Etiological spectrum of clinically diagnosed Japanese. encephalitis cases reported in Guizhou Province, China in

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1 JCM Accepts, published online ahead of print on 0 February 00 J. Clin. Microbiol. doi:0./jcm Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Etiological spectrum of clinically diagnosed Japanese encephalitis cases reported in Guizhou Province, China in 00 Ye Xufang,Wang Huanyu *, Fu Shihong *, Gao Xiaoyan*, Zhao Shuye, Liu Chunting,Li Minghua*, Zhai Yougang*, Liang Guodong* a Institute for Immunization Program, Guizhou province Center for Disease Control and Prevention, Guiyang, People s Republic of China * State Key Laboratory for Infectious Disease Prevention and Control, Department of Viral Encephalitis, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People s Republic of China a Author for correspondence: State Key Laboratory for Infectious Disease Prevention and Control, Department of Viral Encephalitis, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 00 Ying Xin Street, Xuan Wu District, Beijing 000, People s Republic of China. Phone: + 0 0; Fax: + 0 0; gdliang@hotmail.com Number of words in the Abstract: Number of words in the main text: These authors contributed equally to this work.

2 0 0 0 Abstract In this study, the proportion of laboratory-confirmed Japanese encephalitis virus (JEV) infections was compared to JE cases which were reported based on seasonality and clinical symptoms of hospitalized patients in Guizhou Province, in China between April and November 00. Of the, reported JE cases,, patients in nine prefectures were investigated. JE was confirmed in,0 of, (.%) by JEV-specific immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA), hemi-nested RT-PCR, and virus isolation. Two strains of JEV belonging to genotype were isolated. Other viral encephalitis pathogens, including Echovirus, mumps virus, herpes simplex virus, and cytomegalovirus, were identified in of (.%) JE-negative cases. Based on the distribution of the laboratory-confirmed JE cases from different level hospitals, which included provincial, city, county, and township, county hospitals detected the highest number of JE cases (.%) whereas township hospitals detected the least number of JE cases (.%). Provincial and city hospitals had the highest and lowest accuracy rates of JE clinical diagnosis confirmed by laboratory testing (.% and.%, respectively). This study demonstrates that laboratory confirmation improves the accuracy of JE diagnosis and enhanced laboratory capacity is critical for JE surveillance as well as identification of other pathogens that cause encephalitic syndromes with clinical symptoms similar to those of JEV infection.

3 0 0 0 Introduction Japanese encephalitis virus (JEV), a mosquito-borne pathogen of the family Flavivirus (genus Flaviviridae), is the most common cause of epidemic and endemic viral encephalitis in Asia, with the disease being more severe in young children (, ). Japanese encephalitis (JE) is widespread throughout Southeast Asia, the western Pacific region, Australasia (,, ), and China. There are an estimated,000 to 0,000 cases of JE reported annually, with 0-0% of JE patients presenting with neurological and mental sequelae, and approximately 0,000 JE-related deaths (). Before there were 000-0,000 JE cases annually in China, which accounted for >0% of the worldwide total (). Since the introduction of JE vaccine in China, incidence has decreased, with only 0 reported JE cases from China in 00 (). Despite the JE vaccine, JEV infection is still the primary cause of viral encephalitis and continues to be a pressing public health problem in China (, 0). A clinical JE case reporting system has been in place in China since. JE cases and epidemic outbreaks have occurred in all provinces except Xinjiang, Tibet, and Qinghai (). The reported incidence of JE decreased from 0. to 0. per 00,000 between 000 and 00. Five provinces (Guizhou, Sichuan, Chongqing, Henan and Yunnan) have reported the most JE cases, with children under the age of being affected most often (, 0, ). Although, the incidence rate of JE has decreased since 000, the hilly province of Guizhou has the highest annual incidence of JE in China; there were,0 and,0 reported cases of JE in 00 and 00, respectively, accounting for over 0% of the total in China (). JE surveillance in China has consisted of reporting of suspected or clincially diagnosed cases in hospital settings. Seasonalitiy, vaccine status, and clinical tests are factors in diagnosis, but laboratory confirmation has not been part of routine surveillance. We wanted analyze the accuracy of JE diagnosis in Guizhou Province and determine the effect of laboratory testing

4 0 0 0 on enhancing detection of JE cases, as laboratory-based surveillance has been shown to be important for determining the actual JE disease burden (). The JEV immunoglobulin M (IgM)-capture enzyme-linked immunosorbent assay (MAC-ELISA) is highly sensitive and relatively specific, and is therefore the main method for diagnosing JEV infections. However, even with this assay few reported JE cases are confirmed by serological testing in the southwestern provinces of China, especially in Guizhou Province. In 00, only of,0 reported cases were analyzed by JEV MAC-ELISA, and of those (.%) were JE IgM-positive (). No data were available regarding other pathogens known to cause viral encephalitis in Guizhou Province. In the present study, we tested serum and CSF specimens collected from reported JE cases from Guizhou Province during 00 by JEV MAC-ELISA. In addition, CSF specimens underwent nucleic acid detection testing in order to enhance sensitivity. The samples with negative JE results were further tested by IgM ELISA for eight other viral etiologies. The proportion of laboratory confirmed JE cases among the reported JE cases was analyzed by testing method, hospital level, and JE incidence region within Guizhou Province. We found evidence of a mixed etiological spectrum and identified several factors that may affect the assessment of the actual JE disease burden.

5 0 0 0 Patients and Methods Patients and case definition A total of JE cases (including suspected JE cases and those meeting the clinical JE case definition) ages, month to 0 years were reported in Guizhou Province between April and November 00. The urban or rural residence of the patient was not considered in the definition or analysis. For purposes of this study, a reported JE case is defined as a suspected or clinically confirmed JE case, without laboratory confirmation. A suspected case is defined as a hospitalized patient with fever and three or more of the following symptoms: vomiting, headache, focalized or generalized seizure, coma, lethargy, motor deficit, and neck rigidity, who was ill within the defined JE epidemic season (from June to October). A total of 0 suspected JE cases were reported in Guizhou Province between April and November 00. A clinically confirmed JE case is defined as a patient that meets the suspected JE case definition, with clinical laboratory results of CSF pleocytosis (>0 white blood cells/mm ), elevated protein (>0 mg/dl), and normal glucose concentration. Seven hundred thirty-two clinically confirmed JE cases were reported in Guizhou Province between April and November 00. A laboratory confirmed JE case is defined as a suspected or clinically confirmed reported JE case for which there has been laboratory confirmation by the presence of JE virus-specific IgM in a single CSF or serum sample, detection of JE viral RNA in CSF by hemi-nested reverse transcriptase polymerase chain reaction (hnrt-pcr), or isolation and identification of JE virus from CSF. Specimens Specimens were collected from, of the hospitalized reported JE cases in nine prefectures in Guizhou Province (from north to south): Zunyi (ZY), Bijie (BJ), Tongren (TR), Liupanshui (LPS), Guiyang (GY), Qianxinan (QXN), Anshun (AS), Qiannan (QN), and Qiandongnan (QDN). Both serum and CSF

6 0 0 0 samples were obtained from patients. Only serum was obtained from,0 patients; and only CSF was obtained from the remaining 0 patients. From the cases, specimens were collected within the first days of illness onset in (.%), from - days in 00 (.%), and > days in (.%). Specimens were stored at -0 C and transported on dry ice to the Department of Viral Encephalitis and Arbovirus, Institute for Viral Disease Control and Prevention (IVDCP), China CDC in Beijing for testing. Serological testing All the serum and CSF specimens were screened for JEV-specific IgM by MAC-ELISA using a JEV IgM Capture ELISA kit (Shanghai B&C Biological Technology Co., Ltd., Shanghai, China). The JEV IgM-negative serum samples were tested by ELISA IgM kits (Virion/Serion Co., Wurzburg, Germany) for other viral encephalitis pathogens, including Echovirus (ECHOV), Coxsackievirus (COXV), Epstein-Barr virus (EBV), mumps virus, herpes simplex virus types and (HSV+), Varicella-zoster virus (VZV), cytomegalovirus (CMV), and measles virus. All operations were done in accordance with the manufacturer s instructions. All testing was carried out at the Department of Viral Encephalitis and Arbovirus, IVDCP, China CDC. Molecular biological testing Sixty-four of the CSF specimens met the criteria for molecular testing by hemi-nested RT-PCR (hnrt-pcr): ) specimen collection was within days of illness onset, ) the specimen had been appropriately stored and shipped at -0C, ) and sufficient volume of the specimen remained after serological testing was completed. Briefly, RNA was extracted using a QIAamp viral RNA extraction kit (QIAGEN, Valencia, CA, USA) in accordance with the manufacturer s protocol and produced the first strands of cdna by using Ready-To-Go You-Prime First-Strand Beads (Amersham Pharmacia Biotech, Piscatawy, NJ, USA) as described in the manual accompanying the kit. The

7 prm gene of JEV was amplified by hnrt-pcr (, ). One positive and one negative control were included in the reactions, and anti-contamination procedures were strictly enforced. All the operations were carried out according to the manufacturer s specifications Virus isolation and identification Of the CSF specimens examined by hnrt-pcr, had sufficient volume for virus isolation. An aliquot of 00 µl of the CSF specimens was diluted in ml Eagle s minimum essential medium (MEM, Sigma, USA) and inoculated onto confluent BHK- cell monolayers in a T flask. After adsorption for hr at C, fresh medium was added and the cells were incubated at C in % CO. The cells were checked daily for cytopathic effect (CPE). At 0% CPE, the culture supernatants were harvested, cellular debris was removed by centrifugation at,000 rpm. The supernatants were stored at -0 C until testing by JEV by hnrt-pcr. Neighbour-joining phylogenetic analysis on decided the genotype of the JEV strains based on the nucleotide sequences were constructed using MEGA version. Diagnostic criteria A viral pathogen was regarded as etiologic when one of the following criteria was met: ( ) presence of virus-specific IgM antibodies in serum and/or CSF; ( ) virus was isolated from CSF; or ( ) viral RNA was detected in CSF by hnrt-pcr. Statistical analysis Significance was assessed by Pearson s analysis using SPSS (version., SPSS Inc., Chicago, IL, USA)

8 0 0 0 Results Reported JE cases epidemic analysis in Guizhou Province, 00 The nine prefectures were classified into four groups according to the JE incidence rate. The incidence rate was derived from epidemic data of JE cases reported to China CDC in 00, GY, the capital of the Guizhou Province, was in group (JE incidence rate </00,000); the eastern prefectures of TR and QDN were in group (JE incidence rate, -/00,000); most of the prefectures, including LPS, ZY, QXN, and QN, belonged to group (JE incidence rate, -/00,000); and AS and BJ were in group (JE incidence rate, -/00,000) (Fig. ). The hospitals involved in this study are classified in China into four levels: provincial, city, county, and township. Province, city, county and township are the four administrative divisions in China. According to the administrative division, the hospitals involved in this study were divided into provincial hospitals, city hospitals, county hospitals and township hospitals. Provincial hospitals provide medical service for the whole province, while city hospitals mainly serve people within the prefecture, county hospitals mainly serve people within the county, and township hospitals serve as health care centers within the township. Patients with more serious illness in county hospital mostly get transferred to city hospital and/or provincial hospital. In general, the provincial hospital is more comprehensive and capable, followed by city, county and township hospitals. Specimen collection The overall specimen collection rate was.% (,/,) (Table ). In seven prefectures, the collection rate exceeded %. By comparison, the rate in ZY was.%, and that in BJ was.%. Laboratory diagnosis There were, hospitalized patients enrolled in the study. These patients

9 0 0 0 met the suspected or clinically confirmed JE case definition and serum and/or CSF was obtained from each patient. Of the, 0 patients (.%) were laboratory confirmed as JE-positive, (.0%) by JEV MAC-ELISA (0 of,0 serum samples, of 0 CSF samples, of serum/csf samples) and (.0%) by hnrt-pcr only (0 of 0 CSF samples, of serum/csf samples). The JE positivity rates for the prefectures ranged from.% (GY) to.% (LPS). (Table ) Of the CSF specimens tested by hnrt-pcr, cases were confirmed as JE: by JEV-MAC-ELISA alone with negative hnrt-pcr results; which were negative by JEV-MAC-ELISA but positive by hnrt-pcr; and which were positive by both JEV-MAC-ELISA and hnrt-pcr (Tables and ) Virus was isolated from of CSF specimens for which sufficient volume remained (data not shown). Background analysis showed that the CSF sample from one patient (-year-old male; CSF sample collected days after illness onset) was JEV IgM positive, whereas the serum and CSF samples from the other patient (-year-old female; serum and CSF collected and days after onset, respectively) were JEV IgM negative. Neither patient had been vaccinated against JEV. The viral isolates were identified as JEV genotype by phylogentic analysis (data not shown). One hundred seventy-two JE-negative serum specimens were tested for IgM antibodies against other etiological agents. Other viral pathogens were identified in (.%). Mumps virus was the most common pathogen, accounting for. % (/) followed by ECHOV (/,.%), HSV (/,.0%), COXV (/,.0%), and CMV (/,.%). No patient tested positive for EBV, VZV, or measles virus. IgM antibody testing revealed several cases of infection with more than one virus: six cases (.%) of mumps virus and HSV and five cases of ECHOV and COXV (.%) (data not shown). The average rate of diagnosis for all other pathogens was.% (/). Non-JE viral infections accounted for.% of the diagnosed cases overall; the highest frequency (.%) occurred in GY and the lowest (.%), in LPS. The

10 frequency ranged from 0% to % in the other regions (Table ) Sex, age and seasonal distribution From the analysis of reported JE cases for which clinical data was available, among lab-confirmed JE patients the number of males exceeded that of females by a ratio of.:., The < to year-old age group accounted for 0% of the JE-positive patients; this was concordant between hospitalized reported JE cases and JE laboratory confirmed cases. There was no age range associated with any of JE-negative cases with other viral etiologies (Fig. ). The seasonal distribution of reported JE cases showed that JE incidence appears to have a single epidemic peak spanning the months of July and August. Encephalitis caused by other viral pathogens did not show clear seasonal distribution (Fig. ). Distribution of JE cases diagnosed in the laboratory In GY (JE incidence group ), the percentage of laboratory-confirmed cases compared to the suspected or clinically confirmed reported JE cases was relatively low (.%) and differed significantly from the percentages of laboratory confirmed JE cases in the other regions ( = 0., p = 0.00) (Table ). No significant difference was detected among the four incidence groups in Guizhou Province. The hospitals involved in this study were also classified into four different hospital levels, including provincial, city, county and township. County hospitals detected.% of the total laboratory confirmed cases, with the highest and lowest rates in QN (.%) and GY (.%), respectively. This was followed by city (.%), provincial (.%), and township (.%) hospitals (Table ). Accuracy of suspected and clinically diagnosis JE cases, determined by laboratory confirmation, were.%,.%, and.% in the provincial (there were no provincial hospitals in QDN, LPS, QXN, or QN), 0

11 0 0 0 county, and township hospitals, respectively. In comparison, clinical diagnosis accuracy was significantly lower in city hospitals (.%; =., p < 0.00); the lowest accuracy (0.0%) occurred in QDN. The rate of JE laboratory confirmation was lower in the provincial (.0%) and county hospitals (.%) in GY than in the other hospitals. The detection rate for other pathogens was higher in country hospitals in TR prefecture (.%) than in other regions (Table ).

12 0 0 0 Discussion JE is an epidemic disease which occurs during the mosquito transmission season in the summer in mainland China. In previous studies from 000 to 00, the number of cases reported between June and October accounted for over % of the total annual cases, with monthly distribution data showing a single peak spanning July and August (). The incidence of JE decreased steadily after and since has remained at < / 00, 000, which is around the same time that a JE vaccination became widely used in several eastern provinces in China (). A safe and effective live-attenuated JE vaccine made from strain SA-- is routinely administered nationwide in China (, ). Since 00, the prevention and control of JE in China has been advanced through the integration of JE immunization into the Expanded Program of Immunization (EPI) on mainland China, especially in rural and poor areas such as Guizhou Province. The EPI system is aimed mainly at children; however, the number of adult cases of JE being reported in China is increasing; for example, % of the patients in the Yuncheng JE outbreak were adults (). Therefore, it is essential to implement lab-confirmation as part of surveillance for diagnosis of cases hospitalized with suspected JE in order to understand the true JE disease burden. Guizhou Province is a poor, rural area with hilly terrain located in southwestern China. It has had the highest rate of JE epidemics and incidence from to 00 (). It has been hypothesized () that JE incidence in low-income areas such as Guizhou Province would be higher because of the lack of a surveillance system, personal protection, disease and mosquito vector education, and financial resources. We wanted to look at the current incidence rate of JE in Guizhou Province using laboratory-based surveillance, as well as determine the accuracy of JE reporting. We conducted our investigation during the summer JE epidemic season in Guizhou Province in 00. Laboratory testing by virus isolation, serological

13 0 0 0 MAC-ELISA, and hnrt-pcr confirmed.% of the reported JE cases. JEV MAC-ELISA remains the dominant method for lab-confirmation of JE in China, and in our study.0% of cases were identified by JEV MAC-ELISA. Although CSF was considered the best specimen for diagnostic testing, only.% CSF specimens were referred in this study. Of the CSF specimens collected, could be tested by molecular methods; JE cases were confirmed by hnrt-pcr alone, and two JEV isolates were obtained. Among the JE-negative cases, another viral encephalitis etiology was identified in (.%). CMV particularly seems to be an important pathogen among this population (, ). JE was identified most often in children during July and August, whereas the non-je viral infections identified did not show these trends, which is similar to the results of previous studies (, ). Reported JE cases from Guizhou were largely comprised of hospitalized patients meeting the suspected JE case definition. Analysis of the diagnostic accuracy rate at the different level hospitals showed that the misdiagnosis rate was significantly higher in city hospitals than in provincial, county, or township hospitals. Guizhou province is mountainous and economically underdeveloped, with a poor road system. Most encephalitis patients are taken to the closer county hospitals (.%) rather than to provincial (.%) or city (.%) hospitals which are farther away. Few JE cases (.%) were seen at the township level hospital. Because doctors in county hospitals had more contact with JE cases than those from the township or provincial hospitals, they are more familiar with the disease, and it might be assumed, have a higher rate of clinical diagnostic accuracy. Many of the cases in the provincial hospitals, especially in GY, were referred from other hospitals across the province, and their clinical symptoms were complex and serious, resulting in difficult diagnoses and a lower rate of confirmed JE cases. Other studies have also found that severe, rare, or unusual cases are more likely to be found at tertiary care hospitals such as Beijing Children s Hospital compared to other hospitals, where JE may be more likely to be diagnosed by clinicians familiar with the

14 0 0 0 symptoms. Also, because there is no effective treatment for JE, patients at the county hospitals diagnosed with JE based on clinical symptoms would be less likely to be transferred to the tertiarary care provincial hospitals. Anecdotically, this phenomenon also appeared to be true in our study in GY, the capital of Guizhou Province. Despite the comprehensive testing, a large proportion of encephalitis cases remain undiagnosed. Similarly, it was shown in a retrospective study at Beijing Children s Hospital that the etiology could be confirmed by serological and/or molecular biological testing in only.% of cases (). The proportion of acute encephalitis syndrome (AES) related to infection with JEV is about 0-0% in Vietnam, Cambodia, and India (0,, ), where JE is an endemic disease. A separate study on acute undifferentiated fever in Thailand showed that about % of the cases were actually JE (). An investigation into the viral etiology of acute childhood encephalitis in Beijing showed that.% of the cases were caused by JEV (). Given a sample collection rate of.% (, of, reported JE cases), we estimate there were about,0 lab confirmed cases of JE in Guizhou Province in 00. HnRT-PCR can increase the sensitivity of JE detection in very acute JE cases if CSF is collected within the first few days of illness, when IgM may not have reached levels detectable by MAC ELISA. However, molecular methods increase the cost of testing, and the CSF must be stored at -0 C immediately after collection to prevent viral RNA degradation. In addition it is more difficult to establish and maintain the technical skills necessary to conduct the testing in hospitals or public health laboratories. Virus isolation also requires a high-quality CSF specimen, which means that CSF should be collected in a timely manner and stored/shipped in liquid nitrogen and/or dry ice. It would not be possible to do molecular testing routinely for JE surveillance at most public health laboratories. JEV genotype was identified from the isolates. This is the first reported isolation of JEV genotype from humans in China. JEV genotype was first

15 0 0 0 isolated from a human brain in Beijing in (), and genotype was first isolated from mosquitoes in Shanghai in 00 (). Since then, numerous genotype JEV strains have been isolated from mosquitoes in other provinces; in addition, genotypes and have been found to co-circulate across mainland China (, ). Moreover, JEV genotypes and were detected simultaneously in CSF specimens from JE patients and from mosquitoes during the JE outbreak in Yuncheng prefecture, Shanxi province, in 00, while no virus isolates were obtained in this outbreak (). Further investigation and isolations of JEV are needed to determine the role of genotype in JE outbreaks in China. In this study we looked at the accuracy of JE case reporting in Guizhou Province. JE cases were identified primarily from hospitalized patients with suspected and or clinically diagnosed JE. The insight it provides into the disease burden may be useful in further developing a JE vaccination policy in China. This study indicates that the current JE surveillance system in China should be enhanced to include laboratory confirmation of cases, and it is very important that laboratory capacity be improved in these settings in order distinguish JE from other encephalitis viral infections. To determine the true incidence of JE in China, ( ) a system using JE-MAC-ELISA for the diagnosis of JE should be instituted in hospitals and local (county and/or prefecture) CDCs. In addition, in the provincial laboratories with higher technical capacity using molecular detection methods would increase sensitivity. Laboratory-based surveillance for JE among AES patients should also be enhanced, as it would facilitate the identification of cases of JE that might have been missed based on other clinical definitions. In the future, we plan to focus on the etiology of AES, especially JEV, in China.

16 0 0 0 Acknowledgments We thank Dr. Barbara W. Johnson, Diagnostic & Reference Laboratory, Arbovirus Diseases Branch, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO, U.S.A. for her critically reading of the manuscript and helpful discussions about this paper. This work was supported by a grant (No. 00BAA0-0) from the Ministry of Science and Technology of China, Development Grant of State Key Laboratory for Infectious Disease Prevention and Control (00SKLID0) and The Japan Health Science Foundation (00-00).

17 0 0 0 References. Burke D. S., and Leake C. J.. Japanese encephalitis. In Arboviruses: Epidemiology and Ecology, vol., pp. -. Edited by T. P. Monath. Boca Raton: CRC Press.. Chen D., Wang H. Y., Fu S. H., Song H., Deng J., Yang Y. L., Liang G. D. 00. Molecular characteristics of three new isolates of Japanese encephalitis virus in Fujian Province. Chin J Exp Clin Virol. :-.. Chen Y. S., Liang X. F., Wang X. J., Li Y. X., Yang J. F., Li J. H., Yin Z. D., Hu Y. S., Lu W. 00. Epidemiological analysis of the Japanese encephalitis in China during Chin J Vac Immun. :-.. Erlanger, T. E., Weiss, S., Keiser J. 00. Past, Present, and Future of Japanese Encephalitis. Emerg Infect Dis., : -.. Hanna, J. N., Ritchie, S. A., Phillops, D. A.. Japanese encephalitis in north Queensland, Australian, Med J Aust. 0: -.. Hennessy S., Liu Z., Tsai T. F., Strom B. L., Wan C. M., Liu H. L., Wu T. X., Yu H. J., Liu Q. M., Karabatson N., Bilker W. B., Halstead S. B.. Effectiveness of live-attenuated Japanese encephalitis vaccine (SA--): a case-control study. Lancet. :-.. Huang C. H., and Wang Y. M.. Epidemic encephalitis virus isolation and identification in Beijing. Chin Med J. :0-.. Jeffrey, N. H., Scott, A. R., Debra, A. P.. An outbreak of Japanese encephalitis in the Torres Strait, Australia. Med J Aust., : -0.. Knipe D. M., and Howley P. M. Flaviridae: the virus and their replication. In Fields Virology, th Ed., pp. -0. Edited by B.N. Fields. Philadelphia: Lippincott-Raven. 0. Kabilan L., Ramesh S., Srinivasan S., Thenmozhi V., Muthukumaravel S., Rajendran R. 00. Hospital- and laboratory-based investigations of hospitalized children with central nervous system-related symptoms to assess Japanese encephalitis virus etiology in Cuddalore District, Tamil Nadu, India. J Clin Micro. :-.

18 Liu C. Y., Shen Q. L., Liang G. D. 00. Epidemiological analysis of viral encephalitis in children hospitalized in Beijing Children's Hospital from 00 to 00. Chin J Pract Ped. :-0.. Liu W. Clemens J. D., Yang J. Y., Xu Z. Y. 00. Immunization against Japanese encephalitis in China: A policy analysis. Vaccine. : -.. Lowry P. W., Truong D. H., Hinh L. D., Ladinsky J. I., Karabatsons N., Cropp C. B., Martin D., Gubler D. J.. Japanese encephalitis among hospitalized pediatric and adult patients with acute encephalitis syndrome in Hanoi, Vietnam. Am J Trop Med Hyg. :-.. Song C. F., Tao Q., Yu C., Jiang W. J., Zhou J. Z., Wang Z. X. 00. Preventive control strategy and epidemiological analysis on Japanese encephalitis in Guizhou province during -00. Chin J Vector Bio & Cont. :0-.. Srey V. H., Sadones H., Ong S., Mam M., Yim C., Sor S., Grosjean P., Reynes J. M. 00. Etiology of encephalitis syndrome among hospitalized children and adults in Takeo, Cambodia, 000. Am J Trop Med Hyg. : Tsai, F. T New initiatives for the control of Japanese encephalitis by vaccination: Minutes of a WHO/CVI meeting, Bangkok, Thailand,- October. Vaccine, : Suppl :-.. Wang H. Y., Fu S. H., Li X. Y., Song H., Deng J., Yang Y. L., Liang G. D. 00. Isolation and identification of genotype I Japanese encephalitis virus in China. Chin J Microbiol Immunol. : -.. Wang H. Y., Fu S. H., Wang L., Gao X. Y., Li Y. X., Yin Z. D., Zhang S. B., Liang X. F., Chutima S., Liang G. D. 00. Confirm diagnosis for Japanese encephalitis report cases in Shaanxi province Chin J Vac Immun.:-.. Wang Huanyu, Li Yixing, Liang Xiaofeng, Liang Guodong. 00. Japanese Encephalitis in Mainland China. Jpn. J. Infect. Dis. : Wang H. Y., and Liang G. D. 00. Review: Arbovirus in China in recent

19 0 0 0 ten years. Chin J Public Health. :-.. Wang H. Y., Takasaki T., Fu S. H., Sun X. H., Zhang H. L., Wang Z. X., Hao Z. Y., Zhang J. K., Tang Q., Kotaki A., Tajima S., Liang X. F., Yang W. Z., Kurane I., Liang G. D. 00. Molecular epidemiological analysis of Japanese Encephalitis virus (JEV) in China. J Gen Virol. :-.. Wang L. H., Fu S. H., Wang H. Y., Liang X. F., Cheng J. X., Jiang H. M., Cai G. L., Li X. W., Ze W. Y., Lv X. J., Wang H. Q., Zhang D. L., Feng Y., Yin Z. D., Sun X. H., Shui T. J., Li M. H., Li Y. X., Liang G. D. 00. Japanese Encephalitis Outbreak, Yuncheng, China, 00. Emerg Infect Dis. :-.. Wang X. J., Lu Y., Zhang Y. P., Chen Y. S., Liang X. F. 00. Dynamic Tendency of Japanese B Encephalitis in China. Chin J Vacc Immun. 0:0-0.. Wang X. J., Zhang Y. P., Zhang R. Z., Chen Y. S., Liang X. F. 00. Analysis on epidemic trend of Japanese Encephalitis during -00 in China. Chin J Vacc Immun. 0:-.. Watt G., and Jongsakul K. 00. Acute undifferentiated fever caused by infection with Japanese encephalitis virus. Am J Trop Med Hyg. :0-0.. Xin Y. Y., Zhang G. M., Guo Y. P., Ao J., Min L. H.. Safety of a live attenuated Japanese encephalitis virus vaccine (SA--) for children. Am J Trop Med Hyg. :-.. Xu Y. H., Zhao R. G., Sirkka V., Shen K. L., Zhou Y. T., Magnius L. O., Britta W., Annika L.. Viral etiology of acute childhood encephalitis in Beijing diagnosed by analysis of single samples. Ped Infect Dis J. :0-0.. Xu Z. Q., Fu S. H., Zhang Y. P., Li X. L., Gao X. Y., Wang L., Cao Y. X., Xu L. H., Jin Y., Tang Q., Liang G. D. 00. Laboratory testing of specimens from patients with viral encephalitis from regions of China. Chin J Exp Clin Virol. :-00.

20 . Zhuang Y., Wu H., Yu C., Wang Z. X. 00. Epidemiological investigation and analysis of a Japanese encephalitis outbreak in Guizhou Province. Chin J Vector Bio & Control. :

21 TABLE. Specimen collection from reported JE patients in Guizhou Province in 00 JE incidence No. of reported JE No. specimen Specimen collection Area a group cases collected cases rate (%) GY. TR 0 0. QDN. LPS. ZY 0. QXN. QN. AS. BJ. Total. a the abbreviation of the prefectures: Guiyang (GY), Tongren (TR), Qiandongnan (QDN), Liupanshui (LPS), Zunyi (ZY), Qianxinan (QXN), Qiannan (QN), Anshun (AS), and Bijie (BJ) prefectures.

22 TABLE. Laboratory diagnosis of Japanese encephalitis (JE) in reported JE patients in Guizhou Province in 00 JE Serum CSF Serum / CSF Total JE Positive Reported incidence Area ELISA+ / ELISA- / ELISA+ / S CSF S/CSF S/CSF S/CSF S/CSF Positive Rate Cases ELISA+ ELISA+ RT-PCR- RT-PCR+ RT-PCR+ ELISA+ ELISA+ ELISA+ ELISA+/RT-PCR- ELISA-/RT-PCR+ ELISA+/RT-PCR+ cases (%) group GY. TR 0. QDN. LPS 00. ZY 0. QXN 0. QN. AS 0 0. BJ *. Total Abbreviations: S, serum; CSF, cerebrospinal fluid; +, positive; -, negative; ELISA, JEV-specific IgM by MAC-ELISA using a JEV IgM Capture ELISA kit; RT-PCR, molecular testing by hemi-nested RT-PCR (hnrt-pcr) * JEV was isolated from of these samples.

23 TABLE. Positive and Negative result for Japanese Encephalitis (JE) diagnosis by JE- MAC-ELISA and RT-PCR Test JE-MAC-ELISA Total RT-PCR Total 0 TABLE. Laboratory diagnosis of other pathogens in reported JE patients in Guizhou Province in 00 JE-negative Laboratory diagnosis JE incidence No. tested Area cases Other pathogen Negative group cases No. Rate (%) No. Rate (%) No. Rate (%) GY..0. TR 0... QDN..0. LPS... ZY... QXN... QN.. 0. AS... BJ 0... Total.. 0.

24 Group Table. Distribution of lab-confirmed cases of Japanese encephalitis (JE) in Guizhou Province in 00 Area No. tested cases Tested cases JE Province City County Town Other Pathogen Negative Tested cases JE Other Pathogen GY (.) (.0) a 0 (.0) (.) 0 (.) 0 (.) (.) (.) (.) (.) Negative TR 0 (.) (00.0) 0 0 (.) (0.0) 0 (0.0) (.) (.) (.) (.) (.) (0.0) (0.0) (0.0) QDN N b (.) (0.0) (0.0) (0.0) (.) (.) (.) (.) LPS N (.) (0.0) 0 (0.0) (.) (.) (.) (.) (.) (00.0) 0 0 ZY 0 (.) (.0) 0 (.0) (0.) (.) (.) (0.) 0 (.) (.) (.) (.) (.) (00.0) 0 0 QXN N (.) (.) (.) (.) (.) 0 (.) (.) (.) (.) (00.0) 0 0 QN N (.) (00.0) 0 0 (.) (.) (.) 0 (.) AS (0.) (00.0) 0 0 (.) (.) (.) (.0) (.) (.) (.) (.) (.) (00.0) 0 0 BJ (.0) (00.0) 0 0 (.) 0 (.) (.) (.) (.) 0 (.) (.) (.) (.) (00.0) 0 0 Total (.) (.) 0 (.) (.) (.) 0 (.) 0 (.) 0 (.) 00 (.) (.0) (.) (.) (.) (.) (.) a number of cases and the positive rate (%) b no provincial hospitals in this area Tested cases JE Other Pathogen Negative Tested cases JE Other Pathogen Negative

25 Zunyi Bijie Tongren Guiyang Liupanshui Anshun Qiandongnan Qiannan Qianxinan Incidence of JE </00,000 Incidence of JE - /00,000 Incidence of JE - /00,000 Incidence of JE - /00,000 FIGURE. A: The location of Guizhou Province in China. B: The background incidence rate of reported JE cases in nine prefectures within Guizhou Province. Downloaded from on April, 0 by guest

26 0 0 0 Reported JE cases Lab confirmed JE cases Other pathogen infection Negative Number of patients < Age FIGUE. Age distribution of total reported JE cases, lab-confirmed JE cases and other virus infection in Guizhou province, 00 Downloaded from on April, 0 by guest

27 00 Reported JE cases Lab confirmed JE cases 00 Other pathogen infection Negative Number of patients Apr May Jun Jul Aug Sep Oct Nov Months Downloaded from FIGUE. Monthwise distribution of total reported JE cases, lab-confirmed JE cases and other virus infection in Guizhou province, 00 on April, 0 by guest