Drug susceptibility testing of Mycobacterium tuberculosis by a nitrate reductase assay applied directly on microscopy-positive sputum samples.

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.

Rapid and inexpensive drug susceptibility testing of Mycobacterium tuberculosis with a nitrate reductase assay.

Multidrug-resistant tuberculosis is an increasing public health concern in many parts of the world, especially in low-income countries, where most cases occur. Traditional drug susceptibility testing is either time-consuming, such as the proportion method on solid media, or expensive, such as the BACTEC 460 system. We have evaluated a new nitrate reductase assay (NRA) that depends on the ability of Mycobacterium tuberculosis to reduce nitrate to nitrite. The reduction can be detected using specific reagents, which produce a color change. We tested a panel of 57 M. tuberculosis strains with various resistance patterns. The bacteria were inoculated on Löwenstein-Jensen medium, either without drugs or with rifampin, isoniazid, streptomycin, or ethambutol and with potassium nitrate (KNO(3)) incorporated. After incubation for 7, 10, or 14 days, the reagents were added and nitrate reduction, indicating growth, could be detected by a color change. Sensitivities to and specificities for drugs as determined by the NRA method compared to those determined by the BACTEC 460 method were 100 and 100% for rifampin, 97 and 96% for isoniazid, 95 and 83% for streptomycin, and 75 and 98% for ethambutol, respectively. The results were in the majority of the cases available in 7 days. The evaluated method is rapid and inexpensive and could correctly identify most resistant and sensitive M. tuberculosis strains. It has the potential to become an interesting alternative to existing methods, such as the proportion and BACTEC methods, particularly in resource-poor settings.