However, up to now data assessing sensitivity and specificity of specific mutations for the detection of drug resistance phenotypes in our settings is still unavailable. Therefore CANTAM (Central Africa Network for Tuberculosis, HIV/AIDS and Malaria) an EDCTP (European and Developing Clinical BMS345541 mouse Trials Partnership) funded network [19], with the goal to establish a cohort and prepare new sites for conducting future clinical trials of new TB drugs and vaccines in Central Africa countries, carried out a population based study, involving MTBC

strains from Central region of Cameroon, to determine the genetic basis of first line drug resistance. Methods Mycobacterial isolates During this baseline study carried out between April 2010 and March 2011, 725 smear positive pulmonary tuberculosis patients were enrolled at Jamot Hospital and Mbalmayo District Hospital. All positive cultures were tested for drug susceptibility to INH (0.2 μg/ml and 1 μg/ml), SU5402 mouse RIF (40 μg/ml), EMB (2 μg/ml),

SM (4 μg/ml), OFX (2 μg/ml) and KAN (20 μg/ml) by the indirect proportion method on Lowenstein Jensen medium [20]. Phenotypically, 44 isolates were INHR (24 high level and 20 low level), 27 isolates were SMR, 7 isolates were RIFR and 2 isolates were EMBR. The 63 resistant isolates to INH, RIF, SM and EMB or MDR were screened for genetic mutations. In addition, M. tuberculosis strain H37Rv (susceptible) and 100 fully susceptible clinical isolates from the panel of susceptible strains collected during the study period were included to serve as controls. The study was approved by the Cameroon National Ethic Committee and the Cameroonian Ministry of Public Health. Written informed consent was obtained from all

study subjects. DNA extraction Briefly, a loop-full of mycobacterial Astemizole colonies was suspended in 400 μl of 10 mM Tris–HCl, 1 mM EDTA (pH 7.0) buffer and inactivated at 90°C for 30 min. The suspension was then centrifuged at 12,000 g for 1 min and the supernatant, containing nucleic acids, was harvested and transferred into a new eppendorf tube. Crude DNA extracts were stored at -20°C and then shipped to Germany for molecular analysis according to International Air Transport KU-57788 concentration Association guidelines. PCR amplification of target genes The DNA extract was used as a template for PCR with the primers listed in Table 1. Each final PCR volume of 20 μl contained 10× PCR buffer (Qiagen, Germany), 5% DMSO, 20 pmol of forward and 20 pmol of reverse primers, 11.9 μl of distilled water, 0.5 μl MgCl2 25 mM (Qiagen, Germany), dNTPs at a final concentration of 500 μM, 0.2 μl of Taq polymerase 5 U/μL (Qiagen, Germany), and 2 μl of crude DNA extract (≈50 ng). The cycling program included a cycle of an initial denaturation step at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at the temperature and time indicated in Table 1, and elongation at 72°C for 1 min. The final elongation step was set at 72°C for 10 min for one cycle.