Evaluation of Direct Detection of Mycobacterium tuberculosis Rifampin Resistance by a Nitrate Reductase Assay Applied to Sputum Samples in Cotonou, Benin

Abstract

The aim of this study was to evaluate a nitrate reductase assay (NRA) performed on smear-positive sputa for the direct detection of rifampin resistance in Mycobacterium tuberculosis. A total of 213 smear-positive sputa with a positivity score of 1؉ or more (>1 acid-fast bacillus per field by fluorescence microscopy) were used in the study. The samples were decontaminated using the modified Petroff method, and portions of the resulting suspension were used to perform the NRA. The NRA results were compared with the reference indirect proportion method for 177 specimens for which comparable results were available. NRA results were obtained at day 10 for 15 specimens (9%), results for 88 specimens (50%) were obtained at day 14, results for 66 specimens (37%) were obtained at day 18, and results for the remaining 8 specimens (4%) were obtained at day 28. Thus, 96% of NRA results were obtained in 18 days. Of the 177 specimens, there was only one discrepancy (susceptible according to the NRA and resistant according to the indirect proportion method). NRA is simple to perform and provides a rapid, accurate, and cost-effective means for the detection of rifampin resistance in M. tuberculosis isolates.Tuberculosis (TB) remains a major public health problem worldwide. In recent years, the incidence of TB has been rising. There is also an emergence of multidrug-resistant (MDR) tuberculosis (defined as resistance to at least rifampin [RMP] and isoniazid) that is worsening the impact of this disease (1,4,21).Previous studies suggest that RMP resistance could be a surrogate marker for multidrug resistance, especially in settings with a high prevalence of drug resistance (8, 18). Therefore, the detection of resistance to this major anti-TB drug is essential for the optimal control of TB.Conventional tests for the detection of drug resistance require several weeks to yield results (5). Recently, alternative rapid methods have been developed (13). Among them, the nitrate reductase assay (NRA) on Löwenstein-Jensen (LJ) medium is simple to perform and has been successfully implemented in low-income countries (7,13,14). This test is based on the ability of Mycobacterium tuberculosis to reduce nitrate to nitrite, which is revealed as a color change in the culture medium, using the Griess method (10). The indirect (using isolates) NRA yields results in less than 14 days but requires an initial 3 to 4 weeks for cultivation of the isolate (7,13,14).So far, only a few studies have evaluated the NRA applied directly to sputum samples. The results of these studies (which were done in high-incidence settings) were concordant with results obtained by the reference method (15, 17). However, to our knowledge, no direct NRA study has been done in a setting with low resistance prevalence or in Africa, where there is potentially a high frequency of nitrate reductase-negative M. tuberculosis complex strains (11,12).The objective of this study was to evaluate NRA applied directly to smear-positive sputa in the West African country of Benin in orde...

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.…”

“…Such situation is worsened by the increasing incidence of multidrug resistant tuberculosis (MDR-TB), caused by strains resistant to at least isoniazid (INH) and rifampicin (RIF), and of extensively resistant tuberculosis (XDR-TB) caused by a MDR-TB strain also resistant to fluoroquinolone and to at least one of the following antimicrobial agents: kanamycin (KAN), amikacin (AMK) and/or capreomycin (CAP). Tuberculosis caused by resistant strain requires an expensive, longer and less effective treatment (1).…”

Evaluation of Mycobacterium Tuberculosis Drug Susceptibility Testing by Direct Nitrate Reductase Assay

“…So, a cost-effective and rapid drug susceptibility method is required to guide the treatment of TB. 1,2,6,7 We evaluated the results of direct NRA Method for first-line anti-tubercular drugs, i.e. RIF, INH, STR, and EMB in 120 sputum samples (3+).…”

“…1 Drug resistance, multidrug resistance (MDR) and extensive drug resistance (XDR) in tuberculosis is a matter of great concern for TB control programs since there is no cure for some multidrug-resistant TB strains of M. tuberculosis. 2 There is concern that these strains could spread around the world, stressing the need for additional control measures, such as new diagnostic methods, better drugs for treatment, and a more effective vaccine. Hence, there is need for diagnosis of the resistant strains early.…”

Rapid culture-based methods for drug-resistance detection in Mycobacterium tuberculosis

“…An interesting possibility of the NRA is its direct application on sputum samples. This has been accomplished recently in three different studies performed in middle-and low-income settings with encouraging results for detecting resistance to RIF and INH (Musa et al, 2005;Solis et al, 2005;Affolabi et al, 2007).…”