Systematic review and meta-analysis of the nitrate reductase assay for drug susceptibility testing of Mycobacterium tuberculosis and the detection limits in liquid medium

•We selected 20 reports on six major anti-TB drugs and conducted a meta-analysis.•Ethambutol, streptomycin, and kanamycin showed relatively low sensitivity.•In this study, we propose a quantitative standard for determining sample positivity.

Recently, the need for rapid, reliable, and low-cost drug susceptibility testing (DST) methods has increased due to the emergence of multidrug-resistant Mycobacterium tuberculosis. Colorimetric methods of DST provide results more quickly than standard culture methods and are inexpensive than molecular methods. Thus, colorimetric methods, such as the nitrate reductase assay (NRA), are being recommended. We searched Medline PubMed for reports on the NRA for DST of M. tuberculosis written in English and published within the last five years. We selected 20 reports on six major anti-TB drugs and conducted a meta-analysis using Meta-Disc software. The pooled sensitivities for isoniazid, rifampicin, streptomycin, ethambutol, ofloxacin, and kanamycin were 95.4%, 96.4%, 91.5%, 93.1%, 99.3%, and 88.4%, and the pooled specificities were 98.5%, 99.2%, 92.9%, 97.8%, 97.4%, and 99.4%, respectively. The area under the summary receiver operator curve for all drugs was 0.9723-0.9952. The time to results (TTR) for the direct and indirect NRAs was 7-28 days and 6-15 days, respectively. Quality assessments were conducted using the quality of diagnostic accuracy studies tool (QUADAS-2) items, and most reports showed good performance. However, ethambutol, streptomycin, and kanamycin showed relatively low sensitivity. We performed a quantitative NRA in liquid media at various inoculum concentrations. The TTR at 4.94 × 106, 1.67 × 104, and 2.27 × 102 CFU/mL was 4, 14, and 14 days, respectively. The minimum absorbance and nitrite concentration for positive samples were 0.8 and 168 μM, respectively. We propose a quantitative standard to determine sample positivity to address the problems with the current standard NRA which is much less expensive than the conventional assay conducted on solid medium.