Clinical and Etiological Characteristics of Enterovirus 71-Related Diseases during a Recent 2-Year Period in Korea

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1 JOURNAL OF CLINICAL MICROBIOLOGY, July 2010, p Vol. 48, No /10/$12.00 doi: /jcm Copyright 2010, American Society for Microbiology. All Rights Reserved. Clinical and Etiological Characteristics of Enterovirus 71-Related Diseases during a Recent 2-Year eriod in Korea Wi-Sun Ryu, 1,2 Byounghak Kang, 1 Jiyoung Hong, 1 Seoyeon Hwang, 1 Jonghyun Kim, 3 and Doo-Sung Cheon 1 * Division of Enteric and Hepatitis Viruses, National Institute of Health, Korea Center for Disease Control and revention, 1 and Department of Neurology, Seoul National University Hospital, 2 Seoul, and Department of ediatrics, Catholic University College of Medicine, Suwon, 3 Republic of Korea Received 3 December 2009/Returned for modification 3 March 2010/Accepted 3 May 2010 Human enterovirus 71 (EV 71) has caused large-scale outbreaks of hand-foot-and-mouth disease (HFMD), particularly in the Asian-acific region. In this study, we report a major outbreak of EV 71 infection in Korea and describe the clinical differences between EV 71 and non-ev 71 enterovirus infections. We prospectively enrolled patients with suspected viral infections during a recent 2-year period through a nationwide surveillance system. We identified 719 patients with suspected HFMD or herpangina using real-time CR and genotyping based on V1 sequence analysis. The major pathogen causing HFMD changed substantially from 2008 to 2009, with EV 71 becoming the most common cause of HFMD in Korea in We successfully identified the enteroviral genotypes for 218 of the 719 patients. atients with EV 71 infections tended to be younger than those with non-ev 71 enteroviral infections and presented with HFMD and meningoencephalitis. In addition, the occurrence of fever, headache, and neck stiffness was significantly higher in patients with EV 71 infections. Multivariable analysis showed that for patients presenting with HFMD, fever, or a sore throat, each covariate was independently associated with EV 71 infection; the adjusted odds ratios (with 95% confidence intervals in parentheses) for these variables were (10.04 to ), 4.76 (1.71 to 13.25), and 0.18 (0.04 to 0.77), respectively. Our results indicate that EV 71 was a major cause of HFMD in Korea during the study period. In addition, we found that clinical symptoms may be helpful in the early identification of patients with EV 71 infections. Human enterovirus 71 (EV 71) is an important emerging pathogen of hand-foot-and-mouth disease (HFMD) (1, 6, 30). In particular, several major outbreaks of EV 71 have been documented in the Asia-acific region since 1997 (3, 4, 7, 9, 25). EV 71 infection causes HFMD, a common exanthema of young children that is characterized by a fever, rashes on the palms and the bottoms of the feet, and ulcers in the oral cavity. In general, patients with HFMD experience a mild course of disease; however, some patients develop severe neurological complications, especially as a result of EV 71 (18). Given that EV 71 infection could cause severe neurological complications, its early detection in patients with HFMD is an important part of intensive care efforts and efforts to prevent mortality. Unfortunately, current methods for the detection of EV 71 do not enable early detection (23). Here we report an outbreak of EV 71 infection in Korea. As part of this nationwide surveillance study, we examined the clinical differences between EV 71 and non-ev 71 enteroviral infections, and we investigated whether these differences are clinically applicable to the early detection of EV 71 in patients with complications of HFMD. MATERIALS AND METHODS atients. A nationwide surveillance system for infection with enterovirus (EV) in Korea was initiated in Sixty-two clinics (8 primary clinics, 14 secondary * Corresponding author. Mailing address: Division of Enteric and Hepatitis Viruses, National Institute of Health, Korea Center for Disease Control and revention, 194, Tongil-Lo, Eunpyung-Gu, Seoul , Republic of Korea. hone: Fax: cheonds@hanmail.net. ublished ahead of print on 12 May hospitals, and 40 tertiary hospitals located nationwide) participated (Fig. 1). The sentinel clinics sent specimens collected from patients who had a highly suspicious viral disease, and they reported the demographic characteristics and clinical symptoms of the patients via the Internet ( From the beginning of 2008 through 30 October 2009, a total of 4,027 cases of clinically suspected viral disease were reported to the Korean Center for Disease Control and revention (KCDC). Among these cases, we identified 719 (200 in 2008 and 519 in 2009) suspected cases of HFMD or herpangina. Of the 719 patients studied, 1 died of severe neurological complications, while 2 others were in a comatose state. Case definition. The patients were identified as having HFMD or herpangina according to whether skin eruptions were found on the hands, feet, and mouth or only in the oral cavity, respectively. Meningoencephalitis was defined as cerebrospinal fluid (CSF) pleocytosis (white blood cell count, 5/mm 3 ), with or without parenchymal lesions or substantial meningeal enhancement as identified by brain computed tomography or magnetic resonance imaging and/or by the presence of definite neurological dysfunction without CSF pleocytosis. Molecular diagnosis for enterovirus infections and genotyping. Enteroviral genome detection by real-time reverse transcription-cr (RT-CR) using Taq- Man technology was attempted on all throat swabs, CSF samples, and stool specimens collected from patients with EV 71-related symptoms. Briefly, viral RNAs were extracted using the magnetic-bead-based viral nucleic acid purification protocol described by Aertsens et al. (2). Subsequently, one-step real-time RT-CR was performed using a dually labeled fluorogenic EV-specific probe and primers designed on the basis of previous data (26). For genotyping, seminested RT-CR was used to amplify part of the V1 gene of EV based on the U.S. CDC s protocol for the detection of EV by the use of confirmed EV-positive specimens and real-time CR; then, sequences analysis was performed using an ABI 3130XL automatic sequencer and a DNAstar program package (19). Statistical analyses. The means of the continuous variables and the proportions of the categorical variables were compared using Student s t test, 2 tests, and Fisher s exact test, as appropriate. To examine the odds ratios (ORs) for the demographic and clinical characteristics of EV 71 infection, we initially conducted a univariate analysis. Next, the variables for which was 0.1 were selected for a stepwise backward regression analysis. Two-tailed values of 0.05 were considered to be significant. Data analysis was performed using SSS, version 12.0 (SSS Inc., Chicago, IL). 2490

2 VOL. 48, 2010 ENTEROVIRUS 71 INFECTION IN KOREA 2491 FIG. 1. Distribution of the patients in a nationwide surveillance system. RESULTS EV was detected in 447 (62.2%) of the 719 suspected cases of HFMD or herpangina by real-time RT-CR. Among the 447 patients, 314 cases were available for seminested RT-CR, and 252 cases (80.3%) were positive by seminested RT-CR. Of these, we identified the enteroviral genotype in 218 cases (86.5%). The genotype and the frequency of identification of cases as enteroviral infections are shown in Table 1. In 2008, the most common pathogen was CA10, whereas EV 71 was not detected in patients with HFMD or herpangina. In 2009, however, EV 71 was detected most commonly (91 patients; 55.2%). The clinical characteristics of the patients differed accordingly between 2008 and 2009 (Table 2). In 2009, patients were more likely to present with HFMD or with HFMD accompanied by meningoencephalitis than with herpangina, whereas herpangina was more common in Thus, the patients in 2009 tended to have fever, headache, and decreased mental status. Compared to those with herpangina, patients with HFMD tended to be female (44% versus 35%;, 0.04) and were likely to have fever, neck stiffness, and decreased mentality. In addition, meningoencephalitis was more common in patients with HFMD than in those with herpangina (18.6% versus 7%;, 0.002). However, sore throat was more common in patients with herpangina. Other clinical symptoms and age did not differ between the two groups. The demographic and clinical characteristics of the patients according to the presence of the enteroviral genome as determined by real-time RT-CR are given in Table 3. Meningoencephalitis was significantly more common in patients with positive CR results (15.0%) than in those with negative CR results (4.0%). Accordingly, the patients confirmed to be infected with EV by CR were more likely to have decreased mental status and neck stiffness. The other variables considered did not differ between the two groups. We next subdivided the 218 patients with enteroviral infections into two groups: EV 71 (n 91) and non-ev 71 (n 127) infections. The sex distribution and the time interval from symptoms to sampling did not differ between the two groups; however, the patients in the EV 71 group tended to be younger and to have a higher prevalence of HFMD (95.6%) and meningoencephalitis (38.5%) than those in the non-ev 71 group (43.3 and 4.7%, respectively) (Table 4). Accordingly, the patients in the EV 71 group were more likely to have fever ( 37 C), headache, and neck stiffness, which are common symptoms of meningoencephalitis. Interestingly, sore throats were more common in the non-ev 71 group. No other difference in symptoms was noted between the two groups. By univariate analysis, a young age was significantly associated with EV 71 infection (OR, 0.98; 95% confidence interval [95% CI], 0.97 to 1.00). The ORs for presenting with HFMD and herpangina for EV 71 infection were (95% CI, 9.85 to 82.34) and 0.06 (95% CI, 0.03 to 0.14), respectively (Table 5). In addition, fever, headache, neck stiffness, and sore throat were significantly associated with EV 71 infection. By multivariable analysis, presenting with HFMD, fever ( 37 C), and sore throat remained significantly associated with EV 71 infection (adjusted ORs, [95% CI, to ], 4.76 [95% CI, 1.71 to 13.25], and 0.18 [95% CI, 0.04 to 0.77], respectively), whereas headache was marginally associated with EV 71 infection (adjusted OR, 6.51 [95% CI, 0.77 to 55.13]). DISCUSSION In this study, we found that the etiologic causes of HFMD and herpangina changed dramatically in Korea between 2008 and In addition, we demonstrated that patients infected with EV 71 tended to present with HFMD and meningoencephalitis rather than with herpangina, and consequently, presenting with HFMD and presenting with fever were independently associated with EV 71 infection by mul- TABLE 1. revalences of enterovirus genotypes identified in patients with hand-foot-mouth disease or herpangina in South Korea Serotype a No. (%) of cases CA (7.9) CA3 2 (3.8) 0 CA4 4 (7.5) 0 CA (10.3) CA (8.5) CA10 18 (34.0) 0 CA (1.2) CA16 2 (3.8) 15 (9.1) CB1 4 (7.5) 4 (2.4) CB3 4 (7.5) 0 E3 0 4 (2.4) E6 6 (11.3) 1 (0.6) E9 0 2 (1.2) E (2.4) E14 1 (1.9) 0 E30 12 (22.6) 1 (0.6) E (0.6) EV (55.2) Total a CA, coxsackie virus A; CB, coxsackie virus B; E, echovirus; EV, enterovirus.

3 2492 RYU ET AL. J. CLIN. MICROBIOL. TABLE 2. Demographic and clinical comparisons of patients suspected of enterovirus infection between 2008 and 2009 Value 2008 (n 200) 2009 (n 519) Age (mo) (mean SD) Male gender 113 (56.5) 304 (58.6) 0.61 Time interval (days) (mean SD) HFMD 42 (21.0) 371 (71.5) 0.01 Herpangina 162 (81.0) 157 (30.3) 0.01 Meningoencephalitis 8 (4.0%) 79 (15.2) 0.01 resenting symptoms Fever ( 37 C) 37 (18.5) 203 (39.1) 0.01 Headache 11 (5.5) 53 (10.2) 0.04 Vomiting 32 (16.0) 39 (7.5) 0.01 Neck stiffness* 4 (2.0) 12 (2.3) 0.80 Sore throat 59 (29.5) 70 (13.5) 0.01 Abdominal pain* 3 (1.5) 10 (1.9) 0.70 Diarrhea 9 (4.5) 15 (2.9) 0.28 Decreased mental status* 1 (0.5) 12 (2.3) 0.08 Seizure 6 (3.0) 14 (2.7) 0.83 Sample type Stool 94 (47.0) 239 (46.1) 0.82 Respiratory 122 (61.0) 330 (63.6) 0.52 CSF 1 (0.5) 61 (11.8) 0.01 tivariable analysis. To our knowledge, this is the first study to investigate the difference between EV 71 and non-ev 71 enteroviral infections from the perspective of clinical presentation. EV 71 is more contagious than other enteroviruses (18). Based on our current results, no HFMD cases caused by EV 71 were found in 2008, whereas the number of EV 71 infections increased dramatically in 2009, accounting for about 50% of the cases of HFMD and herpangina. Subsequently, the prevalence of accompanying meningoencephalitis also increased substantially, from 4.0 to 15.2%. Moreover, compared to those in 2008, the prevalences of accompanying fever, headache, and decreased mental status increased in These findings are in agreement with those of previous studies in which the viru- TABLE 3. Clinical characteristics of patients suspected of enterovirus infection according to the presence of the enterovirus genome by real-time RT-CR Negative by CR (n 272) Value for patients: ositive by CR (n 447) Age (mo) (mean SD) Male gender 152 (55.9) 265 (59.3) 0.37 Time interval (days) (mean SD) HFMD 152 (55.9) 261 (58.4) 0.51 Herpangina 125 (46.0) 194 (43.4) 0.50 Meningoencephalitis 11 (4.0) 67 (15.0) 0.01 resenting symptoms Fever ( 37 C) 93 (35.7) 143 (32.0) 0.31 Headache 20 (7.4) 44 (9.8) 0.26 Vomiting 23 (8.5) 48 (10.7) 0.32 Neck stiffness* 3 (1.1) 13 (2.9) 0.05 Sore throat 47 (17.3) 82 (18.3) 0.72 Abdominal pain* 6 (2.2) 7 (1.6) 0.53 Diarrhea 13 (4.8) 11 (2.5) 0.09 Decreased mental status* 1 (0.4) 12 (2.7) 0.02 Seizure 7 (2.6) 13 (2.9) 0.79

4 VOL. 48, 2010 ENTEROVIRUS 71 INFECTION IN KOREA 2493 TABLE 4. Clinical characteristics of 218 patients according to EV 71 or non-ev 71 genotype Value for patients with the following genotype: Non-EV 71 (n 127) EV 71 (n 91) Age (mo) (mean SD) Male gender 68 (53.5) 54 (59.3) 0.40 Time interval (days) (mean SD) HFMD 55 (43.3) 87 (95.6) 0.01 Herpangina 74 (58.3) 7 (7.7) 0.01 Meningoencephalitis 6 (4.7) 35 (38.5) 0.01 resenting symptoms Fever ( 37 C) 20 (15.7) 33 (36.3) 0.01 Headache* 5 (3.9) 16 (17.6) 0.01 Vomiting 13 (10.2) 13 (14.3) 0.36 Neck stiffness* 1 (0.8) 6 (6.6) 0.02 Sore throat* 23 (18.1) 4 (4.4) 0.01 Diarrhea 5 (3.9) 0 (0) 0.07 Decreased mental status* 0 (0) 3 (3.3) 0.07 Sample type Stool 68 (53.5) 55 (60.4) 0.31 Respiratory 80 (63.0) 62 (28.1) 0.43 CSF* 3 (2.4) 15 (16.5) 0.01 lence of EV 71 was found to be stronger than that of other enteroviruses (13, 28). During epidemics, enteroviruses are spread by horizontal transmission from child to child and from mother to fetus. Transmission occurs by means of direct contact with nasal and/or oral secretions, fecal material, or aerosolized droplets via a fecal-oral or oral-oral route. Initial viral implantation in the buccal and ileal mucosae is followed by spreading to the lymph nodes within 24 h. Viremia ensues rapidly, with spreading to the oral mucosa and skin (24). Notably, in our study, patients infected with EV 71 were likely to present with HFMD and meningoencephalitis rather than with herpangina, in keeping with previous reports (10, 29). Regarding the pathogenesis of EV, mentioned above, HFMD and meningitis may occur after viremia, whereas herpangina can occur with local oral infections. Thus, it is assumed that EV 71 is more virulent and consequently induces viremia easily, whereas herpangina alone is a rare manifestation of EV 71 infections. This speculation should be investigated further. In contrast to the situation in 2008, EV 71 became a major cause of HFMD and herpangina in Korea in 2009, and in turn, the number of cases with neurological complications increased. However, the reported number of cases of neurological complications was small compared to those in other reports (14, 17, 22, 29). There are several explanations to account for this finding. Given the geographical location of Korea, EV 71 may be imported from China or Taiwan. Indeed, almost all of the EV 71 infections detected in 2009 were nearly identical, in terms of their genetic identities, to those reported in China during the preceding 2 years (32). It has been reported that EV 71 subgenotype C4a causes fewer neurological complications than other subgenotypes, including C1 and B5 (1, 8, 11, 12). An aggressive treatment strategy for EV 71 infection could be another reason. Recent reports of severe neurological complications and their successful treatment using intravenous immunoglobulin (IVIg) have pressed physicians to manage such patients more aggressively (20, 27). In Korea, since EV 71 was identified as a major cause of HFMD, many physicians have TABLE 5. Univariate and multivariable analyses of predictors for EV 71 infection redictor Univariate analysis Multivariate analysis a Unadjusted OR (95% CI) Adjusted OR (95% CI) Age, per yr 0.98 ( ) NA resenting with HFMD ( ) ( ) resenting with herpangina 0.06 ( ) NA Fever ( 37 C) 3.04 ( ) ( ) Headache 5.21 ( ) ( ) Neck stiffness 8.89 ( ) NA Sore throat 0.21 ( ) ( ) a Stepwise backward regression. The variables entered were presenting with HFMD, presenting with herpangina, fever, headache, neck stiffness, and sore throat. NA, not available.

5 2494 RYU ET AL. J. CLIN. MICROBIOL. used IVIg to treat patients with neurologically complicated HFMD. Finally, other factors, including differences in the preexisting immunity of the pediatric population (16) and differences in host genetic susceptibility, may influence (15, 31) the frequency of cases with neurological complications. In a previous study, an older age, attendance at kindergartens/child care centers, contact with HFMD/herpangina, a greater number of children in a family, and rural residence were associated with EV 71 infection (5). In addition, some clinical symptoms and signs have been found to predict central nervous system (CNS) complications in patients with EV 71 infections (21). In the present study, we found that presenting with HFMD, fever, or sore throat was independently associated with EV 71 infection. EV 71 is more likely than other enteroviruses to cause serious neurological complications; moreover, such complications develop a few days after the first symptoms appear (10). Current diagnostic methods are timeconsuming and require special equipment, making their application to general medical practice difficult. Therefore, the early classification of patients according to their symptoms would be helpful. In this regard, our study might provide a means for the initial classification of patients with HFMD or herpangina as having infections due to EV 71 versus non-ev 71 viruses. In conclusion, we demonstrated that EV 71 is an emerging pathogen in Korea, as well as in China and Taiwan. In addition, we found that presenting with HFMD, a fever, or a sore throat was independently associated with EV 71 infection. 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