Comparison of the Eragen Multi-Code Respiratory Virus Panel with Conventional Viral Testing and Real-Time Multiplex PCR Assays for Detection of Respiratory Viruses

High-throughput multiplex assays for respiratory viruses are an important step forward in diagnostic virology. We compared one such assay, the PLx Multi-Code Respiratory Virus Panel (PLx-RVP), manufactured by Eragen Biosciences, Inc. (Madison, WI), with conventional virologic testing, consisting of fluorescent-antibody staining plus testing with the R-mix system and fibroblast tube cultures. The test set consisted of 410 archived respiratory specimens, mostly nasopharyngeal swabs, including 210 that had been positive by conventional testing for a balanced selection of common respiratory viruses. Specimens yielding discrepant results were evaluated using a panel of respiratory virus PCR assays developed, characterized, and validated with clinical specimens. PLx-RVP increased the total rate of detection of viruses by 35.8%, and there was a 25.7% increase in the rate of detection of positive specimens. Reference PCR assay results corroborated the PLx-RVP result for 54 (82%) of 66 discrepancies with conventional testing. Of the 12 specimens with discrepancies between PLx-RVp and the reference PCRs, 6 were positive for rhinovirus by PLx-RVP and the presence of rhinovirus was confirmed by nucleotide sequencing. The remaining six specimens included five in which the PLx-RVP failed to detect parainfluenza virus and one in which the detection of influenza A virus by PLx-RVP could not be confirmed by the reference PCR. Taking the results of the reference PCR assay results into account, the sensitivities of the PLx-RVP for individual viruses ranged from 94 to 100% and the specificities ranged from 99 to 100%. We conclude that PLx-RVP is a highly accurate system for the detection of respiratory viruses and significantly improves the rate of detection of these viruses compared to that by conventional virologic testing.The rapid and accurate detection of respiratory viruses is clinically important. Potential advantages of specific viral detection include obtaining prognostic information, limiting additional diagnostic testing, instituting appropriate infection control precautions, and limiting unnecessary antibiotic usage. The use of respiratory specimens for this purpose is challenging because of the broad range of pathogens that may be present. This task is becoming even more complicated with the recent discoveries of several new viruses in respiratory tract samples, including human metapneumovirus (MPV) (19), coronaviruses (CoVs) NL63 (4, 20) and HKU1 (23), human bocavirus (3), and polyomaviruses KI (2) and WU (5).Molecular methods offer the advantages of rapidity and the ability to detect viruses regardless of the growth requirements or the availability of reagents for rapid diagnostic testing. An exciting recent advance is the development of multiplex molecular assays that allow the simultaneous detection of multiple targets in the same reaction (12, 13, 15, 16). One such test is the xTAG Respiratory Virus Panel, produced by Luminex Molecular Diagnostics (Toronto, ON, Canada). This test, which uses the Luminex-100 or -200 flow cell instrument (12), has been cleared by the Food and Drug Administration for use in the United States for the simultaneous detection and identification of multiple respiratory viruses from nasopharyngeal swab specimens from individuals with suspected respiratory tract infection. In the present study, we have evaluated a different respiratory multiplex PCR test, the PLx MultiCode Respiratory Virus Panel (PLx-RVP), manufactured by Eragen Biosciences, Inc. (Madison, WI). Two important characteristics of the evaluation are, first, that we have included substantial numbers of specimens containing each of the common respiratory viruses detected by the test and, second, that we have designed and validated PCR assays to detect each of the respiratory viruses and used these assays to resolve discrepancies between the results of conventional testing (fluorescent-antibody [FA] staining and culture) and PLx-RVP.