,MIDDLEBROOK 7H11 PLUS

Konfirmasi diagnosis

Follow up pengobatan MDRTBMembedakan MTB dan MOTT/NTM

Bahan untuk uji kepekaan terhadap obat

Cut off value 1000 kuman per ml

S S S

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UJI RESISTENSI

Proportion method

Absolute/break point method

Egg-based media ( LJ ) vs agar-based-media

Solid vs liquid media

Monitoring MDR TB

Panduan terapi definitif

Kultur dan uji resistensi konvensional hasilnya lama

Gold standard

UJI UNTUK KONFIRMASI DIAGNOSTIK LAIN

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UJI UNTUK KONFIRMASI DIAGNOSTIK LAIN

PCR

In house vs commercial kit

Sensitivity vs specificityHigh quality

Tak membedakan mati-hidup

Tak membedakan kolonisasi-infeksi

DETEKSI ANTIBODI

Commercially available kit

Variation of used antigen

Highly influenced by endemicity

Do not differentiate disease & infection

DETEKSI RESPON IMUN

Determination of IFN released by PBL after challenged

with antigen ( Elisa method )

Counting IF producing T cells after challenged with

antigen ( Elispot method )

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SEROLOGY

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SEROLOGY

Antibody responses varies : nutritional status, congenital

and acquired immune deficiencies, endemicity, etc

Specificity depend on the antigen used. Molecular mimicry

is not uncommon

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A varied antibody response- Not all patients with active TB have antibodies

against the same antigens

- HIV co-infection reduced anti-Mtb antibody titers

Confounding statesExposure to mycobacterial antigens

Natural history ofM. tuberculosis

infection

- IgM Ab levels have usually been found to be solow that their reliable measurement has been

difficult.

ANTIBODY DETECTION

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- Antibody takes several months after diagnosis for

patients with pulmonary TB to reach maximum

antibody titers so that serodiagnosis appears to be

more useful in chronic extrapulmonary disease(bone or joint) than in acute forms (miliary TB).- 38-Kd Ag provides serodiagnostic test with most

favorable test characteristics described, but is

limited by the lack of purified Ag.- Serum IgG Ab are observed to rise during the first 3

months of therapy but fall after 12-16 months.

ANTIBODY DETECTION

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Commercial Serologic Antibody Tests ( Steingart 2007 )

Overall, commercial tests vary widely in performance;Sensitivity is higher in smear-positive than smear

negative samples;

Specificity is higher in healthy volunteers than in patientsin whom tuberculosis disease is initially suspected and

subsequently ruled out;

Insufficient data to determine the accuracy of mostcommercial tests in smear microscopy-negative

patients, as well as their performance in children or

persons with HIV infection.

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NAA-BASEDDIAGNOSIS

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GENE TARGETS FOR AMPLIFICATION

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65 Kd antigen (HSPs):Genus-specific gene.Unsuitable for detecting M.tb,particularly in areas where species

like M.avium orM.kansasiiare prevalent.

IS6110 :

IS6110 found in the M.tb complex organisms ( M.tb,

M.africanum, M.microti, M.bovis).IS6110 sequence generally occurs only once in M.bovis

but is found as often as 20 times in certain strains of M.tb,

thus offering multiple targets for amplification. It is atransposon which are self replicating stretches of DNA.

OTHER TARGET : 16S r RNA: genes encoding 38 kda, MPB64,mpt 40,

pmt64

GENE TARGETS FOR AMPLIFICATION

COMMERCIALLY AVAILABLE STANDARD PCR BASED

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COMMERCIALLY AVAILABLE STANDARD PCR BASED

DIAGNOSTIC KIT

The Amplicor MTB Test584 bp fragment of the 16S ribosomal RNA gene,

comprising a species-specific flanked by genus-

specific sequences, is amplified using biotinylated

primers.

Other Amplicor kits are available for detection ofMycobacterium avium and Mycobacteriumintracellulare DNA in clinical samples.

Specificity is close to 100 % while sensitivity rangesfrom 90 % to 100 % in smear-positive samples and

from 50 % to 95.9 % in smear negative ones

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Enhanced-Amplified Mycobacterium Direct Test ( E-MTD )Sensivity for smear-positive cases >95%Sensitivity for smear-negative cases 70-90%

Amplicor Mycobacterium Test

Sensitivity for smear-positive cases >95%Sensitivity for smear-negative cases 60-70%

FDA APPROVED NAAT FOT MTB

The positive predictive value of FDA-approved NAA tests

for TB is >95% in AFB smear-positive cases

when the clinical suspicion of TB is low, the positive

predictive value of the NAA test is

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Approved NAA is less sensitive than culture. Negative results is thusdoes not eliminate a possibility of having TB. NAA tests can reliablydetect ( in 80-90 % cases ) Mycobacterium tuberculosis bacteria in

specimens 1 or more weeks earlier than culture

Sputum specimen may contains NAA inhibitors ( up to 20% ) thatmay decrease or lead to false negative result, although inhibitors

does not cause false negative NAA result in smear positive cases (