Hand, foot and mouth disease (HFMD) is a common disease that mainly infects infants and children.1 On May 2, 2008, the Ministry of Health of the People’s Republic of China listed HFMD as a category C infectious disease. Since then, the number of cases and deaths due to HFMD has ranked first among category C infectious diseases. HFMD can be caused by various enteroviruses. Since the 1970s, enterovirus (EV) 71 and coxsackievirus (CV) A16, which alternately circulate, have served as the major etiologic agents of HFMD.2 However, in recent years, HFMD outbreaks caused by other enteroviruses have been increasingly reported in the USA and several countries in Europe and Asia.3–7 As a result, we analyzed specimens from patients to investigate the pattern, etiologic agents and clinical manifestations of HFMD in Beijing, China during 2013.
MATERIALS AND METHODS
Patients clinically diagnosed with HFMD in Beijing at the Beijing Ditan Hospital, the fixed-point hospital for HFMD treatment, in 2013 were included in the study. Each patient had at least 1 specimen, such as a throat swab or stool specimen, collected within 3 days of diagnosis. Medical records were reviewed retrospectively. Selected patients or their caregivers were interviewed by telephone to collect information about the presence of onychomadesis (nail separation starting at the matrix).4 Informed consent was obtained from the patients or their parents.
Viral RNA was extracted using the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany). Real-time reverse transcription polymerase chain reaction was performed according to the method developed by the Shanghai Kehua Bio-engineering Co., Ltd (KHB), China. To genotype the viruses, one-step reverse transcription polymerase chain reaction (TaKaRa) was performed with primers 486/488, 292/222 or 490/492.8 PCR products were examined by gel electrophoresis, purified using a commercial procedure (Beijing Dingguochangsheng Biotechnology Co., Ltd, China) and subjected to nucleotide sequencing. The sequences were compared with known genotypes in the GenBank database using the BLAST search tool.
SPSS 16.0 software was used for the statistical analyses. Categorical variables were tested using the χ2 or Fisher’s exact test. Continuous variables were tested using the independent-samples t test. A P value of less than 0.05 was considered significant.
A total of 243 HFMD patients, including 22 outpatients and 221 inpatients, were enrolled in this study. The patients included 152 boys and 91 girls, with a sex ratio of 1.7:1. The cases occurred in patients ranging from 2 months to 20 years of age (mean 1.8 years); the cases primarily occurred in children <5 years of age (96.71%, 235/243 patients), especially in those <3 years of age (88.94%, 209/235 patients). Signs of neurological infections were observed in 47 patients (19.34%, 47/243 patients), mainly in children <3 years of age (87.23%, 41/47). The cases were primarily concentrated in the Chaoyang, Shunyi, Tongzhou, Haidian and Changping districts (see Fig., Supplemental Digital Content 1, http://links.lww.com/INF/B951). HFMD was observed throughout the year but peaked from May to August (see Fig., Supplemental Digital Content 2, http://links.lww.com/INF/B952).
A total of 210 throat swabs and 139 stool specimens were collected from the 243 cases. Enterovirus was detected in 210 throat swabs and 44 stool specimens by real-time reverse transcription polymerase chain reaction analysis. The results indicate a considerably higher rate of enterovirus detection in the stool specimens (79.55%, 35/44) compared with the throat swabs (44.76%, 94/210) (P < 0.001). Moreover, 130 stool specimens collected from 130 HFMD cases were randomly selected for genotyping. The results revealed that 100 specimens were typed successfully. In addition, 11 different serotypes were identified, 8 of which belonged to the HEV-A species (CVA2, CVA4, CVA5, CVA6, CVA10, CVA12, CVA16 and EV71) and 3 of which belonged to the HEV-B species [CVB4, echovirus (E) 7 and E9] (Table 1). Notably, the most prevalent serotype was CVA6 (43.08%, 56/130) followed by EV71 (14.62%, 19/130). The other serotypes were only detected with marginal frequencies: CVA10 (4.62%, 6/130), CVA16 (3.85%, 5/130), CVA2 (3.08%, 4/130), CVA5 (2.31%, 3/130), CVA12 (1.54%, 2/130), E9 (1.54%, 2/130), CVA4 (0.77%, 1/130), CVB4 (0.77%, 1/130) and E7 (0.77%, 1/130). In addition, mixed infections were not detected in this study. After a comprehensive analysis of the results from the 2 methods, we concluded that the main causative agent of HFMD in Beijing in 2013 was CVA6 followed by EV71.
Next, we compared the demographic characteristics and laboratory findings of the CVA6 and EV71 HFMD patients. Both serotypes occurred mainly from June to August and primarily in children ≤5 years of age (CVA6, 50/56; EV71, 19/19). Infection with both CVA6 and EV71 caused severe cases of HFMD (CVA6, 6/56; EV71, 10/19). The mean fever duration was 2.67 ± 0.99 and 4.16 ± 1.46 days for CVA6 and EV71 infections, respectively (see Table, Supplemental Digital Content 3, http://links.lww.com/INF/B993). The fever duration of CVA6 patients was significantly reduced compared with that of EV71 patients (P < 0.001). The mean serum C-reactive protein was 32.43 ± 35.84 and 4.56 ± 5.37 mg/L in CVA6 and EV71 infections, respectively, and this difference was significant (P < 0.001). The glucose levels in CVA6 patients were less than those in EV71 patients (P = 0.027), due to the normal glucose levels observed in most CVA6 patients (76.36%) and the increased levels identified in 61.11% of EV71 patients. Significant differences were not observed in other demographic characteristics and laboratory findings from the CVA6 and EV71 patients.
Meanwhile, 100 HFMD patients (Table 1) were interviewed for the presence of onychomadesis. By definition, 13 patients displayed onychomadesis, including 12 with CVA6 infection (22.64%, 12/53) and 1 with CVA10 infection (16.67%, 1/6). Onychomadesis was significantly associated with CVA6 infection (P = 0.002).
In this study, the 243 HFMD cases were mainly concentrated in suburban areas and at the edges of urban areas, and cases were rare in the central cities and outer suburban areas. Dense population and poor sanitation might contribute to the increased morbidity in these locations. Moreover, the duration of HFMD was 1 month longer (May to August) than that reported by Qian et al9 (May to July). However, this finding was not unexpected given that the temperature in August 2013 was approximately 1–2°C higher than in previous years. Since 1981, EV71 and CVA16 have been the major causes of HFMD in China.10 However, in this study, we found that the presence of EV71 and CVA16 decreased dramatically and that CVA6 was the main causative agent of HFMD in Beijing in 2013. To our knowledge, this is the first study to report HFMD caused by CVA6 in mainland China.
CVA6 is one of the major causes of herpangina.4,11 However, the detection rate of CVA6 in HFMD patients is gradually increasing, and it has now become the main etiologic agent of HFMD in Beijing. Moreover, we found that CVA6 could also cause severe cases of HFMD, and the manifestations of these cases are very difficult to distinguish from EV71 infection. It is likely that CVA6 possesses a pathogenic mechanism similar to EV71. Hence, changes in both the HFMD etiologic agent and clinical manifestations caused by CVA6 are worthy of attention.
Onychomadesis is an acute, painless and noninflammatory disease.12 Several onychomadesis outbreaks linked to HFMD have been reported.3–6 In our study, we reported that CVA6 infection was significantly associated with onychomadesis (P = 0.002), which suggested that CVA6 might serve as the main causative agent of onychomadesis after HFMD; however, this hypothesis requires further study.
In addition, both the fever duration and glucose levels of CVA6 patients were significantly reduced compared with EV71 patients, suggesting that the symptoms caused by CVA6 were relatively milder. However, the mean serum C-reactive protein levels of CVA6 patients were considerably higher (P < 0.001), which was not consistent with the illness severity. Hence, the severity of CVA6 HFMD could not be predicted by the elevated C-reactive protein level, which was consistent with a report from Taiwan.13
In all, this study is the first to report a significant change in the HFMD etiologic agents in Beijing in 2013, which is worthy of attention and further research.
The authors thank Qu Pei, Liu Yanan, Zhang Ling, Zhan Yongjing, Zhang Baomin and Ji Tianjiao for excellent technical assistance. The authors also thank the team in the Department of Infectious Diseases and the Department of Pediatrics of Beijing Ditan Hospital.
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