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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Non-human reservoirs of T. cruzi include dogs, cats, rats, mice, raccoons, opossums, and armadillos.1,2 The local pattern of transmission to humans depends mainly on the degree of contact with mammalian reservoirs and on the housing conditions. Thatched houses with cracked or unplastered walls and with a large number of residents, as well as the presence of dogs in bedroom areas, are associated with high risk of transmission of T. cruzi.6–8 Infected triatoma bugs inhabiting niches and cracks in walls and ceilings of such houses emerge at night to feed on the blood of sleeping humans by biting exposed skin areas.
We previously found a high seroprevalence of T. cruzi in Indian populations inhabiting areas of Paraguay and Argentina belonging to the Gran Chaco territory. (see dashed outline in map of South America in Figure 1
).9 This is a vast, low, flat, and partially wooded tropical land with an area of approximately 900,000 km2. We have also shown that these populations are endemically infected with the human T cell lymphotropic virus type II (HTLV-II),10 which has been linked to rare neoplasms of the blood-forming organs and neurodegenerative diseases, and with hantavirus,11 the causative agent of an often fatal pulmonary syndrome. Evidence was reported indicating that in the Gran Chaco Indians transmission of HTLV-II occurs sexually and perinatally. None of these Indians were known to be intravenous drug users.10
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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) of approximately 1,000 km2 in the Department of Boqueron in western Paraguay. These people ranged in age from 2 to 80 years. Of the 680 people examined, 668 were 13–80 years old and 12 were 2–12 years old. All but three of the Indians examined belonged to the Chulupi, Lengua, Guarani, and Ayoreo groups, which represent different linguistic families. The study area included several Mennonite farming colonies, Indian communities, and a main town called Filadelfia. The Indians lived in communities containing varying numbers of families which, as a rule, belonged to a single ethnic group. These communities were situated in the immediate periphery of Filadelfia or in isolated rural sites. The dwellings in the Indian communities had roofs made of mud mixed with thatch and unplastered walls made of adobe or sun-dried mud and branches. In virtually all the Indian communities there was a relatively high concentration of dogs that ramble freely in and out of the dwellings, and the number of household residents was high. The Mennonites resided in Filadelfia, or in farms where they were engaged primarily in agricultural and cattle-raising activities. All but four of the Paraguayans examined lived in Filadelfia. In contrast to the Indian dwellings, the Mennonite and Paraguayan houses were well constructed with manufactured material, had plastered walls and roofs, and suitable sanitation facilities. Many of these houses also had screened windows and doors. Many of the Indians living in the immediate periphery of Filadelfia worked in construction, factories, and other enterprises managed by Mennonites. Indians of both sexes often performed domestic tasks in Mennonite residences. The majority of the Mennonite and Indian men living in rural areas were involved in outdoor agricultural activities, clearing scrubs, planting and harvesting crops, gardening, and caring for livestock. Indian women also performed some of these tasks, but were primarily engaged in cleaning houses and outbuildings used for storage of food, grain, and hay, as well as in milking cows, harvesting, and gardening. The younger Mennonites living in Filadelfia worked in small factories, offices, and shops whereas most of the older Mennonites were retired farmers. The Paraguayan men worked in construction and in offices, and the women were involved in domestic activities
Study plan. The objectives of the study were explained to family heads and other members of the Indian communities through the community leaders or health delegates. The Director of the Hospital of Filadelfia and some of his associates also explained these objectives to members of the Mennonite associations and of Paraguayan groups. Participation was voluntary and based on accessibility. No special selection of the participants was made in terms of ethnicity, sex, age, location of the residence, occupation, or activity. Participants were assembled in local health centers, community-gathering buildings, or in the Hospital of Filadelfia. In addition to blood samples, demographic information was obtained from the participants. This information included age, sex, location, and setting (town, periphery of town, or rural) of the residence, occupation, and, in most cases, family relations. Information on the main risk factors for T. cruzi and HTLV-II infections was also obtained. This information included characteristics of the dwellings (whether they had unplastered walls with cracks, thatched roofs with crevices, and screened windows and doors), presence of dogs in the sleeping areas, and number of household residents. The people examined were also asked whether they observed triatomine bugs in their residences, whether they had been intravenous drug users, and whether they had received blood transfusions. The study also includes plasma samples, demographic information, and information on risk factors for T. cruzi and HTLV-II collected four and a half years earlier from 158 Indians inhabiting the study area.
The study plans and procedures were approved by the Institutional Review Committee (Committee of Studies on Human Beings) of the Office of Regulatory Affairs of the University of Pennsylvania. This is the formal body at the University of Pennsylvania that is responsible for ensuring that research on humans complies with U.S. government regulations on subject, safety, and ethical issues. The study plans and procedures, including those for informed consent, were also approved by the Paraguayan Ministerio de Salud Pública y Bienestar Social, which is the official regulatory institution for research involving humans in Paraguay.
Collection and processing of blood specimens. Heparinized and, in most cases, citrated blood samples were obtained by venipuncture from each individual. The blood samples were centrifuged at 1,500 x g for 20 minutes and the resulting plasma was stored at 4°C for several days and subsequently at -20°C. Peripheral blood leukocytes were obtained and preserved as described.10 Blood samples from children were obtained by fingerprick. Samples were processed within four hours after collection and were kept on ice until that time.
Testing for T.cruzi, HTLV, and hantavirus. As in our previous study,9 plasma samples were analyzed in the Chagas IgG enzyme-linked immunosorbent assay (ELISA) developed by Gull Laboratories (Salt Lake City, UT). The assay was conducted and the results were evaluated according to the manufacturers’ instructions. Comparative studies using a large number of positive and negative samples showed that the sensitivity and specificity of the Gull ELISA are comparable to those of the Chagas 2.0 enzyme immunoassay of Abbott Laboratories (Abbott Park, IL) and the consensus of the indirect immunohemagglutination assay and ELISA developed by Polychaco (Buenos Aires, Argentina).9 approximately 5% of the samples tested gave borderline results in the Gull ELISA. These samples were re-tested twice and scored as positive only if the optical densities were above the cutoff value in two of the tests. There were only five samples that remained indeterminate and were excluded from the study. The HTLV-I and HTLV-II testing was done using the differential Select HTLV ELISA kit (Biochem ImmunoSystems, Montreal, Quebec, Canada) and HTLV-I/II polymerase chain reaction assays. The performance of these assay and the criteria to evaluate the results have been reported.10 Plasma samples were tested for IgG antibodies to hantavirus by a highly sensitive and specific Western blot assay in which a recombinant N protein purified to homogeneity from the CC106 SN virus isolate was used as antigen. This assay was conducted and the results were evaluated as described.16
Statistical analysis. Multivariate logistic regression models, developed with the SAS Logistic program,17 were used to evaluate the associations of the dependent dichotomous variable antibody status to T. cruzi (positive/negative) and HTLV-II infection status (positive/negative) as a function of suggested risk factors for these agents. For reasons explained in the Results, the exposure risk factors for T. cruzi were considered collectively for each of the two main race groups (Indian and non-Indian). In another model, the ethnic group Chulupi was evaluated as a risk factor versus the other Indian groups. Confounders included in both models when examining T. cruzi status were age (continuous variable), sex (male/ female), HTLV-II infection status (positive/negative), and status of antibody to hantavirus (positive/negative). Hantavirus status was included as a confounder because a previous study11 showed that hantavirus seroprevalence in this population was very high. When examining for HTLV-II infection status, the same confounders were included except that the status of antibody to T. cruzi replaced HTLV-II infection status. The general form of the logistic model used was:
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where: P(Y) = probability of an individual being positive for antibody to T. cruzi or HTLV-II infection, Bo = baseline odds, and Bn = risk factors and confounders described earlier.
The multivariate logistic regression approach allows for the evaluation of the results of antibody status to T. cruzi while controlling for age, sex, HTLV-II infection status, and status of antibody to hantavirus. Results for magnitude of effects are reported as odds ratios (ORs). Odds ratios are used to measure the intensity or degree of association between a risk factor and an outcome (e.g., status of antibody to T. cruzi). A ninety-five percent confidence interval (CI) was calculated for each OR. This interval indicates that the true parameter lies between the two end points 95% of the times.
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RESULTS |
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As shown in Table 1, antibodies to T. cruzi were detected in 43.5% of the 519 Indians, in 2.8% of the 140 Mennonites, and in none of the 21 Paraguayan examined. After adjusting for age, sex, HTLV-II status, and hantavirus status, the OR for race (Indians and non-Indians) was 33.82 (95% CI = 12.01–95.22). The prevalence of HTLV-II infection was 22.1% in Indians and 3.7% in the non-Indians (OR = 6.06, 95% CI = 2.49–14.75, after adjusting for age, sex, T. cruzi status, and hantavirus status). Both T. cruzi seroprevalence and prevalence of HTLV-II infection varied among the Indians groups. However, when compared with all the other Indians taken together, Chulupi had the greater prevalence of both T. cruzi (OR = 3.72, 95% CI = 2.50–5.55, after adjusting for age, sex, HTLV-II status, and hantavirus status) and HTLV-II (OR = 3.33, 95% CI = 2.01–5.52, after adjusting for age, sex, T. cruzi status, and hantavirus status). None of the subjects examined was positive for HTLV-I. No association was observed between HTLV-II infection and blood transfusion in the subjects examined and none of them reported intravenous drug use.
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The communities and residences of 658 of the 680 people listed in Table 1 were located in five main zones, which we labeled A, B, C, D, and E (Figure 1) (22 people examined lived in other zones). These communities and residences were either in a rural setting, at the periphery of Filadelfia, or in Filadelfia. As shown in Table 2, antibodies to T. cruzi and HTLV-II infection were detected in people living in all these zones. Communities of Chulupi and Lengua, the two main Indian groups examined, coexisted in the immediate periphery of Filadelfia (Zone C) and in the rural zone of Yalve Sanga (Zone D). After adjusting for age, sex, HTLV-II status, and hantavirus status, in both zones the OR of a Chulupi individual of being seropositive for T. cruzi was 4.88 (95% CI = 1.84–12.93) and 2.50 (95% CI = 1.02–6.16) times greater than that of a Lengua individual, respectively. These differences in T. cruzi seroprevalence were paralleled by differences in the prevalence of HTLV-II infection.
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also show that there were no significant differences in the overall T. cruzi seroprevalence between Chulupi rural communities (118 of 195, 60.5%) and the Chulupi communities located at the periphery of Filadelfia (34 of 61, 55.7%). The same was true of the differences in the overall HTLV-II prevalence between these communities (31.1% versus 33.3%). Likewise, the overall frequencies of T. cruzi and HTLV-II (33.6% and 11.8%, respectively) in the rural Lengua communities did not differ significantly from those (33.3% and 15.3%, respectively) of the Lengua communities located in the periphery of Filadelfia.
The distribution of T. cruzi and HTLV-II in Indians according to sex and age is shown in Table 3. After adjusting for age, sex, HTLV-II status, and hantavirus status, the overall seroprevalence of T. cruzi in males (49.4%) was significantly greater than that in females (40.5%) (OR = 1.61, 95% CI = 1.10–2.36). The overall T. cruzi seroprevalence almost doubled from the 13–17-year-old group (21.7%) to the 18–45-year-old group (42.6%) and increased markedly from this age group to the 46–80-year-old group (61.2%). As in the case of T. cruzi, the prevalence of HTLV-II increased with age in both sexes. However, the overall sex prevalence was the opposite of that of T. cruzi (OR = 0.46, 95% CI = 0.28–0.76 after adjusting for age, sex, T. cruzi status. and hantavirus status).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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The concurrence of high prevalence rates of both T. cruzi and HTLV-II in the same population offers an excellent opportunity to examine the associations between these infectious agents. In a previous study9 of 487 Indians inhabiting the northeastern part of the Salta province of Argentina, of which 209 were also examined for hantavirus, we did not find a statistically significant association between T. cruzi and HTLV-II after adjusting for age, sex, and hantavirus status. However, in the present study, which includes a much larger number of people examined for these agents we found, after adjusting for the same confounders, that a person infected with HTLV-II was 2.28 times more likely to be T. cruzi positive than an HTLV-II negative person.
Whatever its explanation, the finding that an individual infected with HTLV-II may have a significantly greater chance of being seropositive for T. cruzi may open new avenues for the search of yet unidentified risk factors for infections with these agents. The potential public health significance of this search is emphasized by the fact that in the Americas, approximately 18 million people are infected with T. cruzi, and the frequency of HTLV-II infection is on the increase.
Although the study of the reason(s) for the increased risk of T. cruzi infection in people infected with HTLV-II was beyond the scope of the present work, several possible explanations can be postulated on the basis of the information available in the literature. One possibility is that infection with HTLV-II impairs the immune system, thus rendering the host more susceptible to T. cruzi infection. The possibility that HTLV-II infection causes a mild immunosuppression has been proposed as an explanation for the association of HTLV-II with other infectious agents observed in blood donors and intravenous drug users.13–15
A T. cruzi-induced impairment of the immune system could be an alternative explanation for the observed association between this parasite and HTLV-II. Immunosuppression, manifested by altered T and B lymphocyte functions, has been shown to occur in people and laboratory animals acutely infected with T. cruzi.12 It is not known, however, whether the same is true in people chronically infected with the parasite. Other conceivable explanations for the association between T. cruzi and HTLV-II include the possibility that infections with these agents shared a yet unidentified common risk factor(s), and that HTLV-II-infected peripheral blood leukocytes are also transmitted by triatoma bugs.
The finding that T. cruzi seroprevalence is very high in the Indians (43.5%) and low (2.5%) in the non-Indians (Mennonites and Paraguayans) examined in western Paraguay is readily attributable to the distinct differences in exposure of these populations to the known risk factors for T. cruzi. Indeed, unlike the houses of the Mennonites and Paraguayans, the Indian dwellings had cracked or unplastered walls and thatched roofs with crevices that may provide ideal refugia for the triatomine vector. These construction features have been recognized as one of the main risk factors for T. cruzi infection.6–8 Also, the frequent presence of dogs, the main domestic reservoir of T. cruzi, in the sleeping areas and a high number of residents in the dwelling, which are also among the main known risk factors for T. cruzi infection,7,8 were typical of living conditions of the Indians, but not of the non-Indians studied.
The characteristics of the dwellings, the presence of dogs in the sleeping areas, and the number of household residents were similar for the Indians residing in the countryside and in the immediate periphery of Filadelfia. Thus, it is not surprising that the Indians living in these two settings had virtually the same frequency of T. cruzi seropositivity.
The seroprevalence of T. cruzi varied among the Indian groups. Communities of Chulupi and Lengua, which accounted for more than 80% of the Indian population examined, co-existed both in the periphery of Filadelfia and in the countryside of Yalve Sanga, and were close to each other. Even though there were no apparent differences among these communities regarding the characteristic of the dwellings, the presence of dogs in the sleeping areas, number of household residents, and activities of their members, in both settings seroprevalence was significantly greater in the Chulupi communities than in the Lengua communities. It is unlikely that this is due to differences in the application of vector control measures. According to information provided to us by residents, community heads and health workers, antitriatomine spraying was applied to only very few dwellings and only once. Further evidence that the spraying were either not conducted or were ineffective is the observation that in communities examined with an interval of four and a half years was that T. cruzi seroprevalence increased significantly or remained at the same level. Thus, it appears that the greater T. cruzi seroprevalence in Chulupi communities compared with Lengua communities was not due to differences in either exposure to known main risk factors, application of antitriatomide measures, or locations/settings of the communities. On the other hand, the observation that the differences in T. cruzi seroprevalence among the Chulupi and Lenguan communities were paralleled by differences in the prevalence of HTLV-II infection is consistent with the hypothesis that this virus infection constituted a risk factor for T. cruzi infection, or vice versa, or that these infections shared a common risk factor.
A cumulative exposure to the parasite is the most likely explanation for the age-associated increase in T. cruzi seropositivity observed in the Indians. A similar increase was also seen in our previous study9 and in another study.7 For the reasons discussed earlier, it seems unlikely that the observed increase in seroprevalence resulted from a lower exposure in the younger Indians resulting from anti-triatomine campaigns.
Our data show that after adjusting for age, T. cruzi seroprevalence was significantly greater in Indian males compared with Indian females. The same sex difference was observed in our previous study,9 although it was not statistically significant. The explanation for this sex difference is not apparent on the basis of the known domestic and peridomestic risk factors for T. cruzi infection. However, it is conceivable that, while males and females were equally exposed to domiciliary triatomines, some Indian males become infected as a result of exposure to sylvatic triatomines while sleeping in the open during hunting and gathering trips.
The observation that having a T. cruzi-seropositive family member increased the adjusted odds of an individual being seropositive, is consistent with previous data showing that in people inhabiting the Gran Chaco territory9 and other regions,1,2 infection with the parasite typically occurs in household clusters.
The findings that the prevalence of HTLV-II infection increases after puberty and is greater in females than in males confirm previous observations10 and are consistent with the view that in this population the virus is transmitted mainly from male to female via sexual intercourse.
In summary, our study reports the first observation of a strong association between T. cruzi and HTLV-II infections. This observation may provide important leads for studies aimed at the identification of new risk factors for these infections. Such studies may also contribute significantly to the understanding of the biologic properties and epidemiology of both T. cruzi and HTLV-II. The information on the association between these agents and our present data on the main parameters of the distribution of T. cruzi may also contribute to the control and prevention of Chagas’ disease.
Received February 14, 2002. Accepted for publication June 20, 2002.
Acknowledgments: We thank Dr. Andres Vidovich Morales (Ministro de Salud Pública y Bienestar Social of Paraguay) and Dr. Alvin Stahl (Director Médico del Hospital Filadelfia of Paraguay) for their invaluable support and contributions to our studies. We also thank Dr. Stahl for providing us with laboratory space in the Hospital Filadelfia and for facilitating our access to the people studied. The assistance of the personnel of this hospital and of the local health clinics is also gratefully acknowledged.
Financial support: This study was supported in part by a grant from the University of Pennsylvania Research Foundation and the Barbara Kopp Cancer Research Fund.
Authors’ addresses: Jorge F. Ferrer and David Galligan, New Bolton Center, University of Pennsylvania, 382 West Street Road, Kennett Square, PA 19348, Telephone: 610-444-5800, Fax: 610-925-8123. Eduardo Esteban, Alicia Murua, Silvina Gutierrez, and Leonardo Feldman, Area de Virología, Universidad Nacional del Centro de la Provincia de Buenos Aires, Pinto 399, 7000 Tandil, Argentina, Telephone/Fax: 54-2293-441912. Syamalima Dube and Bernard J. Poiesz, Department of Medicine, Hematology/Oncology Laboratories, State University of New York, 750 East Adams Street, Syracuse, NY 13210, Telephone: 315-464-5446, Fax: 315-464-8255. Miguel A. Basombrio, Laboratorio de Patología Experimental, Facultad de Ciencias de la Salud, Universidad Nacional de Salta, Buenos Aires 177, 4000 Salta, Argentina, Telephone/Fax : 54-87-255333.
Reprint requests: Jorge F. Ferrer, New Bolton Center, University of Pennsylvania, 382 West Street Road, Kennett Square, PA 19348, e-mail: jfferrer{at}vet.upenn.edu.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
