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      Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay

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          Abstract

          Early detection of drug resistance in Mycobacterium tuberculosis isolates allows for earlier and more effective treatment of patients. The aim of this study was to investigate the performance of the malachite green decolourisation assay (MGDA) in detecting isoniazid (INH) and rifampicin (RIF) resistance in M. tuberculosis clinical isolates. Fifty M. tuberculosis isolates, including 19 multidrug-resistant, eight INH-resistant and 23 INH and RIF-susceptible samples, were tested. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and agreement of the assay for INH were 92.5%, 91.3%, 92.5%, 91.3% and 92%, respectively. Similarly, the sensitivity, specificity, PPV, NPV and agreement of the assay for RIF were 94.7%, 100%, 100%, 96.8% and 98%, respectively. There was a major discrepancy in the tests of two isolates, as they were sensitive to INH by the MGDA test, but resistant by the reference method. There was a minor discrepancy in the tests of two additional isolates, as they were sensitive to INH by the reference method, but resistant by the MGDA test. The drug susceptibility test results were obtained within eight-nine days. In conclusion, the MGDA test is a reliable and accurate method for the rapid detection of INH and RIF resistance compared with the reference method and the MGDA test additionally requires less time to obtain results.

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          Rapid and effective diagnosis of tuberculosis and rifampicin resistance with Xpert MTB/RIF assay: a meta-analysis.

          Xpert MTB/RIF (Cepheid) assay has been introduced for the diagnosis of tuberculosis (TB) and RIF-resistance. The meta-analysis was used to establish the overall accuracy of Xpert MTB/RIF assay for diagnosing TB and RIF-resistance. Based on comprehensive searches of the Pubmed and Embase, we identified outcome data from all articles estimating diagnostic accuracy with Xpert MTB/RIF assay. A summary estimation for sensitivity, specificity, diagnostic odds ratios (DOR) and the area under the summary ROC curve (AUC) was calculated by using the bivariate random-effects approach. The meta-analysis included 18 studies (10,224 suspected specimens). The summary estimate was 90.4% (95%CI 89.2%-91.4%) for sensitivity, 98.4% (95%CI 98.0%-98.7%) for specificity and 328.3/0.9822 for DOR/AUC in pulmonary tuberculosis (PTB). The sensitivity, specificity and DOR/AUC of detecting RIF-resistance were 94.1%, 97.0% and 177.8/0.9832, respectively. For extrapulmonary tuberculosis, the overall pooled sensitivity was 80.4% and specificity was 86.1%. The findings in subgroup analysis were as follows: the accuracy of Xpert MTB/RIF assay is higher in smear-positive specimens and the sensitivity of diagnosing PTB in adults was higher than that in children (90.8% versus 74.3%). TB and RIF-resistance can be rapidly and effectively diagnosed with Xpert MTB/RIF assay. Copyright © 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
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            Direct susceptibility testing for multi drug resistant tuberculosis: A meta-analysis

            Background One of the challenges facing the tuberculosis (TB) control programmes in resource-limited settings is lack of rapid techniques for detection of drug resistant TB, particularly multi drug resistant tuberculosis (MDR TB). Results obtained with the conventional indirect susceptibility testing methods come too late to influence a timely decision on patient management. More rapid tests directly applied on sputum samples are needed. This study compared the sensitivity, specificity and time to results of four direct drug susceptibility testing tests with the conventional indirect testing for detection of resistance to rifampicin and isoniazid in M. tuberculosis. The four direct tests included two in-house phenotypic assays – Nitrate Reductase Assay (NRA) and Microscopic Observation Drug Susceptibility (MODS), and two commercially available tests – Genotype® MTBDR and Genotype® MTBDRplus (Hain Life Sciences, Nehren, Germany). Methods A literature review and meta-analysis of study reports was performed. The Meta-Disc software was used to analyse the reports and tests for sensitivity, specificity, and area under the summary receiver operating characteristic (sROC) curves. Heterogeneity in accuracy estimates was tested with the Spearman correlation coefficient and Chi-square. Results Eighteen direct DST reports were analysed: NRA – 4, MODS- 6, Genotype MTBDR® – 3 and Genotype® MTBDRplus – 5. The pooled sensitivity and specificity for detection of resistance to rifampicin were 99% and 100% with NRA, 96% and 96% with MODS, 99% and 98% with Genotype® MTBDR, and 99% and 99% with the new Genotype® MTBDRplus, respectively. For isoniazid it was 94% and 100% for NRA, 92% and 96% for MODS, 71% and 100% for Genotype® MTBDR, and 96% and 100% with the Genotype® MTBDRplus, respectively. The area under the summary receiver operating characteristic (sROC) curves was in ranges of 0.98 to 1.00 for all the four tests. Molecular tests were completed in 1 – 2 days and also the phenotypic assays were much more rapid than conventional testing. Conclusion Direct testing of rifampicin and isoniazid resistance in M. tuberculosis was found to be highly sensitive and specific, and allows prompt detection of MDR TB.
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              Comparative evaluation of the nitrate reduction assay, the MTT test, and the resazurin microtitre assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis.

              To evaluate the performance of three rapid low-cost methods for the detection of resistance to first-line drugs in Mycobacterium tuberculosis. One hundred M. tuberculosis clinical isolates were tested by the nitrate reductase assay (NRA), the MTT test and the resazurin microtitre assay (REMA), and the results compared with those obtained with the gold standard proportion method (PM) on Lowenstein Jensen medium. The results using the three methods showed a good sensitivity and specificity between 94% and 100% for the detection of rifampicin and isoniazid resistance. Specificity for ethambutol and streptomycin using MTT and resazurin was low (58-89%). In contrast, NRA showed a good agreement for all first-line drugs tested. This study shows a high level of agreement of these three low-cost methods compared with the PM for rapid detection of rifampicin and isoniazid resistance. However, more standardization is needed for ethambutol and streptomycin using the MTT test and resazurin microtitre assay. The nitrate reductase assay might represent an inexpensive procedure for rapid detection of resistance to first-line drugs in low-resource countries.
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                Author and article information

                Journal
                Mem Inst Oswaldo Cruz
                Mem. Inst. Oswaldo Cruz
                Memórias do Instituto Oswaldo Cruz
                Instituto Oswaldo Cruz
                0074-0276
                1678-8060
                11 October 2013
                December 2013
                : 108
                : 8
                : 1021-1023
                Affiliations
                [1 ]Department of Medical Microbiology, Medical School, Ondokuz Mayis University, Samsun, Turkey
                [2 ]Department of Medical Microbiology, Istanbul Medical School, Istanbul University, Istanbul, Turkey
                Author notes
                + Corresponding author: yilmazden@ 123456hotmail.com
                Article
                0074-0276130697
                10.1590/0074-0276130697
                4005540
                24402143
                b7a68172-b590-466e-b9ce-00ea268e7661

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 12 December 2012
                : 9 September 2013
                Page count
                Figures: 1, Tables: 1, References: 20, Pages: NaN
                Categories
                Articles

                mycobacterium tuberculosis,malachite green decolourisation assay,susceptibility testing,isoniazid,rifampicin

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