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

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.This work was supported by The Joint Science and Technology Office for Chemical and Biological Defense (JSTO-CBD) of the Defense Threat Reduction Agency (DTRA) under plan #CB10246. This study has been partially supported by Fondo de Investigaciones Sanitarias (FIS) – Spanish Health Research Fund Grant PI12/01291, and Instituto de Salud Carlos III (ISCIII), Grant PI15CIII/00028.S

Y155H amino acid substitution in influenza A(H1N1)pdm09 viruses does not confer a phenotype of reduced susceptibility to neuraminidase inhibitors

The Y155H amino acid substitution in the neuraminidase gene (NA) has previously been associated with highly reduced inhibition by neuraminidase inhibitors in the seasonal H1N1 influenza A virus which circulated in humans before the 2009 pandemic. During the 2012/13 epidemic season in Spain, two A(H1N1) pdm09 viruses bearing the specific Y155H substitution in the NA were detected and isolated from two patients diagnosed with severe respiratory syndrome and pneumonia requiring admission to the intensive care unit. Contrary to what was observed in the seasonal A(H1N1) viruses, neither of the Y155H A(H1N1) pdm09 viruses described here showed a phenotype of reduced inhibition by NAIs as determined by the neuraminidase enzyme inhibition assay (MUNANA). High-throughput sequencing of the NA of both Y155H viruses showed that they were composed to >99% of H155 variants. We believe that this report can contribute to a better understanding of the biological significance of amino acid substitutions in the neuraminidase protein with regard to susceptibility of influenza viruses to neuraminidase inhibitors. This is of critical importance for optimal management of influenza disease patients.We would like to acknowledge Dr. Angel Zaballos Sanz at the Genomics Unit of the National Centre for Microbiology of the Carlos III Health Institute and the Next Generation Sequencing service at the Cantoblanco Genomics Unit of the Madrid Scientific Park for their support, and all the members of the Spanish Influenza Surveillance System working in the identification and declaration of patients during the 2012/13 influenza season. We gratefully acknowledge the authors, originating and submitting laboratories of the sequences from GISAID EpiFlu Database which have been included in this research. This work was supported by Instituto de Salud Carlos III (Programa especial de investigación sobre la gripe pandémica) GR09/0040. Sara Monzon received a research contract funding by the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER U758). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.S

Respiratory Infections by Enterovirus D68 in Outpatients and Inpatients Spanish Children.

The incidence of enterovirus D68 (EV-D68) and the spectrum of clinical disease in children are not well known in European countries. We have designed a study with the objective of describing the clinical impact of EV-D68 detected in children with respiratory tract infections. As a part of a prospective study to identify the etiology and clinical characteristics of viral respiratory infections in children in Spain, we performed the analysis of the cases of EV infections in all children hospitalized in a secondary hospital in Madrid, during the epidemic respiratory season 2012-2013. A second group of samples was corresponded to infants of the same area, with ambulatory respiratory infection or asymptomatic. Phylogenetic EV-D68 analysis was made using the viral protein 1 gene (VP1). Clinical data of EV-D68 patients were compared with those infected by rhinovirus in the same period and population. The study population consisted of 720 patients corresponding to 399 episodes of hospitalization for respiratory causes, 44 episodes of ambulatory respiratory infections and 277 children determined as a healthy control group. A total of 22 patients were positive for EVs (3.05%), and 12 of them were specifically typed as EV-D68 (11/443 respiratory infections, 2.5%). The most frequent diagnosis in the 10 hospitalized children with EV-D68 detection was recurrent wheezing. Hypoxia was present in 70% of cases, but admission in the intensive care unit was not required. No neurological signs or symptoms were observed. One patient had an ambulatory mild bronchiolitis and another was asymptomatic. No differences were found with rhinovirus infections except less duration of hypoxia and fever in EV-D68 group. EV-D68 infections were detected in 3.05% of respiratory studied samples (2.5% of admissions). The infection was associated with wheezing episodes with hypoxia. No admissions to intensive care unit or neurological symptoms were found.This study has been partially supported by FIS (Fondo de Investigaciones Sanitarias – Spanish Health Research Fund) Grants No: PI12/01291 and a Research grant from the Spanish Association of Pediatrics.S