Evaluation of Direct Colorimetric Mtt Assay for Detection of Rifampicin and Isoniazid Resistance in Mycobacterium Tuberculosis Under Program Condition
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Date
2007-07-03
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Addis Ababa University
Abstract
The spread of drug resistant tuberculosis especially MDR-TB in the world remains a major public health problem. Early diagnosis of TB and rapid detection of drug resistance is an urgent priority for proper patient management and to control dissemination of resistant strains. Simple, rapid and inexpensive methods of detecting drug resistant TB are essential for effective treatment. The MTT assay has been developed as a rapid, simple and inexpensive method for the detection of rifampicin (RIF) resistant M. tuberculosis with promising results. However, the method was not evaluated under program condition for the detection of RIF resistance and not yet standardized and evaluated for the detection of isoniazid (INH) resistance. The objectives of this study were thus to evaluate the direct MTT assay for detection of RIF resistance in
M. tuberculosis, and to standardize and evaluate the method for INH resistance under program condition. Sputum samples were decontaminated with 4% NaOH and cultured on Lowenstein-Jensen (LJ) media and Middlebrook 7H9 (M7H9) broth media. Drug susceptibility test was done on LJ media using the proportion method. MTT assay was done on M7H9 broth containing 0.2 µg/ml INH, 2 µg/ml RIF and drug free control. Formazan production was quantified by measuring the optical density (OD) at 570 nm and relative optical density unit (RODU) was calculated as a ratio of drug containing tube to drug free control. A strain was defined as resistant if it has a RODU> 0.5 and susceptible if it has RODU< 0.2. Out of 115 isolates tested for RIF resistance, 6 were identified as RIF resistant using the MTT assay but 7 using the proportion method. 0.2 µg/ml INH concentration was taken as a critical concentration to be used for direct MTT assay for detection of INH resistance. Out of 18 isolates tested for INH resistance, 2 were found INH resistant both with proportion method and MTT assay. The direct MTT assay result matched 99% for RIF and 100% for INH with the conventional method. The MTT assay gave 94% interpretable result for rifampicin and 100% for isoniazid at 2nd week. In the conventional method it takes 7-14 weeks to get a final drug susceptibility test result. Therefore, this rapid, simple and inexpensive method could be an alternative method for drug susceptibility test under program condition. Further studies are required in regional labs to validate the method.
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Keywords
Mycobacterium Tuberculosis, Mdr-Tb, Mtt Assay, Proportion Method, Isoniazid, Rifampicin