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.

Desenvolvimento de método rápido de detecção de gene de resistência a claritromicina para o grupo Mycobacterium abscessus por PCR em tempo real / Development of a rapid method for detecting the clarithromycin resistance gene for the Mycobacterium abscessus group by real-time PCR

O grupo Mycobacterium abscessus (MAG) é a principal causa de infecções pulmonares e extrapulmonares entre as micobactérias de crescimento rápido. Para determinar a resistência à claritromicina (CLA), o método de concentração inibitória mínima (CIM) é realizado, no entanto, as leituras são feitas após três e 14 dias de incubação para detectar a resistência induzida (RI). A suscetibilidade a CLA é detectada pela deleção ou polimorfismo no gene erm(41). Quando o genótipo é selvagem (T28), a RI à claritromicina é verificada, enquanto para C28 não é observada. O presente estudo tem como objetivo padronizar um ensaio Taqman de PCR em tempo real (qPCR) para detecção do polimorfismo T28C e compará-lo aos resultados obtidos do sequenciamento do gene erm(41). O total de 207 isolados de MAG que preencheram os critérios bacteriológicos da American Thoracic Society, foi recebido no IAL entre 2010 e 2012. Os isolados foram identificados pelo método PRA-hsp65 como M. abscessus tipo 1 (n=135, 65,2%) e M. abscessus tipo 2 (n=72, 34,8%) e submetidos ao CIM para CLA com leitura nos dias três e 14. A PCR convencional foi utilizada para detecção da deleção; o sequenciamento do gene erm(41) foi realizado para detectar a mutação T28C; o sequenciamento dos genes rpoB e hsp65 foram utilizados para identificação das subespécies e o sequenciamento do gene rrl foi feito para detecção da mutação A2058G (resistência adquirida). A sequência de MAG EU590129 foi submetida ao Centro de Treinamento da empresa Thermo Fisher Scientific, a qual customizou o ensaio e forneceu os parâmetros de corrida. Dos 207 isolados, 33 eram M. a. massiliense, 136 M. a. abscessus e 38 M. a. bolettii pelo sequenciamento do gene rpoB, que apresentou sete (3,38%) isolados discordantes em relação ao sequenciamento do gene hsp65. Nenhum isolado apresentou mutação no gene rrl. No sequenciamento de erm(41), 197 isolados foram T28 e 10 C28, assim como na qPCR. Na comparação dos perfis genéticos com o perfil de suscetibilidade foram encontrados 10 isolados com resultados discordantes (4,8%), sendo nove isolados sensíveis com gene erm(41) selvagem e um...(AU)

The Mycobacterium abscessus (MAG) group is the main cause of pulmonary and extrapulmonary infections among fast-growing mycobacteria. To determine resistance to clarithromycin (CLA), the minimum inhibitory concentration (MIC) method is performed, however, readings are taken after three and 14 days of incubation to detect induced resistance (IR). Susceptibility to CLA is detected by deletion or polymorphism in the erm (41) gene. When the genotype is wild (T28), clarithromycin IR is checked, while for C28 it is not observed. The present study aims to standardize a SNP Taqman real-time PCR (qPCR) assay for the detection of the T28C polymorphism and compare it to the results obtained from the sequencing of the erm(41) gene. A total of 207 MAG isolates that met the bacteriological criteria of the American Thoracic Society, were received at the IAL between 2010 and 2012. The isolates were identified by the PRA-hsp65 method as M. abscessus type 1 (n=135, 65.2%) or M. abscessus type 2 (n=72, 34.8%) and submitted to CIM for CLA with reading on days three and 14. Conventional PCR was used to detect the deletion; sequencing of the erm (41) gene was performed to detect the T28C mutation; the sequencing of the rpoB and hsp65 genes were used to identify the subspecies and the sequencing of the rrl gene was done to detect the A2058G mutation (acquired resistance). The sequence of MAG EU590129 was submitted to the Training Center of the company Thermo Fisher Scientific, which customized the test and provided the running parameters. Of the 207 isolates, 33 were M. a. massiliense, 136 M. a. abscessus and 38 M. a. bolettii by sequencing the rpoB gene, that demonstrated discrepancy between seven isolates (3.38%) compared to sequencing the hsp65 gene. No isolate showed a mutation in the rrl gene. In the erm(41) sequencing, 197 isolates were T28 and 10 C28, as well as in qPCR. When comparing the genetic profiles with the susceptibility profile, 10 isolates were found with discordant results (4,8%), with nine sensitive isolates with wild erm(41) gene and an isolate with erm(41) deletion presenting IR. The qPCR agreement with the sequencing of the erm (41) gene was 100%. The sensitivity and specificity of qPCR was 100%. QPCR has less reaction time, less chance of error and contamination and has easy interpretation of results, decreasing waiting for diagnosis by doctor and patient. (AU)