Andrés Antón

Molecular epidemiology and molecular characterization of respiratory syncytial viruses at a tertiary care university hospital in Catalonia (Spain) during the 2013-2014 season

BACKGROUND Human respiratory syncytial virus (HRSV) is the main cause of lower respiratory tract infections among infants and young children. OBJECTIVES The molecular epidemiology and characterization of HRSV strains detected at a Spanish tertiary hospital during the 2013-2014 season is reported. STUDY DESIGN Phylogenetic analysis and molecular characterization of HRSV laboratory-confirmed respiratory samples were performed, based on coding sequences of G and F proteins. RESULTS HRSV infection was laboratory-confirmed in respiratory samples from 320 patients of which 223 (70%) were less than 2 years of age and none undergoing Palivizumab treatment. HRSV was detected at varying levels throughout the season with a maximum rate in the week 52/2013, right before the beginning of the influenza epidemic. Whilst both HRSV groups were found co-circulating, HRSV-B group clearly predominated. The phylogenetic analyses from 139 HVR-2 sequences revealed that most characterized strains belonged to ON1 and BA9 genotypes. Three different phylogenetic subgroups could be distinguished within these genotypes. In addition, three strains (out of the 52 randomly selected) were carrying amino acid substitutions in the epitope A of the F protein, one of them previously related to Palivizumab resistance. CONCLUSIONS The results of the present study highlight the importance of a continuous HRSV surveillance to monitor not only the introduction of new genotypes on circulation but also the emergence of viral variants with new genetic characteristics that can affect the antigenicity features and the susceptibility to the only current prophylaxis treatment and also for the future development of HRSV vaccines.

First Enterovirus D68 (EV-D68) cases detected in hospitalised patients in a tertiary care university hospital in Spain, October 2014

Abstract Several outbreaks of Enterovirus 68 (EV-D68) have recently been reported in the USA and Canada, causing substantial hospitalisation of children with severe respiratory disease. The acute flaccid paralysis detected in the USA and Canada among children with EV-D68 infection has raised concerns about the aetiological role of this EV serotype in severe neurological disease. The circulation of EV-D68 in the general European population seems to be low, but European Centre for Disease Prevention and Control (ECDC) recommends being vigilant to new cases, particularly in severely ill hospitalised patients. In October 2014, enteroviruses were detected in respiratory samples collected from five hospitalised patients, children and adults. Phylogenetic analysis of partial VP1 sequences confirmed that the detected enteroviruses belonged to the D68 serotype, which were also similar to strains reported in USA (2014). However, all five patients developed respiratory symptoms, but only one required ICU admission. None of the patients described had symptoms of neurological disease. Other considerations related to the detection methods used for the diagnosis of respiratory enteroviruses are also discussed. In conclusion, additional evidence has been provided that supports the role of EV-D68 in respiratory infections in hospitalised patients.

A molecular epidemiological study of human parainfluenza virus type 3 at a tertiary university hospital during 2013-2015 in Catalonia, Spain.

Abstract Human parainfluenza virus type 3 (HPIV-3) is one of the most common respiratory viruses particularly among young children and immunocompromised patients. The seasonality, prevalence and genetic diversity of HPIV-3 at a Spanish tertiary-hospital from 2013 to 2015 are reported. HPIV-3 infection was laboratory-confirmed in 102 patients (76%, under 5 years of age). Among <5 years-old patients, 9 (11.5%) were under any degree of immunosuppression, whereas this percentage was significantly higher (19; 79.2%) among patients older than 5 years. HPIV-3 was detected at varying levels, but mainly during spring and summer. All characterized HN/F sequences fell within C1b, C5 and in other two closely C3a-related groups. Furthermore, a new genetic lineage (C1c) was described. Genetic similarity and epidemiological data confirmed some nosocomial infections, highlighting the importance of the HPIV-3 surveillance, particularly in high-risk patients. This study provides valuable information on HPIV-3 diversity due to the scarce information in Europe.

Circulation of a novel human respiratory syncytial virus Group B genotype during the 2014–2015 season in Catalonia (Spain)

Human respiratory syncytial virus (HRSV) is one of the most common viral aetiological agents in the youngest population. In the present study a novel HRSV-B BA genotype is first described based on the phylogenetic analysis of the coding hypervariable region 2 sequences of G protein from strains detected during the 2014-2015 season. Among all strains detected in the last season, 44% belonged to this new genotype. Therefore, it highlights the importance of a continuous HRSV surveillance to monitor the emergence and spread of new genotypes or variants with genetic changes that may affect antigenic and tropism features.

Evaluation of Seegene Allplex Respiratory Panel 1 kit for the detection of influenza virus and human respiratory syncytial virus

Abstract Background Influenza (FLUV) and human respiratory syncytial (HRSV) viruses are etiological agents of respiratory infections that cause a significant morbidity and mortality worldwide. A rapid and accurate diagnosis of these respiratory viruses is essential for an appropriate patient management. Molecular tests are the best detection option due to their high sensitivity and specificity. Seegene’s Allplex™ Respiratory Panel 1 (Allplex RP1) is a real-time one-step RT-PCR assay for the simultaneous detection of FLUAV, FLUBV, HRSV-A and HRSV-B. In addition, it allows the determination of FLUAV subtype (H1, H3 and H1pdm09). Objectives This study aims to evaluate Allplex RP1 as a rapid molecular test for the detection of FLUAV, FLUBV, HRSV-A and HRSV-B viruses. Study design The Allplex RP1 assay will be compared with other two commercial molecular assays, Prodesse ProFlu+ and ProFAST+ (Hologic, Madison, WI, USA), and GeneXpert Flu/RSV XC (Cepheid, USA). Results Allplex RP1, ProFlu+ and GeneXpert tests showed 95%, 91% and 96% of accuracy; and 94%, 88% and 95% of sensitivity, respectively. Moreover, Allplex RP1 showed a FLUAV subtype sensitivity of 91% and 88% for FLUAV-H1pdm09 and FLUAV-H3 respectively, and ProFAST+ assay showed sensitivities of 100% for both targets. The three assays showed a 100% of specificity and PPV, while the NPV were 84%, 73% and 86% for Allplex RP1, Prodesse and GeneXpert, respectively. Conclusions In this study, Seegene’s Allplex RP1 assay showed to be highly sensitive, specific, and suitable for detection of FLUV and HRSV, including FLUAV subtyping. In addition, it is also a hands-on-time saving assay due to the automated nucleic acid extraction and PCR setup.

Molecular epidemiology of circulating human coronaviruses in children at a tertiary hospital in Catalonia (Spain) from 2014 to 2016

no: 316 Presentation at ESCV 2016: Poster 208 Molecular epidemiology of circulating human coronaviruses in children at a tertiary hospital in Catalonia (Spain) from 2014 to 2016 Javier Ramón ∗, Jorgina Vila, Cristina Andrés, Cintia Castillo, Laura Gimferrer, María Piñana, María Gema Codina, Francisco Fuentes, María del Carmen Martín, Rosario Saiz, Pilar Alcubilla, Carlos Rodrigo, Tomàs Pumarola, Andrés Antón Hospital Universitari Vall d’Hebron, Vall d’Hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain Background: Human Coronaviruses (HCoVs) are singlestranded, positive-sense RNA viruses. Four HCoVs species (229E, OC43, NL63 and HKU1) are currently associated with asymptomatic or mild upper-respiratory tract infections (URTI) in general population, but severe acute respiratory infection (SARI) may occur in patients with high risk of infection, such as immunocompromised patients. The main aim of this study was to describe the seasonality and genetic diversity of HCoVs, and the clinical features related to HCoVs infection, in paediatric patients attended in our hospital from 2014 to 2016. Methods: From October 2014 (week 40) to May 2016 (week 20) respiratory specimens were collected from paediatric patients who were attended at the emergency care unit, outpatient departments or admitted to Hospital Universitari Vall d’Hebron (Barcelona, Spain) for diagnosis of respiratory viruses by Anyplex II RV16 Detection Kit (Seegene, Korea), that is only able to detect HCoV229E, HCoV-OC43 and HCoV-NL63, in addition to other respiratory viruses. Partial RNA-dependent RNA polymerase gene (RdRp) was sequenced from laboratory – confirmed HCoVs specimens for subsequent phylogenetic analysis in order to confirm the routine diagnostic PCR results. In addition, partial coding sequence of the spike (S) glycoprotein was sequenced to identify the different HCoV genotypes. Clinical and epidemiological features of HCoV infected cases were retrospectively reviewed from medical records. Results: A total of 6661 specimens from 3900 patients were received at our laboratory, of which 117 (2%) from 96 patients were positive for HCoVs (11 for HCoV-229E, 12%; 33 for HCoV-NL63, 34% and 52 for HCoV-OC43, 54%). But, phylogenetic analysis of 61 partial RdRp sequences revealed that viruses were belonging to the four species (6 HCoV-229E, 9%; 15 HCoV-NL63, 25%; 22 HCoVOC43, 36%; and 18 HCoV-HKU1, 30%). HCoVs circulated throughout the year, but highest number of detections were shown in autumn months. Based on phylogenetic analysis of 69 S sequences: HCoVNL63 (32) fell into two clusters (16 A, 50%; 16 B, 50%); HCoV-OC43 sequences (19) in two clusters (5 B, 26%; 14 C, 74%); and HCoVHKU1 (18) mainly in other two (16 A, 90%; 1 B, 5%), but one (5%) out of known genetic subgroups. HCoV was more often found in respiratory samples of children with URTI: 58% had URTI, of which 21% were associated with lower respiratory tract infection (LRTI); 20.5% of patients had LRTI without URTI; and, 21.5% were asymptomatic. HCoV-HKU1 (20%) and HCoV-OC43 (29%) URTIs were less associated with LRTI than HCoV229E (50%) and HCoV-NL63 (40%). Most of children admitted with HCoV LRTI required supplemental oxygen (11 out of 17 hospitalised patients), but only 2 required it for more than 4 days. HCoV-229E was related with more oxygen requirements, and HCoV-OC43 with longer hospitalization stays. Only one case was admitted to Paediatric Intensive Care Unit. No fatal cases due to HCoV infection were reported. Conclusions: Simultaneous circulation of the several HCoVs species was shown from 2014 to 2016. Phylogenetic analysis revealed the circulation of viruses belonging to different genetic subgroups. Despite seasonal infection by these four HCoV species is usually related to mild–respiratory disease, little differences in the clinical features per specie were shown. Virological surveillance must be done to detect changes on the virological and clinical features related to circulating viruses. http://dx.doi.org/10.1016/j.jcv.2016.08.248 Abstract no: 319 Presentation at ESCV 2016: Poster 209no: 319 Presentation at ESCV 2016: Poster 209 No substantial circulation of enterovirus D68 in patients with severe respiratory disease in South-eastern Spain (Valencian Community) during the 2015–2016 influenza season Laura Cano 1,∗, Joan Puig-Barberà 2,3, Javier Díez 2, F. Xavier López-Labrador 1,4, for the Valencia Hospital Network for the Study of Influenza and Respiratory Viruses Disease4 1 Virology Laboratory, Genomics and Health Area, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (FISABIO)-Public Health, Valencia, Spain 2 Vaccines Research Area, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (FISABIO)-Public Health, Valencia, Spain 3 Centro de Salud Pública de Castellón, Castellón, Spain 4 Consorcio de Investigación Biomédica de Epidemiología y Salud Públic, Valencia, Spain Background: Enterovirus-D68 (EV-D68) was associated with severe respiratory disease in North America and other geographical regions during the fall of 2014. Methods: We compared the detection rates of EV-D68 in the 2014-2015 influenza season with that of the 2015-2016 season in samples collected in a prospective surveillance scheme for all hospitalizations due to respiratory disease in our region (Valencian Community, South-eastern Spain). Combined nasopharyngeal and nasal (children <14 yr. old) or nasopharyngeal and pharyngeal swabs are analyzed in a single laboratory at FISABIOPublic Health for 16 respiratory viruses by multiplex real-time RT-PCR, including rhinovirus/enterovirus as a single target. All samples positive for rhinovirus/enterovirus were retested with a rhinovirus/enterovirus discriminative real-time RT-PCR, and those enterovirus positive for EV-D68 specific detection as a single target. Results: In the 2014–2015 season, between November 15th and March 31st, 372 of 4472 (8.32%) samples were rhino/enterovirus positive, of which 66 (17.75%) were identified as enterovirus, and 15 (4.03%) confirmed as EV-D68. In the 2015–2016 season, between November 15th and April 30th, 201 of 2700 (7.45%) samples were rhino/enterovirus positive, of which 42 (20.82%) were identified as enterovirus, and only one (0.50%) confirmed as EV-D68.