Hand Foot and Mouth Disease Due to Enterovirus 71 in Malaysia

Hand foot and mouth disease is a febrile sickness complex characterized by cutaneous eruption (exanthem) on the palms and soles with simultaneous occurrence of muco-cutanous vesiculo-ulcerative lesions (enanthem) affecting the mouth. The illness is caused by a number of enteroviruses with coxsackievirus A16 and enterovirus 71 as the main causative agents. Human enterovirus 71 (EV71) belongs to the species Human enterovirus A under the genus Enterovirus within the family Picornaviridae. EV71 has been associa...     

Cyclical Patterns of Hand,Foot and Mouth Disease Caused by Enterovirus A71 in Malaysia

Enterovirus A71 (EV-A71) is an important emerging pathogen causing large epidemics of hand, foot and mouth disease (HFMD) in children. In Malaysia, since the first EV-A71 epidemic in 1997, recurrent cyclical epidemics have occurred every 2–3 years for reasons that remain unclear. We hypothesize that this cyclical pattern is due to changes in population immunity in children (measured as seroprevalence). Neutralizing antibody titers against EV-A71 were measured in 2,141 residual serum samples collected from children ≤12 years old between 1995 and 2012 to determine the seroprevalence of EV-A71. Reported national HFMD incidence was highest in children <2 years, and decreased with age; in support of this, EV-A71 seroprevalence was significantly associated with age, indicating greater susceptibility in younger children. EV-A71 epidemics are also characterized by peaks of increased genetic diversity, often with genotype changes. Cross-sectional time series analysis was used to model the association between EV-A71 epidemic periods and EV-A71 seroprevalence adjusting for age and climatic variables (temperature, rainfall, rain days and ultraviolet radiance). A 10% increase in absolute monthly EV-A71 seroprevalence was associated with a 45% higher odds of an epidemic (adjusted odds ratio, aOR1.45; 95% CI 1.24–1.69; P<0.001). Every 10% decrease in seroprevalence between preceding and current months was associated with a 16% higher odds of an epidemic (aOR = 1.16; CI 1.01–1.34 P<0.034). In summary, the 2–3 year cyclical pattern of EV-A71 epidemics in Malaysia is mainly due to the fall of population immunity accompanying the accumulation of susceptible children between epidemics. This study will impact the future planning, timing and target populations for vaccine programs.     

Immunodominant IgM and IgG Epitopes Recognized by Antibodies Induced in Enterovirus A71-Associated Hand,Foot and Mouth Disease Patients

Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4), and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic in silico were synthesized and used for the characterization of EV-A71 linear B-cell epitopes. In total, we identified 22 IgM and four IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142–156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope. PEP23, mapped to VP1 41–55, was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies, but epitopes against non-structural proteins were also detected. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates.     

致手足口病肠道病毒的病原学研究进展

手足口病是一种多病原体引起的儿童常见传染病,少数患者会进展为重症甚至死亡,对公共卫生造成巨大影响.目前国内虽已研制出EV71灭活疫苗并上市使用,对其它病毒所致手足口病并无交叉保护作用,并不能从总体上降低手足口病的发病率,在应对多种病原体的疫苗问世前,对手足口病病原体的基因进化监测、流行特征和病原学研究仍然是防控手足口病的重要措施.本文就手足口病在病原学方面的研究进展进行综述,为该病的科学防控提供有价值的信息.     

Comparative Study of the Cytokine/Chemokine Response in Children with Differing Disease Severity in Enterovirus 71-Induced Hand,Foot, and Mouth Disease

Background

Enterovirus 71 (EV71) infection can lead to a rapidly progressing, life-threatening, and severe neurological disease in young children, including the development of human hand, foot, and mouth disease (HFMD). This study aims to further characterize the specific immunological features in EV71–mediated HFMD patients presenting with differing degrees of disease severity.

Methodology

Comprehensive cytokine and chemokine expression were broadly evaluated by cytokine antibody array in EV71–infected patients hospitalized for HFMD compared to Coxsackievirus A16-infected patients and age-matched healthy controls. More detailed analysis using Luminex-based cytokine bead array was performed in EV71–infected patients stratified into diverse clinic outcomes. Additionally, immune cell frequencies in peripheral blood and EV71–specific antibodies in plasma were also examined.

Principal Findings

Expression of several cytokines and chemokines were significantly increased in plasma from EV71–infected patients compared to healthy controls, which further indicated that: (1) GM-CSF, MIP-1β, IL-2, IL-33, and IL-23 secretion was elevated in patients who rapidly developed disease and presented with uncomplicated neurological damage; (2) G-CSF and MCP-1 were distinguishably secreted in EV71 infected very severe patients presenting with acute respiratory failure; (3) IP-10, MCP-1, IL-6, IL-8, and G-CSF levels were much higher in cerebrospinal fluid than in plasma from patients with neurological damage; (4) FACS analysis revealed that the frequency of CD19⁺HLADR⁺ mature B cells dynamically changed over time during the course of hospitalization and was accompanied by dramatically increased EV71–specific antibodies. Our data provide a panoramic view of specific immune mediator and cellular immune responses of HFMD and may provide useful immunological profiles for monitoring the progress of EV71–induced fatal neurological symptoms with acute respiratory failure.     

免疫反应及相关细胞因子在EV71 相关手足口病 合并肺水肿中的作用

目的:探讨细胞、体液免疫应答以及相关的细胞因子在EV71相关手足口病合并肺水肿中的作用。方法:将90例经鉴定为EV71感染患儿分为手足口病合并肺水肿组38例,手足口病无并发症组52例,并设查体健康对照组28例。ELISA法检测90例EV71感染引起的手足口病患儿血清IL-6、IL-10、TNF-α及IFN-γ含量;采用流式细胞仪对血液中CD3+T、CD4+及CD8+T细胞百分比进行检测;采用免疫比浊法对90例EV71感染引起的手足口病患儿血清免疫球蛋白(IgG、IgA及IgM)及补体C3、C4含量进行检测。结果:手足口病合并肺水肿组患儿血清IL-6、IL-10及IFN-γ含量明显升高,同时伴随CD4+及CD8+T细胞百分比下降;手足口病合并肺水肿组,无并发症组及健康对照组患儿血清IgM分别为1.85±0.73,1.46±0.790和0.88±0.39,三组之间差别具有统计学意义(F=14.967,P<0.05)。而IgG与IgA在三组之间无明显变化(X2=5.535,P>0.05;F=1.988,P>0.05);手足口病患儿血清C3及C4含量均明显低于健康对照组(F=46.079;62.794,P<0.05)。结论:由IL-10及IFN-γ的异常释放而引起的广泛的中枢及外周神经系统炎症反应和T淋巴细胞衰竭是引起EV71合并肺水肿病程进展的重要原因;在EV71感染后引发的手足口病进程中存在IgM的大量释放,且伴随补体C3、C4的消耗。     

ICAM-1K469E 位点A 等位基因可降低EV71 手足口病发生率

目的:研究ICAM-1基因K469E位点、MCP-1A2518G位点基因多态性及sICAM-1、MCP-1在血清中表达水平与EV71手足口病的关系,探讨EV71型手足口病的遗传易感因素。方法:运用限制性片段长度多态性-聚合酶链反应(PCR-RFLP)检测急性期EV71感染阳性的手足口病患儿和正常儿童中ICAM-1K469E位点及MCP-1A2518G位点碱基变异情况,同时采用双夹心抗体法(ELISA)检测血清sICAM-l和MCP-1水平。结果:EV71手足口病组患儿血清中sICAM-l和MCP-1水平均显著高于正常对照组(P均<0.01)。EV71手足口病组ICAM-1K469E位点中,A等位基因的频率显著低于对照组(x2=6.897,P<0.01)。EV71手足口病组患儿MCP-1基因型分布、等位基因频率与对照组比较均无统计学意义(P>0.05)。结论:sICAM-1表达水平和其基因K469E位点多态性与EV71手足口病有关,A等位基因可降低EV71手足口病发生率。MCP-1表达水平与EV71手足口病感染有关,但MCP-1A-2518G位点基因多态性与EV71手足口病感染无关。     

Genotypes of the Enterovirus Causing Hand Foot and Mouth Disease in Shanghai,China, 2012-2013

Sporadic HFMD (hand foot and mouth disease, HFMD) cases and outbreaks caused by etiologic agents other than EV71 and CA16 have increased globally. We conducted this study to investigate the prevalence and genetic characteristics of enteroviruses, especially the non-EV71 and non-CA16 enteroviruses, causing HFMD in Shanghai. Clinical specimens were collected from patients with a diagnosis of HFMD. A partial length of VP1 was amplified with RT-PCR and subjected to direct sequencing. Phylogenetic analyses were performed using MEGA 5.0. The ages of the HFMD cases ranged from 3 to 96 months, and the male/female ratio was 1.41. The median hospital stay was 2.96 days. Up to 18.0% of patients had neurologic system complications such as encephalitis, meningoencephalitis or meningitis. Of the 480 samples, 417 were positive for enterovirus (86.9%) with RT-PCR. A total of 13 enterovirus genotypes were identified. The most frequent genotypes were CA6 (31.9%), EV71 (30.6%), CA16 (8.8%) and CA10 (7.5%). Infections with CA6, EV71, CA16 and CA10 were prevalent throughout the years of study, while the proportion of CA6 notably increased from Sep. 2012 to Dec. 2013. Phylogenetic analyses showed that EV71 strains belonged to the C4a subgenogroup and CA16 was identified as B1b subgenogroup. The CA6 strains were assigned to genogroup F, whereas the CA10 strains were assigned to genogroup D. Patients infected with CA6 were typically younger, had a shorter hospital stay and had a lower incidence of neurologic system complications when compared to patients infected with EV71. Our study demonstrates that the enterovirus genotypes causing HFMD were diversified, and there was an increasing prevalence of the non-EV71 and non-CA16 enteroviruses from 2012 to 2013. CA6 was the most predominant pathogen causing HFMD from Sep. 2012 to Dec. 2013, and it often caused relatively mild HFMD symptoms. Most severe HFMD cases were associated with EV71 infection.     

Co-Circulation and Genomic Recombination of Coxsackievirus A16 and Enterovirus 71 during a Large Outbreak of Hand,Foot, and Mouth Disease in Central China

A total of 1844 patients with hand, foot, and mouth disease (HFMD), most of them were children of age 1–3-year-old, in Central China were hospitalized from 2011 to 2012. Among them, 422 were infected with coxsackievirus A16 (CVA16), 334 were infected with enterovirus 71 (EV71), 38 were co-infected with EV71 and CVA16, and 35 were infected with other enteroviruses. Molecular epidemiology analysis revealed that EV71 and CVA16 were detected year-round, but EV71 circulated mainly in July and CVA16 circulated predominantly in November, and incidence of HFMD was reduced in January and February and increased in March. Clinical data showed that hyperglycemia and neurologic complications were significantly higher in EV71-infected patients, while upper respiratory tract infection and C-reactive protein were significantly higher in CVA16-associated patients. 124 EV71 and 80 CVA16 strains were isolated, among them 56 and 68 EV71 strains were C4a and C4b, while 25 and 55 CVA16 strains were B1a and B1b, respectively. Similarity plots and bootscan analyses based on entire genomic sequences revealed that the three C4a sub-genotype EV71 strains were recombinant with C4b sub-genotype EV71 in 2B–2C region, and the three CVA16 strains were recombinant with EV71 in 2A–2B region. Thus, CVA16 and EV71 were the major causative agents in a large HFMD outbreak in Central China. HFMD incidence was high for children among household contact and was detected year-round, but outbreak was seasonal dependent. CVA16 B1b and EV71 C4b reemerged and caused a large epidemic in China after a quiet period of many years. Moreover, EV71 and CVA16 were co-circulated during the outbreak, which may have contributed to the genomic recombination between the pathogens. It should gain more attention as there may be an upward trend in co-circulation of the two pathogens globally and the new role recombination plays in the emergence of new enterovirus variants.     

Hand,Foot, and Mouth Disease in China: Modeling Epidemic Dynamics of Enterovirus Serotypes and Implications for Vaccination

Background

Hand, foot, and mouth disease (HFMD) is a common childhood illness caused by serotypes of the Enterovirus A species in the genus Enterovirus of the Picornaviridae family. The disease has had a substantial burden throughout East and Southeast Asia over the past 15 y. China reported 9 million cases of HFMD between 2008 and 2013, with the two serotypes Enterovirus A71 (EV-A71) and Coxsackievirus A16 (CV-A16) being responsible for the majority of these cases. Three recent phase 3 clinical trials showed that inactivated monovalent EV-A71 vaccines manufactured in China were highly efficacious against HFMD associated with EV-A71, but offered no protection against HFMD caused by CV-A16. To better inform vaccination policy, we used mathematical models to evaluate the effect of prospective vaccination against EV-A71-associated HFMD and the potential risk of serotype replacement by CV-A16. We also extended the model to address the co-circulation, and implications for vaccination, of additional non-EV-A71, non-CV-A16 serotypes of enterovirus.

Methods and Findings

Weekly reports of HFMD incidence from 31 provinces in Mainland China from 1 January 2009 to 31 December 2013 were used to fit multi-serotype time series susceptible–infected–recovered (TSIR) epidemic models. We obtained good model fit for the two-serotype TSIR with cross-protection, capturing the seasonality and geographic heterogeneity of province-level transmission, with strong correlation between the observed and simulated epidemic series. The national estimate of the basic reproduction number, R ₀, weighted by provincial population size, was 26.63 for EV-A71 (interquartile range [IQR]: 23.14, 30.40) and 27.13 for CV-A16 (IQR: 23.15, 31.34), with considerable variation between provinces (however, predictions about the overall impact of vaccination were robust to this variation). EV-A71 incidence was projected to decrease monotonically with higher coverage rates of EV-A71 vaccination. Across provinces, CV-A16 incidence in the post-EV-A71-vaccination period remained either comparable to or only slightly increased from levels prior to vaccination. The duration and strength of cross-protection following infection with EV-A71 or CV-A16 was estimated to be 9.95 wk (95% confidence interval [CI]: 3.31, 23.40) in 68% of the population (95% CI: 37%, 96%). Our predictions are limited by the necessarily short and under-sampled time series and the possible circulation of unidentified serotypes, but, nonetheless, sensitivity analyses indicate that our results are robust in predicting that the vaccine should drastically reduce incidence of EV-A71 without a substantial competitive release of CV-A16.

Conclusions

The ability of our models to capture the observed epidemic cycles suggests that herd immunity is driving the epidemic dynamics caused by the multiple serotypes of enterovirus. Our results predict that the EV-A71 and CV-A16 serotypes provide a temporary immunizing effect against each other. Achieving high coverage rates of EV-A71 vaccination would be necessary to eliminate the ongoing transmission of EV-A71, but serotype replacement by CV-A16 following EV-A71 vaccination is likely to be transient and minor compared to the corresponding reduction in the burden of EV-A71-associated HFMD. Therefore, a mass EV-A71 vaccination program of infants and young children should provide significant benefits in terms of a reduction in overall HFMD burden.     

Clinical Features for Mild Hand,Foot and Mouth Disease in China

Background

Mild hand, foot and mouth disease (HFMD) is at a critical stage owing to its ease of communicability and a higher risk of developing severe complications and death. Clinical diagnosis of mild HFMD was made by the presenting symptoms and signs (symptoms in brief) alone. We aim to evaluate the frequencies of symptoms in a retrospective case series study.

Methods

We collected epidemiological, demographic, clinical, and laboratory data from outpatient and inpatient settings on the clinical data warehouse system. We principally described the frequencies of symptoms of mild HFMD. Correlations between symptoms with laboratory-confirmed cases were then analyzed.

Results

The clinical data warehouse system included 3649 probable cases, between 2010 and 2012, of which 956 (26.20%) were laboratory confirmed. The peak incidence was identified in children 2 years of age. A total of 370 of the 956 laboratory confirmed cases (38.70%) were associated with enterovirus 71 (EV71). Logistic regression analysis adjusted for geographical variables, age, sex, month of onset, and time from onset to diagnosis showed that the clinical features constipation (P<0.0001; adjusted OR, 95%CI (2.99, 2.28–3.91)), and blisters (P<0.0001; adjusted OR, 95%CI (2.16, 1.82–2.56)) were positively correlated with the confirmed cases.

Conclusions

This is the largest case series study, including all the guideline-mentioned symptoms of mild HFMD. Our findings suggest that blisters and constipation should be considered as potential warning signs while front-line clinicians manage surges of children diagnosed with mild HFMD during a pandemic.     

EV71相关手足口病患儿T细胞及细胞因子检测及意义

目的:探讨EV71相关手足口病患儿外周血T细胞及细胞因子的水平及其意义.方法:将90例EV71感染手足口病患儿分为脑炎并发肺水肿组8例,单纯脑炎组30例,无并发症组52例.采用间接免疫荧光法检测外周血CD4+T及CD8+T细胞百分比,用双抗体夹心ABC-ELISA法检测血清IL-6、IL-10、TNF-α及IFN-γ含量.结果:并发症组CD4+及CD8+T淋巴细胞亚群的百分比相对无并发症组的降低,差异有统计学意义(F=9.304,P＜0.05;X2=26.075,P＜0.05),并发症组血清1L-10、IFN-γ和IL-6含量较无并发症组增高,差异有统计学意义(F=4.232,P＜0.05;X2=16.975,P＜0.05;X2=15.562,P＜0.05),并发症组TNF-α含量和无并发症组差异无统计学意义.结论:外周血T细胞及细胞因子均参与了EV71相关手足口病重症并发症的发生过程,在手足口病的临床诊疗中可予以参考,早期鉴别出现重症并发症的病例,及时给与相应的治疗措施,降低手足口病的病死率和致残率.     

菏泽地区2009年肠道病毒71型与其他肠道病毒所致手足口病临床差异研究

目的:探讨EV71引起小儿手足口病与其他肠道病毒所致手足口病的临床特点的差异.方法:回顾性分析2009年3月-9月菏泽地区各定点医院收治的病原学检测为阳性的320例小儿手足口病的临床资料,筛出EV71感染的手足口病患儿进行分析,其他肠道病毒所致手足口痛患儿作为对照组.结果:菏泽地区2009年抽取的病原学检测为阳性的320例患儿,肠道病毒71型引起手足口病占106例,其余为其他肠道病毒所致手足口病.(1)男女发病基本相同,发病年龄1～3岁居多,农村患儿占绝大多数.(2)EV71感染的患儿临床主要表现高热、神经系统症状、无皮疹,大多数外周血象高、血糖高,可合并心肌损害,心电图异常、部分发现胸片、脑电图及核磁异常,部分符合重症手足口病标准.(3)其他肠道病毒感染的惠儿临床症状以皮疹、低热、上感症状为主,少数患儿有精神差,基本不出现神经系统症状,少数发现外周血象高、血糖高、合并心肌损害,一般胸片不会发现异常.(4)56例出现神经系统症状的患儿收集脑脊液进行两次RT-PCR扩增,发现50例EV71阳性.结论:EV71导致的手足口病,病情凶险,易致神经损害及肺水肿,早期识别重症病例,及时救治,普及病原体的检测对提高诊治能力,加强疾病的认识,对减少致残率及死亡率很有帮助.     

Time Series Analyses of Hand,Foot and Mouth Disease Integrating Weather Variables

Background

The past decade witnessed an increment in the incidence of hand foot mouth disease (HFMD) in the Pacific Asian region; specifically, in Guangzhou China. This emphasized the requirement of an early warning system designed to allow the medical community to better prepare for outbreaks and thus minimize the number of fatalities.

Methods

Samples from 1,556 inpatients (hospitalized) and 11,004 outpatients (non-admitted) diagnosed with HFMD were collected in this study from January 2009 to October 2013. Seasonal Autoregressive Integrated Moving Average (SARIMA) model was applied to establish high predictive model for inpatients and outpatient as well as three viral serotypes (EV71, Pan-EV and CA16). To integrate climate variables in the data analyses, data from eight climate variables were simultaneously obtained during this period. Significant climate variable identified by correlation analyses was executed to improve time series modeling as external repressors.

Results

Among inpatients with HFMD, 248 (15.9%) were affected by EV71, 137 (8.8%) were affected by Pan-EV+, and 436 (28.0%) were affected by CA16. Optimal Univariate SARIMA model was identified: (2,0,3)(1,0,0)₅₂ for inpatients, (0,1,0)(0,0,2)₅₂ for outpatients as well as three serotypes (EV71, (1,0,1)(0,0,1)₅₂; CA16, (1,0,1)(0,0,0)₅₂; Pan-EV, (1,0,1)(0,0,0)₅₂). Using climate as our independent variable, precipitation (PP) was first identified to be associated with inpatients (r = 0.211, P = 0.001), CA16-serotype (r = 0.171, P = 0.007) and outpatients (r = 0.214, P = 0.01) in partial correlation analyses, and was then shown a significant lag in cross-autocorrelation analyses. However, inclusion of PP [lag -3 week] as external repressor showed a moderate impact on the predictive performance of the SARIMA model described here-in.

Conclusion

Climate patterns and HFMD incidences have been shown to be strongly correlated. The SARIMA model developed here can be a helpful tool in developing an early warning system for HFMD.