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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In Mainland China, an epidemic of dengue caused by DEN-1 and DEN-4 was reported in Shiwan Town, Foshan City, Guangdong Province, in 1978–1979.4 In 1980, a dengue outbreak occurred on Hainan Island. DEN-1 and DEN-3 were isolated from sera of acute-phase patients and pools of adult Ae. aegypti.5 In 1985–1986, the first epidemic of DHF in China occurred on Hainan Island. The morbidity rate was 1,913 per 100,000 residents, with a case fatality rate of 0.25%. DEN-2 was isolated from sera of acute-phase patients.6 Since the 1990s, dengue epidemics have frequently occurred in Guangdong, Guangxi, and Hainan provinces. Aedes aegypti was the vector in coastal areas, and Ae. albopictus was the vector in inland regions of China. The reemergence of dengue in China usually resulted from the introduction of the infection by travelers and refugees from areas of southeast Asia where dengue was endemic.7
In Ningbo, Zhejiang Province of China, no dengue epidemic had been recorded in the past 75 years.8 In early September 2004, families-based clustering cases with acute febrile illness from seven adjacent rural villages of Xiaolin Town, Cixi, Ningbo drew serious attention in two public hospitals in the metropolitan area of Ningbo. The number of cases kept increasing and caused fear in local communities. Our epidemiologic team conducted the investigation on this undifferentiated acute febrile disease. A dengue outbreak was then identified based on the basis of epidemiologic, clinical, and laboratory examinations that met the case definition.9 Efforts to temporarily eradicate mosquito infestation contributed to the cessation of transmission.
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METHODS |
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Characterization of clinical illness. Clinical information was collected from those residents who were admitted to the hospitals in Ningbo between September and October 2004. A standardized questionnaire was used to record the presence or absence of fever, chills, fatigue, rash, myalgia, arthralgia, joint pain, anorexia, cough, and gum bleeding; and the presence or absence of physical signs elicited on examination, including cervical lymphadenopathy, flushing, petechiae, and suffused eyes. Results of laboratory tests performed as part of routine clinical care were recorded from case-sheets, including hematologic and biochemical examination, bacterial cultures of blood, and serologic tests for antibodies to dengue, rickettsioses, typhoparatyphoid, and Japanese encephalitis. Demographic, geographic, and temporal data were collected from residents with febrile disease from the affected villages. An index case and the cases with the disease in August 2004 were retrospectively traced on the basis of clinical, laboratory, and epidemiologic data.
IgM-capture enzyme-linked immunosorbent assay (ELISA). Serum specimens from acute-phase patients were obtained and stored at –20°C. A dengue IgM-capture ELISA (Zhongshan Bioengineering Inc., Guangdong, People’s Republic of China) was performed according to the manufacturer’s instructions.
Virus isolation and RNA extraction. Sera of acute febrile cases were obtained within six days after the onset of disease. Virus isolation was performed by adding the serum samples into the Ae. albopictus cell line C6/36, which was grown in plastic tissue culture flasks (T-25). Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL, Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS) (Gibco-BRL) was used. The cell monolayer was incubated with 200 µL of serum sample at 28° C in an atmosphere of 5% CO2 for one hour before 2 mL of DMEM supplemented with 2% FCS was added. The cultures were incubated for seven days and observed daily for cytopathic effects (CPEs). At the end of the seven-day incubation, the supernatant was harvested and clarified by centrifugation at 2,500 rpm before incubation onto a fresh cell monolayer. The RNA was extracted from 200 µL of sera from 12 acute-phase febrile patients and from viral culture supernatants by using the RNAeasy Mini Kit (Qiagen, Hilden, Germany).
Reverse transcription–polymerase chain reaction (RT-PCR) and sequencing. An RT-PCR developed by Lanciotti and others10 was used for the detection and identification of dengue viral RNA by One Step RNA PCR Kit (TaKaRa Biotech Co., Dalian, People’s Republic of China). The RT-PCR products were analyzed by electrophoresis on a 2% aga-rose gel, stained with ethidium bromide, and visualized under ultraviolet radiation. Genomic RNA of the isolates was reverse transcribed into cDNA using random hexamer oligonucleotides with the SuperScript first-strand synthesis system (Invitrogen, Carlsbad, CA). DNA fragments of the envelope (E) gene and the overlapping DNA fragments that included the genome of the isolate were amplified by PCR with Proof-start (Qiagen) and Taq DNA polymerase (Promega, Madison, WI) and submitted for DNA sequencing to the Shanghai Bioengineering Center of Chinese Academy of Science (Shanghai, People’s Republic of China).
Phylogenetic analysis. A total of 61 DEN-1 strains were retrieved from GenBank with different temporal and geographic origins, and named by the strain number followed by country abbreviation and year of isolation (Supplementary Table). NB01Cho4 (E gene of DQ836632) and the reference sequences were aligned using the CLUSTAL X 1.81 algorithm with default parameters. Phylogenetic trees for the entire sequence of E gene were obtained using different tree building methods either by MEGA (version 3.1) or PHYLIP (version 3.65).11 Bootstrap analysis with 1,000 replicates was used to determine the robustness of the tree and the evolutionary relationship of DEN-1.
Entomology. Cixi (30°02'–30°24'N, 121°02'–121°42'E) has a monsoon climate. The mean annual rainfall is 1272.8 mm, and mean annual temperature 16.0°C. Xiaolin Town is in the center of Cixi. The population in Xiaolin is approximately 82,000 including 41,108 native residents and 41,000 temporary workers from other provinces. Because the dengue outbreak was confirmed on October 5, 2004, we conducted entomologic inspections in Xiaolin to collect and identify mosquito larva in all water-containing receptacles in and around the dwellings on October 6–8, 2004. Devices to trap adult mosquitoes and mosquito larva were installed in a sampling of houses. An estimate was made of infestation by Ae. aegyti and Ae. albopictus using standard larva survey: Breteau index (BI) (number of containers with immature stages per 100 houses), house index (HI) (number of houses containing immature stages per 100 houses), and container index (CI) (number of containers with immature stages per 100 containers with water). Identification of the species of the larva was carried out after the fourth development stage.
To investigate the possibility of dengue virus transovarial transmission, adult mosquitoes and immature forms of Ae. albopictus were collected from households and nearby ponds where residents were diagnosed with DF. After collections, mosquitoes were anesthetized on ice, identified, pooled by sex, date, and place of capture. Size of pools ranged from 5 to 50 mosquitoes. After homogenization, RT-PCR with primers D1 and D2 was used for identification of dengue virus in each pool.10
Comprehensive intervention to reduce mosquito density was enforced from October 8 to 15, 2004. The methods included spraying of pyrethroids and dichlorodiphenyl-trichloroethane (DDT), cleaning up all discard materials in and around the dwellings to eradicate the larva infestation, and personal protection by curtain and repellents. Control efficacy was evaluated by the density of mosquitoes that remained in a subsequent survey. Dengue cases in this area were surveyed in the summer of 2005 and 2006.
Serologic survey for antibodies to dengue virus. In March 2005, a serologic survey antibodies to dengue virus was carried out in the epidemic-stricken area (Xiaolin) and control area (other towns in Cixi without dengue cases). Cluster sampling method was used. The sera from asymptomatic residents during dengue epidemic were sampled for IgG antibody to dengue virus by an indirect immunofluorescence assay, as described.12 The sera from the convalescent dengue patients and normal sera served as positive and negative control, respectively. An IgG titer > 1:80 was considered positive.
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RESULTS |
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The dengue cases clustered from August 25 to October 14, 2004 (Figure 1
). A total of 83 (including the index) hospitalized patients were diagnosed as clinical probable or confirmed cases by epidemiologic and clinical data.9 Sixty-eight cases were laboratory confirmed by isolation of dengue-specific antibodies and nucleic acid in the sera.13 Of the 83 cases (29 male and 54 female), the youngest was 7 years of age and the oldest was 76 years of age. The cases ranged in age from 20 to 55 years old accounted for 78.3% (Table 1). Most cases (49 of 83) clustered within 300 meters2 around Poshan village. Other cases were found in the six surrounding villages. An additional 128 residents with febrile disease from July 23, 2004 to October 15, 2004 were identified by a home-to-home survey in those villages. Dengue was not confirmed in those villagers because they never visited hospitals and refused to provide serum samples for further examination.
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Results of routine microbiologic examinations for other viruses and bacteria by culture and antigen detection were negative in all cases. Serologic tests for Rickettsia typhi, typhoparatyphoid Salmonella, and Japanese encephalitis virus were performed in 12 patients and all results were negative.
IgM-capture ELISA and RT-PCR.
Of 64 proven cases, 34 (53.2%) had IgM antibodies to DEN-1 2–55 days after the onset of disease. DEN-1 RNA was identified in sera of five patients by RT-PCR with consensus primers D1 and D2 within six days after the onset of disease (Figure 2). Seven days after the onset of fever, viral RNA could not be detected. All RT-PCR-positive serum samples were negative for IgM antibodies to dengue virus.
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). RNA isolated from supernatants of cell culture with or without CPE was analyzed by the RT-PCR assay. The correctly sized DNA products (511 basepairs) were obtained after amplification with consensus primers D1 and D2 (Figure 3C). A DNA fragment (482 basepairs), which was the expected size for the DEN-1 target region of the genome, was exclusively obtained after amplification with serotype-specific primer pairs and supernatants of cultures with sera of both patients (Figure 3D). The E gene fragments of the viruses were amplified. Analysis of amplicon DNA sequences showed 100% homology with E gene of viruses from the matched cases. Partial sequence (5,286 basepairs) of the viral genome from patient A was sequenced and submitted to GenBank with accession no. DQ836632.
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).14 Those strains were 0261Th01 (AY732419), 0102Th01 (AY732392), 0499Th01 (AY732389), and 0075Th02 (AY732398), which were obtained in Thailand in 2001–2002, and Hu65Th01 (AB111072) and Hu38Th02 (AB11078), which were obtained in Japan in 2001–2002 from imported cases from Thailand. This analysis suggested that the Ningbo isolate was from Thailand.
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). In May 2005, we collected mosquito larva from households and ponds around dwellings of dengue cases. After growing in the laboratory, Ae. albopictus were homogenized in pools of 5–50 mosquitoes. The RT-PCR and subsequent nested RT-PCR were used for identification of dengue RNA.10 All reactions showed negative results.
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). The number of men was less than the number of women because most men left their hometown for job opportunities in large cities during the survey.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A total of 83 hospitalized cases were diagnosed as having DF according to WHO criteria. Of the 64 cases tested, 34 (53.2%) had IgM antibody to DEN-1. DEN-1 RNA was identified in sera of five patients by RT-PCR. The remaining samples were negative for an etiologic agent, probably because of single sampling in the acute phase of the disease.
To trace the source of infection, an epidemiologic study, serologic examination, and phylogenetic analysis were performed in the cohort of infected persons. Serologic and epidemiologic data strongly suggested that the dengue outbreak was directly linked to the person who returned from Thailand. Antibody tests and a serotype-specific PCR confirmed that DEN-1 was the unique etiologic agent in this outbreak. Phylogenetic analysis showed that the Ningbo isolate and DEN-1 strains from Thailand were grouped together in a well-supported distinct cluster of DEN-1 (Figure 3
). Dengue epidemics caused by DEN-1 were recently reported in other areas.16–20 Dengue serotype 1 infection has also been reported in Guangdong and Hainan Provinces, China.5–7 However, epidemiologic and phylogenetic analysis did not indicate that the DEN-1 strain was from those areas.
The most important prerequisite for this outbreak was the high density of Ae. albopictus, which might have resulted from local climate and lifestyle factors of local residents. The climate in Xiaolin is generally warm and humid. On August 12, 2004, Typhoon Rananim hit the east coast of Zhejiang. In the Ningbo area, the typhoon resulted in a major storm lasted a week. As a result of the storm, tides increased and held back water flow from inland areas to the sea, which resulted in waterlogged areas. The density of mosquitoes increased to new levels in days. In Xiaolin, the economy, mainly family-owned shoe manufacturing, was highly developed, but environmental sanitation was not good. In entomologic surveys, we found that the CI, BI, and HI were extremely high (Table 2). In and around the dwellings, disposable rubber, water tanks, vats, jar-shaped vessels, tires, and earthenware containers were piled up. Sinks, natural receptacles, and grass were frequently found around dwellings in which dengue cases lived. In addition, the residents were accustomed to collecting rain water in tanks, usually 3–5 water tanks per family, for household use in case of insufficient tape water. These factors facilitated mosquito infestation. DEN-1 RNA was also found in a homogenized pool of female Ae. albopictus from households of dengue cases. A high density of mosquitoes contributed to the outbreak of DF after introduction of dengue virus by the traveler.
In the absence of an approved vaccine, control of the mosquito vector is the only effective preventive measure. Although the mosquito infection rate was higher during the dengue epidemic than after the epidemic period, Ae. albopictus can serve as a maintenance vector of dengue in rural areas of dengue-endemic countries.21,22 Transient use of pyrethroids and DDT and cleaning up all discarded material in and around the houses to eradicate mosquito breeding in household water were successful. After mosquito control, the BI for larva of Ae. albopictus was less than 5 (Table 3). The reason why the index of mosquito density continued to decrease might be eradication of larva infestation and cold weather. In May 2005, dengue viral RNA could not be found in the pools of Ae. albopictus collected from households and ponds around dwellings of dengue cases. No new dengue case in the epidemic-stricken area was found from November 2004 to November 2006. These data indicate the cessation of dengue virus transovarial transmission.
Ningbo is not in an area endemic for dengue. A serologic survey for IgG antibody to the four serotypes dengue viruses showed that the background level of IgG antibody was low in the non-epidemic area of Cixi. The percentage of asymptomatic residents with IgG antibody to dengue virus in the epidemic-stricken area was 6.7%, which was significantly higher than that in the non-epidemic area (Table 3). This finding indicated that dengue virus infection was underestimated. The true incidence of imported dengue infection must be higher because dengue may often go undiagnosed in areas where the virus is not endemic.23,24
Because laboratory-based etiologic diagnosis of dengue is often unavailable at the beginning of a disease outbreak, a clinical diagnosis is initially made on the basis of clinical manifestations, laboratory features, and travel experience. Comprehensive analysis of clinical, serologic, epidemiologic, and entomologic data should be an efficient way to quickly identify dengue in febrile cases in the rainy summer season. A dengue surveillance system was therefore established in all medical facilities in the Ningbo area at the end of 2004. In the summer of 2005, two introduced dengue cases from southeast Asia were quickly identified in a metropolitan hospital and isolated in a designated hospital in Ningbo. No secondary dengue cases were found.
Our investigation has several limitations. First, most of the dengue cases described were in hospitalized native residents. Most of non-native employees working in the local shoemaking factories, which accounted for almost half of population, were not admitted to hospitals. Some of the non-natives with dengue returned to their own countries during the early stage of the outbreak, which resulted in loss of data. Second, despite extraordinary efforts to obtain serum specimens, approximately 25% of the residents initially evaluated for dengue refused to provide a convalescent specimen for case definition. Residents in the control area refused to provide specimens for the serologic survey. Third, because of misdiagnosis at the early stage of the outbreak, we lost some opportunities to sample the sera from patients in the acute phase of the disease for virus isolation.
The dengue outbreak in Ningbo is another example of how readily pathogens can be transferred long distances by travelers to cause outbreaks in new areas.3,7,16,18,25 What we learned from this episode includes the need to accurately diagnose and rapidly respond to the developments of communicable disease in the global community, the need to maintain surveillance and control potential disease vectors, and the need to provide sanitation education to the public.
Received November 24, 2006. Accepted for publication March 1, 2007.
Acknowledgments: We thank the participants from Xiaolin town and surrounding towns for providing blood samples and the physicians for their cooperation. We also thank Jie Xu for critically reading the manuscript.
Financial support: This work was supported by grants 04J008 and 06G65 (Guangwen Cao) from the General Logistics of the People’s Liberation Army, and grant 2004063 (Guozhang Xu) from Health Bureau of Ningbo City, Zhejiang Province, People’s Republic of China.
* Address correspondence to Guangwen Cao, Department of Epidemiology, Second Military Medical University, 800 Xiang-yin Road, Shanghai 200433, People’s Republic of China. E-mail: gcao{at}smmu.edu.cn 
Note: A supplementary table, Dengue serotype-1 sequences retrieved from GenBank, appears online at www.ajtmh.org.
Authors’ addresses: Guozhang Xu, Hongjun Dong, Aiming Zhou, Zhihua Cheng, Jianyi Liu, and Ting Fang, Department of Infectious Diseases, Center for Disease Prevention and Control of Ningbo City, 237 Yongfeng Road, Ningbo, Zhejiang Province, People’s Republic of China, E-mails: xugz{at}nbcdc.org.cn, donghj{at}nbcdc.org.cn, zhouam{at}nbcdc.org.cn, chengzh{at}nbcdc.org.cn, liujy{at}nbcdc.org.cn, and fangt{at}nbcdc.org.cn. Nanfeng Shi, Guohua Chen and Jianjie Ye, Department of Infectious Diseases, Center for Disease Prevention and Control of Cixi County, 1141 East Nanerhuan Road, Cixi, Ningbo, Zhejiang Province, People’s Republic of China, E-mails: cxcdc{at}cxcdc.org.cn, hutian11{at}hotmail.com, and yjj852{at}sohu.com. Shijian Liu, Hongwei Zhang, Chunying Gu, Xiaojie Tan, and Guangwen Cao, Department of Epidemiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People’s Republic of China, E-mails: arrow64{at}sohu.com, smmuhongwei{at}yahoo.com.cn, cygu{at}smmu.edu.cn, xjtan2003{at}yahoo.com.cn and gcao{at}smmu.edu.cn. Shuyun Xie, Department of Infectious Disease, Center for Disease Prevention and Control of Zhejiang Province, 17 Laozheda Road, Hangzhou, Zhejiang Province, People’s Republic of China, E-mail: cfetpxsy{at}vip.sina.com.
Reprint requests: Guangwen Cao, Department of Epidemiology, Second Military Medical University, 800 Xiang-yin Road, Shanghai 200433, People’s Republic of China, Telephone: 86-21-25070420, E-mail: gcao{at}smmu.edu.cn.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
