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Does a chill cause a cold?
S4
Abstracts / Journal of Clinical Virology 82S (2016) S1–S142
During the period 2009–2016 we followed a total of 898 patients with either chickenpox or shingles. A complete analysis of anamnestic data was performed and statistically evaluated. Furthermore, in 567 virus isolates from these patients, genotypization of VZV was performed. Results confirmed our previous findings (on a smaller cohort) and showed a higher prevalence of VZV E2 genotype (compared to the E1strain), contrary to the reports from western Europe. In the patients with chickenpox the E1 strain was found in 28,3% of isolates, while the E2 strain in 70,5% of isolates. In patients with shingles/herpes zoster the prevalences of the individual strains were 33.3% of E1 and 66.1% of E2. In general, the E2 strains were more prevalent in the Czech population with 385 positive patients out out of the 567 total (67.9%). As part of this typization study several VZV strains with newly DNA polymorphisms were described. In addition, we have performed Next-Generation-Sequencing (NGS) analysis in 13 VZV isolates. This led to the identification of an European strain with two new mutations in an immunosuppressed child. Several of these strains were also analyzed for their virulence by the real-time monitoring of the infection in cell culture using the xCELLigence system. Taken together we present molecular epidemiology data on a large cohort of VZV positive patients and their correlation with both anamnestic data and disease characteristics. In this study, new variants of the VZV strains were found and several genotypes were correlated to the virulence of the individual strains. Molecular epidemiology is a useful tool for both epidemiological analyses of the individual VZV strain and correlations between the severity of the disease and individual variants. This work was supported by the Institutional Long-Term Developmental Support 1011, the project P304/10/1161 from the Grant Agency of Czech Republic and SV/FVZ201503.
2016. In total, 112 samples were found to be enterovirus positive, 56 of which were positive for EV-D68. As previously described, EVD68 was not found in any CSF samples but was readily detected in respiratory samples during the acute phase of illness. Further genetic characterisation of the enteroviruses (EV-D68 and non EVD68) detected as part of this work is ongoing. Although EV-D68 was found in all age groups, children under the age of 5 years were disproportionally affected, in-keeping with the descriptions from the US outbreak in 2013. Further associated neurological complications were described for two children, including development of a squint in one child and limb weakness in another. However, respiratory complications were the main presenting features with 8 children and one adult requiring intensive care therapy. With the eradication of poliovirus imminent, it has been well documented that increased surveillance of polio and nonpoliovirus enteroviruses should be undertaken importantly, this EV-D68 epidemic in Wales highlights the limitations in current strategies that rely cases of neurological disease and detections in CSF. Since 2014, Public Health Wales routinely tests all respiratory samples submitted through the influenza surveillance scheme from primary care for enteroviruses. One sample from the surveillance scheme tested positive for EV-D68 at the beginning of February 2016 when the number of cases detected in hospital patients was at its peak. We now plan to routinely test all respiratory samples for enteroviruses as part of the routine respiratory panel with the aim to improve detection rates of all enteroviruses and especially those not readily detectable in CSF such as EV-D68. http://dx.doi.org/10.1016/j.jcv.2016.08.008 Abstract no: 180 Presentation at ESCV 2016: Oral 8
http://dx.doi.org/10.1016/j.jcv.2016.08.007 Does a chill cause a cold? Abstract no: 174 Presentation at ESCV 2016: Oral 7 Increased detection of enterovirus type D68 associated with acute flaccid paralysis and severe respiratory illness in Wales, January–February 2016 Catherine Moore 1,∗ , Simon Cottrell 2 , Joanne Watkins 1 , Brendan Mason 2 , Christopher Williams 2 , Rachel Jones 3 1
Public Health Wales Molecular Diagnostics Unit, United Kingdom 2 Public Health Wales CDSC, United Kingdom 3 Wales Specialist Virology Centre, United Kingdom A case of acute flaccid paralysis in a two year old child was notified to Public Health Wales in December 2015. Enterovirus RNA was subsequently detected in a faeces sample by in-house RT-PCR. Investigations for poliovirus were negative and enterovirus type D68 (EV-D68) was detected by specific RT-PCR. Retrospective analysis of respiratory samples collected from children presenting with severe disease during December 2015 were screened by generic enterovirus RT-PCR, this was then followed by testing all those found enterovirus positive and all CSF samples testing enterovirus positive for EV-D68. Two more cases were identified during this time period, both presenting with severe respiratory illness. Prospective testing was then undertaken of all respiratory and CSF samples submitted for respiratory virus detection from across Wales following the same protocols during January and February
R. Price 1,∗ , C. Graham 2 , S. Ramalingam 3 1
University of Edinburgh, UK Wellcome Trust Clinical Research Facility, University of Edinburgh, UK 3 Department of Laboratory Medicine, Royal Infirmary of Edinburgh, UK 2
The common cold is one of the commonest upper respiratory tract infections (URTI) in the world. URTI are caused by a wide range of infectious agents, and often precede more serious lower respiratory tract infections (LRTI). URTI have a seasonal pattern, occurring more frequently during the colder months, with peaks in activity at the start of autumn and spring. The study aimed to examine the seasonality of the causative agents of URTI and the effects of meteorological factors on this seasonal variation. Meteorological data for the Edinburgh Gogarbank area was acquired for five variables: temperature, pressure, relative humidity, wind speed and dew point. Results of respiratory samples tested at the Royal Infirmary of Edinburgh between 2009 and 2015 were analysed. The agents identified were rhinovirus, adenovirus, influenza viruses A and B, parainfluenza viruses (HPIV) 1, 2 and 3, respiratory syncytial virus (RSV), human metapneumovirus (HMPV) and Mycoplasma pneumoniae. Statistical tests were performed with SPSS to assess the relationship between the seasonal variation of these agents and the meteorological factors. These statistical tests included t-tests, ANOVA and post-hoc analysis and the creation of generalised linear models. Seasonal patterns were identified for each agent by graphical representation with meteorological seasonality. T-test and
Abstracts / Journal of Clinical Virology 82S (2016) S1–S142
ANOVA were used to determine the significance of the relationships between agent incidence and the meteorological factors. Significant relationships with temperature, dew point, relative humidity and fluctuation in humidity (humidity-range) were found in many of the infectious agents, with HPIV-3, RSV and Influenza viruses A and B showing the strongest correlations. Influenza viruses A, B and RSV preferred a low temperature, dew point and humidity-range, whilst also preferring a high humidity level. Notably, HPIV-3 showed the opposite relationship. This is the first time, an association between fluctuation in humidity and viral incidence has been described. A generalised linear model was constructed for each agent to establish a statistically rigorous representation of its seasonal pattern and the relationship with temperature, allowing for the seasonality of all the agents to be confirmed. The identification of each agent’s seasonal peaks allowed for the prediction of the order in which each agent will arise in a given year, starting with HPIV-3 in April, followed by rhinovirus, adenovirus, HPIV-1, HPIV-2, M. pneumoniae, RSV, Influenza virus A, Influenza virus B and ending with HMPV in March of the following year. A change either in temperature or humidity or both was associated with transition from one agent to another through the year. Meteorological factors like temperature and humidity have a significant effect on the incidence of the causative agents of the common cold. This information could now possibly be used to predict the transition from one virus to another. By tracking changes in meteorological factors, medical professionals could now be forewarned of an oncoming rise in infections from a particular agent, allowing them to take appropriate preventative measurers. http://dx.doi.org/10.1016/j.jcv.2016.08.009 Abstract no: 285 Presentation at ESCV 2016: Oral 9 Metagenomic analysis of the respiratory virome associated with acute respiratory illness of unknown etiology in infants A. Bal 1,2,∗ , M. Pichon 1,2 , C. Picard 3 , G. Billaud 1 , J.S. Casalegno 1,2 , M. Bouscambert-Duchamp 1 , V. Escuret 1,2 , I. Schuffenecker 1 , M. Valette 1 , B. Lina 1,2 , F. Morfin 1,2 , L. Josset 1,2 1
Virology Department, University Hospital of Lyon, France 2 Virpath, Inserm U1111 – CNRS UMR 5308, Lyon, France 3 Internation Center for Research in Infectiology, INSERM1111, Lyon University, Lyon France Introduction: Acute respiratory infections (ARIs) are the leading cause of illness and death in children under five years old, who experience three to six episodes per year. Viral infections are the main etiology of ARIs but etiologic agents are often not identified. Recent studies have suggested that virome has important effects on human health. Therefore, respiratory virome characterization may help to understand these ARIs of unknown etiology. The aim of the study is to characterize the respiratory virome of children under five with an ARI of undiagnosed etiology in order to identify potential novel respiratory viruses or variants not detected by routine tests. For this purpose, a method for metagenomic analysis of respiratory virome was optimized by implementing a quality control to validate each steps of the process. Methods: A retrospective study was conducted on samples received at the virology laboratory of the University Hospital of Lyon, France, between 2010 and 2015. Upper respiratory samples
S5
from children under five were first selected by an epidemiological approach based on respiratory viruses circulation patterns. As an undiagnosed pathogen could be suspected in numerous infectious diseases with negative biological investigation, “undiagnosed events”, defined as periods with proportion of negative samples > 75% of the total number of samples received in the laboratory, University Hospital of Lyon, were targeted. Samples with a high probability of viral infections were selected according to the following criteria: absence of documented infection with routine techniques used at the time of diagnostic on at least 2 negative respiratory samples collected within 14 days of their admission (hospitalization time > 24 h). These samples were then controlled by a sensitive multiplex nested Polymerase Chain Reaction (FilmArray® Respiratory Panel (FA RP), bioMérieux, Lyon, France). After exclusion of positive samples with this technique, a quality control (viral strain) was added to each sample before metagenomic analysis. Results: 223 patients were identified by targeting “undiagnosed events” as described. Twenty-two patients with high probability of viral infections were selected, 13/22 (59.1%) were under one years old and 14/22 patients (63.6%) had comorbidities (mainly respiratory chronic diseases). Patients developed mostly signs of upper respiratory tract infection, such as cough and rhinorrhea, but two patients developed severe respiratory distress with the need of ventilation. Viruses were detected in most of the samples with FA RP (17/22) (77.3%) mainly Picornaviridae viruses (13/22) (59.1%). A metagenomic analysis with a quality control process was developed. An optimized metagenomic protocol was successfully used for five negative patients. Sequencing analysis are currently in progress. Conclusion: No prior studies performed metagenomics analysis to characterize the respiratory virome involved in ARIs of unknown etiology in infants. Identification of a novel respiratory virus could have a strong impact on ARIs diagnosis and management. In absence of new virus identification, this study has produced useful results describing the respiratory virome of children with ARIs. Characterization of the whole viral communities detectable in the human respiratory tract is key for understanding the role of the virome in respiratory disease. http://dx.doi.org/10.1016/j.jcv.2016.08.010 Abstract no: 37 Presentation at ESCV 2016: Oral 10 Assumption-free improvement of the maxRatio algorithm increases the accuracy of qPCR assays targeting viruses Luigi Marongiu 1,∗ , Eric Shain 2 , Lydia Drumright 1 , Reidun Lillestøl 1 , Donald Somasunderam 3 , Martin D. Curran 3 1
University of Cambridge, United Kingdom Grove Street Technology, United Kingdom 3 Public Health England, Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, United Kingdom 2
Introduction: Quantitative PCR (qPCR) is widely applied in Laboratories of Virology worldwide for screening, diagnostic and research purposes. Analysis of qPCR data is typically performed with the cycle-threshold method (CT), which requires the assignment of the cut-off and baseline range by the operator. These assumptions might impair the reproducibility of the results between laboratories and could underestimate the impact of inhibition of the reaction of amplification. The maxRatio method
Abstracts / Journal of Clinical Virology 82S (2016) S1–S142
During the period 2009–2016 we followed a total of 898 patients with either chickenpox or shingles. A complete analysis of anamnestic data was performed and statistically evaluated. Furthermore, in 567 virus isolates from these patients, genotypization of VZV was performed. Results confirmed our previous findings (on a smaller cohort) and showed a higher prevalence of VZV E2 genotype (compared to the E1strain), contrary to the reports from western Europe. In the patients with chickenpox the E1 strain was found in 28,3% of isolates, while the E2 strain in 70,5% of isolates. In patients with shingles/herpes zoster the prevalences of the individual strains were 33.3% of E1 and 66.1% of E2. In general, the E2 strains were more prevalent in the Czech population with 385 positive patients out out of the 567 total (67.9%). As part of this typization study several VZV strains with newly DNA polymorphisms were described. In addition, we have performed Next-Generation-Sequencing (NGS) analysis in 13 VZV isolates. This led to the identification of an European strain with two new mutations in an immunosuppressed child. Several of these strains were also analyzed for their virulence by the real-time monitoring of the infection in cell culture using the xCELLigence system. Taken together we present molecular epidemiology data on a large cohort of VZV positive patients and their correlation with both anamnestic data and disease characteristics. In this study, new variants of the VZV strains were found and several genotypes were correlated to the virulence of the individual strains. Molecular epidemiology is a useful tool for both epidemiological analyses of the individual VZV strain and correlations between the severity of the disease and individual variants. This work was supported by the Institutional Long-Term Developmental Support 1011, the project P304/10/1161 from the Grant Agency of Czech Republic and SV/FVZ201503.
2016. In total, 112 samples were found to be enterovirus positive, 56 of which were positive for EV-D68. As previously described, EVD68 was not found in any CSF samples but was readily detected in respiratory samples during the acute phase of illness. Further genetic characterisation of the enteroviruses (EV-D68 and non EVD68) detected as part of this work is ongoing. Although EV-D68 was found in all age groups, children under the age of 5 years were disproportionally affected, in-keeping with the descriptions from the US outbreak in 2013. Further associated neurological complications were described for two children, including development of a squint in one child and limb weakness in another. However, respiratory complications were the main presenting features with 8 children and one adult requiring intensive care therapy. With the eradication of poliovirus imminent, it has been well documented that increased surveillance of polio and nonpoliovirus enteroviruses should be undertaken importantly, this EV-D68 epidemic in Wales highlights the limitations in current strategies that rely cases of neurological disease and detections in CSF. Since 2014, Public Health Wales routinely tests all respiratory samples submitted through the influenza surveillance scheme from primary care for enteroviruses. One sample from the surveillance scheme tested positive for EV-D68 at the beginning of February 2016 when the number of cases detected in hospital patients was at its peak. We now plan to routinely test all respiratory samples for enteroviruses as part of the routine respiratory panel with the aim to improve detection rates of all enteroviruses and especially those not readily detectable in CSF such as EV-D68. http://dx.doi.org/10.1016/j.jcv.2016.08.008 Abstract no: 180 Presentation at ESCV 2016: Oral 8
http://dx.doi.org/10.1016/j.jcv.2016.08.007 Does a chill cause a cold? Abstract no: 174 Presentation at ESCV 2016: Oral 7 Increased detection of enterovirus type D68 associated with acute flaccid paralysis and severe respiratory illness in Wales, January–February 2016 Catherine Moore 1,∗ , Simon Cottrell 2 , Joanne Watkins 1 , Brendan Mason 2 , Christopher Williams 2 , Rachel Jones 3 1
Public Health Wales Molecular Diagnostics Unit, United Kingdom 2 Public Health Wales CDSC, United Kingdom 3 Wales Specialist Virology Centre, United Kingdom A case of acute flaccid paralysis in a two year old child was notified to Public Health Wales in December 2015. Enterovirus RNA was subsequently detected in a faeces sample by in-house RT-PCR. Investigations for poliovirus were negative and enterovirus type D68 (EV-D68) was detected by specific RT-PCR. Retrospective analysis of respiratory samples collected from children presenting with severe disease during December 2015 were screened by generic enterovirus RT-PCR, this was then followed by testing all those found enterovirus positive and all CSF samples testing enterovirus positive for EV-D68. Two more cases were identified during this time period, both presenting with severe respiratory illness. Prospective testing was then undertaken of all respiratory and CSF samples submitted for respiratory virus detection from across Wales following the same protocols during January and February
R. Price 1,∗ , C. Graham 2 , S. Ramalingam 3 1
University of Edinburgh, UK Wellcome Trust Clinical Research Facility, University of Edinburgh, UK 3 Department of Laboratory Medicine, Royal Infirmary of Edinburgh, UK 2
The common cold is one of the commonest upper respiratory tract infections (URTI) in the world. URTI are caused by a wide range of infectious agents, and often precede more serious lower respiratory tract infections (LRTI). URTI have a seasonal pattern, occurring more frequently during the colder months, with peaks in activity at the start of autumn and spring. The study aimed to examine the seasonality of the causative agents of URTI and the effects of meteorological factors on this seasonal variation. Meteorological data for the Edinburgh Gogarbank area was acquired for five variables: temperature, pressure, relative humidity, wind speed and dew point. Results of respiratory samples tested at the Royal Infirmary of Edinburgh between 2009 and 2015 were analysed. The agents identified were rhinovirus, adenovirus, influenza viruses A and B, parainfluenza viruses (HPIV) 1, 2 and 3, respiratory syncytial virus (RSV), human metapneumovirus (HMPV) and Mycoplasma pneumoniae. Statistical tests were performed with SPSS to assess the relationship between the seasonal variation of these agents and the meteorological factors. These statistical tests included t-tests, ANOVA and post-hoc analysis and the creation of generalised linear models. Seasonal patterns were identified for each agent by graphical representation with meteorological seasonality. T-test and
Abstracts / Journal of Clinical Virology 82S (2016) S1–S142
ANOVA were used to determine the significance of the relationships between agent incidence and the meteorological factors. Significant relationships with temperature, dew point, relative humidity and fluctuation in humidity (humidity-range) were found in many of the infectious agents, with HPIV-3, RSV and Influenza viruses A and B showing the strongest correlations. Influenza viruses A, B and RSV preferred a low temperature, dew point and humidity-range, whilst also preferring a high humidity level. Notably, HPIV-3 showed the opposite relationship. This is the first time, an association between fluctuation in humidity and viral incidence has been described. A generalised linear model was constructed for each agent to establish a statistically rigorous representation of its seasonal pattern and the relationship with temperature, allowing for the seasonality of all the agents to be confirmed. The identification of each agent’s seasonal peaks allowed for the prediction of the order in which each agent will arise in a given year, starting with HPIV-3 in April, followed by rhinovirus, adenovirus, HPIV-1, HPIV-2, M. pneumoniae, RSV, Influenza virus A, Influenza virus B and ending with HMPV in March of the following year. A change either in temperature or humidity or both was associated with transition from one agent to another through the year. Meteorological factors like temperature and humidity have a significant effect on the incidence of the causative agents of the common cold. This information could now possibly be used to predict the transition from one virus to another. By tracking changes in meteorological factors, medical professionals could now be forewarned of an oncoming rise in infections from a particular agent, allowing them to take appropriate preventative measurers. http://dx.doi.org/10.1016/j.jcv.2016.08.009 Abstract no: 285 Presentation at ESCV 2016: Oral 9 Metagenomic analysis of the respiratory virome associated with acute respiratory illness of unknown etiology in infants A. Bal 1,2,∗ , M. Pichon 1,2 , C. Picard 3 , G. Billaud 1 , J.S. Casalegno 1,2 , M. Bouscambert-Duchamp 1 , V. Escuret 1,2 , I. Schuffenecker 1 , M. Valette 1 , B. Lina 1,2 , F. Morfin 1,2 , L. Josset 1,2 1
Virology Department, University Hospital of Lyon, France 2 Virpath, Inserm U1111 – CNRS UMR 5308, Lyon, France 3 Internation Center for Research in Infectiology, INSERM1111, Lyon University, Lyon France Introduction: Acute respiratory infections (ARIs) are the leading cause of illness and death in children under five years old, who experience three to six episodes per year. Viral infections are the main etiology of ARIs but etiologic agents are often not identified. Recent studies have suggested that virome has important effects on human health. Therefore, respiratory virome characterization may help to understand these ARIs of unknown etiology. The aim of the study is to characterize the respiratory virome of children under five with an ARI of undiagnosed etiology in order to identify potential novel respiratory viruses or variants not detected by routine tests. For this purpose, a method for metagenomic analysis of respiratory virome was optimized by implementing a quality control to validate each steps of the process. Methods: A retrospective study was conducted on samples received at the virology laboratory of the University Hospital of Lyon, France, between 2010 and 2015. Upper respiratory samples
S5
from children under five were first selected by an epidemiological approach based on respiratory viruses circulation patterns. As an undiagnosed pathogen could be suspected in numerous infectious diseases with negative biological investigation, “undiagnosed events”, defined as periods with proportion of negative samples > 75% of the total number of samples received in the laboratory, University Hospital of Lyon, were targeted. Samples with a high probability of viral infections were selected according to the following criteria: absence of documented infection with routine techniques used at the time of diagnostic on at least 2 negative respiratory samples collected within 14 days of their admission (hospitalization time > 24 h). These samples were then controlled by a sensitive multiplex nested Polymerase Chain Reaction (FilmArray® Respiratory Panel (FA RP), bioMérieux, Lyon, France). After exclusion of positive samples with this technique, a quality control (viral strain) was added to each sample before metagenomic analysis. Results: 223 patients were identified by targeting “undiagnosed events” as described. Twenty-two patients with high probability of viral infections were selected, 13/22 (59.1%) were under one years old and 14/22 patients (63.6%) had comorbidities (mainly respiratory chronic diseases). Patients developed mostly signs of upper respiratory tract infection, such as cough and rhinorrhea, but two patients developed severe respiratory distress with the need of ventilation. Viruses were detected in most of the samples with FA RP (17/22) (77.3%) mainly Picornaviridae viruses (13/22) (59.1%). A metagenomic analysis with a quality control process was developed. An optimized metagenomic protocol was successfully used for five negative patients. Sequencing analysis are currently in progress. Conclusion: No prior studies performed metagenomics analysis to characterize the respiratory virome involved in ARIs of unknown etiology in infants. Identification of a novel respiratory virus could have a strong impact on ARIs diagnosis and management. In absence of new virus identification, this study has produced useful results describing the respiratory virome of children with ARIs. Characterization of the whole viral communities detectable in the human respiratory tract is key for understanding the role of the virome in respiratory disease. http://dx.doi.org/10.1016/j.jcv.2016.08.010 Abstract no: 37 Presentation at ESCV 2016: Oral 10 Assumption-free improvement of the maxRatio algorithm increases the accuracy of qPCR assays targeting viruses Luigi Marongiu 1,∗ , Eric Shain 2 , Lydia Drumright 1 , Reidun Lillestøl 1 , Donald Somasunderam 3 , Martin D. Curran 3 1
University of Cambridge, United Kingdom Grove Street Technology, United Kingdom 3 Public Health England, Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, United Kingdom 2
Introduction: Quantitative PCR (qPCR) is widely applied in Laboratories of Virology worldwide for screening, diagnostic and research purposes. Analysis of qPCR data is typically performed with the cycle-threshold method (CT), which requires the assignment of the cut-off and baseline range by the operator. These assumptions might impair the reproducibility of the results between laboratories and could underestimate the impact of inhibition of the reaction of amplification. The maxRatio method