HYBRID-CAPTURE NEXT-GENERATION SEQUENCING FOR VIRAL DETECTION IN ACUTE FEBRILE ILLNESS OF UNKNOWN ETIOLOGY PATIENTS IN NAKHON PHANOM AND TAK PROVINCES, THAILAND

Abstract

An acute febrile illness (AFI) with an unknown etiology is a major diagnostic challenge in tropical settings.  In this study we aimed to evaluate hybrid-capture next-generation sequencing (NGS) for detecting viral pathogens in hospitalized AFI patients with negative routine diagnostic results in order to determine the kinds of viruses causing an AFI in the study locations that were missed by routine testing and the proportion of those cases that could be detected by hybrid-capture NGS testing.  We retrospectively examined previously collected serum samples obtained from patients hospitalized with AFI during 2017-2020 in Nakhon Phanom and Tak Provinces, Thailand in whom no etiology was determined and in which an adequate sample was available to conduct the specified testing.  In each sample, total nucleic acids were extracted and analyzed using a hybrid-capture NGS test with the Twist Comprehensive Viral Research Panel and interpreted using a metagenomics pipeline with predefined background-subtraction and positivity criteria.  The median age of the study subjects in whom the samples were obtained was 36 (range: 2-79) years, 60.0% females.  Viral pathogens were detected by hybrid-capture NGS in 65 (56.5%) of the 115 samples.  Cytomegalovirus (n = 29, 25.2%) and Epstein-Barr virus (n = 27, 23.5%) were the most frequently identified pathogens, followed by enterovirus A (n = 2, 1.7%), enterovirus B (n = 1, 0.9%), hepatitis B virus (n = 2, 1.7%), human herpesvirus 7 (n = 2, 1.7%), influenza A virus (n = 1, 0.9%), and Kaposi’s sarcoma-associated herpesvirus (n = 1, 0.9%).   It is important to remember easily tested for viruses, such as influenza viruses, were already tested for and found to be negative in order to be included in this study.  In summary, hybrid-capture NGS was able to determine more than half of samples previously testing negative with routine testing and the most common viruses detected were CMV and EBV.  We conclude the hybrid-capture NGS may be beneficial for evaluating viral AFI in which the etiology is unclear with routine testing.  Further studies in other populations are needed to determine when and in what situations the hybrid-capture NGS would be of most benefit in guiding management of patients with AFI of unknown etiology.