Introduction
Hand, foot, and mouth disease (HFMD) is a common acute enterovirus (EV) infection, characterized by short-lasting fever, mouth ulcers, and vesicles on the hands, feet, or hips [
1]. In March 2008, a sudden outbreak of HFMD occurred in Anhui Province, China. In May, HFMD was defined as a C-class notifiable disease. HFMD has the highest incidence among communicable diseases since 2009 and has become an important public issue [
2,
3]. Although HFMD is generally a mild clinical syndrome [
4,
5], serious complications may occur [
6]. Although HFMD age of onset is widely variable, ranging from neonatal age to 70 years, children aged 5 years and younger are the most susceptible subjects and may develop severe clinical symptoms [
7,
8]. Reportedly, subjects younger than 3 years have an increased risk of severe HFMD [
2,
8]. However, to date, the age-specific risk of severe HFMD in young children has not been established.
Human EVs belong to the family Picornaviridae, and based on the degree of their genetic relatedness, comprise four species, EV-A to D. Among them, the serotypes EV-A71, Coxsackievirus A16 (CV-A16), CV-A6, and CV-A10, which are frequently associated with HFMD, belong to the EV-A species [
9]. CV-A16 and EV-A71 are responsible for most of the large outbreaks [
10]. Among healthy individuals in Shanghai, 50.5 and 54.2% are positive for neutralising antibodies against EV-A71 and CV-A16, respectively [
11]. Beginning in 2008, CV-A6 has been increasingly reported as a cause of HFMD outbreaks worldwide, and it may be associated with more severe diseases than typical HFMD [
4,
12‐
18].
HFMD can be transmitted both horizontally (faecal-oral/respiratory route) and vertically (prenatal infection). Most new-borns presenting with serious EV disease acquire the infection from a symptomatic mother in the perinatal period; up to 60% of the mothers of infected infants report febrile illness during the last week of pregnancy [
19]. Additionally, serious EV disease may be acquired through nosocomial transmission, spreading throughout nurseries via caregivers engaged in mouth care, gavage feeding, and other activities requiring direct contact. Close contact with infected family members may be also an important route of transmission.
In this prospective cohort study, the neonates with HFMD and their families were recruited in Shanghai in 2016–2017, and the epidemiological features, clinical presentation, pathogens, genes, and immune function were compared in neonates with HFMD and their diseased siblings.
Materials and methods
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 RT-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, 12,608 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 1 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 6 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 RT-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.
Discussion
HFMD is one of the most recognizable viral exanthems in children and adults [
26], but rarely reported in neonates. According to this study, only 0.13% of all HFMD cases were neonates in Shanghai in 2016–2017. All 16 neonates became infected from other family members, mainly their elder siblings. They were all diagnosed with CV-A6 infection and had mild clinical symptoms. Neonatal HFMD cases showed normal immune function. Almost all cytokines exhibited higher plasma levels in cases than in controls.
In this study, the age of neonatal onset ranged between 19 and 28 days, and the mothers had no prenatal infection symptoms; therefore, vertical transmission was not considered. In China, mothers usually rest indoors for one full month after giving birth, avoiding contact with people outside of the family. Therefore, the chances of infection are relatively low for mothers. With the adoption of the two-child policy, the risk of infection is very high for elder siblings, who are generally pre-schoolers in kindergartens [
27]. In addition, according to epidemiological evidence, elder siblings were infected earlier than the neonates, and the nucleotide sequence of CV-A6 viruse similarities between the neonates and elder siblings were 100%, which indicated that the neonates and elder siblings were infected by the same CV-A6 strain. The most likely scenario is that the elder siblings with HFMD acquired the infection from an unknown common source and transmitted the virus to neonates on returning home. This further supported within-family transmission. However, establishing the transmission pathway is still a difficult challenge.
In the literature, significant clinical differences were reported in HFMD manifestations depending on the pathogen. Genetic typing to establish the exact virus strain is usually not necessary to confirm the HFMD diagnosis. However, in some cases of HFMD, identification of the virus type is crucial for appropriate disease management and to reliably assess the risk of potential complications. The sole published case of neonatal EV-A71 infection was quite severe. Another reported case of CV-B3 infection, which was not fatal and self-limited in children, also caused severe disease in a neonatal case. However, none of the five neonates clinically diagnosed with HFMD in southeast China developed brainstem encephalitis or pulmonary oedema, and all recovered well. In our study, CV-A6 was the predominant causative agent of HFMD in all patients. Neonatal HFMD cases were all diagnosed with CV-A6 infection and exhibited mild symptoms. Moreover, the incidence of fever, vomiting, and onychomadesis was lower among neonatal cases compared to elder children. Some HFMD cases exhibited an atypical skin presentation with facial involvement and vesiculobullous lesions throughout the body.
Immunological reactions may be critical for HFMD. Almost all fatal HFMD cases had symptoms of autonomic nervous system dysregulation and increased sympathetic discharge, indicating the involvement of reticular formation [
28]. Systemic inflammatory response also played an important role. Consistently, several studies have reported that virus infection activates the host immune system, causing the release of cytokines, as well as tissue and cell damage [
29]. In our study, cytokine and WBC levels were increased in both neonatal and elder sibling patients. However, neonatal HFMD cases showed significantly lower CD8 T-cell counts compared to diseased elder siblings, which is not uncommon in acute viral infection. The T-cell subset assay is an accurate method to evaluate cellular immunity, and abnormal results may indicate the occurrence or aggravation of viral diseases. Notably, Wang and colleagues previously found that CD4 T-cells, CD8 T-cells, and NK cells are depleted in patients with pulmonary oedema, possibly resulting in impaired EV-A71 clearance [
30]. Another study reported that CD4 T-cells are decreased, while CD8 T-cells are not affected, in patients with HFMD [
31]. There are few reports on neonatal HFMD. We speculate that the low level of CD8 T-cells in neonatal cases is related to the sampling time. Although all samples were collected at admission, the time interval from onset to clinical evaluation may have differed between patients. Since only the symptomatic population was considered, this study may contain a selection bias. In addition, some results, such as the low level of CD8 T cells observed in neonatal HFMD cases, need to be confirmed by performing further studies.
Conclusions
Neonatal HFMD caused by CV-A6 was characterized by mild clinical symptoms and basically normal immune function. Neonatal HFMD is not always a serious condition, and disease severity may depend on the pathogen. In China, with the gradual adoption of the two-child policy, elder brothers or sisters are the main source of infection. In case of infection, control measures should be in place. In addition, as the prevalence of CV-A6 is on the rise, it will be crucial to explore the suitability of CV-A6 as the main component of combined or multivalent vaccines for HFMD prevention and control.
Acknowledgements
The authors thank Heyu Huang, Yingying Jin and Xinxin Zeng for their assistance in preparing this manuscript.
Writing assistance was provided by Elsevier Language Editing Services.
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.