Evaluating the Potential of using Next-Generation Sequencing for Direct Clinical Diagnostics of Faecal Samples from Patients with Diarrhoea

Diarrhoea is a major global disease burden and rapid, precise identification of the causative pathogens is important to initiate patient treatment and to discover and handle potential outbreaks. The current diagnostics involve numerous procedures and often the pathogen is not identified in time to guide clinical management. Furthermore, in many clinical cases pathogens are never identified. With next-generation sequencing (NGS) becoming cheaper it has huge potential in routine diagnostics. The aim of this study was to evaluate the potential of NGS-based diagnostics through direct sequencing of faecal samples. Fifty-eight clinical faecal samples were obtained from patients with diarrhoea as part of the routine diagnostics at Hvidovre University Hospital, Denmark. Ten samples from healthy individuals were also included. DNA was extracted from faecal samples and sequenced on the Illumina MiSeq. Species distribution was determined with MGmapper (http://cge.cbs.dtu.dk/services/MGmapper/) and NGS-based diagnostic prediction was performed based on relative abundance of pathogenic bacteria and Giardia and detection of pathogen-specific virulence genes. NGS-based diagnostic results were compared to conventional findings for 55 of the diarrhoeal samples; 38 conventionally positive for bacterial pathogens, two positive for Giardia, four positive for virus and 11 conventionally negative. The NGS-based approach enabled detection of the same bacterial pathogens as the classical approach in 34 of the 38 conventionally positive bacterial samples, and predicted responsible pathogens in five of 11 conventionally negative samples. Overall, the NGS-based approach enabled pathogen detection comparable to the conventional diagnostics, and the approach has potential to be extended for detection of all pathogens. At present, however, this approach is too expensive and time-consuming for routine diagnostics.