Browsing by Author "Kanade, Swapna"

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    An in-house multiplex PCR test for the detection of Mycobacterium tuberculosis, its validation & comparison with a single target TB-PCR kit.
    (2012-05) Kulkarni, Savita; Singh, P; Memon, Aafreen; Nataraj, Gita; Kanade, Swapna; Kelkar, Rohini; Rajan, M G R
    Background & objectives: The conventional techniques used in TB diagnosis like AFB (acid fast bacilli) smear microscopy lack sensitivity and the gold standard, culture test takes time. A test based on multiplex polymerase chain reaction (PCR) targeting the 38 kDa gene and IS6110 insertion sequence, specific to Mycobacterium tuberculosis was developed to further increase the sensitivity of a TB-PCR kit targeting only 38 kDa gene developed earlier in the same laboratory. The multiplex test was validated using sputum samples from pulmonary TB (PTB) cases. The sensitivity and specificity were compared with AFB smear examination and Lowenstein-Jensen (LJ) culture test. Methods: Multiplex PCR amplifying 340 and 245 bp sequence of 38 kDa gene and IS6110, respectively was standardized and analytical sensitivity was verified. Sputum samples (n=120) obtained from PTB cases were subjected to AFB smear examination, LJ culture and a multiplex as well as single target PCR test. Additionally, 72 non-TB respiratory samples were included in the study as negative controls. Results: Analytical sensitivity of multiplex PCR was found to be 100 fg for 38 kDa gene and 1 fg for IS6110. Multiplex PCR, using both the targets, showed highest sensitivity of 81.7 per cent, followed by 69.2 per cent for L-J culture test and 53.3 per cent for AFB smear when clinical diagnosis was considered as a gold standard. The sensitivity of detection of M. tuberculosis in AFB smear positive and negative samples by multiplex PCR was 93.7 and 67.9 per cent, respectively. Sensitivity of 77.1 per cent observed for the detection of M. tuberculosis with single target PCR increased to 89.2 per cent with multiplex PCR in culture positive samples. Four samples showed positive PCR results only with primers for 38 kDa gene. Interpretation & conclusions: Multiplex PCR increased the sensitivity of single target PCR and will be useful in diagnosing paucibacillary smear negative samples. Further, it can also be used to detect samples with M. tuberculosis strains lacking IS6110.
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    Incremental yield in sputum smear positivity by examining a second early morning sputum specimen in follow-up patients on dots: 7 year analysis of RNTCP laboratory register.
    (2011-04) Nataraj, Gita; Kanade, Swapna; Parikh, Raunak; Khatri, Vijay; Mehta, Preeti; Athavale, Amita; Arun, Bamne
    Background: Patients receiving DOTS undergo periodic follow-up sputum examination, which aids in monitoring response to treatment. Continued or new smear positivity at follow up examination entails extension of intensive phase or change in treatment category and the need for culture and drug susceptibility test. Setting: Tuberculosis microscopy centre at a tertiary care teaching hospital, Mumbai, India. Objective: To determine the incremental yield in sputum smear positivity by examining a second early morning sputum specimen in follow-up patients on DOTS. Design: Retrospective analysis of follow up sputum microscopy results recorded in tuberculosis laboratory register for the period 2002-2008. Results: During the study period, 5015 follow-up patients submitted two early morning sputum specimens, of which 501(9.99 %) patients were detected to be smear-positive. Out of smear positive patients under study, 324 patients had both specimens positive, 79 patients had only first specimen positive and 98 patients had only second specimen positive. The incremental yield was 1.95 % of total and 19.5 % of smear positives. Conclusion: Discordant smears were present in nearly a third of patients detected smear positive during follow-up. More than half of these patients were detected only by examining second specimen. The incremental yield by examining the second early morning specimen was 1.95 % of total and 19.5 % of smear positive specimens. It is important to detect each possible smear positive follow-up patient as they are likely to benefit from altered treatment. The inclusion of a second early morning sputum specimen examination is essential to maximize their detection.
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    Utility of Microscopic Observation of Drug Susceptibility (MODS) assay for mycobacterium tuberculosis in resource constrained settings.
    (2010-10) Limaye, Kashmira; Kanade, Swapna; Nataraj, Gita; Mehta, Preeti
    Introduction: Multidrug Resistant Tuberculosis (MDR TB) is a global health problem. Conventional techniques or automated systems for diagnosis and drug susceptibility testing are either comparatively slow or costly. Microscopic Observation Drug Susceptibility [MODS] assay is a simultaneous detection and direct drug susceptibility test [DST] method which relies on the characteristic growth of Mycobacterium tuberculosis (MTB) in a liquid medium. Aim: Comparison between MODS assay and culture on Lowenstein Jensen (LJ) medium with respect to; i) detection of mycobacterial growth and time taken for culture positivity (ii) to compare concordance of susceptibility results of MTB isolate by MODS with proportion method using LJ medium. Method: A prospective study was carried out on 171 acid fast smear positive sputum specimens. The decontaminated sediment was used for culture and DST using LJ medium (proportion method) and MODS assay plates containing supplemented Middlebrook 7H9 broth with and without critical concentrations of isoniazid and rifampicin. Results: Median time to growth and DST using MODS assay was 10 days and that with LJ medium was 24 and 66 days respectively. The sensitivity and specificity of MODS assay was 100%. All the isolates were characterized as MTB. MODS demonstrated 98.8% and 99.4% concordance for isoniazid and rifampicin respectively and 100% for MDRTB. Positive and negative predictive value for MDRTB was 100%. Conclusion: MODS assay offers a rapid, simple, economical and feasible method for simultaneous culture and DST of MTB. Utility of MODS needs to be ascertained in extrapulmonary TB cases.
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    Utility of MPT 64 antigen detection assay for rapid characterization of mycobateria in a resource constrained setting.
    (2012-04) Kanade, Swapna; Nataraj, Gita; Suryawanshi, Rupali; Mehta, Preeti
    Introduction: Important reasons for the negligible numbers of laboratories performing characterization of Mycobacteria in resource constrained settings are requirement of biosafety measures, longer turnaround time and laborious nature of tests. A rapid, accurate and simple test for characterization is required. “SD BIOLINE TB Ag MPT 64 Rapid®” is a rapid immunochromatographic test for differentiation of Mycobacteria into M. tuberculosis Complex (MTBC) and nontuberculous mycobacteria (NTM). Aim: To evaluate a commercial assay, SD TB Ag MPT64 Rapid® for characterization of Mycobacteria isolated on Lowenstein Jensen (LJ) medium. Material and methods: 150 non duplicate isolates which were previously characterized as MTBC or NTM based on standard phenotypic characteristics were tested by the commercial assay after blinding. The result of the conventional phenotypic test and the commercial assay was compared. Any discordant result was referred for confirmation by genotypic Mycobacterium CM assay (Hain’s life sciences, Germany). Sensitivity and specificity of the commercial assay was calculated using the results of conventional phenotypic and genotypic tests as gold standard. Results: Phenotypically, 124 isolates were characterized as MTBC and 26 as NTM. The commercial assay gave concordant results for 149 isolates. One MTBC isolate did not demonstrate a band. The sensitivity, specificity, PPV and NPV was 99.19%, 100 %, 100% and 97.3% respectively. The total turnaround time for the rapid assay was 30 minutes compared to a few hours to days for phenotypic and genotypic method. Conclusion: “SD BIOLINE TB Ag MPT 64 Rapid®” is a simple, rapid and reliable test to differentiate MTBC from NTM.