lab diag. tb

11

Hi ! Good Morning ! I am Hi ! Good Morning ! I am Mycobacterium Mycobacterium

tuberculosis.tuberculosis.

22

LABORATORY DIAGNOSIS LABORATORY DIAGNOSIS OF OF

MYCOBACTERIUM MYCOBACTERIUM TUBERCULOSISTUBERCULOSIS

33

NEED FOR ACCURATE & EARLY NEED FOR ACCURATE & EARLY DIAGNOSIS OF TUBERCULOSIS.DIAGNOSIS OF TUBERCULOSIS.

• Tuberculosis mainly Tuberculosis mainly caused by M.tuberculosis.caused by M.tuberculosis.

• Is a major health problem Is a major health problem world wide.world wide.

• Found in Neolithic Found in Neolithic remains.remains.

• Largest cause of Largest cause of “DEATHS” from a “DEATHS” from a single infectious single infectious disease.disease.

• HENCE “ACCURATE” & HENCE “ACCURATE” & “EARLY” diagnosis is “EARLY” diagnosis is required for its required for its effective effective “MANAGEMENT”.“MANAGEMENT”.

44

LABORATORY DIAGNOSIS MAY LABORATORY DIAGNOSIS MAY BE ESTABLISHED BY:BE ESTABLISHED BY:

• Demonstration of bacillus by Demonstration of bacillus by microscopy.microscopy.

• Isolation in culture.Isolation in culture.• Transmitting the infection in Transmitting the infection in

experimental animals.experimental animals.• Demonstration of hypersensitivity to Demonstration of hypersensitivity to

tuberculoprotein – Mantoux test.tuberculoprotein – Mantoux test.• Enzyme linked Immunosorbent Spot Enzyme linked Immunosorbent Spot

(ELISPOT) Assay.(ELISPOT) Assay.

55

LABORATORY DIAGNOSIS LABORATORY DIAGNOSIS CONTD.CONTD.• QuantiFeron TB Gold QuantiFeron TB Gold

Test.Test.

• ELISA to detect 38 KDA ELISA to detect 38 KDA Mycobacterial Antigen.Mycobacterial Antigen.

• Molecular methods for Molecular methods for early diagnosis.early diagnosis.

* Amplicor test* Amplicor test * E – MTD* E – MTD * DNA sequencing* DNA sequencing * Line Probe Assay (LiPA)* Line Probe Assay (LiPA) * DNA micro arrays* DNA micro arrays * Molecular beacons.* Molecular beacons. * Single strand conformation * Single strand conformation

Polymorphism (SSCP)Polymorphism (SSCP) * FRET Probes.* FRET Probes.

• Other PCR based Other PCR based Techniques.Techniques.

* Amplification Mutation* Amplification Mutation

{ ARMS }{ ARMS }

* Branch Migration Inhibition* Branch Migration Inhibition

{ BMI }{ BMI }

• Ligase Chain Reaction.Ligase Chain Reaction.

• Diagnosis of TB & Drug Diagnosis of TB & Drug Resistant TB with Resistant TB with Mycobacteriophages.Mycobacteriophages.

66

SPECIMENS COLLECTEDSPECIMENS COLLECTED• Pulmonary secretions – Pulmonary secretions – ** spontaneously produced or induced sputum** spontaneously produced or induced sputum

** gastric lavage** gastric lavage

** transtracheal aspiration** transtracheal aspiration

** bronchoscopy** bronchoscopy

** laryngeal swabbing** laryngeal swabbing

• Gastric lavage specimens.Gastric lavage specimens.

• Urine samples.Urine samples.

• Faecal specimens.Faecal specimens.

• Tissue & Body Fluid specimens.Tissue & Body Fluid specimens.

• Blood specimensBlood specimens

• Wound ,skin lesion aspirates.Wound ,skin lesion aspirates.

• Cervical swab.Cervical swab.

77

SPUTUM EXAMINED BY :SPUTUM EXAMINED BY :

• DIRECT METHOD DIRECT METHOD # Z.N.Staining.# Z.N.Staining.

# Fluorescent Auramine Rhodamine staining# Fluorescent Auramine Rhodamine staining

• AFTER CONCENTRATION AFTER CONCENTRATION

88

Acid-fast bacilliAcid-fast bacilli

99

ZN Smear evaluation & AFB ZN Smear evaluation & AFB ReportReport

(Grading of smear)(Grading of smear)No. of AFBNo. of AFB No. of OIFNo. of OIF ReportReport

00 300300 AFB Not seenAFB Not seen

01 – 0201 – 02 300300 Doubtful; Doubtful; Repeat smearRepeat smear

01 – 0901 – 09 100100 1+1+

01 – 0901 – 09 1010 2+2+

01 – 09 01 – 09 0101 3+3+

10 or more10 or more 0101 4+4+

1010

Indications for Culture Indications for Culture

• Failures of re-treatment casesFailures of re-treatment cases

• Seriously ill cases;Seriously ill cases;– extra-pulmonary casesextra-pulmonary cases – smear negative cases smear negative cases – childhood TB & HIV-TBchildhood TB & HIV-TB

• For DRSFor DRS

• Not for New Smear Positive CasesNot for New Smear Positive Cases

1111

Decontamination ProceduresDecontamination Procedures1946 – Trisodium Phosphate 1946 – Trisodium Phosphate

1955 – Pancreatin Desogen 1955 – Pancreatin Desogen

1958 – Pancreatin + 1% cetrimide1958 – Pancreatin + 1% cetrimide

1915 – Petroff’s NaOH 1915 – Petroff’s NaOH

1962 – Zephiran Trisodium PO1962 – Zephiran Trisodium PO44

1963 – N-acetyl L- cysteine + 2%NaOH1963 – N-acetyl L- cysteine + 2%NaOH

1969 – Swab culture technique + 1% cetrimide1969 – Swab culture technique + 1% cetrimide

1975 – CPC + NaCl1975 – CPC + NaCl22

1212

PETROFF’S METHODPETROFF’S METHOD

Advantages:Advantages:Simple, inexpensive & control the growth of Simple, inexpensive & control the growth of

contaminantscontaminantsTwenty samples can be processed in 2 Hrs, with Twenty samples can be processed in 2 Hrs, with

centrifuge capacity being the limiting factorcentrifuge capacity being the limiting factorSterilized NaOH can be kept for several weeksSterilized NaOH can be kept for several weeks

Limitations:Limitations:The specimen exposure times must be strictly The specimen exposure times must be strictly

followed to prevent over kill of tubercle bacilli. followed to prevent over kill of tubercle bacilli. The initial kill is independent of additional The initial kill is independent of additional contributory factors such as heat build-up in the contributory factors such as heat build-up in the centrifuge and centrifugal efficiencycentrifuge and centrifugal efficiency

1313

Processing of sputum with CPC Processing of sputum with CPC MethodMethod

If delay of more than 48 hours If delay of more than 48 hours between collection and processing is between collection and processing is anticipated, the sputum should be anticipated, the sputum should be collected with 1%CPC and 2%NaCl2collected with 1%CPC and 2%NaCl2

CPC acts as homogenizing and CPC acts as homogenizing and decontaminating agent decontaminating agent

It helps in retaining viability of It helps in retaining viability of Tubercle bacilli up to 7 daysTubercle bacilli up to 7 days

These specimens should not be These specimens should not be treated with NaOH ( Petroff’s)treated with NaOH ( Petroff’s)

1414

MYCOBACTERIAL CULTUREMYCOBACTERIAL CULTUREAdvantagesAdvantages::

Increases number of cases foundIncreases number of cases foundDetects cases among smear negative patientsDetects cases among smear negative patientsEstablishes viability of organismsEstablishes viability of organismsDistinguishing between Mycobacterial speciesDistinguishing between Mycobacterial speciesHelps in performing DSTHelps in performing DSTHelps in diagnosing cases of failureHelps in diagnosing cases of failure

LimitationsLimitations::

Expensive Expensive Require enriched mediaRequire enriched mediaRequire considerable expertise Require considerable expertise Time consumingTime consuming

1515

Specimen

Sterile Non - Sterile

Centrifuge & use sediment Liquefaction (N-acetyl-L- cystein)

Decontamination NaOH

Neutralization Buffer or H2O

Centrifugation > 3000 X g

Screen by AFB smear & inoculate media (one liquid & one solid)

FLOW CHART OF SPECIMEN PROCESSING FOR ISOLATION OF MYCOBACTERIA

1616

FLOW CHART CONTD.

Screen by AFB smear & inoculate media (one liquid & one solid)

Liquid Medium Solid Media

MGIT BACTEC SEPTI-CHEK

CMS

IncubateAt 37ºCFor 6 wks


IncubateinvertingAt 37ºCFor 8 wks


Fluoresc--ence detected

GrowthIndex>10

Colonies orturbidity

Growthdetected

Confirm by AFB smear Reinoculate onSolid media

L J

L J with RNA

LJ with Pyruvic acid

Incubate

At 37ºCFor 8 wks

If growthConfirm onAFB smear

1717

Reading and ReportingReading and Reporting

Characteristics of Tubercle bacilliCharacteristics of Tubercle bacilliGrowth of Primary culture takes 2 – 4 Growth of Primary culture takes 2 – 4

weeks to obtain visible coloniesweeks to obtain visible coloniesColonies are buff colored and rough, Colonies are buff colored and rough,

having the appearance of bread crumbs or having the appearance of bread crumbs or cauliflowercauliflower

Not easily emulsified but give a granular Not easily emulsified but give a granular suspensionsuspension

Microscopically frequently arranged in Microscopically frequently arranged in serpentine cords of varying length or serpentine cords of varying length or show linear clumpingshow linear clumping

1818

Culture Media : SolidCulture Media : Solid

LJLJ

LJ with Na pyruvateLJ with Na pyruvate

LJ with out asparagineLJ with out asparagine

Middlebrook’s 7H10 & 7H11Middlebrook’s 7H10 & 7H11

Selective 7H10 & 11Selective 7H10 & 11

Ogawa Ogawa

Tarshi’s Blood AgarTarshi’s Blood Agar

1919

Slow growthSlow growth

3-4 wk

2020

24-hr generation time24-hr generation time

AFB

AFB

AFB

24 hr

2121

MYCOBACTERIAL COLONIES ON SOLID MYCOBACTERIAL COLONIES ON SOLID MEDIAMEDIA

2222

Colonies growing on mediaColonies growing on media

2323

Laboratory diagnosisLaboratory diagnosis

• Traditional (slow)Traditional (slow)– Growth on solid mediaGrowth on solid media– Biochemical tests to speciateBiochemical tests to speciate

• RecentRecent– Radiometric growth detectionRadiometric growth detection– BACTECBACTEC– SEPTI – CHEKSEPTI – CHEK– MGITMGIT– Gene probes to speciateGene probes to speciate

2424

Flow ChartFlow ChartIdentification of Tubercle bacilli & Identification of Tubercle bacilli &

relatedrelatedGrowth on LJ Medium

Rapid growth within 7 daysGrowth on MacConkey Agar

Aryl-sulphataes test

Slow GrowthNiacin test

-VeTellurite

Reduction

+ veM. smegmatis

- veM. phlei

- veType of growth

+ veM.

tuberculosis+ ve

M. Fortuitumcomplex

Pigment Scanty Smooth Flat Colonies

In LightGroup I

Photochromogens

In DarkGroup II

Scotochromogens

No PigmentGroup III

Non - Chromogens

- veBCG

+ veM. bovis

M.KansasiM.intermedium

M.SzulgaiM.scrofulaceum

M.Avium complexM.gastri

2525

Other Culture MethodsOther Culture Methods

• Septi-check AFBSepti-check AFB

• MGIT 960MGIT 960

• Backtec/MB/BactBacktec/MB/Bact

• ESP Culture iiESP Culture ii

• MMicroscopic icroscopic OObservation of bservation of BBroth roth CCulture ulture

• MODS: MMODS: Micro icro CColony olony DDetection etection SSystemystem

2626

BACTEC CONTINUOUS MONITORING BACTEC CONTINUOUS MONITORING SYSTEMSYSTEM

2727

BLOOD CULTURE BOTTLES FOR BLOOD CULTURE BOTTLES FOR BACTEC 9240,9120,9050.BACTEC 9240,9120,9050.

2828

SEPTI - CHECKSEPTI - CHECK

2929

Radiolabeled palmitic acidRadiolabeled palmitic acid

AFB*C___

*C___ *C___

*C___

3030

Detect growth with CODetect growth with CO22

AFBAFB

AFBAFB

AFB

*CO2

*CO2

*C___

*C___

3131

BIOCHEMICAL REACTIONS:BIOCHEMICAL REACTIONS:

SPECIES NIACIN TEST

ARYL-SULPH---ATASE TEST

NITRATE REDUC---TION TEST

HOT CATAL---ASE TEST

PEROX---IDASE TEST

TWEEN 80

HYDRO--LYSIS TSET

TELLURI--TE REDUCTION TEST

GROWTH ON TCH

PYRAZI-NAMIDASE TSET

UREASE TEST

M tuberculosis

+ - + - + - +/- + + +

M bovis

- - - - - - +/- - - +

M African

um

- - - - - - - +/- - +

3232

BIOCHEMICAL REACTIONSBIOCHEMICAL REACTIONS

3333


3434


3535


3636

ANIMAL INOCULATIONANIMAL INOCULATION

• Intramuscular injection of the bacterial Intramuscular injection of the bacterial

concentrated material into two healthy concentrated material into two healthy

guinea pigs of 12 week and autopsied, guinea pigs of 12 week and autopsied,

one after four weeks & second after one after four weeks & second after eight eight

weeks of inoculation.weeks of inoculation.

3737

ANTIGEN PROTEIN DETECTIONANTIGEN PROTEIN DETECTION• Tuberculostearic acid :Tuberculostearic acid : a fatty extracted from a fatty extracted from

the cell wall of M.tuberculosis detected by gas the cell wall of M.tuberculosis detected by gas

chromatography/ mass spectrometry in clinical chromatography/ mass spectrometry in clinical

samples. samples. M.tuberculosis is unique to release M.tuberculosis is unique to release

tuberculostearic acid .tuberculostearic acid .

• Host enzyme Adenosine deaminase :Host enzyme Adenosine deaminase : Host Host

enzyme. It is increased in infection caused by enzyme. It is increased in infection caused by

M.tuberculosis.M.tuberculosis.

3838

CHROMATOGRAPHIC ANALYSISCHROMATOGRAPHIC ANALYSIS

Analysis of mycobacterial lipids by ----Analysis of mycobacterial lipids by ---- * Thin paper chromatography* Thin paper chromatography * Gas liquid chromatography (GLC)* Gas liquid chromatography (GLC) * Reverse phase high performance liquid chromatography * Reverse phase high performance liquid chromatography

( HPLC)( HPLC)

HPLC of extracted mycobacteria -HPLC of extracted mycobacteria -

* A very rapid & specific method for identification of species.* A very rapid & specific method for identification of species.

3939

Why Measure Interferon-Why Measure Interferon-??

• TB infection induces T-cell response (CMI)TB infection induces T-cell response (CMI)• IFN- IFN- is the ‘classic’ CMI cytokine is the ‘classic’ CMI cytokine• Produced Produced in vitroin vitro in response to specific in response to specific

antigenantigen• Secreted in measurable and stable Secreted in measurable and stable

amountsamounts• Absent from normal circulationAbsent from normal circulation• Extensive literature showing importance Extensive literature showing importance

of IFN-of IFN- in TB infection in TB infection

4040

What is Quanti-FERONWhat is Quanti-FERON®®-TB -TB GoldGold

•Blood assay for Blood assay for M. tuberculosis > M. tuberculosis > Interferon Interferon γγ release assay release assay

•In vitroIn vitro test using whole blood test using whole blood specimen for the diagnosis of TB specimen for the diagnosis of TB infection, whether latent or activeinfection, whether latent or active

•Does not distinguish between latent TB Does not distinguish between latent TB infection or TB diseaseinfection or TB disease

4141

Quanti-FERONQuanti-FERON®®-TB Gold – -TB Gold – Scientific BasisScientific Basis

•This recognition process involves the This recognition process involves the generation of interferon-generation of interferon-γγ, a specific cytokine , a specific cytokine for cell mediated immune responsefor cell mediated immune response

•Individuals infected with Individuals infected with M. tuberculosisM. tuberculosis complex organisms have lymphocytes in their complex organisms have lymphocytes in their blood that recognize mycobacterial antigensblood that recognize mycobacterial antigens

•The detection and subsequent quantification The detection and subsequent quantification of IFN-of IFN-γγ is the basis of this test is the basis of this test

•The test uses synthetic peptide antigens The test uses synthetic peptide antigens (ESAT-6, CFP-10) that simulate mycobacterial (ESAT-6, CFP-10) that simulate mycobacterial proteins to generate the immune responseproteins to generate the immune response

4242

Interferon Gamma ReleaseInterferon Gamma Release

4343

Whole Blood IFN-Whole Blood IFN- Assay AssayQuantiFERON-TB TestQuantiFERON-TB Test

ESAT-6 CFP 10MitogenControl

TMBTMB

COLORCOLOR

Stage 1 Whole Blood CultureStage 1 Whole Blood Culture

Stage 2 IFN-gamma ELISAStage 2 IFN-gamma ELISA

NilControl

Incubate Incubate →→ INF- INF- from sensitized T-from sensitized T-

cellscells

Draw blood Draw blood + heparin+ heparin

Aliquot blood Aliquot blood & add antigen& add antigen

Harvest plasma Harvest plasma from above settled from above settled

cellscells

Measure [ IFN-Measure [ IFN-] in ] in ‘Sandwich’ ELISA‘Sandwich’ ELISA

ComputerizedComputerizedinterpretationinterpretation

Cellestis

4444

Species Specificity of ESAT-6 and Species Specificity of ESAT-6 and CFP-10CFP-10

4545

QFT AssayQFT Assay

4646

In VivoIn Vivo and and In VitroIn Vitro Diagnostic TestsDiagnostic Tests

Antigenpresenting

cell

MemoryT-cell

Presentation ofmycobacterial antigens

IFN-

IFN-

IL-8, etc.

IL-8, etc.

TNF-

TNF-

4747

Results and InterpretationResults and InterpretationRESULTRESULT INTERPRETATIONINTERPRETATION

POSITIVEPOSITIVE ESAT-6 and/or CFP-10 ESAT-6 and/or CFP-10 responsiveness detectedresponsiveness detected

M. tuberculosisM. tuberculosis infection infection likelylikely

NEGATIVENEGATIVE No ESAT-6 or CFP-10 No ESAT-6 or CFP-10 responsiveness detectedresponsiveness detected

M. tuberculosis M. tuberculosis unlikelyunlikely

INDETERMINATEINDETERMINATE MTB infection status cannot MTB infection status cannot be determined as a result of be determined as a result of impaired immunity and/or impaired immunity and/or incorrect performance of incorrect performance of the testthe test

4848

QFT and TSTQFT and TSTQFTQFT

•in vitroin vitro test test

•Specific antigensSpecific antigens

•No boostingNo boosting

•1 patient visit1 patient visit

•Lab variabilityLab variability

•Results possible in 1 Results possible in 1 dayday

•Requires phlebotomyRequires phlebotomy

•Includes + controlIncludes + control

TSTTST

•in vivoin vivo test test

•Less specific PPDLess specific PPD

•BoostingBoosting

•2 patient visits2 patient visits

•Inter-reader variabilityInter-reader variability

•Results in 2-3 daysResults in 2-3 days

•No phlebotomy No phlebotomy requiredrequired

•No + controlNo + control

4949

T-Spot.T-Spot.TBTB: : “Six“Six easy Steps”easy Steps”

Nil Control

Positive Control

Infection

Infection

Oxford Immunotec

5050

ENZYME LINKED IMMUNOSORBENTENZYME LINKED IMMUNOSORBENT SPOT (ELISPOT) TEST.SPOT (ELISPOT) TEST.

• It is based on ELISA testIt is based on ELISA test

• Allows visualization of secretory Allows visualization of secretory products of individual activated cells.products of individual activated cells.

• Provides information about type of Provides information about type of immune protein (qualitative)& immune protein (qualitative)& number of responding cells number of responding cells (quantitative)(quantitative)

5151

USING ELISA TO DETECT USING ELISA TO DETECT 38kDA MYCOBACTERIAL 38kDA MYCOBACTERIAL

ANTIGENANTIGEN• 38 kDA secretory protein being one of 38 kDA secretory protein being one of the the

most important specific antigens of MTBmost important specific antigens of MTB

• It induses B & T cell responses with high It induses B & T cell responses with high

specificity to MTBspecificity to MTB

• Detected by Direct & Sandwich ELISA.Detected by Direct & Sandwich ELISA.

5252

Mantoux Tuberculin Skin Test

•Preferred method of testing for TB infection in adults and children

•Tuberculin skin testing useful for

- Examining person who is not ill but may be infected

- Determining how many people in group are infected

- Examining person who has symptoms of TB

5353

Administering the Tuberculin Skin Test

•Inject intradermally 0.1 ml of 5TU PPD tuberculin

•Produce wheal 6 mm to 10 mm in diameter

•Do not recap, bend, or breakneedles, or remove needles from syringes

•Follow universal precautions for infection control

5454

Reading the Tuberculin Skin Test

•Read reaction 48-72Hours after injection

•Measure only induration

•Record reaction inmillimeters

5555

Classifying the Tuberculin Reaction

>5 mm is classified as positive in

• HIV-positive persons

• Recent contacts of TB case • Persons with fibrotic changes on chest

radiograph consistent with old healed TB

• Patients with organ transplants and other immunosuppressed patients

5656

Classifying the Tuberculin Reaction (cont.)

>10 mm is classified as positive in• Recent arrivals from high-prevalence countries

• Injection drug users

• Residents and employees of high-risk congregate settings

• Mycobacteriology laboratory personnel

• Persons with clinical conditions that place them at high risk

• Children <4 years of age, or children and adolescentsexposed to adults in high-risk categories

5757

Classifying the Tuberculin Reaction (cont.)

>15 mm is classified as positive in

• Persons with no known risk factors for TB

• Targeted skin testing programs should only be conducted among high-risk groups

5858

BCG and TST (1)BCG and TST (1)• General teaching is that reactivity from General teaching is that reactivity from

BCG wanes after a few years and is BCG wanes after a few years and is unlikely to persist > 10 years, but may be unlikely to persist > 10 years, but may be boosted by PPD.boosted by PPD.

• Study done in Switzerland* suggests that Study done in Switzerland* suggests that false positives due to BCG may be much false positives due to BCG may be much more common than we thought:more common than we thought:– 40% of 5000 HCW had positive TST40% of 5000 HCW had positive TST– Prior BCG strongest risk factor for positive TST Prior BCG strongest risk factor for positive TST

among those less than age 40 with TSTs among those less than age 40 with TSTs <<18 18 mm (was not as strong a risk factor for those mm (was not as strong a risk factor for those > 40 years old and those with TSTs > 40 years old and those with TSTs >> 20 mm) 20 mm)

5959

BCG and TST (2)BCG and TST (2)

• Review of studies that compared TST Review of studies that compared TST responses to BCG during and after infancyresponses to BCG during and after infancy

• Vaccination during infancy estimated to Vaccination during infancy estimated to cause false-positive TST in 6.3% overall, cause false-positive TST in 6.3% overall, but only 1% of those tested more than 10 but only 1% of those tested more than 10 years after vaccinationyears after vaccination

• Vaccination at 2 years of age or older Vaccination at 2 years of age or older estimated to cause false-positive TST in estimated to cause false-positive TST in 40% of persons overall, 20% of those 40% of persons overall, 20% of those tested 10 years or more after vaccinationtested 10 years or more after vaccination

Farhat M et al, Int J Tuberc Lung Dis 2006; 10: 1192-204

6060

Nucleic acid amplification Nucleic acid amplification for mycobact. diagnosisfor mycobact. diagnosis

Genus specific protocolsTargeting genes code for 16S rRNA

65KDa hsp M.TB Complex specific is 6110Other targets: Genes encoding 38 KDa MPB 64 mtp 40 PMT 64Methods:Target amplification - PCR(TMA, LCR, SDA or signal amplification EG: QB amplification)

PFYFFER G.E. J.INF. 1999, 39, 21-26. TC/ICM 30

6161

MOLECULAR TECHNIQUES TO MOLECULAR TECHNIQUES TO DETECT M.TUBERCULOSIS - 1DETECT M.TUBERCULOSIS - 1

•Amplicor TestAmplicor Test

** Detects the presence of the ** Detects the presence of the

mycobacterial 16S ribosomal mycobacterial 16S ribosomal ( rRNA)( rRNA)

gene by the PCR amplification gene by the PCR amplification

followed by ELISA reaction.followed by ELISA reaction.

6262

Mycobacteria (AFB) sampleMycobacteria (AFB) sample

AFB

6363

Extract 16S RNAExtract 16S RNA

16SRNA

6464

Probe for specific 16S RNAProbe for specific 16S RNA

16SRNA

ProbeProbe

6565

Probe remains in sampleProbe remains in sample

16SRNA

Probe

6666

Detector binds to probeDetector binds to probe

16SRNA

Probe**************

6767

Detector remains in sampleDetector remains in sample

16SRNA

Probe*******

6868

Detector identifies 16S RNADetector identifies 16S RNA

16SRNA

Probe*******

6969

MOLECULAR TECHNIQUES TO MOLECULAR TECHNIQUES TO DETECT M.TUBERCULOSIS - 2DETECT M.TUBERCULOSIS - 2

•E – MTDE – MTD ** The assay is based on the

transcription mediated amplification system ( TMA ).The mycobacterial rRNA from target cells is released by sonication & amplified a billion fold by TMA.

7070

Conventional method for Conventional method for diagnosis of drug resistant diagnosis of drug resistant M.tuberculosisM.tuberculosis• Biochemical tests : Biochemical tests : Catalase & Catalase &

Peroxidase Peroxidase tests tests

*** BOTH ARE NEGATIVE IN INH *** BOTH ARE NEGATIVE IN INH RESISTANT M.TUBERCULOSIS.RESISTANT M.TUBERCULOSIS.

7171

SENSITIVITY TESTSSENSITIVITY TESTS

• Absolute conc. Method : Absolute conc. Method : No.of media No.of media containing serial conc. Of the drugs are containing serial conc. Of the drugs are inoculated & minimum inhibitory inoculated & minimum inhibitory conc.calculated.conc.calculated.

• Resistance Ratio Method : Resistance Ratio Method : Two sets of Two sets of media containing graded conc.of drugs media containing graded conc.of drugs inoculated. One with test strain & other inoculated. One with test strain & other with standard strain of known sensitivity.with standard strain of known sensitivity.

• Proportion Method : Proportion Method : Average sensitivity of Average sensitivity of the strain.the strain.

7272

Various Mycobacterial gene Various Mycobacterial gene mutations involved in drug mutations involved in drug

resistance.resistance.

• rpoB gene mutation --- rpoB gene mutation --- RifampicinRifampicin

• katG & inhA mutation --- katG & inhA mutation --- INHINH

• rpsL gene mutation --- rpsL gene mutation --- StreptomycinStreptomycin

• pncA gene mutation --- pncA gene mutation --- PyrazinamidePyrazinamide

• embB gene mutation --- embB gene mutation --- EthambutolEthambutol

7373

Molecular diagnostic methods Molecular diagnostic methods for drug resistant for drug resistant

M.tuberculosisM.tuberculosis

• DNA Sequencing : DNA Sequencing : reliable & accurate.reliable & accurate.

• Line Probe Assay (LiPA) : Line Probe Assay (LiPA) : to identify mutations in the to identify mutations in the rpoB core gene.rpoB core gene.

• DNA microarrays :DNA microarrays : based on principle of hybridization based on principle of hybridization

• Molecular beacons :Molecular beacons :they are hair-pin shaped probes to they are hair-pin shaped probes to detect presence of specific nucleic acids.detect presence of specific nucleic acids.

• Single strand conformation polymorphism :Single strand conformation polymorphism :determines determines presence of mutations in specific DNA regionspresence of mutations in specific DNA regions

• FRET ( Fluorescent resonance energy transfer) probes: FRET ( Fluorescent resonance energy transfer) probes: used to detect presence of mutations in real time PCR.used to detect presence of mutations in real time PCR.

7474

Other PCR based tests:Other PCR based tests:• Amplification Refractory Mutation Amplification Refractory Mutation

System: System: has been applied to know has been applied to know mutations in rpoB, katG, embB, genes.mutations in rpoB, katG, embB, genes.

• Branch Migration Branch Migration Inhibition :Inhibition :spontaneous strand spontaneous strand exchange is inhibited by sequence exchange is inhibited by sequence difference between two DNA molecules.difference between two DNA molecules.

7575

Diagnosis of drug resistance Diagnosis of drug resistance with Mycobacteriophageswith Mycobacteriophages

• Phage amplified biological assay Phage amplified biological assay (phaB)(phaB)

• Luciferase reporter phages (LRPs)Luciferase reporter phages (LRPs)

7676

Antituberculosis Drugs Antituberculosis Drugs Currently in UseCurrently in Use

• First-line DrugsFirst-line Drugs– IsoniazidIsoniazid– RifampinRifampin– RifapentineRifapentine– RifabutinRifabutin– EthambutolEthambutol– PyrazinamidePyrazinamide

• Second-line DrugsSecond-line Drugs– CycloserineCycloserine– EthionamideEthionamide– LevofloxacinLevofloxacin– MoxifloxacinMoxifloxacin– GatifloxacinGatifloxacin– PP-Aminosalicylic acid-Aminosalicylic acid– StreptomycinStreptomycin– Amikacin/kanamycinAmikacin/kanamycin– CapreomycinCapreomycin– LinezolidLinezolid

7777

• -BCG-BCG

PREVENTION CHALLENGESPREVENTION CHALLENGES

WHO Jan 07

“BCG vaccine should not be used in children

known to be

HIV-infected”

lab diag. tb

Education