Mycobacterium tuberculosis AND BASIC TO DRUG RESISTANCE

BIOLOGICAL CHARACTER

CLASIFICATION
OPPORTUNIS-SAPROFIT M gordonae M terrae-triviae complex M gastri M nonchromogenicum M paratuberculosis M flavescens

M smegmatis
M vaccae M parafortuitum complex M phlei

HUMAN PATHOGEN M tuberculosis M bovis M leprae M avium-intracellulare M kansasii M scrofulaceum M xenopi M szulgai M marinum M ulcerans M haemophilum M africanum M malmoense M simiae M asiaticum M fortuitum-chelonae complex M thermoresistible

CLASIFICATION AND MYCOBACTERIOSIS

HABITUS
Tuberculosis complex M tuberculosis M bovis Human Human, livestock water, livestock fish, water Primate Primate land,water,food

LESSION
Bronchopulmonal Soft tissue & GI tract Skeletal skin & Soft tissue Bronchopulmonal Pulmonary Limfadenitis

Photochromogen
M kansasii M marinum M simiae M asiaticum Scotochromogen M scrofulaceum

M szulgai
M gordonae M Flavescens M xenopi

unclear
water land,water water

Bronchopulmonal
Pulmonary Pulmonary Bronchopulmonal

HABITUS Nonphotochromogen M avium complex M ulcerans M gastrii M terrae land,water,pig livestock,bird unclear land,water land,water land,water, animal, sea biotic M abscessus the same above

LESSION Pulmonary, KGB,systemic skin & soft tissue Pulmonary Pulmonary skin,soft tissue, sistemic The same above, skeletal

Rapid grower
M fortuitum

M chelonae
M semgmatis M leprae

the same above

moisture surface Human,armadillo

The same above, skeletal

Pulmonary skin,soft tissue, systemic

Clinical Manifestation of M. avium complex

Pediatric
Common limfadenitis superficial ulcus, skin abses Rare pulmonary systemic

Adult
colonization pulmonary skin lession limfadenitis systemic

M. avium complex in HIV cases

Pediatric
Common systemic pulmonary

Adult
colonization systemic

Rare

single limfadenitis single skin lession

pulmonary osteomielitis peritonitis oral lesion

appendicitis

Mycobacterium tuberculosis

1. Intracellular parasites

2. Do not produce endo nor exotoxin

2. Prefer aerobic condition 3. Highly adapted to various environmental condition

3. Varieties of virulent factor

a. secreted vs membrane-bound b. interfere with various steps of immune responses

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)

Stucture of M. tuberculosis cell wall

TRANSMITION AND REPLICATION

Spreading of M. tuberculosis through microdroplet Infectious dose 10 cells Spreading of M. bovis through contaminated milk

INHALATION OF MTB

IMMEDIATE KILLING

PRIMARY COMPLEX

STABILIZATION (LATENCY)

LOCAL DISEASE (PRIMARY TB)

DISSEMINATION

STABILIZATION (LATENCY)

ACUTE DISEASE (MENINGITIS MILIARY TB)

REACTIVATION (POST PRIMARY)

Organs most commonly affected are : Kidney,Suprarenal gland , Fallopian tube , Epididymis.,Brain and meninges, Bone and joints.

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.

Mtb dormancy

M. tuberculosis-derived glycolipids promote survival in the host M and evasion of the host immune response. M. tuberculosis (MTB) resident in the host 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 contributing to M. tuberculosis survival . Tf, transferrin.

RESISTANT MECHANISM TO DRUG

- Anti tuberculosis drug is not resistant inducers -No evidence that plasmid nor transposon play a role 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

FACTORS FAVOURING 0CCURENCE OF RESISTANT M. tuberculosis

Poor compliance Low quality drugs Inappropiate drug regimens and treatment duration Co-morbid : Cellular Immunodeficiency Disorder that interfere with pharmacokinetic and pharmacodynamic of drugs

RELATED GENE FOR RESISTANCE TO ANTI TB

DRUG H GENE katG inhA Nadh ahpC rpoB pncA embCAB rpsl, rrs rrs gyrA gyrB lfrA Et etaA/ethA inhA FUNCTION Catalase-peroxidase Enoyl ACP reductase NADH dehydrogenase Alkyl hydroperoxidase RNA polymerase Nicotinamidase S12 ribosomal protein 16s rRNA 16s rRNA DNA gyrase A DNA gyrase B Efflux protein Flavinmonooxidase Prodrug conversion Drug target ROLE Prodrug conversion Drug target Modulator of INH activity Marker of resistance Drug target Prodrug conversion Drug target Drug target Drug target Drug target Participate in drug binding

R Z E S A/K Q

MECHANISM OF RESISTANCE

Drug Isoniazid

Genes katG, inhA,Nadh.ahpC

Notes 50-90% in katG, 20-35 in inhA gene,1015% in ahpC-oxyR gene >95%) of R-resistant Mtb is due to mutation at 81 base pair region ( codon 507-533

Rifampicin rpoB

EMB interacts with the EmbCAB proteins encoded by the embC, embA, and embB genes, leading to inactivation of arabinogalactan synthesis. Mutations

in the embB 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).

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 being responsible 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

AMPLIFICATION OF DRTB

SPONTANEOUS MUTATION

SELECTION BY ANTI TB DRUGS

Rise and Fall phenomenon

Emergence of drug resistance due to mono-therapy
10 10
10

Resistant mutants
8

10

10 4

Susceptible organisms
10
2

Months after start of treatment

CHEMICAL AGENT AND ENVIRONMENTAL FACTOR

- Fresh disinfectant everyday - Derivat of amonium quaterner can not be used - Natrium hypochlorit 1g/l or concentration of chlor active 1000 ppm (part per million), and pour contamination 10.000 ppm. Exposure time must 15-30 minutes - Karbol dan formalin 5 %, - Iodofor 3-5% or 3-5 g/100 ml (not 1 % as comercial disinfectant) . Exposure time 15-30 minutes - Ethanol 70 90%. - UV 40 280 nm, power 1800 6400 microwatt/cm2

MICROBIOLOGY DIAGNOSTIC OF M. tuberculosis

AVAILABLE DIAGNOSIS METHODS

MICROSCOPY, direct and after cetrifugation at 3000g CULTURE AND DST

SEROLOGY
INTERFERON DETECTION PROBE AND NAAT SKIN TEST CHEST X RAY
Primary examination for diagnosis of M. tuberculosis is microscopy, culture, and histopatologi ( international standard for tuberculosis care 2008 ). Since 2008, standardized nucleic acid amplification was recommended by WHO

Sample
-Immediately process , especially sample is contaminated by normal 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

BAHAN PEMERIKSAAN SPUTUM ( PROGRAM ) 1. 3 Spesimen dalam 2 kunjungan. Dahak pagi sewaktu 2. 3-5 ml dalam botol tutup ulir,tahan pecah, mulut lebar 3. Diambil di tempat terbuka 4. induksi : a. Gliseril guaikolat 200 mg malam

b. Olah raga ringan, tarik nafas panjang

c. Inhalasi larutan garam dapur steril Sediaan dibuat dari bagian perkijuan,nekrotik,berdarah. Tambah sama banyak 5 % Na hipoklorit sebelum dibuat sediaan jika tidak akan dikultur

MIKROSKOPIS
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

ZIEHL NEELSEN (HOT) MODIFIED-ZIEHL NEELSEN ( HOT ) KINYOUN ( COLD ) KINYOUN-GABBETT ( TAN THIAM HOK ) ( COLD ) AURAMINE FLUOROCHROME ( COLD )

PELAPORAN PEMERIKSAAN MIKROSKOPIK ( IUADLT,PROGRAM )

TEMUAN 0/100 lp 1-9/100 lp 10-99/100 lp 1-10/lp,min 50 lp >10/lp,min 20 lp LAPORAN tulis jumlah + ++ +++

Jika 1-3/100 lp, ulang dg dahak baru. Jika tetap : laporkan negatif. Jika 4-9/100 lp laporkan positif Potensi keparahan dan tingkat penularan Evaluasi terapi

ISOLASI DAN IDENTIFIKASI

Konfirmasi diagnosis Follow up pengobatan MDRTB Membedakan MTB dan MOTT/NTM Bahan untuk uji kepekaan terhadap obat Cut off value 1000 kuman per ml

NON SELECTIVE MEDIA :

LJ, ATS, MIDLDLEBROOK 7H10, MIDDLEBROOK 7H11,PETRAGANI

SELECTIVE MEDIA : MODIFIED LJ, LJ PLUS, MIDDLEBROOK 7H10 PLUS, MIDDLEBROOK 7H11 PLUS

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

PCR In house vs commercial kit Sensitivity vs specificity High 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 )

SEROLOGY

SEROLOGY

Antibody responses varies : nutritional status, congenital and acquired immune deficiencies, endemicity, etc

Specificity depend on the antigen used. Molecular mimicry is not uncommon

ANTIBODY DETECTION 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 states Exposure to mycobacterial antigens Natural history of M. tuberculosis infection - IgM Ab levels have usually been found to be so low that their reliable measurement has been difficult.

ANTIBODY DETECTION - 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.

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 patients in whom tuberculosis disease is initially suspected and subsequently ruled out; Insufficient data to determine the accuracy of most commercial tests in smear microscopy-negative patients, as well as their performance in children or persons with HIV infection.

EVALUATION OF SEROLOGY TEST ( WHO 2008 )

Product of 19 companies : Mycodots 9 Easy Step, TB Rapid Screen Test, TBSTAT PAK II, Immune Sure TB Plus, SD TB Tapid test, TB-Spot Ver 2.0, TB Rapid test, dBest One Step TB Test, BIOLINE Tuberculosis test, etc Specimen : TB(+)HIV(+); TB(+)HIV(-);TB(-)HIV(+);TB(-)HIV(-). N 355 Collected from TB non endemic and endemic countries Analysis : Specicity, Sensitivity, Reproducibility, etc

EVALUATION OF SEROLOGY TEST ( WHO 2008 )

OVERALL SENSITIVITY RANGED FROM 1% TO 60% AND WAS HIGHER IN SPUTUM SMEAR POSITIVE THAN SPUTUM SMEAR NEGATIVE AND AMONGST HIV POSITIVE SAMPLES. THE MAJORITY OF PRODUCTS HAD POOR SPECIFICITY ( <80% ) WHEN TESTED IN TB SUSPECTS FROM ENDEMIC SETTING. TEST WITH SPECIFICITY OVER 90% DETECTED LESS THAN 30% OF ALL TB PATIENTS
HIV CO-INFECTION MIGHT DIMINISHES THE PERFORMANCE OF THE ASSAYS

SOME PRODUCTS SHOW HIGH LOT TO LOT, RUN TO RUN, OPERATOR TO OPERATOR AND INTER-READER REPRODUCIBILITY

EVALUATION OF SEROLOGY TEST ( WHO 2008 )

NONE OF THE ASSAYS PERFORM WELL TO REPLACE MICROSCOPY. SMEAR MICROSCOPY COMBINED WITH MOST RAPID TESTS IMPROVED OVERALL SENSITIVITY FROM 75% TO 89 %, but had an association with unacceptably high false positive rate of 42%.

THESE TEST ARE SOLD AND USED IN MANY ENDEMIC COUNTRIES

At present, commercial antibody detection tests for extrapulmonary tuberculosis have no role in clinical care or case detection ( Steingardtl,K.R et al. Post Graduate Med. J. 2007 )

NAA-BASED DIAGNOSIS

Conventional PCR
Commercially available
Inhibitors in the specimens.false negative Easily contaminated..false positive High technical skills

Equal sensitivity with 3 sputum smears Can also be used to detect NTM

In house NAAT is not recommended by W.H.O

META ANALYSIS OF 14 STUDY FOR MENINGITIS TB : SENSITIVITY 0.56; SPECIFICITY 0.98

Lancet Inf Dis 2003

GENE TARGETS FOR AMPLIFICATION

65 Kd antigen (HSPs):

Genus-specific gene.
Unsuitable for detecting M.tb,particularly in areas where species like M.avium or M.kansasii are 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 a transposon which are self replicating stretches of DNA. OTHER TARGET : 16S r RNA: genes encoding 38 kda, MPB64,mpt 40, pmt64

COMMERCIALLY AVAILABLE STANDARD PCR BASED DIAGNOSTIC KIT

The Amplicor MTB Test
584 bp fragment of the 16S ribosomal RNA gene, comprising a species-specific flanked by genusspecific sequences, is amplified using biotinylated primers. Other Amplicor kits are available for detection of Mycobacterium avium and Mycobacterium intracellulare DNA in clinical samples.

Specificity is close to 100 % while sensitivity ranges from 90 % to 100 % in smear-positive samples and from 50 % to 95.9 % in smear negative ones

LAMP
- Loop-mediated isothermal amplification. It is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. - LAMP is used for detection of M.tb complex, M.avium,and M.intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT) or on a solid medium (Ogawas medium).

Species-specific primers were designed by targeting the gyrB gene.

FDA APPROVED NAAT FOT MTB

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%

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 <50%

Approved NAA is less sensitive than culture. Negative results is thus does not eliminate a possibility of having TB. NAA tests can reliably detect ( 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% ) that may decrease or lead to false negative result, although inhibitors does not cause false negative NAA result in smear positive cases ( < 3%)

Currently available FDA approved NAA tests are not sufficiently sensitive (detecting 50%--80% of AFB smear-negative, culture-positive pulmonary TB cases) to exclude the diagnosis of TB in AFB smear-negative patients suspected to have TB .
NAA results often remain positive after culture results become negative during therapy. Application of NAA for TB in children and extrapulmonary TB await further clarification NAA does not replace the need for AFB smear and culture as standard diagnostic

INNOLIPA MYCOBACTERIA
The line probes of INNO LiPA Mycobacteria are speciesspecific fragments of the internal transcribed spacer (ITS) region interposed between 16S and 23S ribosomal RNA genes. The system includes a genus Mycobacterium-specific probe, two complex-specific probes (M. tuberculosis complex and M. avium complex) and 23 other probes suitable for identifying 18 species and several intraspecific variants (within the Mycobacterium chelonaeabscessus group and in species M. kansasii)

GENOTYPE MTB

GenoType Mycobacteria Direct test is based on the NASBA and the DNASTRIP technology, direct from decontaminated pulmonary and extrapulmonary specimens (except for blood )

W.H.O RECOMMENDED SCREEINING TEST FOR MDRTB SCREENING

LINE PROBE ASSAY Amplification of targetted genes followed by hybridization with immobilized probes. INNO LiPa Rif, GenotypeMTBDR

INNOLIPA Rif

SCREENING MDRTB

GENOTYPE MDRTB

GenoType MTBDRsl

GENEXPERT

FULLY AUTOMATIC-MULTIPLEX REAL TIME PCR. USE FOR DETECTION OF MTB FROM SPUTUM AND SCREENING OF MDRTB

Mtb detection

Ripamficin resistant detection

POWER OF NEW DIAGNOSTICS