Evaluation of direct microplate nitrate reductase assay as a rapid method for the detection of multiple and extensively tuberculosis drug resistance

Abstract

Introduction: Reports of Mycobacterium tuberculosis resistant to multiple drugs are increasing globally and laboratories are becoming increasingly aware of the need for drug susceptibility testing. In recent years, due to the long time required by conventional drug susceptibility testing, new approaches have been proposed for faster detection of drug resistance, such as the nitrate reductase assay, considered fast and inexpensive, making it a good diagnostic tool for low resource countries.

Objective: The present study proposed a fast direct colorimetric drug susceptibility testing method in a microplate format using solid medium.

Materials and methods: The diagnostic accuracy was evaluated by comparing the proportion method with the direct nitrate reductase assay in plates. Frozen sputum samples, known to be positive, were decontaminated and processed by Petroff method. The decontaminated suspension was used to perform direct nitrate reductase assay in 7H11 medium using 1 μg/ml rifampicin (RIF), 0.2 μg/ml isoniazid (INH), 2 μg/ml ofloxacin (OFX), 6 μg/ml kanamycin (KAN), 2 μg/ml amikacin (AMK) and 10 μg/ml capreomycin (CAP). Eighty-four samples were tested and the results for 69% of them were available within 21 days.

Results: The sensitivity and specificity compared to the proportion method, was 98.5% and 100% for INH, 98.3% and 96.2% for RIF, 91.7% and 100% for KAN, 78.8% and 97.3% for OFX, 100% and 100% for AMK and CAP, respectively.

Conclusion: The results lead to the conclusion that direct nitrate reductase assay, in this new format, is an accurate, quick and inexpensive method to determine the susceptibility profile of M. tuberculosis and may become an alternative for countries with limited resources.