Epidemiological Characteristics Of Children With Hand-foot And Mouth Disease In Shanghai And The Early Laboratory Diagnose Of EV71Infection

Objective:Retrospectively analyzed the clinical features and epidemiology of hospitalized children with hand-foot and mouth disease (HFMD) during2009and2010in Shanghai to investigate some risk factors with fatal cases and the molecular epidemiological characteristic. Establish the RT-LAMP (reverse-transcription and loop-mediated isothermal amplification) technology to detect enterovirus71from clinical patients with HFMD.Methods:A total of3208HFMD patients in Shanghai were analyzed from2009to2010, and437clinical specimens were collected for the determination of causative pathogens. Seven of the isolated EV71strains were sequenced and phylogenetically analyzed. Exploit the software of Primerexplorer and manually search primer groups for RT-LAMP detecting EV71, and then make it comparison with the efficacy of detecting EV71by regular nested RT-PCR.Results:A total of3208patients were enrolled with the ratio of male to female was about1.8:1.2458patients were categorized into stage1;726patients of stage2;24patients of stage3with17in2009and7in2010.7patients of stage3died, with the fatality29.2%, which was more higher than in stage2(P<0.01). The children aged between2months10days to12years7months with onset median age of25months. Among them,62.4%patients aged between1to4years. Rashes (99.2%), fever (88.3%), vomiting (25.4%), cough (22.5%) and myoclonus jerk (10.2%) were the most frequent symptoms occurred, while vomiting and myoclonus jerk can most indicate the central nervous system involving. The mean peak body temperature and the duration of fever in children of stage3was (39.61±0.46)℃and (5.0±1.73) d respectively, which indicated significant difference when compared to stage2and1. The average values of white-blood-cell counts and blood glucose in severe patients were (15.33±8.18)×109/L and (8.63±3.51)mmol/L. They were higher when compared to those in stage2and1(P<0.05). But there was no significant difference when come to C-reaction protein or cerebrospinal fluid white-blood-cell counts.182patients were enrolled for MRI study during the acute stage with37(37/182,20.3%) presented abnormal. Among them, most frequent findings are hyperintense lesions varied in brain stem (11cases).The widespread outbreak of HFMD in Shanghai, was caused predominantly by EV71(86.5%), and partially by Coxsackievirus A16(CA16)(6.9%). The high incidence of mixed infections with EV71and CA16(17.6%of the total CA16-infected cases)has never been observed. Phylogenetical analysis on the basis of VP1gene sequences revealed that the EV71strains that circulated in Shanghai belong to the C4subgenotype. The phylogenetical dendrogram of the complete genome indicated that these Shanghai strains could be grouped into two clusters, one of which possibly originated from other cities in China. The other cluster was similar to isolates in Korea and Malaysia.68clinical fecal specimens from HFMD patients were detected by RT-LAMP and RT-PCR, and results showed that92.6%(63/68) of specimens detected by RT-LAMP was EV71positive, while73.5%(50/68) of it was EV71positive by RT-PCR. RT-LAMP showed higher sensitivity than RT-PCR, and both methods did not find false positive. However, the EV71negative samples detected by RT-LAMP showed80%(1/5) accuracy, which was higher than that of detected by RT-PCR only with the accuracy of22.2%(4/18).Conclusion:The epidemic of HFMD has some correlation with the area, season, health condition of the family and gender of the children. Children under4years especially from rural area are susceptible to the HFMD infection. Frequent vomiting or myoclonus jerk may indicate the central nervous system involving. But persistent high fever may indicate severe tendency. Some laboratory examinations can help us find the fatal cases at an early time.The subgenotype C4of EV71was the major causative agent of the HFMD outbreak in Shanghai, which could be grouped into two clusters. The high incidence of mixed infections with EV71and CA16was also observed.RT-LAMP technology applied to EV71detection is more sensitive than RT-PCR and the negative results from RT-LAMP were more reliable than that of RT-PCR. The ways to determine the EV71-positive or negative results were also more convenient and easier by naked eye or single staining. Consequently, it is a more promising technology for application in the small county institutes of medical service.