Onychomadesis and potential association with HFMD outbreak in a kindergarten in Hubei province, China, 2017

Background In 2017, an outbreak of onychomadesis occurred in kindergarten H, Hubei province, China. We investigated the field to learn about the magnitude and reason of the outbreak. Methods The case definition was that a child with onychomadesis or transverse ridging (Beau’s line) in fingernails and toenails without previous traumatic or systemic disease in kindergarten H from Sep. 1st to Nov. 30th, 2017. A retrospective cohort study was carried out to analyze the epidemiological relationship between onychomadesis and the hand-foot-mouth disease (HFMD) in Primary Class #2, kindergarten H. We also performed a serological survey for neutralizing antibodies against coxsackie virus A6 (CVA6), coxsackie virus A10 (CVA10) among 15 cases and six healthy children in the kindergarten. Meanwhile, some children were carried out with routine blood, fungal microscopic and microelement tests. Indoor environment examinations had been done for all classes. Results A total of 20 cases were identified in Kindergarten H. Seventy-five percent (15/20) cases occurred in Primary Class #2. Fifty-five percent of the cases (11/20) had suffered from HFMD within two months. The median time between onychomadesis and HFMD was 45 days (ranging from 31 to 58 days). A retrospective cohort study in Primary Class #2 showed the attack rate was 90.0% among 10 children who suffered from HFMD in the past two months compared to 30.0% among 20 children who didn’t (Rate Ratio [RR] =3.0, 95% Confidence Interval [CI] =1.5–6.0). The positive rates of neutralizing antibodies were 66.7% for CVA6 and 26.7% for CVA10 in tested cases. The result of routine blood, fungal microscopic, microelements tests were normal in cases. The indicators of environment were within the normal range. Conclusion The results of this study suggested that the outbreak of onychomadesis in Hubei province was probably associated with HFMD epidemic within two months.


Epidemiological investigations
A case was defined as a child with onychomadesis or Beau's line in fingernails and toenails without previous  traumatic or systemic disease in Kindergarten H, Songzi County, Hubei province from Sep. 1st to Nov. 30th, 2017. The diagnosis was conducted through on-site checking of nails by pediatricians and dermatologists, combined with interviewing kindergarten staffs, and reviewing routine health and absence records of children. Questionnaires were used to collect the information about the onset, diagnosis and treatment, illness in the last two months, behavior habits and nutrition condition. Meanwhile, we asked if similar cases had occurred in four nearby kindergartens in the county. A retrospective cohort study was under-taken to analyze the relationship between onychomadesis and the prevalence of HFMD in recent two months in Primary Class #2 in Kindergarten H. EXCEL2007 was used to collate data and draw the epidemic curve, and SPSS13.0 was used to analyze the data.

Laboratory tests
Blood samples of six cases and six healthy children were collected to identify their microelement status including calcium, iron, zinc, lead, cadmium, copper and magnesium. The indoor environment of all classes in Kindergarten H was examined with the indicators of formaldehyde, benzene, toluene, and xylene. Six pieces of plasticines used by cases were detected for the lead. Fungal microscopic examinations of nail smear samples were taken. We collected the anal swabs and serum specimens from 15 cases and 6 healthy children. Viral RNA was extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA). We tested RNA from each sample for sequencing VP1 (viral protein 1) gene of enterovirus including EV71, CA16, CA6 and CA10 by realtime reverse transcriptase-polymerase chain reaction (RT-PCR) using fluorescent PCR detection kits for HFMD viral RNA (Shuoshi Biotechnology company, Jiangsu, China). Neutralizing antibody tests of CA6 and CA10 were performed for serum specimens as described [23]. A neutralizing titer of≥1:16 was considered as a threshold for positivity [23,24].

Descriptive epidemiology
A total of 20 cases were identified in Kindergarten H which has 250 children in our study. The attack rate was 8.0% (20/250), and there was no severe or death case. All 20 cases (100.0%) has occurred onychomadesis and 4 cases (20.0%) also had Beau's line in their nails. Onychomadesis started from the proximal deck and gradually developed to the distal deck. The numbers of affected fingers or toes were between one and nine with the average two. Eighteen and two cases had unhealthy fingernails and toenails respectively, while two cases had unhealthy fingernails and toenails simultaneously. The onychomadesis cases occurred between Nov. 4th and Nov. 22th as shown in Fig. 1. The attack rates of male and female children in kindergartens were 6.7% (8/118) and 9.1% (12/132) respectively, without statistical difference (x 2 = 0.45, P > 0.05). The median age of the 20 patients was 3 years old (range, 3-4 years). There were seven classes in kindergarten H, and three of them had cases with onychomadesis. 75.0% (15/20) cases occurred  in Primary Class #2. According to the retrospective interview, 55.0% of the cases (11/20) had suffered from HFMD characterized by fever (72.8%) and vesicular eruptions (100.0%) on hands or feet or in the mouth, within two months before the onset of onychomadesis. The median time between onychomadesis and HFMD was 45 days (ranging from 31 to 58 days) (Figs. 2, 3).

Analysis of risk factors
A retrospective cohort study in Primary Class #2 showed the attack rate was 90.0% among ten who suffered from HFMD in recent two months compared to 30.0% among 20 who didn't (Rate Ratio [RR] = 3.0, 95% Confidence Interval [CI] = 1.5-6.0) ( Table 1).
Enterovirus co-infections and onychomadesis after hand, foot, and mouth disease, Spain, 2008.
Outbreak of hand, foot and mouth disease with onychomadesis caused by Coxsackie virus A16 in Granada.
An outbreak of hand-foot-and-mouth diseasemimicking chicken pox, with a frequent association of onychomadesis in Japan in 2009: a new phenotype caused by coxsackievirus A6.
Onychomadesis after hand-foot-and-mouth diseaseoutbreak in northern Greece: case series and brief review of the literature.

Northern Greece
Kao QJ et al. 2016 [17]. An outbreak of coxsackievirus A6 hand, foot, and mouth disease associated with onychomadesis in Hangzhou.

Hangzhou, China
Li J et al. 2018 [18]. An outbreak of Coxsackievirus A6-associated hand, foot, and mouth disease in a kindergarten in Beijing in 2015.
Beijing, China tested cases were within the normal range (Additional file 1 Table S1).

Discussion
8.0% (20/250) of children suffered from onychomadesis in kindergarten H, Songzi county, Hubei province, from Sep 1 to Nov 30 in 2017. Fifty-five percent of the cases (11/20) had suffered from HFMD within two months before onychomadesis. The median time between onychomadesis and HFMD was 45 days (ranging from 31 to 58 days). A retrospective cohort study in primary class #2 showed that children who had suffered from HFMD within two months had three times of the risk of onychomadesis. The positive rates of neutralizing antibodies were 66.7% for CVA6 and 50.0% for CVA10. The results of this study suggested that the onychomadesis epidemic occurred in Hubei province was probably associated with HFMD within two months prior to the onset of the disease. In our study, the factors of idiopathic, medicine, fungal microscopic, microelement, and environment for onychomadesis were almost excluded. The symptoms of cases were similar with other onychomadesis outbreak after HFMD (Table 3) [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. The median time between onychomadesis and HFMD in this outbreak was 45 days (ranging from 31 to 58 days), which was also similar with other references except for a little shorter than 36-69 days in an onychomadesis outbreak, Spain, 2009 [7]. 66.7% (10/15) of the cases were positive for CVA6 or CAV10 neutralization test, which was similar with 65.9% (29/44) positive in an onychomadesis outbreak, Spain, 2008 [9] The positive rate of neutralizing antibodies was 58% for CVA6 and 43% for CVA10 in healthy children aged 4-6, China [24]. In our study, the positive rate of neutralizing antibodies was 66.7% for CVA6 and 26.7% for CVA10 in cases, while 66.7% for CVA6 and 50.0% for CVA10 in healthy children. The high positively rates probably caused by the concentration of Hand-foot-andmouth disease within two months before the onset of the disease, which also supported our conclusion. Reports about the mechanism of onychomadesis after HFMD were limited. Onychomadesis may occur after inflammation of the nail matrix or maceration associated finger blisters because of traumatic nail loss [19]. The shed nail fragments of a patient who suffered onychomadesis after HFMD were positive for CVA6 nucleic acid test in an outbreak of onychomadesis, which suggested that CVA6 virus replication caused the damage of nail matrix, resulting in onychomadesis [12]. HFMD outbreak frequently occurs in kindergarten, but there were few reports of onychomadesis outbreak in China [17,18]. The pathogens of the HFMD kept drifting in different countries [25][26][27][28][29][30]. The EV71 inactivated vaccine successfully entered the market in China since June 2016 [31]. Other enterovirus (except EV71 and CVA16) in the confirmed cases of HFMD were the predominate pathogen (accounted for 76%) in Hubei province in 2017, which was different from the past pathogen trends and showed the change of major pathogen [20,21]. An outbreak of HFMD in 2016 showed that the positive rate was 76% (146/192) for CVA6 and 2.6% for CVA10 in Chongqing [32]. In our study, the HFMD outbreak before onychomadesis outbreak only affected Primary Class #2 by the passive and active surveillance in Kindergarten H, which may related to the contact transmission. Our study is the first reported onychomadesis outbreak in Hubei province and it was rare in China, which means further research is necessary to learn about its etiology, epidemic trend, prevention and control, and the relationship with onychomadesis. There are several limitations in our study. The positive rate of HFMD nucleic acid test was low, and the results of neutralizing antibody test only suggested previous infection. The HFMD nucleic acid test was delayed and we collected anal swabs rather than feces or shed nail described in previous literatures [10,12,13]. The results of zero prevalence of HFMD virus in anal swabs by using nucleic acid tests were predictable regarding the clearance of HFMD virus after recovery. In addition, we only tested the neutralizing antibodies CVA6 and CVA10 which were common pathogens related to onychomadesis. The emergency stocks of the neutralizing antibodies for HFMD were insufficient. The surveyed pathogenic types of HFMD were EV71, CVA16 and other enteroviruses, and further classification of other enteroviruses was not official requirement yet in Hubei province.

Conclusions
In conclusion, the first onychomadesis outbreak occurred in Hubei province was probably associated with HFMD epidemic within two months after onset.
Additional file 1: Table S1. Results of blood and environment tests in Kindergarten H, Hubei province, 2017.