INTRODUCTION
Dengue (DEN) virus belongs to the genus Flaviviruses, and consists of four serotypes: serotype 1 (DEN-1), serotype 2 (DEN-2), serotype 3 (DEN-3), and serotype 4 (DEN-4). Infection with DEN virus has been a major concern in DEN-endemic societies for decades worldwide.1,2 An infection with this virus usually causes self-limiting diseases; fever, rash, and joint pain (dengue fever [DF]). However, a more severe form of DEN virus infection accompanied by hemorrhagic fever (dengue hemorrhagic fever [DHF]) and shock (dengue shock syndrome [DSS]) has occurred in DEN-endemic areas where all four serotypes co-circulate.3 According to investigations conducted by the World Health Organization, the worldwide incidence of DF is estimated to range from 25 million to 50 million and that of DHF or DSS to be more than 500,000.4 Dengue virus causes outbreaks and major epidemics in most tropical and subtropical areas, where Aedes aegypti and Ae. albopictus are abundant. These mosquito species are the principal vectors responsible for the human-to-human transmission and spread of DEN virus infection.5
Various factors are related to the introduction and spread of DEN virus infection in Santo Domingo, the capital of the Dominican Republic, i.e., increased human transportation by airplane, lack of effective mosquito control, an explosive increase in the population, and uncontrolled urbanization, which has resulted in substandard housing and an inadequate water supply, sewage system, and waste management systems for millions of residents.
DEN virus infection has been reported to be epidemic in Caribbean countries where Ae. aegypti has infested and uncontrolled urbanization has occurred.6 In the Dominican Republic, laboratory-confirmed DEN outbreaks, related to epidemics in the neighboring Caribbean islands, have been reported since the 1960s, and each of the four DEN virus serotypes has been attributed as causative. The first appearance of each of DEN virus serotype in the Dominican Republic is as follows: DEN-3 virus in 1963, DEN-4 virus in 1982, DEN-1 virus in 1984, and DEN-2 virus in 1985.7 In 2000, more than 3,400 cases of DF and DHF caused by each of the four serotypes were reported in the Dominican Republic, with an incidence of 40.75 per 100,000 population (Pan American Health Organization, 2000, available at www.paho.org/english/hcp/hct/vbd/dengue-cases-2000.htm).
Exposure to the virus generally occurs in the infantile to juvenile period among residents in DEN-endemic areas, and the prevalence of DEN virus-specific IgG increases with age and reaches its peak before adolescence.8 Collecting information on the prevalence of DEN virus-specific IgG among residents of a DEN-endemic area would be an initial step in estimating the magnitude of DEN virus infection in a population.
In the present study, the prevalence of IgG specific for DEN virus was surveyed and determined among residents of Santo Domingo who visited blood banks for blood donation, and among hospital-visiting children, to estimate the magnitude of DEN virus circulation in this city and the age at which residents of Santo Domingo become initially exposed to DEN virus. Serotypes of DEN virus circulating among the residents of Santo Domingo were also determined by focus reduction neutralization tests.
MATERIALS AND METHODS
Sample collection from adults.
All adult samples were collected at seven blood banks of public hospitals located in Santo Domingo from June 17 to July 23, 2002. Study subjects were restricted to volunteers who resided in the Santo Domingo district (Distrito Nacional). One to two milliliters of blood was collected from an inlet tube connecting the needle to a blood reservoir bag prior to screening for infectious agents (hepatitis B virus, hepatitis C virus, human immuno-deficiency virus, syphilis, human T cell lymphotropic virus 1), as well as for detection of elevated levels of transaminases. Basic epidemiologic information such as age, sex, and current residential area was also collected.
Sample collection from children.
Blood samples were collected from children ≤10 years old visiting the outpatient department of Centro de Gastroenterología Hospital in Santo Domingo from July 30 to August 27, 2002. Study subjects generally visited the hospital seeking care for gastrointestinal diseases. Patients with fever were carefully excluded by measuring their axillary temperature to minimize the number of patients with acute DEN virus infections, which would augment the prevalence of DEN virus-specific IgG among the study subjects. Data on age and sex were collected by questionnaire from the guardians of the study subjects.
Ethical considerations.
Written consent to participate in the study was obtained from each subject or their guardians of the subject after a full explanation of the study was provided. This study was reviewed and approved by ethics committee of the Centro de Educación Médica de Amistad Dóminico-Japonesa. All data were handled confidentially and anonymously. The maintenance and care of animals used in the study complied with National Institute of Health (Bethesda, MD) guidelines.
Dengue virus-specific IgG assay.
The DEN virus-specific IgG titers of the samples were determined by an enzyme-linked immunosorbent assay (ELISA) system (Microwell ELISA Dengue IgG; Diagnostic Automation, Inc., Calabasas, CA) or a dengue fever virus IgG ELISA (Focus Technologies, Inc., Herndon, VA) within one week after sample collection. Analysis of the samples and interpretation of positive or negative ELISA reactions were made according to the manufacturer’s instructions.
Preparation of anti-DEN virus sera.
Each serotype of DEN virus strain in our laboratory (DEN-1, Hawaiian strain; DEN-2, New guinea B strain; DEN-3, H-87 strain; and DEN-4, H-241 strain) was proliferated on C6/36 cells and concentrated with a sulfate cellulofine resin (Seikagaku Kogyo Co., Tokyo, Japan) column purification method.9 Four rabbits were immunized subcutaneously with approximately 500 μg each of purified DEN-1, DEN-2, DEN-3, and DEN-4 viruses emulsified in Freund’s complete adjuvant and then given booster immunizations every two weeks. Harvested rabbit sera were adsorbed completely with C6/36 cells if necessary.
Focus reduction neutralizing test for DEN virus.
Sixty-two samples were chosen from adult samples collected at blood banks by random number generation method using Excel® software (Microsoft, Redmond, WA) software, and assayed for neutralizing antibody to each of DEN-1, DEN-2, DEN-3, and DEN-4 virus stocks on baby hamster kidney (BHK)-21 cells and compared. Briefly, a serotype of a DEN virus suspension (approximately 1 × 107 focus-forming units/mL) was incubated with 1:20, 1:100, and 1:500 dilutions of serum for one hour at 37°C, then added to BHK-21 cells cultured in 96-well flat-bottom plates. After incubation for one hour at 37°C, the infected cells were overlayed with minimum essential medium (GIBCO-BRL, Gaithersburg, MD) with 2% fetal bovine serum plus 1% gum tragacanth (Sigma Aldrich, St. Louis, MO), then incubated for 48 hours at 37°C in an atmosphere of 5% CO2. After fixing with methanol and washing, the cells were reacted sequentially with rabbit anti-dengue serum (1:1,000 dilution), goat anti-rabbit IgG (1:1,000 dilution) (Cappel MP Biomedicals, Inc., Aurora, OH), and rabbit peroxidase anti-peroxidase (1:1,000 diluted) (Chemicon, Temecula, CA). Immunostained foci were visualized with diaminobenzidine (Wako, Osaka, Japan), and counted microscopically. Focus reduction neutralization test endpoints were determined at 50% (FRNT50) and at 90% (FRNT90) using an optimal curve obtained from the reciprocal titers of serum at each dilution. Reciprocal titers obtained at the dilution less than 20 by FRNT50 were referred to as zero.
Statistical analysis.
The prevalence of DEN virus-specific IgG determined by ELISA was analyzed by the chi-square test and compared on the basis of age, sex, and residential area.
RESULTS
Samples from adults.
A total of 1,008 adult samples were collected in the study. The male to female ratio of the subjects was 1:0.056. The age of the study subjects ranged from 17 to 79 years and the median age was 32 years (Table 1). The residence of the study subjects were divided into five areas, as designated by the Ministry of Public Health and Welfare of the Dominican Republic. The number of study subjects in each area was area 1 = 196, area 2 = 355, area 3 = 142, area 4 = 236, and area 5 = 79.
Samples from children.
Two hundred one samples were collected from children in the study. The male to female ratio of the study subjects was 1:0.69. The age of the subjects ranged from 3 months to 10 years, and the median age was 6 years (Table 2). One hundred ninety-five of the study subjects resided in Santo Domingo.
Prevalence of DEN virus-specific IgG among samples from adults.
Among adult subjects, 987 (98%) of 1,008 samples showed a positive reaction to DEN virus by ELISA. The prevalence of IgG reacting with DEN virus among the subjects in each age group was as follows: < 20 years old, 97% (60 of 62); 20–29 years old, 97% (346 of 357); 30–39 years old, 98% (328 of 333); 40–49 years old, 99% (182 of 184); 50–59 years old, 98% (49 of 50); and ≥60 years old, 100% (22 of 22) (Figure 1). The prevalence of IgG among male and female subjects was 98% (935 of 955) and 98% (52 of 53), respectively. The prevalence of IgG by residential areas of the subjects was as follows: area 1, 97% (191 of 196); area 2, 97% (345 of 355); area 3, 99% (140 of 142); area 4, 98% (232 of 236), and area 5, 100% (79 of 79). There were no significant differences in the prevalence among each group when compared.
Prevalence of DEN virus-specific IgG among samples from children.
Among samples obtained from children, 113 (56%) of 201 samples showed a positive reaction to DEN virus by ELISA. The prevalence of IgG specific for DEN virus among each age group is shown in Figure 2. The IgG prevalence increased with age except for that seen in the group less than one year of age (50%, 4 of 8). The prevalence of IgG among male and female subjects was 56% (67 of 119) and 56% (46 of 82), respectively.
Dengue virus focus reduction neutralization test.
Sixty-two samples from adults were tested for DEN virus focus reduction neutralization consisted of 60 ELISA-determined DEN virus-specific IgG positive samples and two negative samples. Fifty-three of 62 serum samples showed an FRNT50 titer to any of the four DEN virus serotypes. Two DEN virus-specific IgG negative samples showed no neutralizing activity against any of the DEN virus serotypes at a dilution less than 20. Twenty-eight serum samples showed a high FRNT90 titer to any of the four DEN serotypes (Table 3). Among the 28 samples, 3 had monotypic antibody to DEN-4 virus. However, the remaining 25 serum samples showed antibody titers to more than two dengue virus serotypes simultaneously when the FRNT50 criteria were used, with 5 samples reactive with two DEN virus serotypes, 11 samples reactive with three DEN serotypes, and 9 samples reactive with all four DEN virus serotypes.
DISCUSSION
The Dominican Republic, which occupies the east two-thirds of the island of Hispaniola, the second largest island in the Caribbean area, has been a DEN-endemic region since 1960 when a laboratory-confirmed epidemic with DEN-virus was initially reported.7 It is likely that infection with DEN virus has been epidemic for long time in Santo Domingo, the most urbanized city in the Dominican Republic. However, there have been few reports describing the magnitude of circulation of the virus in this city. Information on the seroprevalence of DEN virus-specific IgG among the inhabitants of Santo Domingo would provide a clue as to the extent of prevalence of dengue in the Dominican Republic.
The objectives of the current study were to determine the seroprevalence of DEN virus-specific IgG among adult volunteers recruited at blood banks in Santo Domingo, and among children ≤10 years old who visited a hospital in Santo Domingo, and to identify the DEN virus serotypes that have been circulating in this city, through which the extent of trans mission of DEN virus among adults and children and their causative serotypes among adults can be estimated.
According to a census conducted by the Ministry of Public Health and Welfare of the Dominican Republic, 56,242 blood donations were made nationwide in 1999, 66% (37,144 cases) of which were made in the Santo Domingo district (Secretaría de Estado de Salud Pública y Asistencia Social, Estadísticas de Salud, unpublished data). In the Dominican Republic, blood for transfusions is collected principally by a repayment system (81.3%) in which blood recipients or their families are supposed to donate blood depending on the amount they had received, followed by a voluntary donation (17.9%) and blood deposition for autologous transfusion (0.8%) (Secretaría de Estado de Salud Pública y Asistencia Social, Estadísticas de Salud, unpublished data). Sample collection at blood banks is advantageous because subjects from various backgrounds from broad areas of the city can be recruited, although the low number of women due to local habits was predicted as a study limitation. In fact, the residential areas of the adult subjects were distributed almost all over the Santo Domingo area.
Among the samples obtained from adults, 98% (987 of 1,008) showed a positive reaction to DEN virus in the ELISA. There were no significant differences among all age groups when they were compared (Figure 1). It could be interpreted that the prevalence of DEN virus-specific IgG is high and almost saturated among adult inhabitants of Santo Domingo, probably indicating that an extremely high magnitude of DEN virus infection has been present in this region for a long time. In the present study, IgG detected was considered to be specific for DEN virus because no cases of epidemics caused by flaviviridae other than DEN virus, which may elicit cross-reacting antibody to DEN virus, have been reported in the Santo Domingo region. In addition, nationwide vaccinations for yellow fever virus or Japanese encephalitis virus, which may induce cross-reacting antibody to DEN virus,10 have not been implemented among the inhabitants of the city.
Fifty-six percent (113 of 201) of the samples from children showed a positive reaction to DEN virus. Seropositivity was strongly associated with age, i.e., the prevalence of DEN virus-specific IgG increased with age except for that seen in the group less than one year of age, which was probably due to the reaction of transferred maternal DEN virus-specific IgG.11 The ages of the children were categorized into three groups in view of the seroprevalence of DEN virus-specific IgG: ≤2 years old, low prevalence group (12.8% [5 of 39] including the data of the group less than one year of age; 3.22% [1 of 31] not including the data of the group less than one year of age); 3–6 years old, intermediate prevalence group (46.5% [34 of 73]); and ≥7 years old, high prevalence group (83.1% [74 of 89]). From these results, a possible interpretation of the epidemiology of DEN virus infection among children in Santo Domingo is as follows: 1) children ≤2 years old are rarely exposed to DEN virus; 2) between three and six years of age the risk of viral infection increases; and 3) by seven years of age almost all children have been infected at least once since the prevalence of DEN virus-specific IgG is almost saturated. In Bangkok, Thailand, another area endemic for DEN virus, 53% of the children had antibodies to at least one DEN viral serotype by the age of 7–9 years.8 This indicates that a higher magnitude of DEN virus infection has present in Santo Domingo than that in Bangkok in the late 1980s. Since school begins officially at six years of age in Santo Domingo, this may account for the accelerated spread of DEN virus infection among ≥7 years of age as a result of daytime transmission in the schools.12
To determine the serotype of DEN virus circulating among the inhabitants of Santo Domingo, neutralization tests were used in this study. These tests show the greatest virus serotype specificity, and thus represent an acceptable approach to obtain information about previous virus exposure and present immunity from serologic information compared with other studies including hemmaglutination inhibition or complement fixation tests.13 In the current study, relatively stringent criteria with a focus reduction endpoint of 90% (FRNT90) was used for identifying virus serotypes because information on previous DEN virus infections were not intensively collected from the subjects and because the FRNT90 shows increased serotype specificity, making it possible to reduce uncertainty arising from low-titer antibodies.14 Among 62 samples tested, 28 samples showed a relatively high neutralizing antibody FRNT90 to any of four DEN virus serotypes (Table 3). There were three samples with monotypic antibody to DEN-virus 4 in the group. However, the remaining 25 samples had antibody titers to more than two dengue virus serotypes when the FRNT50 was used, indicating that DEN virus infections with different serotypes probably have occurred sequentially or simultaneously in the Santo Domingo area, which is supported by a previous report.7 When the FRNT50 was determined, 53 (85.5%) of 62 samples showed a neutralizing antibody titer to any of the four DEN virus serotypes, which was similar to the result obtained in the ELISA assay in this study.
Evidence indicates that most cases of severe dengue, DHF, and/or DSS occur following a secondary infection with a DEN virus serotype different from that of the primary infection.8,15 Areas endemic for dengue where four serotypes co-circulate, or where a DEN virus serotype that is different from its antecedents has been newly introduced are at risk of having a severe outbreak of infection with DEN virus. According to the data in the report from PAHO (Pan American Health Organization, 2000, available at www.paho.org/english/hcp/hct/vbd/dengue-cases-2000.htm), there were 29, 58, and 4 cases of sporadic DHF in the Dominican Republic in 1999, 2000, and 2001, respectively. Thus, Santo Domingo is an area at risk for an outbreak of severe DEN infection.
Distribution of the adult subjects by age and sex (Jun 17 to July 23, 2002; 37 days)
| Samples collected | 1,008 |
| Male:female ratio | 1:0.056 |
| Median age (years) | 32 |
| Age range (years) | |
| < 20 | 62 |
| 20–29 | 357 |
| 30–39 | 333 |
| 40–49 | 184 |
| 50–59 | 50 |
| ≥ 60 | 22 |
Distribution of the children subjects by age and sex (July 30 to August 27, 2002; 29 days)
| Samples collected | 201 |
| Male:female ratio | 1:0.69 |
| Median age (years) | 6 |
| Age (years) | |
| <1 | 8 |
| 1 | 12 |
| 2 | 19 |
| 3 | 16 |
| 4 | 25 |
| 5 | 15 |
| 6 | 17 |
| 7 | 12 |
| 8 | 29 |
| 9 | 17 |
| 10 | 31 |
Neutralizing-antibody responses to each serotype of dengue (DEN) virus in adult volunteers from Santo Domingo, Dominican Republic
| FRNT90* reciprocal antibody titer | |||||
|---|---|---|---|---|---|
| Sample no. | Age (years) | DEN-1 | DEN-2 | DEN-3 | DEN-4 |
| *FRNT90 = 90% focus reduction neutralizing antibody titer. | |||||
| †Reciprocal titers < 20 by 50% focus reduction neutralizing antibody titer (FRNT50). Values in parentheses are FRNT50. | |||||
| 154 | 17 | 0† | 0 | 0 | 30 |
| 391 | 17 | 0 | 75 | 30 | 0 |
| 121 | 20 | 90 | (30) | 0 | (50) |
| 224 | 20 | (35) | 20 | (20) | 0 |
| 272 | 22 | 280 | 120 | (200) | 0 |
| 575 | 22 | 0 | (150) | (400) | 20 |
| 428 | 23 | 0 | 0 | 0 | 45 |
| 640 | 23 | 0 | 0 | 0 | 25 |
| 648 | 23 | (45) | (20) | (20) | 150 |
| 8 | 24 | 0 | (150) | 20 | 0 |
| 902 | 24 | 0 | 0 | 20 | 30 |
| 18 | 28 | 0 | 25 | (400) | (70) |
| 114 | 28 | 0 | 200 | (35) | (100) |
| 414 | 29 | (20) | 0 | (250) | 45 |
| 153 | 30 | 60 | 26 | (150) | (450) |
| 848 | 30 | 140 | (80) | 0 | 45 |
| 302 | 32 | 0 | 0 | (100) | 40 |
| 436 | 32 | 0 | (40) | (25) | 65 |
| 604 | 32 | (30) | (100) | (25) | 85 |
| 811 | 34 | (40) | (160) | 35 | (80) |
| 551 | 38 | 35 | (140) | (20) | 200 |
| 19 | 39 | (40) | (80) | (500) | 20 |
| 495 | 39 | (60) | (180) | 250 | 120 |
| 395 | 41 | (25) | 150 | (80) | 200 |
| 232 | 49 | 0 | 20 | (100) | (20) |
| 251 | 52 | (20) | (150) | (400) | 60 |
| 220 | 60 | 0 | 65 | 0 | (20) |
| 485 | 61 | (250) | 0 | 130 | (400) |
Age distribution of adult subjects (columns) and the prevalence of dengue (DEN) virus-specific IgG of each age group (line) assayed by an enzyme-linked immunosorbent assay. The subjects were recruited at blood banks in Santo Domingo, Dominican Republic from June 17 to July 23, 2002. Recruitment was restricted to residents of Santo Domingo.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 2; 10.4269/ajtmh.2004.71.138
Age distribution of adult subjects (columns) and the prevalence of dengue (DEN) virus-specific IgG of each age group (line) assayed by an enzyme-linked immunosorbent assay. The subjects were recruited at blood banks in Santo Domingo, Dominican Republic from June 17 to July 23, 2002. Recruitment was restricted to residents of Santo Domingo.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 2; 10.4269/ajtmh.2004.71.138
Age distribution of adult subjects (columns) and the prevalence of dengue (DEN) virus-specific IgG of each age group (line) assayed by an enzyme-linked immunosorbent assay. The subjects were recruited at blood banks in Santo Domingo, Dominican Republic from June 17 to July 23, 2002. Recruitment was restricted to residents of Santo Domingo.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 2; 10.4269/ajtmh.2004.71.138
Age distribution of children ≤10 years old) (columns) and the prevalence of dengue (DEN) virus-specific IgG among each age group (line). The subjects were recruited from patients seeking medical care for gastrointestinal diseases at a hospital from July 30 to August 27, 2002. Patients with fever were excluded by measuring their axillary temperature.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 2; 10.4269/ajtmh.2004.71.138
Age distribution of children ≤10 years old) (columns) and the prevalence of dengue (DEN) virus-specific IgG among each age group (line). The subjects were recruited from patients seeking medical care for gastrointestinal diseases at a hospital from July 30 to August 27, 2002. Patients with fever were excluded by measuring their axillary temperature.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 2; 10.4269/ajtmh.2004.71.138
Age distribution of children ≤10 years old) (columns) and the prevalence of dengue (DEN) virus-specific IgG among each age group (line). The subjects were recruited from patients seeking medical care for gastrointestinal diseases at a hospital from July 30 to August 27, 2002. Patients with fever were excluded by measuring their axillary temperature.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 2; 10.4269/ajtmh.2004.71.138
Authors’ addresses: Tetsu Yamashiro, Institute of Scientific Research, Faculty of Medicine, Oita University, Hasama-machi, Oita 879-5593, Japan, Telephone: 81-97-586-5712, Fax: 81-97-586-5719, E-mail: tetyam@med.oita-u.ac.jp. Mildre Disla, Angela Petit, Delfis Taveras, Mercedez Castro-Bello, Miguel Lora-Orste, Sonia Vardez, Ana Julia Cesin, and Barbara Garcia, Centro de Educación Médica de Amistad Dominico-Japonesa, Calle Federico Velazquez No.1, María Auxiladora, Santo Domingo, Dominican Republic, Telephone: 809-684-0411, Fax: 809-245-5148. Akira Nishizono, Department of Infectious Diseases, Faculty of Medicine, Oita University, Hasama-machi, Oita 879-5593, Japan, Telephone: 81-97-586-5712, Fax: 81-97-586-5719, E-mail: a24zono@oita-med.ac.jp.
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