Novel mutations in the resistome of a new sequence type (ST262) of clarithromycin resistant Mycobacterium abscessus subsp. massiliense.

OBJECTIVES: The aim of this study was to evaluate the relationship between antimicrobial susceptibility and genotype of a Mycobacterium abscessus subsp. massiliense isolate obtained from the respiratory tract of a patient in southern Brazil. METHODS: The isolate (named Myco1POA) was submitted to whole-genome sequencing using an Illumina MiSeq platform. Data were analysed using Trim Galore!, SPAdes Genome Assembler, Geneious and BioEdit software. Multilocus sequence typing (MLST) was performed by in silico analysis of seven housekeeping genes according to the Institut Pasteur database. The antimicrobial susceptibility profile was determined by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: Several mutations in genes related to antimicrobial resistance were identified in Myco1POA. MLST indicated that the isolate belonged to a novel sequence type (ST), which was designated ST262. Phenotypic susceptibility and genotypic findings were concordant, except for clarithromycin [erm(41) and rrl genes]. CONCLUSION: Here we describe the genome sequence of M. abscessus subsp. massiliense Myco1POA identified as a novel sequence type (ST262) and indicate possible new gene mutations leading to clarithromycin resistance.

Evaluation of respiratory syncytial virus group A and B genotypes among nosocomial and community-acquired pediatric infections in Southern Brazil.

BACKGROUND: Respiratory syncytial virus (RSV) is the main cause of lower respiratory tract illness in children worldwide. Molecular analyses show two distinct RSV groups (A and B) that comprise different genotypes. This variability contributes to the capacity of RSV to cause yearly outbreaks. These RSV genotypes circulate within the community and within hospital wards. RSV is currently the leading cause of nosocomial respiratory tract infections in pediatric populations. The aim of this study was to evaluate the G protein gene diversity of RSV amplicons. METHODS: Nasopharyngeal aspirate samples were collected from children with nosocomial or community-acquired infections. Sixty-three RSV samples (21 nosocomial and 42 community-acquired) were evaluated and classified as RSV-A or RSV-B by real-time PCR. Sequencing of the second variable region of the G protein gene was performed to establish RSV phylogenetics. RESULTS: We observed co-circulation of RSV-A and RSV-B, with RSV-A as the predominant group. All nosocomial and community-acquired RSV-A samples were from the same phylogenetic group, comprising the NA1 genotype, and all RSV-B samples (nosocomial and community-acquired) were of the BA4 genotype. Therefore, in both RSV groups (nosocomial and community-acquired), the isolates belonged to only one genotype in circulation. CONCLUSIONS: This is the first study to describe circulation of the NA1 RSV genotype in Brazil. Furthermore, this study showed that the BA4 genotype remains in circulation. Deciphering worldwide RSV genetic variability will aid vaccine design and development.

Optimization of one-step duplex real-time RT-PCR for detection of influenza and respiratory syncytial virus in nasopharyngeal aspirates.

Viruses are major contributors to acute respiratory infection-related morbidity and mortality worldwide. The influenza (IF) viruses and human respiratory syncytial virus (RSV) play a particularly important role in the etiology of acute respiratory infections. This study sought to standardize a one-step duplex real-time RT-PCR technique to optimize diagnosis of IFA/IFB and RSVA/RSVB infection. Viral RNA was extracted with the commercially available QIAamp Mini Kit according to manufacturer instructions. RT-PCR was performed with primers to the matrix protein gene of IFA, the hemagglutinin gene of IFB and the N gene of RSVA and RSVB. The limits of detection were 1 copy/µL for IFA, 10 copies/µL for IFB, 5 copies/µL for RSVA, and 250 copies/µL for RSVB. The specificity of RT-PCR was determined by comparison against a panel of several respiratory pathogens. RT-PCR and indirect immunofluorescence (IIF) were compared in a sample of 250 nasopharyngeal aspirates (NPAs) collected during the year 2010. RT-PCR was more sensitive than IIF and able to detect viral co-infections. In summary, RT-PCR optimized for IFA/IFB and RSVA/RSVB is sensitive and specific for these viral agents and is therefore useful for assessment of the etiology of respiratory infections, whether for clinical or epidemiological purposes.