Proficiency Testing of Metagenomics-Based Detection of Food-Borne Pathogens Using a Complex Artificial Sequencing Dataset

Metagenomics-based high-throughput sequencing (HTS) enables comprehensive detection of all species comprised in a sample with a single assay and is becoming a standard method for outbreak investigation. However, unlike real-time PCR or serological assays, HTS datasets generated for pathogen detection do not easily provide yes/no answers. Rather, results of the taxonomic read assignment need to be assessed by trained personnel to gain information thereof. Proficiency tests are important instruments of validation, harmonization, and standardization. Within the European Union funded project COMPARE [COllaborative Management Platform for detection and Analyses of (Re-) emerging and foodborne outbreaks in Europe], we conducted a proficiency test to scrutinize the ability to assess diagnostic metagenomics data. An artificial dataset resembling shotgun sequencing of RNA from a sample of contaminated trout was provided to 12 participants with the request to provide a table with per-read taxonomic assignments at species level and a report with a summary and assessment of their findings, considering different categories like pathogen, background, or contaminations. Analysis of the read assignment tables showed that the software used reliably classified the reads taxonomically overall. However, usage of incomplete reference databases or inappropriate data pre-processing caused difficulties. From the combination of the participants\u2019 reports with their read assignments, we conclude that, although most species were detected, a number of important taxa were not or not correctly categorized. This implies that knowledge of and awareness for potentially dangerous species and contaminations need to be improved, hence, capacity building for the interpretation of diagnostic metagenomics datasets is necessary

Metagenomics-based proficiency test of smoked salmon spiked with a mock community

An inter-laboratory proficiency test was organized to assess the ability of participants to perform shotgun metagenomic sequencing of cold smoked salmon, experimentally spiked with a mock community composed of six bacteria, one parasite, one yeast, one DNA, and two RNA viruses. Each participant applied its in-house wet-lab workflow(s) to obtain the metagenomic dataset(s), which were then collected and analyzed using MG-RAST. A total of 27 datasets were analyzed. Sample pre-processing, DNA extraction protocol, library preparation kit, and sequencing platform, influenced the abundance of specific microorganisms of the mock community. Our results highlight that despite differences in wet-lab protocols, the reads corresponding to the mock community members spiked in the cold smoked salmon, were both detected and quantified in terms of relative abundance, in the metagenomic datasets, proving the suitability of shotgun metagenomic sequencing as a genomic tool to detect microorganisms belonging to different domains in the same food matrix. The implementation of standardized wet-lab protocols would highly facilitate the comparability of shotgun metagenomic sequencing dataset across laboratories and sectors. Moreover, there is a need for clearly defining a sequencing reads threshold, to consider pathogens as detected or undetected in a food sample

Plasmodium

Malaria is the most important parasitic disease worldwide in terms of numbers of affected people and mortality. It is caused by parasites of the genus Plasmodium, which have a complex life cycle including insect vectors that are in the case of human malaria exclusively Anopheles mosquitoes. To date the genomes of several Plasmodium species have been sequenced. The overall genome organization is rather conserved, but highly divers species-specific gene families have been identified as well. The different life cycle stages exhibit a very variable morphology reflecting their respective needs. The change in cell shape during development is genetically inherited, but epigenetic factors also appear to play an important role. In the vertebrate host cell invasion and egress are crucial steps for the survival of the parasite and have evolved to highly orchestrated events, and some molecular details have been deciphered to date. Invasion occurs by invagination of the host cell membrane, and the parasite finally resides in a parasitophorous vacuole. From there it controls the behavior of its host cell by secretion of proteins into the host cell cytoplasm and to its surface. Exposed parasite proteins at the surface of an infected red blood cell allow cytoadherence and are responsible for the pathogenicity of a Plasmodium infection. Egress is a two-step process initiated by the rupture of the parasitophorous vacuole membrane and followed by disintegration of the host cell membrane that involves the activation of proteases, kinases, and membrane lytic enzymes. Recent discoveries revealed completely new parasite strategies to switch from asexual to sexual development during the blood stage and to avoid elimination by cytosolic immune responses of host cells during infection of hepatocytes