Rapid identification, capsular typing and molecular characterization of Streptococcus pneumoniae by using whole genome nanopore sequencing.

BACKGROUND: Whole genome sequencing has emerged as a useful tool for identification and molecular characterization of pathogens. MinION (Oxford Nanopore) is a real-time third generation sequencer whose portability, affordability and speed in data production make of it an attractive device for whole genome sequencing. The objective of this study is to evaluate MinION sequencer for pathogen identification and molecular characterization of Streptococcus pneumoniae isolated at a children's Hospital. Whole genome sequencing of 32 Streptococcus pneumoniae invasive isolates, previously characterized by standard methods (Quellung reaction, Multiplex PCR and Sanger-MLST), were performed. DNA was extracted using ZymoBIOMICS DNA Microprep kit. Quantification and purity of DNA was assessed by Qubit and Nanodrop, respectively. Library preparation was performed using the Rapid Barcoding Kit. Real-time workflow EPI2ME platform "What's it in my pot" was used for species identification. Fast5 sequences were converted into FASTQ by Albacore software. Reads were assembled using CANU software. PathogenWatch, genomic epidemiology and pubmlst online tools were used for capsular typing and/or whole genome-MLST profile. RESULTS: Rapid identification of Streptococcus pneumoniae was achieved by "What's in my pot". Capsular typing was correctly assigned with PathogenWatch in all 32 isolates at serogroup level and 24 at serotype level. Whole genome-MLST results obtained by genomic epidemiology and pubmlst were consistent with double locus variant clonal complex obtained by Sanger-MLST in 31 isolates. CONCLUSION: MinION sequencer provides a rapid, cost-effective and promising pathway for performing WGS by a pocked-sized device for epidemiological purposes but improving its sequencing accuracy will make it more appealing to be used in clinical microbiology laboratories.

Evaluation of the eazyplex MRSA assay for the rapid detection of Staphylococcus aureus in pleural and synovial fluid.

OBJECTIVE: Loop-mediated isothermal amplification (LAMP) is a simple and sensitive technique for rapid microbiological diagnosis. The aim of this study was to evaluate the analytical and diagnostic performance of the LAMP eazyplex MRSA test for the direct detection and differentiation of methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) in synovial/pleural fluid. METHODS: Analytical validation included the determination of the limit of detection (LoD) and analytical specificity of the eazyplex MRSA test. A diagnostic evaluation of the eazyplex test against bacterial culture was performed on routine pleural/synovial samples collected prospectively from patients aged less than 18 years with complicated pneumonia with empyema or arthritis admitted to the Children's Hospital Sant Joan de Déu in Barcelona, Spain, between April 2015 and May 2016. RESULTS: The new system appropriately detected a quality control panel of clinical samples with DNA of MSSA, MRSA, and other pathogens. The LoD was established at 6.4×103 CFU/ml for S. aureus and 1.0×104 CFU/ml for MRSA. Diagnostic validation of the eazyplex MRSA assay was performed on 51 prospective clinical invasive samples, resulting in sensitivity and specificity values of 83.3% and 97.8%, respectively, for S. aureus detection. The mean turnaround time was 70min. CONCLUSIONS: The eazyplex MRSA assay was found to be a useful test for the rapid detection of S. aureus in invasive samples such as pleural/synovial fluid.