AbstractIntroductionUkraine’s ability to respond to the spread of viruses that causepandemics and reduce economic losses from influenza, can bestrengthened only in the presence of a developed surveillance networkincluding the monitoring of virus circulation in humans. Specialistsof Dnipropetrovsk Oblast have great experience in virologicalsurveillance on the circulation of influenza virus A/California/H1N1and timely determination of the etiology of outbreaks caused by thevirus.MethodsLaboratory diagnostics of influenza was performed usingserological methods, PCR, and virological studies in the cell culture.During the last seven epidemic seasons, including the flu pandemicof 2009-2010, most of samples came from four health-care facilitiesof Dnipropetrovsk, which were determined as basic hospitals forthe sentinel center. Patients with severe acute respiratory infections(SARI) were examined. Nasopharyngeal washouts and swabs werecollected into cryo-tubes with a transport medium. The samples werestored at hospitals in Dewar flasks.The delivery of the samples to the laboratory was performedaccording to cold-chain rules. After sample preparation stage, thesamples were tested for the presence of influenza A/B virus RNA byPCR using Bio-Rad CFX-96 cycler and the following commercialtest-kits AmpliSens® Influenza virus A/B-FL, AmpliSens® Influenzavirus A/H1-swine, and AmpliSens® Influenza virus A-type-FL.All positive samples with detected RNA of influenza virusA/H1-swine were tested using MDSK cell cultures (Canine KidneyEpithelial Cells). Flu viruses caused cytopathic changes in the cellcultures in the form of poppy-sand-like degeneracy not earlier thanin 72 hours after the infection of the cells followed by cell monolayerfragmentation.Fig. 1 MDSK cell cultureFig. 2 MDSK cell culture 72 hours after infection with influenzavirus A (H1N1)Express immunochromatic tests «Cito test influenza A+B» oragglutination test (AT) using erythrocyte suspension of human 0 (I)group blood were used for the determination of haemagglutinatingagents.ResultsDuring the seven epidemic seasons, 5,467 people were examinedfor flu and acute respiratory viral infections. During the swine flupandemic in 2009-2010, 1,217 severely ill patients were tested.Positive results were found in 50% of cases (607 persons). Fromthose, pandemic influenza virus (RNA of influenza A/H1-swinevirus) was detected in 100% of positive cases.Fig.3 Data on the determined pandemic flu virus strains(RNA of influenza A/H1-swine virus) using PCR duringepidemiological seasons from 2009 to 2016 in DnipropetrovskOblast, UkraineFrequency of pandemic influenza virus detection declined to zeroin the following epidemic seasons (2010-2011 and 2011-2012).However, incidence of the virus variant (influenza A/H1-swine)began to grow slowly during the last four epidemic flu seasonsfrom separate cases (6 in 2012-2013, 1 in 2012-2013) to 26 cases in2014-2015. During the last epidemic season (2015-2016), the numberof pandemic influenza cases increased dramatically to 166, accounting29% of all examined persons.Fig. 4 Results of isolation of pandemic strains of influenzaviruses in cell culture MDSK flu epidemic seasons from 2009-2010to 2015-2016 in Dnipropetrovsk Oblast, UkraineMost of the virus isolates were sent for confirmation and furtheridentification to the Ukrainian Center for Influenza and to theworld influenza centers (Atlanta, USA and London, UK) in order tosupport Ukraine’s participation in the worldwide pandemic influenzasurveillance. The world flu centers confirmed the isolates to beinfluenza virus strain A/California/(H1N1)/07/2009.Conclusions1. Circulation of the pandemic type of influenza virus A/California/(H1N1)/ 07/2009 among the population of Dnipropetrovsk oblast isof sporadic character.2. The return of the virus A/California/(H1N1)/07/2009 after the2009-2010 pandemic occurred during the last 2015-2016 epidemicseason.3. Application of PCR can significantly shorten the examination ofpatients with severe course of influenza, but cannot help with virusisolation.4. The use of express immunoassay tests accelerates theidentification of viruses isolates.5. The employment MDSK cell culture for influenza virusisolation allows obtaining of a spectrum of influenza strainscirculating during an epidemic period including the strainA/California/(H1N1)/07/2009.
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