Solunum virusları ve influenza A virus alt tiplerinin multipleks PCR yöntemi ile aynı anda saptanması
Küçük Resim Yok
Tarih
2014
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Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu çalışmada, influenza A (H1N1)pdm09 virusu ile oluşan salgın sonrasında, grip semptomu olan hastalarda solunum viruslarının araştırılması ve influenza A virusu saptanan hastalarda alt tiplerin multipleks PCR yöntemiyle tiplendirilmesi amaçlanmıştır. Çalışmaya, Eylül 2010-Ocak 2013 tarihleri arasında, 38oC’nin üzerinde ateş, halsizlik, rinit, baş ve boğaz ağrısı, öksürük, miyalji gibi Dünya Sağlık Örgütü’nün tanımladığı grip semptomları olan 700 olgudan (313 kadın, 387 erkek; yaş aralığı: 24 gün-94 yıl, ortanca yaş: 1 yıl) alınan nazofaringeal sürüntü örnekleri dahil edilmiştir. Örneklerden nükleik asit ekstraksiyonu (Viral DNA/RNA Extraction Kit, iNtRON, Güney Kore) ve cDNA sentezi (RevertAid First Strand cDNA Synthesis Kit, Fermentas, ABD) üretici firmaların önerileri doğrultusunda yapılmıştır. İnfluenza A (INF-A), influenza B (INF-B), solunum sinsityal virusu (RSV) ve diğer solunum viruslarına ait nükleik asitlerin multipleks amplifikasyonu, DPO (dual priming oligonucleotide) primerler ve RV5 ACE Screening Kit (Seegene, Güney Kore) kullanılarak gerçekleştirilmiştir. PCR ürünleri, Screen Tape çoklu tanımlama sistemi kullanılarak otomatize poliakrilamid jel elektroforezi ile saptanmıştır. Pozitifliğin saptandığı örnekler, özgül DPO primerleri, FluA ACE Subtyping ve RV15 Screening (Seegene, Güney Kore) kitleri ile tekrar çalışılmış; bu kitlerle INF-A virusunun dört alttipi [human H1 (hH1), human H3 (hH3), swine H1 (sH1), avian H5 (aH5)] ile diğer 11 solunum virusu [Adenovirus, parainfluenza virus (PIV) tip 1-4, insan bokavirusu (HBoV), insan metapnömovirusu (HMPV), rinovirus tip A ve B, koronavirus (HCoV) OC43 ve 229E/NL63] araştırılmıştır. Çalışılan örneklerin %53.6 (375/700)’sında en az bir solunum virusuna ait nükleik asit pozitifliği saptan- mış; pozitif olguların %15.7 (59/375)’sinde çoklu etken tespit edilmiştir. Çoklu virus enfeksiyonlarının %83 (49/59)’ü pediatrik grupta görülmüştür. İkili etken enfeksiyonlarında en sık RSV ve rinovirus birlikteliği (18/29, %62.7) izlenmiştir. Hastalarda (n= 375) saptanan toplam 436 solunum virusunun 189 (%43.3)’u RSV, 93 (%21.4)’ü rhinovirus, 86 (%19.8)’sı INF-A, 7 (%1.6)’si INF-B, 22 (%5)’si PIV tip 1-3, 14 (%3.2)’ü HMPV, 11 (%2.5)’i HCoV, 9 (%2)’u HBoV ve 5 (%1.2)’i adenovirus olarak tanımlanmıştır. Toplam 86 INF-A virusunun 55 (%64)’i hH3, 24 (%27.9)’ü sH1 ve 7 (%8.1)’si hH1 olarak tiplendirilmiş; aH5 hiç bir örnekte tespit edilmemiştir. INF-A, INF-B, RSV ve diğer solunum viruslarının prevalansı sırasıyla; %12, %1, %27 ve %14.6 olarak bulunmuştur. RSV çocuklarda (161/313, %51), INF-A ise erişkinlerde (24/62, %38.7) en sık saptanan solunum virusları olmuştur. Grip semptomları olan hastaların %13.3 (93/700)’ünde sorumlu etkenin influenza virusları olduğu görülmüştür. Olgulardan, 1 aylık bir bebeğin üç virus tipi (INF-A hH1 + INF-A sH1 + HCoV OC43), 82 yaşında bir olgunun ise iki ayrı INF-A alt tipi (hH3 + sH1) ile enfekte olduğu belirlenmiştir. INF-A viruslarının en sık aralık-mart ayları arasında (79/86) saptandığı izlenmiştir. INF-A sH1, Şubat 2011 tarihine kadar grip olgularında en sık saptanan alt tip olmuş (22/86) ve daha sonra yerini hH3’e bırakmıştır. Şubat 2012 tarihinden itibaren grip olgularında INF-A hH3 alt tipinde belirgin bir artış (39/86) görülmüştür. Sonuç olarak, toplumda dolaşan influenza virus tip ve alt tiplerinin belirlenmesi ve sürveyansının, hem epidemiyolojik verilere katkı sağlamak hem de aşı tiplerinin seçimine yardımcı olmak açısından önemli olduğu düşünülmüştür.
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.
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.
Açıklama
Anahtar Kelimeler
Mikrobiyoloji
Kaynak
Mikrobiyoloji Bülteni
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Cilt
48
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4
