Molecular Detection Of Respiratory Viruses Research Articles

Aetiology of influenza-like illnesses in the pre-COVID-19 season 2019-2020: role of coronaviruses

Abstract Background Only a proportion of seasonal influenza-like illnesses (ILIs) can de facto be attributed to influenza viruses. This study investigated the aetiology of ILIs to estimate the prevalence of human coronaviruses (CoVs) and to analyse their clinical-epidemiological traits. Methods A sample of 613 outpatients (253 adults, 360 children) with ILI in Veneto Region, Italy, was included. ILI was defined according with the EU Decision 2018/945. Sigma-Virocult nasopharyngeal swab were used. Nucleic acids were extracted with the QiaAmp Viral RNA Mini Kit (Qiagen). Molecular detection of respiratory viruses was performed with commercial One-step RT qPCR reagents (Allplex® Respiratory Panels, Seegene). Information on age, sex, symptoms, co-infections and comorbidities was collected. Results CoVs were the 3rd most frequent pathogen in adults (7.5%, after influenza and rhinovirus) and the 4th in children (4.7%, after influenza, rhino- and adenovirus). Subtype distribution was similar, with OC43 the most frequent. Probability of CoV involvement was twice in males (AOR=2.16; 95%CI: 1.05-4.39), whereas no association with age was noted. Co-infection with other viruses was frequent in children (65% of cases). CoV symptoms were not peculiar, although respiratory tract involvement was less likely than influenza (AOR=0.13; 95%CI: 0.04-0.41). Among CoV outpatients, 36% had one or more chronic diseases, compared with 5.6% among influenza (p = 0.001). Conclusions Even before the COVID-19 pandemic, CoVs had a substantial role in ILI aetiology: 1 case of CoV every 3 influenza infections in adults. The higher prevalence of comorbidities among CoV positives compared to influenza indirectly shows the benefits of flu vaccines in individuals at higher risk. Careful surveillance of the viruses responsible for ILI continues to be desirable, including, but not limited to, detecting a possible change in the aetiology of ILI after the administration of SARS-CoV-2 vaccines in the population. Key messages • Pre-pandemic virological surveillance of influenza-like illnesses (ILIs) reveals how seasonal coronaviruses were the third most frequent respiratory pathogen in adults. • Prevalence of comorbidities was significantly higher in patients with a coronavirus-related ILI compared to influenza, supporting the benefits of flu vaccination for high risk groups.

Lack of clinical significance for molecular detection of respiratory viruses in bronchoalveolar lavage samples.

The clinical significance of molecular detection of respiratory viruses in bronchoalveolar lavage (BAL) samples is poorly defined. We performed an observational retrospective study including all patients who underwent a BAL procedure in our institution, regardless of the reason for bronchoscopy, from January 2015 to December 2018. Respiratory viruses were detected by real-time polymerase chain reaction with a commercial multiplex panel, and a cell culture was performed to detect cytomegalovirus and herpes simplex virus. Positive results were correlated with clinical symptoms and patients' characteristics. Of 540 BAL samples analyzed, 113 (20.9%) were positive for any respiratory virus. Viral detection was significantly associated with respiratory symptoms (83.2% vs. 68.9%, p = .004) and radiological infiltrates (67.3% vs. 52.2%, p = .006). The most frequent viruses detected were rhinovirus (42/113, 37.2%), influenza virus (20/113, 17.7%), and parainfluenza virus (PIV) (16/113, 14.2%). Respiratory pathogens codetections were found in 51/113 (45.1%) BAL samples, including more than one virus (16/51, 31.4%), fungi (8/51, 15.7%), and bacteria (9/51, 17.6%). Viral detection was significantly higher in immunocompromised patients (26.5% vs. 16.9%; p = .022). PIV and human metapneumovirus were mostly observed in lung (50.0%, 8/16) and hemopoietic transplant recipients (25%, 2/8), respectively, with clinical repercussions. Our data underline that molecular diagnosis allows identification of viral agents as the etiology of respiratory infections; however, the high frequency of codetections hinders identification of the agent responsible for the current respiratory symptomatology. Immunocompromised patients are the target population in whom to investigate the presence of respiratory viruses in their BAL samples.

Prevalence of respiratory viruses using polymerase chain reaction in children with wheezing, a systematic review and meta-analysis.

Wheezing is a major problem in children, and respiratory viruses are often believed to be the causative agent. While molecular detection tools enable identification of respiratory viruses in wheezing children, it remains unclear if and how these viruses are associated with wheezing. The objective of this systematic review is to clarify the prevalence of different respiratory viruses in children with wheezing. We performed an electronic in Pubmed and Global Index Medicus on 01 July 2019 and manual search. We performed search of studies that have detected common respiratory viruses in children ≤18 years with wheezing. We included only studies using polymerase chain reaction (PCR) assays. Study data were extracted and the quality of articles assessed. We conducted sensitivity, subgroup, publication bias, and heterogeneity analyses using a random effects model. The systematic review included 33 studies. Rhinovirus, with a prevalence of 35.6% (95% CI 24.6-47.3, I2 98.4%), and respiratory syncytial virus, at 31.0% (95% CI 19.9-43.3, I2 96.4%), were the most common viruses detected. The prevalence of other respiratory viruses was as follows: human bocavirus 8.1% (95% CI 5.3-11.3, I2 84.6%), human adenovirus 7.7% (95% CI 2.6-15.0, I2 91.0%), influenza virus6.5% (95% CI 2.2-12.6, I2 92.4%), human metapneumovirus5.8% (95% CI 3.4-8.8, I2 89.0%), enterovirus 4.3% (95% CI 0.1-12.9, I2 96.2%), human parainfluenza virus 3.8% (95% CI 1.5-6.9, I2 79.1%), and human coronavirus 2.2% (95% CI 0.6-4.4, I2 79.4%). Our results suggest that rhinovirus and respiratory syncytial virus may contribute to the etiology of wheezing in children. While the clinical implications of molecular detection of respiratory viruses remains an interesting question, this study helps to illuminate the potential of role respiratory viruses in pediatric wheezing. PROSPERO, CRD42018115128.

Self-Sampling of Oropharyngeal Swabs Among Healthcare Workers for Molecular Detection of Respiratory Viruses: A Valuable Approach for Epidemiological Studies and Surveillance Programs.

This study aimed at assessing the validity of self-collected (self-sampled) oropharyngeal (OP) swabs among healthcare workers compared to those collected by trained sentinel general practitioners (GP-sampled) from individuals with influenza-like illness (ILI), to be implemented in epidemiological studies and/or surveillance programs of viral pathogens involved in community respiratory infections. In our study, OP swabs were collected from adults (>18 years) with ILI during the 2018–2019 influenza season. Two groups of samples were considered: group 1−131 self-sampled OP swabs collected by healthcare workers after being trained on the sampling procedure; group 2−131 GP-sampled OP swabs collected from outpatients by sentinel GPs operating within the Italian Influenza Surveillance Network. To assess swabbing quality, following RNA extraction, each sample was tested for the presence of the human ribonuclease P gene (RNP) by in-house real-time reverse transcriptase–polymerase chain reaction (RT-PCR). Samples with a cycle threshold (Ct) <35 were considered adequate for further virological analysis. Influenza viruses (IVs), respiratory syncytial virus (RSV), and rhinovirus (RV) genomes were detected by in-house real-time RT-PCR. All samples were positive to RNP detection with Ct <35. The mean Ct value was similar in the two groups (group 1 vs. group 2: 25.93 ± 2.22 vs. 25.46 ± 2.40; p = 0.10). IVs, RSV, and RV positivity rates were 26.7 vs. 52.7% (p < 0.01), 7.6 vs. 9.9% (p = 0.52), and 21.4 vs. 19.9% (p = 0.76), respectively. Self-sampled OP swabs resulted as valid as GP-sampled OP swabs for molecular detection of respiratory viruses. Self-swabbing can thus be a worthwhile strategy for sample collection to implement molecular surveillance of respiratory pathogens and carry out epidemiological studies, easily reaching a larger population size.

PF766 T CELL EXHAUSTION PROFILE (CD4+/CD28/PER+/GRA+ CD8+/CD28/PER+/GRA+) IN STEM CELL TRANSPLANTATION, CMV RE ACTIVATIONS AND RESPIRATORY VIRUS INFECTION.CELLULAR DYNAMICS IN THE FIRST 100 DAYS POST SCT

Background:Recipients of allogeneic stem cells grafts have clonally expanded CD8+/CD28‐ T lymphocytes during the early period after SCT, this cellular dynamic is associated with the acquisition of a cytoxic phenotype. This scenario predisposes to continuous inflammation. On the other hand, respiratory viruses (RV) are recognized as the predominant leading to pneumonia after SCT, and CMV re activations were associated with morbi‐mortality in SCT patients.RVI and CMV re‐activations might contribute to the emergence of exhausted T cells phenotype.Aims:The present study aimed to evaluate the dynamics of T cell exhaustion profile during the +100d after SCT(auto, allo/patients with RVI and CMV re activations)Methods:Informed consent was obtained from all patients. PBs were obtained previous SCT and at day+100 post SCT from 37 patients (11 auto, 26 allo). To evaluate the expression of CD4+/CD28Per+/Gran+, CD8+/CD28Per+/Gran+. Flow cytometry analysis was performed: 100 μL of PB was labeled, with a panel of 8 AbMos: PERFORIN FITC, GRANZIME PE, CD4PerCP, CD28 APC, CD8 APCH7, CD16/56 V450 and CD45 V500. The molecular detection of RV was tested with theCLART® Pneumovir assay (Clinical Array Technology, Genomica, Spain). CMV detection was performed by quantitative CMV PCR (COBAS®Amplicor® CMV MonitorTM test) (qPCR CT). Statistical analysis was performed with IBM SPSS v24Results:Thirty seven patients were evaluated from November 2016 to December 2017 at the University Hospital of La Princesa, Madrid Spain. Patient characteristics:Table 1.The median percentages of baseline CD8+/CD28‐ cell line was 8,81%(range, 0,6–49,5) and the median percentages of CD8+/CD28‐ cellline at +100d were 29,2%(range, 1,2–60,8).Likewise, significant differences were found(p = 0.001).The group of patients who had an RVI and the group without RVI: in the CD8+/CD28+100d cell line, the median percentages of the RVI patients were 58.43%(range, 42.7–68.4)and the median percentages in the uninfected were 29.26%(range, 13.2–38.9)(p = 0.002). CMV re activations,the median percentages at +100d of CD8+/CD28‐ cell line with CMV‐ was 5,7%(range, 0,6–20,6)and the median percentage of CD8+/CD28‐with CMV+ at +100d was 36,7%(range, 13,5–68,7)p = 0,001.CMV reactivations and RVI, the median percentages at +100d of CD8+/CD28‐cell line with CMV/RVI was 6,82%(range, 0,6–20,6)and the medianpercentage of CD8+/CD28‐ with CMV+/RVI+ at +100d was 34,6%(range, 1,4–49,8)p = 0,012.Summary/Conclusion:We show a dynamic change of the T cell exhausted phenotype CD8+/CD28‐throughout the SCT, an also ID interactions (RVI, CMV re activations).There is a statistically significant difference when analyzing patients with RVI+ vs RVI‐ (baseline vs +100d), also might exist a synergic effect RVI+/CMV+.The CD8+/CD28‐Tcell population has been shown to contain virus specific memory CTL that respond to CMV and to other antigens.Thus, clonally expanded CD8+CD28‐ Tcells following STC may be derived from the T cells recognizing antigens that persistently exist in the host (CMV) or to the exposure to certain virus antigens in the first 100d after STC (synergic effect), this may contribute to the appearance of populations of exhausted T cells CD8+/CD28‐ favoring an inflammatory environment and triggering certain immunological complications (immunedysregulation).We need to follow up these patients and correlate with other clinical epidemiological variables in SCT(aGVHD,cGVHD,pulmonary functional tests,ID complications)image

Impact of Rapid Molecular Detection of Respiratory Viruses on Clinical Outcomes and Patient Management.

To determine if rapid molecular testing for respiratory viruses in patients with respiratory illnesses can provide advantages to patients and hospitals, rigorous investigations on the impacts of using these assays are required. Well-conducted studies are needed to inform decisions about implementation of new rapid assays to replace standard molecular testing or to initiate testing in laboratories that are currently not doing molecular tests for respiratory viruses due to the complex nature of standard panels. In this issue of the Journal of Clinical Microbiology, N. Wabe et al. (J Clin Microbiol 57:e01727-18, 2019, https://doi.org/10.1128/JCM.01727-18) report the results of their evaluation of the impact of using a rapid molecular test for influenza A/influenza B and RSV on outcomes for adults hospitalized with respiratory illness. The median time from admission to test result of the rapid test was 7.5 h compared to 40.3 h for the standard PCR assay. Compared to the use of the standard molecular assay, use of a rapid test significantly shortened time in the hospital and reduced the number of other microbiology tests performed. The authors concluded that rapid PCR testing of adults hospitalized with respiratory illnesses could provide benefits to both the patients and the hospital. Patients were able to leave the hospital earlier and a greater proportion of them had received their test results before discharge, which would allow appropriate treatment to be provided more quickly.

Multiple molecular detection of respiratory viruses and associated signs of airway inflammation in racehorses.

BackgroundThe potential involvement of viruses in inflammatory airway disease (IAD) was previously investigated through either serology or PCR from nasopharyngeal swabs (NS). The aims of this study were to determine the prevalence and incidence of viral genome detection by qPCR in the equine airways, and their association with respiratory clinical signs.MethodsBoth NS and tracheal washes (TW) were collected monthly on 52 Standardbred racehorses at training, over 27 consecutive months (581 samples). Equid herpesviruses (EHV)-1, −4, −2 and −5, equine rhinitis virus-A and -B (ERBV), equine adenovirus-1 and −2, equine coronavirus and equine influenza virus were systematically investigated in both NS and TW. Nasal discharge, coughing, tracheal mucus score and TW neutrophil proportions were simultaneously recorded.ResultsGenome for 7/10 viruses were detected at least once throughout the study; up to 4 different viruses being also concomitantly detected. Monthly incidence in TW was respectively 27.9% (EHV-5), 24.8% (EHV-2), 7.1% (ERBV), 3.8% (EHV-4), 1.9% (EAdV1) and 0.2% (EHV-1; ERAV). Neither agreement nor correlation between NS and TW was found for respectively genome detection and viral loads. Detection of viral genome in NS was not associated with any clinical sign. Coughing was significantly associated with TW detection of EHV-2 DNA (OR 3.1; P = 0.01) and ERBV RNA (OR 5.3; P < 0.001). Detection of EHV-2 DNA in TW was also significantly associated with excess tracheal mucus (OR 2.1; P = 0.02).ConclusionsDetection and quantification of EHV-2 and ERBV by qPCR in TW, but not in NS, should be considered when investigating horses with IAD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0657-5) contains supplementary material, which is available to authorized users.

False-negative detection of respiratory syncytial virus as an example that regular update of RT-PCR is required for reliable molecular detection of respiratory viruses

False-negative detection of respiratory syncytial virus as an example that regular update of RT-PCR is required for reliable molecular detection of respiratory viruses

Development of an external quality assessment panel for the molecular detection of respiratory viruses

Development of an external quality assessment panel for the molecular detection of respiratory viruses

Comparison of nasopharyngeal flocked swabs and nasopharyngeal wash collection methods for respiratory virus detection in hospitalized children using real-time polymerase chain reaction

This paper describes the molecular detection of respiratory viruses from nasopharyngeal flocked swabs (flocked swabs) and nasopharyngeal washes (washes) in a clinical setting. Washes and flocked swabs collected from children<3 years old hospitalized with a lower respiratory tract infection were tested for parainfluenza virus 1-3, respiratory syncytial virus, influenza A and B and metapneumovirus (Group 1) and adenovirus, rhinovirus and coronavirus (Group 2) using real-time reverse transcriptase PCR (rRT-PCR). A consensuses standard was used to determine sensitivity and compare cycle thresholds (C(T)) of washes and flocked swabs for each virus and group of viruses. Sensitivities ranged from 79 to 89% and 69 to 94% for flocked swabs and washes, respectively, excluding AdV which had a sensitivity of 35% for flocked swabs. When the flocked swabs and washes of Group 1 viruses were collected on the day of admission, the sensitivity of both sample types was 100%. Wash specimens had a lower C(T) value and higher sensitivity than flocked swabs; however there was no statistical difference in the sensitivity of a flocked swab (89%) versus wash (93%) for the detection of Group 1 viruses, particularly when samples were collected on the same day. Flocked swabs may be a useful alternative to washes for detection of respiratory viruses in clinical settings.

Molecular detection of respiratory viruses in immunocopromised ICU patients: Incidence and meaning

PurposeProspective single-center study to assess the sensitivity and clinical relevance of molecular testing for respiratory viruses in critically ill immunocompromised patients with acute respiratory failure (ARF).Methods100 consecutive critically ill immunocompromised patients with ARF in 2007–2009. Among them, 65 had hematologic malignancies (including 14 hematopoietic stem cell transplant recipients), 22 had iatrogenic immunosuppression, and 13 had solid malignancies. A multiplex molecular assay (MMA) was added to the usual battery of tests performed to look for causes of ARF.ResultsNasopharyngeal aspirates and/or bronchoalveolar lavage fluid were tested for respiratory viruses using both the MMA and immunofluorescence. A virus was detected in 47 (47%) patients using the MMA and 8 (8%) patients using immunofluorescence (P = 0.006). MMA-positive and MMA-negative patients had similar clinical and radiographic presentations and were not significantly different for the use of ventilatory support (58% vs. 76%, P = 0.09), occurrence of shock (43% vs. 53%, P = 0.41), use of renal replacement therapy (26% vs. 23%, P = 0.92), SAPS II (35 [26–44] vs. 38 [27–50], P = 0.36), time spent in the ICU (6 vs. 7 days, P = 0.35), or ICU mortality (17% vs. 28%, P = 0.27). Using MMA, a virus was found in 6 of the 12 patients with no diagnosis at the end of the etiologic investigations.ConclusionsIn critically ill immunocompromised patients, an MMA was far more sensitive than immunofluorescence for respiratory virus detection. Patients with RVs detected in the respiratory tract had the same clinical characteristics and outcomes as other patients.

Molecular Detection of Respiratory Viruses in Clinical Specimens From Children With Acute Respiratory Disease in Iran

Viruses are the most common causes of respiratory tract infections in children. The implementation of rapid virology assays can identify the most common pathogens involved. This study was undertaken on Iranian children less than 6 years old with respiratory infection. A total of 202 specimens were tested for the presence of 9 respiratory viruses by developing 3 multiplex reverse transcription-polymerase chain reaction and 1 uniplex polymerase chain reaction assays. Viral pathogens were detected in 92 samples (45.5%) with 5.4% having dual infections. Overall, respiratory syncytial virus was the most frequently identified virus (16.8%), followed by adenovirus (14.4%), influenza A virus (4.9%), parainfluenza virus-3 (4.4%), parainfluenza virus-1 (2.9%), and both influenza B virus and hMPV (0.49). Respiratory tract infection is a frequent cause of pediatric morbidity and mortality and a common reason for admission in acute care hospitals and outpatients visits. Appropriate diagnostic testing is important for specific diagnosis at an early stage of the illness because of the similarity in clinical presentation of patients with different viral infections caused by several respiratory pathogens.

OP4-3 Molecular detection of respiratory viruses: routine application on 522 samples taken in children less than 2 year old

OP4-3 Molecular detection of respiratory viruses: routine application on 522 samples taken in children less than 2 year old

Comparison of conventional and molecular detection of respiratory viruses in hematopoietic cell transplant recipients

Sensitive detection of respiratory viruses is important for early diagnosis of infection in patients following hematopoietic cell transplantation (HCT). To evaluate the relative sensitivity of respiratory virus detection in specimens from HCT recipients, we compared the results of conventional and quantitative molecular methods. We tested 688 nasal wash samples collected prospectively from 131 patients during the first 100 days after HCT by viral culture, fluorescent antibody staining (FA), and real-time quantitative reverse transcription-polymerase chain reaction (PCR) assay for detection of respiratory syncytial virus (RSV), influenza virus types A (FluA) and B (FluB), and parainfluenza virus types 1 (PIV1) and 3 (PIV3). Testing for human metapneumovirus (MPV) was performed only by PCR. Data regarding 10 respiratory symptoms were collected with each sample. By any method 37 specimens were positive for a respiratory virus; 34 were positive by PCR, 15 by culture, and 6 by FA. Four specimens were positive by all 3 methods (3 RSV, 1 FluA). One specimen was positive for PIV1, and 2 were positive for rhinovirus by culture alone. Specimens positive by PCR alone included 2 RSV, 2 PIV1, 8 PIV3, and 8 MPV. In 10 specimens positive for RSV, PIV, or influenza virus collected from patients reporting no respiratory symptoms, 9, 4, and 1 specimen were positive by PCR, culture, and FA, respectively. Overall, specimens positive only by PCR had significantly fewer viral copies/mL (mean log(10)=4.32) than specimens positive by both PCR and culture (mean log(10)=5.75; P=0.002) or PCR and FA (mean log(10)=6.83; P<0.001). FA testing alone did not detect a significant proportion of respiratory virus-positive samples in HCT recipients, especially in patients with no respiratory symptoms and patients with PIV detection. PCR increased the yield of positive specimens 2 times relative to culture and more than 4 times relative to FA. Detection of respiratory viruses by PCR alone was associated with lower virus quantities and with fewer reported respiratory symptoms compared with concomitant detection by both PCR and conventional methods, indicating that PCR may be important to detect asymptomatic or mildly symptomatic stages of respiratory viral infections.

Dry cotton or flocked respiratory swabs as a simple collection technique for the molecular detection of respiratory viruses using real-time NASBA

This paper describes the molecular detection of influenza A, influenza B, respiratory syncytial virus and human metapneumovirus using real-time nucleic acid sequence based amplification (NASBA) from respiratory samples collected on simple dry cotton swabs, non-invasively and in the absence of transport medium. Viral RNA was detectable on dry cotton and flocked swabs for at least 2 weeks at room temperature and was readily extracted using magnetic silica extraction methods. Dry cotton respiratory swabs were matched with traditionally collected respiratory samples from the same patient, and results of traditional laboratory techniques and real-time NASBA were compared for all four viral targets. The results not only showed a significant increase in the detection rate of the viral targets over traditional laboratory methods of 46%, but also that dry swabs did not compromise their recovery. Over two subsequent winter seasons, 736 dry cotton respiratory swabs were collected from symptomatic patients and tested using real-time NASBA giving an overall detection rate for these respiratory virus targets of 38%. The simplicity of the method together with the increased detection rate observed in the study proves that transporting a dry respiratory swab to the laboratory for respiratory virus diagnosis using molecular methods is a suitable and robust alternative to traditional sample types.

A multi-centre pilot proficiency programme to assess the quality of molecular detection of respiratory viruses

Objectives To assess the quality of molecular detection of respiratory viruses in clinical diagnostic laboratories. Study design Respiratory virus proficiency panels were produced from diluted stocks of respiratory viruses provided and tested by four reference laboratories. The panels consisted of strong positive, positive, low positive and negative samples for influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses 1 and 3, adenovirus serotypes 4 and 7, human rhinovirus serotypes 16, 72 and 90, human coronaviruses OC43 and 229E. The panels were sent to 17 participants; results and information on methodology was collected. Results All laboratories returned results, of which five submitted complete data sets. So, for analysis all results were combined. Samples were correctly identified by participants in 93.75%, 76.75% and 47.03% for the high positive, positive and low positive samples, respectively. One false positive was reported for all data sets (1.1%). The overall score for all assays using different methodologies was 78.8%. Laboratory performance was not dependant on methodology as all in-house methodologies could achieve optimal results, but dependant on careful optimisation and procedures specific to the laboratory. Conclusions The first proficiency panel showed that in general all participants performed well. Although, it also highlights areas for improvement for all participants in order to generate robust results for use in clinical diagnostics.