Application and Comparison of Laboratory Parameters for Forecasting Severe Hand-Foot-Mouth Disease Using Logistic Regression, Discriminant Analysis and Decision Tree.

BACKGROUND: In recent years, the prevalence of hand-foot-mouth disease (HFMD) in China and some other countries has caused worldwide concern. Mild cases tend to recover within a week, while severe cases may progress rapidly and tend to have bad outcome. Since there is no vaccine for HFMD and anti-inflammatory treatment is not ideal. In this study, we aimed to establish a valid forecasting model for severe HFMD using common laboratory parameters. METHODS: Retrospectively, 77 severe HFMD cases from Zhengzhou Children's hospital in the peaking period between years 2013 to 2015 were collected, with 77 mild HFMD cases in the same area. The study recorded common laboratory parameters to assist in establishment of the severe HFMD model. After screening the important variables using Mann-Whitney U test, the study also matched the logistic regression (LR), discriminant analysis (DA), and decision tree (DT) to make a comparison. RESULTS: Compared with that of the mild group, serum levels of WBC, PLT, PCT, MCV, MCH, LCR, SCR, LCC, GLO, CK-MB, K, S100, and B in the severe group were higher (p < 0.05), while MCR, EOR, BASOR, SCC, MCC, EO, BASO, NA, CL, T, Th, and Th/Ts were lower (p < 0.05). Five indicators including MCR, LCC, Th, CK-MB, and CL were screened out by LR and the same for DA, and five variables including EO, LCC, CL, GLO, and MCC screened out by DT. The area under the curve (AUC) of LR, DA, and DT was 0.805, 0.779 and 0.864, respectively. CONCLUSIONS: The findings were that common laboratory indexes were effectively used to distinguish the mild HFMD cases and severe HFMD cases by LR, DA, and DT, and DT had the best classification effect with an AUC of 0.864.

[Expression and activity determination of recombinant capsid protein VP2 gene of enterovirus type 71].

OBJECTIVE: To clone and express the recombinant capsid protein VP2 of enterovirus type 71 (EV71) and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen. METHODS: VP2 gene of EV71 was amplified by PCR, and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E.coli TB1, the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside ( IPTG) , and the expression products were analyzed by SDS-PAGE and western blotting method. EV71 IgM antibody detection method by ELISA was set up, and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease (HFMD) patients were determined, of which 52 samples were positive and 8 samples were negative; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected. RESULTS: VP2 gene of 762 bp was obtained by PCR, the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion. The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE. The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting. The sensitivity and specificity of ELISA was 87% and 83%, respectively. Of the 88 acute phase serum samples from children with HFMD, 48 samples (55%) were positive by the ELISA assay. CONCLUSIONS: VP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study. The recombination EV71-VP2 has well antigenicity, which could be useful for developing diagnose reagent or vaccine of EV71.

Detection of human enterovirus 71 and Coxsackievirus A16 in an outbreak of hand, foot, and mouth disease in Henan Province, China in 2009.

During 2009, an outbreak of hand, foot, and mouth disease (HFMD) enrolled 490 people in Henan Province, causing the death of two children. In order to investigate the pathogens responsible for this outbreak and characterize their genetic characteristics, a total of 508 clinical specimens (stool, throat swab, and vesicle fluid) were collected from the Center for Disease Control and Prevention of Henan Province. Virological investigations (virus isolation, conventional reverse transcription PCR, and real-time reverse transcription PCR) and phylogenetic analysis were performed. It was found that human enterovirus 71 (EV71) was the main pathogen causing this outbreak, while Coxsackievirus A16 (CoxA16) played only a subsidiary role. Phylogenetic analysis of 24 EV71 isolates collected during the period from March 11 to July 24, 2009 showed that they belonged to subgenotypes C4 and C5. Our study for the first time characterizes the epidemiology of HFMD and EV71 infection in Henan Province in 2009 and provides the first direct evidence of the genotype of EV71 circulating in Henan Province at that time. Our study should facilitate the development of public health measures for the control and prevention of HFMD and EV71 infection in at-risk individuals in China.

[Etiology surveillance of hand-foot-mouth disease in Henan province between 2008 and 2011].

OBJECTIVE: To understand etiological types and distribution features of hand-foot-mouth disease (HFMD) in Henan province between 2008 and 2011. METHODS: A total of 30 486 specimens of feces, rectal swabs or throat swabs from HFMD patients were collected by each Municipal CDC in Henan from 2008 to 2011. The enterovirus 71 (EV71), coxsackie virus A16 (CA16) and other enterovirus (EV) were detected by RT-PCR or real time RT-PCR. The VP1 gene of EV71 was amplified and the sequences were analyzed by bioinformatics software. A genetic evolution tree of the sequence was constructed as well. RESULTS: The positive rates of EV71, CA16 and other EV were 62.70% (11 209/17 876), 12.03% (2150/17 876), 25.27% (4517/17 876) in 17 876 laboratory diagnosed cases, respectively. The differences were statistically significant (χ(2) = 157.17, P < 0.05). The positive rates of EV71, CA16 and other EV were 63.40% (7370/11 624), 11.58% (1346/11 624) and 25.02% (2908/11 624) in male patients and 61.40% (3839/6252), 12.86% (804/6252) and 25.74% (1609/6252) in female patients, respectively. The differences were statistically significant (χ(2) = 4.06, P < 0.05). The children under 5 years old were high-risk population of HFMD, accounting to 97.67% (17 459/17 876) of the laboratory-diagnosed patients.86.92% (15 537/17 876) cases were children between 1 to 3 years old. Constituent ratio of EV71 changed seasonally during a year, there was a high infection ratio of EV71 between April and June, especially in May, the infection ratio reached 69.34% (2384/3438). The positive rates of EV71, CA16 and other EV were 82.48% (5715/6929), 1.76% (122/6929) and 15.76% (1092/6929) among the 6929 laboratory-diagnosed severe cases, respectively. The positive rates of EV71 was higher than CA16 and other EV (χ(2) = 9259.17, 6170.81, P < 0.05, respectively). There were 117 deaths because of severe HFMD, 55 (47.01%) of which were laboratory confirmed. 50 death cases were infected by EV71, and according to the genetic evolution analysis, the VP1 gene of EV71 strain was belonged to subtype C4 of gene C. CONCLUSION: The EV71 and CA16 were the main pathogens which caused HFMD in Henan province, and EV71 virus was the dominant strain, belonging to C4 subtype of gene C.

Chinese guidelines for the diagnosis and treatment of hand, foot and mouth disease (2018 edition).

BACKGROUND: Hand, foot, and mouth disease (HFMD) is a common infectious disease in childhood caused by an enterovirus (EV), and which is principally seen in children under 5 years of age. To promote diagnostic awareness and effective treatments, to further standardize and strengthen the clinical management and to reduce the mortality of HFMD, the guidelines for diagnosis and treatment have been developed. METHODS: National Health Commission of China assembled an expert committee for a revision of the guidelines. The committee included 33 members who are specialized in diagnosis and treatment of HFMD. RESULTS: Early recognition of severe cases is utmost important in diagnosis and treatment of patients with HFMD. The key to diagnosis and treatment of severe cases lies in the timely and accurate recognition of stages 2 and 3 of HFMD, in order to stop progression to stage 4. Clinicians should particularly pay attention to those EV-A71 cases in children aged less than 3 years, and those with disease duration less than 3 days. The following indicators should alert the clinician of possible deterioration and impending critical disease: (1) persistent hyperthermia; (2) involvement of nervous system; (3) worsening respiratory rate and rhythm; (4) circulatory dysfunction; (5) elevated peripheral WBC count; (6) elevated blood glucose and (7) elevated blood lactic acid. For treatment, most mild cases can be treated as outpatients. Patients should be isolated to avoid cross-infection. Intense treatment modalities should be given for those severe cases. CONCLUSION: The guidelines can provide systematic guidance on the diagnosis and management of HFMD.

[Etiology study on severe cases caused by hand-foot-mouth disease in children from Henan province, 2014].

OBJECTIVE: To investigate the etiology of severe hand-foot-mouth disease (HFMD) in children in Henan province. METHODS: A total of 244 HFMD cases admitted to a hospital in Zhengzhou from April to June of 2014 were recruited for research sampling, Real-time RT-PCR, virus isolation, VP1 sequencing and alignment methods were used to test the enterovirus-related etiology. SPSS 17.0 was used in performing statistical analysis. RESULTS: There were 109 severe and 135 mild cases among all the 244 HFMD cases. The number of enterovirus positive stool samples was 229, with positive rate as 93.85%. EV71, Cox A16 and Cox A10 made up 83.84%, 5.68% and 8.30% of the enterovirus etiologicy, strains, respectively. EV71 infection caused 8 HFMD cases with heart-lung failure and 2 death, Cox A10 infection led to 1 HFMD case with heart-lung failure and death. There were statistically differences seen regarding the enterovirus infection rates between severe and the mild HFMD cases (χ(2)=5.312,P=0.021). Statistically significant difference was seen in the constituent ratio of EV71, Cox A16 and the others by Fisher' s exact test (P=0.048). There was statistically significant difference seen between the cardiorespiratory failure rate and the fatality rate by EV71 and Cox A10 infection (χ(2)=0.051,P=0.821; χ(2)=2.198,P=0.138). Cox A10 strains idenfied in Henan in 2014 belonged to genotype 6. The rates on homology of nucleotide and amino acid among the Cox A10 strains in Henan in 2014 were 94.3%-99.7% and 96.3%-100.0% respectively. CONCLUSIONS: EV71 still remained the most common pathogen that causing severe HFMD in children, with the increasing Cox A10 percentage in the pathogens spectrum of HFMD infection. Cox A10 strains in Henan in 2014 belonged to genotype 6. Genotype 6 Cox A10 had appeared and widely distributed in Henan for long time, but not yet variated or reconstructed. Cox A10 infection could lead to cardio-respiratory failure thus called for the monitoring program on non-EV71 and non-Cox A16 enterovirus, especially Cox A10 to be strenthened.

Clinical Efficacy of Therapy with Recombinant Human Interferon α1b in Hand, Foot, and Mouth Disease with Enterovirus 71 Infection.

UNLABELLED: A rapid expansion of HFMD with enterovirus 71 infection outbreaks has occurred and caused deaths in recent years in China, but no vaccine or antiviral drug is currently available for EV71 infection. This study aims to provide treatment programs for HFMD patients. We conducted a randomized, double-blind, controlled trial and evaluated clinical efficacy of therapy with rHuIFN-α1b in HFMD patients with EV71 infection. There were statistical differences in outcomes including the fever clearance time, healing time of typical skin or oral mucosa lesions, and EV71 viral load of the HFMD patients among ultrasonic aerosol inhalation group, intramuscular injection group and control group. rHuIFN-α1b therapy reduced the fever clearance time, healing time of typical skin or oral mucosa lesions, and EV71 viral load in children with HFMD. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR-TRC-14005153.

Epidemiological and etiological characteristics of hand, foot, and mouth disease in Henan, China, 2008-2013.

Hand, foot, and mouth disease (HFMD) is a common childhood illness caused by enteroviruses. HFMD outbreaks and reported cases have sharply increased in China since 2008. Epidemiological and clinical data of HFMD cases reported in Henan Province were collected from 2008 to 2013. Clinical specimens were obtained from a subset of these cases. Descriptive epidemiological methods were used to analyze the time, region and population distribution. The VP1 gene from EV71 and CA16 isolates was amplified, and the sequences were analyzed. 400,264 cases of HFMD were reported in this study, including 22,309 severe and 141 fatal cases. Incidence peaked between April and May. Laboratory confirmation was obtained for 27,692 (6.9%) cases; EV71, CA16, and other enteroviruses accounted for 59.5%, 14.1%, 26.4%, respectively. Phylogenetic analysis revealed that EV71 belonged to the C4a evolution branch of C4 sub-genotype and CA16 belonged to subtype B1a or B1b. The occurrence of HFMD in Henan was closely related to season, age and region distribution. Children under five were the most affected population. The major pathogens causing HFMD and their genotypes have not notably changed in Henan. The data strongly support the importance of EV71 vaccination in a high population density area such as Henan, China.

[Genomic characteristics of coxsakievirus A16 isolated in Henan Province].

To reveal the genomic sequence characteristics of coxsackievirus A16 (CoxA16) strain isolated from patients with hand-foot-mouth disease (HFMD) in Henan province. A total of 406 samples were detected by reverse-transcription polymerase chain reaction (RT-PCR) and cell-culture-based isolation of coxsackievirus A16. The whole genome of CoxA16 isolate was amplified using 10 pairs of primers, the sequences were analyzed and phylogenetic tree was generated by bioinformatics software. The full length of HN1162/HN/CHN/2010 genome was 7411bp. Compared with the other CoxA16 strains released in GenBank, the nucleotide similarities were 87.0-97.9%, 77.0%-95.4%, 80.3%-96.9%, 77.9% 96.2%, 80.5-100% in 5'UTR, P1, P2, P3, 3'UTR region, respectively; The similarities of nucleotide and amino acid sequences in VP1 region were 91.4%-96.4% and 99.3%-99.7%, respectively. Phylogenetic tree analysis showed that CoxA16 strains isolated from Henan, Shenzhen, Guangzhou and Fujian belonged to the same cluster. The newly isolated CoxA16 from Henan province belonged to subgenotype C2/B-2. These results will have great significance in monitoring CoxA16 and for prevention and control of hand-foot-mouth disease.

Simultaneous detection and identification of enteric viruses by PCR-mass assay.

Simultaneous detection of enteric viruses that cause similar symptoms (e.g. hand, foot and mouth disease) is essential to the prevention of outbreaks and control of infections. In this study, a novel PCR-Mass assay combining multiplex polymerase chain reaction (PCR) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was developed and used for simultaneous detection of eight distinct human enteric viruses. Enteric viral isolates and standard viral RNAs were examined to determine the sensitivity and specificity of the PCR-Mass assay. Clinical performance was evaluated with a total of 101 clinical specimens from patients suspected of having hand, foot and mouth disease (HFMD). The results were compared to those of previous analyses using real-time RT-PCR. The identification of specific viruses and clinical specimens shows that the PCR-Mass assay performed as well as or better than standard methods with respect to indicating the presence of multiplex pathogens in a single specimen.

[Complete genome sequencing of enterovirus 71 (EV71) HENAN08 strain isolated in Henan province].

OBJECTIVE: To sequence the whole-genome of enterovirus 71 (EV71) srtain isolated from patient with hand, foot and mouth in Henan province in 2008. METHODS: Eight overlapping clones covering the whole viral genome were obtained by RT-PCR and the sequences were determined by Sanger dideoxg-mediated chain termination method. RESULTS: Data it showed that the full length of enterovirus 71 (EV71) HENAN08 genome (not including Poly A tail) is 7405 bp. No deletion or insertion was detected in the coding region. There were several deletions and insertions in 5'UTR and 3'UTR regions. In P1 region, HENAN08 strain shared high homology with AnhuiFY08 strain, Zhejiang08 strain and SHZH strains (SHZH98, SHZH03) but low homology with Cox. A16. In P2 and P3 regions, HENAN08 strain shared higher nucleotide homology with Cox. A16 (81.7% and 83.7%) than that with BrCr and TW2086 strains. The phylogenetic analysis based on P1 region demonstrates that HENAN08 strain had the nearest genetic relationship with AnhuiFY and Zhejiang strains (isolated in 2008). CONCLUSION: The HENAN08 strain might belong to the same genogroup with AnhuiFY08 and Zhejiang08 strains as C4 gene subtypes.