Drug Susceptibility Testing of Mycobacterium tuberculosis by a Nitrate Reductase Assay Applied Directly on Microscopy-Positive Sputum Samples

Abstract

Current methods for drug susceptibility testing of Mycobacterium tuberculosis are either costly or slow. As the prevalence of multidrug-resistant strains increases, the need for fast, reliable, and inexpensive methods that can also be applied in settings with scarce resources is obvious. We evaluated a rapid colorimetric nitrate reductase assay (NRA) for direct drug susceptibility testing of M. tuberculosis directly from clinical sputum samples with positive microscopy results for acid-fast bacilli with more than 10 acid-fast bacilli per high-power field. We have saved valuable time by omitting the preisolation step. The sensitivity (ability to detect true drug resistance) and specificity (ability to detect true drug susceptibility) of the direct NRA, using the direct proportion method as the reference, were 100 and 100%, 93 and 100%, 76 and 100%, and 55 and 99% for rifampin, isoniazid, streptomycin, and ethambutol, respectively, when tested on M. tuberculosis strains present in 121 samples. The results were in most cases available in 14 days. The direct NRA could be used as a rapid, inexpensive, and accurate method to determine rifampin and isoniazid susceptibility directly from sputum. The technique might become a valid alternative to traditional methods, especially in low-income countries.There is no doubt that the global tuberculosis epidemic remains a problem for public health, and there is also an emergence of multidrug-resistant tuberculosis that is worsening the impact of this disease (10,19). In order to fight this situation, we need a rapid and inexpensive drug susceptibility test to allow a prompt initiation of correct antibiotic therapy.With traditional methods, such as the indirect or direct proportion method, it could take 3 to 4 weeks to obtain susceptibility results (4, 8). The time needed to obtain these results represents a potential danger to patients, health workers, and the community (11, 16). Using liquid medium-based culture systems such as the BACTEC 460 TB system (Becton Dickinson, Sparks, Md.), the Mycobacteria Growth Indicator Tube MGIT 960 (Becton Dickinson, Cockeysville, Md.), BacT/ ALERT 3D (bioMérieux, Durham, NC), or ESP Culture System II (Trek Diagnostics, Inc., Westlake, Ohio) to perform indirect susceptibility tests (which require a pure culture of Mycobacterium tuberculosis), the results are available anywhere after 9 to 30 days. If susceptibility tests are performed directly from clinical specimens with these systems, the time needed for results is between 4 and 23 days (2,3,6,15,18). Although rapid, these methods require expensive substrates and equipment and are therefore not feasible in most developing countries. A recent article (1) described a rapid and inexpensive nitrate reductase assay (NRA) for the drug susceptibility testing (DST) of M. tuberculosis. This technique is based on the property of M. tuberculosis to reduce nitrate to nitrite, which is revealed as a color change of the culture media, using the Griess method (1, 7). With the NRA, it was possible to obtain ...

Evaluation of the Commercial Kit SIRE Nitratase for detecting resistant Mycobacterium tuberculosis in Brazil

“…The concordance (Kappa) between the gold standard and the Kit SIRE Nitratase was very good for streptomycin, isoniazid, and rifampicin and moderate for ethambutol. These discrepant results for ethambutol have been reported in previous studies 8,10 . The Kit SIRE Nitratase was easy to perform and provided an affordable alternative method for the rapid detection of resistance to first-line antituberculous drugs in the 40 clinical M. tuberculosis strains.…”

“…Additionally, the analytical performance of the Kit SIRE Nitratase demonstrates that it could reduce the turnaround time for drug susceptibility testing if applied directly to sputum smear-positive samples, as previously described 10 . Thus, studies evaluating the direct application of the SIRE Nitratase assay to sputum smear positive samples are necessary and will be performed in the near future.…”

“…The accuracy values determined in this study are similar to those reported by other studies using noncommercial NRA 2,4,8,11 . A recent meta-analysis 11 comprised of 38 studies showed that the SIRE Nitratase method exhibits high sensitivity and specificity in identifying isoniazid and rifampicin resistance 10 . Both the sensitivity and specificity values for ethambutol (variation of 55%-75% and 75%, respectively) and streptomycin (variation of 92-100% and 78%-99%, respectively) 11 were lower in the present study.…”

Evaluación de la prueba de la nitrato reductasa directa en microplaca para la detección rápida de tuberculosis multirresistente y extensivamente resistente a fármacos

“…Our results confirm previous findings with direct nitrate reductase assay carried out with INH, RIF, STR and ethambutol (EMB) (1,6,12,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). The sensitivity and specificity varied between 87-100%, 87.5-100%, 66.6-100%, 55-100%, respectively.…”

Rapid detection of multidrug-resistant Mycobacterium tuberculosis in Cotonou (Benin) using two low-cost colorimetric methods: resazurin and nitrate reductase assays

“…However, these strains are rare in our setting (Affolabi et al, 2007b) and could be tested by REMA. NRA has recently been applied directly in sputum samples with good results (Musa et al, 2005;Solis et al, 2005;Affolabi et al, 2007b). Application of REMA directly on sputa is not described so far; however, it could also represent a good option for the rapid detection of MDR-TB.…”