Neonatal hand, foot, and mouth disease: a case-control study in Shanghai

Background Evidence of hand, foot, and mouth disease (HFMD) in neonates is limited. This study aimed to report the clinical symptoms, possible transmission routes, and prognosis of neonatal HFMD in Shanghai. Methods This was a case-control study based on registry system of HFMD. All neonates and infected family members were enrolled between 2016 and 2017 in Shanghai. Neonates with HFMD were followed for at least half a year. The detailed questionnaires, medical history and physical examination were recorded. Routine blood examination, liver and renal function, immunophenotypes of peripheral blood lymphocytes (CD3, CD4, and CD8 T-cells; NK cells), immunoglobulin (Ig) M, IgG, and IgA and cytokine interleukin (IL-1β, IL-2R, IL-6, IL-8, IL-10, and TNF-α) levels were detected. All rectal swab specimens were collected and genotyped for enterovirus. T-test or nonparametric test was used to evaluate the differences. Logistic analysis was applied to find the risk of clinical symptoms in the group of neonates and their HFMD paired siblings. Results There were 16 neonates among the 12608 diagnosed patients with HFMD, accounting for 1.3‰. All the neonates were transmitted within-family, mainly by the elder sibling, with different types of coxsackievirus A6 infection. Coxsackievirus A6 was also the emerging and predominant causative agent of HFMD in Shanghai. None of the neonates with HFMD suffered fever, onychomadesis, or severe complications. However, two elder sibling patients showed lethargy, and one developed hypoperfusion. The white blood cells in the elder siblings with HFMD were generally higher than the neonates with HFMD. The immunologic function of the neonates was basically normal. The inflammatory response was high regardless of the neonates or elder siblings. The clinical symptoms receded at about one week. None of the neonates had sequalae.

Introduction 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 ttest 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.

Clinical presentations of the neonates with HFMD
There were 16 neonates among the 12608 diagnosed cases with HFMD at Xinhua Hospital affiliated to Shanghai Jiao Tong University in Shanghai 2016-2017, accounting for 1.3‰.
Among them, there were 14 cases of severe HFMD, and most of which were EV71 infection. A total of 259 patients were sampled from this sentinel hospital for routine enterovirus surveillance, among which 206 cases were positive, with a positive rate of 79.54%. CV-A6 was the predominant causative agent of HFMD ( Figure 1). All of the neonatal cases were not severe, while two elder siblings developed severe HFMD cases.
Half of the neonates with HFMD were diagnosed in the summer.
The median age of the neonates with HFMD was 25 days, ranging from 21 to 27 days. All the neonates were full term with a normal birth weight. The median age of their elder siblings was 4.1 years with a range of between 2.4 years and 6.3 years. Among them, 37.5% of neonates and 62.5% of elder siblings were boys. As shown in Table 1, the symptoms of neonates with HFMD cases were milder than their elder siblings. None of the neonates developed complications of hypoperfusion, lethargy, onychomadesis, or other complications. Most of the elder siblings had fever, whereas none of the neonates did. Ten elder sibling patients had vomiting symptoms, two had lethargy, and one developed hypoperfusion. The prevalence of vomiting was 5 times higher in the elder siblings than in the neonates. Cutaneous lesions were more common in elder siblings than in neonatal cases, especially intraoral erosions. The site of the rash of neonate cases was not typical, mainly in the peri-oral and upper limb ( Figure 2). After symptomatic treatment, the clinical symptoms receded at about one week, and no sequelae occurred within half a year.
Interestingly, all infected neonates had an elder sibling affected, and two parents in the Discussion Neonatal HFMD is rarely reported in the literature. In this study, we found the percent of neonatal HFMD among all cases was only 1.3‰ in Shanghai, 2016-2017. All 16 neonates were infected within families, mainly from their elder siblings. They were diagnosed with different subtypes of CV-A6 infection aetiologically. Neonatal HFMD cases showed normal immune function. Almost all plasma levels of cytokines were significantly higher in cases than in their controls.
HFMD is a common acute enterovirus infection, characterized by a short-lasting fever, mouth ulcers, and vesicles on the hands, feet, or hips [18]. It can be transmitted both horizontally (fecal-oral/ respiratory route) and vertically (prenatal infection). Most newborns presenting with serious enterovirus disease acquire the infection from a symptomatic mother in the perinatal period; up to 60 percent of mothers of infected infants report a febrile illness during the last week of pregnancy [19]. Additionally, serious enterovirus disease is also acquired via nosocomial transmission, with spread throughout nurseries occurring via caregivers engaged in mouth care, gavage feeding, and other activities requiring direct contact. Close contact with infected family members or caregivers is also an important route of transmission. In our study, the age range of neonatal onset was 19-28 days, and the mothers had no prenatal infection symptoms, so vertical transmission is not considered. In China, mothers traditionally are expected to rest indoors for one full month after giving birth and avoid contact with people outside the family, so the infection chances are very low for mothers. With the adoption of the twochild policy, the potential of cross contamination is very high for elder siblings, who are generally pre-schoolers in kindergarten [20]. In addition, according to the epidemiological investigation, there were children in the family infected earlier than the neonates, and the pathogen was the same epidemic strain, so it is speculated that neonatal cases are mainly transmitted by the family. However, it's still hard to verify the transmitted pathway. increasingly reported as a cause of outbreaks of HFMD around the world, and it may also be associated with more severe disease than typical HFMD [4, 26-32]. It was reported that sporadic cases and epidemics in patients with CV-A6 appear, principally affecting elder children, adolescents, and adults rather than young infants. CV-A6 has replaced EV71 and CV-A16 as the most common pathogen causing HFMD in Shanghai [12]; thus, we inferred that most pregnant women had been infected asymptomatically, but the proportion of detected neutralizing antibodies in maternal blood was too low to protect their child.
In the literature, significant clinical differences were reported in the HFMD depending on the pathogen. Genetic typing to distinguish the exact virus strain is usually not necessary to confirm the HFMD diagnosis. However, in some cases of HFMD, information on the exact type of the virus is crucial for appropriate disease management and reliable assessment of the risk of potential complications. The sole published EV71 infection neonate case was quite severe. Another reported CV-B3, which was not fatal and was a self-limited pathogen in children, also caused severe conditions in a neonate case. However, none of the five neonates clinically diagnosed with HFMD developed brainstem encephalitis or pulmonary oedema, and they all recovered well in southeast China. CV-A6 had a broader spectrum of manifestations [33]. In our study, the symptoms of neonate HFMD cases were mild. The risk of neonate cases with symptoms of fever, vomiting, and onychomadesis was lower than that for elder children. They may have an atypical skin presentation with facial involvement and vesiculobullous lesions on the body.
Immunology reaction may be important to HFMD. Almost all the fatal HFMD cases had symptoms of autonomic nervous system dysregulation and increased sympathetic discharge, indicating the involvement of reticular formation. Immune activation, such as higher levels of cytokines and WBC, were the early biomarkers to measure the condition of the autonomic nervous system. In our study, the levels of these biomarkers were  P-value 1 : T-test between neonate HFMD and neonate control.