Evaluation of the Seegene Allplex™ RV master assay for one-step simultaneous detection of eight respiratory viruses in nasopharyngeal specimens

• Published in: Journal of virological methods 2025-01, Vol.331, p.115042, Article 115042
• Main Authors: Mdunyelwa, Anele, Seema, Colette, Mabaso, Anna, Mlambo, Khamusi, Mtsweni, Mandisa, Maphanga, Mathapelo, Rammutla, Elizabeth, Tempelman, Hugo A., Umunnakwe, Chijioke N.
• Format: Article
• Language: English
• Subjects: Adolescent; Adult; Aged; automation; Child; Child, Preschool; COVID-19 - diagnosis; COVID-19 - virology; diagnostic sensitivity; Female; genome; genotyping; Human orthopneumovirus; Humans; Infant; influenza; Influenza viruses; Laboratory Testing; Male; Middle Aged; Molecular Diagnostic Techniques - methods; Molecular Diagnostics; Multiplex Polymerase Chain Reaction - methods; Multiplex RT-PCR; Nasopharynx - virology; nucleic acids; Performance evaluation; Real-time PCR; Real-Time Polymerase Chain Reaction - methods; Respiratory infections; Respiratory Tract Infections - diagnosis; Respiratory Tract Infections - virology; Respiratory viruses; reverse transcriptase polymerase chain reaction; SARS-CoV-2; SARS-CoV-2 - genetics; SARS-CoV-2 - isolation & purification; Seegene Allplex RV Master; Sensitivity and Specificity; Severe acute respiratory syndrome coronavirus 2; temperature; viruses; Viruses - classification; Viruses - genetics; Viruses - isolation & purification; Young Adult
• ISSN: 0166-0934; 1879-0984; 1879-0984
• DOI: 10.1016/j.jviromet.2024.115042

The Seegene Allplex™ RV Master (RVM) assay is a one-step multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) system for detecting eight viral respiratory pathogens from nasopharyngeal swab, aspirate, and bronchoalveolar lavage specimens. The eight RVM targets are: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Influenza A (Flu A), Influenza B (Flu B), Human respiratory syncytial virus (RSV), adenovirus (AdV), rhinovirus (HRV), parainfluenza virus (PIV), and metapneumovirus (MPV). The assay is based on Seegene’s multiple detection temperature (MuDT) technology and provides cycle threshold (Ct) values for each of its viral targets upon PCR completion. We aimed to evaluate the diagnostic performance of the RVM assay by calculating sensitivity, specificity, accuracy, Positive Predictive Value (PPV), Negative Predictive Value (NPV), Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), and Overall Percent Agreement (OPA) compared to definite diagnosis and analogous reference assays. Diagnostic sensitivity, specificity, accuracy, PPV, and NPV were calculated by comparing the results of the RVM assay to that of definite diagnosis assays; while PPA, NPA, and OPA were calculated by comparing results of the RVM assay to that of analogous reference products. Definite diagnosis and reference methods comprised whole genome sequencing and PCR genotyping, the Allplex™ SARS-CoV-2/FluA/FluB/RSV and Respiratory Panels 1, 2, and 3 assays (Seegene), and the Xpert® Xpress SARS-CoV-2/FluA/FluB/RSV Plus assay (Cepheid). Reproducibility of the RVM assay using fully-automated and semi-automated nucleic acid (NA) extraction workflows and as performed by independent operators was also assessed. In total, 249 positive respiratory specimens and at least 50 negative specimens for each target tested were used for this evaluation study. Sensitivity, specificity, accuracy, PPV, NPV, PPA, NPA, and OPA ranged from 95.7 % to 100 % for detecting all eight targets tested on the RVM assay. Reproducibility PPA, NPA, and OPA between automated and semi-automated NA extraction workflows were all >97.9 %, while the reproducibility PPA, NPA and OPA between independent operators were all 100 %. These results demonstrate a high level of sensitivity, specificity, accuracy and diagnostic predictive value of the RVM assay and high agreement with comparable reference assays in identifying all eight of its targets. Taken together, our study underscores t