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Diagnosis of Tuberculosis


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Laboratory diagnosis of Tubercular Infections

Published in: Health & Medicine

Diagnosis of Tuberculosis

  3. 3. Routine Lab Anaemia Lymphocytosis/ Leukopenia Raised ESR Detection of adenosine deaminase (ADA) An enzyme present in almost all mammalian cells, principally in the lymphocytes, being directly related to lymphocyte activation. In diseases presenting greater lymphocyte activation and participation, elevated levels of ADA are usually detectable. Other disease conditions that cause pleural effusion with predominance of lymphocytes such as SLE, lymphoma etc, high levels of ADA may also be seen. Pleural TB - an ADA cut off value of 35 U/L
  4. 4. Chest Radiographs
  5. 5. Collection of Right Clinical Sample Pulm TB Sputum (Min requirement - At least 02 samples 01 hr apart) Induced Sputum Laryngeal swabs Transtracheal aspiration Gastric aspiration BAL Transbronchial lung biopsy Blood samples are of no value Extra pulm TB – from site of disease Pleural effusion – Pleural tap/ biopsy TB Lymphadenitis - FNA/ LN Biopsy TB Meningitis – CSF tap Urine Body fluids Tissue biopsy Smear microscopy for AFB Nucleic acid amplification (NAAT) Culture
  6. 6. Sputum Microscopy Direct smears of unconcentrated sputum - Fast, simple, inexpensive, widely applicable. Extremely specific for M. tuberculosis in high incidence areas. Ziehl-Neelsen staining (carbol fuchsin type) most common. Auramine Rhodamine stain Fluorescence Microscopy - More accurate: 10% more sensitive than light microscopy, with specificity comparable to ZN staining. Sputum processing - Concentration +/- Chemical pretreatment  Higher sensitivity (15-20% increase) and higher smear positive rate Steingart KR, et al. Lancet Infect. Dis. 2006; 6 (9):570-81 Steingart KR, et al. Lancet Infect. Dis. 2006; 6 (10):664-74
  7. 7. Sputum Microscopy > 10 bacilli/ field +++ 1 – 10 / field ++ 10 – 99 / 100 field + 1 – 9 / 100 field Write the no No bacilli seen Negative Oil immersion Field 5000 – 10000 bacilli per ml sputum
  8. 8. Culture Higher sensitivity than smear microscopy - If TB suspected and sputum smears are negative, culture may provide diagnosis Allows for identification of mycobacterial species. Allows for drug susceptibility testing. Tuberculosis culture room of the Lala Ram Swawrup (LRS) Institute of Tuberculosis and Respiratory Diseases in New Delhi 10 – 100 bacilli per ml sputum
  9. 9. Culture Solid media Lowenstein-Jensen: egg-based Middlebrook 7H 10 or 7H11: agar-based Liquid media Middlebrook 7H 12 Liquid media More sophisticated equipment Faster detection of growth Higher sensitivity than solid media Can also be used for drug-susceptibility testing BACTEC 460 TB MGIT MGIT Incubator Conventional Automated
  10. 10. Smooth, buff-colored colonies suggestive of Mycobacterium avium complex Rough, buff-colored colonies suggestive of Mycobacterium tuberculosis Visual assessment of colony morphology: Culture – Identification of MTb Nitrate reduction and niacin production are definitive for M.tb
  11. 11. Mycobacteriophage Based Assay Rapid - Results available within 24 hr of sample preparation From clinical samples Detects patient positive samples missed by smear No instrumentation required Safe - no culturing of pathogen Sensitive - assay sensitivity 100 - 300 cells/ml Only detects live bacilli - reduces the possibility of false positives Technology can be extended for antibiotic susceptibility testing FastPlaque ® TB
  12. 12. Molecular Methods of Diagnosis Direct detention of MTB from Specimen Identification of MTB from Cultures Genotypic PCR LAMP NAATs Ligase chain reaction Phenotypic - FAST Plaque TB PCR based sequencing 1 – 10 bacilli per ml sputum Rapid – 3 – 4 hr Cannot differentiate between dead and living MTB Expensive
  13. 13. NAATs Nucleic acid amplification tests (NAAT) Not recommended to be used on blood samples NAA assays amplify M. tuberculosis-specific nucleic acid sequences using a nucleic acid probe. Sensitivity - 80% in most studies - Require as few as 10 bacilli in a given sample. Specificity - 98% to 99%. Official statement of ATS and CDC, July 1999
  14. 14. LAMP (Loop Mediated Isothermal Amplification) for TB A manual simplified NAAT for TB Particularly applicable to resource-poor settings. Amplifies target DNA with high specificity, efficiency and rapidity under isothermal conditions DNA can be amplified 10 9 -10 10 times in 15-60 min Visually detect DNA directly from clinical samples, in less than two hours and with minimal instrumentation.
  15. 15. Xpert MTB/RIF Endorsed by WHO (2010). Updated WHO recommendations for diagnosis of pulmonary TB, paediatric TB, extrapulmonary TB and rifampicin resistance (2013). Implementation in endemic countries is ongoing. Cost ~ Rs 3500/- A recent Cochrane review has shown that the Xpert MTB/RIF test has 88% sensitivity and 98% specificity when compared to culture Xpert MTB/RIF can detect rifampicin resistance with a sensitivity of 94% and specificity of 98%.
  16. 16. Line Probe Assay DNA strip test - rapid, manual NAAT Use PCR and reverse hybridization methods Can diagnose MDR-TB directly from smear-positive sputum samples, providing results in just 05 hrs. 2008 - WHO issued a recommendation for the use of molecular LPA for the rapid diagnosis of MDR-TB in high TB-burden, low-income settings. Highly accurate in detecting MDR-TB and cost-effective when compared with TB culture and DST GenoType MTBDRplus® by Hain Lifescience, Germany
  17. 17. Liquid culture significantly faster: the average time-to-growth detection with liquid culture is 10 to 14 days, as opposed to four to six weeks with traditional solid culture. Up to 20% more sensitive than solid culture. Considered the gold standard for TB diagnosis and the only technology that can detect resistance to all major TB drugs. Liquid cultures are also very useful for smear-negative TB and extrapulmonary TB. Liquid Cultures MGIT (Mycobacterium Growth Indicator Tube method) by BD, USA BacT/Alert by BioMerieux, France
  18. 18. Liquid Cultures Rs 35,00,000 + Rs 120 per MGIT vial. BACTEC™ MGIT™ 960 Mycobacterial Detection System
  19. 19. Role of Blood Tests in TB ELISA to detect IgG, IgM, IgA Abs Rapid strip or card tests ‘Mycodot’ TB IgG/IgM Serological Tests
  20. 20. June 2012 - Govt of India, acting on the 2011 WHO policy against serological tests, banned the use, import, sale, and manufacture of Ab - based blood tests for TB, and discouraged the use of tests like "TB Gold" for active TB Role of Blood Tests in TB
  21. 21. Interferon-γ Release AssaysIGRAs Based on the detection of IFN-γ - Released by sensitized T cells on stimulation with very specific Ags – Early secretory antigen target-6 (ESAT 6) Culture filtrate protein-10 (CFP 10) Both derived from a very specific region of MTb, the region of difference 1 (RD1). This segment (RD1) is deleted from all strains of BCG and the majority of environmental mycobacteria (except M. kansasii, M. szulgai, M. marinum, M. flavescens, and M. gastrii). Advantage - Discriminate between MTB infection and previous use of BCG vaccine. T-Spot TB test - Directly count the number of IFN-γ- secreting T cells Quantiferon TB Gold In-Tube test - Measures the concentration of IFN-γ secretion.
  22. 22. Role in Indian ScenarioIGRAs Like Mantoux test - Do not differentiate between active pulmonary TB disease and latent TB infection. High False positive results – unnecessary treatment with ATT Cross-reactivity - L-ESAT, a M. leprae Ag homolog to the T-ESAT-6 seen in M. tuberculosis, may induce the production of IFN-γ, therefore making the test less useful in populations in which leprosy is endemic. Expensive. Meant for detection of Latent MTB Infection (Important in non endemic countries) LTBI – 6-9 months single agent Isoniazid Active TB – 6 months short course High negative predictive value - A negative test in a healthy patient excludes tuberculosis infection. (40% Indian population latently infected)
  23. 23. Role of Tuberculin (Montaux) test in Indian Scenario Indicates previous exposure and carriage of T.B. Tuberculin positive persons may develop reactivation type of T.B. False positive reactions are mainly due to Infection with nontuberculous mycobacteria/ BCG vaccination Children below 5 years of age with no exposure history - Positive test must be regarded suspicious Even the induration of 5 mm to be considered positive when tested on HIV patients. (40% Indian population latently infected) Excludes infection in suspected persons Tuberculin negative persons are at risk of gaining new infection False negative reactions may be due to Severe tuberculosis infection (Miliary T.B.) Hodgkin’s disease Corticosteroid therapy Malnutrition AIDS Positive test Negative test
  24. 24. Histopathological Examination Central Ceseation Necrosis Epitheloid Cell Granuloma Multinucleated Langhans Giant Cells
  25. 25. Aptamers Aptamers (Oligonucleotides) recognise a target with high affinity and specificity, but can also discriminate between very subtle structural differences. The main competitive advantage of aptamers over conventional approaches includes their high specificity, high sensitivity, relatively low production costs, convenience and simplicity, which allows for rapid point-of-care diagnosis. MODS (Microscopic Observation Drug Susceptibility) ROAD AHEAD