Risk Factors Associated With Serious Conditions Of Novel Influenza A (H1N1) Infection

(Background]The 2009 influenza A (H1N1) pandemic was characterized by mild in the majority of cases, maybe milder than the 1918 influenza pandemic. However, severe and fatal cases had been emerging continuously. Past studies from western counties have proved that there exist several relevant risk factors which are associated with advanced age and infants, chronic illnesses (chronic lung disease, diabetes, obesity), immunosuppression, pregnancy and delayed admission to hospital. These findings hold great significance to investigation that whether these risk factors are associated with development of serious condition in China. Moreover, we were also interested to probe into other possible risk factors. Until now, no studies on the causes and risk factors of pandemic 2009 influenza A (H1N1) in China have been reported. Therefore, we seeked to investigate the risk factors of serious conditions of H1N1 infection in this study.To explore whether these factors mentioned above would also result in severe and fatal influenza A in China and whether there is any other kind of potential risk factors, this study evaluated possible influential factors related with severe H1N1 cases based on case-control study from epidemiology aspect. According to the genetic sequence identification of hemagglutinin (HA) and neuraminidase (NA) for representive isolates, it aims to analysed the variation features of influenza A (H1N1) strains and differences between severe and mild strains on molecular levels and to assess the pathogen features and risk factors among severe/fatal cases.L Objectives]1. To analyze the correlation factors of H1N1 influenza and investigate the risk factors of serious conditions of H1N1 infection in this study, in order to provide a scientific basis for the future influenza surveillance, prevention and control strategies.2. The objectives of this research are molecular and phylogenetic analysis of pandemic influenza A (H1N1) strains that circulated in Shandong province in China, focusing on severe or fatal infections, identification of sequence variations of hemagglutinin and neuraminidase gene in relation with the severity of the illness and comparison of circulating viruses with the vaccine strain.[Methods]We designed a case-control study with 343 severe patients and 343 randomly selected mild controls included in this study. First of all, collection of the general information, epidemiological history and clinical data of confirmed cases by unified questionnaires designed by the Minnistry of Health. Nasopharyngeal swabs from fever patients were collected and diagnosed by real time quantitative RT-PCR method. Throat specimens from confirmed patients were isolated and cultured in MDCK cells. To isolate and identify the influenza virus in Shandong Province in 2009-2010 and randomly selected isolated pandemic influenza A (H1N1) virus. HA and NA genes of this selected virus were sequenced and performed genetic evolution and amino acid substitutions.A database was established using Microsoft Office Excel 2007. For initial screening of potential risk factors, we applied x2 test or Fisher's exact test to compare the distribution of different variables between severe and mild patients, including age, sex, race, occupation, BMI, time from symptom onset to hospitalization, chronic coexisting illness, pregnant status, received seasonal influenza vaccination one year before onset. For those variables with statistical significance (P<0.05). We carried out univariate and multivariable regression analysis to estimate the odds ratio (ORs) and corresponding 95% confidence intervals (CIs) for association study of potential risk factors with serious conditions of H1N1 influenza. For patients with renal disease, chronic liver disease, chronic cardiac disease, cancer or immunosuppressive disorder, we found no cases with mild H1N1 influenza therefore these variables were excluded from the multivariable regression analysis. All statistical analysis was performed using SAS 9.1 software. The 2-tailed and P value of 0.05 was considered significant for all statistical tests. Homology analysis using program EditSeq and MegAlign in DNASTAR software package. Genetic evolution and amino acid substitutions of the gene sequences were analyzed by using MEGA 4.0 software.[Results]1. In a multivariable logistic model, the overweight or obese subjects were more likely to be infected with severe H1N1 influenza, the risk increased to 3.01 (95% CI 1.53-5.91) and 41.02 (95% CI 5.29-318.13) fold respectively, compared with subjects having BMI less than 25. This study compared the severity of H1N1 influenza disease among subjects with different ages. Compared with subjects with age ranged between 5-60 years, subjects with age less than 5 years or elder than 60 years had a markedly increased risk of severe conditions (OR 22.18,95% CI 6.18-79.63). We also found markedly increased risk of severe disease among subjects with longer time interval from system onset to hospitalization (OR 3.12,95% CI 1.90-5.11) or peasants (OR 9.44,95% CI 4.47-19.95). In a multivariable logistic model, patients from our following analysis having chronic respiratory disease, diabetes and pregnancy were associated with increased risk of severe H1N1 influenza the ORs were 26.33 (95% CI 3.23-214.69),15.54 (95% CI 1.81-133.47),8.39 (95% CI 2.36-29.89) respectively. To testify our results furthermore, we also applied univariate logistic analysis for these potential risk factors and the analysis reached similar results. However, no significant two-way interaction terms were observed to be significant during the model-building process.2. Phylogenetic analysis for hemagglutinin and neuraminidase showed they were related to their vaccine strain, with an average of 96.4-99.6% and 99.1-100% sequence identity, respectively. The phylogenetic tree of HA nucleotide sequences of the Shandong pandemic strains and the vaccine strain is that the majority of the early, obviously imported viruses appear at the above of the tree and the domestic strains evolved further from the early imported strains. No clear distribution of viruses according to geographical could be observed. Also viruses identified in patients suffering from severe infections were scattered throughout the phylogenetic tree of HA gene. Most viruses obtained from severe infections were gathered throughout the phylogenetic tree of the NA gene.3. The number of amino positions changed for HA protein was 49,13 of whose were located in the antigenic site. Amino acid mutations of one viruse identified in a patient suffering from severe infection, which were located in four antigenic sites, respectively Sa,Sb,Cal,Ca2. The number of amino positions changed for NA protein was 24, which didn't lead to change the enzyme sites. Amino acid residues at the enzymatically active sites the NA genes were strictly conserved in all influenza virus strains. Furthermore, one glycosylation site of HA protein and NA protein were changed respectively, at residue 40 of HA protein and residue 386 from Asparagine to aspartic acid (N-D). Antigenic drift of HA gene occurred in a late virus identified in a patient suffering from severe infection. One of the characteristic differencies between the epidemic viruses and the A/California/07/2009 vaccine virus occurred at residue V106I N248Då'ŒY351F. All Shandong epidemic viruses had these changes at above residues. Two severe infections were detected with two mutations at NA residue V83A, A138T. None of the analyzed viruses had the amino acid change from histidine (H) to tyrosine (Y) at residue 275, which would render the virus resistant to oseltamivir.[Conclusions]1. The results of this research suggest that the risk factors of serious conditions of H1N1 infection include Chronic respiratory disease, obesity and overweight, age less than 5 years or elder than 60 years, diabetes, peasant, pregnancy and longer time interval from system onset to hospitalization.2. Phylogenetic and molecular analysis showed that viruses isolated from the patients suffering from severe infections still belonged to the classical swine lineages and did not have the genetic characteristics of highly pathogenic influenza virus.3. The results showed that the HA and NA genes of the epidemic strains were high homology, some mutations in the HA and NA proteins were found, the antigenic site and glycosylation site of some strains were changed on the Epidemic process, all the samples were sensitivity to oseltamivir.4. Several amino acid residue mutations on HA and NA protein were detected, which seemed not to affect the virulence and pathogenicity of the viruses. We do not see a trend in the composition of severe versus mild cases in our dataset that could lead to affect the virulence and pathogenicity of the viruses. Therefore, risk factors with serious conditions of HlNl influenza may be related to individual differences or promptly treatment of patients.