title: Simultaneous detection and high-throughput identification of a panel of RNA viruses causing respiratory tract infections.
abstract: Clinical presentations for viral respiratory tract infections are often nonspecific, and a rapid, high-throughput laboratory technique that can detect a panel of common viral pathogens is clinically desirable. We evaluated two multiplex reverse transcription-PCR (RT-PCR) products coupled with microarray-based systems for simultaneous detection of common respiratory tract viral pathogens. The NGEN respiratory virus analyte-specific assay (Nanogen, San Diego, CA) detects influenza A virus (Flu-A) and Flu-B, parainfluenza virus 1 (PIV-1), PIV-2, and PIV-3, and respiratory syncytial virus (RSV), while the ResPlex II assay (Genaco Biomedical Products, Inc., Huntsville, AL) detects Flu-A, Flu-B, PIV-1, PIV-2, PIV-3, PIV-4, RSV, human metapneumovirus (hMPV), rhinoviruses (RhVs), enteroviruses (EnVs), and severe acute respiratory syndrome (SARS) coronavirus (CoV). A total of 360 frozen respiratory specimens collected for a full year were tested, and results were compared to those obtained with a combined reference standard of cell culture and monoplex real-time TaqMan RT-PCR assays. NGEN and ResPlex II gave comparable sensitivities for Flu-A (82.8 to 86.2%), Flu-B (90.0 to 100.0%), PIV-1 (87.5 to 93.8%), PIV-3 (66.7 to 72.2%), and RSV (63.3 to 73.3%); both assays achieved excellent specificities (99.1 to 100.0%) for these five common viruses. The ResPlex II assay detected hMPV in 13 (3.6%) specimens, with a sensitivity of 80.0% and specificity of 99.7%. The ResPlex II assay also differentiated RSV-A and RSV-B and gave positive results for RhV and EnV in 31 (8.6%) and 19 (5.3%) specimens, respectively. PIV-2, PIV-4, and SARS CoV were not detected in the specimens tested. The two systems can process 80 (NGEN) and 96 (ResPlex II) tests per run, with a hands-on time of approximately 60 min and test turnaround times of 6 h (ResPlex II) and 9 h (NGEN). Multiple-panel testing detected an additional unsuspected 9 (3.4%) PIV-1 and 10 (3.7%) PIV-3 infections. While test sensitivities for RSV and PIV-3 need improvement, both the NGEN and ResPlex II assays provide user-friendly and high-throughput tools for simultaneous detection and identification of a panel of common respiratory viral pathogens in a single test format. The multiplex approach enhances diagnosis through detection of respiratory viral etiologic agents in cases in which the presence of the agent was not suspected and a test was not ordered by the clinicians.
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Haijing
authors.initials: H
authors.lastname: Li
authors.forename: Melinda A
authors.initials: MA
authors.lastname: McCormac
authors.forename: R Wray
authors.initials: RW
authors.lastname: Estes
authors.forename: Susan E
authors.initials: SE
authors.lastname: Sefers
authors.forename: Ryan K
authors.initials: RK
authors.lastname: Dare
authors.forename: James D
authors.initials: JD
authors.lastname: Chappell
authors.forename: Dean D
authors.initials: DD
authors.lastname: Erdman
authors.forename: Peter F
authors.initials: PF
authors.lastname: Wright
authors.forename: Yi-Wei
authors.initials: YW
authors.lastname: Tang
mesh_terms: D000293:Adolescent; D000328:Adult; D005260:Female; D006801:Humans; D008297:Male; D012327:RNA Virus Infections / Q000821:virology*; D012328:RNA Viruses / Q000145:classification / Q000302:isolation & purification*; D012140:Respiratory Tract Diseases / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014777:Virus Diseases / Q000821:virology*