Participants and specimens
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 hours via the surveillance system. Basic epidemiologic and clinical information was recorded for each HFMD patient [12]. Sixteen local CDC representing 16 districts are responsible for collecting samples and transporting them in their own districts. However, 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. The specimens of patients were sampled for pathogen test at local sentinel hospitals in each district, at least ten outpatients diagnosed with HFMD per month. The clinicians can also test the specimens as the condition requires. Throat swabs or/and faecal samples (rectal swabs) are then sent directly to microbiology labs at the local CDCs, where EV71, CV-A16, CV-A6, CV-A10 and other EV can be confirmed by real-time PCR [13].
All cases were diagnosed according to the criteria of HFMD Prevention and Treatment Guidelines [14]. Patients who had a rash, with or without fever, and no other organ damage, were classified as having common HFMD. Those with any complications (i.e., aseptic meningitis, brainstem encephalitis, encephalitis, encephalomyelitis, acute flaccid paralysis or autonomic nervous system dysregulation, pulmonary edema, pulmonary hemorrhage, or cardiorespiratory failure), or those who died, were classified as having severe HFMD. 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 the 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 were manifest as small vesicles, papulovesicular lesions or macular rashes on the palms, soles, buttocks and oral mucosas, or presenting on the limbs, trunks or facial areas. All of the family members were also included for screening. Because the prognosis of neonatal HFMD is unknown, they were all admitted to the hospital for observation, including routine clinical blood examination, biochemical function, immune function, and virus detection. Infected family members were also fully evaluated and treated. The clinical specimens (e.g., rectal swabs and plasma) were collected from each patient within one days of diagnosis. To compare the differences of immunological function, we also recruited age- and birth weight-matched non-infected neonates (e.g., breast milk jaundice) as neonatal controls, the age-matched preoperative patients without infection (e.g., hypospadias) as elder sibling’s controls. Finally, 16 neonates with HFMD and their infected families were included, and all of them were 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 subject were required to sign a written informed consent form.
Data collection
Demographic data, clinical manifestations, and laboratory findings of every participant were recorded. Among the clinical manifestations, fever, timing of skin lesions, and distribution of skin lesions were evaluated. The skin lesions were classified into 8 sites: peri-nasal, peri-oral, 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 each case and control. The immunophenotypes of peripheral blood lymphocytes (CD3, CD4, and CD8 T-cells; NK cells) were determined by flow cytometry (Becton Dickinson Immunocytometry Systems) and were analysed by Cell Quest software (Becton Dickinson). The serum levels of immunoglobulin (Ig) M, IgG, and IgA were detected by turbidimetric immunoassay. ELISA for quantitative determination (Quantikine; R&D Systems) was used to analyse the levels of 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 rectal swab specimens from each patient were genotyped for enterovirus. Viral RNA was extracted directly from clinical specimens using a QIAamp Viral RNA Mini Kit (Qiagen, Santa Clara, CA) and was stored at -80 °C. The identification of EV and serotyping of EV71 and CV-A16 from samples were performed by real-time reverse transcription-polymerase chain reaction (RT-PCR) as previously described [15, 16]. To further identify EV serotypes other than EV71 and CV-A16, semi-nested RT-PCR and sequencing were conducted as previously described [17]. The serotype was determined by comparison of the viral sequences with corresponding sequences of the EV prototype strains using blast online (https:// blast.ncbi.nlm.nih.gov/ Blast.cgi).
Statistical analysis
We calculated the means and their standard deviations of normal distributed variables and medians (interval of quartiles) of skewed distributed variables. T-test was used only if it was normally distributed, and the nonparametric test were applied for those abnormally distributed when pair-wise comparisons were made. The cytokine levels all showed abnormal distributions; thus, they were log-transformed into normal distributions in the t-test analysis. Frequency and percent values were calculated for categorical variables, and chi-square test was used to test the difference in the categorical variables between the neonatal HFMD and paired sibling HFMD. Logistic analysis was applied to identify the risk of clinical symptoms in these two groups. All statistical analyses were conducted using SPSS 17.0 software. P-value <0.01 was regarded as statistically significant.