title: Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza a and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4.
abstract: Laboratory diagnosis of viral respiratory infections is generally performed by virus isolation in cell culture and immunofluorescent assays. Reverse transcriptase PCR is now recognized as a sensitive and specific alternative for detection of respiratory RNA viruses. A rapid real-time multiplex PCR assay was developed for the detection of influenza A and influenza B viruses, human respiratory syncytial virus (RSV), parainfluenza virus 1 (PIV1), PIV2, PIV3, and PIV4 in a two-tube multiplex reaction which used molecular beacons to discriminate the pathogens. A total of 358 respiratory samples taken over a 1-year period were analyzed by the multiplex assay. The incidence of respiratory viruses detected in these samples was 67 of 358 (19%) and 87 of 358 (24%) by culture and real-time PCR, respectively. Culture detected 3 influenza A virus, 2 influenza B virus, 57 RSV, 2 PIV1, and 2 PIV3 infections. All of these culture-positive samples and an additional 5 influenza A virus, 6 RSV, 2 PIV1, 1 PIV2, 1 PIV3, and 3 PIV4 infections were detected by the multiplex real-time PCR. The application of real-time PCR to clinical samples increases the sensitivity for respiratory viral diagnosis. In addition, results can be obtained within 6 h, which increases clinical relevance. Therefore, use of this real-time PCR assay would improve patient management and infection control.
authors.affiliation: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Kate E
authors.initials: KE
authors.lastname: Templeton
authors.forename: Sitha A
authors.initials: SA
authors.lastname: Scheltinga
authors.forename: Matthias F C
authors.initials: MF
authors.lastname: Beersma
authors.forename: Aloys C M
authors.initials: AC
authors.lastname: Kroes
authors.forename: Eric C J
authors.initials: EC
authors.lastname: Claas
mesh_terms: D002460:Cell Line; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification; D007251:Influenza, Human / Q000175:diagnosis / Q000821:virology; D010222:Parainfluenza Virus 1, Human / Q000235:genetics / Q000302:isolation & purification; D010223:Parainfluenza Virus 2, Human / Q000235:genetics / Q000302:isolation & purification; D010224:Parainfluenza Virus 3, Human / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D018357:Respiratory Syncytial Virus Infections / Q000175:diagnosis / Q000821:virology; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics / Q000302:isolation & purification; D012141:Respiratory Tract Infections / Q000175:diagnosis* / Q000821:virology*; D010253:Respirovirus Infections / Q000175:diagnosis / Q000821:virology; D019351:Rubulavirus Infections / Q000175:diagnosis / Q000821:virology; D012680:Sensitivity and Specificity; D013997:Time Factors; D014776:Virus Cultivation