Viruses are the main cause of respiratory tract infections. Metagenomic next-generation sequencing (mNGS) enables unbiased detection of all potential pathogens. To apply mNGS in viral diagnostics, sensitive and simultaneous detection of RNA and DNA viruses is needed. Herein, were studied the performance of an in-house mNGS protocol for routine diagnostics of viral respiratory infections with potential for automated pan-pathogen detection. The sequencing protocol and bioinformatics analysis were designed and optimized, including exogenous internal controls. Subsequently, the protocol was retrospectively validated using 25 clinical respiratory samples. The developed protocol using Illumina NextSeq 500 sequencing showed high repeatability. Use of the National Center for Biotechnology Information's RefSeq database as opposed to the National Center for Biotechnology Information's nucleotide database led to enhanced specificity of classification of viral pathogens. A correlation was established between read counts and PCR cycle threshold value. Sensitivity of mNGS, compared with PCR, varied up to 83%, with specificity of 94%, dependent on the cutoff for defining positive mNGS results. Viral pathogens only detected by mNGS, not present in the routine diagnostic workflow, were influenza C, KI polyomavirus, cytomegalovirus, and enterovirus. Sensitivity and analytical specificity of this mNGS protocol were comparable to PCR and higher when considering off-PCR target viral pathogens. One single test detected all potential viral pathogens and simultaneously obtained detailed information on detected viruses. (J Mol Diagn 2020, 22: 196e207; https://doi.

IntroductionExacerbations are major contributors to morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD), and respiratory bacterial and viral infections are an important trigger. However, using conventional diagnostic techniques, a causative agent is not always found. Metagenomic next-generation sequencing (mNGS) allows analysis of the complete virome, but has not yet been applied in COPD exacerbations.ObjectivesTo study the respiratory virome in nasopharyngeal samples during COPD exacerbations using mNGS.Study design88 nasopharyngeal swabs from 63 patients from the Bergen COPD Exacerbation Study (2006–2010) were analysed by mNGS and in-house qPCR for respiratory viruses. Both DNA and RNA were sequenced simultaneously using an Illumina library preparation protocol with in-house adaptations.ResultsBy mNGS, 24/88 samples tested positive. Sensitivity and specificity, as compared with PCR, were 96% and 98% for diagnostic targets (23/24 and 1093/1120, respectively). Additional viral pathogens detected by mNGS were herpes simplex virus type 1 and coronavirus OC43. A positive correlation was found between Cq value and mNGS viral normalized species reads (log value) (p = 0.002). Patients with viral pathogens had lower percentages of bacteriophages (p<0.001). No correlation was found between viral reads and clinical markers.ConclusionsThe mNGS protocol used was highly sensitive and specific for semi-quantitative detection of respiratory viruses. Excellent negative predictive value implicates the power of mNGS to exclude any pathogenic respiratory viral infectious cause in one test, with consequences for clinical decision making. Reduced abundance of bacteriophages in COPD patients with viral pathogens implicates skewing of the virome during infection, with potential consequences for the bacterial populations, during infection.

Rapid diagnosis of respiratory infections is of great importance for adequate isolation and treatment. Due to the batch-wise testing, laboratory-developed real-time polymerase chain reaction (PCR) assays (LDT) often result in a time to result of one day. Here, LDT was compared with rapid ePlex® Respiratory Pathogen (RP) Panel testing of GenMark Diagnostics (Carlsbad, CA, USA) with regard to time to result, installed isolation precautions, and antibacterial/antiviral treatment. Between January and March 2017, 68 specimens of 64 patients suspected of an acute respiratory infection were tested with LDT and the ePlex® RP panel. The time to result was calculated as the time between sample reception and result reporting. Information regarding isolation and antibacterial/antiviral treatment was obtained from the patient records. Thirty specimens tested LDT positive (47%) and 29 ePlex® RP panel positive (45%). The median time to result was 27.1 h (range 6.5–96.6) for LDT versus 3.4 h (range 1.5–23.6) for the RP panel, p-value < 0.001. In 14 out of 30 patients, isolation was discontinued based on the ePlex® RP panel results, saving 21 isolation days. ePlex® RP panel test results were available approximately one day ahead of the LDT results in the 19 patients receiving antiviral/antibacterial treatment. In addition, two bacterial pathogens, not requested by the physician, were detected using the RP panel. Analysis of respiratory infections with the ePlex® RP panel resulted in a significant decrease in time to result, enabling a reduction in isolation days in half of the patients. Furthermore, syndromic RP panel testing increased the identification of causative pathogens.

BackgroundDifferences in clinical impact between rhinovirus (RVs) species and types in adults are not well established. The objective of this study was to determine the epidemiology and clinical impact of the different RV species.MethodsWe conducted a prospective study of RVs infections in adults with acute cough/lower respiratory tract infection (LRTI) and asymptomatic controls. Subjects were recruited from 16 primary care networks located in 11 European countries between 2007 and 2010. RV detection and genotyping was performed by means of real time and conventional reverse-transcriptase polymerase chain reaction assays, followed by sequence analysis. Clinical data were obtained from medical records and patient symptom diaries.ResultsRVs were detected in 566 (19%) of 3016 symptomatic adults, 102 (4%) of their 2539 follow-up samples and 67 (4%) of 1677 asymptomatic controls. Genotyping was successful for 538 (95%) symptomatic subjects, 86 (84%) follow-up infections and 62 (93%) controls. RV-A was the prevailing species, associated with an increased risk of LRTI as compared with RV-B (relative risk (RR), 4.5; 95% CI 2.5 to 7.9; p<0.001) and RV-C (RR 2.2; 95% CI 1.2 to 3.9; p=0.010). In symptomatic subjects, RV-A loads were higher than those of RV-B (p=0.015). Symptom scores and duration were similar across species. More RV-A infected patients felt generally unwell in comparison to RV-C (p=0·023). Of the 140 RV types identified, five were new types; asymptomatic infections were associated with multiple types.InterpretationIn adults, RV-A is significantly more often detected in cases with acute cough/LRTI than RV-C, while RV-B infection is often found in asymptomatic patients.

Introduction: Exacerbations are major contributors to morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD), and respiratory bacterial and viral infections are an important trigger for the occurrence of such exacerbations. However, using conventional diagnostic techniques, a causative agent is not always found. Metagenomic next-generation sequencing (mNGS) allows analysis of the complete virome, but has not yet been applied in COPD exacerbations. Objectives: To study the respiratory virome in nasopharyngeal samples during COPD exacerbations using mNGS. Study design: 88 nasopharyngeal swabs from 63 patients from the Bergen COPD Exacerbation Study (2006-2010) were analysed by mNGS and in-house qPCR for respiratory viruses. Both DNA and RNA were sequenced simultaneously using an Illumina library preparation protocol with in-house adaptations. Results: By mNGS, 23/88 samples tested positive. Sensitivity and specificity were both 96% for diagnostic targets (23/24 and 1067/1120, respectively). Viral pathogens only detected by mNGS were herpes simplex virus type 1 and coronavirus OC43. A positive correlation was found between Cq value and mNGS viral species reads (p=0.008). Patients with viral pathogens had lower percentages of bacteriophages (p<0.000). No correlation was found between viral reads (species and genus level) and clinical markers. Conclusions: The mNGS protocol used was highly sensitive and specific for semi-quantitative detection of respiratory viruses. Excellent negative predictive value implicates the power of mNGS to exclude any infectious cause in one test, with consequences for clinical decision making. Reduced abundance of bacteriophages in COPD patients with viral pathogens implicates skewing of the virome, and speculatively the bacterial population, during infection.