Simultaneous detection of respiratory viruses and influenza A virus subtypes using multiplex PCR

Abstract

This study was conducted to investigate the respiratory viruses and subtyping of influenza A virus when positive by multiplex PCR in patients with flu-like symptoms, after the pandemic caused by influenza A (H1N1)pdm09. Nasopharyngeal swab samples collected from 700 patients (313 female, 387 male; age range: 24 days-94 yrs, median age: 1 yr) between December 2010 - January 2013 with flu-like symptoms including fever, headache, sore throat, rhinitis, cough, myalgia as defined by the World Health Organization were included in the study. Nucleic acid extractions (Viral DNA/RNA Extraction Kit, iNtRON, South Korea) and cDNA synthesis (RevertAid First Strand cDNA Synthesis Kits, Fermentas, USA) were performed according to the manufacturer's protocol. Multiplex amplification of nucleic acids was performed using DPO (dual priming oligonucleotide) primers and RV5 ACE Screening Kit (Seegene, South Korea) in terms of the presence of influenza A (INF-A) virus, influenza B (INF-B) virus, respiratory syncytial virus (RSV), and the other respiratory viruses. PCR products were detected by automated polyacrylamide gel electrophoresis using Screen Tape multiple detection system. Specimens which were positive for viral nucleic acids have been further studied by using specific DPO primers, FluA ACE Subtyping and RV15 Screening (Seegene, South Korea) kits. Four INF-A virus subtypes [human H1 (hH1), human H3 (hH3), swine H1 (sH1), avian H5 (aH5)] and 11 other respiratory viruses [Adenovirus, parainfluenza virus (PIV) types 1-4, human bocavirus (HBoV), human metapneumovirus (HMPV), rhinovirus types A and B, human coronaviruses (HCoV) OC43, 229E/NL63] were investigated with those tests. In the study, 53.6% (375/700) of the patients were found to be infected with at least one virus and multiple respiratory virus infections were detected in 15.7% (59/375) of the positive cases, which were mostly (49/59, 83%) in pediatric patients. RSV and rhinovirus coinfections were the most prevalent (18/29, 62.7%) dual infections. The distribution of 436 respiratory viruses identified from 375 patients were as follows; 189 (43.3%) RSV, 93 (21.4%) rhinovirus, 86 (19.8%) INF-A, seven (1.6%) INF-B, 22 (5%) PIV types 1-3, 14 (3.2%) HMPV, 11 (2.5%) HCoV, nine (2%) HBoV, and five (1.2%) adenovirus. Fifty-five (64%) out of 86 INF-A viruses were subtyped as hH3, 24 (27.9%) were sH1 and seven (8.1%) were hH1. Avian H5 was not detected in any samples. The overall prevalence rates of INF-A, INF-B, RSV and other respiratory viruses were 12%, 1%, 27%, and 14.6%, respectively. RSV was the most prevalent respiratory agent in pediatric (161/313, 51%) cases, while INF-A virus in adult (24/62, 38.7%) patients. Influenza viruses were detected as responsible pathogens in 13.3% (93/700) of the patients with flu-like symptoms. Among the cases, a 1-month-old baby was infected with three virus strains (INF-A hH1+INF-A sH1+HCoV OC43) and a 82-year-old patient was infected with two INF-A virus subtypes (hH3 + sH1). INF-A viruses were mostly detected (79/86) in winter period, from December to March. INF-A virus sH1, was the most prevalent subtype in flu cases till February 2011 (22/86), after replaced by INF-A virus hH3. Beginning from February 2012, a significant increase observed in the cases infected with INF-A virus subtype hH3 (39/86). In conclusion, the identification and surveillance of influenza virus types and subtypes circulating in populations have importance both for epidemiological data and selection of vaccine strains.