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

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

Xpert MTB/RIF from different samples

SAMPLE SENSITIVITY SPECIFICITYSputum 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)