Epidemiological And Etiological Characteristics Investigation Of Hand, Foot And Mouth Disease In Zhengjiang Province

OBJECTIVE:Hand, foot, and mouth disease (HFMD) is a common childhood illness caused by enteroviruses, and HFMD was classified as a class"C" notifiable disease in 2008.The majority of cases detected had mild to moderate symptoms, most HFMD presents with fever, sores on the hands, feet, mouth and buttocks. some patients are associated with severe complications, including encephalitis, aseptic meningitis, respiratory tract infections and myocarditis. HFMD causes a more serious threat to the health of children. The purpose of this study is:1. To survey the enterovirus infection of clinically diagnosed cases of hand, foot and mouth disease from 2010 to 2013, understand the epidemiology characteristics of enterovirus infections, and define the effective factors of detection rate of enterovirus virus, for providing the scientific basis of effective prevention and control of HFMD.2. To analyze the genetic characteristic of isolated strains of various enterovirus by detecting nucleotide sequence of VP1 genes of various enterovirus, understand the molecular biological characteristics of various enterovirus and the genetic evolution of various enterovirus strains, investigate the molecular basis of variations of enterovirus, for providing the basis of variations of enterovirus.METHODS:1. The detection of clinical specimens and isolation of virus:four kinds of enterovirus of samples from clinical diagnosis cases, including enterovirus71, coxackievirus A16, coxsackievirus A6 and coxsackievirus A10, were detected by One-step quantitative RT-PCR with probe, PCR or RT-PCR. Part of positive samples of EV71、CoxA16、CoxA6 virus testing from our laboratory were inoculated into the human malignant embryonal rhabdomyoma cells (RD) for viral isolation and culture.2. molecular biological technology:isolated virus strains, were validated by the nucleic acid detection kit for EV71, CoxA16、CoxA6 virus, and were amplified by specific primers of EV7、CoxA16、CoxA6 designed by self. The other positive specimens of viral nucleic acid testing were amplified by RT-PCR, the sequences of products were then detected by Genomics Institute Corporation in Hangzhou.3. Bioinformatics analysis:biological softwares, such as DNASTAR and DANMAN, were applied for analyzing the nucleotide sequences of VP1 genes of the Cox A6 strains in Zhejiang province, with reference of the representative strains of Cox A6, and the Cox A6 sequences in other areas of mainland in China.RESULTS:1.Demographic characteristicsFrom 1 January 2010 to 31 December 2013, a total of 4218 HFMD clinical diagnosis cases were collected, among these cases,2686 were males and 1532 were females, with an average male-to-female sex ratio 1.75 (range:1.58 to 2.16). Most were in the 1-< 3 year age group (52%),93% of cases were aged< 5 years. And the majority of cases occurred in scattered children, followed by children in kindergarten.2. SeasonalityAlthough HFMD cases occurred throughout the year, an increase in case reporting over background was evident from April to June (2013 was March to June),peaks were seen in May(2010 and 2011)and June(2012 and 2013).The time distribution of the incidence of HFMD in 2010 was similar to the time distribution 2011, the peak was seen one time in a year. The second peak occurred in September in 2012 and 2013.3. Pathogen DistributionThe total detection rate of enterovirus was 71.08%.873 enterovirus nucleic acid positive samples were detected in 2010, and EV71、CoxA16 and other enteroviruses accounted for 52.0%,26.9% and 21.1% of the enterovirus nucleic acid positive samples respectively,839 enterovirus nucleic acid positive samples were detected in 2011, and EV71. CoxA16 and other enteroviruses accounted for 45.1%、30.0% and 24.9% of the enterovirus nucleic acid positive samples respectively,595 enterovirus nucleic acid positive samples were detected in 2012, and EV71、CoxA16. other enteroviruses and EV71 and CoxA16 both positive cases accounted for 40.3%、31.3%、22.5% and 5.9% of the enterovirus nucleic acid positive samples respectively, The results showed that EV71 was the predominant HFMD pathogen during 2010 to 2012, followed by CoxA16. 711 enterovirus nucleic acid positive samples were detected in 2013, and EV71、 CoxA16 and other enteroviruses accounted for 20.0%.2.8% and 77.2% of the enterovirus nucleic acid positive samples respectively, and the predominant HFMD pathogens in 2013 were the other HFMD viruses (not EV71 or CoxA16), the detection rate of EV71 and CoxA16 were decreased. We selected 267 samples which belong to the other enterovirus-positive samples in 2013 and detected the CoxA6 and CoxA10 nucleic acid, the results showed that CoxA6 accounted for the major part of these enterovirus(51.3%),CoxA10 accounted for 6.0% of the sampling.4.The enterovirus detection rate of different types of samples.From 2010 to 2013, a total of 4246 cases were collected, among these samples,1421 were throat swabs,2803 were stooland 22 were vesicle fluid. The detection rate of enterovirus in the vesicle fluid、stool and throat swabs samples were 100%、76.2% and 65.1% respectively. The detection rate of vesicle fluid was the highest, followed by stool, the detection rate of throat swab was lowest.5.15 strains of CoxA6 were isolated, and 4 strains of them were selected for detecting genetic sequence of VP1 genes, which were then used for homologous comparison by online Nucleotide BLAST analysis in the NCBI Database. The identities of the VP1 genes from the isolates were from 89.3% to 99.6%. And the referenced 34 strains from GenBank were acquired, all these sequences were aligned for phylogenetic analysis. From the generated tree, one strain named CoxA6-14-3 was found to be a far distance in the tree topology from other 3 CoxA6 strains.CONCLUSION:1.2010-2012,the predominant pathogen of clinical diagnosis cases was EV71, followed by CoxA16. and the predominant HFMD pathogens in 2013 were the other HFMD viruses (not EV71 or CoxA16), the positive rate of CoxA6 increased significantly, the predominant pathogens of HFMD were changed.2. The peaks of incidence were seen in May to June during 2010-2013, and the second peaks occurred in September in 2012 and 2013, the results suggested that outbreaks of HFMD were seasonality-dependent. HFMD cases declined in summer and winter, suggesting that virus transmission was impacted by extremes in temperature. The majority of cases occurred in scattered children who were aged<5, the incidence of male was higher than females.3.The homologous and phylogenetic analysis of CoxA6 strain demonstrated that the Cox A6 strains in Zhejiang province had some differences between each other, the identities between Cox A6-1-43 and the other three strains were below 90%, so the Cox A6-1-43 may be transmitted from other places.