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

TLDR

Laboratory diagnosis of viral respiratory infections is traditionally performed by cell‑culture isolation and immunofluorescent assays, but reverse‑transcriptase PCR is now recognized as a more sensitive and specific alternative. The study aimed to develop a rapid real‑time multiplex PCR assay to detect influenza A and B, RSV, and PIV1–4. The assay uses a two‑tube multiplex reaction with molecular beacons to discriminate the pathogens and was applied to 358 respiratory samples collected over one year. The multiplex PCR detected 24 % of samples versus 19 % by culture, identifying all culture‑positive cases plus additional infections, yielding results within 6 h and thereby improving patient management and infection control.

Abstract

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.