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RECENT ADVANCES IN
DIAGNOSIS OF TUBERCULOSIS
CHAIRPERSON: PROF. KRISHNENDU ROY
SPEAKER: ANGAN KARMAKAR
(JUNIOR RESIDENT)
Approach towards microbiological
diagnosis of TB
FLUORESCENT MICROSCOPY
• increased sensitivity
• LED microscopy
advantages?
• low power consumption
• no dark room requirement
disadvantages?
• dye toxicity
RAPID CULTURE METHODS
• BACTEC 460 (Middle
brook7H9 broth)
• Mycobacteria growth
indicator tube (MGIT)
ADVANTAGES OF MGIT
• all type of specimens
• continuously monitored
• positive signals over 10-12 days
• non-radiometeric
• cheaper than BACTEC
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%
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
RECENT ADVANCES IN DIAGNOSIS OF TUBERCULOSIS
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.
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
MOLECULAR METHODS
• uncultivable or difficult to culture
• HIV infected patients
• rapid result
• quantitative information (viral load)
• susceptibility testing (drug resistance) without
culture
• may not differentiate active infection as DNA
from a dead organism during antibiotic
treatment can be detected and amplified by
PCR
• High cost
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%.
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
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
RECENT ADVANCES IN DIAGNOSIS OF TUBERCULOSIS
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)
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
GenoType®MTBDR/MTBDRplus
Detects:
• Genotype
• RIF resistance
• INH resistance
• 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
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
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
WHO policies
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)
“Unite to end TB”
(World TB day 2016 theme)

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RECENT ADVANCES IN DIAGNOSIS OF TUBERCULOSIS

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