Drug Susceptibility Testing and Molecular Characterization of Mycobacterium tuberculosis Isolates from Pulmonary TB Patients at the End of Two Month Intensive Therapy in Addis Ababa, Ethiopia

Tuberculosis is one of the major health concerns worldwide which continue to exert a heavy toll on public health. Now a days, the AIDS epidemic and the growing problem of resistance to antituberculosis drugs have further complicated management of the disease. Thus, early detection of drug resistance constitutes one of the priorities of TB control programs. This project was designed to assess the drug susceptibility pattern of Mycobacterium tuberculosis isolates among patients who remained culture positive at the end of the intensive phase treatment in order to investigate the correlation between being culture positive at the second month of treatment in relation to final treatment outcome. Another aim was to establish a quality controlled method for the detection of antiTB drug resistance including second line drugs used for MDR-TB. In total, 80 consecutive clinical samples that were on treatment with first line anti-TB drugs for the intensive phase were included in the study. Susceptibility testing for firstand second line drugs was done based on a proportion method adopted for 24-well plates containing 7H10 medium. The result showed that 49 (61.2%) isolates were resistant to one or more of the first line drugs tested. MDR-TB was identified in 20% of the study participants. It was also observed that all isolates resistant to rifampicin were also isoniazid resistant supporting that rifampicin resistance could be used as surrogate marker for MDR-TB. Out of the 80 patients, 51 (63.8%) were cured and 28.7% were defined as treatment failure. A majority of MDR-TB cases (87.5%) detected at the second month were found to be treatment failures which serves as an external validation of the DST method. Moreover, it was also found that all isolates were susceptible to the major second line drugs except for ethionamide (13.8 % resistant) as expected where a cross resistance to isoniazid has been described. The quality of the 24-well 7H10 agar based method was assessed using intraand inter laboratory controls which showed that the method was highly reproducible. Spoligotyping analysis revealed that the T3_ETH (ST 149, n=23) and the CAS1_KILLI (ST 21, n=9) subfamilies were the predominant genotypes observed. One Beijing strain was also reported. MDR-TB isolates exhibited more clustering compared to drug susceptible and other drug resistant cases. Based on the above finding, culture and drug susceptibility testing for first and second line drugs using the newly developed DST method is advised for patients who remained culture or smear positive at the end of the intensive phase. The 24-well DST method was found to be easy to perform with relatively low cost and with minimal training necessary. It offers the production of hundreds of plates a day. In particular, it seems suitable for suspected MDR cases where both first line and second line drugs are necessary for providing the best therapy for the patient. This method is found to be advantageous in that it can test as many drugs as per the number of wells in a 24-well, single plate. It can also be used in determining the exact minimum inhibitory concentrations (MICs) of the drugs in addition to discriminating between resistance and susceptibility. Thus, in view of these advantages we suggest that the method should be further validated so that it could be used for routine drug susceptibility testing.