Retrospective use of next-generation sequencing reveals the presence of Enteroviruses in acute influenza-like illness respiratory samples collected in South/South-East Asia during 2010–2013

BACKGROUND: Emerging and re-emerging respiratory pathogens represent an increasing threat to public health. Etiological determination during outbreaks generally relies on clinical information, occasionally accompanied by traditional laboratory molecular or serological testing. Often, this limited testing leads to inconclusive findings. The Armed Forces Research Institute of Medical Sciences (AFRIMS) collected 12,865 nasopharyngeal specimens from acute influenza-like illness (ILI) patients in five countries in South/South East Asia during 2010–2013. Three hundred and twenty-four samples which were found to be negative for influenza virus after screening with real-time RT-PCR and cell-based culture techniques demonstrated the potential for viral infection with evident cytopathic effect (CPE) in several cell lines. OBJECTIVE: To assess whether whole genome next-generation sequencing (WG-NGS) together with conventional molecular assays can be used to reveal the etiology of influenza negative, but CPE positive specimens. STUDY DESIGN: The supernatant of these CPE positive cell cultures were grouped in 32 pools containing 2–26 supernatants per pool. Three WG-NGS runs were performed on these supernatant pools. Sequence reads were used to identify positive pools containing viral pathogens. Individual samples in the positive pools were confirmed by qRT-PCR, RT-PCR, PCR and Sanger sequencing from the CPE culture and original clinical specimens. RESULTS: WG-NGS was an effective way to expand pathogen identification in surveillance studies. This enabled the identification of a viral agent in 71.3% (231/324) of unidentified surveillance samples, including common respiratory pathogens (100/324; 30.9%): enterovirus (16/100; 16.0%), coxsackievirus (31/100; 31.0%), echovirus (22/100; 22.0%), human rhinovirus (3/100; 3%), enterovirus genus (2/100; 2.0%), influenza A (9/100; 9.0%), influenza B, (5/100; 5.0%), human parainfluenza (4/100; 4.0%), human adenovirus (3/100; 3.0%), human coronavirus (1/100; 1.0%), human metapneumovirus (2/100; 2.0%), and mumps virus (2/100; 2.0%), in addition to the non-respiratory pathogen herpes simplex virus type 1 (HSV-1) (172/324; 53.1%) and HSV-1 co-infection with respiratory viruses (41/324; 12.7%).