Drug susceptibility test of Mycobacterium tuberculosis by nitrate reductase assay

Abstract

Background: Drug susceptibility testing for Mycobacterium tuberculosis (M. tuberculosis) is especially required in difficult cases of tuberculosis (TB) chemotherapy and in cases of multidrug resistance (MDR-TB; combined resistance to isonizid and rifampicin with or without resistance to any other drug). The methods for in vitro cultivation and drug susceptibility testing (DST) of M. tuberculosis are cumbersome and not readily adaptable in most routine laboratories, particularly those in the developing world due to limited resources and lack of political will in those countries. A simple and cost effective method, the nitrate reductase assay (NRA), was compared with the gold standard proportion (egg bases Lowenstein Jensen’s [LJ]) method for DST of M. tuberculosis in order to substantiate its suitability for routine use in Nigeria and in other countries of the developing world with high TB endemicity. Method: Drug susceptibility test was performed for 70 pulmonary isolates of M. tuberculosis (Indirect DST) and 20 sputum (10 acid fast bacilli [AFB] positive and 10 AFB negative) specimens (direct DST) by the NRA and the proportion method using 0.2μg isoniazid (INH), 2μg ethambutol (EMB), 40 μg rifampicin (RIF) and 4 μg streptomycin STR). Results: The indirect NRA showed sensitivity and specificity for INH: 100% and 100%, EMB: 75% and 100% RIF: 90% and 96.6%, STR: 66.6% and 91.8%. The results of direct NRA and proportion method for INH, EMB RIF and STR agreed 10/10 (100%) for AFB negative specimens and 9/10 (90%) with AFB positive specimens. Conclusion: Drug susceptibility test of M. tuberculosis by the NRA is simple and sensitive with shorter turn around time of 10 to 1 4 days compared to 42 days by the LJ proportion method. The direct use of AFB positive sputum specimens is likewise reproducible and excludes about 3 – 8 weeks period required for isolation of M. tuberculosis