title: Rapid multiplex nested PCR for detection of respiratory viruses.
abstract: Respiratory tract infections can be caused by a heterogeneous group of viruses and bacteria that produce similar clinical presentations. Specific diagnosis therefore relies on laboratory investigation. This study developed and evaluated five groups of multiplex nested PCR assays that could simultaneously detect 21 different respiratory pathogens: influenza A virus (H1N1, H3N2, and H5N1); influenza B virus; parainfluenza virus types 1, 2, 3, 4a, and 4b; respiratory syncytial virus A and B; human rhinoviruses; human enteroviruses; human coronaviruses OC43 and 229E; severe acute respiratory syndrome coronavirus; human metapneumoviruses; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Legionella pneumophila; and adenoviruses (A to F). These multiplex nested PCRs adopted fast PCR technology. The high speed of fast PCR (within 35 min) greatly improved the efficiency of these assays. The results show that these multiplex nested PCR assays are specific and more sensitive (100- to 1,000-fold) than conventional methods. Among the 303 clinical specimens tested, the multiplex nested PCR achieved an overall positive rate of 48.5% (95% confidence interval [CI], 42.9 to 54.1%), which was significantly higher than that of virus isolation (20.1% [95% CI, 15.6 to 24.6%]) and that of direct detection by immunofluorescence assay (13.5% [95% CI, 9.7 to 17.4%]). The improved sensitivity was partly due to the higher sensitivity of multiplex nested PCR than that of conventional methods in detecting cultivatable viruses. Moreover, the ability of the multiplex nested PCR to detect noncultivatable viruses, particularly rhinoviruses, coronavirus OC43, and metapneumoviruses, contributed a major gain (15.6%) in the overall positive rate. In conclusion, rapid multiplex nested PCR assays can improve the diagnostic yield for respiratory infections to allow prompt interventive actions to be taken.
authors.affiliation: Department of Microbiology, The Chinese University of Hong Kong, 1/F Clinical Science Building, Prince of Wales Hospital, Shatin, New Territories, Hong Kong.
authors.forename: W Y
authors.initials: WY
authors.lastname: Lam
authors.forename: Apple C M
authors.initials: AC
authors.lastname: Yeung
authors.forename: Julian W
authors.initials: JW
authors.lastname: Tang
authors.forename: Margaret
authors.initials: M
authors.lastname: Ip
authors.forename: Edward W C
authors.initials: EW
authors.lastname: Chan
authors.forename: Mamie
authors.initials: M
authors.lastname: Hui
authors.forename: Paul K S
authors.initials: PK
authors.lastname: Chan
mesh_terms: D005455:Fluorescent Antibody Technique; D006801:Humans; D009980:Influenza A virus / Q000302:isolation & purification; D009981:Influenza B virus / Q000302:isolation & purification; D029121:Metapneumovirus / Q000302:isolation & purification; D010222:Parainfluenza Virus 1, Human / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012136:Respiratory Syncytial Viruses / Q000302:isolation & purification; D012141:Respiratory Tract Infections / Q000175:diagnosis / Q000821:virology*; D012680:Sensitivity and Specificity; D013997:Time Factors