Detection Of Multiple Respiratory Viruses Research Articles

Microfluidic qPCR for detection of 21 common respiratory viruses in children with influenza-like illness.

Multiple respiratory viruses lead to high morbidity and mortality, yet global surveillance platforms focus primarily on seasonal influenza viruses. The COVID-19 pandemic and new RSV vaccines highlight the importance of a broader approach. Upper respiratory tract swabs from children aged 24-59months presenting with influenza-like illness in The Gambia were collected during follow-up of a live-attenuated influenza vaccine randomised controlled trial in 2017-18. A microfluidic quantitative polymerase chain reaction (qPCR) assay was established and used to detect 21 respiratory viruses. 76.6% of samples had one or more viruses detected (n = 121/158). The viruses detected most frequently were rhinovirus (n = 37/158, 23.4%) and adenovirus (n = 34/158, 21.5%), followed by parainfluenza virus 3, influenza B and human metapneumovirus B. A third of positive samples had multiple viruses detected (two n = 31/121, 25.6%; three n = 9/121, 7.4%). Our data demonstrates how microfluidic qPCR is a useful tool for high-throughput, comprehensive detection of multiple respiratory viruses in surveillance platforms. Rapidly changing epidemiology exemplifies the need for new, broader approaches to virus surveillance in low-resource settings to respond to future epidemics and to guide the need for and use of new prevention and therapeutic measures.

Salivary polyreactive antibodies and Haemophilus influenzae are associated with respiratory infection severity in young children with recurrent respiratory infections.

Recurrent respiratory tract infections (rRTIs) are a common reason for immunodiagnostic testing in children, which relies on serum antibody level measurements. However, because RTIs predominantly affect the respiratory mucosa, serum antibodies may inaccurately reflect local immune defences. We investigated antibody responses in saliva and their interplay with the respiratory microbiota in relation to RTI severity and burden in young children with rRTIs. We conducted a prospective cohort study including 100 children aged <10 years with rRTIs, their family members and healthy healthcare professionals. Total and polyreactive antibody concentrations were determined in serum and saliva (ELISA); respiratory microbiota composition (16S rRNA sequencing) and respiratory viruses (quantitative PCR) were characterised in nasopharyngeal swabs. Proteomic analysis (Olink) was performed on saliva and serum samples. RTI symptoms were monitored with a daily mobile phone application and assessed using latent class analysis and negative binomial mixed models. Serum antibody levels were not associated with RTI severity. Strikingly, 28% of salivary antibodies and only 2% of serum antibodies displayed polyreactivity (p<0.001). Salivary polyreactive IgA was negatively associated with recurrent lower RTIs (adjusted OR 0.80, 95% CI 0.67-0.94) and detection of multiple respiratory viruses (adjusted OR 0.76, 95% CI 0.61-0.96). Haemophilus influenzae abundance was positively associated with RTI symptom burden (regression coefficient 0.05, 95% CI 0.02-0.08). These results highlight the importance of mucosal immunity in RTI severity and burden, and suggest that the level of salivary polyreactive IgA and H. influenzae abundance may serve as indicators of infection severity and burden in young children with rRTIs.

Influenza A, Influenza B, human respiratory syncytial virus and SARSCoV-2 molecular diagnostics and epidemiology in the post COVID-19 era

BackgroundThe concurrent circulation of SARS-CoV-2 with other respiratory viruses is unstoppable and represents a new diagnostic reality for clinicians and clinical microbiology laboratories. Multiplexed molecular testing on automated platforms that focus on the simultaneous detection of multiple respiratory viruses in a single tube is a useful approach for current and future diagnosis of respiratory infections in the clinical setting.MethodsTwo time periods were included in the study: from February to April 2022, an early 2022 period, during the gradual lifting of COVID-19 prevention measures in the country, and from October 2022 to April 2023, the 2022/23 respiratory infections season. We analysed a total of 1,918 samples in the first period and 18,131 respiratory samples in the second period using a multiplex molecular assay for the simultaneous detection of Influenza A (Flu-A), Influenza B (Flu-B), Human Respiratory Syncytial Virus (HRSV) and SARS-CoV-2.ResultsThe results from early 2022 showed a strong dominance of SARS-CoV-2 infections with 1,267/1,918 (66.1%) cases. Flu-A was detected in 30/1,918 (1.6%) samples, HRSV in 14/1,918 (0.7%) samples, and Flu-B in 2/1,918 (0.1%) samples. Flu-A/SARS-CoV-2 co-detections were observed in 11/1,267 (0.9%) samples, and HRSV/SARS-CoV-2 co-detection in 5/1,267 (0.4%) samples. During the 2022/23 winter respiratory season, SARS-CoV-2 was detected in 1,738/18,131 (9.6%), Flu-A in 628/18,131 (3.5%), Flu-B in 106/18,131 (0.6%), and HRSV in 505/18,131 (2.8%) samples. Interestingly, co-detections were present to a similar extent as in early 2022.ConclusionThe results show that the multiplex molecular approach is a valuable tool for the simultaneous laboratory diagnosis of SARS-CoV-2, Flu-A/B, and HRSV in hospitalized and outpatients. Infections with Flu-A/B, and HRSV occurred shortly after the COVID-19 control measures were lifted, so a strong reoccurrence of various respiratory infections and co-detections in the post COVID-19 period was to be expected.

High Accuracy of Clinical Verification of Electrohydrodynamic-Driven Nanobox-on-Mirror Platform for Molecular Identification of Respiratory Viruses.

The molecular detection of multiple respiratory viruses provides evidence for the rational use of drugs and effective health management. Herein, we developed and tested the clinical performance of an electrohydrodynamic-driven nanobox-on-mirror platform (E-NoM) for the parallel, accurate, and sensitive detection of four respiratory viral antigens. The E-NoM platform uses gold-silver alloy nanoboxes as the core material with the deposition of a silver layer as a shell on the core surfaces to amplify and enable a reproducible Raman signal readout that facilitates accurate detection. Additionally, the E-NoM platform employs gold microelectrode arrays as the mirror with electrohydrodynamics to manipulate the fluid flow and enhance molecular interactions for an improved biosensing response. The presence of viral antigens binds the nanobox-based core-shell nanostructure on the gold microelectrode and creates the nanocavity with extremely strong "hot spots" to benefit sensitive analysis. Significantly, in a large clinical cohort with 227 patients, the designed E-NoM platform demonstrates the capability of screening respiratory infection with achieved clinical specificity, sensitivity, and accuracy of 100.0, 96.48, and 96.91%, respectively. It is anticipated that the E-NoM platform can find a position in clinical usage for respiratory disease diagnosis.

Outcomes of viral coinfections in infants hospitalized for acute bronchiolitis

Background and ObjectiveThe clinical relevance of the detection of multiple respiratory viruses in acute bronchiolitis (AB) has not been established. Our goal was to evaluate the effect of viral coinfections on the progression and severity of AB.MethodsA retrospective observational study was conducted in a tertiary hospital in Spain from September 2012 to March 2020. Infants admitted for AB with at least one respiratory virus identified by molecular diagnostic techniques were included. A comparison was made between single-virus infections and viral coinfections. The evolution and severity of AB were determined based on the days of hospitalization and admission to the pediatric intensive care unit (PICU).ResultsFour hundred forty-five patients were included (58.4% male). The median weight was 5.2 kg (IQR 4.2–6.5), and the median age was 2.5 months (IQR 1.4–4.6). A total of 105 patients (23.6%) were admitted to the PICU. Respiratory syncytial virus (RSV) was the most frequent etiological agent (77.1%). A single virus was detected in 270 patients (60.7%), and viral coinfections were detected in 175 (39.3%), of which 126 (28.3%) had two viruses and 49 (11%) had three or more viruses. Hospital length of stay (LOS) increased in proportion to the number of viruses detected, with a median of 6 days (IQR 4–8) for single infections, 7 days (IQR 4–9) for coinfections with two viruses and 8 days (IQR 5–11) for coinfections with ≥ 3 viruses (p = 0.003). The adjusted Cox regression model showed that the detection of ≥ 3 viruses was an independent risk factor for a longer hospital LOS (HR 0.568, 95% CI 0.410–0.785). No significant association was observed between viral coinfections and the need for PICU admission (OR 1.151; 95% CI 0.737–1.797).ConclusionsViral coinfections modified the natural history of AB, prolonging the hospital LOS in proportion to the number of viruses detected without increasing the need for admission to the PICU.

Linear spot Raman detector for the bi-channel immunochromatographic assay to simultaneously detect respiratory viruses

The rapid and accurate measurement of Raman signals on the test line (T-line) of surface-enhanced Raman scattering immunochromatography assay (SERS-ICA) strip is necessary for on-site detection based on SERS. Here, a novel linear spot Raman detector which can directly collect the SERS signals of the whole T-line on the dual-diagnostic SERS-ICA strip is developed for detection of multiple respiratory viruses via two magnetic SERS tags within 26 min. The tags modified with two Raman reporter molecules can specifically recognize two viruses, thus enabling the simultaneous detection of both viruses on one T-line. Measured by the linear Raman detector, the limits of detection of the proposed dual-diagnostic SERS-ICA strip is as low as 870 and 263 copies/mL for severe acute respiratory syndrome coronavirus 2 and H1N1, respectively. The dual-diagnostic strip combined with the novel Raman detector herein are of great importance in the differential diagnosis of respiratory pathogens and show potential in the rapid and accurate diagnosis and control of emerging acute respiratory diseases.

Clinical and Virologic Impacts of Respiratory Viral Co-infections in Children With Influenza.

Advances in multiplex polymerase chain reaction (PCR) methods have enabled the simultaneous detection of multiple respiratory viruses. We aimed to estimate the clinical and virologic impacts of influenza and other respiratory virus co-infection in children. We enrolled 38 and 35 children diagnosed with influenza and treated with baloxavir marboxil (baloxavir) and oseltamivir, respectively. We performed quantitative reverse transcription-PCR to detect and measure the levels of noninfluenza viruses from 3 nasopharyngeal swab samples collected before and on days 3 and 5 after the initial antiviral dose. We assessed patients' clinical information using questionnaires. One or more respiratory viruses other than influenza virus were detected in 26 (35.6%) of 73 children before antiviral treatment. The influenza virus load and clinical characteristics on the day of influenza onset were similar between children with and without virus co-infections. Of the 26 and 32 children without the emergence of the reduced baloxavir and oseltamivir susceptible variants after treatment, 8 (30.8%) and 7 (21.9%) children were dually co-infected with human rhinovirus only, respectively. The level of human rhinovirus RNA on day 0 in these children was less than -3 log 10 that of influenza virus RNA, and the human rhinovirus co-infection had no impact on the disease course either clinically or virologically. When multiple respiratory viruses are detected in the same patient, it is necessary to assess clinical symptoms as well as the levels of detected viruses to determine which virus contributes to the development of illness.

Simultaneous Detection of Multiple Respiratory Viruses Among SARS-CoV-2-Positive and Negative Patients by Multiplex TaqMan One-Step Real-Time PCR

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has clinical manifestations similar to other common respiratory viral infections. There are limited data on the frequency of viral respiratory coinfection among patients infected with SARS-CoV-2 in Iran. Objectives: This cross-sectional study investigated the prevalence of multiple respiratory viruses among SARS-CoV-2-positive and negative patients during the coronavirus disease 2019 (COVID-19) pandemic in Iran. Methods: We included oropharyngeal/nasopharyngeal swab specimens of patients suspected of COVID-19 from December 2020 to March 2021. A reliable multiplex TaqMan one-step real-time PCR method was employed to detect 17 viral respiratory pathogens simultaneously. Descriptive analyses were performed to characterize the specimens concerning age, gender, clinical manifestations, and underlying disease. Results: Multiple respiratory viruses with a frequency of 18.78% were detected in 197 studied patients. Human metapneumovirus was the most prevalent pathogen detected in both SARS-CoV-2-positive (n = 7, 7.7%) and negative (n = 7, 6.6%) patients. Moreover, the frequency rate of viral infection was almost the same in both SARS-CoV-2-positive (18.68%) and negative (18.86%) patients. Altogether, there were no differences in baseline demographic characteristics such as age, sex, clinical symptoms, and comorbidities between the two groups (P &gt; 0.05). Conclusions: The data presented here expand our understanding of the epidemiology of multiple types of viral respiratory pathogens in suspected COVID-19 patients. Therefore, simultaneous screening of other viral respiratory pathogens will be helpful for clinicians and researchers interested in the control of viral respiratory tract infections.

Comparative Evaluation of Allplex Respiratory Panels 1, 2, 3, and BioFire FilmArray Respiratory Panel for the Detection of Respiratory Infections.

Multiplex nucleic acid amplification assays that simultaneously detect multiple respiratory pathogens in a single nasopharyngeal swab (NPS) specimen are widely used for rapid clinical diagnostics. We evaluated Allplex Respiratory Panel (RP) 1, 2, 3, and the BioFire FilmArray RP assay for detecting respiratory pathogens from NPS specimens. In all, 181 NPS specimens obtained from patients suspected of having respiratory infections during the non-influenza season (August–December 2019) were included. The Allplex RP 1, 2, and 3 detected 154 samples positive for respiratory viruses, whereas the BioFire FilmArray detected viruses in 98 samples. Co-infection with two or more viruses was detected in 41 and 17 NPS specimens by Allplex RP and the BioFire FilmArray RP, respectively. For adenoviruses, Allplex RP 1 detected 31 specimens, compared to 34 by the BioFire FilmArray. In all, 64 NPS specimens were positive for human enterovirus (HEV) and human rhinovirus (HRV) on the Allplex RP, in contrast to 39 HEV/HRV on the BioFire FilmArray. The parainfluenza virus (PIV-1–4) detection rate differed between the two systems. Most discrepant results were observed for NPS specimens with high cycle threshold values obtained by Allplex RP. This study showed concordant performance of the Allplex RP 1, 2, 3, and the BioFire FilmArray RP for the simultaneous detection of multiple respiratory viruses.

Recent advances in the detection of respiratory virus infection in humans.

Respiratory tract viral infection caused by viruses or bacteria is one of the most common diseases in human worldwide, while those caused by emerging viruses, such as the novel coronavirus, 2019‐nCoV that caused the pneumonia outbreak in Wuhan, China most recently, have posed great threats to global public health. Identification of the causative viral pathogens of respiratory tract viral infections is important to select an appropriate treatment, save people's lives, stop the epidemics, and avoid unnecessary use of antibiotics. Conventional diagnostic tests, such as the assays for rapid detection of antiviral antibodies or viral antigens, are widely used in many clinical laboratories. With the development of modern technologies, new diagnostic strategies, including multiplex nucleic acid amplification and microarray‐based assays, are emerging. This review summarizes currently available and novel emerging diagnostic methods for the detection of common respiratory viruses, such as influenza virus, human respiratory syncytial virus, coronavirus, human adenovirus, and human rhinovirus. Multiplex assays for simultaneous detection of multiple respiratory viruses are also described. It is anticipated that such data will assist researchers and clinicians to develop appropriate diagnostic strategies for timely and effective detection of respiratory virus infections.

Multiplex Real-Time PCR Detection of Respiratory Viruses in Lower Respiratory Tract Infections in Children

Acute respiratory infections cause significant morbidity and mortality in children. Several new respiratory viruses have been identified and co-detection of multiple viruses is commonly reported. This is part of a prospective study which aims to detect respiratory viruses by multiplex molecular method and conventional methods. Nasopharyngeal aspirate specimens were taken from hospitalised children aged less than 5 years with lower respiratory tract infections. These were tested using viral culture, immunofluorescence and Seegene Anyplex™ II RV16 real-time polymerase chain reaction. From 102 samples, 69 (67.6%) were positive by PCR, 12 (11.8%) positive by culture method and 13 (12.7%) positive by IF. A single viral pathogen was detected in 48 samples (47.1%), while 21 samples (20.6%) had co-detection of 2 to 4 viral pathogens. Respiratory syncytial virus (RSV) was most common, detected in 17 samples (16.7% of all samples), followed by adenovirus and rhinovirus in 16 (15.7%), respectively. Bocavirus was detected in 15, enterovirus in 15, influenza A in 8 and parainfluenza-4 in 4 samples, with highest occurrences in co-detection (12/15, 10/15, 5/8 and 3/4, respectively). RSV was the least likely detected in co-detection (3/17). In PCR-positive samples, 54/69 (78.3%) were patients aged up to 24 months. Molecular methods detect more viral aetiologies than conventional methods, with simultaneous detection of multiple respiratory viruses. More sensitive, specific and rapid tools to determine aetiological agents could be incorporated into diagnostic algorithms of respiratory tract infections. Interpretations, significance, and applicability in clinical practice could be further explored, particularly for patients up to 2 years old.

Rapid detection of multiple respiratory viruses based on microfluidic isothermal amplification and a real-time colorimetric method.

Respiratory viruses are major threats causing development of acute respiratory tract infections, which are common causes of illness and death throughout the world. Here, an integrated microsystem based on real-time colorimetry was developed for diagnosing multiple respiratory viruses. The microsystem employed magnetic beads for nucleic acid extraction and an eight-channel microfluidic array chip integrated with a loop-mediated isothermal amplification system for point-of-care screening of respiratory viruses. The overall detection process (including sample collection, nucleic acid extraction, sample loading, real-time detection, and signal output) could be completed within 1 h. Our results show that the developed method could specifically recognize influenza A virus subtypes (H1N1, H3N2, H5N1, and H7N9), influenza B virus, and human adenoviruses. The results obtained with 109 clinical samples indicate that the developed method has high specificity (100%, confidence interval 94.9-100.0) and sensitivity (96%, confidence interval 78.1-99.9). The integration of magnetic bead-based pre-treatment techniques and microfluidic isothermal amplification provides an effective solution for rapidly detecting etiological agents of respiratory diseases. The strategy of using a closed chip system and real-time colorimetry reduced aerosol contamination and ensured the accuracy of the results. The developed method provides an effective alternative for rapid point-of-care screening for viruses that cause respiratory disease syndromes and further aids in accurate and timely detection to control and prevent the spread of respiratory diseases caused by such pathogens.

Evaluation of custom multiplex real - time RT - PCR in comparison to fast - track diagnostics respiratory 21 pathogens kit for detection of multiple respiratory viruses

BackgroundSevere acute respiratory infections in children can be fatal, rapid identification of the causative agent and timely treatment can be life saving. Multiplex real time RT-PCR helps in simultaneous detection of multiple viruses saving cost, time and labour. Commercially available multiplex real time RT-PCR kits are very expensive. Therefore the aim of the present study was to develop a cost effective multiplex real time RT-PCR for the detection of 18 respiratory viruses and compare it with an in-vitro diagnostics approved Fast Track Diagnostic Respiratory Pathogens 21 Kit (FTD).MethodsNasopharyngeal aspirates and throat swabs were collected and processed for extraction of nucleic acid using an automated extraction system and multiplex real time RT-PCR was performed using the FTD kit and a custom assay on 356 samples.ResultsCustom and FTD assays detected one or more respiratory viruses in 268 (75.29 %) and 262 (73.60 %) samples respectively. The concordance between the custom assay and the FTD assay was 100 % for HCoV OC43, HCoV 229E, HPIV-1, HPIV-2, HBoV, HPeV, Flu A, and Influenza A(H1N1)pdm09 and 94.66 – 99.71 % for the remaining viruses; Flu B (99.71 %), HRV (99.71 %), HPIV-3 (98.87 %), HPIV-4 (99.43 %), HCoV NL63 (99.71 %), HMPV A/B (99.71 %), RSV A/B (94.66 %), EV (98.31 %), HCoV HKU1 (99.71 %), HAdV (99.71 %). Major discrepancy was observed for RSV A/B, which was over detected in 18 samples by the custom assay as compared to the FTD assay. The custom assay was much cheaper than the FTD assay and the time taken was only 29 min more.ConclusionThe custom primer and probe mix was found to be comparable to the FTD assay with good concordance but was much cheaper and the time taken for reporting was only 29 min more. The low cost custom multiplex RT-PCR can be a useful alternative to the costly FTD kit for rapid identification of viral aetiology in resource limited settings.

Detection of Multiple Respiratory Viruses Associated With Mortality and Severity of Illness in Children.

Respiratory viral infection is a common source of morbidity and mortality in children. Coinfection with multiple viruses occurs frequently; however, the clinical significance of concomitant viral pathogens is unclear. We hypothesized that presence of more than one respiratory virus is associated with increased morbidity and mortality when compared with children with a single respiratory virus. Retrospective cohort study. A tertiary care hospital. All children at Duke Children's Hospital over a 2-year period with isolation of a virus on an extended viral respiratory panel result. Demographic data, comorbidities, and details of hospital encounter were recorded. None. Two hundred thirty-five hospital encounters demonstrated positive extended viral respiratory panels. Immunocompromised status (37%) and respiratory comorbidities (23%) were common. Twenty-eight patients (12%) tested positive for multiple viruses, with adenovirus (23/28) and respiratory syncytial virus (15/28) most prevalent in patients with multiple viruses. Viral codetection was associated with increased use of noninvasive ventilation (p = 0.02), extracorporeal membrane oxygenation (p = 0.02), increased likelihood of moderate or severe illness (p = 0.005), and increased mortality (p = 0.01). Subgroup analysis demonstrated that this mortality association persisted for children with normal immune function (p = 0.003) and children with no comorbidities (p = 0.007). Children with multiple respiratory viruses may be at increased risk of moderate or severe illness and mortality, with previously healthy children potentially being at greatest risk. Further studies are indicated to determine the significance and generalizability of this finding and to better understand the pathophysiology of viral coinfection.

Field evaluation of TaqMan Array Card (TAC) for the simultaneous detection of multiple respiratory viruses in children with acute respiratory infection

BackgroundMultipathogen reverse transcription real-time PCR (RT-qPCR) platforms have proven useful in surveillance for acute respiratory illness (ARI) and study of respiratory outbreaks of unknown etiology. The TaqMan® Array Card (TAC, Life Technologies™), can simultaneously test 7 clinical specimens for up to 21 individual pathogens (depending on arrangement of controls and use of duplicate wells) by arrayed singleplex RT-qPCR on a single assay card, using minimal amounts of clinical specimens. A previous study described the development of TAC for the detection of respiratory viral and bacterial pathogens; the assay was evaluated against well-characterized analytical materials and a limited collection of human clinical specimens. ObjectivesWe wished to compare TAC assay performance against standard individual RT-qPCR assays for respiratory viral detection, focusing on 10 viruses (adenovirus, human metapneumovirus, human parainfluenza viruses 1–4, influenza viruses A and B, respiratory syncytial virus, and rhinovirus) from a larger collection of human specimens. Study designWe used specimens from 942 children with ARI enrolled systematically in a population-based, ARI surveillance study (New Vaccine Surveillance Network, NVSN). ResultsCompared with standard individual RT-qPCR assays, the sensitivity of TAC for the targeted viruses ranged from 54% to 95% (54%, 56%, and 75% for adenovirus, human parainfluenza viruses-1 and -2, respectively, and 82%–95% for the other viruses). Assay specificity was 99%, and coefficients of variation for virus controls ranged from 1.5% to 4.5%. ConclusionThe TAC assay should prove useful for multipathogen studies and rapid outbreak response.

Comparison of four multiplex PCR assays for the detection of viral pathogens in respiratory specimens

Multiplex PCR has become the test of choice for the detection of multiple respiratory viruses in clinical specimens. However, there are few direct comparisons of different PCR assays. This study compares 4 different multiplex PCR assays for the recovery of common respiratory viruses. We tested 213 respiratory specimens using four different multiplex PCR assays: the xTAG respiratory viral panel fast (Abbott Molecular Laboratories), Fast-track Respiratory Pathogen assay (Fast-track Diagnostics), Easyplex respiratory pathogen 12 kit (Ausdiagnostics), and an in-house multiplex real-time PCR assay. The performance of the four assays was very similar, with 93–100% agreement for all comparisons. Other issues, such as through-put, technical requirements and cost, are likely to be as important for making a decision about which of these assays to use given their comparative performance.

Influenza A/H1N1 2009 pandemic and respiratory virus infections, Beijing, 2009-2010.

To determine the role of the pandemic influenza A/H1N1 2009 (A/H1N1 2009pdm) in acute respiratory tract infections (ARTIs) and its impact on the epidemic of seasonal influenza viruses and other common respiratory viruses, nasal and throat swabs taken from 7,776 patients with suspected viral ARTIs from 2006 through 2010 in Beijing, China were screened by real-time PCR for influenza virus typing and subtyping and by multiplex or single PCR tests for other common respiratory viruses. We observed a distinctive dual peak pattern of influenza epidemic during the A/H1N1 2009pdm in Beijing, China, which was formed by the A/H1N1 2009pdm, and a subsequent influenza B epidemic in year 2009/2010. Our analysis also shows a small peak formed by a seasonal H3N2 epidemic prior to the A/H1N1 2009pdm peak. Parallel detection of multiple respiratory viruses shows that the epidemic of common respiratory viruses, except human rhinovirus, was delayed during the pandemic of the A/H1N1 2009pdm. The H1N1 2009pdm mainly caused upper respiratory tract infections in the sampled patients; patients infected with H1N1 2009pdm had a higher percentage of cough than those infected with seasonal influenza or other respiratory viruses. Our findings indicate that A/H1N1 2009pdm and other respiratory viruses except human rhinovirus could interfere with each other during their transmission between human beings. Understanding the mechanisms and effects of such interference is needed for effective control of future influenza epidemics.

Detection of Respiratory Viruses by Molecular Methods

Clinical laboratories historically diagnose seven or eight respiratory virus infections using a combination of techniques including enzyme immunoassay, direct fluorescent antibody staining, cell culture, and nucleic acid amplification tests. With the discovery of six new respiratory viruses since 2000, laboratories are faced with the challenge of detecting up to 19 different viruses that cause acute respiratory disease of both the upper and lower respiratory tracts. The application of nucleic acid amplification technology, particularly multiplex PCR coupled with fluidic or fixed microarrays, provides an important new approach for the detection of multiple respiratory viruses in a single test. These multiplex amplification tests provide a sensitive and comprehensive approach for the diagnosis of respiratory tract infections in individual hospitalized patients and the identification of the etiological agent in outbreaks of respiratory tract infection in the community. This review describes the molecular methods used to detect respiratory viruses and discusses the contribution that molecular testing, especially multiplex PCR, has made to our ability to detect respiratory viruses and to increase our understanding of the roles of various viral agents in acute respiratory disease.

Detection of multiple respiratory viruses by real-time polymerase chain reaction in infants attending an outpatient clinic

The detection of respiratory viruses in many prospective clinical studies has relied upon detection with standard laboratory assays, such as antigen detection, viral culture, or serology. Data describing the relative importance of community-acquired respiratory viruses, such as rhinovirus, human metapneumovirus (HMPV) [1], and newly described human coronavirus (HCV) subtypes [2], which are not detectable by these standard assays, in healthy young children who are not admitted to the hospital is limited. We developed a novel respiratory real-time reverse transcription (RT) polymerase chain reaction (PCR) panel to detect the presence of HMPV, respiratory syncytial virus (RSV), influenza viruses A and B, parainfluenza virus (PIV) 1, 2, and 3, adenovirus, human coronaviruses Group 1 (229E and NL63) and Group 2 (OC43 and HKU1), and rhinovirus. We conducted a one-year prospective outpatient community-based study of respiratory tract infections (RTIs) in a healthy infant cohort. Our goals were to determine the proportion of viral RTIs caused by these 13 viruses in symptomatic but otherwise healthy infants, as well as to characterize the clinical features of the identified viral infections and quantify the decline in viral load of patients infected with HMPV in this outpatient population. This cohort surveillance study in a population of healthy infants followed for 13 months was conducted between 1 April 2004 and 1 May 2005. Infants between the ages of 0– 6 months were enrolled from the Well Child Clinic at the Madigan Army Medical Center (MAMC) in Tacoma, WA. All subjects were evaluated at each RTI visit by one of three study physicians. Infections were determined to be lower RTIs if bronchiolitis or pneumonia were diagnosed clinically or radiographically by a study physician. We also assessed the presence of a secondary infection requiring antibiotics. During the 13 months of the study, parents called for an appointment in our study clinic if their infant had any respiratory tract symptoms. The clinical data obtained at the study visits for RTIs and by phone follow-up included the duration and complications of the illness, and missed days of work or daycare. Those infants with new onset of at least 2 of 5 respiratory tract symptoms (cough, rhinorrhea, wheezing, fever, and nasal congestion) had a physical Eur J Clin Microbiol Infect Dis (2008) 27:1245–1248 DOI 10.1007/s10096-008-0558-7