Prevalence of human respiratory viruses in adults with acute respiratory tract infections in Beijing, 2005–2007

To determine the aetiological role and epidemiological profile of common respiratory viruses in adults with acute respiratory tract infections (ARTIs), a 2-year study was conducted in Beijing, China, from May 2005 to July 2007. Nose and throat swab samples from 5808 ARTI patients were analysed by PCR methods for common respiratory viruses, including influenza viruses (IFVs) A, B, and C, parainfluenza viruses (PIVs) 1–4, enteroviruses (EVs), human rhinoviruses (HRVs), respiratory syncytial virus (RSV), human metapneumovirus (HMPV), human coronaviruses (HCoVs) OC43, 229E, NL63, and HKU1, and adenoviruses (ADVs). Viral pathogens were detected in 34.6% of patient samples, and 1.6% of the patients tested positive for more than one virus. IFVs (19.3%) were the dominant agents detected, followed by HRVs (6.5%), PIVs (4.3%), EVs (3.2%), and HCoVs (1.1%). ADVs, RSV and HMPV were also detected (<1%). The viral detection rates differed significantly between infections of the lower and upper respiratory tracts in the sample population: PIVs, the second most commonly detected viral agents in lower acute respiratory tract infections (LRTIs), were more prevalent than in upper acute respiratory tract infections, indicating that the pathogenic role of PIVs in LRTIs should be investigated. Currently, this study is the largest-scale investigation of respiratory virus infections in China with multiple agent detection, providing baseline data for further studies of respiratory virus infections in adults with ARTIs.     

Simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-PCR assays

There is a need for rapid, sensitive, and accurate diagnosis of lower respiratory tract infections in children, elderly, and immunocompromised patients, who are susceptible to serious complications. The multiplex RT-nested PCR assay has been used widely for simultaneous detection of non-related viruses involved in infectious diseases because of its high specificity and sensitivity. A new multiplex RT-PCR assay is described in this report. This approach includes nested primer sets targeted to conserve regions of human parainfluenza virus haemagglutinin, human coronavirus spike protein, and human enterovirus and rhinovirus polyprotein genes. It permits rapid, sensitive, and simultaneous detection and typing of the four types of parainfluenza viruses (1, 2, 3, 4AB), human coronavirus 229E and OC43, and the generic detection of enteroviruses and rhinoviruses. The testing of 201 clinical specimens with this multiplex assay along with other one formerly described by our group to simultaneously detect and type the influenza viruses, respiratory syncytial viruses, and a generic detection of all serotypes of adenovirus, covers the detection of most viruses causing respiratory infectious disease in humans. The results obtained were compared with conventional viral culture, immunofluorescence assay, and a third multiplex RT-PCR assay for all human parainfluenza viruses types described previously. In conclusion, both multiplex RT-PCR assays provide a system capable of detecting and identifying simultaneously 14 different respiratory viruses in clinical specimens with high sensitivity and specificity, being useful for routine diagnosis and survey of these viruses within the population.     

Metagenomic analysis of viral diversity in respiratory samples from patients with respiratory tract infections in Kuwait

A metagenomic approach based on target independent next‐generation sequencing has become a known method for the detection of both known and novel viruses in clinical samples. This study aimed to use the metagenomic sequencing approach to characterize the viral diversity in respiratory samples from patients with respiratory tract infections. We have investigated 86 respiratory samples received from various hospitals in Kuwait between 2015 and 2016 for the diagnosis of respiratory tract infections. A metagenomic approach using the next‐generation sequencer to characterize viruses was used. According to the metagenomic analysis, an average of 145, 019 reads were identified, and 2% of these reads were of viral origin. Also, metagenomic analysis of the viral sequences revealed many known respiratory viruses, which were detected in 30.2% of the clinical samples. Also, sequences of non‐respiratory viruses were detected in 14% of the clinical samples, while sequences of non‐human viruses were detected in 55.8% of the clinical samples. The average genome coverage of the viruses was 12% with the highest genome coverage of 99.2% for respiratory syncytial virus, and the lowest was 1% for torque teno midi virus 2. Our results showed 47.7% agreement between multiplex Real‐Time PCR and metagenomics sequencing in the detection of respiratory viruses in the clinical samples. Though there are some difficulties in using this method to clinical samples such as specimen quality, these observations are indicative of the promising utility of the metagenomic sequencing approach for the identification of respiratory viruses in patients with respiratory tract infections.

     

Development and implementation of a molecular diagnostic platform for daily rapid detection of 15 respiratory viruses

Acute respiratory tract infections are caused by a large number of viruses. Diagnostic methods have until recently been available only for a limited number of these viruses. With the objective to achieve sensitive assays for all respiratory viruses, a rational workflow in the laboratory, and a short turn‐around time, a real‐time PCR diagnostic platform for daily rapid detection of 15 respiratory viruses was developed. The system was evaluated on 585 stored nasopharyngeal aspirates from hospitalized children. Previous analysis by immunofluorescence and virus isolation identified viruses in 37% of the samples while the new PCR diagnostic panel detected 57% virus positive samples. The new platform was introduced in the laboratory in October 2007 and has then fully replaced the standard immunofluorescence assay for rapid detection of viruses and virus isolation. J. Med. Virol. 81:167–175, 2009. © 2008 Wiley‐Liss, Inc.     

Diagnosis of human coronavirus infection by immunofluorescence: method and application to respiratory disease in hospitalized children.

Rabbit antisera were prepared against coronavirus strains 229E and OC43 and used successfully to detect viral antigen in epithelial cells shed from the nasopharynx of symptomatic volunteers who had received coronavirus inocula three to four days before. The same serologic reagents were applied to nasopharyngeal secretion cells obtained from 106 infants and children hospitalized with respiratory tract disease and apparently not infected with conventional respiratory viruses. No coronavirus infections were detected by this method. It appears that coronavirus OC43 or 229E infections were not common in children in Tyneside hospitals during the period of study. However, fluorescence is a useful method for detection of coronavirus infections in symptomatic human subjects.     

Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients

A panel of 23 real-time PCR assays based on TaqMan technology has been developed for the detection and monitoring of 16 different viruses and virus families including human polyomaviruses BK virus and JC virus, human herpesviruses 6, 7, and 8, human adenoviruses, herpes simplex viruses 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, parvovirus B19, influenza A and B viruses, parainfluenza viruses 1 to 3, enteroviruses, and respiratory syncytial virus. The test systems presented have a broad dynamic range and display high sensitivity, reproducibility, and specificity. Moreover, the assays allow precise quantification of viral load in a variety of clinical specimens. The ability to use uniform PCR conditions for all assays permits simultaneous processing and detection of many different viruses, thus economizing the diagnostic work. Our observations based on more than 50,000 assays reveal the potential of the real-time PCR tests to facilitate early diagnosis of infection and to monitor the kinetics of viral proliferation and the response to treatment. We demonstrate that, in immunosuppressed patients with invasive virus infections, surveillance by the assays described may permit detection of increasing viral load several days to weeks prior to the onset of clinical symptoms. In virus infections for which specific treatment is available, the quantitative PCR assays presented provide reliable diagnostic tools for timely initiation of appropriate therapy and for rapid assessment of the efficacy of antiviral treatment strategies.     

Comparison of the FilmArray RP,Verigene RV+, and Prodesse ProFLU+/FAST+ Multiplex Platforms for Detection of Influenza Viruses in Clinical Samples from the 2011-2012 Influenza Season in Belgium

Respiratory tract infections (RTIs) are caused by a plethora of viral and bacterial pathogens. In particular, lower RTIs are a leading cause of hospitalization and mortality. Timely detection of the infecting respiratory pathogens is crucial to optimize treatment and care. In this study, three U.S. Food and Drug Administration-approved molecular multiplex platforms (Prodesse ProFLU+/FAST+, FilmArray RP, and Verigene RV+) were evaluated for influenza virus detection in 171 clinical samples collected during the Belgian 2011-2012 influenza season. Sampling was done using mid-turbinate flocked swabs, and the collected samples were stored in universal transport medium. The amount of viral RNA present in the swab samples ranged between 3.07 and 8.82 log₁₀ copies/ml. Sixty samples were concordant influenza A virus positive, and 8 samples were found to be concordant influenza B virus positive. Other respiratory viruses that were detected included human rhinovirus/enterovirus, respiratory syncytial virus, parainfluenza virus type 1, human metapneumovirus, and coronavirus NL63. Twenty-five samples yielded discordant results across the various assays which required further characterization by sequencing. The FilmArray RP and Prodesse ProFLU+/FAST+ assays were convenient to perform with regard to sensitivity, ease of use, and low percentages of invalid results. Although the limit of sensitivity is of utmost importance, many other factors should be taken into account in selecting the most convenient molecular diagnostic assay for the detection of respiratory pathogens in clinical samples.     

Viral respiratory infections in hospitalized and community control children in Alaska

Respiratory syncytial virus (RSV) in Alaska Native children from the Yukon Kuskokwim (YK) Delta is associated with a hospitalization rate five times higher than that reported for the general US child population. The role of other viral respiratory pathogens has not been studied in this population. YK Delta children <3 years of age hospitalized with respiratory infections and same aged community control children were prospectively enrolled between October 2005 and September 2007. Polymerase chain reaction detection of viruses was performed on nasopharyngeal samples. Characteristics of hospitalized and asymptomatic control children were analyzed. From October 2005 to September 2007, 440 hospitalized and 425 control children were analyzed. Respiratory viruses were detected in 90% (395) of hospitalized children: 194 (44%) rhinovirus, 131 (30%) adenovirus, 102 (23%) RSV, 77 (18%) para influenza viruses (PIV), 66 (15%) human metapneumovirus (hMPV), 23 (5%) influenza, and 25 (6%) coronavirus. Fifty‐two percent (221) of control children had a virus detected, most commonly rhinovirus (33%), and adenovirus (16%). RSV, PIV, hMPV, and influenza were significantly more common in hospitalized cases than control children, but rhinovirus, adenovirus, and coronavirus were not. RSV and hMPV were associated with higher severity of illness. In this study, RSV remains the most important virus associated with respiratory hospitalization, although hMPV and PIV were also common. RSV and hMPV were associated with more severe illness. Rhinovirus and adenovirus were detected in two‐thirds of hospitalized children, but their frequent detection in control children made their role in respiratory hospitalization uncertain. J. Med. Virol. 82:1282–1290, 2010. © 2010 Wiley‐Liss, Inc.     

Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections

Viruses are the major cause of pediatric acute respiratory tract infection (ARTI) and yet many suspected cases of infection remain uncharacterized. We employed 17 PCR assays and retrospectively screened 315 specimens selected by season from a predominantly pediatric hospital-based population. Before the Brisbane respiratory virus research study commenced, one or more predominantly viral pathogens had been detected in 15.2% (n = 48) of all specimens. The Brisbane study made an additional 206 viral detections, resulting in the identification of a microbe in 67.0% of specimens. After our study, the majority of microbes detected were RNA viruses (89.9%). Overall, human rhinoviruses (HRVs) were the most frequently identified target (n = 140) followed by human adenoviruses (HAdVs; n = 25), human metapneumovirus (HMPV; n = 18), human bocavirus (HBoV; n = 15), human respiratory syncytial virus (HRSV; n = 12), human coronaviruses (HCoVs; n = 11), and human herpesvirus-6 (n = 11). HRVs were the sole microbe detected in 37.8% (n = 31) of patients with suspected lower respiratory tract infection (LRTI). Genotyping of the HRV VP4/VP2 region resulted in a proposed subdivision of HRV type A into sublineages A1 and A2. Most of the genotyped HAdV strains were found to be type C. This study describes the high microbial burden imposed by HRVs, HMPV, HRSV, HCoVs, and the newly identified virus, HBoV on a predominantly paediatric hospital population with suspected acute respiratory tract infections and proposes a new formulation of viral targets for future diagnostic research studies.     

Lower respiratory tract infection in the community: associations between viral aetiology and illness course

ObjectivesThis study determined associations between respiratory viruses and subsequent illness course in primary care adult patients presenting with acute cough and/or suspected lower respiratory tract infection.MethodsA prospective European primary care study recruited adults with symptoms of lower respiratory tract infection between November 2007 and April 2010. Real-time in-house polymerase chain reaction (PCR) was performed to test for six common respiratory viruses. In this secondary analysis, symptom severity (scored 1 = no problem, 2 = mild, 3 = moderate, 4 = severe) and symptom duration were compared between groups with different viral aetiologies using regression and Cox proportional hazard models, respectively. Additionally, associations between baseline viral load (cycle threshold (Ct) value) and illness course were assessed.ResultsThe PCR tested positive for a common respiratory virus in 1354 of the 2957 (45.8%) included patients. The overall mean symptom score at presentation was 2.09 (95% confidence interval (CI) 2.07–2.11) and the median duration until resolution of moderately bad or severe symptoms was 8.70 days (interquartile range 4.50–11.00). Patients with influenza virus, human metapneumovirus (hMPV), respiratory syncytial virus (RSV), coronavirus (CoV) or rhinovirus had a significantly higher symptom score than patients with no virus isolated (0.07–0.25 points or 2.3–8.3% higher symptom score). Time to symptom resolution was longer in RSV infections (adjusted hazard ratio (AHR) 0.80, 95% CI 0.65–0.96) and hMPV infections (AHR 0.77, 95% CI 0.62–0.94) than in infections with no virus isolated. Overall, baseline viral load was associated with symptom severity (difference 0.11, 95% CI 0.06–0.16 per 10 cycles decrease in Ct value), but not with symptom duration.ConclusionsIn healthy, working adults from the general community presenting at the general practitioner with acute cough and/or suspected lower respiratory tract infection other than influenza impose an illness burden comparable to influenza. Hence, the public health focus for viral respiratory tract infections should be broadened.     

Changes in the etiology of viral lower respiratory tract infections in hospitalized children in Wenzhou,China: 2008-2017

This study investigated the seasonality and secular trends in the etiology of viral lower respiratory tract infections (LRTIs) among hospitalized children in Wenzhou, southeastern China. A retrospective review was conducted concerning viral LRTIs in children hospitalized at a university hospital between January 1, 2008 and December 31, 2017. Direct immunofluorescence was used to detect respiratory syncytial virus (RSV), adenovirus (AdV), influenza A virus (Inf A), influenza B virus (Inf B), and human parainfluenza virus types 1 to 3 (hPIV1-3). Of 89 898 children tested, at least one viral respiratory pathogen was identified in 25.6% and multiple pathogens were identified in 0.4%. RSV (17.6%), hPIV3 (4.0%), and AdV (2.2%) were the most frequently detected pathogens. The proportion of positive samples varied with age and was the highest in children aged <6 months (36.2%). Seasonal differences were observed in RSV, AdV, Inf A, Inf B, hPIV1, and hPIV3 infections. There was a declining trend in the proportion of positive samples over time, primarily due to a decrease in RSV and hPIV3 infections. RSV, hPIV3, and AdV were the most common viral respiratory pathogens identified among hospitalized children with LRTIs. The distribution of viruses varied with age and season.     

Molecular epidemiology and disease severity of respiratory syncytial virus in relation to other potential pathogens in children hospitalized with acute respiratory infection in Jordan

Human respiratory syncytial virus (HRSV) is the major viral cause of acute lower respiratory tract infections in children. Few data about the molecular epidemiology of respiratory syncytial virus in developing countries, such as Jordan, are available. The frequency and severity of infections caused by HRSV were assessed in hospitalized Jordanian children <5 years of age compared with other potential etiological agents. Overall a potential pathogen was detected in 78% (254/326) of the children. HRSV was detected in 43% (140/326) of the nasopharyngeal aspirates. HRSV was found more frequently during the winter (January/February), being less frequent or negligible by spring (March/April). Analysis of 135 HRSV-positive strains using restriction fragment length polymorphism showed that 94 (70%) belonged to subgroup A, and 41 (30%) to subgroup B. There were also two cases of mixed genotypic infection. Only four of the six previously described N genotypes were detected with NP4 predominating. There were no associations between subgroup or N-genogroup and disease severity. HRSV was significantly associated with more severe acute respiratory infection and the median age of children with HRSV was lower than for those without. Next in order of frequency were adenovirus (116/312: 37%), human bocavirus (57/312: 18%), rhinovirus (36/325: 11%), Chlamydia spp. (14/312: 4.5%), human metapneumovirus (8/326: 2.5%), human coronavirus NL63 (4/325: 1.2%), and influenza A virus (2/323: 0.6%). Influenza B; parainfluenza viruses 1-4, human coronavirus HKU1 and Mycoplasma pneumoniae were not detected.     

Etiology of acute viral respiratory infections common in Pakistan: A review

Respiratory infections, especially those of the lower respiratory tract, remain a foremost cause of mortality and morbidity of children greater than 5 years in developing countries including Pakistan. Ignoring these acute‐level infections may lead to complications. Particularly in Pakistan, respiratory infections account for 20% to 30% of all deaths of children. Even though these infections are common, insufficiency of accessible data hinders development of a comprehensive summary of the problem. The purpose of this study was to determine the prevalence rate in various regions of Pakistan and also to recognize the existing viral strains responsible for viral respiratory infections through published data. Respiratory viruses are detected more frequently among rural dwellers in Pakistan. Lower tract infections are found to be more lethal. The associated pathogens comprise respiratory syncytial virus (RSV), human metapneumovirus (HMPV), coronavirus, enterovirus/rhinovirus, influenza virus, parainfluenza virus, adenovirus, and human bocavirus. RSV is more dominant and can be subtyped as RSV‐A and RSV‐B (BA‐9, BA‐10, and BA‐13). Influenza A (H1N1, H5N1, H3N2, and H1N1pdm09) and Influenza B are common among the Pakistani population. Generally, these strains are detected in a seasonal pattern with a high incidence during spring and winter time. The data presented include pneumonia, bronchiolitis, and influenza. This paper aims to emphasise the need for standard methods to record the incidence and etiology of associated pathogens in order to provide effective treatment against viral infections of the respiratory tract and to reduce death rates.     

Epidemiology of viral respiratory infections in a tertiary care centre in the era of molecular diagnosis,Geneva, Switzerland, 2011–2012

Few studies have examined the epidemiology of respiratory viral infections in large tertiary centres over more than one season in the era of molecular diagnosis. Respiratory clinical specimens received between 1 January 2011 and 31 December 2012 were analysed. Respiratory virus testing was performed using a large panel of real-time PCR or RT-PCR. Results were analysed according to sample type (upper versus lower respiratory tract) and age group. In all, 2996 (2469 (82.4%) upper; 527 (17.6%) lower) specimens were analysed. Overall positivity rate was 47.4% and 23.7% for upper and lower respiratory samples, respectively. The highest positivity rate was observed in patients under 18 years old (p <0.001); picornaviruses were the most frequent viruses detected over the year. Influenza virus, respiratory syncytial virus, human metapneumovirus and coronaviruses showed a seasonal peak during the winter season, while picornaviruses and adenoviruses were less frequently detected in these periods. Multiple viral infections were identified in 12% of positive cases and were significantly more frequent in children (p <0.001). In conclusion, we observed significant differences in viral infection rates and virus types among age groups, clinical sample types and seasons. Follow-up of viral detection over several seasons allows a better understanding of respiratory viral epidemiology.     

Etiology of viral respiratory infections in Northern Lao People's Democratic Republic

In Lao People's Democratic Republic (PDR), acute respiratory infections overburden the health care system, but viral etiology, genetic diversity, and seasonality, especially in light of the introduction of influenza vaccination in the country, are poorly understood. From August 2010 to April 2011, 309 outpatients were recruited at the Luang Prabang Provincial Hospital covering highland Lao communities. Nasopharyngeal swabs were screened for the presence of 13 respiratory viruses. At least one virus was detected in 69.6% and dual/triple viral infections in 12.9%/1.9% of the patients. Influenza A and B viruses combined were the most frequently detected pathogens, followed by human adenovirus and respiratory syncytial virus (RSV). The other viruses were detected in less than 10% of the patients. Phylogenetic analyses on a representative set of RSV strains revealed that, while otherwise very rare, the RSV‐B CB1/THB genotype cocirculated with other common genotypes. A single wave of influenza virus and RSV activity was observed during the rainy season, providing further support to influenza vaccination before the onset of the rains. This study provides recommendations for influenza vaccination that still needs optimization and highlights the need for revised guidelines for treatment and prevention of respiratory infections in Lao PDR, as well as for increased surveillance efforts.     

Broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex RT-PCR DNA microarray system

Newly available molecular tools allow a sensitive detection of a broad panel of viruses in respiratory tract specimens. In the present study, the application of a multiplex RT-PCR DNA microarray in diagnosis and epidemiological survey of viral infections in infants hospitalized for bronchiolitis was assessed. One hundred and thirty-eight nasopharyngeal aspirates collected from October 2007 to September 2008 were tested by direct immunofluorescence and viral culture, a combination of referenced RT-PCRs and the DNA microarray. One or more viruses were detected in 96, 126 and 126 of the specimens by direct immunofluorescence and viral culture, RT-PCRs and DNA microarray, respectively (70 vs. 91 vs. 91%, P < 10(-3)). The RT-PCRs and the DNA microarray yielded concordant results for 99% of specimens and identified mixed viral infections in 85 (62%). The most common associations were: human bocavirus and respiratory syncytial virus (32%), adenovirus and respiratory syncytial virus (30%), and parainfluenza virus type 3 and respiratory syncytial virus (23%). None of the bronchiolitis severity parameters including intensive care unit admission, O(2) supply, O(2) saturation percentage, O(2) length and length of stay at the hospital appeared to be significantly increased in multiple viral infections compared to single viral infections (P > 0.1). In conclusion, the use of this DNA microarray in clinical virology practice allows rapid and accurate identification of common and uncommon viral respiratory pathogens in infants hospitalized for bronchiolitis. It should improve the clinical management, the epidemiological survey, and the prevention of the nosocomial transmission of respiratory viruses in pediatric wards.