Pathogenic Mechanism And Etiological Characteristics Of Hand-Foot-and-Mouth Disease In Guangdong Province During 2010~2015

Objective1.Analysis of the change of enterovirus genotype and clinical symptoms in children with hand foot mouth disease in Guangdong during 2010²015;2.Objective to explore the clinical characteristics and related factors of children with hand foot mouth disease complicated with convulsion;3.To study the effects of different enteric viruses on the expression of MAVS,an antiviral signaling protein,and explore the pathogenesis.Materials/methods1.We performed fluorescence real-time RT – PCR for testing EV,EV71,CVA16 with rectal / pharyngeal swab from HFMD patients admitted by Guangzhou Women and Children’s Medical Center during 2010²015,and genotypes of non-EV71/non-CVA16 isolated from inpatients were determined by amplifying partial-lengths of VP1 and/or5’-UTR,summarizing and analyzing the clinical features.2.Case control study using 1:1pairing,123 Hand Foot and Mouth Disease with seizures in Guangzhou Women’s and Children’s Medical Center from 2015 were selected as seizure group,123 children with Hand Foot and Mouth Disease who had no convulsion were selected as the control group by the time of admission ± 3 and the age ± 2 months,the basic information,clinical symptoms,auxiliary examination results and pathogenic variables were analyzed by Logistic regression,to explore the risk factors and related intensity of convulsion in children with Hand Foot and Mouth Disease.3.Effects of different types of enterovirus on the expression of MAVS protein: The peripheral blood mononuclear cells of children with different types of enterovirus（CVA10/CVA6/EV71/other EV）were extracted,and the differential expression of MAVS protein was detected by Western Blot method.Results1.Epidemiology and genotype of enterovirus related Hand Foot and Mouth Disease in Guangdong during 2010²015A total of 7176 patients with EV related Hand Foot and Mouth Disease were admitted by Guangzhou Women and Children Medical Center From 2010 to 2015.The constituent ratio of EV71,CVA16 and other EVs was significantly different from other in different years,data show the prevalence of enterovirus genotypes changed significantly in the period of 2010²015 in Guangdong: the positive rates of EV71 and CVA16 in children with Hand Foot and Mouth Disease show a trend of decreasing,and decreased significantly,EV71-children with Hand Foot and Mouth Disease accounted for 53.3% of all children with Hand Foot and Mouth Disease in 2010 down to 8.6% in 2015,from the main cause of Hand Foot and Mouth Disease change into secondary pathogens;CVA16-children with Hand Foot and Mouth Disease accounted for 24.7% of all children with Hand Foot and Mouth Disease in 2010 down to 7.5% in 2015,from the main cause of Hand Foot and Mouth Disease change into secondary pathogens;other EVs-children with Hand Foot and Mouth Disease accounted for 19.5% up to 75.1% in 2015,from the secondary pathogens change into the main cause of Hand Foot and Mouth Disease.The number of Hand Foot and Mouth Disease in 2011 was significantly lower than that in 2010（40.1%）,and the total number of cases declined slowly in the following years.Hand Foot and Mouth Disease in Guangdong has entered the epidemic period since March,and reached the peak in May and June.There were two peaks in 2010²012,but there was only one peak in 2013²015,but it has the characteristics of higher peak value and longer platform period.In order to understand the composition and prevalence of enterovirus genotypes in children with Hand Foot and Mouth Disease in Guangdong,we sequenced the genes of enterovirus from Hand Foot and Mouth Disease in 2015,143 cases of Hand Foot and Mouth Disease were successfully classified finally,a total of 6 enteroviruses were identified.The infection rate of Coxsackievirus A 6 was highest（56.1%）,the second was Coxsackievirus A 10（20.3%）,the rest included Coxsackievirus A 2 and Coxsackievirus A8,even some children with mixed infection: CVA6 & CVA10（1 case）,CVA6 & CVA 16（3 cases）,CVA 10 & CVA 16（1 case）.2.Clinical features of Hand Foot and Mouth Disease With the prevalence of different genotypes of enterovirus,the clinical characteristics of Hand Foot and Mouth Disease have changed accordingly : the ratio of male to female was about 1.65:1;Children mainly below the age of five years old,accounting for about 97.1%,of which 0³ years old children with a total of 5877 cases,up to 81.9%;The youngest case was one month old and the oldest case was 12 years old,mean age was（2.54±1.42）years old;Fever mainly manifested as medium high fever（41.7%）,about（3.48 ± 1.83）d;Central nervous system involvement: startle（41.8%⁴5.2%,ρ =0.444）、 limb jitter（35.3%³9.5%,ρ =0.052）and vomit（20.5%²3.6%,ρ=0.075）,there was no obvious fluctuation.However the incidence of convulsion in children with Hand Foot and Mouth Disease showed an upward trend,namely 1.9%、3.7%、3.9%、10.1%、4.5% and 12.4%ρ<0.05),the difference was statistically significant.Objective to summarize the clinical features of 123 children with Hand Foot and Mouth Disease in 2015,seizure symptoms occurred in the course of firstly thirdly day,up to 5 times,average of 1.42 times,94.3% with fever,loss of consciousness（99.2%）,gaze/strabismus（100%）,cyanosis of lips（84.6%）and twitching limbs（92.8%）,average persistence 3.1 min,after remission show mental fatigue（97.8%）and poor appetite（88.6%）,some with limb movement disorder（0.81%）,all the patients were cured without sequelae.3.Analysis of related factors in children with Hand Foot and Mouth Disease There were 123 cases（15.7%）with seizure among 988 Hand Foot and Mouth Disease in 2015.In this study,62 cases of Hand Foot and Mouth Disease with seizure were selected to identified the genotypes of enterovirus,and 57 cases were successfully identified : CVA6 31 cases（50.0%）,CVA10 9 cases（14.5%）,EV71 8 cases（12.9%）,CVA16 5 cases（8.1%）and CVA2 4 cases（6.5%）.The result of paired test about the seizure group and the control group show that: the blood ammonia of seizure group is higher than control group（t=4.98,ρ< 0.01）;the Ca²⁺（t=-4.92,ρ<0.01）and the Mg²⁺（t=-2.32,ρ <0.02）of seizure group were less than the control group,all the differences were statistically significant.Multivariate conditional logistic regression analysis show: compare with < 1.25 mmol/L Ca²⁺ group,the risk of seizure in 1.25¹.30mmol/L Ca²⁺ group（OR = 0.41;95% CI = 0.36⁰.51）and > 1.30 mmol/L Ca²⁺ group（OR =0.23,95% CI = 0.18⁰.30）was significantly reduced.Compare with < 0.7mmol/L Mg²⁺group,the risk of seizure in 0.70¹.00 mmol/L（OR = 0.23,95% CI=0.001⁰.117）or > 1.00 mmol/L Mg²⁺group（OR =0.003,95% CI = 0.000⁰.066）was significantly reduced.The result of CSF test about 31 children with seizure show half patient（15/31）existence abnormal : 4 case WB increased,4 cases Glu reduced,3 cases Glu increased,2 cases chloride increased or reduced.4.Enterovirus can cause degradation of MAVS protein Compared with normal children,the expression of MAVS in children with hand,foot and mouth disease was significantly reduced,and there were differences in the degree of cleavage of MAVS.Conclusion:1.Significant changes in the genotype of enterovirus related Hand Foot and Mouth Disease occurred in Guangdong during 2010²015,EV71 and CVA16 from dominant enterovirus genotype to minor enterovirus genotype,CVA6 and CVA10 from minor enterovirus became dominant enterovirus,and various genotype of enterovirus prevalent in the same time.2.Hand Foot and Mouth Disease in children with central nervous system symptoms include vomiting,trembling limb etc in Guangdong during 2010²015,but there was no significant change trend.However,seizure gradually increased,it may be associated with the CVA6 pandemic.3.CVA6 is the main pathogen of Hand Foot and Mouth Disease,low serum Ca²⁺ or serum Mg²⁺ are independent risk factors for seizures in children with Hand Foot and Mouth Disease,even the lower serum Ca²⁺ or serum Mg²⁺,the higher the risk of convulsion in children with Hand Foot and Mouth Disease.4.Enterovirus can inhibit the RLRs signal transduction pathway,escape the innate immunity of the host,and the occurrence and development of severe hand foot and mouth disease may be associated with the expression of MAVS,the less expression of MAVS,the more severe hand foot and mouth disease.