A Critique of the Proposed National Education Policy Reform
Molecular diagnosis of Mycobacterium tuberculosis TB - Second MBBS -
1. MOLECULAR DIAGNOSIS OF MYCOBACTERIUM
TUBERCULOSIS
Dr. R. Someshwaran, MD, Assistant professor, Dept. of Microbiology,
KFMS&R, Othakalmandapam.
12/17/15
10. 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
12/17/15
11. DIRECT DETECTION OF MYCOBACTERIUMFROMCLINICAL
SAMPLES
I. In house PCR– IS 6110 /16S rDNA
II. AMPLICOR MTB TEST (ROCHE)-16S rRNA
III. Amplified Mycobacteriumtuberculosis Direct test (AMTD,
Genprobe, USA) - 16S rRNA
IV. BDProbeTec 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 12/17/15
12. PRINCIPLES of Commercial Tests forMTBdetection
I. In house PCR
II. AMPLICOR MTB TEST – PCRbased
III. Amplified Mycobacteriumtuberculosis Direct test – TMA based
IV. BDProbeTec 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 PCRbased
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13. COMMERCIAL TESTS FORDIRECT DETECTION
OF MTBC fromclinical samples
ASSAYS COBAS
AMPLICOR,
ROCHE
AMTD
Genprobe
BD
PROBETEC
BD
Genotype MDA,
HAIN
LCHX
ABBOT
Amplification
Technology
PCR TMA SDA NASBA LCR
Target 16 s rDNA r RNA IS6110 23SrRNA Protein ag B
Detection Colorimetric Chemiluminiscenc
e
Fluorimetric Colorimetric Fluorimetric
TAT (hrs) 6.5 3.5 4 4 5-6
INSTRUMENT
AL USE
Thermocycler
photometer
Heat block
luminometer
Probetec
instrument
Twin cubator
thermocycler
Lcx
fluorimetric
analyser
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14. MOST COMMON (FDA approved)
COBAS AMPLICORPCR(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 MTDassay (genprobe)
- PCR amplification of r RNA
- Detect MTBC By Hybridization
- With Acridium ester labeled DNA probe
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15. RNA target RNA
Primer RT
RNA polymerase
promoter
First strand
synthesis
RNA ase H activity
RNA degradation
RT Primer
Second strand
synthesis
RNA synthesis
RNA polymerase
Nucleic Acid Sequence Based Amplification
(NASBA)
18. Identification of Mycobacterial species from
culture by molecularmethods
1 ) PCRbased sequencing- Gold standard
2 ) DNA probe technology - AccuProbe(Genprobe)
- SS DNA with Acridiumester
- Target rRNA
3 ) Line probe technology(hybridisation in strips )
- PCR
- Reverse hybridisation
- Different specific probes
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19. 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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20. 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
12/17/15
21. Rapid identification of cultured MTBC
LATERAL FLOWASSAYS(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 )
12/17/15
23. 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 Bgene – beta sub unit of RNA polymerase(96%)
12-24%
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24. 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 for12 S ribosomal subunit
- rrs – codes for16 S rRNA
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25. 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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26. 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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28. 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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30. 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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31. PCR– SSCP
Single Strand Conformation Polymorphisms
100% specificity for both RMP & INH
96% for RMP & 87% for INH
Steps:
a. PCR amplification
b. Denaturation
c. PAGE
d. With Wild type reference control
e. Electrophoretic mobility differences observed
Nested PCRSSCP:
Can detect MTB and its Resistance from samples directly
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32. PYROSEQUENCING
Target: 180 bp region of rpo Bgene
PCR& PYROSEQUENCING
Full agreement with BACTEC 460 phenotypic method
RT-PCRmethod
Xpert MTB
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33. Newer NAAT - Xpert MTB/RIF
test
PROCESS
12/17/15
42. 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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43. 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
Myco bacte rium sz ulg ai, Myco bacte rium kansasii &
Myco bacte rium m arinum .
NOT
FOR CHILDREN LESS THAN 17 YRS
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44. SERODIAGNOSIS
SEROLOGY
Antigens – 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 FORSEROLOGICAL
TESTS
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45. SUMMARY
12/17/15
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.