Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses

Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95–100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID₅₀/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2–1280.8 copies/μL (0.08–3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2–8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4–1280.8 copies/μL, 2.50–3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6–94.8 copies/μL, 0.20–1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID₅₀/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported.     

Clinical severity of pediatric respiratory illness with enterovirus D68 compared with rhinovirus or other enterovirus genotypes

Background:

Enterovirus D68 (EV-D68) resulted in a reported increase in the number of children needing hospital or critical care admission because of respiratory insufficiency during 2014. It remains unclear, however, whether EV-D68 infections were more severe than rhinovirus or non–EV-D68 enterovirus infections.

Methods:

We evaluated consecutive children presenting to a pediatric hospital between Aug. 1 and Oct. 31, 2014, with positive nasopharyngeal swabs for rhinovirus or enterovirus that were sent automatically for EV-D68 testing. We compared characteristics and outcomes of patients with EV-D68 with those with rhinovirus or non–EV-D68 enterovirus in a matched cohort study.

Results:

A total of 93/297 (31.3%) of rhinovirus or enterovirus samples tested positive for EV-D68, and it was possible to compare 87 matched pairs. Children with EV-D68 infection were more likely to have difficulty breathing (odds ratio [OR] 3.00, 95% confidence interval [CI] 1.47–6.14). There was no significant difference in admission to the critical care unit or death among children with EV-D68 infection compared with those with other rhinovirus or enterovirus infections (adjusted OR 1.47, 95% CI 0.61–3.52). Children with EV-D68 infection were more often admitted to hospital, but not significantly so (adjusted OR 2.29, 95% CI 0.96–5.46).

Interpretation:

Enterovirus D68 seems to be a more virulent pulmonary pathogen than rhinovirus or non–EV-D68 enterovirus, but we did not find a significant difference in death or need for critical care.Rhinoviruses and enteroviruses are closely related, small RNA viruses in the genus Enterovirus. Rhinovirus species are the leading cause of upper respiratory tract disease, whereas enterovirus species generally infect the gastrointestinal tract and may then disseminate systemically. In August of 2014, the first reports of severe respiratory illness caused by enterovirus D68 (EV-D68), described in the media as a “new” pathogen, came from the American Midwest.¹ However, it should be emphasized that EV-D68 has been circulating for decades. It was first detected in 1962 in California,² and has since been detected in several countries, including the Philippines, Japan and the Netherlands, as well as in previous clusters in the United States.^3–5 However, by the end of 2014, a total of more than 1000 cases from 49 states in the US were reported.⁶ Almost 40% of samples submitted to the Centers for Disease Control and Prevention (CDC) for suspected EV-D68 were positive for EV-D68,⁶ and the genotype spread further north into Canada.⁷Subsequent to media reports, numerous clinicians perceived an “increase” in the number of children who required hospital or intensive care admission for respiratory disease, leading to the hypothesis that EV-D68 was more pathogenic than other respiratory viral pathogens. However, this has not yet been determined. The CDC documented a total of 12 deaths under investigation in 1152 confirmed cases for an estimated mortality of 1%,⁶ which does not appear to greatly exceed expected mortality associated with rhinovirus or non–EV-D68 enterovirus infection.⁸ Furthermore, the CDC estimates may have been artificially elevated by spectrum bias, as patients with severe illness would be more likely to have specimens forwarded to reference laboratories for EV-D68 testing.The goal of this study was to compare the severity of illness in children with EV-D68 infection with that of children with rhinovirus or non–EV-D68 enterovirus infection.     

Epidemiology of Enterovirus D68 in Ontario

In August 2014, children’s hospitals in Kansas City, Missouri and Chicago, Illinois notified the Centers for Disease Control and Prevention (CDC) about increased numbers of pediatric patients hospitalized with severe respiratory illness (SRI). In response to CDC reports, Public Health Ontario Laboratories (PHOL) launched an investigation of patients being tested for enterovirus D-68 (EV-D68) in Ontario, Canada. The purpose of this investigation was to enhance our understanding of EV-D68 epidemiology and clinical features. Data for this study included specimens submitted for EV-D68 testing at PHOL from September 1, 2014 to October 31, 2014. Comparisons were made between patients who tested positive for the virus (cases) and those testing negative (controls). EV-D68 was identified in 153/907 (16.8%) of patients tested. In the logistic regression model adjusting for age, sex, setting and time to specimen collection, individuals younger than 20 years of age were more likely to be diagnosed with EV-D68 compared to those 20 and over, with peak positivity at ages 5–9 years. Cases were not more likely to be hospitalized than controls. Cases were more likely to be identified in September than October (OR 8.07; 95% CI 5.15 to 12.64). Routine viral culture and multiplex PCR were inadequate methods to identify EV-D68 due to poor sensitivity and inability to differentiate EV-D68 from other enterovirus serotypes or rhinovirus. Testing for EV-D68 in Ontario from July to December, 2014 detected the presence of EV-D68 virus among young children during September-October, 2014, with most cases detected in September. There was no difference in hospitalization status between cases and controls. In order to better understand the epidemiology of this virus, surveillance for EV-D68 should include testing of symptomatic individuals from all treatment settings and patient age groups, with collection and analysis of comprehensive clinical and epidemiological data.     

Enterovirus D-68 Infection,Prophylaxis, and Vaccination in a Novel Permissive Animal Model,the Cotton Rat (Sigmodon hispidus)

In recent years, there has been a significant increase in detection of Enterovirus D-68 (EV-D68) among patients with severe respiratory infections worldwide. EV-D68 is now recognized as a re-emerging pathogen; however, due to lack of a permissive animal model for EV-D68, a comprehensive understanding of the pathogenesis and immune response against EV-D68 has been hampered. Recently, it was shown that EV-D68 has a strong affinity for α2,6-linked sialic acids (SAs) and we have shown previously that α2,6-linked SAs are abundantly present in the respiratory tract of cotton rats (Sigmodon hispidus). Thus, we hypothesized that cotton rats could be a potential model for EV-D68 infection. Here, we evaluated the ability of two recently isolated EV-D68 strains (VANBT/1 and MO/14/49), along with the historical prototype Fermon strain (ATCC), to infect cotton rats. We found that cotton rats are permissive to EV-D68 infection without virus adaptation. The different strains of EV-D68 showed variable infection profiles and the ability to produce neutralizing antibody (NA) upon intranasal infection or intramuscular immunization. Infection with the VANBT/1 resulted in significant induction of pulmonary cytokine gene expression and lung pathology. Intramuscular immunization with live VANBT/1 or MO/14/49 induced strong homologous antibody responses, but a moderate heterologous NA response. We showed that passive prophylactic administration of serum with high content of NA against VANBT/1 resulted in an efficient antiviral therapy. VANBT/1-immunized animals showed complete protection from VANBT/1 challenge, but induced strong pulmonary Th1 and Th2 cytokine responses and enhanced lung pathology, indicating the generation of exacerbated immune response by immunization. In conclusion, our data illustrate that the cotton rat is a powerful animal model that provides an experimental platform to investigate pathogenesis, immune response, anti-viral therapies and vaccines against EV-D68 infection.     

Rhinovirus genome variation during chronic upper and lower respiratory tract infections

Routine screening of lung transplant recipients and hospital patients for respiratory virus infections allowed to identify human rhinovirus (HRV) in the upper and lower respiratory tracts, including immunocompromised hosts chronically infected with the same strain over weeks or months. Phylogenetic analysis of 144 HRV-positive samples showed no apparent correlation between a given viral genotype or species and their ability to invade the lower respiratory tract or lead to protracted infection. By contrast, protracted infections were found almost exclusively in immunocompromised patients, thus suggesting that host factors rather than the virus genotype modulate disease outcome, in particular the immune response. Complete genome sequencing of five chronic cases to study rhinovirus genome adaptation showed that the calculated mutation frequency was in the range observed during acute human infections. Analysis of mutation hot spot regions between specimens collected at different times or in different body sites revealed that non-synonymous changes were mostly concentrated in the viral capsid genes VP1, VP2 and VP3, independent of the HRV type. In an immunosuppressed lung transplant recipient infected with the same HRV strain for more than two years, both classical and ultra-deep sequencing of samples collected at different time points in the upper and lower respiratory tracts showed that these virus populations were phylogenetically indistinguishable over the course of infection, except for the last month. Specific signatures were found in the last two lower respiratory tract populations, including changes in the 5'UTR polypyrimidine tract and the VP2 immunogenic site 2. These results highlight for the first time the ability of a given rhinovirus to evolve in the course of a natural infection in immunocompromised patients and complement data obtained from previous experimental inoculation studies in immunocompetent volunteers.     

Evolution,geographic spreading,and demographic distribution of Enterovirus D68

Worldwide outbreaks of enterovirus D68 (EV-D68) in 2014 and 2016 have caused serious respiratory and neurological disease. We collected samples from several European countries during the 2018 outbreak and determined 53 near full-length genome (‘whole genome’) sequences. These sequences were combined with 718 whole genome and 1,987 VP1-gene publicly available sequences. In 2018, circulating strains clustered into multiple subgroups in the B3 and A2 subclades, with different phylogenetic origins. Clusters in subclade B3 emerged from strains circulating primarily in the US and Europe in 2016, though some had deeper roots linking to Asian strains, while clusters in A2 traced back to strains detected in East Asia in 2015-2016. In 2018, all sequences from the USA formed a distinct subgroup, containing only three non-US samples. Alongside the varied origins of seasonal strains, we found that diversification of these variants begins up to 18 months prior to the first diagnostic detection during a EV-D68 season. EV-D68 displays strong signs of continuous antigenic evolution and all 2018 A2 strains had novel patterns in the putative neutralizing epitopes in the BC- and DE-loops. The pattern in the BC-loop of the USA B3 subgroup had not been detected on that continent before. Patients with EV-D68 in subclade A2 were significantly older than patients with a B3 subclade virus. In contrast to other subclades, the age distribution of A2 is distinctly bimodal and was found primarily among children and in the elderly. We hypothesize that EV-D68’s rapid evolution of surface proteins, extensive diversity, and high rate of geographic mixing could be explained by substantial reinfection of adults. Better understanding of evolution and immunity across diverse viral pathogens, including EV-D68 and SARS-CoV-2, is critical to pandemic preparedness in the future.     

Human rhinovirus C infection is associated with asthma in children determined by xTAG respiratory viral panel FAST

<正>Dear Editor,Cumulative evidence supports the role of early-life viral infections,especially respiratory syncytial virus(RSV)and human rhinovirus(HRV),as major antecedents of childhood asthma(Lemanske,2002;Jackson et al.,2008).In this study,the x TAG respiratory viral panel FAST(RVP FAST)assay,a multiplex polymerase chain reaction(PCR)-based method(Arens et al.,2010;BaladaLlasat et al.,2011;Gharabaghi et al.,2011;Selvaraju,2012),was used to investigate the association of infec-     

Global prevalence and case fatality rate of Enterovirus D68 infections,a systematic review and meta-analysis

A substantial amount of epidemiological data has been reported on Enterovirus D68 (EV-D68) infections after the 2014 outbreak. Our goal was to map the case fatality rate (CFR) and prevalence of current and past EV-D68 infections. We conducted a systematic review (PROSPERO, CRD42021229255) with published articles on EV-68 infections in PubMed, Embase, Web of Science and Global Index Medicus up to January 2021. We determined prevalences using a model random effect. Of the 4,329 articles retrieved from the databases, 89 studies that met the inclusion criteria were from 39 different countries with apparently healthy individuals and patients with acute respiratory infections, acute flaccid myelitis and asthma-related diseases. The CFR estimate revealed occasional deaths (7/1353) related to EV-D68 infections in patients with severe acute respiratory infections. Analyses showed that the combined prevalence of current and past EV-D68 infections was 4% (95% CI = 3.1–5.0) and 66.3% (95% CI = 40.0–88.2), respectively. The highest prevalences were in hospital outbreaks, developed countries, children under 5, after 2014, and in patients with acute flaccid myelitis and asthma-related diseases. The present study shows sporadic deaths linked to severe respiratory EV-D68 infections. The study also highlights a low prevalence of current EV-D68 infections as opposed to the existence of EV-D68 antibodies in almost all participants of the included studies. These findings therefore highlight the need to implement and/or strengthen continuous surveillance of EV-D68 infections in hospitals and in the community for the anticipation of the response to future epidemics.     

Total infectome characterization of respiratory infections in pre-COVID-19 Wuhan,China

At the end of 2019 Wuhan witnessed an outbreak of “atypical pneumonia” that later developed into a global pandemic. Metagenomic sequencing rapidly revealed the causative agent of this outbreak to be a novel coronavirus denoted SARS-CoV-2. To provide a snapshot of the pathogens in pneumonia-associated respiratory samples from Wuhan prior to the emergence of SARS-CoV-2, we collected bronchoalveolar lavage fluid samples from 408 patients presenting with pneumonia and acute respiratory infections at the Central Hospital of Wuhan between 2016 and 2017. Unbiased total RNA sequencing was performed to reveal their “total infectome”, including viruses, bacteria and fungi. We identified 35 pathogen species, comprising 13 RNA viruses, 3 DNA viruses, 16 bacteria and 3 fungi, often at high abundance and including multiple co-infections (13.5%). SARS-CoV-2 was not present. These data depict a stable core infectome comprising common respiratory pathogens such as rhinoviruses and influenza viruses, an atypical respiratory virus (EV-D68), and a single case of a sporadic zoonotic pathogen–Chlamydia psittaci. Samples from patients experiencing respiratory disease on average had higher pathogen abundance than healthy controls. Phylogenetic analyses of individual pathogens revealed multiple origins and global transmission histories, highlighting the connectedness of the Wuhan population. This study provides a comprehensive overview of the pathogens associated with acute respiratory infections and pneumonia, which were more diverse and complex than obtained using targeted PCR or qPCR approaches. These data also suggest that SARS-CoV-2 or closely related viruses were absent from Wuhan in 2016–2017.     

Rhinovirus Load Is High despite Preserved Interferon-β Response in Cystic Fibrosis Bronchial Epithelial Cells

Lung disease in cystic fibrosis (CF) is often exacerbated following acute upper respiratory tract infections caused by the human rhinovirus (HRV). Pathophysiology of these exacerbations is presently unclear and may involve deficient innate antiviral or exaggerated inflammatory responses in CF airway epithelial cells. Furthermore, responses of CF cells to HRV may be adversely affected by pre-exposure to virulence factors of Pseudomonas (P.) aeruginosa, the microorganism that frequently colonizes CF airways. Here we examined production of antiviral cytokine interferon-β and inflammatory chemokine interleukin-8, expression of the interferon-responsive antiviral gene 2’-5’-oligoadenylate synthetase 1 (OAS1), and intracellular virus RNA load in primary CF (delF508 CFTR) and healthy airway epithelial cells following inoculation with HRV16. Parallel cells were exposed to virulence factors of P. aeruginosa prior to and during HRV16 inoculation. CF cells exhibited production of interferon-β and interleukin-8, and expression of OAS1 at levels comparable to those in healthy cells, yet significantly higher HRV16 RNA load during early hours post-inoculation with HRV16. In line with this, HRV16 RNA load was higher in the CFBE41o- dF cell line overexpessing delF508 CFTR, compared with the isogenic control CFBE41o- WT (wild-type CFTR). Pre-exposure to virulence factors of P. aeruginosa did not affect OAS1 expression or HRV16 RNA load, but potentiated interleukin-8 production. In conclusion, CF cells demonstrate elevated HRV RNA load despite preserved interferon-β and OAS1 responses. High HRV load in CF airway epithelial cells appears to be due to deficiencies manifesting early during HRV infection, and may not be related to interferon-β.     

Genetics,Recombination and Clinical Features of Human Rhinovirus Species C (HRV-C) Infections; Interactions of HRV-C with Other Respiratory Viruses

To estimate the frequency, molecular epidemiological and clinical associations of infection with the newly described species C variants of human rhinoviruses (HRV), 3243 diagnostic respiratory samples referred for diagnostic testing in Edinburgh were screened using a VP4-encoding region-based selective polymerase chain reaction (PCR) for HRV-C along with parallel PCR testing for 13 other respiratory viruses. HRV-C was the third most frequently detected behind respiratory syncytial virus (RSV) and adenovirus, with 141 infection episodes detected among 1885 subjects over 13 months (7.5%). Infections predominantly targeted the very young (median age 6–12 months; 80% of infections in those <2 years), occurred throughout the year but with peak incidence in early winter months. HRV-C was detected significantly more frequently among subjects with lower (LRT) and upper respiratory tract (URT) disease than controls without respiratory symptoms; HRV-C mono-infections were the second most frequently detected virus (behind RSV) in both disease presentations (6.9% and 7.8% of all cases respectively). HRV variants were classified by VP4/VP2 sequencing into 39 genotypically defined types, increasing the current total worldwide to 60. Through sequence comparisons of the 5′untranslated region (5′UTR), the majority grouped with species A (n = 96; 68%, described as HRV-Ca), the remainder forming a phylogenetically distinct 5′UTR group (HRV-Cc). Multiple and bidirectional recombination events between HRV-Ca and HRV-Cc variants and with HRV species A represents the most parsimonious explanation for their interspersed phylogeny relationships in the VP4/VP2-encoding region. No difference in age distribution, seasonality or disease associations was identified between HRV-Ca and HRV-Cc variants. HRV-C-infected subjects showed markedly reduced detection frequencies of RSV and other respiratory viruses, providing evidence for a major interfering effect of HRV-C on susceptibility to other respiratory virus infections. HRV-C''s disease associations, its prevalence and evidence for interfering effects on other respiratory viruses mandates incorporation of rhinoviruses into future diagnostic virology screening.     

The role of human rhinovirus in immunology, COPD, and corresponding treatments

The common cold is most often a result of human rhinovirus (HRV) infection. Common cold symptoms including rhinorrhea and nasal obstruction frequently occur during HRV infection of the upper respiratory tract. Conversely, HRV may also infect the epithelial cells of the lower respiratory tract. Symptom severity associated with HRV infection ranges from mild to potentially serious depending on a person’s susceptibility and pre-existing condition, such as chronic obstructive pulmonary disease. An over active host immune response is believed to be the primary contributor to HRV pathogenesis. Enhanced activity of various host cell cytokines and granulocytes mediate specific cellular pathways inducing many of the symptoms associated with HRV infection. There are over 100 serotypes of HRV which can be further categorized based on the specific characteristics of each type. The two main categories of HRV consist of the major and minor groups. The unique host cell receptor is the distinguishing factor between these two groups. Yet, these viruses may also differ in mechanism of infection and replication. Due to the high frequency of hospital and clinical visits and the corresponding economic burden, novel therapies are of interest. Several different treatment options varying from herbal remedies to anti-viral drugs have been studied. However, the vast number of HRV serotypes complicates the progress of developing a universal treatment for attenuating HRV infection.     

Response to Rhinovirus Infection by Human Airway Epithelial Cells and Peripheral Blood Mononuclear Cells in an In Vitro Two-Chamber Tissue Culture System

Human rhinovirus (HRV) infections are associated with the common cold, occasionally with more serious lower respiratory tract illnesses, and frequently with asthma exacerbations. The clinical features of HRV infection and its association with asthma exacerbation suggest that some HRV disease results from virus-induced host immune responses to infection. To study the HRV-infection-induced host responses and the contribution of these responses to disease, we have developed an in vitro model of HRV infection of human airway epithelial cells (Calu-3 cells) and subsequent exposure of human peripheral blood mononuclear cells (PBMCs) to these infected cells in a two-chamber trans-well tissue culture system. Using this model, we studied HRV 14 (species B) and HRV 16 (species A) induced cytokine and chemokine responses with PBMCs from four healthy adults. Infection of Calu-3 cells with either virus induced HRV-associated increases in FGF-Basic, IL-15, IL-6, IL-28A, ENA-78 and IP-10. The addition of PBMCs to HRV 14-infected cells gave significant increases in MIP-1β, IL-28A, MCP-2, and IFN-α as compared with mock-infected cells. Interestingly, ENA-78 levels were reduced in HRV 14 infected cells that were exposed to PBMCs. Addition of PBMCs to HRV 16-infected cells did not induce MIP-1β, IL-28A and IFN-α efficiently nor did it decrease ENA-78 levels. Our results demonstrate a clear difference between HRV 14 and HRV 16 and the source of PBMCs, in up or down regulation of several cytokines including those that are linked to airway inflammation. Such differences might be one of the reasons for variation in disease associated with different HRV species including variation in their link to asthma exacerbations as suggested by other studies. Further study of immune responses associated with different HRVs and PBMCs from different patient groups, and the mechanisms leading to these differences, should help characterize pathogenesis of HRV disease and generate novel approaches to its treatment.     

Human Rhinovirus Induced Cytokine/Chemokine Responses in Human Airway Epithelial and Immune Cells

Infections with human rhinovirus (HRV) are commonly associated with acute upper and lower respiratory tract disease and asthma exacerbations. The role that HRVs play in these diseases suggests it is important to understand host-specific or virus-specific factors that contribute to pathogenesis. Since species A HRVs are often associated with more serious HRV disease than species B HRVs, differences in immune responses they induce should inform disease pathogenesis. To identify species differences in induced responses, we evaluated 3 species A viruses, HRV 25, 31 and 36 and 3 species B viruses, HRV 4, 35 and 48 by exposing human PBMCs to HRV infected Calu-3 cells. To evaluate the potential effect of memory induced by previous HRV infection on study responses, we tested cord blood mononuclear cells that should be HRV naïve. There were HRV-associated increases (significant increase compared to mock-infected cells) for one or more HRVs for IP-10 and IL-15 that was unaffected by addition of PBMCs, for MIP-1α, MIP-1β, IFN-α, and HGF only with addition of PBMCs, and for ENA-78 only without addition of PBMCs. All three species B HRVs induced higher levels, compared to A HRVs, of MIP-1α and MIP-1β with PBMCs and ENA-78 without PBMCs. In contrast, addition of CBMCs had less effect and did not induce MIP-1α, MIP-1β, or IFN-α nor block ENA-78 production. Addition of CBMCs did, however, increase IP-10 levels for HRV 35 and HRV 36 infection. The presence of an effect with PBMCs and no effect with CBMCs for some responses suggest differences between the two types of cells possibly because of the presence of HRV memory responses in PBMCs and not CBMCs or limited response capacity for the immature CBMCs relative to PBMCs. Thus, our results indicate that different HRV strains can induce different patterns of cytokines and chemokines; some of these differences may be due to differences in memory responses induced by past HRV infections, and other differences related to virus factors that can inform disease pathogenesis.     

Viral and Atypical Bacterial Etiology of Acute Respiratory Infections in Children under 5 Years Old Living in a Rural Tropical Area of Madagascar

Background

In Madagascar, very little is known about the etiology and prevalence of acute respiratory infections (ARIs) in a rural tropical area. Recent data are needed to determine the viral and atypical bacterial etiologies in children with defined clinical manifestations of ARIs.

Methods

During one year, we conducted a prospective study on ARIs in children between 2 to 59 months in the community hospital of Ampasimanjeva, located in the south-east of Madagascar. Respiratory samples were analyzed by multiplex real-time RT-PCR, including 18 viruses and 2 atypical bacteria. The various episodes of ARI were grouped into four clinical manifestations with well-documented diagnosis: “Community Acquired Pneumonia”(CAP, group I), “Other acute lower respiratory infections (Other ALRIs, group II)”, “Upper respiratory tract infections with cough (URTIs with cough, group III)”and “Upper respiratory tract infections without cough (URTIs without cough, group IV)”.

Results

295 children were included in the study between February 2010 and February 2011. Viruses and/or atypical bacteria respiratory pathogens were detected in 74.6% of samples, the rate of co-infection was 27.3%. Human rhinovirus (HRV; 20.5%), metapneumovirus (HMPV A/B, 13.8%), coronaviruses (HCoV, 12.5%), parainfluenza virus (HPIV, 11.8%) and respiratory syncytial virus A and B (RSV A/B, 11.8%) were the most detected. HRV was predominantly single detected (23.8%) in all the clinical groups while HMPV A/B (23.9%) was mainly related to CAP (group I), HPIV (17.3%) to the “Other ALRIs” (group II), RSV A/B (19.5%) predominated in the group “URTIs with cough” (group III) and Adenovirus (HAdV, 17.8%) was mainly detected in the “without cough” (group IV).

Interpretation

This study describes for the first time the etiology of respiratory infections in febrile children under 5 years in a malaria rural area of Madagascar and highlights the role of respiratory viruses in a well clinically defined population of ARIs.     

Epidemiology and Clinical Characteristics of Respiratory Infections Due to Adenovirus in Children Living in Milan,Italy, during 2013 and 2014

To evaluate the predominant human adenovirus (HAdV) species and types associated with pediatric respiratory infections, nasopharyngeal swabs were collected from otherwise healthy children attending an emergency room in Milan, Italy, due to a respiratory tract infection from January 1 to February 28 of two subsequent years, 2013 and 2014. The HAdVs were detected using a respiratory virus panel fast assay (xTAG RVP FAST v2) and with a HAdV-specific real-time polymerase chain reaction; their nucleotides were sequenced, and they were tested for positive selection. Among 307 nasopharyngeal samples, 61 (19.9%) tested positive for HAdV. HAdV was the only virus detected in 31/61 (50.8%) cases, whereas it was found in association with one other virus in 25 (41.0%) cases and with two or more viruses in 5 (8.2%) cases. Human Enterovirus/human rhinovirus and respiratory syncytial virus were the most common co-infecting viral agents and were found in 12 (19.7%) and 7 (11.5%) samples, respectively. Overall, the HAdV strain sequences analyzed were highly conserved. In comparison to HAdV-negative children, those infected with HAdV had a reduced frequency of lower respiratory tract involvement (36.1% vs 55.2%; p = 0.007), wheezing (0.0% vs 12.5%; p = 0.004), and hospitalization (27.9% vs 56.1%; p<0.001). Antibiotic therapy and white blood cell counts were more frequently prescribed (91.9% vs 57.1%; p = 0.04) and higher (17,244 ± 7,737 vs 9,565 ± 3,211 cells/μL; p = 0.04), respectively, in children infected by HAdV-C than among those infected by HAdV-B. On the contrary, those infected by HAdV-B had more frequently lower respiratory tract involvement (57.1% vs 29.7%) but difference did not reach statistical significant (p = 0.21). Children with high viral load were absent from child care attendance for a longer period of time (14.5 ± 7.5 vs 5.5 ± 3.2 days; p = 0.002) and had higher C reactive protein levels (41.3 ± 78.5 vs 5.4 ± 9.6 μg/dL; p = 0.03). This study has shown that HAdV infections are diagnosed more commonly than usually thought and that HAdVs are stable infectious agents that do not frequently cause severe diseases. A trend toward more complex disease in cases due to HAdV species C and in those with higher viral load was demonstrated. However, further studies are needed to clarify factors contributing to disease severity to understand how to develop adequate preventive and therapeutic measures.     

Risk factors for lower respiratory complications of rhinovirus infections in elderly people living in the community: prospective cohort study.

OBJECTIVE: To assess the role of rhinoviruses in elderly people living in the community. DESIGN: Prospective community based surveillance of elderly people, without intervention. Subjects were telephoned weekly to identify symptomatic upper respiratory tract infections. Symptoms and impact of illnesses were monitored, and specimens were collected for diagnostic serology and human rhinovirus polymerase chain reaction. SETTING: Leicestershire, England. SUBJECTS: 533 subjects aged 60 to 90. MAIN OUTCOME MEASURES: Symptoms, restriction of activity, medical consultations, and antibiotic use during 96 rhinovirus infections. Adjusted odds ratios for lower respiratory syndromes with respect to smoking and health status. RESULTS: A viral cause was established in 211 (43%) of 497 respiratory illnesses; rhinoviruses were identified in 121 (24%) and as single pathogens in 107. The median duration of the first or only rhinovirus infection in the 96 people with 107 rhinovirus infections was 16 days; 18 of the 96 patients were confined to bed and 25 were unable to cope with routine household activities. Overall, 60 patients with rhinovirus infections had lower respiratory tract syndromes; 41 patients consulted their doctor, 31 of them (76%) receiving antibiotics. One patient died. Logistic regression analysis showed that chronic medical conditions increased the estimated probability of lower respiratory rhinovirus illness by 40% (95% confidence interval 17% to 68%) and smoking by 47% (14% to 90%). There were almost six times as many symptomatic rhinovirus infections as influenza A and B infections. CONCLUSIONS: Rhinoviruses are an important cause of debility and lower respiratory illness among elderly people in the community. Chronic ill health and smoking increase the likelihood of lower respiratory complications from such infections. The overall burden of rhinovirus infections in elderly people may approach that of influenza.     

Lower Respiratory Tract Infections Associated with Rhinovirus during Infancy and Increased Risk of Wheezing during Childhood. A Cohort Study

Background and Objectives

Although association between respiratory syncytial virus infection and later asthma development has been established, little is known about the role of other respiratory viruses. Rhinovirus was considered a mild pathogen of the upper respiratory tract but current evidence suggests that rhinovirus is highly prevalent among children with lower respiratory tract infections (LRTI). The aim of the study was to evaluate whether LRTI hospitalization associated with rhinovirus during infancy was associated with an increased risk of wheezing – a proxy measure of asthma – during childhood.

Methods

During a 12 months period, all infants <1 year admitted to Manhiça District Hospital with symptoms of LRTI who survived the LRTI episode, were enrolled in the study cohort. Nasopharyngeal aspirates were collected on admission for viral determination and study infants were classified according to presence or not of rhinovirus. The study cohort was passively followed-up at the Manhiça District Hospital for up to 4 years and 9 months to evaluate the association between LRTI associated with rhinovirus in infancy and wheezing during childhood.

Findings and Conclusions

A total of 220 infants entered the cohort; 25% of them had rhinovirus detected during the LRTI episode as opposed to 75% who tested negative for rhinovirus. After adjusting for sex and age and HIV infection at recruitment, infants hospitalized with LRTI associated with rhinovirus had higher incidence of subsequent visits with wheezing within the year following hospitalization [Rate ratio=1.68, (95% confidence interval=1.02-2.75); Wald test p-value = 0.039]. No evidence of increased incidence rate of visits with wheezing was observed for the remaining follow-up period. Our data suggest a short term increased risk of wheezing after an initial episode of LRTI with RV.     

Characterization of the Upper Respiratory Tract Microbiomes of Patients with Pandemic H1N1 Influenza

The upper respiratory tract microbiome has an important role in respiratory health. Influenza A is a common viral infection that challenges that health, and a well-recognized sequela is bacterial pneumonia. Given this connection, we sought to characterize the upper respiratory tract microbiota of individuals suffering from the pandemic H1N1 influenza A outbreak of 2009 and determine if microbiome profiles could be correlated with patient characteristics. We determined the microbial profiles of 65 samples from H1N1 patients by cpn60 universal target amplification and sequencing. Profiles were examined at the phylum and nearest neighbor “species” levels using the characteristics of patient gender, age, originating health authority, sample type and designation (STAT/non-STAT). At the phylum level, Actinobacteria-, Firmicutes- and Proteobacteria-dominated microbiomes were observed, with none of the patient characteristics showing significant profile composition differences. At the nearest neighbor “species” level, the upper respiratory tract microbiomes were composed of 13-20 “species” and showed a trend towards increasing diversity with patient age. Interestingly, at an individual level, most patients had one to three organisms dominant in their microbiota. A limited number of discrete microbiome profiles were observed, shared among influenza patients regardless of patient status variables. To assess the validity of analyses derived from sequence read abundance, several bacterial species were quantified by quantitative PCR and compared to the abundance of cpn60 sequence read counts obtained in the study. A strong positive correlation between read abundance and absolute bacterial quantification was observed. This study represents the first examination of the upper respiratory tract microbiome using a target other than the 16S rRNA gene and to our knowledge, the first thorough examination of this microbiome during a viral infection.