Detection Of Viral Respiratory Pathogens Research Articles

Metagenomics characterization of respiratory viral RNA pathogens in children under five years with severe acute respiratory infection in the Free State, South Africa.

Prompt detection of viral respiratory pathogens is crucial in managing respiratory infection including severe acute respiratory infection (SARI). Metagenomics next-generation sequencing (mNGS) and bioinformatics analyses remain reliable strategies for diagnostic and surveillance purposes. This study evaluated the diagnostic utility of mNGS using multiple analysis tools compared with multiplex real-time PCR for the detection of viral respiratory pathogens in children under 5 years with SARI. Nasopharyngeal swabs collected in viral transport media from 84 children admitted with SARI as per the World Health Organization definition between December 2020 and August 2021 in the Free State Province, South Africa, were used in this study. The obtained specimens were subjected to mNGS using the Illumina MiSeq system, and bioinformatics analysis was performed using three web-based analysis tools; Genome Detective, One Codex and Twist Respiratory Viral Research Panel. With average reads of 211323, mNGS detected viral pathogens in 82 (97.6%) of the 84 patients. Viral aetiologies were established in nine previously undetected/missed cases with an additional bacterial aetiology (Neisseria meningitidis) detected in one patient. Furthermore, mNGS enabled the much needed viral genotypic and subtype differentiation and provided significant information on bacterial co-infection despite enrichment for RNA viruses. Sequences of nonhuman viruses, bacteriophages, and endogenous retrovirus K113 (constituting the respiratory virome) were also uncovered. Notably, mNGS had lower detectability rate for severe acute respiratory syndrome coronavirus 2 (missing 18/32 cases). This study suggests that mNGS, combined with multiple/improved bioinformatics tools, is practically feasible for increased viral and bacterial pathogen detection in SARI, especially in cases where no aetiological agent could be identified by available traditional methods.

2020. Concordance of Direct vs. Indirect Pathogen Detection Using the BioFire® System

BackgroundPolymerase chain reaction (PCR) is a highly sensitive and specific method for pathogen detection. While direct methods enable rapid identification, they are limited by pathogen titer, available assays, or sample matrix. Transcriptomic analysis addresses these limitations by measuring systemic host gene expression changes to infections. The BioFire System uses sample-to-answer multiplex PCR that was adapted to detect 42 transcripts differentially expressed during viral and bacterial infections. Here we report concordance between indirect detection of viral respiratory pathogens and the FDA-cleared BioFire® Respiratory Panel 2 (RP2).MethodsPaired nasal pharyngeal swabs and blood samples were obtained by informed consent from patients with suspected acute respiratory illness. Swabs (COPAN FLOQSwab™) were collected and stored in viral transport media (BD) for BioFire RP2 testing and peripheral blood samples were collected in PAXgene tubes (Qiagen) for testing with the research use only human response (HR) panel. A logistic regression model was developed to classify viral and nonviral positive samples using normalized quantification cycles for each assay. Probabilities of viral infection for each sample were calculated via cross-validation and an optimal threshold of positivity was identified.ResultsOverall accuracy of the HR panel relative to BioFire RP2 was 86% (CI95% 80%, 91%) for all viral infections with an area under the receiver operating characteristic curve of 0.87 (CI95% 0.76, 0.95). Accuracy varied by infection etiology: Influenza virus 100% (CI95% 88%, 100%) Human Rhinovirus/Enterovirus 63% (CI95% 35%, 85%), other viral infections 50% (CI95% 25%, 75%). While most of the BioFire RP2 negative results exhibited low viral probabilities, strong viral probabilities were measured in a few samples; this may be indicative of an infection at a low titer or the presence of a viral pathogen not on the BioFire RP2 Panel.ConclusionThese results demonstrate that indirect transcriptomic analysis resulted in similar accuracy to direct viral pathogen detection in acute respiratory patients; however, additional research is needed to elucidate the relationship between transcriptional results and infectious etiologies.Disclosures C. Gritzen, BioFire Diagnostics, LLC.: Employee, Salary. T. Wilson, BioFire Diagnostics, LLC.: Employee, Salary. J. Nawrocki, BioFire Diagnostics, LLC.: Employee, Salary. M. Deneris, BioFire Diagnostics, LLC.: Employee, Salary. C. Baird, BioFire Diagnostics, LLC.: Employee, Salary. E. Ott, BioFire Diagnostics, LLC.: Employee, Salary. J. Jones, BioFire Diagnostics, LLC.: Employee, Salary. J. Bastar, BioFire Diagnostics, LLC.: Employee, Salary. H. Kim, BioFire Diagnostics, LLC.: Employee, Salary. S. House, Washington University School of Medicine: Research Contractor, Research support. D. Cohen, Nationwide Children’s Hospital: Research Contractor, Research support. A. Leber, Nationwide Children’s Hospital: Research Contractor, Research support. R. Crisp, BioFire Diagnostics, LLC.: Employee, Salary. A. Hemmert, BioFire Diagnostics, LLC.: Employee and Investigator, Salary.

Detection of viral respiratory pathogens in mild and severe acute respiratory infections in Singapore

To investigate the performance of laboratory methods and clinical case definitions in detecting the viral pathogens for acute respiratory infections (ARIs) from a prospective community cohort and hospital inpatients, nasopharyngeal swabs from cohort members reporting ARIs (community-ARI) and inpatients admitted with ARIs (inpatient-ARI) were tested by Singleplex Real Time-Polymerase Chain Reaction (SRT-PCR), multiplex RT-PCR (MRT-PCR) and pathogen-chip system (PathChip) between April 2012 and December 2013. Community-ARI and inpatient-ARI was also combined with mild and severe cases of influenza from a historical prospective study as mild-ARI and severe-ARI respectively to evaluate the performance of clinical case definitions. We analysed 130 community-ARI and 140 inpatient-ARI episodes (5 inpatient-ARI excluded because multiple pathogens were detected), involving 138 and 207 samples respectively. Detection by PCR declined with days post-onset for influenza virus; decrease was faster for community-ARI than for inpatient-ARI. No such patterns were observed for non-influenza respiratory virus infections. PathChip added substantially to viruses detected for community-ARI only. Clinical case definitions discriminated influenza from other mild-ARI but performed poorly for severe-ARI and for older participants. Rational strategies for diagnosis and surveillance of influenza and other respiratory virus must acknowledge the differences between ARIs presenting in community and hospital settings.

Equivalence of Self- and Staff-Collected Nasal Swabs for the Detection of Viral Respiratory Pathogens

BackgroundThe need for the timely collection of diagnostic biosamples during symptomatic episodes represents a major obstacle to large-scale studies on acute respiratory infection (ARI) epidemiology. This may be circumvented by having the participants collect their own nasal swabs. We compared self- and staff-collected swabs in terms of swabbing quality and detection of viral respiratory pathogens.Methodology/Principal FindingsWe conducted a prospective study among employees of our institution during the ARI season 2010/2011 (December-March). Weekly emails were sent to the participants (n = 84), reminding them to come to the study center in case of new symptoms. The participants self-collected an anterior nasal swab from one nostril, and trained study personnel collected one from the other nostril. The participants self-collected another two swabs (one from each nostril) on a subsequent day. Human β-actin DNA concentration was determined in the swabs as a quality control. Viral respiratory pathogens were detected by multiplex RT-PCR (Seeplex RV15 kit, Seegene, Eschborn, Germany). Of 84 participants, 56 (67%) reported at least one ARI episode, 18 participants two, and one participant three. Self-swabbing was highly accepted by the participants. The amount of β-actin DNA per swab was higher in the self- than in the staff-collected swabs (p = 0.008). β-actin concentration was lower in the self-swabs collected on day 1 than in those collected on a subsequent day (p<0.0001). A respiratory viral pathogen was detected in 31% (23/75) of staff- and in 35% (26/75) of self-collected swabs (p = 0.36). With both approaches, the most frequently identified pathogens were human rhinoviruses A/B/C (12/75 swabs, 16%) and human coronavirus OC43 (4/75 swabs, 5%). There was almost perfect agreement between self- and staff-collected swabs in terms of pathogen detection (agreement = 93%, kappa = 0.85, p<0.0001).Conclusions/SignificanceNasal self-swabbing for identification of viral ARI pathogens proved to be equivalent to staff-swabbing in this population in terms of acceptance and pathogen detection.

Molecular Diagnostic Assays for Detection of Viral Respiratory Pathogens in Institutional Outbreaks

Outbreaks of viral respiratory disease in institutions may be associated with high morbidity and mortality, depending upon the viral etiology and the age and immune status of the affected patients. Control of outbreaks may include isolation and/or cohorting, and prohylaxis or treatment with specific antiviral agents may be indicated, all dependent upon the specific cause of the outbreak. Conventional methods of viral diagnosis detect only a limited number of the viruses that are known to cause outbreaks. The availability of sensitive and specific molecular assays has facilitated rapid diagnosis of a wider range of viruses from respiratory outbreaks. Molecular methods have distinct advantages over conventional methods, including the ability to rapidly develop assays for emerging viruses and new variants of existing viruses. In addition, molecular testing allows rapid detection of resistance to antiviral agents or mutations leading to increased virulence. However, high-throughput molecular testing requires batch processes that may compromise the ability to respond quickly to urgent testing demands.