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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)
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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.
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THANK YOU
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