Evaluation of Unbiased Next-Generation Sequencing of RNA (RNA-seq) as a Diagnostic Method in Influenza Virus-Positive Respiratory Samples

Abstract

e Unbiased nontargeted metagenomic RNA sequencing (UMERS) has the advantage to detect known as well as unknown pathogens and, thus, can significantly improve the detection of viral, bacterial, parasitic, and fungal sequences in public health settings. In particular, conventional diagnostic methods successfully identify the putative pathogenic agent in only 30% to 40% of respiratory specimens from patients with acute respiratory illness. Here, we applied UMERS to 24 diagnostic respiratory specimens (bronchoalveolar lavage [BAL] fluid, sputum samples, and a swab) from patients with seasonal influenza infection and 5 BAL fluid samples from patients with pneumonia that tested negative for influenza to validate RNA sequencing as an unbiased diagnostic tool in comparison to conventional diagnostic methods. In addition to our comparison to PCR, we evaluated the potential to retrieve comprehensive influenza virus genomic information and the capability to detect known superinfecting pathogens. Compared to quantitative real-time PCR for influenza viral sequences, UMERS detected influenza viral sequences in 18 of 24 samples. Complete influenza virus genomes could be assembled from 8 samples. Furthermore, in 3 of 24 influenza-positive samples, additional viral pathogens could be detected, and 2 of 24 samples showed a significantly increased abundance of individual bacterial species known to cause superinfections during an influenza virus infection. Thus, analysis of respiratory samples from known or suspected influenza patients by UMERS provides valuable information that is relevant for clinical investigation. Influenza has a severe impact on our health system, not only owing to its potential to cause worldwide pandemics but also due to the high number of seasonal infections. Bacterial and/or viral coinfections and subsequent pneumonia can lead to enhanced illness in elderly and immunosuppressed patients. Approximately 0.5% of all influenza A infections in healthy younger adults and 2.5% of influenza A infections in the elderly and younger children are accompanied by severe bacterial-induced pneumonia (1, 2). These numbers are significantly higher during pandemic episodes (3-5). The most common causes of coinfections observed in both pandemic and seasonal episodes of influenza A infections are Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, and Streptococcus pyogenes (6, 7). This relationship between influenza virus and bacterial pathogenicity is underlined by several studies using animal models. For example, mice infected with influenza virus or Streptococcus pneumoniae alone showed mortality rates of 35% and 15%, respectively, whereas mice coinfected with influenza virus and Streptococcus pneumoniae displayed a 100% mortality rate (8). Furthermore, several studies in humans indicate that colonization with Streptococcus pneumoniae increases the risk of severe complications associated with influenza A viral infection (9, 10), thus highlighting the importance of rapid diagnosis of bacterial coinfecti...

Chronic equine hepacivirus infection in an adult gelding with severe hepatopathy

“…After depletion of host sequences, de novo assembly of contigs was performed (Fischer et al . , ; Baechlein et al . ).…”

Metagenomics Study of Viral Pathogens in Undiagnosed Respiratory Specimens and Identification of Human Enteroviruses at a Thailand Hospital

“…Frequent emergence, reemergence, migration, and even unpredicted introduction of novel or significantly mutated viral pathogens are a considerable challenge to clinical diagnostics which target known pathogens. The robust next-generation sequencing (NGS) technology has the potential to improve underdiagnoses and identify unknown pathogens cost-effectively 6,7. NGS is advancing toward practical clinical use for sequence-based diagnostics 8–10…”

Translational metagenomics and the human resistome: confronting the menace of the new millennium

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