Analysis Of Clinical Characteristics And Pathogenic Genetic Characteristics Of Hand,foot And Mouth Disease In Qingdao Area In Autumn And Winter Of 2019

BackgroundHand-foot-mouth disease(HFMD)is an acute febrile disease with characteristic skin lesions on the hands,feet,and mouth,which can be infected by a variety of enteroviruses(EV).It mainly affects children under the age of five,but can also affect adults.HFMD has long been considered to be a mild viral infection with typical clinical manifestations that resolve spontaneously within a few days without complications.However,in the past two decades,HFMD has received new attention as evidence has emerged that the clinical,epidemiological,and etiological characteristics of the disease are very different from those initially recognized.In China,Shandong Province is one of the regions with more severe HFMD.As Qingdao is located near the sea and the air humidity is conducive to the prevalence of HFMD.In recent years,there have been multiple outbreaks of HFMD,and its incidence rate far exceeds the average incidence in Shandong Province.Therefore,clarifying the clinical and pathogenic features,molecular epidemiological characteristics,and gene recombination of HFMD in Qingdao area is of great significance for not only the diagnosis and treatment of the disease but also the formulation of effective prevention and control measures.PurposesIn this study,we collected and analyzed clinical data and throat swab specimens of 104 patients with clinical diagnosis of HFMD and complete medical records from Qingdao Women and Children’s Hospital and Central Hospital from September to December 2019,to understand the clinical characteristics and pathogen spectrum composition of patients with HFMD in autumn and winter.In addition,the genetic characteristics and recombination of CV-A6 and CV-A16 were analyzed to study their evolutionary relationships and the current popular recombination forms,so as to provide help for the prevention and treatment of HFMD.Methods1.After collecting clinical data and throat swab specimens from 104 patients with a clinical diagnosis of HFMD in Qingdao Women and Children’s Hospital and Central Hospital from September to December 2019,we performed statistical description and analysis of these 104 clinical data,and then compared the clinical features,laboratory and biochemical test results caused by CV-A6 and CV-A16 viruses.2.EV positive samples were screened by universal real-time fluorescence quantitative PCR of this virus.For positive samples,we further identified virus types by PCR amplification and sequencing of part of the VP1 gene.Then the next step,amplified the full length of CV-A6 and CV-A16 VP1 fragments,to analyze their homology,evolutionary relationship and amino acid changes.3.According to the phylogenetic trees,we amplified the full length of CV-A6 and CV-A16 3D genes,and downloaded representative reference sequences from the GenBank database,then constructed a phylogenetic tree to analyze the recombination form of its 3D region.4.For CV-A6 and CV-A16,we selected 8 samples each with CT value<30 for virus isolation,and TCID50 was used to test the virulence of the virus.Results1.The data collected this time for the autumn and winter of 2019 shows that the HFMD population in Qingdao is mainly infants and children under 5 years old,accounting for 76.9%;male patients are more than female,with a ratio of 2.47:1;scattered children account for patients 72.1%of the total;in addition,all 104 patients had fever with characteristic skin lesions on the hands,feet,mouth and other parts,and some of the children had skin lesions involving the limbs and trunk;77 patients had neurological symptoms,accounting for 74.04%.2.Etiological tests showed that 60 samples were EV-positive,with a positive rate of 57.7%,including 28 samples of CV-A6 and 32 of CV-A16,no other types of pathogens were found.Besides,no significant difference was found in the clinical characteristics of the two groups.3.The CV-A6 epidemic strain in Qingdao area belongs to the D3 gene subtype,the CV-A16 epidemic strain belongs to the B1a gene subtype and the B1b gene subtype;the nucleotide and amino acid homology between CV-A6 is 93.6-100%,98-100%,respectively;the nucleotide and amino acid homology with the prototype strain Gdula(GenBank Sequence Number:AY421764)is 82.5-83.8%and 95.4-96.4%,respectively.the nucleotide and amino acid homology between CV-A16 is 86.5-100%and 98.0-100%,respectively;the nucleotide and amino acid homology with the prototype strain G-10(GenBank sequence number:U05876.1)is 74.6-76.5%and 91.2-92.3%,respectively.The amino acid variation analysis with the prototype strain showed that there were 12 sites that were changed in CV-A6.Gln98Leu was located in the BC loop,and Gly160Ser and Ile174Val were located in the EF loop.There are 23 sites where CV-A16 changes.Ala213Glu,Pro215Leu,Ser217Ala,and Ala220Leu are in the GH loop.4.The popular recombination form of CV-A6 in Qingdao is RF-A,while RF-B and RF-D are popular recombination forms of CV-A16.5.Successfully isolated 3 strains of CV-A16 and 2 strains of CV-A6;the TCID50 titers of the 3 strains of CV-A16 were:5 × 106.875/ml,5 × 107/ml,5 × 106/ml;The TCID50 titers of the 2 strains of CV-A6 were 5 × 106/ml and 5 × 106.25/ml respectively.Conclusions1.In the autumn and winter of 2019,the HFMD patients in Qingdao were mainly caused by CV-A6 and CV-A16 infection.Among them,the patients were mainly infants under 5 years old,and mainly scattered children.There were more males than females in children.74.04%of patients with HFMD developed neurological symptoms.The prevalence of HFMD in the autumn and winter needs to be closely monitored.2.There were two subtypes prevalent in Qingdao in the autumn and winter of 2019.CV-A6 is the D3 gene subtype,and its nucleotides and amino acids are relatively conservative;the CV-A16 epidemic strain belongs to the Bla gene subtype and the B1b gene subtype.It is consistent with the current prevalence of CV-A6 and CV-A16 in China.Amino acid sequence variation analysis shows that the amino acid positions of CV-A6 VP1 BC loop,EF loop and CV-A16 VP1 GH loop have changed.3.In the autumn and winter of 2019,the popular CV-A6 recombination form in Qingdao area is RF-A,and the recombination form of CV-A16 is RF-B and RF-D,which is consistent with the current popular recombination form in China.