Severe Hand, Foot, And Mouth Disease Patients Research Articles

A multiplex one-step fluorescence quantitative differential diagnosis method for severe hand, foot and mouth disease caused by coxsackievirus A16

Hand foot and mouth disease (HFMD) is a common childhood infectious disease which is caused by human enterovirus. The objective of this study was to develop a rapid, sensitive, and accurate method for detecting severe HFMD caused by coxsackievirus A16 (CV-A16). A closed-tube sensitive multiplex one-step reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was applied to detect CV-A16 in the early stage of severe HFMD. This assay targeted the CV-A16 structure protein VP1 to distinguish CV-A16 from other coxsackieviruses The 5’UTR region of enteric viruses was used for detecting the enterovirus and ribonuclease P (RNaseP) was adopted as the internal reference gene. The multiplex MGB probe assay system was used to detect PCR amplicons with different fluorescence reporters in the same system. The limit of detection (LOD) of the RT-qPCR assay for the CV-A16 VP1 gene was 125.893 copies/μl, for the 5′ UTR was 50.1187 copies/μl and for the RNaseP gene was 158.49 copies/μl. Furthermore, specificity analysis showed that the multiplex RT-PCR had no cross-reactivity with the influenza virus, herpangina virus and SARS-COV-2. In correlation analysis, the sensitivity of the multiplex RT-qPCR assay for CV-A16 detection was 100 % (288/288) and the specificity of the multiplex RT-qPCR assay was 99.94 % (3395/3397). The overall agreement between the multiplex RT-qPCR and the results of clinical diagnosis was 99.95 % (3683/3685) and kappa value was 0.996 (p<0.001). The entire procedure, from specimen processing to result reporting, could be completed within 1.5 hours. The one-step multiplex RT-qPCR assay for detecting CV-A16 developed in this study is a good laboratory diagnostic tool for rapid and reliable distinguished detection of CV-A16, especially for severe HFMD patients at an early stage in the disease with low virus load of CV-A16.

Local immune dysregulation and subsequent inflammatory response contribute to pulmonary edema caused by Enterovirus infection in mice.

Pulmonary edema that comes on suddenly is the leading cause of mortality in hand-foot-and-mouth disease (HFMD) patients; however, its pathogenesis is still largely unclear. A range of research suggest immunopathogenesis during the occurrence of pulmonary edema in severe HFMD patients. Herein, to investigate the potential mechanism of immune dysregulation in the development of pulmonary edema upon Enterovirus (EV) infection, we established mouse infection models for Enteroviruses (EVs) including Coxsackievirus (CV) A6, Enterovirus A71 (EVA71), and CVA2 exhibiting a high incidence of pulmonary edema. We found that EVs infection induced an immune system disorder by reducing the numbers of pulmonary and circulatory T cells, B cells, macrophages, and monocytes and increasing the numbers of lung neutrophils, myeloid-derived suppressor cells (MDSCs), and activated T cells. In addition, the concentrations of C-X-C motif chemokine ligand 1 (CXCL-1), tumor necrosis factor-alpha, monocyte chemoattractant protein-1, and interleukin 6 were increased in EV-infected lungs. Moreover, we found that EVs replication in mice lungs lead to apoptosis of lung cells and degradation of tight junction proteins. In conclusion, EVs infection likely triggered a complexed immune defense mechanism and caused dysregulation of innate immune cells (MDSCs, neutrophils, monocytes, and macrophages) and adaptive cellular immunity (B cells, T cells). This dysregulation increased the release of cytokines and other inflammatory factors from activated immune-related cells and caused lung barrier damage and pulmonary edema.

Enterovirus 71 non-structural protein 3A hijacks vacuolar protein sorting 25 to boost exosome biogenesis to facilitate viral replication.

Human enterovirus 71 (EV71) is one of the major agents of the hand, foot, and mouth disease (HFMD), and occasionally causes severe neurological complications. There is clinical evidence that EV71 infection increases the exosomes in the serum of severe HFMD patients, suggesting a role of exosomes in EV71 pathogenesis. However, the relationship between exosomes and EV71 replication remains elusive. In this study, we initially found that EV71 infection elevated exosome biogenesis in the cultured cells. Among EV71 non-structural proteins, we identified EV71 3A, but not 3B, constitutively promoted exosome secretion. In detail, EV71 3A protein interacted with vacuolar protein sorting 25 (VPS25), while knock-down of VPS25 reduced EV71 3A protein- and EV71-induced exosome production. Further studies revealed VPS25 located on exosomes and its expression correlated to the exosome production. During EV71 infection, knock-down of VPS25 decreased exosome biogenesis to attenuate viral replication. Consistently, GW4869, an exosome inhibitor, exerted an obviously antiviral activity against EV71 replication companied with the decrease of exosome secretion or formation. These findings suggest the binding of EV71 3A and VPS25 benefited exosome biogenesis, thereby boosting viral replication. This study uncovers a novel mechanism underlying EV71-mediated exosomes in the regulation of viral replication, which provides potential anti-viral strategies against the EV71 infection and transmission in HFMD.

Neurocognitive deficits and sequelae following severe hand, foot, and mouth disease from 2009 to 2017, in JiangSu Province, China: a long-term follow-up study

BackgroundThe aim of this study was to evaluate the long-term sequelae and cognitive profiles resulting from severe hand, foot, and mouth disease (HFMD) with central nervous system (CNS) involvement. Methods294 HFMD cases were included in a retrospective follow-up study. Physical examinations were conducted. The Chinese Wechsler Preschool and Primary Scale of Intelligence, Fourth Edition (WPPSI-IV) was used to assess intelligence. Results58 mild HFMD cases and 99 severe HFMD cases with mild CNS involvement did not present any neurological sequelae. In comparison, the sequelae incidence for severe HFMD with more severe CNS complications was 50.0%. The proportion of full-scale intelligence quotient (FSIQ) impairment was 45.0%. In the 2:6–3:11 age group, severe HFMD with more severe CNS complications and lower maternal education level were risk factors for verbal comprehension disorder. Urban–rural residence and lower paternal education level were risk factors for FSIQ disorder. Furthermore, in the 4:0–6:11 age group, severe HFMD with more severe CNS complication was a risk factor for visual spatial disorder and fluid reasoning disorder. Lower paternal education level was a risk factor for FSIQ disorder. ConclusionEarly assessment and intervention among severe HFMD patients with more severe CNS involvement at a very young age will prove beneficial for their future performance.

Searching for Risk Factors and Establishing Predictive Models for Severe and Critical Hand-Foot-and-Mouth Disease

Objectives: Severe and critical hand-foot-and-mouth disease (HFMD) patients have an acute onset and poor prognosis. This study intended to establish an appropriate risk prediction model by analyzing the blood biochemical indicators of patients. Methods: A total of 3,204 patients with HFMD were enrolled in this study, including 2,131 mild patients, 962 severe patients, and 111 critical patients. We first analyzed the data of each group through multivariate statistics based on SIMCA-P and screened out the variables that had important contributions to the discrimination of each group. Furthermore, the risk factors and predictors were screened out by comparison with the results of univariate statistical analysis. Finally, binary logistic regression analysis was used to establish a suitable prediction model. Results: With the aggravation of HFMD patients' conditions, the blood content and risk warning ability of seven indicators of SP, DP, NEUT%, TP, GLB, RBP, and Glu were significantly increased. We found for the first time that the more severe the HFMD patients, the lower the levels of Chr, %MRETIC, and %HRETIC in their blood. The average prediction accuracy of the established models for Mild/Severe, Severe/Critical, and Severe/Critical was 82.89, 96.16, and 89.37%, respectively, and the AUROC was 0.8722 (95%CI, 0.8583 - 0.8861), 0.9499 (95%CI, 0.9339 - 0.9659), and 0.7913 (95% CI, 0.7471 - 0.8356), respectively. Conclusions: Multivariate statistical analysis based on SIMCA-P could be used to analyze the clinical data of HFMD patients. Besides, SP, DP, NEUT%, TP, GLB, RBP, and Glu could be used as risk factors for severe and critical HFMD patients. The abnormal changes of Chr, %MRETIC, and %HRETIC reflected the possible damage to bone marrow hematopoietic function in HFMD patients. The predictive model established by us could be used for the differential diagnosis of Mild/Severe, Mild/Critical, and Severe/Critical.

Prognostic Value of Neutrophil-to-Lymphocyte Ratio in Predicting Death Risk in Patients with Severe Hand, Foot and Mouth Disease.

IntroductionSevere hand, foot, and mouth disease (HFMD) may lead to serious complications, which cause child mortality during outbreaks. The aim of this study was to determine whether neutrophil-to-lymphocyte ratio (NLR) can predict death risk in severe HFMD.MethodsMedical records for 664 severe HFMD patients were retrospectively examined, and NLR was calculated from blood counts. Youden’s index was calculated to determine the optimal NLR cutoff. Uni- and multivariate logistic regression were used to determine death risk factors associated with severe HFMD.ResultsAn NLR cutoff value of 2.01 and 2.50 respectively predicted mortality among all 664 severe HFMD and 137 critical HFMD. Among all 664 patients, the multivariate model identified the following as independently associated with death risk: high fever (OR 3.342, 95% CI 1.736–6.432), EV71 infection (OR 3.200, 95% CI 1.529–6.698), fasting glucose (OR 37.343, 95% CI 18.616–74.909), and NLR (>2.01) (OR 2.142, 95% CI 1.125–4.079). Among 137 critical HFMD, EV71 infection (OR 3.441, 95% CI 1.132–10.462), fasting glucose (OR 14.173, 95% CI 4.920–40.827), and NLR (>2.50) (OR 4.166, 95% CI 1.570–11.051) were associated with death risk.ConclusionIn conclusion, NLR (>2.01) in severe HFMD and NLR (>2.50) in critical HFMD patients may be associated with increased death risk.

IFI27 as a potential indicator for severe Enterovirus 71-infected hand foot and mouth disease

The pathogenesis of Enterovirus 71 (EV71)-induced severe hand foot and mouth disease (HFMD) has not yet been clearly established. Further study into the mechanisms underlying host immune responses to EV71 infection and identifying important predictors will be crucial to antiviral treatment and early recognition of severe HFMD. The present study establishes that T help (Th)1 type, Th2 type, and Th17 type cytokine levels in serum of peripheral blood from patients with severe HFMD is higher than in peripheral blood from healthy subjects. The most significant increase occurred as the IL-6. In order to identify the important molecules in peripheral blood mononuclear cells (PBMCs) from severe HFMD patients, we performed transcriptome sequencing analysis of PBMC from severe HFMD patients and compared them to healthy controls. Interferon α-inducible protein 27 (IFI27) and cluster of differentiation 27 (CD27) were found to be the most significant differentially expressed gene. Finally, IFI27 was proved to be present at higher levels in patients with severe HFMD than in patients with mild HFMD. Our results suggest that IFI27 may be an indicator of the severity of cases EV71-induced HFMD.

Sequence analysis of VP1 and VP4 genes of enterovirus 71 strains isolated from children with severe and mild hand-foot and mouth disease

Objective To analyze and compare VP1 and VP4 genetic characteristics of human enterovirus 71(EV71) isolated from children with severe and mild hand-foot and mouth disease(HFMD). Methods EV71 strains isolated from severe HFMD patients (6 cases) and mild HFMD patients (6 cases) in Taizhou district, Zhejiang province were included during 2016.Total virus RNA was extracted by Viral RNA Mini Extraction Kit, and reverse transcription polymerase chain reaction(RT-PCR) was used to amplify the sequence of the VP1 and VP4 genes of EV71.And then the sequencing results were compared with those of A, B, C genotype reference EV71 strains from GenBank by nucleotide alignment and amino acid alignment analysis. Results There were no statistically significant differences in sex and age between the two groups(χ2=14.51, t=2.82, all P<0.05). The homogeneity between EV71 strains from severe patients and mild patients was 95.8%~99.6% and 99.1%~100.0% for VP1 nucleotide sequences and amino acid sequences, respectively, while 95.0%~99.9% and 99.0%~100.0% for VP4 nucleotide sequences and amino acid sequences, respectively.The twelve EV71 strains isolated from HFMD patients in Taizhou shared the highest identity with EV71-genotype C, especially genotype C4a.In addition, compared with the mild patients, three respective strains from severe HFMD patients showed mutations at the residue 170 in the VP1 protein(V→L), residue 293 in the VP1 protein(A→S) and residue 7 in the VP4 protein(T→A). Conclusion All EV71 strains isolated from severe and mild HFMD patients in Taizhou district share high homology of nucleotide and amino sequence, and all of them belong to subgenogroup C4a.The mutations in the VP1 and VP4 of EV71 might be related to HFMD disease severity. Key words: Hand-foot and mouth disease; Enterovirus 71; Genotype; Disease severity; Mutation

Scavenger receptor class a, member 3 is associated with severity of hand, foot, and mouth disease in a case-control study.

Hand, foot, and mouth disease (HFMD) spreads rapidly and has been recognized as a public health problem in recent years in China. Unfortunately, there is no effective vaccine or antiviral drug currently for EV71 infection. In this study, we aim to identify biomarker which are associated with for severity of EV71 infection cases using high-throughput RNA sequencing approach.RNA sequencing of samples from severe HFMD (S) patients group (n = 10) and control HFMD (C) patients group (n = 10) were performed and the results were verified by qPCR. mRNA with the highest expression level was selected to be validated in an independent cohort comprising of 45 severe EV71 infected patients and 45 control by qPCR assay.Seventeen significant differentially expressed genes were identified. Scavenger receptor class A, member 3 (SCARA3) was one of the significantly upregulated genes with the highest expression level and was selected for validation. The mean relative expression levels in severe HFMD and control HFMD patients were 10.1-fold and 5.0-fold, respectively, P value <.001.We found that SCARA3 is associated with severity of HFMD, and it may be a potential prognostic marker to predict the HFMD progression in EV71 infected patients.

Increased effector γδ T cells with enhanced cytokine production are associated with inflammatory abnormalities in severe hand, foot, and mouth disease

BackgroundAlthough γδ T cells have been reported to be closely related to the immunopathogenesis of some viral infectious diseases, the changes or roles of γδ T cells in the development of hand, foot, and mouth disease (HFMD) remain unclear. MethodsPeripheral γδ T cells and their subsets were determined by surface (γδ TCR, Vδ1 TCR, Vδ2 TCR, CD45RA, and CD27) or intracellular (IFN-γ, TNF-α, CD107a, and Granzyme B) markers in healthy controls (HCs) and HFMD patients with FACS. The plasma levels of IFN-γ, TNF-α, IL-6, and MCP-1 were measured by ELISA. Differences in γδ T cells or their subsets and correlations between γδ T cells and inflammation indicators were statistically analyzed. ResultsCompared to HCs, HFMD patients showed increased effector γδ T and TNF-α+γδ T cells and plasma TNF-α levels, especially in severe cases. In addition, significantly increased Vδ1 T and IFN-γ+γδ T cells and other plasma inflammatory cytokines were further found in severe patients. Furthermore, EV71+ severe patients showed significantly increased effector and cytokine-producing γδ T cells, while the EV71− severe patients displayed significantly greater plasma cytokine levels. The percentage of IFN-γ+γδ T or TNF-α+γδ T cells was positively correlated with that of effector γδ T cells. There was a positive correlation between the proportion of Vδ1 T cells and white blood cell (WBC) count or the proportion of IFN-γ+γδ T or TNF-α+γδ T cells and neutrophil (N) count, while there was a negative correlation between Vδ2 T cells and WBC or N count. Moreover, the percentages of Vδ1 T and effector γδ T cells in the acute phase of disease declined significantly to normal levels during the recovery phase. ConclusionsIncreased effector γδ T cells with enhanced cytokine production were remarkably observed in severe HFMD patients, which was also associated with clinical inflammation parameters. These data indicated that γδ T cells might be involved in inflammatory abnormalities in severe HFMD.

Low frequency, weak MCP-1 secretion and exhausted immune status of peripheral monocytes were associated with progression of severe enterovirus A71-infected hand, foot and mouth disease.

A minority of hand, foot and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) results in severe neural complications. However, whether monocyte-mediated immunity is involved in the disease progression of HFMD remains unknown. One hundred and twenty mild and 103 severe HFMD patients were recruited and enzyme-linked immunosorbent assay (ELISA), flow cytometry and Transwell culture were performed in the study. Peripheral monocyte counts were lower in both absolute counts and frequencies in severe cases compared to mild cases. After screening 10 monocyte-related cytokines by ELISA, only monocyte chemoattractant protein-1 (MCP-1) was found at higher levels in sera of mild cases compared to those with severe symptoms. Monocytes purified from mild cases produced more MCP-1 than the cells from severe patients when stimulated in vitro. We observed that immune exhaustion markers programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) were highly regulated on the surface of monocytes from severe cases compared to mild cases. PD-L1 blockade induced a higher production of MCP-1 in the supernatant of a Transwell system. The production of MCP-1 also increased following PD-L1 blockade of purified monocytes activated by granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with R848 or EV-A71 virus. Our results indicate that absolute count, frequency and levels of MCP-1 secretion of peripheral monocytes, together with their immune status, probably contribute to differential disease prognosis in EV-A71-associated HFMD.

Surveillance for severe hand, foot, and mouth disease from 2009 to 2015 in Jiangsu province: epidemiology, etiology, and disease burden

BackgroundSevere hand, foot, and mouth disease (HFMD) is a common childhood illness caused by various enteroviruses. The disease has imposed increased burden on children younger than 5 years old. We aimed to determine the epidemiology, CNS complication, and etiology among severe HFMD patients, in Jiangsu, China.MethodsEpidemiological, clinical, and laboratory data of severe HFMD cases were extracted from 2009 to 2015. The CNS complication, annually severe illness rates, mortality rates, severity-PICU admission rates, severity-hospitalization rates, and so on were analyzed to assess the disease burden of severe HFMD. All analyses were stratified by time, region, population, CNS involvement and serotypes. The VP1 gene from EV-A71, CV-A16, CV-A6, CV-A10 and other enteroviruses isolates was amplified. Phylogenetic analysis was performed using MEGA5.0.ResultsSeven thousand nine hundred ninety-four severe HFMD cases were reported, of them, 7224 cases were inpatients, 611 were PICU inpatients, and 68 were fatal. The average severe illness rate, mortality rate, severity−fatality rate, severity-PICU admission rate, and severity-hospitalization rate were 14.54, 0.12,8506, 76,430, and 903,700 per 1 million, respectively. The severe illness rate was the highest in the 12–23 months age group, and the greatest mortality rate was in the 6–11 months age group. Geographical difference in severe illness rate and mortality were found. Patients infected with EV-A71 were at a higher proportion in different CNS involvement even death. EV-A71, CV-A16 and other enteroviruses accounted for 79.14, 6.49, and 14.47%, respectively. A total of 14 non-EV-A71/ CV-A16 genotypes including CV-A2, CV-A4, CV-A 6, CV-A9, CV-A10, CV-B1, CV-B2, CV-B3, CV-B4, CV-B5, E-6, E-7, E-18, and EV-C96 were identified. Phylogentic analyses demonstrated that EV-A71 strains belonged to subgenotype C4a, while CV-A16 strains belonged to sub-genotype B1a and sub-genotype B1b of genotype B1. CV-A6 strains were assigned to genogroup F, and CV-A10 strains belonged to genogroup D.ConclusionsFuture mitigation policies should take into account the age, region heterogeneities, CNS conditions and serotype of disease. Additional a more rigorous study between the mild and severe HFMD should be warranted to elucidate the difference epidemiology, pathogen spectrum and immunity patterns and to optimize interventions in the following study.

Significance of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 levels in evaluation of severe hand, foot, and mouth disease complicated with neurogenic pulmonary edema

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

Abstract P-135: MULTICENTER CLINICAL EPIDEMIOLOGIC STUDY OF SEVERE HAND FOOT AND MOUTH DISEASE.

Aims & Objectives: We conducted a multicenter epidemiological survey of critical hand foot and mouth disease (HFMD), to find out the treatment outcomes and disease burden in severe HFMD. Methods A collaborative study group was established including children’s hospital of Fudan university, Jiangxi provincial children’s hospital, Anhui provincial children’s hospital, Linyi people’s hospital and No. 2 people’s hospital of Fuyang city. Clinical dates of severe HFMD were prospective collected. Results We collected 226 severe HFMD cases from April 2014 to October 2016. EV71 virus positive accounted for 70.9%. 97.8% were given mannitol, 40.3% glycerol fructose. 87.6% were given IVIG, IVIG use in stage 2, 3, 4 was 82.8%, 96.7%, 89.2% respectively. 84.1% were given antivirus therapy, antivirus drug use in stage 2, 3, 4 was 78.9%, 93.4, 86.5% respectively. 64.2% were given hormone therapy.47.8% needed milrinone, 27.4% needed Phentolamine, vasoactive agent use in stage 2, 3, 4 was 46.1%, 93.4, 91.9% respectively. 30.5% received CPAP, 40.3% with mechanical ventilation,The mean mechanical ventilation time was 125.9 ± 101.8 hours. Hormone, IVIG, antivirus agent did not reduce the mortality of severe HFMD patients. 11 cases died or died after giving up. Length of stay in hospital were 9.18 ± 5.16 (1–37) days. Multivariate analysis found that respiratoy rhythm abnormality, CRT more than 3 seconds were risk factors for the deaths in the severe HFMD. Conclusions The children in stage 3, 4 needed more IVIG, antivirus drugs, vasoactive drugs, mechanical ventilation compared with stage 2. The lenth of mechanical ventilation and hospital stay was higher in stage 3, 4 compared to stage 2.

Crosstalk between Toll-like receptor 3 and Notch signaling contributes to CD14+ monocytes activity in enterovirus 71 infected hand, foot, and mouth disease

Interaction between Toll-like receptor (TLR) and Notch signaling contributes to inflammatory response in nephropathy and fungicidal infection, however, the role of this crosstalk remains not fully elucidated in enterovirus 71 (EV71)-induced hand, foot, and mouth disease (HFMD). The aim of this study was to investigate the crosstalk between TLR and Notch in inflammatory regulation in EV71 infection. Thirty-seven EV-71-indcued HFMD (16 mild and 21 severe cases) and eleven normal control (NC) were enrolled. CD14+ monocytes were purified, and were stimulated with either TLR3/4 agonists [poly(I: C) or LPS] or Notch signaling inhibitor. TLRs and Notch receptors expression, proinflammatory cytokines production, and important molecules in signaling pathways were measured by real-time PCR, ELISA, and Western blot. TLR3 and TLR4 was significantly elevated in CD14+ monocytes from HFMD patients than NC. Notch1 and Notch2 mRNA was also remarkably increased in CD14+ monocytes from severe HFMD. Poly(I: C) stimulation resulted in robust increase of IL-8, IL-6, and TNF-α by CD14+ monocytes in severe HFMD compared to NC. Activation of Notch1, Notch2, and target genes, Hes1 and Hes5 was also enhanced upon ploy(I: C) treatment. Although inhibition of Notch signaling did not affect TLR3 expression, poly(I: C)-induced inflammatory response was robustly attenuated, which was accompanied by silencing Src phosphorylation in CD14+ monocytes from severe HFMD patients. The current data indicated that crosstalk between TLR3 and Notch signaling modulated CD14+ monocytes function and inflammatory responses in the progression of EV71-induced HFMD.

Derivation and Validation of a Mortality Risk Score for Severe Hand, Foot and Mouth Disease in China

Outbreaks of hand, foot and mouth disease (HFMD) have increased recently, as has the case fatality rate in severe cases. No scoring system currently exists to predict mortality risk for severe HFMD in previous study. We retrospectively collected laboratory parameters for 546 patients with severe HFMD (a derivation and a validation cohort) at Hunan Children’s Hospitals between January 2012 and December 2014. We developed a mortality risk score comprising four laboratory parameters: blood glucose (GLU), white blood cells (WBC), lactate (LAC), and N-terminal-probrain natriuretic peptide (NT-proBNP). Using an “optimal” cutoff score of 4, the sensitivity, specificity, positive predictive value and negative predictive value was 88.00%, 96.14%, 62.86% and 99.08%, respectively, in the derivation cohort. Among severe HFMD patients with low- and high-risk scores in the validation cohort, case fatality rates were 1.49% and 74.07%, respectively. According to the “optimal” cut-off in the derivation cohort, the sensitivity, specificity, positive predictive value, and negative predictive value were 80.95%, 93.83%, 62.96% and 97.44%, respectively, in the derivation cohort. The mortality risk score demonstrated good discrimination (AUC > 0.9) and calibration (P > 0.05) in both cohorts. The mortality risk score, comprising WBC, GLU, LAC and NT-proBNP, has been demonstrated good discrimination and calibration in the both cohorts.

Association study of inflammatory cytokine and chemokine expression in hand foot and mouth disease.

ObjectiveTo determine the relationship of cytokine/chemokine expression with the clinical presentation of hand, foot and mouth disease (HFMD).ResultsAll cytokine/chemokine levels were higher in severe HFMD patients than in mild HFMD patients or controls (P < 0.01). RANTES, MCP-1, IL-4, IL-12 and IL-18 levels were higher in mild HFMD patients than in the controls (P < 0.05). In severe HFMD, all levels (except IL-8 and IL-4) were higher in patients with encephalitis plus pulmonary edema than in those with encephalitis alone (P < 0.05). All levels (except IL-8) were higher in EV71-positive patients than in EV71-negative patients (P < 0.05). In mild HFMD, all levels (except IL-8 and IL-4) were higher in EV71-positive patients than in EV71-negative patients (P < 0.05). In severe HFMD, only RANTES, IP-10 and IFN-γ levels were higher in EV71-positive patients than in EV71-negative patients (P < 0.05). In the EV71-negative group, all levels were higher in severe HFMD than in mild HFMD (P < 0.01). In the EV71-positive group, all levels (except IL-8) were higher in severe HFMD than in mild HFMD (P < 0.01).Materials and MethodsThis study involved 28 mild HFMD patients, 44 severe HFMD patients and 26 healthy children. Venous blood was tested for cytokines (IL-4, IL-12, IL-18, TNF-α, IFN-γ) and chemokines (IL-8, RANTES, MCP-1, IP-10). Stool samples from the patients were tested for EV71 nucleic acid using reverse transcription polymerase chain reaction.ConclusionsCytokines/chemokines participate in HFMD pathogenesis, and could have potential value in monitoring disease progression and predicting prognosis.

Clinical stages and outcomes of severe cases on hand, foot and mouth disease

Objective: To understand the characteristics and relation of clinical stage and outcome of severe cases on hand, foot and mouth disease (HFMD) and to establish the evaluation method for understanding severity of this disease. Methods: According to factors as geographical location, economic and epidemic levels, five provinces (Henan, Shandong, Yunnan, Zhejiang and Sichuan provinces) were selected. Reported severe cases of HFMD from the National Notifiable Diseases Reporting System were selected randomly in the five provinces. Basic epidemiological information, clinical data, and pathogen testing results in the involved hospitals were collected. Clinical stages on all the patients were decided in accordance with"the clinical expert consensus on diagnosis and treatment for severe case of enterovirus type 71 (EV71) infections (2011 edition)" . Data were analyzed using SPSS software 18.0 and other epidemiological methods. Results: A total of657 severe HFMD cases were investigated, with 326 cases positive of EV71, accounting for 91.3% (326/357) among all the laboratory-confirmed cases. Of the 657 cases, 542 cases (82.5%, 95%CI: 79.4%-85.3%) were diagnosed as in stage 2 (with nervous system involvement), 99 cases (15.1%, 95%CI: 12.4%-18.0%) in stage 3 (early phase of function failure on heart and lung), and 16 cases (2.4%, 95%CI: 1.4%-3.9%) were in stage 4 (function failure of heart and lung). 11 cases (1.7%, 95%CI: 0.9%-3.0%) were with squeal when discharged from hospital with 8 cases (1.2%, 95%CI: 0.6%-2.3%) died. When comparing the proportions among stage 2, stage 3 and stage 4, significant differences were found between age groups (χ(2)=22.632, P=0.012). The younger the patient was the lower the proportions of stage 2 and the more proportion of stage 3 appeared. When comparing the proportions of clinical stages among the five provinces, significant differences (χ(2)=41.481, P=0.000) were noticed. Proportions of different clinical stages in gender, ethnicity, occupation, place of residence types and the type of pathogen appeared no significant differences, respectively. However, the proportions of squeal and death in stage 2, stage 3 and stage 4 showed significant differences (sequela: χ(2)=12.960, P=0.001; Death: χ(2)=16.850, P=0.001), respectively. Conclusions: The percentage of clinical stages of severe HFMD patients related to the rate of squeal and death. Clinical staging can be used for assessing the clinical severity of complications and the effectiveness of treatment, of HFMD.

Health related quality of life on severe hand, foot and mouth disease patients

Objective: To evaluate the health related quality of life (HRQoL) for severe hand, foot and mouth disease (HFMD) patients with different complications. Methods: A national telephone interview under the EQ-5D proxy2 questionnaire (EQ-SD and EQ-VAS), was conducted to obtain the HRQoL of lab-confirmed severe HFMD patients, aged between six months and five-year-olds from the national communicable disease surveillance system from January 1, 2012 to December 31, 2013. Results: A total of 685 severe HFMD cases were included in the study. A total of 456 (66.6%) of them were males with 75.8% of them younger than three years old. A total of 337 (49.2%) and 407 (59.4%) of the participants reported that they had problems in mobility or daily activities. A total of 569 (83.1%) and 616 (89.9%) of the cases reported having problems in pain/discomfort or anxiety/depression. The average EQ-5D and EQ-VAS scores were 0.58±0.23 and 53.6±25.7, both were positively associated with the duration of illness. Mean quality adjusted life years loss during the HFMD episode for the severe patients was (15.45±13.75) years/1 000 persons. The QALY losses for severe patients with each of below complication were: respiratory diseases (11.17±8.83) years/1 000 persons, aseptic meningitis (13.56±11.99) years/1 000 persons, encephalitis/brainstem encephalitis/acute flaccid paralysis (AFP) (15.31±12.63) years/1 000 persons, Myocarditis (17.28±18.16) years/1 000 persons, pulmonary hemorrhage/pulmonary edema (17.34±14.98) years/1 000 persons, cardiopulmonary failure (25.47±20.53) years/1 000 persons. Among patients with lab confirmed Entero virus A71 (EV71) (16.51±14.48) years/1 000 persons, the QALY loss was seen higher than Coxsackie virus A16 (Cox A16) (13.02±11.45) years/1 000 persons and other Enter virus (14.74±14.22) years/1 000 persons (Z=11.83, P=0.003). Conclusion: The HRQoL loss for severe HFMD patients substantially increased under complications exacerbation and related to the duration of illness.

A Case-control Study on Risk Factors for Severe Hand, Foot and Mouth Disease

The objective of this study was to identify potential risk factors for severe hand, foot and mouth disease (HFMD). In this case-control study, 459 severe HFMD patients and 246 mild HFMD patients from Guangdong province and Henan province, China were included. Data comprising demographic characteristics, clinical symptoms and signs, laboratory findings and other factors were collected. Univariate analysis revealed 30 factors associated with severe cases. Further multivariate analysis indicated four independent risk factors: fatigue (p < 0.01, odd ratio [OR] = 204.7), the use of glucocorticoids (p = 0.03, OR = 10.44), the use of dehydrant drugs (p < 0.01, OR = 73.7) and maculopapular rash (p < 0.01, OR = 84.4); and one independent protective factor: herpes or ulcers in mouth (p = 0.01, OR = 0.02). However, more systematic research and validation are needed to understand the underlying risk factors for severe HFMD.