Development of identification and fast detection of clarithromycin resistance genes method for Mycobacterium abscessus group by real time PCR

Abstract

Mycobacterium abscessus is the fast-growing mycobacteria (MCR) specie that most cause lung and extrapulmonary infections. This microorganism has the ability to persist in hospital settings and is one of the most resistant species. The drug clarithromycin is the basis of combined therapy against M. abscessus infections, However, therapeutic response is associated with mechanisms of induced and acquired resistance. For phenotypic resistance to clarithromycin to be determined, is necessary to perform the standardized test of the minimum inhibitory concentration (MIC) method, with three-day reading. However, since reading in three days does not detect induced resistance and this result may lead to false sensitivity to clarithromycin, a second extended reading is required for 14 days. Extended reading increases the waiting time of the diagnosis by the patient under combination therapy. In clinical laboratories, testing should be rapid and accurate, with the lowest possible cost. In order to reduce the time to perform these tests and provide laboratory bases for the establishment of correct treatment, this work aims to develop a method based on real-time PCR (multiplex) for identification of the group M. abscessus and rapid detection of erm (41) and rrl resistance genes from clinical isolates referred to the Adolfo Lutz Institute between 2010 and 2012. The susceptibility tests for clarithromycin will be performed by MIC method. The isolates identified as M. abscessus by the PRA-hsp65 method will be submitted to rpoB gene sequencing to identify the three subspecies of the M. abscessus group and the erm (41) and rrl genes to identify mutations. The real-time PCR will be developed based on the analysis of erm (41) and rrl gene sequences by the design of primers and probes and validation will be performed by comparing the results between conventional PCR and real-time PCR. (AU)