Real Time PCR Assay for the Differentiation of Mycobacterial Species in Bronchial Washings

Abstract

Pulmonary infections caused by Nontuberculous Mycobacteria (NTM) species has to be carefully interpreted due to their ubiquitous nature. NTM infections are more common than before in nonimmunosuppressed hosts. Real-time PCR designed for Mycobacterium species, allows precise identification through melting point analysis. This study was designed for identification of Mycobacterium species present in bronchial washings. Ethical clearance was obtained from the PostGraduate Institute of Science, University of Peradeniya. Bronchial washings (n=150) were collected from patients, suspected of having pulmonary diseases, attending the General Hospital Kandy. The samples were processed according to modified Petroff's method and inoculated onto Löwenstein-Jensen medium. Culture positives were subjected to Ziehl-Neelsen (ZN) staining, DNA were extracted from AFB isolates using the standard CTAB (N Cetyl-N, N, N-trimethyl ammonium bromide) method. SYBR green mediated real-time PCR assay was conducted to identify rapid and slow growers in two parallel reactions. Primers specific for Mycobacterium genus, Mycobacterium tuberculosis complex (MTC), M. avium complex (MAC), M. chelonae-M. abscessus group (MCAG) and M. fortuitum group (MFG) were used. Among the 26 AFB isolates 25 were found to be belonging to the Mycobacterium genus. Two MTC isolates and three MAC isolates were confirmed; following reaction I. Reaction II confirmed the presence of Mycobacterium genus and the presence of MCAG for two isolates. Application of SYBR green mediated real time PCR assay in clinical microbiology could improve the diagnostics due to the increased specificity. Moreover, it is a tool that can be used for the rapid detection of pathogenic NTM species.