Mycobacterium Tuberculosis Klinik İzolatlarında İlaç Direncinin Multipleks Real-Time Pcr Yöntemiyle Saptanması

Abstract

Tuberculosis (TB) is stil one of the most serious threats to human health around the world. Increasing of the drug resistance is one of the main cause of this state. Early diagnosis of drug-resistant TB cases is urgently needed to prevent the transmission of resistant strains and to optimize treatment regimens. The purpose of this study is to detect the mutations causing resistance to rifampicin (RIF), isoniazide (INH) and ethambutol (EMB) in M. tuberculosis complex (MTBC) isolates by using multiplex real-time PCR and melting curve analysis. By using this method, resistance to RIF, INH and EMB can be detected by three PCR assays in just two hours. In this study 50 MTBC strains were included. For detection of RIF resistance we used two probes (rpoP1 and rpoP2) for rpoB 81-bp RIF resistance determining region in a tube. For detection of INH resistance, we used two dually labeled probes for katG315 and inhA promoter sides in a tube. For detection of EMB resistance, we used one probe in another tube. For detecting the mutation types, DNA sequencing procedure was performed for 16 isolates. Comparision of the results with MGIT and sequencing data showed that all mutations in rpoB, katG and inhA covered by this four probes were detected with 100% sensitivity and 100% specifity. For detecting EMB resistance, our method showed 92% sensitivity and 97% specificity. This method provided a new way to rapidly detect drug-resistant mutations in MTBC. On the other hand, the susceptibility of 50 clinical isolates of MTBC to pyrazinamide (PZA) was assessed by the MGIT 960 system. Detection of PZA resistance was followed by a repeat testing using a reduced inoculum of 0.25 ml instead of 0.5 ml. According to the first MGIT 960 analysis, resistance was observed in 41 samples. In the reduced inoculum assay, 2 samples turned out to be susceptible and 39 proved to be resistant (5% different results). Further studies are required to confirm our findings.