Evaluation of a Multiplexed PCR Assay for Detection of Respiratory Viral Pathogens in a Public Health Laboratory Setting

Abstract

There are numerous viral and bacterial causes of respiratory disease. To enable rapid and sensitive detection of even the most prevalent causes, there is a need for more-simplified testing systems that enable researchers and clinicians to perform multiplexed molecular diagnostics quickly and easily. To this end, a new multiplexed molecular test called the MultiCode-PLx respiratory virus panel (PLx-RVP) was developed and then implemented in a public health laboratory setting. A total of 687 respiratory samples were analyzed for the presence of 17 viruses that commonly cause respiratory disease. As a comparator, the samples were also tested using a standard testing algorithm that included the use of a real-time influenza virus A and B reverse transcription-PCR test and routine viral culture identification. The standard testing algorithm identified 503 (73%) samples as positive and 184 as negative. Analyzing the same 687 samples, the PLx-RVP assay detected one or more targets in 528 (77%) samples and found 159 samples negative for all targets. There were 25 discordant results between the two systems; 14 samples were positive for viruses not routinely tested for by the Wisconsin State Laboratory of Hygiene, and 13 of these were confirmed by real-time PCR. When the results of the standard testing algorithm were considered "true positives," the PLx-RVP assay showed an overall sensitivity of 99% and an overall specificity of 87%. In total, the PLx-RVP assay detected an additional 40 viral infections, of which 11 were mixed infections.

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

“…The two viruses which were associated with the largest numbers of discrepancies were RV and PIV. As was seen in the other evaluations (8,13,15), the PLx-RVP detected many more RVs than conventional testing. The presence of RV was confirmed in the large majority of the discrepant specimens by the reference PCR assay, supporting the enhanced sensitivity of PLx-RVP for RV.…”

“…The newly developed multiplex molecular assays to detect respiratory viruses (12,13,15,16) are a major step forward in diagnostic virology. In the present study, we evaluated one such assay, the PLx-RVP, by comparing it to conventional virologic testing, consisting of FA staining and culture as performed in a routine diagnostic virology laboratory.…”

“…The PLx-RVP detects the presence of nucleic acids from the following viruses: InfA and -B; RSV types A and B; PIV types 1, 2, 3, 4a, and 4b; RV; human CoVs OC43, 229E, and NL63; MPV; and AdV species B, C, and E. The specific procedures for performing the PLx-RVP have been described previously (13,15). Testing with the PLx-RVP was performed at the Eragen Biosciences laboratory in Madison, WI, using nucleic acid extracts prepared from specimens at Washington University, as described above.…”

“…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).…”

Comparison of Viral Isolation and Multiplex Real-Time Reverse Transcription-PCR for Confirmation of Respiratory Syncytial Virus and Influenza Virus Detection by Antigen Immunoassays

“…The improved sensitivity of PCR over antigen testing and virus isolation for RSV (14,16,44,45) and influenza virus (17,20,40,41,48) has been previously demonstrated by uniplex, as well as multiplex, testing (4,23,28,32). Studies have also demonstrated improved sensitivity of PCR in combination with culture as a composite "gold standard" or as the alternate reference test for RSV or influenza virus (1,19,33,36,38).…”