title: Deep sequencing approach for investigating infectious agents causing fever.
abstract: Acute undifferentiated fever (AUF) poses a diagnostic challenge due to the variety of possible aetiologies. While the majority of AUFs resolve spontaneously, some cases become prolonged and cause significant morbidity and mortality, necessitating improved diagnostic methods. This study evaluated the utility of deep sequencing in fever investigation. DNA and RNA were isolated from plasma/sera of AUF cases being investigated at Cairns Hospital in northern Australia, including eight control samples from patients with a confirmed diagnosis. Following isolation, DNA and RNA were bulk amplified and RNA was reverse transcribed to cDNA. The resulting DNA and cDNA amplicons were subjected to deep sequencing on an Illumina HiSeq 2000 platform. Bioinformatics analysis was performed using the program Kraken and the CLC assembly-alignment pipeline. The results were compared with the outcomes of clinical tests. We generated between 4 and 20 million reads per sample. The results of Kraken and CLC analyses concurred with diagnoses obtained by other means in 87.5 % (7/8) and 25 % (2/8) of control samples, respectively. Some plausible causes of fever were identified in ten patients who remained undiagnosed following routine hospital investigations, including Escherichia coli bacteraemia and scrub typhus that eluded conventional tests. Achromobacter xylosoxidans, Alteromonas macleodii and Enterobacteria phage were prevalent in all samples. A deep sequencing approach of patient plasma/serum samples led to the identification of aetiological agents putatively implicated in AUFs and enabled the study of microbial diversity in human blood. The application of this approach in hospital practice is currently limited by sequencing input requirements and complicated data analysis.
authors.affiliation: College of Medicine and Dentistry, James Cook University, PO Box 902, Cairns Hospital, Cairns, QLD, Australia. tri.susilawati@my.jcu.edu.au.|Department of Microbiology, Faculty of Medicine, Sebelas Maret University, Jl. Ir. Sutami 36 A, Surakarta, Central Java, Indonesia. tri.susilawati@my.jcu.edu.au.
authors.forename: T N
authors.initials: TN
authors.lastname: Susilawati
authors.affiliation: Population of Health and Immunity Division, Walter and Eliza Hall Institute, Parkville, VIC, Australia.|Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, Australia.|Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, Australia.
authors.forename: A R
authors.initials: AR
authors.lastname: Jex
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, UK.
authors.forename: C
authors.initials: C
authors.lastname: Cantacessi
authors.affiliation: Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Pearson
authors.affiliation: Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, Australia.
authors.forename: S
authors.initials: S
authors.lastname: Navarro
authors.affiliation: Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, Australia.
authors.forename: A
authors.initials: A
authors.lastname: Susianto
authors.affiliation: Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, Australia.
authors.forename: A C
authors.initials: AC
authors.lastname: Loukas
authors.affiliation: College of Medicine and Dentistry, James Cook University, PO Box 902, Cairns Hospital, Cairns, QLD, Australia.
authors.forename: W J H
authors.initials: WJ
authors.lastname: McBride
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001315:Australia / Q000453:epidemiology; D003141:Communicable Diseases / Q000175:diagnosis / Q000453:epidemiology* / Q000209:etiology* / Q000628:therapy; D019295:Computational Biology / Q000379:methods; D005334:Fever / Q000175:diagnosis / Q000453:epidemiology* / Q000209:etiology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008875:Middle Aged; D015203:Reproducibility of Results; D057188:Workflow; D055815:Young Adult