Identification of Mycobacterium Tuberculosis Complex, Using Molecular Methods

Abstract Background and Objective: A high level of homogeneity observed within all bacteria in the Mycobacterium tuberculosis complex makes a property that seriously challenges traditional biochemical-based identification methods of these pathogens in the laboratory. The work presented here was conducted to characterize Mycobacterium tuberculosis complex isolates in Golestan, Northern Iran. Material and Methods: Between 2008 and 2010, 42 mycobacterial isolates were collected from clinical tuberculosis-suspected patients in Golestan province. The isolates were sub-cultured on fresh Mycobacterium-specific culture media including glycerinated and pyruvated Lowenstein-Jensen slopes. The isolates were subsequently subjected to a PCR-based identification scheme coined Huard-Warren method. This strategy consisted of three individual algorithms namely, 16SrRNA RV typing (Rv0577, Rv3877.8, Rv1970, Rv3120, Rv1510 and IS1561) and RD typing (RD1, RD 4, RD9 and RD12). Results: All isolates were proved to be M. tuberculosis. Furthermore, none of the patients were being infected with any other member of the M. tuberculosis complex or simultaneously co-infected with two mycobacteria. This fundamental observation was independently obtained by specific culture media, RV typing and also RD typing. Conclusion: Considering the fact that cattle and sheep farming play an important role in the economy of the region, absence of Mycobacterium bovis in the studied isolates can be unexpected to some extent. Huard-Warren which is a simple and cost-effective identification method can be used in both reference and regional laboratory for differential diagnosis of tuberculosis. Keywords: Mycobacterium Tuberculosis Complex, Huard-WarrenMethod, 16SrRNA, Golestan Province, RD Typing, RV Typing