Development of multiplex real-time PCR for detection of clarithromycin resistance genes for the Mycobacterium abscessus group.

Introduction. The M. abscessus molecular identification and its drug-resistance profile are important to choose the correct therapy.Aim. This work developed a multiplex real-time PCR (mqPCR) for detection of clarithromycin resistance genes for the Mycobacterium abscessus group.Methodology. Isolates received by Adolfo Lutz Institute from 2010 to 2012, identified by PCR restriction enzyme analysis of a fragment of the hsp65 gene (PRA-hsp65) as M. abscessus type 1 (n=135) and 2 (n=71) were used. Drug susceptibility test (DST) for CLA were performed with reading on days 3 and 14. Subespecies identification by hsp65 and rpoB genes sequencing and erm(41) and rrl genes for mutation detection and primer design were performed. erm(41) gene deletion was detected by conventional PCR. Primers and probes were designed for five detections: erm(41) gene full size and with deletion; erm(41) gene T28 and C28; rrl gene A2058.Results. In total, 191/206 (92.7 %) isolates were concordant by all methods and 13/206 (6.3 %) were concordant only between molecular methods. Two isolates (1.0 %) were discordant by mqPCR compared to rrl gene sequencing. The mqPCR obtained 204/206 (99.0 %) isolates in agreement with the gold standard, with sensitivity and specificity of 98 and 100 %, respectively, considering the gold standard method and 92 and 93 % regarding DST.Conclusion. The mqPCR developed by us proved to be an easy-to-apply tool, minimizing time, errors and contamination.

Evaluation of drug susceptibility in nontuberculous mycobacteria using the SLOMYCO and RAPMYCO sensititre plates.

Background: Non-tuberculous Mycobacteria (NTM) cause different forms of diseases. According to recent guideline by ATS/ERS/ESCMID/IDSA, drug susceptibility test (DST) is an important requirement to choose adequate treatment. The minimum inhibitory concentration (MIC) test is the recommended method. Sensititre SLOMYCO and RAPMYCO commercial panels were developed to perform mycobacteria DST easier. However, there are only two comparative studies between SLOMYCO and the MIC method and none for the RAPMYCO panel. The present study aimed to evaluate the Sensititre SLOMYCO and RAPMYCO plates in determining drug susceptibility compared to the gold standard method (MIC). Methods: The tests were carried out with clinical isolates received in the diagnostic routine of the Tuberculosis Laboratory at Institute Adolfo Lutz from the most frequent species in the state of São Paulo, Brazil. Reference strains were tested for repeatability and reproducibility analyses. MIC and Sensititre plates readings were compared with and without resazurin stain. Agreement between results was defined as MIC within the same dilution or dilution variation resulting the same category in both tests. Results were classified by categorical errors. Results: The RAPMYCO panel had 100% agreement for the drugs amikacin, doxycycline, ciprofloxacin and trimethoprim/sulfamethoxazole, 83.3% for clarithromycin and moxifloxacin and 60% for cefoxitin. The SLOMYCO panel had 80% agreement for amikacin and moxifloxacin and 60% for clarithromycin, rifabutin, rifampicin and ciprofloxacin. The repeatability and reproducibility with RAPMYCO and SLOMYCO plates showed a high level of agreement for the drugs tested, being higher with the use of resazurin. However, an evaluation on routine condition is needed. Conclusions: The present study found that the fewer steps in the tests with Sensititre plates and reading with resazurin allow its use with greater safety and efficiency in the laboratory routine. The results presented here will facilitate the execution of a validation for complete incorporation of Sensititre plates into a diagnostic routine.