Comparison of Singleplex qPCR and the Luminex MAGPIX Platform for the Detection of Viral Pathogens

ABSTRACT

Background: Various respiratory molecular assays are available, each with different characteristics and advantages that make them uniquely valuable. The objective of this study was to compare rates of viral detection using singleplex and multiplex platforms in a research setting. Methods: A prospective viral surveillance study was conducted in Davidson County, TN. Infants under one year who presented with fever and/or respiratory symptoms were enrolled from the outpatient, emergency department and inpatient settings. Nasal swabs were collected and tested for influenza A (FluA), influenza B (FluB), human metapneumovirus (MPV), respiratory syncytial virus A and B (RSVA and RSVB), human adenovirus (AdV), parainfluenza 1, 2, 3, and 4 (PIV1-4) and SARS-2-CoV by both singleplex qPCR and the Luminex NxTAG Respiratory Pathogen and NxTAG CoV Extended panels. The rhinovirus/enterovirus, human bocavirus, Chlamydophila pneumoniae, Mycoplasma pneumoniae and coronavirus HKU1, NL63, 229E and OC43 results from the Luminex panel were excluded because singleplex qPCR was not performed on those targets. For singleplex qPCR results, cycle threshold (Ct) values were used as a surrogate for viral load, with a higher Ct value indicating a lower viral load. Results: A total of 112 nasal specimens were tested by both singleplex qPCR and Luminex, of which 65 were positive for at least one virus by either platform and 56 had a virus detected on both platforms (Figure 1). Seven specimens were positive by singleplex qPCR only and two were positive by Luminex only (Figure 1). The targets positive by singleplex qPCR only included FluB, RSVA, AdV and PIV2 and those positive by Luminex only included FluA H1N1 and RSVB (Figure 2). Specimens that were positive only on the singleplex assay had a higher average Ct value than those that were positive on both assays, indicating a lower viral load (Figure 3). Figure 1 Figure 2 Figure 3 Conclusion: The multiplex assay identified 89% of the total viruses detected while singleplex qPCR identified 97% of the total viruses detected. Lower viral loads may contribute to false negative results on the multiplex platforms. Future studies with larger sample sizes are needed in order validate our findings.