| Objective:1 To master the infection of clinically diagnosis cases with HFMD and the severe cases with EV71 in Hebei Province to provide scientific evidence for the epidemiogical investigation and the prevention and control of HFMD.2 To analyze the genotype to provide scientific evidence for adopting targeted measures for prevention and control and improving the data of genotypes and subgenotypes in china,monitoring the epidemic strains in hebei province to lay a good foundation for the future vaccine development.Methods:1 According to epidemiological investigation,specimens were collected,including throat swab specimens,vesicle fluid specimens,stool specimens,cerebrospinal fluid specimens and serum specimens from clinically diagnosis cases with HFMD.2 Two specific primer pairs for EV and EV71 were designed respectively according to the conservative gene sequences of 5′-UTR of EV and the VP1 gene sequences of EV71,specimens from clinically diagnosis cases with HFMD were detected by RT-PCR,and PCR products were identified by agarose gel electrophoresis. 3 EV71 was isolated with RD cells from EV71 positive specimens from laboratorially diagnosed cases with HFMD; specimens with CPE appearance were detected by RT-PCR assay with the primer pairs for EV71, and 18 positive EV71 strains were selected as representative strains of Hebei Province.4 The VP1 of 18 EV71 representative strains were amplificatied by RT-PCR assay with specific primer pairs for VP1 entire gene sequences of EV71,and then sequenced the purified PCR amplification products to perform nucleotide sequence homology analysis and construction of the phylogenetic tree with DNASTAR software for analyzing the gene subtype and its variation of the EV71 representative strains in Hebei province.Results:1 1005 specimens from clinically diagnosis cases with HFMD were detected by RT-PCR,of which 594 were EV positive with EV positive detection rate 59.1%;of which 293 specimens were EV71 positive with EV71 positive detection rate 29.2%.138 vesicle fluid specimens were detected by RT-PCR,of which 59 specimens were EV71 positive with EV71 positive detection rate 42.8%;7 stool specimens were detected by RT-PCR,of which 2 were EV71 positive with EV71 positive detection rate 28.6%;840 throat swab specimens were detected by RT-PCR,of which 232 were EV71 positive with EV71 positive detection rate 27.6%;there was no EV71 positive detected in cerebrospinal fluid specimens and serum specimens. 2 EV71 was detected from specimens from clinically diagnosis cases with HFMD collected at different times,and the time distribution of its acquisition was as follows:time distribution from the throad swab specimens was 0-10 days and the median of time distribution was 2 days;that of from the vesicle fluid specimens was 0-11 days,and the median was 2 days;that of from the stool specimens was 3-12 days,and the median was 3 days.3 EV71 was isolated with RD cells from 201 EV71 positive specimens from laboratorially diagnosed cases with HFMD,and specimens with CPE appearance were tested by RT-PCR,of which 74 was EV71 positive cases,and separation rate was 36.8%.95 stool specimens were inoculated,and 38 were isolated with separation rate 40.0%;70 throat swab specimens were inoculated,and 19 were isolated with separation rate 27.1%;31 vesicle fluid specimens were inoculated,17 were isolated with separation rate 54.8%;5 cerebrospinal fluid specimens were inoculated without EV71 were isolationed.4 522 laboratorially diagnosis cases with HFMD,244 cases were positive for EV71 infected with positive infection rate 46.7%.217 cases were mild,accounting for 88.9% of the total EV71 infected cases,with the symptoms of fever and rash;27 were severe,accounting for 11.1%,with the complications of nervous system and respiratory system.5 522 laboratorially diagnosis cases with HFMD,482 cases were mild,of which 217 were positive for EV71 infected with infection rate 45.0%;40 were severe cases,of which 27 were positive for EV71 infected with infection rate 67.5%.6 In the 244 EV71 positive cases,156 were male, account- ting for 63.9%,and 88 were female,accounting for 36.1%, male: female 1.77:1,with 46.4% infection rate for male and 47.3% of that for female.The youngest one infected with EV71 was four months old and the oldest one was 17 years old.An EV71 case was in 0-year-old group,accounting for 0.4% of the total EV71 infected cases;167 was in 1～3-year-old group,accounting for 68.4%;65 was in 4～6-year-old group, accounting for 26.7%;11 was in 7～17-year- group,accounting for 4.5%.In occupation distribution,children in the diaspora hit the most,accounting for 78.81% of the total EV71 infected cases,preschools for children accounting for 16.02%,students accounting for 4.83%,and the others for 0.34%.7 The homogeneity of VP1 nucleotide sequence of the 18 selected EV71 representative strains from Hebei Province was between 95.2%～100% themselves;with isolates from Taiwan was between 88.8%～90%;with isolates from Zhejiang,Shaihai, Chongqin,Shenzhen,Beijing and Fuyang was between 94.6%～99.4%;with the representative isolates of A and B genotypes was between 81.4%～83.1%,83.8%～85.1% respectively;with the representative isolates of C genotypes was between 88.1%～98.4%;with the representative isolates of C1,C2,C3 and C4 sub-genotypes was between 89.5%～91.4%,89.1%～90.2%, 88.1%～89%,96.3%～98.4% respectively. 8 The EV71 strains selected had been confirmed to be a C4 sub-genotypes as same as the strains isolated from mainland China by analyzing of the EV71 genetic characteristics and phylogenetic tree.Conclusion:1 Specimens from clinically diagnosis cases were tested by RT-PCR,and RT-PCR can be used for early rapid diagnosis of HFMD pathogen for its higher feasibility and specificity.2 EV71 positive detection rate was different according to their different specimens.Positive detection rate of EV71 from vesicle fluid specimens was higher than that from throat swab specimens.It is advisable to collect vesicle fluid specimens, throat swab specimens and stool specimens at incidence of one week.3 EV71 was isolated by RD cells from vesicle fluid specimens,throat swab specimens and stool specimens,with vesicle fluid specimens the higher separation rate than the rate of throat swab specimens.4 EV71,being one of the pathogens for HFMD, can cause mild and severe cases with HFMD,mainly mild.EV71 infection rate of severe cases with HFMD was higher than that of mild cases with HFMD.5 Both men and women can be lead to HFMD symptoms for being infected by EV71,there is no difference between the two rates;Childen in 1～3-year-old group were the major infectious target of EV71,followed by 4～6-year-old group, mainly children in the diaspora.6 The EV71 strains selected had been confirmed to be a C4 sub-genotypes as same as the strains isolated from mainland China by analyzing of the EV71 genetic characteristics and phylogenetic tree. |
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