title: Evaluating next-generation sequencing for direct clinical diagnostics in diarrhoeal disease.
abstract: The accurate microbiological diagnosis of diarrhoea involves numerous laboratory tests and, often, the pathogen is not identified in time to guide clinical management. 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 system. Species distribution was determined with MGmapper and NGS-based diagnostic prediction was performed based on the 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 the responsible pathogens in five of the 11 conventionally negative samples. Overall, the NGS-based approach enabled pathogen detection comparable to conventional diagnostics and the approach has potential to be extended for the detection of all pathogens. At present, however, this approach is too expensive and time-consuming for routine diagnostics.
authors.affiliation: National Food Institute, Division for Epidemiology and Microbial Genomics, Technical University of Denmark, Søltofts Plads, Building 221, 2800, Kgs. Lyngby, Denmark.|Department of Microbiology and Infection Control, Statens Serum Institute, Copenhagen, Denmark.
authors.forename: K G
authors.initials: KG
authors.lastname: Joensen
authors.affiliation: Department of Clinical Microbiology, Hvidovre University Hospital, Copenhagen, Denmark.|Department of Medical Gastroenterology, Køge University Hospital, Køge, Denmark.
authors.forename: A L Ø
authors.initials: ALØ
authors.lastname: Engsbro
authors.affiliation: National Food Institute, Division for Epidemiology and Microbial Genomics, Technical University of Denmark, Søltofts Plads, Building 221, 2800, Kgs. Lyngby, Denmark.
authors.forename: O
authors.initials: O
authors.lastname: Lukjancenko
authors.affiliation: National Food Institute, Division for Epidemiology and Microbial Genomics, Technical University of Denmark, Søltofts Plads, Building 221, 2800, Kgs. Lyngby, Denmark.
authors.forename: R S
authors.initials: RS
authors.lastname: Kaas
authors.affiliation: Center for Biological Sequence Analysis, Department of System Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.
authors.forename: O
authors.initials: O
authors.lastname: Lund
authors.affiliation: Department of Clinical Microbiology, Hvidovre University Hospital, Copenhagen, Denmark.|Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: H
authors.initials: H
authors.lastname: Westh
authors.affiliation: National Food Institute, Division for Epidemiology and Microbial Genomics, Technical University of Denmark, Søltofts Plads, Building 221, 2800, Kgs. Lyngby, Denmark. fmaa@food.dtu.dk.
authors.forename: F M
authors.initials: FM
authors.lastname: Aarestrup
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D002675:Child, Preschool; D003718:Denmark; D003967:Diarrhea / Q000175:diagnosis*; D005243:Feces / Q000382:microbiology / Q000469:parasitology / Q000821:virology; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006785:Hospitals, University; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D055815:Young Adult