Comparative evaluation of the Nitrate Reductase Assay and the Resazurin Microtitre Assay for drug susceptibility testing of Mycobacterium tuberculosis against first line anti-tuberculosis drugs

Abstract

Tuberculosis remains as a serious infection disease of worldwide distribution, with high morbidity and mortality, mainly in low socio-economic condition countries. The state of emergency of tuberculosis caused by the resistant and multidrug-resistant (MDR) strains, became the main threat to the tuberculosis treatment and control programs. A fast detection method for the resistant strains will allow the implementation of an adequate treatment and contribute for controlling the dissemination of these resistant strains. This study evaluated the performance of the nitrate reductase assay in solid (NRA-LJ) and liquid (NRA-7H9) media, to determine the susceptibility to first line anti-tuberculosis drugs: isoniazid (INH), rifampicin (RMP), ethambutol (EMB) and streptomycin (SMR). Both methods NRA-LJ and NRA-7H9 were evaluated among 18 strains with a known susceptibility profile. The resazurin microtiter assay (REMA) was performed as a reference method. One hundred percent of accordance was observed between NRA-7H9 and REMA for the four tested drugs. When the NRA-LJ method was compared to REMA, the sensitivity and the specificity to INH, RMP, EMB and SMR were 100%, 100 %, 85.7%, 76.9% and 80%, 100%, 75% and 80%, respectively. From the 57 clinical isolates of M. tuberculosis evaluated by NRA-7H9 and REMA, 56 (98.2%) were sensitive to all antibiotics tested (INH, RMP, EMB and SMR) by the NRA-7H9 method, while three of these strains were resistant to INH by REMA. One strain showed resistance to INH and RMP for both methods, and MIC of 1.0 μg/ml to INH for both methods, while MIC of 1.0 and 2.0 μg/ml to RMP for REMA and NRA-7H9, respectively. The three assays showed a high level of agreement for rapid detection of rifampicin and isoniazid resistance. Regarding rapidness, the detection of color change in the NRA method is within instants as compared to the overnight incubation required for the REMA test. NRA might represent an inexpensive and alternative assay for rapid detection of resistance in low-income countries.

Growth kinetics of Mycobacterium tuberculosis measured by quantitative resazurin reduction assay: a tool for fitness studies

“…These strains originated from the Laboratory of Mycobacteria at the Universidade Federal do Rio Grande in Rio Grande, Brazil. The profile of resistance to the first-line drugs (rifampicin, isoniazid, ethambutol and streptomycin) was determined by nitrate reductase assay (12). One strain (06-235) was found multidrug-resistant (MDR), that is, resistant to rifampicin and isoniazid.…”

Comparative evaluation of the microplate nitrate reductase assay and the rezasurin microtitre assay for the rapid detection of multidrug resistant Mycobacterium tuberculosis clinical isolates

“…Syre et al (2003) performed the nitrate reductase assay using the macrodilution method and reported that the sensitivity and specificity were 100% and 94% for INH and 95% and 100% for RIF; their results were obtained on the 5th day. Sanchotene et al (2008) performed the nitrate reductase assay using the microdilution method and suggested that this assay could be used as a rapid and low-cost method, particularly in developing countries.…”

Characterization of Mycobacterium tuberculosis complex isolated from iranian and afghani patients by spoligotyping method

“…Multidrug-resistance TB, defined as simultaneous resistance to the two most important drugs, INH and RIF, is a potential threat to tuberculosis control (29). Patients infected with strains resistant to multiple drugs are extremely difficult to cure, and the necessary treatment is much more toxic and expensive (27).…”