TRADITIONAL TESTS AND RECENT DIAGNOSTIC MODALITIES FOR TUBERCULOSIS WITH EMPHASIS TO MOLECULAR DETECTION TECHNIQUES, DRUG SENSITIVITY ASSESMENT IN INDIAN PERSPECTIVE

1. RECENT ADVANCES IN
DIAGNOSIS OF TUBERCULOSIS
CHAIRPERSON: PROF. KRISHNENDU ROY
SPEAKER: ANGAN KARMAKAR
(JUNIOR RESIDENT)

2. Approach towards microbiological
diagnosis of TB

3. FLUORESCENT MICROSCOPY
• increased sensitivity
• LED microscopy
advantages?
• low power consumption
• no dark room requirement
disadvantages?
• dye toxicity

4. RAPID CULTURE METHODS
• BACTEC 460 (Middle
brook7H9 broth)
• Mycobacteria growth
indicator tube (MGIT)

5. ADVANTAGES OF MGIT
• all type of specimens
• continuously monitored
• positive signals over 10-12 days
• non-radiometeric
• cheaper than BACTEC

6. DST using rapid culture method
MODS
• microscopic-observation drug-susceptibility assay
• antituberculous drug containing Middle brook
7H9 broth
• growth (cord formation) detected using inverted
light microscope
• time to detection- 7 days
• sensitivity 95% & specificity 100%

7. IFN-γ release assays
• in vitro assays
• whole blood incubated with TB antigens
• measures interferon (IFN-γ) released by
sensitized T cells
• measures immune reactivity to M.tb

9. QuantiFERON TB Gold
• combines IFN-γ release
technology with the
diagnostic power of
synthetic TB-specific
peptides (ESAT-6 and
CFP-10) to provide the
best available method
of diagnosing TB
infection
• FDA-approved for the
detection of LTBI, 2001.

10. Analysis of IGRA report
Positive IGRA
• person has been infected with TB bacteria.
additional tests are needed to determine if
the person has latent TB infection or TB
disease
Negative IGRA:
• latent TB infection or TB disease is not likely

11. MOLECULAR METHODS
• uncultivable or difficult to culture
• HIV infected patients
• rapid result
• quantitative information (viral load)
• susceptibility testing (drug resistance) without
culture

12. • may not differentiate active infection as DNA
from a dead organism during antibiotic
treatment can be detected and amplified by
PCR
• High cost

13. Nucleic acid amplification tests
• amplify M. tuberculosis-specific nucleic acid
sequences using a nucleic acid probe
• sensitivity at least 80% in most studies
• Require 10 bacilli/ml of given sample
• specificity 98% to 99%.

14. Molecular methods used in NAAT
For amplification
• Polymerase chain reaction
• Transcription mediated amplification
• Loop mediated isothermal amplification
• Ligase chain reaction
• Real time PCR
For detection:
• Electrophoresis
• Line probe assay
• Real time detection

15. Xpert MTB/RIF
• detects M. tuberculosis & rifampicin
resistance conferring mutations
• unprecedented sensitivity-smear negative,
culture positive specimens
• provides results directly from the sputum
within 100 minutes
• Endorsed by WHO since 2011

17. Xpert MTB/RIF from different samples
SAMPLE SENSITIVITY SPECIFICITY
Sputum 88% 99%
Lymph node aspirate 84.9% 92.5%
CSF 79.5% 98.6%
Pleural fluid 43.7% 98.1%
Gastric lavage/aspirate 83.8% 98.8%
Source: WHO policy update for Xpert MTB/RIF(2013)

18. Line probe assays
• PCR/hybridization
technique to identify
Mycobacterium
Tuberculosis
• identify drug-resistant
strains by detecting the
most common single
nucleotide
polymorphorisms
associated with
resistance

19. GenoType®MTBDR/MTBDRplus
Detects:
• Genotype
• RIF resistance
• INH resistance

20. • In-house PCR tests are widely used in the
developing countries
• The line probe assays (LPA) are suitable only
for national or regional level laboratories
because of its complexity and bio safety
requirements

21. Molecular DST- candidates?
• Patients suspected or at high risk of having drug-
resistant TB
• very ill patients
• HIV infected patients
• who do not get better while taking standard first-
line therapy

22. Lateral flow urine lipoarabinomannan
(LF-LAM)assay
• From urine of active TB patients
• Endorsed by WHO in 2015
• Only for HIV patients- seriously ill/ low CD4
count

23. WHO policies

24. Cost of diagnostic tests
TESTS
ZN microscopy
LED-FM microscopy
Gene Xpert MTB/RIF
Line probe assay
Solid culture
Liquid culture
COST in INR
50
100
600
1500
250
600
Source: Medicine update 2016 (Association of Physicians, India)

25. “Unite to end TB”
(World TB day 2016 theme)