Evaluating next-generation sequencing for direct clinical diagnostics in diarrhoeal disease

European Journal of Clinical Microbiology & Infectious Diseases, Mar 2017

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.

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Evaluating next-generation sequencing for direct clinical diagnostics in diarrhoeal disease

Eur J Clin Microbiol Infect Dis Evaluating next-generation sequencing for direct clinical diagnostics in diarrhoeal disease K. G. Joensen 0 1 2 3 5 6 A. L. ?. Engsbro 0 1 2 3 5 6 O. Lukjancenko 0 1 2 3 5 6 R. S. Kaas 0 1 2 3 5 6 O. Lund 0 1 2 3 5 6 H. Westh 0 1 2 3 5 6 F. M. Aarestrup 0 1 2 3 5 6 0 Center for Biological Sequence Analysis, Department of System Biology, Technical University of Denmark , Kgs. Lyngby , Denmark 1 Department of Medical Gastroenterology, K?ge University Hospital , K?ge , Denmark 2 Faculty of Health Sciences, University of Copenhagen , Copenhagen , Denmark 3 National Food Institute, Division for Epidemiology and Microbial Genomics, Technical University of Denmark , S?ltofts Plads, Building 221, 2800 Kgs. Lyngby , Denmark 4 F. M. Aarestrup 5 Department of Clinical Microbiology, Hvidovre University Hospital , Copenhagen , Denmark 6 Department of Microbiology and Infection Control, Statens Serum Institute , Copenhagen , Denmark 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. Introduction Diarrhoea has a global disease burden estimated to encompass 1.7 billion cases each year, with 1.5 million deaths worldwide attributed in 2012, and is the second most common cause of death in young children [ 1, 2 ]. Diarrhoea is typically a symptom of a gastrointestinal infection, but may also be a symptom of several medical conditions or a result of drug treatment, e.g. antibiotic-associated diarrhoea [3]. Diarrhoea of different infectious origin (bacterial, viral and some parasitic) may be difficult to distinguish based on history or clinical observations and, thus, rapid laboratory analyses are important, since treatment and patient care depends on the pathogen [ 1, 4, 5 ]. In addition, rapid and accurate diagnostics, characterisation and comparison of pathogens are essential to identify both nosocomial and foodborne outbreaks. However, diagnostic results are often not available in a timely fashion and current methods employed are labourious, timeconsuming, costly, require significant expertise and result in the detection of pathogens in only a small fraction of examined samples [ 4, 5 ]. Thus, conventional diagnostics typically only result in the identification of a minority of diarrhoeacausing microbial agents, while up to 80% of cases remain unresolved [6]. It is also well -known that some bacterial pathogens are difficult to grow or are even non-culturable, while still being viable [ 7 ]. Polymerase chain reaction (PCR)-based methods for the detection of enteropathogens from stool samples that are more rapid and more sensitive than the conventional culturing procedures have been described [ 8?11 ]. The disadvantage of using PCR may be that we are only detecting those agents we are looking for and it is normally not possible to obtain phylogenetic information. Next-generation sequencing (NGS) has started to gain ground in public health and clinical microbiology. NGS provides cost-efficient analysis and rapid turnaround time [ 12, 13 ]. It has already been used in clinical settings for elucidating bacterial outbreaks [ 14?16 ] and it has been proposed for the real-time typing and surveillance of pathogens [ 16?18 ]. NGS has, until recently, been employed mainly on bacterial isolates. However, as demonstrated for urinary tract infections [ 19 ], the technology can be applied directly (...truncated)


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K. G. Joensen, A. L. Ø. Engsbro, O. Lukjancenko, R. S. Kaas, O. Lund, H. Westh, F. M. Aarestrup. Evaluating next-generation sequencing for direct clinical diagnostics in diarrhoeal disease, European Journal of Clinical Microbiology & Infectious Diseases, 2017, pp. 1325-1338, Volume 36, Issue 7, DOI: 10.1007/s10096-017-2947-2