Rapid drug-susceptibility testing of Mycobacterium tuberculosis clinical isolates to first-line antitubercular drugs by nitrate reductase assay: A comparison with proportion method

Abstract

Objective/background: Early initiation of therapy in patients with tuberculosis is imperative for its control. Conventional methods of susceptibility testing such as the proportion method (PM) require visual detection and counting of colonies that takes up to 6 weeks. Rapid and simple phenotypic methods that have been endorsed by the World Health Organization can serve as alternatives. Methods: In this study, we evaluated the colorimetric nitrate reductase assay, which utilizes the detection of nitrate reduction as an indicator of growth much earlier compared with PM (within 7–14 days). The susceptibility of 75 clinical isolates of Mycobacterium tuberculosis to four first-line antitubercular drugs was tested by nitrate reductase assay and compared with the standard PM. In this assay, inoculation was done on both drug-free and drug-containing Löwenstein–Jensen medium containing sodium nitrate. After incubation for 7–14 days, reduction to nitrite was taken as an indicator of growth, which was detected by color change on addition of Griess reagent. Results: Agreement between nitrate reductase assay and PM was 100% for rifampicin, 97.30% for isoniazid, 93.30% for streptomycin, and 98.60% for ethambutol. Cost/isolate with this assay was found to be approximately two times lesser than that of PM. All results were obtained in 7–14 days by nitrate reductase assay, which was significantly rapid compared with 42 days taken for obtaining results by PM. Conclusion: Nitrate reductase assay can be used as a rapid and inexpensive method for drug-susceptibility testing of M. Tuberculosis for first-line antitubercular drugs without compromising accuracy of standard methods.

Keywords

• Drug-susceptibility testing
• Indirect nitrate reductase assay
• Indirect proportion method
• Mycobacterium tuberculosis