The Etiological Diagnosis Of The Outbreak Of Hand, Foot, And Mouth Disease In Shandong, 2007

An outbreak of Hand,Foot,and Mouth Disease(HFMD) happened from March to May,2007 in Linyi,Shandong Province.First HFMD case in Linyi was reported on 15^th March.The cases increased from then on,apparently in May.The total number of cases reached 1718 on 31^st May.Most of the cases had mild clinical manifestations, with vesicular lesions on hand,feet,mouth,and sometimes buttocks.Lesions in the mouth were often ulcerated,and fever was also common.Three cases developed severe clinical complications to death.HFMD is an usual infectious disease of children,caused by many types of human enteroviruses(EV).Laboratory diagnosis is essential for identification of the etiological agent.To identify the agent associated with this outbreak,clinical specimens from HFMD cases collected during the outbreak was presented to the National Lab for Poliomyelitis to make laboratory diagnosis on 26^th May.A total of 182 specimens collected from 101 cases,including specimens of stool, CSF,throat swab,serum,and lung aspirates,virus isolation was performed on,using RD and HEp-2.58 enteroviruses were isolated from 52(51%) of the 101 HFMD patients.Serotype was identified by RT-PCR and sequence analysis.Isolates were 50 enterovirus 71(EV71) from 44 cases,7 Coxsackievirus A16(CV-A16) from 7 cases, and 1 Coxsackievirus B2 from 1 case.33 pairs of acute and convalescent serum were collected from 33 HFMD cases to titer the specific neutralization antibody.Micro-neutralization tests were undertaken in RD cell line,attacked by EV71 and CV-A16 isolated in this outbreak,also ECHO3 isolated by Shandong centers for disease control and prevention(CDC) during early period of the outbreak.The results indicated that 67%(22/33) got acute infection of EV71,30%(6/20) of echovirus 3(ECHO3),9%(2/22) of CV-A16.There were 7 cases infected at least two kinds of EV recently according to virus isolation and neutralization test.IgM antibody to Echovirus and coxsackievirus were tested separately in 34 serum and 6 CSF specimens collected 3 days later after disease onset.The results also suggested that many types EV were epidemic during this HFMD outbreak.In conclusion,64 out of 103(62%) HFMD cases were identified to get EV infection according to virus isolation and serum neutralization test.53%(55/103) cases got EV71 infection(11 cases identified by neutralization test),so EV71 was identified as the major etiological cause of this HFMD outbreak,also other types EV (ECHO3,CV-A16,and CV-B2) were epidemic at the same time.According to molecular epidemiological analysis,EV71 epidemic during this outbreak had the largest nucleotide sequence identity(96.1%) with C4 subgenogroup and clustered with C4 references in phylogenetic tree(95 bootstrap),belonged to C4 subgenogroup.Excluding SD/07/521-09/stool and SD/07/523-11/swab,other isolates had a larger nucleotide sequence identity,98.6%～100%.Nucleotide sequence identity between SD/07/521-09/stool and SD/07/523-ll/swab was 96.0%,between this two and other isolates was 96.0%～97.1%and 95.3%～96.1%separately.So,EV71 belonged to different clades were circulating during this outbreak.