Epidemiologic Studies on Dengue in Santiago de Cuba, 1997
Maria G. Guzmán1,
Gustavo Kouri1,
Luis Valdes2,
Jose Bravo1,
Mayling Alvarez1,
Susana Vazques1,
Iselys Delgado1 and
Scott B. Halstead3
1 Institute of Tropical Medicine "Pedro Kouri," Havana, Cuba.
2 Provincial Public Health and Epidemiology Center, Santiago de Cuba, Cuba.
3 Department Molecular Microbiology and Immunology, Johns Hopkins School of Hygiene and Public Health, Baltimore, MD.
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ABSTRACT
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A small, isolated outbreak of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) due to dengue virus type 2 (DEN-2) was documented in Santiago de Cuba on the island of Cuba beginning in January 1997. There were 205 DHF/DSS cases, all in persons older than age 15 years. All but three had evidence of a prior dengue infection, with the only known opportunity being the islandwide dengue virus type 1 (DEN-1) epidemic of 19771979. Virtually complete clinical and laboratory surveillance of overt disease was achieved. From December 1997 to January 1998, a random, age-stratified serum sample was obtained from 1,151 persons in 40 residential clusters in Santiago. Sera were tested for DEN-1 and DEN-2 neutralizing antibodies. The prevalence of DEN-2 antibodies in children age 15 years and under, born after the 1981 DEN-2 epidemic, was taken as the 1997 DEN-2 infection rate. This was adjusted slightly to accommodate observed cases, resulting in an estimated infection rate of 4.3%. Dengue fever and DHF/DSS attack rates were calculated from estimated total primary and secondary DEN-2 infections. Only 3% of 13,116 primary infections were overt. The DHF/DSS attack rate for adults of all ages was 420 per 10,000 secondary DEN-2 infections. Am J Epidemiol 2000;152:7939.
dengue; dengue hemorrhagic fever; dengue virus; immunologic factors
Abbreviations:
DEN-1, dengue virus type 1; DEN-2, dengue virus type 2; DHF/DSS, dengue hemorrhagic fever/dengue shock syndrome; EIM, enzyme-linked immunosorbent assay inhibition method; ELISA, enzyme-linked immunosorbent assay; IgM, immunoglobulin M; IPK, Institute Pedro Kouri.
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INTRODUCTION
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From the end of World War II until 1977, Cuba remained free of dengue transmission. In that and the following year, dengue virus type 1 (DEN-1) virus infected nearly 50 percent of the population islandwide (1
). In 1981, dengue virus type 2 (DEN-2) was introduced, producing an epidemic of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (2
). For the next decade, it was possible to attain strict and nearly complete Aedes aegypti control in the republic (2
, 3
). A long-established national dengue surveillance system detected febrile illnesses serologically documented as dengue in Santiago de Cuba on the island of Cuba in early 1997. This city, located in the eastern part of Cuba, has a population of 475,569 and a population density of 453.2 inhabitants per km2. This municipality eradicated A. aegypti in 1987. The species was reestablished in 1992, and at the end of 1996, moderate vector densities were reported in some localities. The domestic index case had an onset date of January 22, 1997. He was a White male age 23 years resident in the "30 de Noviembre" health area (figure 1) with no history of travel outside the country. Within 2 weeks, an additional eight cases of dengue fever, all persons known by the index case, were confirmed. DEN-2 was detected by polymerase chain reaction and by viral isolation in C6/36 cells 9 (4
, 5
). Gene-sequencing data place the 1997 Cuban DEN-2 strain in the Southeast Asian genotype, closely related to DEN-2 Jamaica 1982 (M. G. Guzmán and R. Rico-Hesse, Southwest Foundation, San Antonio, Texas, unpublished data). Despite early detection and augmented vector control measures, transmission continued in Santiago de Cuba. Extension of the outbreak to municipalities outside Santiago did not occur. In the small outbreak that ensued, 15,431 febrile cases were reported to health authorities (3.2 percent of the population). Sera were obtained from 9,747 of these (63.2 percent of the total).

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FIGURE 1. Map of Santiago de Cuba showing health areas and the distribution of clusters selected for the seroepidemiologic study. Reported 1997 suspected cases of dengue per 100,000 population are shown for each health area.
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An immunoglobulin M (IgM)-capture enzyme-linked immunosorbent assay (ELISA) confirmed recent dengue infection in 3,012 of 9,747 tested cases. Applying the proportion of positives among those tested to all reported cases, there were an estimated 5,208 clinically overt febrile illnesses caused by DEN-2 infection in 1997 (table 1). These are referred to as adjusted, laboratory-confirmed cases. Among these were 205 DHF/DSS cases with 12 deaths, all in adults. All but three cases had unequivocal evidence of a recent secondary DEN-2 infection. The predominant clinical picture was dengue fever. Because of the age of the patients, the limited experience with dengue infections, and the absence of other flaviviruses in Cuba, the only known opportunity for an initial dengue infection was the 19791999 epidemic of DEN-1. The 1997 epidemic was largely controlled in August, and the last case was detected in November (table 1).
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TABLE 1. Suspected dengue cases and sera tested by IgM* capture ELISA,* showing number and percent positive for IgM dengue antibodies, Santiago de Cuba, 1997
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Recognizing the uniqueness of secondary DEN-2 DHF/DSS occurring at an interval of 20 years after DEN-1 infection and understanding the opportunity to define the "virulence" of this outbreak, we thought it important to characterize dengue infection rates by using a retrospective seroepidemiologic study format. Here we report measurements of primary and secondary DEN-2 infections during the 1997 outbreak and calculate infection-specific dengue fever and dengue hemorrhagic fever attack rates.
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MATERIALS AND METHODS
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Dengue surveillance prior to 1997
After the 1981 DHF/DSS epidemic, a passive dengue surveillance system was established in the entire country. Until 1997, all confirmed dengue cases had a history of recent travel outside Cuba. Because of increasing travel between Cuba and nearby dengue-endemic countries, it was recognized that there was an increased possibility of the reintroduction of dengue viruses. For this reason, a proactive dengue surveillance system was established in January 1997 (4
, 5
). In this system, sera were collected 5 to 6 days after onset of a fever from persons seen in the primary health care system or emergency rooms of hospitals and polyclinics. Dengue syndromes were as defined by using the Guidelines for Control and Prevention of Dengue and Dengue Hemorrhagic Fever in the Americas (6
).
To study whether dengue transmission occurred prior to the recognition of the index case in 1997, charts of 60,000 cases admitted to emergency rooms from November 1, 1997 to January 28, 1998, in Santiago de Cuba were reviewed. Of these, 592 described illnesses that were compatible with dengue fever. Home interviews reduced this number to 154. However, no IgM dengue antibody was detected in blood samples from 143 of these individuals (4
, 5
).
Surveillance during the 1997 epidemic
Once an outbreak was detected, surveillance was intensified. Training courses that emphasized methods to search for suspected cases were offered to family doctors. The outbreak generated intense public awareness. In Cuba, one family practitioner is assigned to each 120 families. During the dengue outbreak, these practitioners made regular home visits searching for dengue-like illnesses. Single or paired serum specimens were collected from nearly all reported cases with the exception of the month of June (table 1). Owing to the volume of cases reported, no sera were collected for a period of 2 weeks. From clinical syndromes reported in June, there appears to have been a concurrent epidemic of respiratory disease accounting for up to two-thirds of "dengue" cases.
Epidemiologic methods
Population-based seroepidemiologic study.
From December 1997 to January 1998, a representative cluster sample was selected by using household registries of family doctors (figure 1). Using an equal probabilistic random sampling method and a sample size sufficient to detect a dengue infection rate of 5 percent, we selected 40 clusters from 722 family practice household registries in Santiago de Cuba. From these clusters, 375 families and 1,151 individuals gave blood samples. In this sample, 58.1 percent (n = 669) were females, 41.9 percent (n = 482) were males, 35.1 percent (n = 404) were White, 36.3 percent (n = 418) were mulattos, and 28.6 percent (n = 329) were Black. From census data, in the city as a whole, 48.8 percent were males, 30.1 percent were White, 42.8 percent were mulattos, and 26.8 percent were Black. The distributions between sample and city populations did not differ (p > 0.05).
Using a protocol approved by the Institute Pedro Kouri (IPK) Human Use Committee, trained survey teams made one or two visits to each selected household. After signed consent was obtained from each participant, a questionnaire was completed for every family member, recording general demographic data, history of chronic diseases, and history of dengue-like illnesses during 1977, 1981, or 1997. Blood was collected by finger prick on two Nobuto type A filter papers (Toyo Roshi International, Inc., Tokyo, Japan).
Supplemental serosurvey.
Because the 1997 DEN-2 infection rate was very low, an effort was made to remeasure this rate. Sera were tested first by ELISA Inhibition Method (EIM) (7
), and positives were retested by plaque-reduction neutralization test (8
).
Serologic tests
Surveillance sera and filter paper eluates were screened for dengue antibodies by IgM-capture ELISA (7
). Filter paper eluates were tested for DEN-1 and DEN-2 neutralizing antibodies on BHK-21, clone 15 cells at a 1:30 dilution using exactly the same methods used in our laboratory to measure antibodies after the 1981 epidemic (8

11
). Until 1987, paired sera from clinically suspect dengue cases were sent from polyclinics and hospitals to the IPK, where dengue antibodies were measured in paired sera by using an hemagglutination-inhibition test (12
). From 1987, sera were sent to the Centro Provincial de Higiene y Epidemiología of Santiago Province, where they were tested to detect dengue IgM antibodies by an UltramicroELISA (13
).
Antibody positive samples were retested at IPK using the EIM (14
). For patients, primary and secondary dengue antibody responses were characterized on sera collected 6 or more days after onset of fever. A secondary type response was defined as a sample with a titer equal to or greater than 1:10,240 or 1:2,560 by EIM or hemagglutination-inhibition, respectively (12
, 15
).
Statistical analysis
Data were analyzed by means of Mann-Whitney and chi-square tests.
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RESULTS
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DEN-1 and DEN-2 antibodies in the population of Santiago de Cuba
The distribution of monotypic DEN-1 and DEN-2 virus antibodies and bivalent DEN-1 and DEN-2 antibodies by age group in individuals from Santiago de Cuba is shown in table 2. From December 1997 to January 1998, of 1,151 individuals sampled, 59.1 percent had no dengue antibodies, 24.8 percent were immune to DEN-1 only, and 6.3 percent were immune to DEN-2 only, while 9.9 percent had antibodies to both viruses. These antibodies have cumulated from dengue epidemics in the 1940s, 19771979, 1981, and 1997. Of 251 individuals younger than age 16 years and therefore born after the 1981 DEN-2 epidemic, only five (2.0 percent) had neutralizing antibodies to DEN-2 (95 percent confidence interval: 0.17, 4.17 percent). None had antibodies to DEN-1. Because the measured 1997 DEN-2 infection rate was so low, the prevalence of DEN-2 neutralizing antibodies was remeasured in 205 children who gave blood samples in October 1998 (table 3). In this supplemental sample, DEN-2 antibody prevalence was even lower than in the cluster sample. Only two (0.97 percent) of these children (ages 4 and 11 years) had monotypic DEN-2 antibodies.
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TABLE 2. Prevalence of DEN-1 only, DEN-2 only, and DEN-1 and DEN-2 neutralizing antibodies by age group, Santiago de Cuba, December 1997 to January 1998
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TABLE 3. Outpatient opportunity sample: distribution of DEN-2* neutralizing antibodies in children aged 116 years, Santiago de Cuba, 1997
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Estimating the preepidemic serologic status
To calculate the secondary DEN-2 infection rate, it is necessary to know how many individuals had monotypic DEN-1 antibody before the 1997 epidemic. Assuming a 2 percent DEN-2 infection rate in 1997, the number of individuals immune to DEN-1 and DEN-2 prior to the epidemic in the postepidemic sample can be reduced by 1.022, increasing slightly the number of DEN-1 immunes (table 4). Using this adjustment, we estimated total secondary DEN-2 infections in 1997 at 2,221. However, there were 4,810 adjusted, laboratory-confirmed cases caused by secondary DEN-2 infections (a subset of the 5,208 adjusted total clinical cases), a number that is 2.166-fold higher than this estimate (table 4).
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TABLE 4. DEN-1* antibody status by age group in a 19971998 cluster sample with estimates of secondary DEN-2* infections at 2.0% and 4.2% infection rates, Santiago de Cuba, 1997
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Calculating clinical attack rates during primary and secondary DEN-2 infections
To reconcile the apparent undermeasurement of the 1997 DEN-2 infection rate, secondary DEN-2 infections were calculated by using the adjusted rate of 4.23 percent (table 4) and the estimated prevalence of preepidemic DEN-1 immunes (table 5). Age-specific DHF/DSS attack and death rates were calculated to the base 10,000 secondary DEN-2 infections (table 5). The number of primary DEN-2 infections in the Santiago de Cuba population was estimated for each relevant age group (table 6). Finally, the number of adjusted, laboratory-confirmed primary or secondary DEN-2 and DHF/DSS cases were compared with the estimated total number of residents who experienced primary and secondary DEN-2 infections in Santiago in 1997 (table 7).
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TABLE 6. Estimated preepidemic dengue susceptibles and primary DEN-2* infections by age groups, using adjusted DEN-2 infection rate, Santiago de Cuba, 1997
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TABLE 7. Distribution of clinically overt, laboratory-confirmed dengue illnesses by type of infection compared with estimated total number of residents experiencing primary and secondary DEN-2* infections, Santiago de Cuba, 1997
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DISCUSSION
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The outbreak in Santiago de Cuba in 1997 was monitored by an exceptional surveillance system. Only during the peak epidemic month of June was there a brief period when serum samples were not taken from suspected cases. This system and the high rate of submission of specimens for laboratory testing provide credibility that an accurate estimate of overt dengue illnesses has been obtained. During 1997, there were an adjusted 5,208 laboratory-confirmed dengue illnesses, 205 DHF/DSS cases, and 5,003 dengue fever cases, of which 202 cases of DHF/DSS (98.5 percent) and 4,608 cases of dengue fever (92.1 percent) were secondary dengue infections. These conservative numbers, based upon EIM titers in postfever sera, would tend to underestimate the number of secondary dengue infections. However, EIM results were confirmed on a random sample of 252 of these sera by the neutralization test (relatively monospecific DEN-2 vs. broadly reactive high-titered DEN-1 and DEN-2; data not shown).
There is sufficient congruence between the serologically studied sample and the general population (p > 0.05) to warrant projection of serosurvey neutralization test data to the entire population (table 2). We measured DEN-2 infections from the prevalence of DEN-2 neutralizing antibodies in 251 children from the cluster sample who were born after the 1981 DEN-2 epidemic. This rate was measured at 2.0 percent, yielding an estimated total of 2,221 secondary DEN-2 infections in the city (table 4). However, the clinical surveillance system (table 1) reported 2.17 times more residents with confirmed secondary DEN-2 dengue illnesses (n = 4,810). Explanations for this underestimate include 1) the physician-diagnosed "adjusted" dengue cases might have come from a population larger than that of Santiago de Cuba, 2) DEN-2 infections were undermeasured in the cluster sample, or 3) the DEN-2 infection rate may have been higher in persons with DEN-1 antibodies than in the population as a whole. For the first possibility, no confirmed dengue cases were reported from persons with residence outside the Santiago de Cuba municipality. Further, a serologic survey in 1998 of the nearby city of Palma Soriano (population, 114,283 persons), where no cases were reported, showed no evidence of recent DEN-2 infection (data not shown). It is likely that the undermeasurement of the 1997 DEN-2 infection rate is due to a combination of possibilities 2 and 3. Figure 1 shows marked heterogeneity in dengue attack rates in different health areas. It may be surmised that the cluster sample might not be capable of measuring focal dengue transmission accurately, although the adjusted rate adopted is close to the 95 percent confidence intervals of the measurement of DEN-2 antibody prevalence in the sample. The opportunity sample of pediatric outpatients apparently also underestimated the 1997 DEN-2 transmission rate. Hospital-based serum collection is subject to socioeconomic, neighborhood, or sample size biases. In Singapore, where the citywide A. aegypti house index was low, dengue transmission occurred, but was focal (16
). It is quite likely that DEN-2 transmission in Santiago also was focal. Focal transmission is likely to result from the heterogeneous distribution of A. aegypti. If, as is likely, households infested with A. aegypti in 1977 were again infested in 1997, then DEN-2 infection rates among DEN-1-immunes would be expected to be higher than that among susceptibles. The phenomenon of higher dengue infection rates among immunes than nonimmunes has been described previously (9
, 11
). The ""true" primary DEN-2 infection rate could be 2 percent or even lower.
The numbers of primary DEN-2 infections calculated by age group are displayed in table 7. As shown, only 398 of 13,116 (3.0 percent) estimated primary DEN-2 infections were overt. As noted above, the primary DEN-2 infection rate might be 2 percent. If so, there would be an estimated 6,402 primary DEN-2 infections, of which 6.2 percent were overt. This is the first evidence that primary DEN-2 infections in adults can be predominantly unapparent. Pre-viously, Vaughn et al. (17
) noted that in Thai children primary DEN-2 infections were mainly silent. According to our data, virtually all secondary DEN-2 infections in adults resulted in clinical disease, dengue fever through DHF/DSS. This can be contrasted with Thai children who developed mild or severe febrile illnesses in only a small percent of secondary DEN-2 infections (9
, 17
). If the Cuban population had been without prior DEN-1 infection experience, it is unlikely that a small outbreak with this particular DEN-2 strain would have produced any recognizable disease. This phenomenon is likely to be occurring in areas of the American tropics outside of Cuba and contributing to the relatively silent transmission of dengue viruses in the region. The mild nature of the observed primary DEN-2 infections in adults is in sharp contrast to virgin soil epidemics caused by DEN-1 (18
).
Age-specific DHF/DSS attack and death rates per 10,000 secondary DEN-2 infections are given in table 5. For affected adults of all ages, the 1997 DEN-2 epidemic resulted in a DHF/DSS attack rate of 420 per 10,000 secondary DEN-2 infections (4.2 percent). This rate is similar to secondary dengue infection DHF/DSS attack rates measured in Southeast Asian children (19
). The unique dengue infection experience of the Cuban population has made it possible to apply DEN-1 and DEN-2 antibody prevalence data from age-stratified serologic samples to calculate age-specific secondary DEN-2 infection rates (M. G. Gurmán et al., "Pedro Kouri" Tropical Medical Institute, Havana, Cuba, unpublished data). We plan to reanalyze data from the 1981 DEN-2 epidemic to obtain age and secondary DEN-2 specific death and DHF/DSS attack rates that will make it possible to compare disease severity in the same age group at 4- and 20-year intervals after DEN-1 infection. Such measurements should facilitate future assessments of risk factors for DHF/DSS.
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ACKNOWLEDGMENTS
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The authors thank Dr. Mayra Serret and Lic. María E. Galano of the Centro Provincial de Higiene y Epidemiología of Santiago de Cuba province for providing active support throughout this study and Lucía Iturralde for the preparation of the manuscript.
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NOTES
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Reprint requests to Dr. Scott B. Halstead, 5824 Edson Lane, N. Bethesda, MD 20852 (e-mail: halsteads{at}erols.com).
Editor's note: An invited commentary on this paper appears on page 800, and the author's response appears on page 804.
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Received for publication October 12, 1999.
Accepted for publication June 19, 2000.
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