Participants and specimens
This was a case-control study based on the National Registry of HFMD. The Chinese government established a network-based national surveillance system for HFMD since 2009. In Shanghai, local health providers and physicians are required to report clinically diagnosed HFMD cases to the Shanghai Municipal Centre for Disease Control and Prevention (CDC) within 24 h via the surveillance system. Basic epidemiologic and clinical information is recorded for each HFMD patient [20]. Sixteen local CDCs, representing as many districts, are responsible for sample collection and transport. The specimens of patients were sampled for pathogen testing at local sentinel hospitals in each district. At least ten outpatients were diagnosed with HFMD each month. The clinicians could also test the specimens as the conditions required. Throat and/or faecal swabs were sent directly to microbiology laboratories at the local CDCs, where the presence of EV-A71, CV-A16, CV-A6, CV-A10, and other EVs was confirmed by real-time PCR [11]. The vast majority of children with HFMD are treated in two designated hospitals, the Children’s Hospital of Fudan University and the Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine.
All cases were diagnosed according to the criteria specified by the HFMD Prevention and Treatment Guidelines [21]. Patients who had a rash, with or without fever, and no other organ damage, were classified as having common HFMD. Those with any complication (i.e., aseptic meningitis, brainstem encephalitis, encephalitis, encephalomyelitis, acute flaccid paralysis or autonomic nervous system dysregulation, pulmonary oedema, pulmonary haemorrhage, or cardiorespiratory failure), or those who died, were classified as severe HFMD cases. From January 2016 to December 2017, 12608 patients were diagnosed with HFMD at Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine. The distribution of patients covered all 16 municipal districts in Shanghai.
Patients who met the following criteria were recruited in our study: 1. neonates diagnosed less than 28 days after birth; 2. skin lesions manifested as small vesicles, papulovesicular lesions or macular rashes on the palms, soles, buttocks, and oral mucosa, or were present on the limbs, trunks or facial areas. All family members were included in the screening. Since the prognosis of neonatal HFMD is unknown, they were all admitted to the hospital for observation, including routine clinical blood examination, evaluation of biochemical and immune function, and virus detection. The clinical specimens (e.g., rectal swabs and plasma) were collected from each patient within one day of diagnosis. To evaluate alterations in specific parameters, such as in immune function, we also recruited age- and birth weight-matched non-infected neonates (e.g., infants with breast milk jaundice) as neonatal controls, and age-matched preoperative patients without infection (e.g., subjects with hypospadias) as elder sibling controls. The control subjects had no symptoms of HFMD and tested negative for enteroviruses. Finally, 16 neonates with HFMD and their infected families were included in the study and followed up for at least six months for sequelae.
This study was approved by the Ethics Committee of Xinhua Hospital, affiliated to Shanghai Jiao Tong University School of Medicine (XHEC-C-2018-082), and the procedures were carried out in accordance with the Helsinki Declaration. Parents or guardians of each case or control were required to sign a written informed consent form. The relevant tests were paid by the research group.
Data collection
Demographic data, clinical manifestations, and laboratory findings of each participant were recorded. Fever, as well as timing and distribution of skin lesions, were evaluated. The skin lesions were classified into 8 groups based on the site: perinasal, perioral, scalp, palms/soles, lower limbs, upper limbs, abdomen, and intraoral lesions.
Complete blood cell count, liver and kidney function, and the levels of myocardial enzymes, immunoglobulins, lymphocyte subsets, and cytokines were assessed in cases and controls. The immunophenotypes of peripheral blood lymphocytes (CD3, CD4, and CD8 T-cells, NK cells) were determined by flow cytometry (Becton Dickinson Immunocytometry Systems) and analysed by Cell Quest software (Becton Dickinson). The serum levels of immunoglobulin (Ig) M, IgG, and IgA were detected by turbidimetric immunoassay. ELISA (Quantikine; R&D Systems) was used for quantitative determination of the cytokines IL-1β, IL-2R, IL-6, IL-8, IL-10, and TNF-α. The assays were performed according to the manufacturer’s instructions.
The EVs were genotyped from rectal swab specimens. Viral RNA was extracted directly from the clinical specimens using a QIAamp Viral RNA Mini Kit (Qiagen, Santa Clara, CA) and stored at -80 °C. A commercial real-time PCR Kit panel (Jiangsu Bioperfectus Technologies Co., Ltd., China, http://en.s-sbio.com/) was used to determine enterovirus type and subtype, including EV-A71, CV-A16, CV-A6, and CV-A10, as previously described [22, 23]. A partial VP1 gene sequence was amplified using one-step reverse transcription polymerase chain reaction (TaKaRa) with primers 292/222 as previously described [24], and the amplicons were sequenced directly. EVs were genotyped by sequence comparison by using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequenced DNA fragments were assembled into complete genomes using ContigExpress project in Vector NTI version 11.5. Multiple-sequence alignments were performed using the MAFFT software (http://www.ebi.ac.uk/Tools/mafft/). Phylogenetic trees were constructed by the maximum likelihood (ML) method using the MEGA version 7 software [25].
Statistical analysis
We calculated the means and standard deviations for normally distributed variables, and the medians (interval of quartiles) for variables with skewed distribution. For pairwise comparisons, Student’s t-test and nonparametric tests were applied in case of normal and non-normal distributions, respectively. Frequency and percent values were calculated for categorical variables, and the chi-square test was used to determine the differences in these variables between neonatal and paired siblings with HFMD. Logistic analysis was applied to calculate the risk of clinical manifestations in these two groups. All statistical analyses were conducted using SPSS 17.0 software. A p-value <0.01 was regarded as statistically significant.