Introduction

Hand, foot, and mouth disease (HFMD) is a common childhood infectious disease with high morbidity,1 which always leads to mouth sores and rash on the hands and feet.2 Although this disease is mostly a self-limiting illness, a significant number of patients develop neurological and/or cardiopulmonary complications, such as encephalitis, aseptic meningitis, encephalomyelitis, acute flaccid paralysis and fatal cardiopulmonary complications.3 4 A long-term follow-up study suggests that 75% of severe HFMD cases have sequelae after discharge.5 HFMD sequelaes refer to some persistent or long-term symptoms or lesions of patients infected with HFMD virus and after treatment. The most common sequelae include but not limited to the following: nail loss, muscle weakness and atrophy of the limbs, facial nerve palsy, central hypoventilation, ptosis, neurodevelopmental disability and cognitive impairment.6 A large population-based study also suggests that enterovirus infections are associated with speech and language impairment in child survivors.7 The crucial period for humans' cognitive development is the first several years of early life; therefore, the deleterious neurocognitive effects of neurological complications (eg, encephalitis and encephalomyelitis) caused by severe HFMD are likely to be substantial and long lasting.8 There is sufficient evidence to show that neurological abnormalities in patients with HFMD can affect the patients' prognosis and future quality of life (QoL).9 As more paediatric patients survive HFMD, exploring the long-term outcomes is important for clinical management in the acute phase, follow-up programmes after the acute phase and health economics evaluation.

As a result, we pay more attention to the long-term sequelae of child survivors and conduct a hospital-based cohort study of patients with HFMD. The purpose of this cohort is to gain a comprehensive overview of the potential harm caused by this common infectious disease to assess the social burden caused by the disease and to make recommendations for the rehabilitation of survivors and the prevention of further disability.

Cohort description study design

The main content and specific objectives of this study include a retrospective cohort study to analyse the incidence of sequelae of surviving HFMD cases in Henan Province from 2014 to 2022. The most important part of the study is to establish a prospective cohort with long-term follow-up (in-person interviews, physical examination, laboratory tests and biosample collection) to ultimately understand the aetiology of the development of HFMD sequelae and the disease burden. The study design is shown in figure 1.

Figure 1

Flowchart of this cohort study.

Source data

This HFMD sequelae cohort study (HNHFMDCS) was conducted in Henan Province (Central China) as Henan Province has a large population base of children. According to the seventh census data released by the National Bureau of Statistics of China, Henan Province has the third largest population in the country, among which the 0–14 age group ranks the fourth in the country, accounting for 23.14% of the population. Children’s Hospital Affiliated to Zhengzhou University, Henan Children’s Hospital and Zhengzhou Children’s Hospital is the largest paediatric hospital in Henan Province, with 2200 beds. The hospital is the designated referral hospital for severe HFMD, and since 2008, when HFMD was officially listed as a legal Category C infectious disease, the number of reports of HFMD has been ranked first in Henan. At the same time, the hospital was approved as the National Children’s Regional Medical Centre and the Henan Children’s Medical Centre. In addition to the Henan Children’s Hospital and Zhengzhou Children’s Hospital, baseline data for this study were also obtained from The First Affiliated Hospital of Zhengzhou University, The Third Affiliated Hospital of Zhengzhou University (Maternal and Child Health Hospital of Henan Province), The Fifth Affiliated Hospital of Zhengzhou University, Henan Infectious Disease Hospital and The First Affiliated Hospital of Xinxiang Medical University, which are all tertiary-level hospitals that are key hospitals for the diagnosis and treatment of HFMD in Henan Province. We select above six hospitals as follow-up sites to conduct a long-term cohort on the occurrence of sequelae after HFMD recovery. Their geographical locations in Henan Province are shown in figure 2.

Figure 2

Geographical locations of the hospital sites.

Participants

Patients or the public were not involved in the design, recruitment, conduct, reporting or dissemination plans of our research. However, our researchers asked them whether they would be willing to participate in this study and they were told the approximate time required to participate, including length of survey response time and the number of potential years of involvement. In this cohort, children diagnosed with HFMD were selected as subjects from January 2014 to December 2023. Among them, severe cases with neurological and cardiorespiratory dysfunction during hospitalisation are the main targets for follow-up. Children will not be eligible for study enrollment if they have one or more of the following criteria: (1) ≥14 years of age at admission; (2) the presence of other viral and parasitic infections; (3) have physical/mental disability at admission; (4) have any underlying neurological, systematic, hereditary or metabolic diseases; (5) lack of written informed parental/guardian consent. First, the investigator introduced the participants to the information about this cohort study (HNHFMDCS). Second, the investigator asked the study participants if they would accept a questionnaire, a physical examination, a biospecimen collection and a search of their health information from a medical database. Finally, the subjects were given the option to withdraw at any time during the survey.

Case definition criteria

According to Chinese guidelines for the diagnosis and treatment of HFMD (2018 edition),10 we categorised clinical cases into mild cases and severe cases. Mild cases refer to the appearance of herpes or maculopapular rash on the patient’s hands, feet, buttocks and mouth with or without fever, while severe cases are defined as the presence of either central nervous system (CNS) complication (aseptic meningitis, encephalitis, encephalomyelitis, acute flaccid paralysis or autonomic nervous system dysregulation) or cardiopulmonary complications (pulmonary oedema, pulmonary haemorrhage or cardiorespiratory failure), or both.

Follow-up period

For participants enrolled in 2014–2022, we backtrack the patient’s hospitalisation records through the registration number and investigate the occurrence of sequelae through telephone follow-ups. For participants enrolled in 2023, the patient needs to be reviewed in the hospital within 1 month after healing; thus, we initially follow up monthly and then every 1–2 years when the cohort is stable. The first follow-up will be finished at the end of December 2023. The next follow-ups for the cohort will be held in 2024–2025 and 2026–2027.

Definition of sequelae

Developmental disorders are a very large group of syndromes in which the typical sequences or patterns of development are disrupted with delays in developmental steps and/or deviations in development processes. Delay in development is generally determined by a child who does not attain developmental milestones as compared with peers from the same population.11 Neurological delays include motor delays, language delays, communicative delays, adaptive delays and cognitive delays. We define muscle weakness as critical illness polyneuropathy, with primary sensorimotor axonal involvement with preservation of the CNS, and exhibiting a course usually characterised by progressive improvement.12 Intellectual impairment refers to a range of neuropsychiatric syndromes that share the common characteristics of early onset, cognitive deficits and resulting learning and adaptive dysfunction.13 Chronic respiratory diseases (CRDs) are characterised by exertional dyspnoea, exercise limitation and reduced health-related QoL.14 15 CRDs are associated with abnormalities in the airways and other structures of the lung. The most common CRDs are asthma and impaired pulmonary ventilation function. Asthma is a chronic inflammatory disease of the airways. In susceptible individuals, this inflammation causes recurrent episodes of wheezing, breathlessness, chest tightness and coughing, particularly at night or in the early morning.16 Impaired lung ventilation may be caused by bronchitis, pneumonia, bronchiectasis, etc. Speech and language impairments in childhood are variously described as children who do not talk, have difficulty in speaking, unable to understand others' speech or have stutter problems. The International Classification of Disease (ICD), Revision 9, Clinical Modification differentiates several types of speech and language problems, including stuttering, developmental speech or language disorder, aphasia, voice disturbance and other speech disturbance.17 Obstructive sleep apnea is an increasingly common disorder, characterised by a recurrent partial or complete collapse of the upper airway during sleep.18 Mental disorders are diagnosed according to the ICD-10 classification of mental and behavioural disorders: diagnostic criteria for research. Mental disorders mainly include anxiety disorders, depression, etc. Anxiety disorders are one of the most common types of mental disorders and usually develop before or in early adulthood. Core features include excessive fear and anxiety or avoidance of perceived threats that are persistent and impairing.19 Depression often presents with physical symptoms, primarily fatigue, pain or sleep disturbance.20 Brain fog is defined as an altered state of consciousness in which a person is less wakeful, aware, alert and focused than usual.21 22 Fatigue is a complex biological, psychosocial and behavioural phenomenon,23 which is classified as physical (central or peripheral) and mental. Cardiomyopathies lead to some of the worst cardiological outcomes, such as dilated cardiomyopathy conventionally defined as the presence of left ventricular or biventricular dilatation or systolic dysfunction in the absence of abnormal loading conditions.24 Large amount of studies confirm that the occurrence of dilated cardiomyopathy is associated with myocardial damage (or myocarditis) caused by enteroviruses infection.25 HFMD-related eye involvement presents variable signs, including conjunctivitis, outer retinitis, etc. Inflammation or infection of the conjunctiva is known as conjunctivitis and is characterised by dilatation of the conjunctival vessels, resulting in hyperemia and oedema of the conjunctiva, typically with associated discharge.26 Retinitis syndrome consists of an arteritis and phlebitis of the retinal and choroidal vasculature, confluent, necrotising retinitis that preferentially affects the peripheral retina and moderate to severe vitritis.27 28

Measurements

For the retrospective phase of the cohort study, we have collected clinical information and demographic information on inpatients with HFMD from 2014–2022 through a self-developed questionnaire. The self-developed questionnaire includes the general situation survey of patients, family structure, family history, disease history, behaviour and lifestyle scale, etc., all tailored to meet the objectives of our study. The content within the scale was derived from established sources and adhered to international standards. For instance, it included instruments such as the Pittsburgh sleep quality index, the Chinese short version of the International Physical Activity Questionnaire, the generalised anxiety disorder seven-item scale and the pupil rating scale revised for screening learning disabilities. For the prospective phase of the cohort study, we will recruit hospitalised patients with HFMD to participate in our survey and follow up the enrolled cases over time after completing the baseline survey, which includes four parts: in-person interview, comprehensive physical examination, clinical laboratory-based tests and biosample collection. Table 1 details what is measured at baseline and at first follow-up. Figure 3 shows the protocol of a cohort study on the sequelae of children with HFMD in Henan, China.

Table 1

Summary of measurements at baseline and follow-up in the prospective cohort study

Figure 3

Cohort programme of children with sequelae of HFMD in Henan, China. CNS, central nervous system; HFMD, hand, foot and mouth disease

Findings to dateCohort characteristics

In the long-term work, the Molecular Epidemiology Group of Zhengzhou University has applied epidemiological principles and methods to study HFMD, and has carried out many basic and prospective studies in the areas of virus evolution, epidemiology, disease prevention and vaccine development. For the retrospective sequelae analysis of the cohort, a total of 1 8 705 HFMD cases (11 834 males and 6871 females) were observed between 2014 and 2022, of which 17 202 were mild cases (10 839 males and 6363 females) and 1503 were severe cases (995 males and 508 females) (table 2). The number of children with severe HFMD was the highest in 2014 (407 cases, 14.01%).

Table 2

Number of hospitalised HFMD cases from 2014 to 2022

Of all patients enrolled, 748 (4.00%) had encephalitis and encephalomyelitis; 672 (3.59%) cases were accompanied by symptoms of convulsions; 2945 (15.74%) cases had sepsis; 827 (4.42%) cases had pneumonia; 1122 (6.00%) cases had bronchitis; 512 (2.74%) cases were accompanied by myocardial damage; 81 (0.43%) cases suffered from autonomic dysfunction; 305 (1.63%) cases suffered from pharyngitis; 48 (0.26%) cases were accompanied by epilepsy; 48 (0.26%) cases had respiratory failure, 276 (1.48%) cases had liver injury, 27 (0.14%) cases had infection of the CNS and 186 (0.99%) cases had anaemia (online supplemental table 1).

Levels of clinical examination indicators

We conducted detailed statistical analysis of the collected clinical examination data and found that there were statistical differences in many clinical indicators between mild and severe patients with HFMD. Online supplemental table 2 shows detailed clinical examination indicators and statistical analysis among mild and severe patients.

Pathogen typing and phylogenetic analysis of HFMD

The biological characteristics of enteroviruses play a crucial role in understanding the sequelae associated with HFMD. These characteristics not only enhance our comprehension of the pathogenesis and epidemiological features of the disease but also provide a scientific foundation for developing effective prevention and control strategies. Furthermore, they guide clinical treatments and facilitate the evaluation of vaccine efficacy. From 1 January 2019 to 20 October 2023, we performed a cumulative total of 3709 pan-enterovirus nucleic acid tests, with 2599 positive cases, of which 6 were EVA71 (0.2%), 116 were CVA10 (4.5%), 982 were CVA6 (37.8%), 290 were CVA16 (11.2%) and 1205 (46.4%) were other EVs (figure 4A). The pathogen surveillance data from 2016 to 2021 in Henan Province are shown in figure 4B. Therefore, CVA6 becomes the main pathogen of HFMD in Henan Province. In order to analyse genome-wide characteristics and patterns of genetic evolution of isolated strains, we downloaded representative strains from different regions of the world for comparative analyses (figure 5). As mentioned earlier, CVA6 serves as the primary pathogen during the study period, and to fully understand the phylogenetic analysis of CVA6, we constructed a phylogenetic tree of four laboratory CVA6 isolates with globally uploaded representative strains. CVA6 strains circulating worldwide can be divided into six genotypes designated as A–F, and the D genotype can be further subdivided into D1–D3 subgenotypes. Our results revealed that these four CVA6 strains were subgrouped into the subgenotype D3 (figure 6). The D3 subgenotype is the predominant genotype that circulates all over the world, particularly in Southeast Asia and Europe in recent years.29

Figure 4

Pathogen spectrum of patients with HFMD in Henan Province. (A) Laboratory typing results in Henan Children’s Hospital, 2019–2023. (B) Pathogen surveillance data in Henan Province, 2014–2021. HFMD, hand, foot and mouth disease.

Figure 5

Genetic evolutionary characteristics of enterovirus isolated in Henan Province. Maximum likelihood phylogenetic trees for enteroviruses were constructed with partial sequences from Sanger sequencing. The blue stars marked the strains that have been isolated in our lab since the study began.

Figure 6

Maximum likelihood tree of CVA6 strains. (A) Phylogenetic tree of GenBank reference and Chinese sequences. (B) Zoom of the labelled subtrees in (A). The Chinese sequence is labelled in purple. All the strains are labelled using the following format: accession number/country of origin/year of isolation. The prototype strain of EVA71 is marked with green star. The prototype strain of CVA6 is marked with green triangle. The green squares mark the representative strains causing atypical HFMD. HFMD, hand, foot and mouth disease.

Until 20 October 2023, the cumulative collection of biological samples for the cohort included 2863 blood samples, 503 stool samples and 516 oral swab samples from patients with HFMD. And 70 blood samples, 26 stool samples and 48 oral swab samples were collected from healthy controls. All biological samples were stored at −80 °C.

Discussion

In retrospective cohort studies, many clinical indicators differ between mild and severe cases. Some of the results based on the baseline data of the cohort have also been published.30 By a case-control study, we found the concentrations of interleukin (IL)−4, IL-6, IL-10 and tumour necrosis factor (TNF) studies; many clinical indicators differ between mild and severe cases.31 Another work addressing the involvement of neutrophil activation underlying CVA6-induced myocardial injury has also been published recently.32 Our observational study suggests that rural living, hyperpyrexia, immune indicators (eg, decreased number of eosinophils and increased number of neutrophils and the levels of IgG and globulin) and metabolic alterations (eg, decreased levels of Na+, Cl- and CK, and the increased level of ALP) are predictors of severe HFMD.31 We also found that some laboratory indicators were effective in differentiating mild from severe HFMD cases. The PLT and CRP are sensitive indicators of inflammation, and their changes may reflect the different degrees of influence of the disease on the inflammatory response in patients. The AST and ALT may indicate liver damage, especially in severely ill patients. The changes of %MONO and %EOS may be related to the immune response and allergic status of patients, while the increase or decrease of the absolute number of MONO directly reflects the changes in the number of monocytes in patients, which is of great significance for evaluating the severity and prognosis of the disease. Changes in the number of T lymphocyte subsets (CD3+ and CD3+CD4+) and B lymphocytes (CD19+) reflect the immune function status of the patient, which is critical to understanding the impact of the disease on the patient’s immune system. An accelerated ESR may indicate an acute inflammatory response in the patient. Abnormal liver and kidney function indices (gamma glutamyl transferase, TP, ALB and GLO) may indicate the injury or dysfunction of related organs in patients, while elevated lactate dehydrogenase may be related to tissue injury or abnormal energy metabolism. In addition, the maintenance of electrolyte balance (Na+, Cl− and Ca2+) is essential for the patient’s physiological function, and its abnormalities may reflect the impact of the disease on the patient’s electrolyte balance in the body. IgM and complement C3 are key components of the immune system, and their changes may reflect how active the immune response is in the patient.

The genotyping and genetic evolution analysis of HFMD pathogens showed that CV-A6 was the main pathogen of HFMD in Henan Province. The D3 subgenotype is the dominant CV-A6 genotype that has been circulating around the world in recent years. Since 2010, there have been large-scale HFMD outbreaks in several countries, including Brazil,33 China,34 India,35 Japan36 and Vietnam,37 which have been attributed to CV-A6 D3 strains. Analysing the phylogenetic tree of the sequences of enteroviruses, we found evidence of coevolution between CV-A6 strains prevalent in Henan and CV-A6 strains sourced from other parts of China. This indicates that there may be multiple transmission chains for the strains studied. Given the vast topography and climatic diversity across China, the transmission and replication capacity of CV-A6 may vary by region. It is worthy to note that the D3 subgenotype of CV-A6 might be highly infectious and virulent, which might also be the cause of the large-scale epidemic of CV-A6. For the prospective analysis, the cohort study is currently ongoing, primarily recruiting hospitalised patients with HFMD to participate in our investigation. After completing the baseline survey, we will conduct long-term follow-up on the enrolled cases. Within the next year, we anticipate obtaining preliminary data on the survival rates of patients with HFMD from 1 to 10 years postdischarge, along with information on the occurrence of sequelae. These data will be updated and expanded annually to facilitate the continuous monitoring of patient health and disease progression.

For the prospective part of this study, we made inferences based on its pathological mechanisms and clinical manifestations. Enteroviruses can invade the CNS in severe cases with HFMD. Viral infections can lead to damage in the CNS, subsequently impacting the functionality of multiple organs throughout the body.9 Some severe patients may experience severe complications, including encephalitis, encephalomyelitis, meningitis and myocarditis, as well as pulmonary oedema and haemorrhage. As a result, the sequelae of HFMD include but are not limited to the following. (1) Limb weakness: enteroviruses may invade the anterior horn cells, nerve roots, nerve plexus and other regions of the spinal cord. This invasion can lead to obstruction of nerve conduction, subsequently resulting in limb weakness.38 Limb weakness typically manifests in the lower extremities; however, it may also extend to the upper extremities. This condition is characterised by instability during ambulation and challenges with maintaining an upright posture. (2) Muscle atrophy: in severe cases, patients may experience neurological complications that can lead to neurogenic muscle atrophy. Typically, muscle atrophy initiates in the lower limbs and progressively ascends to involve the trunk and upper limbs. This condition results in muscle weakness, wasting and potentially sagging skin.39 (3) Neurodevelopmental delay: in cases where severe manifestations are not addressed with timely and effective treatment, the persistent replication of the virus can lead to damage in the CNS, thereby disrupting the normal developmental processes of the brain.40 Subsequently, this condition may further affect cognitive abilities, learning capacity, motor coordination and social skills. (4) Myocardial injury: some patients may experience myocardial damage as a result of viral invasion of cardiomyocytes, leading to arrhythmias, decline in cardiac function and other associated symptoms.41 (5) Sleep disorders: The CNS involvement may result in abnormal sleep regulation, such as difficulty falling asleep, frequent awakenings or daytime sleepiness.

This cohort study is specifically designed to conduct a comprehensive investigation into the long-term sequelae experienced by individuals who have survived HFMD. The objective is to provide an in-depth understanding of the potential hazards and adverse effects associated with this infectious disease, thereby illuminating the broader implications and consequences of HFMD on the health and well-being of those affected. The study seeks to contribute to the existing body of knowledge regarding HFMD and inform future interventions, treatments and preventive measures aimed at mitigating its impact.

Strengths and limitations

This cohort study has several important strengths. First, this cohort is mainly conducted in Children’s Hospital Affiliated to Zhengzhou University (approved as the designated hospital for severe HFMD in Henan Province). In addition, we also included other important provincial hospitals as study sites, and these hospitals are all designated hospitals for HFMD in Henan Province. This study is also based on Henan Province Engineering Research Center of Diagnosis and Treatment of Pediatric Infection and Critical Care. Second, data are constantly subject to quality control procedures that involve data collection, request, extraction and processing to improve their reliability, validity, quality and research applicability. In addition, long-term follow-up may be adequate for investigating the aetiology and prognosis of specific chronic diseases, as well as elucidating the causal relationship between multimorbidity and the development of HFMD.42–44

There were several limitations in our study. First, we could not eliminate the possibility of unmeasured confounding variables influencing the results, such as environmental factors, parental influences, socioeconomic status or family history within the retrospective cohort. Second, it is possible that these sequelae may develop beyond the observation period of this study. Despite these aforementioned limitations, the strengths of this research include its national representativeness, large sample size and longitudinal data.

Collaboration

The study has facilitated cooperation between various professionals and fields of medicine. Future collaborations are appreciated. Data are accessible on reasonable request. Although the study data are not freely available, we welcome specific proposals for future collaborations.