BACKGROUND: Severe hand-foot-and-mouth disease (HFMD) is a life-threatening contagious disease among young children and infants. Although enterovirus A71 has been well acknowledged to be the dominant cause of severe HFMD, there still remain other unidentified risk factors for severe HFMD. Previous studies mainly focused on identifying the individual-level risk factors from a clinical perspective, while rare studies aimed to clarify the association between regional-level risk factors and severe HFMD, which may be more important from a public health perspective. METHODS: We retrieved the clinical HFMD counts between 2008 and 2014 from the Chinese Center for Disease Control and Prevention, which were used to calculated the case-severity rate in 143 prefectural-level cities in mainland China. For each of those 143 cities, we further obtained city-specific characteristics from the China City Statistical Yearbook (social and economic variables) and the national meteorological monitoring system (meteorological variables). A Poisson regression model was then used to estimate the associations between city-specific characteristics (reduced by the principal component analysis to avoid multicollinearity) and the case-severity rate of HFMD. The above analysis was further stratified by age and gender to examine potential modifying effects and vulnerable sub-populations. RESULTS: We found that the case-severity rate of HFMD varied dramatically between cities, ranging from 0 to 8.09%. Cities with high case-severity rates were mainly clustered in Central China. By relating the case-severity rate to city-specific characteristics, we found that both the principal component characterized by a high level of social and economic development (RR = 0.823, 95%CI 0.739, 0.916) and another that characterized by warm and humid climate (RR = 0.771, 95%CI 0.619, 0.960) were negatively associated with the case-severity rate of HFMD. These estimations were consistent across age and gender sub-populations. CONCLUSION Except for the type of infected pathogen, the case-severity rate of HFMD was closely related to city development and meteorological factor. These findings suggest that social and environmental factors may also play an important role in the progress of severe HFMD.
Adolescent ; Child ; Child, Preschool ; China/epidemiology* ; Cities/epidemiology* ; Female ; Hand, Foot and Mouth Disease/virology* ; Humans ; Incidence ; Infant ; Infant, Newborn ; Male ; Risk Factors

Objective: To estimate the serotype and age-specific hospitalization burden associated with hand, foot and mouth disease (HFMD) in Anhua county of Hunan province, between October 2013 and September 2016. Methods: We collected hospitalization records of HFMD patients from 6 virological surveillance hospitals, and reimbursement records through new rural cooperative medical system from 23 township health centers to estimate the age-specific hospitalization burden of HFMD in Anhua. Combined with the results of virological surveillance, the serotype-specific hospitalization burden of HFMD in Anhua, was estimated. Results: During the three years, it was estimated that 3 541 clinical diagnosed HFMD cases, including 3 146 laboratory-confirmed HFMD cases, were hospitalized in Anhua, but only one was diaguosed as being severe. The estimated average hospitalization rate was 723/100 000(95%CI: 699/100 000-747/100 000) for clinical diagnosed HFMD and 642/100 000 (95%CI: 620/100 000-665/100 000) for laboratory-confirmed HFMD between October 2013 and September 2016. The cases caused by Cox A16 (208/100 000) and Cox A6 (202/100 000) had higher hospitalization rates compared with the cases caused by EV71 (130/100 000), Cox A10 (38/100 000) and other enterovirus (64/100 000), and the difference was statistically significant (P<0.001). HFMD-associated hospitalization rates peaked in children aged 1 year (3 845/100 000), and then decreased with age. Compared with the hospitalized HFMD caused by EV71 and Cox A16, Cox A6-associated hospitalizations mainly occurred in younger age groups (P<0.001). Conclusion: Our study revealed a substantial hospitalization burden associated with mild HFMD caused by EV71, Cox A16, Cox A6 and Cox A10, especially in young children, in Anhua.
Child ; China/epidemiology* ; Enterovirus ; Enterovirus A, Human/isolation & purification* ; Enterovirus Infections/virology* ; Hand, Foot and Mouth Disease/virology* ; Hospitalization/statistics & numerical data* ; Hospitals/statistics & numerical data* ; Humans ; Infant ; Serogroup

OBJECTIVEKnowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes.

METHODSIn this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing.

RESULTSWithin the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run.

CONCLUSIONMinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.

Child, Preschool ; Enterovirus ; genetics ; Enterovirus A, Human ; genetics ; Enterovirus Infections ; virology ; Feces ; Genome, Viral ; Hand, Foot and Mouth Disease ; virology ; Humans ; Nucleic Acid Amplification Techniques ; instrumentation ; methods

OBJECTIVETo investigate the etiology of severe hand-foot-mouth disease (HFMD) in children in Henan province.

METHODSA 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.

RESULTSThere 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.

CONCLUSIONSEV71 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.

Amino Acids ; genetics ; Biometry ; Child ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; epidemiology ; virology ; Evolution, Molecular ; Genotype ; Hand, Foot and Mouth Disease ; epidemiology ; prevention & control ; virology ; Hospitals ; statistics & numerical data ; Humans ; Real-Time Polymerase Chain Reaction

OBJECTIVETo study the molecular epidemiology of hand-foot-mounth disease (HFMD) associated Coxsackievirus A10 (Cox A10) identified in Fujian province.

METHODSA total of 1 525 specimens from non-EV71 non-Cox A16 HFMD patients were collected during 2011-2014. Isolated virus strains were identified and sub-typed. Full-length coding regions for the VP1 gene of the predominant serotype Cox A10 isolates were amplified and sequenced.

RESULTSAmong the 407 non-EV71 non-Cox A16 HFMD cases confirmed by virus isolation and molecular subtyping, 103 (25.3%) were caused by Cox A10, accounting for 11.0%, 6.0%, 18.4% and 9.2% among the HFMD-associated entero-viruses identified in 2011, 2012, 2013 and 2014, respectively, in Fujian province. Compared to the general features observed in the HFMD epidemics, no differences on the Cox A10-specificity rates were observed among factors as geographical origins, gender or age groups, but all with high rates of severity. Data from the nucleotide sequence analyses on VP1 genes showed low homology levels of 76.0%-77.1% among Cox A10 strains from Fujian province, in contrast to the prototype Cox A10 strain, but with high levels of homology in the amino acid sequences (91.9%-93.6%). RESULTS from the Phylogenetic analysis also indicated that Cox A10 isolates from Fujian province were distinct from the prototype strain or other isolates from other countries but was homologous to domestic strains, but the Fujian isolates clustered into multiple branches.

CONCLUSIONSCox A10 remained one of the predominant serotypes of HFMD in Fujian province. Cox A10 isolates identified in Fujian province were co-circulating and co-evolving with other domestic strains.

Benzeneacetamides ; Child ; Child, Preschool ; China ; epidemiology ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Epidemics ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; genetics ; virology ; Humans ; Infant ; Male ; Molecular Epidemiology ; Molecular Sequence Data ; Open Reading Frames ; Phylogeny ; Piperidones ; Serogroup

Hand, foot, and mouth disease (HFMD) is a viral infectious disease regarded to be a public-health problem worldwide. Since the 1990s, HFMD began to spread in the Asia-Pacific region (especially in South-East Asia). HFMD outbreaks have occurred in mainland China frequently since 2008, and the morbidity and mortality of HFMD has continued to increase in recent years. In mainland China, enterovirus A serotype enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) have been the major pathogens of HFMD during these years. However, the etiological spectrum of HFMD changes with time. This review focuses mainly on the etiological spectrum of HFMD and changes in epidemic patterns in mainland China.
China ; epidemiology ; Disease Outbreaks ; Enterovirus ; classification ; genetics ; isolation & purification ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Prevalence

OBJECTIVETo investigate the genetic characteristics of coxsackievirus A10(CV-A10) strains isolated from hand, foot and mouth disease (HFMD) cases in Ningxia province.

METHODSBased on the HFMD laboratory network surveillance system, 2 470 patients clinical specimens including 450 faeces and 2 020 throat swaps were collected from various regions people's hospital in Ningxia Hui Autonomous Region during January, 2013 to December, 2014. All specimens were isolated using rhabdomyosarcoma cells. VP1 regional gene of isolated strains was amplified by RT-PCR using degenerate primers and sequenced. Sequences were compared with the database of GenBank by the Blast algorithm to identify the enterovirus genotypes. All the CV-A10 strains were performed the homology and phylogenetic evolution analysis.

RESULTS450 specimens identified as non-EV-A71, non-CV-A16 enterovirus were collected and 36 CV-A10 strains were isolated, 6 strains were isolated in 2013 and 30 strains were isolated in 2014. The homology of nucleotides and amino acids among 36 CV-A10 strains were 90.6%-100.0% , and 90.2%-100.0%, respectively. Compared 36 strains with genotype A, B, C, D representative strains, it has the highest homology with the genotype C, the nucleotide and amino acids homogeneity were 90.2%-98.9% and 95.7%-99.7%. The phylogenetic tree showed 36 strains and genotype C representative strains located in the same evolutionary branch.

CONCLUSIONCV-A10 was one of the most common pathogen of HFMD in Ningxia Hui Autonomous Region. All CV-A10 strains belonged to genotype C and contained wide homology range.

China ; Enterovirus ; genetics ; Genotype ; Hand, Foot and Mouth Disease ; virology ; Humans ; Phylogeny ; Sequence Homology, Amino Acid ; Sequence Homology, Nucleic Acid

OBJECTIVETo develop a simple, rapid and sensitive colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of coxsackievirus A6 (CV-A6) based on the colour chang of hydroxy naphthol blue (HNB).

METHODSThe method employed a set of six primers that recognized sequences of VP1 gene for amplification of nucleic acid under isothermal conditions at 63 °C for 50 min. The products were detected through visual inspection of color change by the pre-addition of HNB dye. The specificity was validated by detecting a collection of different human enteroviruses. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of CV-A6 VP1 gene, and compared with real-time RT-PCR (rRT-PCR) in parallel. This assay was evaluated with 92 clinical specimens from patients with hand-foot-mouth disease.

RESULTSA positive color (sky blue) was only observed in the preparation of CV-A6, whereas none of the other 23 kinds of human enteroviruses showed a color change. The HNB based RT-LAMP showed a sensitivity of 100 copies/reaction, which was at the same level as that of the rRT-PCR. The result of RT-LAMP in analysis of 92 clinical specimens was consistent with that of the rRT-PCR. The kappa correlation between the two methods was 1 and both of the sensitivity and specificity of the RT-LAMP assay were 100%.

CONCLUSIONThe established RT-LAMP assay had good specificity and sensitivity and thus demonstrated to be a promising screening tool for CV-A6 infection. It also has the potential to be used in resource-limited clinical sites and field study.

Colorimetry ; Coloring Agents ; chemistry ; DNA Primers ; Enterovirus ; isolation & purification ; Hand, Foot and Mouth Disease ; virology ; Humans ; Indicators and Reagents ; chemistry ; Naphthalenesulfonates ; chemistry ; Nucleic Acid Amplification Techniques ; Real-Time Polymerase Chain Reaction ; Reverse Transcription ; Sensitivity and Specificity

OBJECTIVETo analyze the genotype, epidemic pattern and the characteristics of the disease of enteroviruses during the epidemic season of hand, foot and mouth disease (HMFD) in children from 2013 to 2014 in Beijing to provide the scientific evidence for prevention and treatment of HFMD.

METHODDuring April to September in 2013 and March to October in 2014, a total of 977 throat swabs were collected from children who visited the Children's Hospital Affiliated to Capital Institute of Pediatrics, including 147 from patients with HFMD in 2013, 343 with HFMD, 201 with atypical HFMD, 83 with herpangina, 25 with fever with convulsions, 64 fever with rash and 114 with rash in 2014. Enteroviruses universal type (EV), Enteroviruses type 71 (EV71) and Coxsackievirus group A 16 (CA16) were detected by real-time RT-PCR respectively. The nucleic acid of specimens which were identified with non-EV71, non-CA16 was tested by nested PCR and analyzed by VP1 sequencing. The detection rate and epidemic pattern of different genotypes of enterovirus were analyzed among different age groups and between 2013 and 2014.

RESULTOf 977 throat swabs, 80. 1% samples were detected positive for enteroviruses. The positive rates of CA16, EV71, CA6, CA10, CA4 and other EVs were 25. 6% (250/977), 18. 9% (185/977), 20. 0% (195/977), 5. 0% (49/977), 1.5% (15/977) and 9.1% (89/977), respectively. Twenty six of the 89 other EVs included CA2, CA5, CA8, CA9, CA12, CA14, CB2, CB5, E6, E9 and E25, each genotype of which was no more than 3. The nucleotide homologies shared among CA6, CA10 and CA4 strains between 2013 and 2014 were 94. 3% - 100%, 93. 8% - 99. 1% and 92.7% - 99. 8%, respectively. The positive rates of ≤1 year group were 71. 1% (106/149), which was lower than that of other age groups (all P <0. 05), but similar to that of >5 year group (χ2 =1. 181,P = 0. 277). In 2013, the positive rate of EV was 85. 7% (126/147) and the predominant genotype was CA6 54. 8% (69/126), followed by CA16 20. 6% (26/126) and EV71 11. 9% (15/126). In 2014, the positive rate of EV was 85. 4% (293/343) in the 343 children with HFMD, the predominant genotypes were CA16 with the positive rate of 42. 7% (125/293), EV71 with 38. 2% (112/293) and CA6 with only 11. 3% (33/293). In 2014, the positive rates of EV in 201 atypical HFMD, 83 herpangina, 25 fever with convulsions, 64 fever with rash and 114 rash were 83. 6% (168/201), 80. 7% (67/83), 76. 0% (19/25), 64. 1% (41/64) and 60. 5% (69/114), respectively. All genotypes of enteroviruses peaked mainly during May to August every year, but there were no obvious epidemiological pattern about each genotype.

CONCLUSIONCA6 became the main causative agent of HFMD in 2013, however, CA16 and EV71 predominated again in 2014 in Beijing. The clinical manifestations caused by CA6, CA10, CA4 and other genotype of enteroviruses differed from EV71 and CA16. Besides EV71 and CA16, more attention should be paid to CA6, CA10, CA4 and other type of enteroviruses.

Beijing ; epidemiology ; Child, Preschool ; Enterovirus A, Human ; classification ; Enterovirus Infections ; epidemiology ; virology ; Exanthema ; Fever ; Genotype ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Infant ; Real-Time Polymerase Chain Reaction

OBJECTIVETo investigate the etiology of hand, foot and mouth disease (HFMD) in Beijing during 2013, and study the clinical characteristics of HFMD caused by the main serotypes of enterovirus in the study.

METHODClinical data and 128 stool samples were collected from 128 hospitalized children with HFMD in Beijing Ditan Hospital during 2013. One step RT-PCR method was used for enterovirus genotyping to investigate the etiology of HFMD. Clinical characteristics of HFMD caused by the main serotypes of enterovirus were analyzed. And VP1 segments of the main virus were amplified to construct phylogenetic tree for the phylogenetic analysis.

RESULTA total of 128 hospitalized children with HFMD were included. HFMD was more likely developed in children under 2 years of age (81.6%, 102/125); 11 different enteroviruses were genotyped, with a total enterovirus positive rate of 76.6% (98/128); the positive rate of coxsackievirus A6 (CA6), 43.0% ( 55/128), was the highest, followed by enterovirus 71 (EV71), accounting for 14.8% (19/128). HFMD caused by CA6 was atypical, the rashes of which involved the perioral, trunk, limbs, face and neck (47%, 26/55), besides the common parts. Of the 55 cases caused by CA6, 6 children had clinical manifestations of nervous system involvement, one of whom even displayed type 2 respiratory failure. Mental status change more likely to occur in EV71-infected children than in CA6-infected ones (42% (8/19) vs. 11% (6/55) (χ(2)=7.041, P=0.008)); 13 children displayed onychomadesis, including 12 CA6 cases (23%, 12/53) and 1 CA10 cases (17%, 1/6), in the convalescence of hand, foot and mouth disease, and the correlation between onychomadesis and CA6 infection was significant (χ(2)=9.297, P=0.002). Phylogenetic analysis of 33 CA6 VP1 showed that the CA6 isolates of this study were highly similar to that of Taiwan and the nucleotide similarity was 95.91%-98.89%.

CONCLUSIONCA6 was the major pathogen of hospitalized children with hand, foot and mouth disease in Beijing during 2013, followed by EV71. The rashes caused by CA6 involved a wide range of skin sites and patients with CA6 infection displayed manifestations of neurological involvement or pulmonary edema similar to EV71 infection. Mental status change more likely occurred in EV71-infected children when neurological system was involved..

Child, Hospitalized ; Child, Preschool ; Enterovirus ; classification ; Enterovirus Infections ; diagnosis ; virology ; Exanthema ; pathology ; Genotype ; Hand, Foot and Mouth Disease ; diagnosis ; virology ; Hospitals ; Humans ; Infant ; Phylogeny ; Pulmonary Edema ; pathology ; Skin ; pathology ; Taiwan