Direct Detection of Multidrug Resistant Tuberculosis on Smear Positive Pulmonary Specimens by Using Crystal Violet Decolorization Assay

Abstract

Background: Emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) tuberculosis (TB) highlights the need for rapid, cost effective and reliable test for drug susceptibility testing. Crystal violet has antibacterial activity however; mycobacteria reduce crystal violet by its sequestration into lipid contents of cell wall. Therefore, this study was conducted to evaluate the utility of a crystal violet decolorization assay (CVDA) for the detection of isoniazid (INH) and rifampicin (RIF) resistance in smear positive pulmonary specimens. Methods & Materials: The study was carried out at department of microbiology, Armed Forces Institute of Pathology, Rawalpindi Pakistan from June 2017 through Sept 2017. 50 smear positive pulmonary specimens were simultaneously tested for INH and rifampicin RIF resistance by CVDA and MGIT 960 system (taken as gold standard). In CVDA, three tubes were used; one growth control (GC) without drug and other two were drug labeled having INH and RIF. The samples were incubated for 14 days and then on 14th day, 100 μL of CV (25 mg/L stock solution) was then added to all tubes. The tubes were then again left for incubation for an additional 48-72 hrs. Specimens sensitive to INH and RIF failed to decolorize CV where as viable isolates in specimens resistant to the respective drug decolorized CV resulting in change of color from blue/purple to colorless. Results: Out of total 50 specimens, one specimen in GC tube did not decolorize CV at the end of protocol (30 days) and therefore was excluded from the final data analysis. The sensitivity, specificity, positive predictive value, negative predictive value and agreement for INH were 82%, 100%, 100%, 100% and 100%, respectively, and 85.7%, 100%, 100%, 91.6% and 94.7%, respectively for RIF. Mean time to obtain results was 18.5 days with a range of 17–21 days. Overall agreement of CVDA was 95% when compared with MGIT 960 system. Conclusion: CVDA is rapid, cost effective, reliable as well as easy to perform drug susceptibility method for detection of MDR-TB on smear positive pulmonary specimens especially in resource starved countries. However, further studies on large scale are needed to know its usefulness in our setup.