MOLECULAR DIAGNOSIS of MYCOBACTERIUM TUBERCULOSIS TB PPT - SECOND MBBS STUDENTS - UNDERGRADUATE STUDENTS - MEDICAL COLLEGE - POWERPOINT

1. MOLECULAR DIAGNOSIS OF MYCOBACTERIUM
TUBERCULOSIS
Dr. R. Someshwaran, MD, Assistant professor, Dept. of Microbiology,
KFMS&R, Othakalmandapam.
12/17/15

2. ???????????????
12/17/15

3. 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
12/17/15

4. Microscopy
Overall sensitivity varies 22 –77%
 Carbol fuchsin based
 Ziehl Neelsen
 Kinyoun
 Fluorochrome
 Auramine/ rhodamine
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5. RNTCP GRADING OF SMEARS
EXAMINATIONEXAMINATION RESULTRESULT GRADINGGRADING NO. OF FIELDSNO. OF FIELDS
TO BE EXAMINEDTO BE EXAMINED
>10 AFB /oil immersion fields>10 AFB /oil immersion fields PositivePositive 3 +3 + 2020
1-10 AFB /oil immersion1-10 AFB /oil immersion
fieldsfields
PositivePositive 2 +2 + 5050
10-99 AFB /100 oil10-99 AFB /100 oil
immersion fieldsimmersion fields
PositivePositive 1 +1 + 100100
1-9AFB /100 oil immersion1-9AFB /100 oil immersion
fieldsfields
ScantyScanty Record exactRecord exact
number seennumber seen
200200
No AFB /100 oil immersionNo AFB /100 oil immersion
fieldsfields
NegativeNegative -- 100100
12/17/15

6. 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
12/17/15

7. Culture - Gold standard for diagnosis
 Egg based
Lowenstein Jensen
 Agar based
Middlebrook 7H10/11
 Liquid/Broth based
Middlebrook 7H9
12/17/15

8. Automated cultures – WHOrecommended
 Radiometric
 BACTEC 460
 Non Radiometric
 MGIT 960
 MB/BacT
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9. 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
12/17/15

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
12/17/15

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
12/17/15

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)

16. Line probe assay
 Genotype mycobacterium CM assay
12/17/15

17. GenoType® MTBDRplus assay
12/17/15

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
12/17/15

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
12/17/15

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

22. Molecularmethods fordetecting
Drug resistance in Mycobacterial
strains
 Phenotypic methods:
1. Solid culture methods: Proportion method etc.,
2. Liquid culture methods
-Bactec TB460 /Bactec MGIT 960 (BD)
-Bact/Allert 3D(Biomerieux)
 Genotypic methods:
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%
12/17/15

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
12/17/15

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
12/17/15

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)
12/17/15

27. Inno-LiPA Rif TB
Test strip
12/17/15

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%)
12/17/15

29. GenoType MTBDRplus Test
12/17/15

30. Hybridisation on DNA chips
 DNA Microarray
 Combi Chip Mycobacteria (South
Korea)
 rpo B– 7 codons (100% identified)
 kat G & inh A – (84%)
12/17/15

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
12/17/15

32. PYROSEQUENCING
 Target: 180 bp region of rpo Bgene
 PCR& PYROSEQUENCING

Full agreement with BACTEC 460 phenotypic method
RT-PCRmethod
 Xpert MTB
12/17/15

33. Newer NAAT - Xpert MTB/RIF
test
PROCESS
12/17/15

34. PHAGE BASED TESTS
12/17/15

35. PHAGE BASEDTESTS
Two methods are commercially available:
 FASTPlaque – TB
 FastPlaqueMDRi
12/17/15

36. MYCOBACTERIOPHAGE BASED
TECHNIQUES
 FAST plaque TBassay
- 48-72 hrs
- Economical
- Smearpositive: Senitivity - 87.4%
Specificity - 88.2%
- Smearnegative: Sensitivity - 67.1%
Specificity - 98.4%
12/17/15

37. Fast plaque TB test
12/17/15

38. PHAGE ….
Luciferase reportermycobacteriophage –
Phage D29 - Carries luciferase gene
- organism grown in with &with out drug
- add reporter (phage)
- add luciferin(subsrate)
- light + Resistant
 Fortesting INH&RIF sensitivity
 Sensitivity& specificity - 95%
12/17/15

39. 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
12/17/15

40. 12/17/15

41. Interferon γ assay
 QuantiFERON TB-GOLD
 T-SPOT.TB assay
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
12/17/15

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
12/17/15

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
12/17/15

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.

46. THANKYOU
12/17/15