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.

Fig. 1

Abundance of bacterial, parasitic and human DNA among faecal samples. For each group of samples, healthy, patients with bloody diarrhoea and patients with non-bloody diarrhoea (or unknown), the fraction of reads mapping to bacteria, parasites and human reference genomes is shown. The abundance is normalised according to the total number of reads in each specific sample

Fig. 2

Relative abundance of pathogens in samples positive by conventional diagnostics. For each pathogen (Giardia, Salmonella, Y. enterocolitica, E. coli, C. jejuni, C. difficile and Shigella), the fraction of reads mapping to the pathogen is plotted for all samples positive by conventional diagnostic methods. The orange dots indicate the presence of pathogen-specific virulence genes as determined by NGS analysis, while the green dots indicate the absence. The upper fence (Q₃ + 1.5×IQR) of the relative abundance for the healthy controls and for the diarrhoea samples where the particular pathogen was not detected by conventional methods are shown

Fig. 3

Relative abundance of pathogens in samples negative by conventional diagnostics. For samples that were either negative or virus-positive by conventional diagnostics, the fraction of reads mapping to each pathogen (Giardia, Salmonella, Y. enterocolitica, E. coli, C. jejuni, C. difficile and Shigella) was plotted. The orange dots indicate the presence of pathogen-specific virulence genes, while the green dots indicate the absence. The upper fence (Q₃ + 1.5×IQR) of the relative abundance for healthy controls and for the diarrhoea samples where the particular pathogen was not detected by conventional methods are shown

Fig. 4

Isolate detection within metagenomic samples. Reference mapping of reads from the metagenomic sample against the isolate sequence from the specific sample was employed to assess the percentage of the isolate covered by the metagenomic sequencing. Also, the fraction of reads within the metagenomic sample that were used in mapping is illustrated, as well as the total number of reads present in the metagenomic sequences

Fig. 5

Phylogenetic relationships among metagenomic samples and isolates. An NDtree is shown for E. coli. The tree was constructed by mapping isolate WGS sequences and complete metagenomic sequences against the reference E. coli O157:H7 str. Sakai. Escherichia coli pathotypes are shown in parentheses on isolates