Metagenomic sequencing of clinical human samples for zoonotic viral etiology in Madagascar

Institut Pasteur of Madagascar is the national reference laboratory for influenza, measles, rubella, polioviruses, rabies, arboviruses, mycobacteria, and plague, receiving clinical specimens from public hospitals across 114 districts. We present results from an initial screening using metagenomic Next Generation Sequencing (mNGS) techniques of for 323 samples (104 nasopharyngeal swabs, 86 serum samples, and 95 whole blood samples) collected in part with routine surveillance in Madagascar. The bulk of these samples (>90%) represent undiagnosed fevers, which [reviousy assayed negative using standard clinical PCR- and ELISA-based screening techniques. We assayed this subset of samples as part of our training as recent recipients of the Nill & Melinda Gates Foundation Grand Challenges Explorations award “Applications of Next Generation Sequencing for Pathogen Detection.” Nasopharyngeal (NP) swabs returned the highest quality results in our dataset, yielding high quality viral hits (including several complete genomes) for common human respiratory fevers, including rhinoviruses, respiratory syncytial viruses (RSV), and betacoronaviruses. Ten of eleven positive control samples were accurately identified from the NP swabs. Serum and whole blood samples were severely degraded as a result of poor sample storage procedures and multiple thaw-refreeze cycles upstream of acquisition of the samples. As a result, no RNA-virus positive controls were accurately identified from these samples, but DNA pathogens, including multiple Plasmodium spp. infections were reliably sequenced. Additionally, previously unrecognized pegiviruses were identified in these samples. In part with this Gates Grand Challenges award, we are also simultaneously sequencing blood, urine, and fecal samples derived from known bat reservoirs of circulating henipaviruses and filoviruses in Madagascar. Though no zoonotic agents have yet been identified in human or bat samples, betacoronavirus sequences from both taxa cluster together in phylogenetic analysis. Metagenomic Next Generation Sequencing offers a promising platform for unbiased assay and identification of the etiology of disease in undiagnosed clinical cases.