String-Powered Microfluidic Chip Integrating Heparin-Mediated One-Pot RT-RPA/CRISPR-Cas12a for Multiplex Detection of HFMD Viruses.

Objective To study the clinical characteristics of children with hand, foot and mouth disease (HFMD) in Maternal & Child Health Hospital of Guangxi Zhuang Autonomous Region. Methods A total of 45 793 children with HFMD in Maternal & Child Health Hospital of Guangxi Zhuang Autonomous Region from January 1, 2013 to December 31, 2016, were selected as research subjects. After obtaining informed consents of all cases, throat swabs or (and) herpes fluid samples were collected from 363 mild type of HFMD cases, and throat swabs samples from 732 severe type of HFMD cases to detect enterovirus (EV) including EV71, coxsackievirus (Cox)A16 and other types of EV. Clinical data of all children with HFMD were analyzed retrospectively, and the characteristics of HFMD cases in our hospital from 2013 to 2016 were analyzed statistically including: ①rates of severe type and fatality rates of HFMD in each year; ②distribution characteristics of HFMD including months, ages, gender, crowd and region, and rates of severe type of HFMD of these factors; ③etiologic characteristics of HFMD cases. The study protocol was approved by the Ethical Review Board of Investigation in Human Being of Maternal & Child Health Hospital of Guangxi Zhuang Autonomous Region. Results ①Among 45 793 HFMD children, 98.40% (45 061/45 793) of them were with mild type of HFMD, and 1.60%(732/45 793) were with severe type of HFMD. Fatality rate of children with severe type of HFMD was 1.64%(12/732). In 2013, rate of severe type of HFMD (1.13%) was the lowest compared to that of 2014 (1.85%), 2015 (1.69%) and 2016 (1.65%), respectively, and all the differences were statistically significant (P 1-3 years old. 94.96% of HFMD cases were age of children ≤5 years old.Rates of severe type of HFMD in children ≤1 year old or >1-3 years old were higher than those of children >3-5 years old and >5 years old, respectively, and all the differences were statistically significant (P<0.05). ④The proportion of male children with HFMD was 61.47% (28 149/45 793). Rate of male with severe type of HFMD (1.80%, 508/28 149) was higher than that of female (1.27%, 224/17 644), and the difference was statistically significant (χ2=19.745, P<0.001). HFMD proportion of scattered children, children in kindergarten and students were 73.11%, 25.56% and 1.33%, respectively, and rates of severe type of HFMD were 1.92%, 0.67% and 1.97%, respectively. HFMD constituent ratio of living in Nanning city, other counties in Nanning and other districts outside Nanning were 91.48%, 8.31% and 0.21%, respectively, and rates of severe type of HFMD children were 1.09%, 7.25% and 1.03%, respectively. ⑤EV positive rate of mild type of HFMD cases (87.05%, 316/363) was higher than that of severe type of HFMD cases (78.28%, 573/732), and the difference was statistically significant (χ2=12.272, P<0.001). The preponderant EV from mild type of HFMD cases were CoxA16 in 2013, EV71 in 2017, other types of EV in 2015 and CoxA16 in 2016, and the preponderant EV from severe type of HFMD cases were other types of EV in 2013, EV71 in 2014, other types of EV in 2015 and other types of EV in 2016. There were 12 death cases, and 83.34% (10/12) of them were tested positive of EV71. Conclusions From 2013 to 2016 in our hospital, the most HFMD cases occurred annually from April to June, or from August to October, boys were more affected of HFMD than that of girls, and high risk populations were in scattered children≤ 5 years old in Nanning city. The mild type of HFMD cases were mainly caused by Cox A16, and the severe type of HFMD cases were mainly caused by other types of EV. Key words: Hand, foot and mouth disease; Coxsackievirus infections; Enterovirus A, human; Child

Adenovirus in EV71-associated hand, foot, and mouth disease

Hand, foot, and mouth disease (HFMD) is a common childhood infection that causes a substantial disease burden in the Asia-Pacific region. Various climate variables, such as humidity and temperature, have been associated with HFMD. However, few studies have assessed the impact of PM10 on childhood HFMD. This study investigated the association between PM10 and HFMD. We fitted a standard distributed lag non-linear model to investigate the temporal lagged relationship between PM10 and HFMD, and then further assessed whether this relationship varied by gender and pathogen. Between 2011 and 2015, a total of 122,564 HFMD cases under 15 years of age were reported in Chengdu. The PM10-HFMD associations were shown to be non-linear in all subgroups, with the peak at 101–218 μg/m3. Male children were more sensitive to PM10 effects. For pathogen-specific relative risks, we found that the risk estimates were generally higher in cases of CVA16 infection. Our study provides evidence that PM10 increases the risk of HFMD. Authorities and parents should be fully aware of the impact of PM10 on childhood HFMD. Furthermore, appropriate protective measures should be taken to reduce risks.

Objective To investigate the significance of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in serum and cerebrospinal fluid for evaluation of severe hand, foot, and mouth disease (HFMD) complicated with neurogenic pulmonary edema (NPE). Methods A total of 140 patients diagnosed with HFMD in Henan Children′s Hospital were enrolled and divided into three groups including mild group, severe HFMD group without NPE , severe HFMD group with NPE .These severe HFMD patients were also divided into survival group and death group according to the 28-day prognosis. Meanwhile, 50 age-matched healthy children were selected as controls. Serum MMP-9 and TIMP-1 levels were measured in all enrolled children. At the same time, MMP-9, TIMP-1 and ratio of MMP-9/TIMP-1 in cerebrospinal fluid were measured in the severe HFMD group with and without NPE. Quantitative data were compared using one-way analysis of variance, and means comparisons between samples were conducted using LSD- t test. Results Among 140 children with HFMD, 66 were in mild group, 42 in severe HFMD without NPE group, and 32 in severe HFMD with NPE group. And 50 healthy children were in control group. After 28 days, 14 cases died in severe HFMD groups. MMP-9, TIMP-1 and MMP-9/TIMP-1 in serum of severe HFMD group with NPE increased significantly greater than those in the other three groups (F=269.356, 121.301 and 101.502, respectively, all P <0.05). MMP-9, TIMP-1 and MMP-9/TIMP-1 in cerebrospinal fluid of severe HFMD group with NPE were (57.24±8.92) μg/L, (35.26±8.14) μg/L and (1.66±0.23) μg/L, respectively, while those in cerebrospinal fluid of severe HFMD group without NPE were (30.57±3.89) μg/L, (26.25±0.32) μg/L and (1.17±0.61) μg/L, respectively. The differences between the two groups were all statistically significant (t=62.485, 37.680 and 169.387, respectively, all P<0.01). MMP-9, TIMP-1 and MMP-9/TIMP-1 in serum and cerebrospinal fluid of death group increased significantly greater than those in survival group, the difference were statistically significant (all P<0.01). The maximum area under curve (AUC) was reached when the MMP9/TIMP-1 ratio in cerebrospinal fluid was 0.890 (95% CI: 0.801-0.978). Conclusions MMP-9 and TIMP-1 may be involved in the pathogenesis of HFMD complicated with NPE. The detection of MMP-9 and TIMP-1 levels may be beneficial for the early diagnosis of severe HFMD with NPE. The imbalance of MMP-9/TIMP-1 ratio can be used as one of the predictors of severe HFMD combined with NPE. Key words: Matrix metalloproteinase 9; Hand, foot and mouth disease; Pulmonary edema; TIMP-1

Objective To analyze the epidemiological characteristics of the severe cases of hand, foot and mouth disease (HFMD) in Danzhou and to provide a scientific evidence for the prevention of severe HFMD. Methods Descriptive epidemiological analysis was used to analyze the characteristics of severe cases of HFMD occurred from 2010 to 2014. Results A total of 18 960 cases of HFMD were reported in Danzhou City from 2010 to 2014. The death rate (annual deaths/1000 persons) was 0.13%. One hundred and eighty-eight cases (0.99%) were diagnosed as severe HFMD with a male to female ratio of 2.4∶1 and 96.28% of them were scattered inhabiting children. Six cases were died from severe HFMD and all of them were under 2 years of age. In total 87.77% of the severe cases were occurred in children under 2 years of age. The severe cases were mainly occurred in June and July. Children form the countryside showed higher rates of severe HFMD than those from cities and towns. No significant differences in the time between the initial diagnosis and treatment for children with HFMD in countryside and urban areas were found. However, the differences in the duration from initial diagnosis to severe HFMD between children in countryside and urban areas were statistically significant (M-W test, P<0.05). Among the 188 severe cases, 82.44% were initially diagnosed as common HFMD cases by the county-level medical institutions and 90.96% were diagnosed as severe cases by the municipal and above medical institutions. The cases positive for EV71 strains accounted for 44.15%. Conclusion Most of the severe HFMD were developed in scattered inhabiting children under 2 years of age in the countryside of Danzhou during 2010 to 2014. In order to decrease the morbidity and mortality of severe HFMD in children, it is necessary to implement health education for residents, to improve the professional skills of medical staffs in the early diagnosis of HFMD, and to strengthen etiological surveillances and warning system for HFMD. Key words: Hand, foot and mouth disease; Severe case; Epidemiology

Hand, foot, and mouth disease (HFMD) is a common childhood infection caused by human enteroviruses and is clinically characterised by fever with vesicular rash on the hands, feet, and mouth. While enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) were the major etiological agents of HFMD in India earlier, the data on recently circulating enteroviruses associated with HFMD are sparse. Here, we describe the molecular epidemiology of enteroviruses associated with HFMD in South India from 2015 to 2017. We used archived enterovirus real-time reverse transcription (RT) PCR-positive vesicle swab and/or throat swab specimens from clinically suspected HFMD cases collected from four secondary-care hospitals in South India between July 2015 and December 2017. PCR amplification and sequencing were done based on the 5’VP1, 3’VP1, VP2, or 5´NCR regions to identify enterovirus types. Genetic diversity among enteroviruses was inferred by phylogenetic analysis. Of the 107 enterovirus RNA real-time RT-PCR-positive HFMD cases, 69 (64%) were typed as CVA6, 16 (15%) were CVA16, and one (1%) was CVA10, whereas in 21 (20%) cases, the virus was not typeable by any of the methods used in the study. The majority of HFMD cases (89, 83%) were in children less than five years old, while 11 (10.3%) were in adults. 5’VP1 yielded the maximum number of enteroviruses genotyped, and phylogenetic analysis showed that the CVA6 strains belonged to subclade D3, while the subclades of CVA16 and CVA10 were B1c and D, respectively. The predominant etiological agent of HFMD in South India during 2015-2017 was CVA6, followed by CVA16 and CVA10.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00705-022-05561-0.

Objective To study the significance of level changes of serum hydrogen sulfide(H2S)and interleukin-6(IL-6) in children with hand, foot and mouth disease(HFMD). Methods Nity-two cases with HFMD were enrolled and divided into severe HFMD group(48 cases) and common HFMD group(44 cases). Forty-six healthy children were enrolled as healthy control group.The serum H2S and IL-6 were detected. Results Acute phase: compared to the healthy control group[H2S(55.76±7.80) μmol/L, IL-6(61.31±13.43) ng/L], the level of serum H2S significantly reduced and the level of IL-6 significantly increased in severe HFMD group[H2S(21.72±7.52) μmol/L; IL-6(131.33±17.82) ng/L] and common HFMD group[H2S(31.86±8.41) μmol/L; IL-6(95.48±15.07) ng/L](P<0.01), and there was significantly difference between the severe HFMD group and common HFMD group(P<0.01). Recovery phase: compared to the healthy control group, the serum H2S level[(34.54±13.21) μmol/L] was lower and IL-6[(92.73±15.25) ng/L]was higher in severe HFMD group(P<0.01). The serum H2S level was negatively correlated with IL-6 in severe HFMD group and common HFMD group(r=-0.31, P<0.01; r=-0.45, P<0.01). Conclusion The serum H2S and IL-6 participate in the pathological process of HFMD, and the level change can be used as one of indicators of early diagnosis. Key words: Hand, foot and mouth disease; Hydrogen sulfide; Interleukin-6,

To understand the epidemiological profiles of hand, foot and mouth disease (HFMD) and the major enteroviruses causing the epidemics of HFMD in Shanghai from 2010 to 2014. The city-wide surveillance data between 2010 and 2014 were used to analyze the epidemiologic characteristics of the HFMD outbreaks in Shanghai. The annual incidence of HFMD was estimated based on the 2010 Shanghai Census data. From 2010 to 2014, the reported HFMD cases were 41 080, 37 323, 51 172, 42 198, and 65 018, respectively; the severe cases (case-severity ratio) were 469 (1.14%), 456 (1.22%), 318 (0.62%), 104 (0.25%) and 248 (0.38%), respectively. Based on Shanghai census data by the end of 2010, the attack rates of HFMD in Shanghai were 0.16%-0.28% in the entire population. In terms of the proportion of HFMD cases and severe cases in the specific population, male accounted for 59.62%-61.48% and 62.26%-73.08%, migrant population accounted for 51.86%-62.40% and 72.01%-80.38%; children aged 1.0-1.9 years comprised the highest proportion, up to 22.70%-27.00% and 32.08%-36.40%. HFMD peaked from April to July, in parallel with the peak circulation of enterovirus (EV) 71, and a small peak usually occurred in autumn and winter. All the critically severe and fatal cases were caused by EV71. The detection rates of EV71 and Coxsackievirus A (CA) 16 were 73.08%-88.09% and 1.12%-2.90% in severe HFMD cases, 19.75%-48.74% and 2.02%-23.69% in uncomplicated inpatients, and 16.78%-40.08% and 8.36%-33.39% in mild community cases, respectively. The detection rates of CA6 and CA10 in the mild community cases in 2014 were 18.38% and 1.43%, respectively. In 2013 non-EV71 and non-CA16 enteroviruses comprised 74.86% in the community cases. The annual HFMD outbreaks occurred in Shanghai during 2010-2014. Children under 5 years of age, migrant population and male were the major susceptible population. EV71 and CA16 were the predominant pathogens causing the epidemics of HFMD except in 2013, and CA6 was prevalent in the community cases in 2014. The major peak season of HFMD usually overlapped with the peak of EV71 circulation and the majority of severe HFMD cases were associated with EV71 infection.

Objective To investigate the clinical implication of the neutrophil to lymphocyte ratio (NLR) in predicting the severity of hand, foot and mouth disease (HFMD). Methods From May 2008 to December 2017, a total of 1 593 children with HFMD were collected from The Second Affiliated Hospital of Xi’an Jiaotong Univesity and Xi’an Children’s Hospital. According to the degree of illness, they were divided into mild HFMD group (929 cases) and severe HFMD group (664 cases). The best critical value of neutrophil/lymphocyte ratio (NLR) for predicting the severity of HFMD was calculated by receiver operating characteristic curve (ROC curve). According to NLR, 1 593 children were divided into high NLR group and low NLR group. Multivariate two-classification Logistic regression analysis was used to analyze the correlation between the factors and the severity of children with HFMD. Results Fever, hyperpyrexia, nervous system symptom, circulatory system symptom, leukocyte (WBC), NLR, procalcitonin (PCT), blood sugar, enterovirus 71 IgM antibody (EV71-IgM) positive and infection with Epstein-Barr virus (EBV) were all with significant differences in severe HFMD group compared with those of mild HFMD group (all P 39 ℃), positive EV71-IgM, complicated with EBV infection, blood glucose > 8.3 mmol/L, PCT > 0.1 ng/ml and NLR > 1.59 were all risk factors for severe HFMD. The ratio of odds ratio (OR) were 1.557, 5.124, 3.533, 7.323, 1.727 and 2.031, respectively (all P < 0.01). Conclusions NLR > 1.59 is closely related to the severity of HFMD, which has important predictive value. NLR is one of the clinical indexes to guide the clinical decision and treatment of children with HFMD. Key words: Hand, foot and mouth disease; Neutrophil-to-lymphocyte ratio; Severity; Risk factor

Objective To analyze the risk factors of hand, foot and mouth disease (HFMD) in scattered children in the northern area of Guangzhou, and provide scientific evidences for HFMD control and prevention. Methods The questionnaire survey of risk factors was conducted in scattered children from May to July 2017 in the northern part of Guangzhou city by case-control study. The study included 462 confirmed HFMD cases and 485 healthy children. Non-conditional logistic regression was used for multiple factors analysis. Results Multiple factors analysis showed that family having other children in kindergarten (OR=2.621, 95% CI: 1.829-3.755) , children had contacted with HFMD cases within 2 weeks before the disease onset (OR=29.285, 95% CI: 11.421-75.092) , had contacted with herpangina cases (OR=8.223, 95%CI: 3.275-20.647) , had played in public facilities (OR=2.488, 95% CI: 1.821-3.399) , had received transfusion (OR=4.216, 95% CI: 1.510-11.770) , and had visited emergency department in the hospital (OR=4.203 95%CI: 2.003-8.816) were risk factors of HFMD. Children living in city area (OR=0.369, 95%CI: 0.027-0.502) , permanent resident population (OR=0.022, 95%CI: 0.003-0.167) , and children had contacted with flu cases within 2 weeks before the disease onset (OR=8.657, 95% CI: 0.364-0.817) were protective factors of HFMD. Conclusions Children who contact with HFMD or herpangina cases, play in public facilities, visit emergency department in the hospital or receive infusion are risk factors of HFMD. Home quarantine after onset of HFMD and herpangina; cleaning and disinfection in public places and medical institutions; health education of HFMD prevention in county and migrant population are key prevention measures of HFMD in scattered children. Key words: Hand, foot and mouth disease; Scattered children; Risk factors

To determine the circular DNA level of patients with hand foot and mouth disease (HFMD) and evaluate its potential clinical value. Venous blood in 30 healthy children and 78 patients with HFMD within 3 days of onset of illness and convalescent period was collected. The level of plasma circular DNA was detected by duplex real-time polymerase chain reaction assay. Blood sugar, high-sensitive CRP(hs-CRP) and leucocyte were also detected. The level of circular DNA in control group was (6.57 +/- 4.67) ng/ml. The level of circular DNA in ordinary and severe HFMD patients was (11.51 +/- 7.75) ng/ml and (20.59 +/- 10.67) ng/ml before treatment, respectively. The levels of circular DNA in ordinary and severe HFMD patients were significantly higher than that in control group (P = 0.021; 0.000); the level of circular DNA in severe HFMD patients was significantly higher than that in ordinary HFMD patients (P = 0.011). The level of circular DNA in severe HFMD patients after treatment were significantly lower than that before treatment (P = 0.033). The level of circular DNA before treatment and after treatment in ordinary HFMD patients had no significant difference. The levels of blood sugar and hs-CRP in severe HFMD patients were higher than those in ordinary before treatment (P = 0.045; 0.011). The levels of blood sugar and hs-CRP before treatment and after treatment in ordinary HFMD patients had no significant change. There was significantly positive correlation between the level of circular DNA and that of hs-CRP in HFMD patient (P = 0.021), but there was no correlation between the level of circular DNA and that of blood sugar and leucocyte. The level of circular DNA not only become an early identification marker of severe HFMD patients, but also become monitoring marker of effect of treatment.

Hand, foot, and mouth disease (HFMD) is a highly contagious disease that primarily affects children under five years of age. It is mainly caused by serotypes of Enterovirus A (EVA): EVA71, Coxsackievirus A types 6 (CVA6), 10 (CVA10), and 16 (CVA16). Despite being highly prevalent in Japan and other countries in the Asia-Pacific region, few studies have investigated HFMD pathogens in wastewater. The present study aimed to develop a highly sensitive and broadly reactive quantitative polymerase chain reaction (qPCR) assay of dominant serotype CVA6, to revise previously developed CVA6, CVA10, and CVA16 assays, and to test these assays in wastewater samples from Yamanashi Prefecture, Japan. The new-CVA6 qPCR assay was developed with maximal nucleotide percent identity among CVA6 isolates from Japan. The new-CVA6 and revised assays were highly sensitive and had the ability to quantify respective positive controls at levels as low as 1 copy/μL. Among the 53 grab influent samples collected between March 2022 and March 2023, EVA71, CVA10, and CVA16 RNA were not detected in any samples, whereas the new-CVA6 assay could detect CVA6 RNA in 38 % (20/53) of samples. CVA6 RNA was detected at a significantly higher concentration in the summer season (3.3 ± 0.8 log10 copies/L; 79 % (11/14)) than in autumn (2.7 ± 0.6 log10 copies/L; 69 % (9/13)). The seasonal trend of CVA6 RNA detection in wastewater aligned with the trend of HFMD case reports in the catchment of the wastewater treatment plant. This is the first study to report the detection and seasonal trends of the EVA serotypes associated with HFMD in wastewater samples in Japan. It provides evidence that wastewater-based epidemiology is applicable even for diseases that are prevalent only in specific population groups.

Hand, foot, and mouth disease (HFMD), a common childhood infection caused by enterovirus, poses a serious public health concern in China. We collected and analyzed epidemiological data on 62,133 HFMD cases in Shenyang City, Liaoning Province, from 2013 to 2023. The average annual incidence was 76.12 per 100,000 person-years; 99.45% of cases were mild, while 0.55% were severe. Only one patient died. HFMD infections peaked annually in July. Children in kindergartens and scattered children accounted for 44.6% and 42.2% of cases, respectively. Real-time RT-PCR detection of enteroviruses in 5534 patient samples revealed 3780 positives, of which 25.1% were CVA16-positive. Positives were randomly sampled, yielding 240 VP1 sequences of CVA16. Phylogenetic tree results showed that all VP1 sequences belonged to the B1 sub-genogroup. However, the sub-genogroup prevalence varied over time: from 2013 to 2014 and 2019 to 2021, the predominant sub-genogroup was B1a, while it was B1b from 2015 to 2018. Further phylogenetic analyses showed substantial divergence between B1a branches in CVA16, suggesting possible turnover of the B1a sub-genogroup in CVA16 due to evolution. This study provides epidemiological data on HFMD in Shenyang, and provides a phylogenetic analysis of CVA16, offering a theoretical basis for preventing and controlling HFMD in Shenyang City.

Objective To discuss the change trends of pathogen of severe hand, foot and mouth disease(HFMD) in Chaoshan area during 2011 to 2015. Methods All 1 410 throat swabs of cases who were diagnosed as HFMD were collected from children hospitalized in our hospital during May 2011 to August 2015.Enterovirus were detected by nest RT-PCR, and the results of these positive cases diagnosed as severe HFMD were analyzed. Results (1) There were 216 positive cases(67.29%, 216/321) diagnosed as severe HFMD, including 53.70%(116/216)enterovirus 71(EV71), 19.91%(43/216)coxsackievirus A16(CA16), 12.04%(26/216)CA6, 8.80%(19/216)CA10, 3.24%(7/216)CA4, 0.93%(2/216)coxsackievirus B5, 0.46%(1/216)enteric cytopathogenic human orphan virus and 0.93%(2/216)unclassified samples were unclassified to species.(2)Five cases of critical HFMD were all caused by EV71.(3)The EV71 positive samples were given priority to severe cases(51.79%, 116/224)and the non EV71 positive samples were given priority to mild cases(82.08%, 458/558), the difference was statistically significant(χ2=91.68, P<0.001). (4)The change trends of severe HFMD year by year were consistent with the change trends of EV71 composition, and were highly correlated(Rs=0.9, P=0.037). (5) Severe HFMD caused by non EV71 virus gradually increased. Conclusion Severe HFMD in Chaoshan area during 2011 to 2015 were mainly caused by EV71, non EV71 viruses including CA16, CA6, CA10, CA4, coxsackievirus B5, enteric cytopathogenic human orphan virus 6 could also develop to severe HFMD.The composition ratio of severe HFMD increased accordingly in the year of EV71 as the dominant pathogen.The proportion of severe HFMD caused by non EV71 virus gradually increased after 2013 year. Key words: Severe hand, foot and mouth disease; Pathogen; Enterovirus 71; Non enterovirus 71 virus

Objective To investigate the association of serum 25-(OH) D levels with severity of children with enterovirus 71 (EV71) related hand, foot and mouth disease (HFMD) and to predict the clinical value of 25-(OH) D in severe HFMD of type EV71. Methods A total of 255 children with EV71 HFMD admitted to Xi’an Children’s Hospital from January 2017 to December 2018 were selected as case group, including 150 mild cases (mild group) and 105 severe cases (severe group). Meanwhile, 50 healthy children who were randomly selected for physical examination in the same period were selected as control group. Serum 25-(OH) D levels were measured by chemiluminescence. The clinical data of case groups were collected by a retrospective research method, a unified survey and analysis tables were designed, the data of possible influencing factors were recorded. The univariate and multivariate Logistic regression analysis on factors that may affect the conversion of mild HFMDinto severe HFMD were done. The risk factors of mild EV71 type HFMD into severe EV71 type HFMD and the relationship between serum 25-(OH) D level and severe EV71 related HFMD were investigated, respectively. Results The levels of 25-(OH) D between cases with EV71 HFMD and control group [(35.66 ± 11.60) ng/mlvs. (49.63 ± 8.86) ng/ml] were significantly different (t= 8.062,P 15 × 109/L (χ2= 26.616, P 8.3 mmol/L (χ2= 26.616, P 8.3 mmol/L (OR=2.682, P= 0.028), WBC > 15 × 109/L (OR= 2.090,P= 0.019) and 25-(OH) D < 30.88 ng/ml (OR= 3.792,P< 0.001) were all independent risk factors for severe illness, with significant differences. Conclusions 25-(OH) D <30.88 ng/ml is significantly related to the severity of EV71 HFMD and has important predictive value, which is an early warning factor for severity of EV71 HFMD. Key words: Enterovirus 71; Hand, foot and mouth disease; 25-(OH) D; Risk factor