Epidemiology And Clinical Feature Of Hand,Foot,and Mouth Disease In Shenzhen,China,2009-2013

BackgroundHand,foot,and mouth disease(HFMD)is caused by human enteroviruses,mainly infecting 3 year old young children.HFMD can occur throughout the year,especially in summer.Patients infected with different enteroviruses show varied clinical symptom,similar clinical symptoms may be caused by different enteroviruses.HFMD is a self-limited and recovers in 7 days characterized by fever and vesicular eruptions on the hand,feet,mouth,buttock and knee.However,some severe cases deteriorate quicklly,it may appear severe nervous system complication such as aseptic encephalitis,meningitis,brainstem encephalitis,acute flaccid paralysis(AFP),neurological pulmonary edema or even some patients dead,survivals may leave sequela.EV71(Enterovirus 71)infection tends to cause severe HFMD and death,other enteroviruses infection few occur.EV71 has replaced poliovirus become the most remarkable enterovirus.Recently,CA16(Coxsackievirus A16)and other enteroviruses could also result in severe nervous system complications,and CA6 caused atypical,widespread manifestions worldwide.However,further detailed study about the relationship between pathogen of HFMD and clinical feature was few,it needs further study.HFMD is caused by enterovirus which is RNA virus,Family Picornaviridae.EV consists of A,B,C,D grounp,including poliovirus,coxsackievirus,echovirus and new enterovirus.There are about 20 kinds of enteroviruses that can cause HFMD,it includes coxsackievirus A 7,9,10,16,coxsackievirus B 2,4,5,encho 13,19,30 and EV71,of which EV71 and CA16 are the most common serotypes.Renctly,CA6 outbreak was reported in Siganpore,Taiwan,Japan,America and Britain.What is more,according to the last five-year surveilence data from Shenzhen Center for Disease Control and Prevention(CDC),the proportion of CA6 is increading.The study about HFMD etiology mainly focuses on total HFMD,few about severe and mild HFMD etiology.Shenzhen is an immigrant metropolis connecting mainland China and Hong Kong,located on the southern coast of China.The city has a high population density and mobility,as well as a subtropical environment.These features make Shenzhen a HFMD-prone area.Due to the severity of HFMD epidemic,it needs to monitor the changes of pathogen of HFMD,especially the severe HFMD.MethodClinical specimens were collected from patients suffering from HFMD,along with clinical report forms during 2009-2013.The clinical report forms were obtained with information on patient demographics and clinical findings.Specimens were extracted RNA,then,Real-time RT-PCR was used to screen EV71,CA16,CA6,CA10,CA4 and enterovirus.Specimens that were positive for EV71 and CA16 were randomly selected to amplify the entre VP1 gene for phylogenetic analysis.Specimens that were positive for EVs but negative for both EV71 and CA16,three rounds of independent reverse transcription-seminested PCR(RT-snPCR)based on the VP1 gene was performed to detect other unidentified EVs-positive specimens.Visible PCR products were subjected to nucleotide sequencing.Multiple sequence alignments were performed with ClustalW.Phylogenetic analysis of entreVP1 gene of EV71 and CA6 was conducted using the maximum-likelihood method with the Kimura two-parameter model of nucleotide substitution in MEGA 5.05.We conducted a study to describe the etiological spectrum of mild and severe HFMD,and the relationship between pathogen of HFMD and clinical feature.Result1.Etiological spectrum of HFMD in Shenzhen,2009-2013From 2009 to 2013,a total of 2,299 clinical specimens of HFMD patients were collected including 494 severe case and 1805 mild cases.1,862 of the specimens were tested positive for EVs,which was comprised of 1,464 mild and 398 severe cases of HFMD.For the severe HFMD cases,the number of EV71,CA16 and CA6 was 331(83.17%),19(4.77%),and 25(6.28%),respectively;for the mild HFMD cases,the number of EV71,CA16 and CA6 was 522(35.66%),224(15.30%),and 387(26.43%),respectivelyFor the HFMD in Shenzhen,2009-2013?Female were more than male,Approximately 99.5%of cases occurred in children under the age of 5 years,and more than three-quarters occurred in children less than 3 years.Two seasonal peaks were observed in both the total and mild HFMD cases,with the first peak occurring in May and the second peak between July and September.EV71 infection mainly occurred in summer,EV71 infection mainly occurred during autumn and winter.There was a dramatic change of the etiological spectrum of total HFMD during 2009-2013.The proportion of EV71 decreased.The proportion of EV71 was more than half during 2009-20111,but it dropped to 31.87%and 18.35%in 2012 and 2013,respectively.The proportion of CA16 fluctuated,it decreased in general.However,the proportion of CA6 detected in patient samples steadily increased each year,surpassing the CA16 in 2010 and EV71 in 2013,and become the most dominant causative agent of HFMD.The etiological spectrum of severe HFMD during 2009-2013 did not change according.EV71 still was the main agent of severe HFMD.The proportion of EV71 was always beyond 70%.Overall,the proportion of CA16 was decreasing.It was 10.00%in 2009,but fell to 1.85%in 2010,and 3.55%in 2011.The proportion of CA6 was rising except for 2010.It was only 1.85%in 2010,but quiclly climbed to 17.65%in 2013,with a large increase of 15.80%.The etiological spectrum of mild HFMD during 2009-2013 was similar with the total HFMD.The proportion of EV71 was 46.38,55.46%and 46.90%,respectively,but rapidly declined to 24.84%in 2012 and 15.68%in 2013.The proportion of CA16 decreased from 32.27%in 2009 to 4.59%in 2013.The proportion of CA6 was quiklly increasing with 5.00-8.00%each year,surpassing the CA16 in 2010 and EV71 in 2012.The difference between CA6 and EV71 was up to 31.89 in 2013.2.Sequence comparisons and phylogenetic analysis of EV71 and CA6The sequences of EV71 were highly nucleotide and amino acids homologous within and between mild and severe strains infection.The nucleotide homology of the VP1 gene of these strains was calculated to be 92.8-100.0%.The amino acids sequence homology was 96.2-100.0%,with high homology observed in severe HFMD patients.The sequences of CA6 were highly nucleotide and amino acids homologous within and between mild and severe strains infection.The nucleotide and amino acid identities of the VP1 gene from mild CA6 strains were 73.2-100.0%,and the amino acid identity of the VP1 gene from mild and severe CA6 strains were 78.3-100.0%,with high homology observed in severe HFMD patients.Irrespective of whether the strain was isolated from mild or severe patients,all EV71 strains collected in this study were closely grouped into genotype C4,which displayed a close genetic relationship with Shandong strain,Fujian strains,Jiangsu strains,Lanzhou strains,Yunnan strains,but had a far genetic relationship with orther countries strains.Phylogenetic analysis of CA6 sequences showed that all the CA6 strains were segregated into six different clusters that all included China strains.The CA6 strains isolated from both mild and severe HFMD patients were dispersed into two sub-clusters(cluster A and cluster C).Some CA6 strains isolated from mild HFMD cases,as well as other regions of China,formed two different sub-clusters(cluster B and cluster D).3.The relationship between pathogen of HFMD and clinical featureOur study found the mean age of patients with CA6 or CA10 infection was younger than those with EV71 or CA16 infection.Fever,vesicles on the hand,foot and mouth were the most common.Approximately less than 10.0%of patients displayed with vesicles on the knee or elbow,coughing and rhinorrfea.Patients infected with CA10 were more likely to develop fever and pharyngalgia;and less likely to develop vesicles on the hand,foot,knee and buttock.Vesicles on the mouth more frequently occurred in the patients infected with CA6,but less in those infected with EV71.Myoclonic jerk and vomiting were the most frequent severe symptom.Severe nervous system complication was mainly caused by EV71;meanwhile,CA6,CA16,CA10,CA4,and E-9 could also cause aseptic encephalitis.The most common severe nervous system complication was aseptic encephalitis,following by meningitis and brainstem encephalitis.Acute flaccid paralysis and neurological pulmonary edema were few.During the five-year study period,20 patients died as a result of EV71 infection in Shenzehn.The most common symptoms were fever,rash and myoclonic jerk and vomiting,all fatal cases had fever.None of these patients had vesicles on elbow.Some fatal cases did not display rash or vesicle of only have subtle vesicles on hand and foot.Nervous system complications were noted in eighr patients.Of these seven patients,four had brainstem encephalitis including one complicated neurogenic pulmonary edema,three patients had aseptic encephalitis,and one had neurogenic pulmonary edema.During 2009-2013,10 patients co-infect EV71 and CA16.9 patients appeared viscle on hand,foot,and mouth,none of these cases had elbow viscle,rhinorrfea and coughing,two cases developed into severe HFMD,but both of them had not severe nervous system complications,and only had myoclonic jerk and vomiting.There was no fatal case.During 2009-2013,a total of 18 serotype enteroviru B,46 cases was detected.Most of these patients had fever,viscle on hand,foot,and mouth,but other location viscle,rhinorrfea and coughing was few.Only one or two CB2,CB5,E-9 and E-11 infecton developed into severe casws,one E-9 infection had aseptic encephalitis.There was no fatal case.We isolated CA24 belong to enterovirus C from two mild HFMD patients.They included one female and one male,and both 1 year old.Both of them were mild and had viscle on hand,foot,and mouth,one patients had fever,button rash and pharyngitis.Conclusion1.During 2009-2013,the etiological spectrum of total and mild HFMD changed dramatically.CA6 has replaced EV71 and CA16 become the major agent.However,the etiological spectrum of severe HFMD did not change according.EV71 was still the dominant agent,but CA6 has increased.2.The sequences of EV71 and CA6 were highly nucleotide and amino acids homologous within and between mild and severe strains infection.It could not distinguish the mild and severe strains isolated from EV71 and CA6 infection.3.Vesicles on the hand,foot and mouth were the most common symptom;myoclonic jerk and vomiting were the most frequent severe symptom.Patients infected with different enteroviruses show varied clinical symptom.Severe nervous system complication was mainly caused by EV71,but other enterovviruses could also cause.