MultiCode-PLx system for multiplexed detection of seventeen respiratory viruses

The MultiCode-PLx system (EraGen Biosciences, Inc., Madison, WI) for the detection of respiratory viruses uses an expanded genetic alphabet, multiplex PCR chemistry, and microsphere flow cytometry to rapidly detect and specifically identify 17 different respiratory viruses directly in clinical specimens. The MultiCode-PLx system was tested in parallel with direct fluorescent-antibody (DFA) staining and rapid shell vial culture (R-mix cells; Diagnostic Hybrids, Inc. Athens, OH) with 354 respiratory specimens from adult patients that were submitted to the clinical virology laboratory at the Emory University Hospital. Single-target PCRs were performed with retained samples to confirm the positive results obtained with the MultiCode-PLx system for viruses not covered by DFA and R-mix culture (metapneumovirus, coronaviruses [CoV], parainfluenza viruses 4a and 4b, and rhinoviruses) and to resolve any discrepancies between the DFA and R-mix culture and the MultiCode-PLx results for viruses common to both systems. Respiratory viruses were detected in 77 (21.8%) and 116 (32.7%) specimens by DFA and R-mix culture and with the MultiCode-PLx system, respectively. Among the viruses common to both systems, the MultiCode-PLx system detected significantly more influenza A viruses (P = 0.0026). An additional increased diagnostic yield with the MultiCode-PLx system resulted from the detection of human metapneumovirus (HMPV) in 9 specimens, human CoV (HCoV) in 3 specimens, and human rhinovirus (HRV) in 16 specimens. Also, two mixed viral infections were detected by the MultiCode-PLx system (HCoV OC43 and HRV infections and HMPV and HRV infections), but none were detected by DFA and R-mix culture. Single-target PCRs verified the results obtained with the MultiCode-PLx system for 73 of 81 (90.1%) specimens that had discordant results or that were not covered by DFA and R-mix culture. The MultiCode-PLx system provides clinical laboratories with a practical, rapid, and sensitive means for the massively multiplexed molecular detection of common respiratory viruses.