Pathogen Composition Causing Hand Foot And Mouth Disease(HFMD) And Seroepidemiology Of Enterovirus71Infection In Shanghai, China

Objective Hand foot and mouth disease (HFMD) is a globally common infec-tious disease caused by enterovirus in young children, particularly in those less than5years old. In this study, we aimed to gain insight into the main compositions of patho-gens and their pathogenic characteristics by analyzing enterovirus genotypes in pa-tients with HFMD. On the other hand, the seroepideimology of EV71infections was assessed by detecting EV71specific neutralizing antibodies from HFMD patients and non-HFMD individuals respectively. This study will enhance our understanding in the area of HFMD, and provide basic data and evidence for prevention and therapy of HFMD.Methods1. From May2010to April2011, a total of290stool specimens were collected from patients suffering from HFMD who were hospitalized at children’s Hospital of Fudan University, Shanghai, China. Enterovirus was detected by nested reverse transcripted PCR (RT-PCR), and then directly genotyped by sequencing the PCR products. Phylogenetic analysis of the nucleotides of VP1partial gene was con-ducted with MEGA4.1. Clinical data of all the patients were collected and difference of clinical manifestations were compared among groups infected by different enterovirus genotypes.2. A total of95blood specimens from HFMD patients hospitalized at Children’s Hospital of Fudan University were obtained from July2010to January2011. During the same period,164blood specimens from children without HFMD and23serums from maternities at1week after delivery at Obstetrics&Gynecology Hospital of Fudan University were collected. EV71specific neutralizing antibody in serums was determined using microneutralization assays.3. All the data were analyzed by SPSS17.0statistical software.Results1. Molecular characterization of pathogens and their pathogenic cha-racteristics were as following. Of the290samples tested,277were positive (95.5%). Except that8specimens were untypable, a total of nine different genotypes were iden-tified, including EV71(63.8%,185/290), CA10(9.0%,26/290), CA6(8.3%,24/290), CA16(6.9%,20/290), CA12(2.4%,7/290), CA4(1.4%,4/290), CA14(0.3%,1/290), Echo6(0.3%,1/290), and HEV-C(0.3%,1/290). Only EV71and CA16were detected in patients over6years old, however at least6different genotypes could be seen in the other4age groups. Phylogenetic analysis indicated that EV71strains belonged to C4a subtype and CA16was identified to be B type. CA6was closely related to strains de-tected previously in Japan, Taiwan and China, and CA10, CA12, CA4, CA14were phyogentically close to the strains circulating in China. The age of all the HFMD cases ranged from6to153months (31.6±19.2M), and63.1%(183/290) of them were boys. Up to49%patients had complications like encephalitis, meningoencephalitis or me-ningitis. The patients infected with EV71had an older age (33.8±20.3M vs27.8±16.3M, P<0.05), a longer hospital stay (4.1±2.4d vs3.2±1.1d, P<0.05) and a higher rate of complications (57.8%vs33.3%, P<0.001). The hospitalization days and the probability of complications in CA6, CA10and CA16infection groups were sig-nificantly lower than that in EV71infection group (P<0.05). CA12infection group had a minimum age, and most likely have a higher risk of complications compared with other non-EV71infection groups.2. Seroepidemiology of EV71infections was as following. The EV71seropreva-lence in non-HFMD individuals was51.8%and there is no statistical difference be-tween genders. EV71seroprevalence in infants<6M was64.3%(18/28) and dropped to28.6%(8/28) in children of1-2years old, then it stably increased with age and reached76.5%(26/34) after6years of old, close to the high levels in the maternities. These individuals had a low median titers of EV71neutralizing antibody (1:8) and similar levels between infants less than6months and the maternities. EV71antibody titers decreased to the lowest in children of12-23M but increased slowly and peaked after6years of age. The EV71neutralizing antibody seroprevalence in HFMD patients was35.8%which was significantly lower than that in non-HFMD children (P<0.05). The seroprevalence of male patients was significantly higher than the females (43.1%vs20%, P<0.05). HFMD patients had much lower levels of anti-EV71neutralizing antibody than the non-HFMD people, however they showed similar trends changing with ages, and the lowest seroprevalence and antibody levels were found in24-47M age group.Conclusions Besides EV71and CA16, CA10, CA6and CA12were the other three prevalent enterovirus genotypes that caused HFMD during the year of2010-2011 in Shanghai. EV71infection has emerged as the most important pathogen causing se-vere HFMD, while the relationship of CA12with the severity of HFMD deserved fur-ther attention. The levels of EV71neutralizing antibodies were low in children, espe-cially those of1-2Y age group. The lower seroprevalence and neutralizing antibody titers in HFMD patients compared with that in non-HFMD children of the same age group were possibly one of the major reasons leading to HFMD.