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Am. J. Trop. Med. Hyg., 68(6), 2003, pp. 678–682 Copyright © 2003 by The American Society of Tropical Medicine and Hygiene
SEROPREVALENCE OF TRYPANOSOMA CRUZI INFECTION AMONG SCHOOL-AGE CHILDREN IN THE ENDEMIC AREA OF GUATEMALA NIDIA R. RIZZO, BYRON A. ARANA, ANAITE DIAZ, CELIA CORDON-ROSALES, ROBERT E. KLEIN, AND MALCOLM R. POWELL Center for Health Studies, Universidad del Valle de Guatemala/Medical Entomology Research and Training Unit/Guatemala, Division of Parasitic Diseases, Centers for Diseases Control and Prevention, Guatemala City, Guatemala; Medical Entomology Research and Training Unit/Guatemala, Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Diseases Control and Prevention. Public Health Service, U. S. Department of Health and Human Services, American Embassy, Miami, Florida; Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Diseases Control and Prevention, Atlanta, Georgia
Abstract. In support of the National Program for Chagas Disease Control, we conducted a cross-sectional study to estimate the seroprevalence rate of Trypanosoma cruzi infection across the five Departments (Chiquimula, Jalapa, Zacapa, Jutiapa, and Santa Rosa) that are believed to comprise the entire principal endemic area in Guatemala. Also, so that the results could be used to identify areas of active transmission, we conducted the survey in school-aged children. We used an experimental enzyme-linked immunosorbent assay with blood samples obtained by finger prick to estimate the seroprevalence of T. cruzi. This assay has been previously tested and showed good sensitivity and specificity. Overall, the seropositivity rate for T. cruzi infection was 5.28% (235 of 4,450). Of 173 communities evaluated, 35 (20.23%) had a seropositive rate ranging from 10% to 45%. A number of parameters, including but not limited to living conditions, were examined for possible association with seropositivity. While there are several associations, the strongest association with seropositivity is living in a house with a thatch roof. The survey results will permit the Ministry of Health to stratify T. cruzi-endemic communities, enabling local health authorities to efficiently focus on vector control operations. the Andean Initiative, including Bolivia, Colombia, Ecuador, Peru, and Venezuela, was launched in February 1997, followed in October 1997 by the Central American Initiative, which includes Belize, Costa Rica, El Salvador, Guatemala, Honduras, Nicaragua, and Panama. Although there has been no representative sample survey to evaluate the public health impact of Chagas disease in Guatemala, this country is thought to have one of the highest Chagas disease risk levels outside the Southern Cone.5 Indicative information presented at the Tropical Disease Research/World Health Organization (TDR/WHO)−sponsored meeting in Tegucigalpa, Honduras in October 1997 summarized the epidemiologic situation in Guatemala. It is estimated that 3,400,000 persons, 34% of the total population, is at risk of infection. In the endemic regions, house infestation rates range from 10% to 34% with Triatoma dimidiata and from 3% to 18% with Rhodnius prolixus. The prevalence of human infection in the general population approaches 10%, and the prevalence of infected blood in blood banks is 0.97%. We conducted a cross-sectional study to estimate the seroprevalence rate of Chagas disease in the five Departments of Guatemala thought to comprise the principal endemic area in this country. An additional goal of the study was to determine which areas have active vector-borne transmission. Since infection with T. cruzi and associated seropositivity is life long, the most efficacious way to determine where transmission is ongoing is to evaluate seropositivity in young people. Thus, in collaboration with the Ministry of Education and the Ministry of Health, we conducted the serosurvey in school-age children. These results will be used by the National Program for Chagas Disease Control to stratify the endemic region for a vector control program.
INTRODUCTION Infection with Trypanosoma cruzi, the causative agent of Chagas disease, is an important public health problem in Latin America. An estimated 18 million persons are infected, and Chagas disease is the leading cause of cardiomyopathy and sudden cardiac-related death.1 However, even though most people are unaware that they are infected, approximately 30% eventually develop the clinical manifestations of chronic Chagas disease. Trypanosoma cruzi is naturally transmitted by reduviid bugs. Generally, infection is seen in persons who live in the lower economic rural or suburban areas of Latin America, in poor housing conditions, and who have had repeated exposure to the vector. However, a second and increasingly important route of transmission of T. cruzi is by blood transfusion when blood from infected asymptomatic donors is transfused due to poor screening practices. Although not all recipients of T. cruzi-seropositive blood become infected, lack of effective screening poses a substantial risk to populations in endemic areas.2 In addition, congenital transmission has also been documented in Guatemala.3 National vector control programs aimed at the interruption of disease transmission by vector and by transfusion mediated routes, mainly through the application of residual insecticides in house holds and screening practices of blood donors, have proven to be feasible and effective. Based on these principles, the Southern Cone Initiative for the Control of Chagas Disease was launched in 1991 with the participation of several countries (Argentina, Bolivia, Brazil, Chile, Paraguay, and Uruguay).4 Current epidemiologic and entomologic data show that the incidence of T. cruzi transmission has been reduced by 70% in these countries as a result of the control measures. The interruption of transmission of Chagas disease in Central American countries and Andean countries will likely be achieved by adapting the same vector control strategies successful in the Southern Cone Initiative to the local entomologic conditions of these geographic areas. Towards this goal,
MATERIALS AND METHODS Study design and sites. This cross-sectional study was conducted in five Departments in Guatemala: Chiquimula, Jalapa, Zacapa, Jutiapa, and Santa Rosa. Previous studies indicate high seroprevalence in this region.3 In the five de-
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partments where the study was conducted, there are 4,659 communities within 58 Municipios. The study was reviewed and approved by the Institutional Review Board of the Universidad del Valle, Guatemala. Sample size. Sample size was calculated using the format provided by the TDR/WHO Manager on Applied Research on Chagas Disease. Sample units were the rural schools within each Municipio. The total sample size/Municipio was divided by the number of schools present in the communities located less than 2,000 meters above sea level. A weighted sample of children was then randomly selected from each school. The statistical parameters used to calculate the sample size were 95% confidence level, 90% power, and 5% precision. The a priori estimated rate of seroprevalence for T. cruzi was 5%, based on seroprevalence data from previous studies.3 Using these criteria, 4,450 children were randomly selected from the 58 municipios. Inclusion/exclusion criteria. Children who met the following criteria were eligible: 1) Apparently healthy male or female children enrolled in elementary schools; 2) residents of the area for at least 12 months before the initiation of the study; and 3) provision of written consent from a parent or legal guardian to participate in the study. Children were excluded from the study for any of the following reasons: presence of severe medical conditions such as severe malnutrition or cardiac, renal, hematologic, or endocrinologic problems. Data collection. A structured questionnaire was used to obtain basic data on individual and demographic characteristics, living conditions, and children’s knowledge about Chagas disease. Questionnaires were coded with a unique identification (ID) number for each participant. In addition, three drops of blood were collected onto Whatman No. 1 filter paper (Fisher Scientific, Pittsburgh, PA) from each child by the finger prick method. Samples were coded using the same unique ID number assigned during the questionnaire, and stored at 4°C until processed. Laboratory methods. Antibodies were eluted from the filter paper samples and analyzed by an enzyme-linked immunosorbent assay (ELISA) essentially as described.6 Briefly, 6-mm diameter pieces of filter paper containing dried blood were incubated in phosphate-buffered saline (PBS) containing 0.5% Tween 20 (Sigma, St. Louis, MO). Samples were then diluted in PBS containing 5% non-fat dry milk approximating a 1:500 dilution of plasma, and placed in 96-well plates that had been previously coated with T. cruzi antigens prepared from culture forms of the Brazil strain (primarily epimastigotes) as previously described.7 Antibodies to T. cruzi were detected using goat anti-human IgG conjugated to horseradish peroxidase (Biosource, Sunnyvale, CA), developed with 3,3⬘,5,5⬘-tetramethylbenzidine (Kirkegaard and Perry Laboratories, Gaithersburg, MD), and read using an automated ELISA reader (Molecular Devices, Sunnyvale, CA) This assay had been previously tested in our laboratory, and showed a close correlation (r2 ⳱ 0.96) with results obtained using a radioimmunoprecitation assay (RIPA)8 and a commercial ELISA (Abbott Laboratories, Abbott Park, IL). Compared with the RIPA and the commercial ELISA, using a standardized panel of sera, we showed that our assay has a specificity of 85% and 95%, respectively, a sensitivity of 100%, and a positive predictive value of 97%. Infection with Leishmania or Trypanosoma rangeli has not been reported in the study area.
Data analysis. The questionnaire data were entered into a database and response frequencies were calculated for each question. Data from the serologic analysis were integrated into the database and the associations between the questionnaire data and the serology results were established. The chisquare test was used to assess differences between proportions (seronegative versus seropositive persons). Results were considered significant at P < 0.05. When appropriate, relative risk, with 95% confidence limits, was calculated using EpiInfo Version 6.04b software (Centers for Disease Control and Prevention, Atlanta, GA). RESULTS Enrollment. From the 4,659 communities in the endemic area, 1,814 (38.9%) were initially selected because they were located in a rural area less than 2,000 meters above sea level and had a school. The total enrollment in these schools, according to data provided by the Ministry of Education, was 164,051. Following the sample selection methodology, 4,450 students were selected for the study. Demographic and household characteristics of the study subjects. Fifty-one percent (2,272) of the participants were male and 49% (2,175) were female. The mean ± SD age for both males and females was 10.1 ± 2.2 years. Most (84.7%, 3,765) had lived in their current house since birth, while only a small proportion (15.3%, 682) had lived less than five years in their current house. Household construction characteristics are shown in Table 1. Knowledge of Chagas disease. Few children (5.35%, 238) had heard the term Chagas disease (Enfermedad de Chagas). Preserved specimens of the different stages of T. dimidiata and R. prolixus were mounted in petri dishes and presented to the participants. When shown the reduviids and asked if “they had seen one of these insects inside their house,” 31.5% (1,402) responded affirmatively. There were no significant differences between the proportion of children recognizing TABLE 1 House characteristics of the study volunteers (type of material used for house construction) in Guatemala Material
Walls Cane Bajareque* Adobe† Wood Corrugated metal Cement Other Roof Straw Palm tree Teja‡ Wood Corrugated metal Cement Other Floor Dirt floor Cement Other
Number
%
86 416 2,528 254 22 984 157
1.9 9.4 56.8 5.7 0.5 22.1 3.6
73 214 1,322 25 2,598 20 195
1.6 4.8 29.7 0.6 58.4 0.4 4.5
2,268 2,112 67
51.0 47.5 1.5
* Mixture of sand and straw that covers a stick wall. † Mixture of mud and straw shaped brick-like. ‡ Tile.
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TABLE 2 Chagas disease seroprevalence by department in the endemic area of Guatemala Department
Jutiapa Chiquimula Santa Rosa Jalapa Zacapa Total
No. positive/no. of samples
%
60/1,441 57/848 47/997 60/757 11/407
4.16 6.72 4.71 7.93 2.70
235/4,450
5.28
either T. dimidiata or R. prolixus. Most (93.4%, 1,309) of those who recognized the insects called them “chinche” or “chinche picuda.” When asked if they had been bitten by one of these insects, only 4.3% (192) responded affirmatively. Finally, we asked if they knew if any member of their family had been diagnosed with Chagas disease, and only 2.4% (106) responded affirmatively. Serologic survey. The seropositivity rates by department are shown in Table 2. The overall seropositivity rate for Chagas disease obtained in the survey was 5.28% (235 of 4,450). Of 173 communities evaluated, 35 (20.23%) had a seropositivity rate of 10−45%. The geographic seroprevalence rate by Municipio is shown in Figure 1. The association of seropositivity with the different parameters assessed during the survey is shown in Table 3.
DISCUSSION We conducted a survey to determine the seroprevalence of Chagas disease among school-age children in the endemic area of Guatemala in support of the National Chagas Disease Control Program to permit epidemiologic stratification of disease-endemic communities for vector control operations. The overall seroprevalence rate was found to be similar and not significantly different (5.28% versus 4.70%; P ⳱ 0.33) from the seroprevalence rate found among blood donors in the same endemic area (Huang S and others, unpublished data). The age-specific prevalence rates, expressed in five-year intervals, are similar to those reported in epidemiologic surveys in Mexico and Venezuela, and in a selected area of Guatemala, where a consistent increase in seroprevalence with chronologic age was observed.6 However, because the sample size in the present study was small in the youngest and oldest age brackets, this relationship was not statistically significant. As in many previous studies, we identified several factors, especially those related to house construction (Table 3), to be associated with human infection, including type of roof, walls, and floor.9–11 Other investigators have also reported that T. cruzi infection is associated with the presence or evidence of triatomines inside dwellings. We also found a correlation between the risk of testing seropositive with the antecedent of “seeing a chinche inside the house.” Similarly, in our previous study conducted with blood donors, this variable was found to be significantly associated with seropositivity (relative risk ⳱ 16.2; Huang S and others, unpublished data).
FIGURE 1. Seroprevalence of Chagas disease among schoolchildren in the endemic area of Guatemala, by municipality. Values in the box are percentages.
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TABLE 3 Comparison between seropositive and seronegative persons among the variables evaluated during the survey in Guatemala Variable*
Sex Male Female Age (years) 6–10 11–15 >16 Time living in the house
