Target-independent high-throughput sequencing methods provide evidence that already known human viral pathogens play a main role in respiratory infections with unexplained etiology

Abstract

Despite the advanced PCR-based assays available, a fraction of the pediatric respiratory infections remain unexplained every epidemic season, and there is a perception that novel viruses might be present in these specimens. We systematically collected samples from a prospective cohort of pediatric patients with respiratory infections, that returned negative results by validated molecular RT–PCR assays, and studied them with a target-independent, high-throughput sequencing-based approach. We also included a matched cohort of children with no symptoms of respiratory infection, as a contrast study population. More than fifty percent of the specimens from the group of patients with unexplained respiratory infections were resolved. However, the higher rate of detection was not due to the presence of novel viruses, but to the identification of well-known viral respiratory pathogens. Our results show that already known viral pathogens are responsible for the majority of cases that remain unexplained after the epidemic season. High-throughput sequencing approaches that use pathogen-specific probes are easier to standardize because they ensure reproducible library enrichment and sequencing. In consequence, these techniques might be desirable from a regulatory standpoint for diagnostic laboratories seeking to benefit from the many advantages of these sequencing technologies.

A Founder Effect Led Early SARS-CoV-2 Transmission in Spain

“…The first of these methods was a hybrid capture-based enrichment protocol. Clinical samples were processed and amplified by sequence-independent, singleprimer-amplification reverse transcription-PCR (SISPA RT-PCR) as described in a previous work (28), and the preparation of libraries and probe hybridization reactions were performed according to the Twist SARS-CoV-2 research panel instructions (Twist Bioscience Corporation, CA, USA). The second approach employed was genome amplification using the ARTIC network's PCR protocol based on the use of the pool of primers "ARTIC n-CoV-2019 v3" (29).…”

Detection of respiratory viruses directly from clinical samples using next‐generation sequencing: A literature review of recent advances and potential for routine clinical use

“…Six out of 52 (11.5%) studies used Sanger's sequencing methods to confirm the NGS results. 16,37,38,44,50,77 Three studies (5.8%) performed real-time quantitative reverse transcription PCR (qRT-PCR) assays on the same specimens to confirm the NGS results. 39,73,78 Two studies (3.8%) attempted to perform two NGS platforms on same specimens and use the result of one platform to validate the result of the other.…”

“…47 The first strategy was to employ ultracentrifugation, sample filtration, or nuclease-treatment method to enrich viral particles before the nucleic acid extraction step. 44,[48][49][50][51] One study attempted to utilise combinations of filtration and nuclease treatment to obtain higher genome sequence coverage and enough reads to make a single nucleotide polymorphism (SNP) analysis of the H7N9 virus. 44 In contrast, another study selected the centrifugation approach instead of the filtration approach to increase the proportion of viral reads (H3N2 virus) derived from clinical samples because of its low cost and easy handling.…”

“…Studies have documented that the metagenomic sequencing method based on NGS technologies usually can be used to detect multiple respiratory viruses simultaneously from clinical samples. 16,37,48,50,63,[67][68][69][70][71][72][73][74][75][76] These include RSV, HPIV, HAdV, HRV, HMPV, and HBoV. Researchers can obtain partial or even complete genome sequences of each above virus and provide detailed subtyping information by using the NGS technologies.…”

Current Trends in Diagnostics of Viral Infections of Unknown Etiology

“…Furthermore, some respiratory viruses (e.g., Rhinoviruses) frequently appear to be the only or the most abundant pathogens in samples from patients with respiratory infections [64]. This pattern implies that either the diagnostic tools are erroneous [65][66][67][68] or the groups of related viruses cause infections with a variable clinical manifestation [69]. Metagenomics reveals actual etiological factors, allowing for genotyping, identifying antibiotic resistance markers [70] and supplying data molecular epidemiology [71].…”