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.
Highlights
Acute undifferentiated fever (AUF) is caused by a variety of causes, producing a range of clinical manifestations with acute fever as a unifying symptom
Analysis with the CLC Genomics Workbench revealed that Kraken-unclassified reads still contained human sequences, accounting for 18.9– 81.7 % of the total contigs in each sample
Non-host contigs classified by Basic Local Alignment Search Tool (BLAST) analysis included viruses, bacteria and other organisms, such as archaea (e.g. Sulfolobus sp.), fungi (e.g. Saccharomyces sp., Cryptococcus sp., Penicillium sp.), algae (e.g. Navicula gregaria), plants, protozoa (e.g. Toxoplasma gondii, Plasmodium berghei), human parasites and larger animals
Summary
Acute undifferentiated fever (AUF) is caused by a variety of causes, producing a range of clinical manifestations with acute fever as a unifying symptom. Most clinicians and researchers define acute fever as evidence of raised body temperature to ≥38 °C for ≤3 weeks, without detection of systemic disease or the focus of infection or inflammation after initial clinical evaluation and basic laboratory investigations [1]. This condition poses a diagnostic challenge for clinicians due to nonspecific clinical features and the indistinctive profile of routine blood tests. The limitation of the current diagnostic approach causes a significant proportion of fever to go undiagnosed. The frequencies of undiagnosed AUFs in Asian tropical countries ranges from 8 to 80 % [1]
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