UTILITY OF PCR ASSAY IN DIAGNOSIS OF PULMONARY TUBERCULOSIS AND IDENTIFICATION OF MUTATIONS ASSOCIATED WITH ANTIMYCOBACTERIAL RESISTANCE BY DNA SEQUENCING

Abstract

Tuberculosis describes an infectious disease that has plagued humans since the ancient times. It has been affecting man for at least 5.000 years. (Stark, 2000). The emergence of anti-tuberculous drug resistance, especially multidrug resistant tuberculosis (MDR-TB), poses a serious threat to the success of TB control programs. [Suresh et al., 2006]. This study aimed to evaluate peripheral blood based PCR technique in the diagnosis of active pulmonary tuberculosis . It also aimed to use manual DNA sequencing technique for sequencing 3 selected genes. To achieve this goal, 40 patients were selected from those who were diagnosed to have pulmonary tuberculosis by their history ,clinical examination , radiological finding and laboratory criteria. The main laboratory criteria for selection was their +ve smear stained by Z.N for sputum samples showing AFB. All patients had history of treatment failure with RIF and INH for at least 6 months. 20 healthy blood donors were chosen as a control group. PCR results showed 100% specificity and 100% sensitivity.15 samples from those which were PCR +ve for IS6110 gene were randomly selected where manual DNA sequencing was done for 3 selected genes : rpoB , katG and inh-A gene. Our results revealed that mutant rpoB genes were 93% versus 7% which were non mutant. The sequenced katG genes showed 100% point 87% of manually sequenced inh-A genes were mutant versus only 13% which were non mutant.