Identification And Epidemic Characteristic Of Enterovirus Inducing HFMD In Lu'an

Objectives Throat swabs and vesicle swabs will be taken from HFMD in epidemic area, they were detected by RT-PCR and virus isolation; Compare with the results of different samples and methods, it will provide data of electing fit samples and methods; To sequence the VP1 gene of EV71 isolated, it'll be analyzed the nucleotide identity with other strains at home and abroad. At the same time it will provide information on molecular epidemiology of EV71 local strain in Lu'an.Methods (1) Collecting 22 clinical samples of throat and vesicle swabs from patients of HFMD or them associated with central nervous system involvement. Throat and vesicle swabs were inoculated into the RD and HEp-2 cell, we got virus identified by microneutralization test and RT-PCR when CPE occurred. At the same time the samples were centrifugaled, the sediments were detected by RT-PCR. The use of statistical methods for the two specimens and two methods to compare the positive rate, give us provide reference for the selection of methods and specimen in practical work.(2) As well as the complete VP1 gene sequences for 4 strains of EV71 isolated in Lu'an were determined and analyzed. A phylogenetic was constructed by comparison of the sequences with other EV71 strains isolated from Shenzhen, Wuhan, Taiwan, Fuyang, as well as Korea,Singpore, Malaysia and the United States. Genotype and subgenotype of EV71 isolated Lu'an were analyzed.(3) Positive patients of virus isolation and RT-PCR were analyzed by children's age, sex, time of onset, fever, skin rash, with or without clinical manifestations of neurological complications. Results (1) Six strains of enteroviruses were isolated from 22 specimens of throat swabs and vesicular swabs, the positive rate was 27.3%. The enteroviruses confirmed by neutralization test, RT-PCR and the VP1 sequence determination are EV71. Molecular analyses of 4 strains of EV71 by sequencing the VP1 differed from BrCr-ts by 89%~96%. In contrast, it exhibited a nucleotide variability of 74%~ 82% when compared with EV71 isolated respectively genotype B and C. It shows epidemic strains of HFMD in Lu'an belong to genotype A, differed from genotypes of EV71 isolated in the other regions of our mainland. Genotype A wasn't reported till the date in our country.(2) We collected 22 clinical samples including throat and vesicular swabs from 16 HFMD patients, but 16 specimens were determined by RT-PCR and 22 were done by viral culture. The positive rates of RT-PCR and viral culture were 62.5% (10/16) and 27.3% (6 / 22) respectively, the two methods have significant difference(χ2=6.35,P<0.05). Because RT-PCR method has its own limitations, can not rule out the possibility of false positive, and viral culture is "gold standard" for detection of EV71, so we selected detection method of EV71 in accordance with the actual conditions.(3)Through patients confirmed as EV71 infection by RT-PCR and virus isolation, accounting for 57.1% (8 / 14), male and female ratio was 5:3. From the age point of view, RT-PCR detection of positive cases in addition to two cases of age over 4 years (separately for the 5-year-old and 7 years old), the rest of children are all less than 4 years old, accounting for 75% (6 / 8). Children with HFMD mainly appear in early May. 8 cases of EV71 infection were caused HFMD with hand, foot, hip and knee in skin rash or herpes varying degrees, one of cases of encephalitis have mild symptoms. 6 cases had be fever, and 3 cases of patients were fever prior to rash, and 2 were rash before fever, but 1 was fever and rash simultaneously. Conclusion (1)This study showed that EV71 are pathogens of HFMD in Lu'an region in 2008, and nucleotide sequences of VP1 of EV71 were amplificated by RT-PCR before the sequences were determined. This showed epidemic strains of EV71 were genotype A in Lu'an. As genotype A of EV71 occurred in the mainland of China, and it had not prevailed all over world for many years, we must pay close attention to. (2) Because throat swab specimens have higher detectable rate than other specimen in RT-PCR and virus isolation for detection of EV71, it requires carrying out RT-PCR and virus isolation, at least throat swab should be collected in order to improve detection rate.(3) EV71 can cause not only HFMD but also complication of central nervous system. Genotype A of EV71 were isolated in Lu'an, it hasn't been isolated since 1970. EV71 strains isolated in the other mainland of China belonged to genotype C, which put forward a new topic in prevention of HFMD in our country. 8 cases of EV71 infection were caused HFMD with hand, foot, hip and knee in skin rash or herpes varying degrees, one of cases of encephalitis have mild symptoms. 6 cases had be fever, and 3 cases of patients were fever prior to rash, and 2 were rash before fever, but 1 was fever and rash simultaneously.