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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
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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 (