MOLECULAR DIAGNOSIS OF MYCOBACTERIUM TUBERCULOSIS

Dr. R. Someshwaran, MD, Assistant professor, Dept. of Microbiology, KFMS&R, Othakalmandapam.05/01/23

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Direct Indirect Microscopy Culture Molecular nucleic

acid techniques Antigen detection Phage based assays Liquid

chromatographic tests

Tuberculin skin testing (TST)

Interferon γ assays

Serological tests

Diagnostic tests

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Microscopy

Overall sensitivity varies 22 –77% Carbol fuchsin based

Ziehl Neelsen Kinyoun

Fluorochrome Auramine/ rhodamine

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RNTCP GRADING OF SMEARS

EXAMINATIONEXAMINATION RESULTRESULT GRADINGGRADING NO. OF FIELDS NO. OF FIELDS TO BE EXAMINEDTO BE EXAMINED

>10 AFB /oil immersion >10 AFB /oil immersion fieldsfields

Positive Positive 3 +3 + 2020

1-10 AFB /oil immersion 1-10 AFB /oil immersion fieldsfields

Positive Positive 2 +2 + 5050

10-99 AFB /100 oil 10-99 AFB /100 oil immersion fields immersion fields

PositivePositive 1 +1 + 100100

1-9AFB /100 oil immersion 1-9AFB /100 oil immersion fieldsfields

Scanty Scanty Record exact Record exact number seennumber seen

200200

No AFB /100 oil immersion No AFB /100 oil immersion fieldsfields

Negative Negative -- 100100

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LED –Fluorescent microscopy

GRADE ZN stain ( 100x) FM(40x)

Scanty 1-9 1-19/ 40 fields

1+ 10-99 20-199/40 fields

2+ 1-10/ field 5-50/field

3+ >10/ field >50/ field

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Culture - Gold standard for diagnosis

Egg based Lowenstein Jensen

Agar based Middlebrook 7H10/11

Liquid/Broth based Middlebrook 7H9

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Automated cultures – WHO recommended

Radiometric BACTEC 460

Non Radiometric MGIT 960 MB/BacT

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Molecular nucleic acid techniques

Culture confirmation and species identification by probes

Direct detection in clinical samples by nucleic acid amplification

Detection of drug resistance

DNA finger-printing and strain typing

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DIRECT DETECTION OF MYCOBACTERIUM FROM CLINICAL SAMPLES

I. In house PCR – IS 6110 / 16S r DNA

II. AMPLICOR MTB TEST (ROCHE)-16S r RNA

III. Amplified Mycobacterium tuberculosis Direct test (AMTD , Genprobe, USA) - 16S r RNA

IV. BD ProbeTec ET (BD)-IS 6110

V. Genotype Mycobacteria direct assay (Hain lifesciences) - 23S r RNA

VI. LCx MTBC assay (Abbot ) – Protein antigen b

VII. Gene pert- Xpert MTB/RIF test

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PRINCIPLES of Commercial Tests for MTB detection

I. In house PCR

II. AMPLICOR MTB TEST – PCR based

III. Amplified Mycobacterium tuberculosis Direct test – TMA based

IV. BD ProbeTec ET (BD) – SDA based

V. Genotype Mycobacteria direct assay (Hain lifesciences) - NASBA

VI. LCx MTBC assay (Abbot ) – LCR

VII. Gene Xpert - Xpert MTB/RIF test – Real Time PCR based

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COMMERCIAL TESTS FOR DIRECT DETECTION OF MTBC from clinical

samples

ASSAYS COBAS AMPLICOR,

ROCHE

AMTD Genprobe

BD PROBETEC

BD

Genotype MDA, HAIN

LCHXABBOT

AmplificationTechnology PCR TMA SDA NASBA LCR

Target 16 s rDNA r RNA IS6110 23SrRNA Protein ag B

Detection Colorimetric

Chemiluminiscence

Fluorimetric

Colorimetric Fluorimetric

TAT (hrs) 6.5 3.5 4 4 5-6INSTRUMEN

TAL USEThermocycl

erphotometer

Heat blockluminometer

Probetec instrument

Twin cubator

thermocycler

Lcx fluorimetric

analyser

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MOST COMMON (FDA approved)

COBAS AMPLICOR PCR (ROCHE) - PCR Amplification of 16s r RNA (585 BP) - Amplified product Biotin labeled - CAPTURED BY PROBE IN Micro titre well - TAT-6.5 hrs Amplified MTD assay (genprobe) - PCR amplification of r RNA - Detect MTBC By Hybridization - With Acridium ester labeled DNA probe

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RNA target RNA

Primer RT

RNA polymerasepromoter

First strandsynthesis

RNA ase H activity

RNA degradation

RT Primer

Second strandsynthesis

RNA synthesis

RNA polymerase

Nucleic Acid Sequence Based Amplification (NASBA)

Line probe assay Genotype mycobacterium CM assay

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GenoType® MTBDRplus assay

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Identification of Mycobacterial species from culture by molecular methods

1 ) PCR based sequencing- Gold standard

2 ) DNA probe technology - AccuProbe(Genprobe) - SS DNA with Acridium ester - Target rRNA3 ) Line probe technology(hybridisation in strips ) - PCR - Reverse hybridisation - Different specific probes

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Line probe technology - Hybridisation in strips

a) Inno LiPA Mycobacterium v2: - Target Mycobacterial spacer region 16S-23S r RNA - 17 species - Sensitivity-100% & specificity-94.4% - Cross reactions seen b) Genotype Mycobacterium (Hain) 1.Genotype MTBC— gyr B polymorphism 2.genotype Mycobacterium CM (com- mycobact)- 23s r DNA 3.genotype Mycobacterium AS (addl sps) - 23s r DNA

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Identification………4 ) PRA method (PCR with Restriction enzyme analysis) - Amplification of 65 –kDa heat shock protein - RFLP ( bst E II /Hae III )

- TAT-1 day cost effective /reliable

5) Pyrosequencing (biotage,sweden) - for short sequences of 20-30 bp

6) DNA Micro arrays (DNA chips ) -fluorescent labeled amplicons hybridised on DNA arrays -16S r RNA and rpo B loci -2Hrs

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Rapid identification of cultured MTBC

LATERAL FLOW ASSAYS(ICT)

- Antigen detection from Culture

- detects MTBC specific antige MPT64

- detection limit 105 CFU/ml

1.Capilia TB rapid 2.TB AG MPT64 rapid test –SD bioline 3.BD MGIT TB c identification test (BD

)

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Molecular methods for detecting Drug resistance in Mycobacterial strains

Phenotypic methods: 1. Solid culture methods: Proportion method etc.,

2. Liquid culture methods -Bactec TB 460 / Bactec MGIT 960 (BD) -Bact/Allert 3D (Biomerieux)

Genotypic methods:

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Mechanismof drug resistance

Isoniazid (INH) –mutations in the following genes

- kat G – catalase (peroxidase) – 30 -90% mutation in codon 315 - inh A – Mycolic acid synthesizing protein – 32% - ahp C – alkyl peroxidase reductase - kas A - Nil in 10 -15%

Rifampicin - rpo B gene – beta sub unit of RNA polymerase(96%)

12-24%

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Mechanism of drug resistance

Pyrazinamide - pnc A –pyrazinamidase/nicotinamidase 70% - 30% unknown

Ethambutol - emb CAB – membrane proteins -70%

Streptomycin - modification of 30 S sub unit - rps L – codes for 12 S ribosomal subunit - rrs – codes for 16 S rRNA

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Genotypic methods

1. PCR – DNA sequencing

2. Hybridisation based techniques

3. Hybridisation on DNA chips

4. PCR – SSCP (Single Strand Conformation Polymorphisms)

5. Pyrosequencing

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Hybridisation based techniques

Line probe technology:a. Inno-LiPA Rif TB: Culture (100% sensitivity) Direct specimens (80% Sensitivity)

10 oligo nucleotide probes - 1 for MTBC - 5 for wild type probes(S1-S5) - 4 for Rifampicin resistance(R2, R4, R4b &

R5)

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Inno-LiPA Rif TB Test strip

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Hybridisation based techniques

Line probe technology:

b. GenoType MTBDR plus (Hain Life sciences)- In culture and Direct specimens - Detects rpo B, Kat G, inh A genes- Rifampicin resistance(98.7% correlation)- Isoniazid resistance(92%)

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GenoType MTBDR plus Test

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Hybridisation on DNA chips

DNA Microarray Combi Chip Mycobacteria

(South Korea) rpo B – 7 codons (100%

identified) kat G & inh A – (84%)

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PCR – SSCP Single Strand Conformation Polymorphisms 100% specificity for both RMP & INH 96% for RMP & 87% for INHSteps:a. PCR amplificationb. Denaturationc. PAGE d. With Wild type reference controle. Electrophoretic mobility differences observed Nested PCR SSCP : Can detect MTB and its Resistance from samples

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PYROSEQUENCING

Target: 180 bp region of rpo B gene PCR & PYROSEQUENCING

Full agreement with BACTEC 460 phenotypic method

RT-PCR method Xpert MTB

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Newer NAAT - Xpert MTB/RIF test PROCESS

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PHAGE BASED TESTS

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PHAGE BASED TESTS

Two methods are commercially available:

FASTPlaque – TB

FastPlaqueMDRi

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MYCOBACTERIOPHAGE BASED TECHNIQUES FAST plaque TB assay - 48-72 hrs - Economical - Smear positive: Senitivity - 87.4% Specificity - 88.2% - Smear negative: Sensitivity - 67.1% Specificity - 98.4%

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Fast plaque TB test

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PHAGE ….

Luciferase reporter mycobacteriophage – Phage D29 - Carries luciferase gene - organism grown in with &with out

drug - add reporter (phage) - add luciferin(subsrate) - light + Resistant For testing INH&RIF sensitivity Sensitivity& specificity - 95%

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Liquid Chromatographic tests

Direct detection on clinical samples – CSF Gas Liquid Chromatography-Mass

spectrometry (GLC-MS) - Detects presence of Tuberculostearic acid (TBSA)

Species identification of culture isolates High Performance Liquid Chromatography

(HPLC) - Mycolic acid is analyzed

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Interferon γ assay

QuantiFERON TB-GOLD

T-SPOT.TB assay

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Interferon γ assay

T cells sensitized with M.tuberculosis

Re-encounter Mycobacterial antigens(ESAT 6, CFP 10)

Release interferon γ (a Th1 cytokine)

IFN γ can be used in all settings where TST is used

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Igra ….

Higher sensitivity/specificity

Better correlation with exposure to mtbc

Low cross reactivity with BCG/ATYPICAL MYCOBACTERIA

Detects latent mycobacterial infections False positive results can occur with Mycobacterium szulgai, Mycobacterium kansasii &

Mycobacterium marinum. NOT FOR CHILDREN LESS THAN 17 YRS

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SERODIAGNOSIS

SEROLOGYAntigens – 38kDa, LAM, 35kDa, Kp90

40 PLUS TESTS AVAILABLE SENSITIVITY ----------------------1-----60% SPECIFICITY ------------------------53---98.7% PERFORMANCE POOR WITH SPUTUM NEGATIVE SAMPLES LOT to LOT VARIATION OPERATOR TO OPERATOR RUN TO RUN

WHO --- NO ROLE FOR SEROLOGICAL TESTS

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SUMMARY

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Nucleic acid techniques

Best when used for culture confirmation, species identification & detection of resistance

Results on clinical samples to be interpreted in light of clinical parameters and culture results.

THANK YOU

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