Diagnosis of respiratory pathogen viruses by the methods of multiplex PCR and IFAT

XIVSUMMARYAccurate detection of respiratory viruses is important to guide antiviraltherapy, prevent nosocomial spread, provide surveillance and in some cases, decreasehospital costs and lengths of stay. By using standard laboratory methods, such asstaining with fluorescent antibodies (FAT) and isolation by culture, viruses have beendetected in 13 to 45 % of children with symptoms of respiratory illness. In this study;two diagnostic methods used for respiratory viruses have shown acute respiratorytract infection symptoms in childrens.Detection of IgM antibodies from blood by fluorescent-antibody (FAT-Euroimmun GmbH Germany) and Multiplex RT PCR assays (Hexaplex Plus,Prodesse Inc. USA) for detection of respiratory syncytial virus (RSV), influenza virustype A (FluA), influenza virus type B (FluB), parainfluenza virus types 1, 2, and 3(PIV1, PIV2, and PIV3), human metapneumovirus (MPV) and adenovirus (AdV)from nasopharyngeal samples. A multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (RT-PCR) was developed andused to rapidly detect and quantitative RNA of seven viruses in nasopharyngealspecimens in a single test. Primers and probes originated from highly conservedregions of each viral genome. Six and a half primer pairs were mixed for thesimultaneous detection and quantitation of RNA from seven different respiratoryviruses. This was carried out in 46 nasopharyngeal and bloods specimens fromchildren with respiratory illnesses collected over a 1-year period.The median age of the patients from whom the specimens were collected was16.4 ± 4.5 months (range 2 months to 13 years); Fifty-two percent of samples werefrom male patients, and 48 % were from female patients. Of these 46 bloodspecimens, 13 (28.3 %) were positive by FAT (2 for RSV, 9 for FluA-H1N1, H3N2,8 for FluB, 3 for PIV3). Of these 46 nasopharyngeal specimens, 8 (17.4 %) werepositive by Multiplex PCR (1 for RSV, 8 for FluA, 1 for FluB, 3 for PIV3, 5 forMPV, and 1 for both FluA and 5 for hMPV, AdV absent in Hexaplex Plus). Thenumber of specimens positive only by PCR among specimens positive by PCR and/orFAT was 1 (50.0 %) of 2 for RSV, 8 (88.9 %) of 9 for FluA, 3 (100 %) of 3 for PIV3(P < 0.001).Use of both Multiplex PCR and FAT to identify viral respiratory pathogens inchildren will lead to improved diagnosis of respiratory illness.Key Words: Respiratory viruses, IFAT, Multiplex PCR