Proficiency Testing of Virus Diagnostics Based on Bioinformatics Analysis of Simulated In Silico High-Throughput Sequencing Data Sets.

Quality management and independent assessment of high-throughput sequencing-based virus diagnostics have not yet been established as a mandatory approach for ensuring comparable results. The sensitivity and specificity of viral high-throughput sequence data analysis are highly affected by bioinformatics processing using publicly available and custom tools and databases and thus differ widely between individuals and institutions. Here we present the results of the COMPARE [Collaborative Management Platform for Detection and Analyses of (Re-)emerging and Foodborne Outbreaks in Europe] in silico virus proficiency test. An artificial, simulated in silico data set of Illumina HiSeq sequences was provided to 13 different European institutes for bioinformatics analysis to identify viral pathogens in high-throughput sequence data. Comparison of the participants' analyses shows that the use of different tools, programs, and databases for bioinformatics analyses can impact the correct identification of viral sequences from a simple data set. The identification of slightly mutated and highly divergent virus genomes has been shown to be most challenging. Furthermore, the interpretation of the results, together with a fictitious case report, by the participants showed that in addition to the bioinformatics analysis, the virological evaluation of the results can be important in clinical settings. External quality assessment and proficiency testing should become an important part of validating high-throughput sequencing-based virus diagnostics and could improve the harmonization, comparability, and reproducibility of results. There is a need for the establishment of international proficiency testing, like that established for conventional laboratory tests such as PCR, for bioinformatics pipelines and the interpretation of such results.

Meta-genomic analysis of toilet waste from long distance flights; a step towards global surveillance of infectious diseases and antimicrobial resistance.

Human populations worldwide are increasingly confronted with infectious diseases and antimicrobial resistance spreading faster and appearing more frequently. Knowledge regarding their occurrence and worldwide transmission is important to control outbreaks and prevent epidemics. Here, we performed shotgun sequencing of toilet waste from 18 international airplanes arriving in Copenhagen, Denmark, from nine cities in three world regions. An average of 18.6 Gb (14.8 to 25.7 Gb) of raw Illumina paired end sequence data was generated, cleaned, trimmed and mapped against reference sequence databases for bacteria and antimicrobial resistance genes. An average of 106,839 (0.06%) reads were assigned to resistance genes with genes encoding resistance to tetracycline, macrolide and beta-lactam resistance genes as the most abundant in all samples. We found significantly higher abundance and diversity of genes encoding antimicrobial resistance, including critical important resistance (e.g. blaCTX-M) carried on airplanes from South Asia compared to North America. Presence of Salmonella enterica and norovirus were also detected in higher amounts from South Asia, whereas Clostridium difficile was most abundant in samples from North America. Our study provides a first step towards a potential novel strategy for global surveillance enabling simultaneous detection of multiple human health threatening genetic elements, infectious agents and resistance genes.