Application Of Nasal Swab In The Diagnosis Of Acute Lower Respiratory Tract Infections In Children And Analysis Of Important Sites Of HA Gene In Influenza A Virus

BackgroundAcute respiratory tract infection（ARTI）is a common disease in human.The symptom of ARTI can varies from mild amygdalitis to severe pneumonia or even die from complications,which cause high hospitalization rate and mortality.Thus,fast and accurate method for pathogen detetion contributes to better diagnosis and clinical care.Nasopharyngeal aspirates（NPA）or nasal swabs（NS）are regarded as the ideal detection of upper respiratory tract viruses by polymerase chain reaction（PCR）.Upper airway sampling can be performed on the bedside and is minimally invasive.In contrast,bronchoalveolar lavage fluid（BALF）as lower airway sampling in young children is invasive.At present,the consistency of the pathogens in NS specimens and BALF specimens in ALRTI children is not clear.Whether NS specimens can achieve the diagnostic effect of BALF specimens has not been systematically studied.Influenza A virus（IAV）is a common pathogen detected in acute respiratory infection.IAV causes 300,000–500,000 deaths worldwide each year.Influenza A H1N1virus caused an influenza pandemic in the 21st century for the first time since its outbreak in Mexico in April 2009.Vaccination is the primary method employed to prevent infection by H1N1 virus and its associated complications.Vaccine efficacy depends on the difference between the vaccine strain and the circulating strains.World Health Organization（WHO）show that since the outbreak of H1N1 2009 in China there have been two large-scale H1N1 virus epidemics in 2014 and 2016.Here we detect the accumulation of antigenic variations in the influenza virus and assess the efficacy of the vaccine strain.In March 2013,a novel H7N9 avian influenza virus（AIV）emerged and caused severe disease in humans in China.There have been five infection waves since H7N9emerged.Notably,the fifth wave started earlier than the previous four,with a sudden and steep increase in the number of human cases.More importantly,an insertion in the cleavage site of H7N9 was observed.The presence of multiple amino acids in the cleavage site indicates that the H7N9 has evolved from low-pathogenic avian influenza（LPAI）to high-pathogenic avian influenza（HPAI）.Therefore,more strategies are needed against the further spread and damage of H7N9 in the world.Objective1.To evaluate the value of nasal swab specimens in the diagnosis of acute lower respiratory tract infections in children.2.To analyze the genetic characteristics and evolution of HA1 gene of the influenza A/H1N1 virus circulating in Guangzhou between 2014 and 2017,as well as determine the vaccine efficacy.3.To analyze the HA gene cleavage sites in 2 patients infected avian influenza（H7N9）in Guangzhou,2017Methods1.One hundred and twenty paired nasal swab（NS）and bronchoalveolar lavage fluid（BALF）were obtained from sixty ALRTI children during 2016 to 2017.NS and BALF specimens were tested for pathogens using the Filmarray respiratory pathogen detection platform and laboratory developed qPCR method.2.Forty two cases of H1N1-positive nasal swabs were selected from the First Affiliated Hospital of Guangzhou Medical University.The HA1 gene were amplified by PCR.Phylogenetic trees and the mutations of amino acids were constructed and analyzed.Vaccine efficacy was calculated using the P_epitopemethod.3.The sputum of 2 patients with avian influenza（H7N9）infection in the First Affiliated Hospital of Guangzhou Medical University were collected in 2017.The sputum of first patient collected on the 19th and 26th days disease onset and that second patient collected on the 9th and 16th days disease onset.The fragment at the cleavage site of the H7N9 virus were amplified by PCR.The amplified products were sequenced,and the mixed sequences were conduct TA Clones.RNA structure was predicted using the RNAfold Web Server.A minimum free energy approach without base pairing constraints was applied.Results1.Both detection methods showed that no statistical difference in the detection rate of pathogens between NS and BALF（P>0.05）.The detection of RV,RSV,IFB,OC43 and IFA in NS and BALF was almost identical（Kappa≥0.75）.The results of Filmarray method showed that 83.3%（50/60）of children had one or more virus detected from NS and 86.7%（52/60）had one or more pathogens from BALF.The pathogens in NS from 68.3%（41/60）of cases included the pathogens detected in the BALF from the same individual,50%of which had exactly the same results.Laboratory developed qPCR method showed83.3%（50/60）of children had one or more virus detected from NS and 76.7%（46/60）had one or more pathogens from BALF.The pathogens in NS from 68.3%（41/60）of cases included the pathogens detected in the BALF from the same individual,53.3%of which had exactly the same results.2.The results of H1N1 epidemic strains in Guangzhou in 2014-2016 compered with the 2014-2016 H1N1 vaccine strain show that amino acid substitutions P83S（Epitope E）,S203T（Epitope B）,S185T（Epitope B）,K163Q（Epitope D）were observed in circulating strains.The vaccine efficacy of the vaccine strain in 2017 was 36.7%,which was more effective than the 2014-2016 vaccine strain（36.7%VS 24.5%）3.In the sputum of the first patient we detected the amino acid sequence of A（H7N9）virus cleavage site was mixed on the 19th day disease onset.Results of TA clone show that,in total 85 sequences,36（42.4%）of sequences had amino acid insert at the cleavage site,the sequence is GKRTAR/G and 49（57.6%）of sequences cleavage site was GR/G,which has a mix of high and low pathogenic H7N9 virus.After 7 days the sequence of virus in sputum was GKRTAR/G.The amino acid sequence at the cleavage site of the H7N9 virus detected in the sputum of the second patient on the 9th day disease onset was RKRAAR/G,and the virus was negative on the 16th day.G989T,A995G,A1023G substitutions appeared in all sequences had nucleotide insertion at the cleavage site,G1031A substitution also observed in sequence of sputum collected from second patient.Variations at the G989T,A995G,and A1023G sites can cause changes in the secondary structure of the RNA at the HACS of the virus.Conclusions1.In ALRTI children,the detection of virus RV,RSV,IFB,OC43,and IFA in NS can be a good reflection of the pathogens in lower respiratory tract.2.By analysis of HA1 gene we have found that the H1N1 influenza virus in Guangzhou has had an antigenic drift compared with the vaccine strains of the same year in 2014-2016.The influenza strains in 2017 were more consistent with the vaccine strains in the same year,and the vaccine efficacy was higher.3.Our study first reported the high and low pathogenic mixed infection of H7N9virus in humans.The mechanism and pathogenicity of highly pathogenic avian influenza（HPAI）H7N9 viruses need to be further studied.