Trypanosoma cruzi infection in the human population of the Bolivian Chaco: four serosurveys over a 26-year period (1987-2013)

Introduction: Chagas disease (CD) remains a public health concern in several Latin American countries. At global level, Bolivia has the highest CD burden and the Chaco region, in the southeast of the country, is the most affected area. We report the results of four serosurveys for Trypanosoma cruzi antibodies, carried out approximately ten years apart from each other, during the lapse 1987-2013, in different localities of the Bolivian Chaco. Methodology: Four cross-sectional surveys were conducted in various localities, mostly rural, of the Bolivian Chaco, during the period 1987-2013. Results: Although a reliable analysis of CD epidemiological trend is challenging, a partial reduction of anti- T. cruzi seroprevalence over the past four decades in the Bolivian Chaco may be assumed. In particular, in 1987 the exposure to T. cruzi in rural setting was universal since the first years of life, while it resulted gradually lower and age-dependent thereafter. Moreover, T. cruzi seroprevalence among women of reproductive age (15-45 years) has been persistently high in rural areas. Conclusions: T. cruzi transmission is still active and CD remains a concern throughout the Bolivian Chaco. More efforts are needed in order to achieve a sustainable interruption of vector-borne CD transmission in this area.


Introduction
Chagas disease (CD) or American trypanosomiasis, caused by the infection with the protozoan parasite Trypanosoma cruzi, is a public health concern in several Latin American countries. An estimated 6-7 million people worldwide are infected by T. cruzi, and the most part remains unsuspected (it is named "the invisible disease"), undiagnosed or untreated [1]. Thanks to regional intergovernmental control programs, launched during the 1990s, e.g. the Southern Cone Initiative (1991), the Andean Pact Initiative, and the Central America Initiative (1997), vector-borne transmission due the main vector, Triatoma infestans (Reduviidae, Triatominae), has been interrupted in Uruguay, Chile, Brazil, Eastern Paraguay, and few provinces in Argentina [2]. By contrast, Bolivia has still the highest prevalence over the planet (6.1%), with an estimated 607,186 prevalent infections and an endemic area covering 60% of the country [3,4]. For this reason, Bolivian Health Authorities declared CD prevention and control a national priority in Law 3374 dated March 23, 2006 [5]. Control campaigns are promoted and managed by the National Program for Chagas Disease Control (NPCDC), with the main goal of reducing the proportion of infested houses to less than 3%. A considerable reduction was achieved in the last decades at national level, from an estimated 47.7% infestation rate (IR) in 1999 to 1.9% in 2016 [6]. However, control interventions obtained a limited success in the Bolivian Chaco, southeast of the Plurinational State of Bolivia, where the risk for T. infestans transmission continues to be high, and the house IR is still above 3% in the great part of the municipalities of the three Departments (Santa Cruz, Chuquisaca, and Tarija) [2,6].
Among the research activities, realized since the late 1980s within the longstanding collaboration between the Infectious and Tropical Diseases Unit, University of Florence, Italy and the Bolivian Ministry of Health, four seroprevalence studies for T. cruzi antibodies have been carried out, in different rural and urban/semi-urban localities of the Bolivian Chaco. In this paper, we report the results of those T. cruzi surveys, conducted approximately ten years apart from each other during the lapse 1987-2013, in order to set out CD prevalence trend and to strengthen the awareness on the current CD burden in the extremely affected Bolivian Chaco region.

Study area
The Bolivian Chaco region is a semi-arid, homogeneous ecological zone, situated between the latitudes 17°59' -22°21' South and the longitudes 64°31' -58°51' West, and is approximately 127 755 km² in size. The region is sparsely-populated and consists of three Departments and five provinces (Cordillera, Luis Calvo, Hernando Siles, Gran Chaco, and O'Connor) ( Figure 1). The inhabitants live in few small urban centres, and, for the most part, in rural communities, where households are poor dwellings, with walls of sticks, straw and clay and thatched roofs. People are mainly of the Guaraní ethnic group. Local economy is based on agriculture and animal farming [7].

Study populations
We carried out four cross-sectional surveys throughout the territories of the Bolivian Chaco region, in 1987, 1997, 2006, and 2013, respectively. In all cases, prevalence of anti-T. cruzi antibodies was obtained as part of broader epidemiological surveys in human population of the Chaco region.
Each study was firstly explained during a preparatory meeting, involving local health care providers and community leaders. With their collaboration, the invitation to participate, directed to all individuals of the communities, was disseminated during public meetings. Therefore, the surveyed populations consisted of healthy volunteers, consecutively enrolled among those who consented to participate (with the exception of the studies carried out in Camiri and Boyuibe in 1987, which included a number of hospitalized patients).
In 1987, the study population included individuals from three different settings of the Cordillera Province (Department of Santa Cruz): Camiri, (a little urban centre of approximately 30,000 inhabitants); Boyuibe, (a semi-urban municipality of about 2,500 inhabitants), and Javillo (an extremely isolated rural community of about 110 people). In Camiri and Boyuibe, the study population consisted of primary and secondary school children and in-and out-patients of the local hospitals (178 people in Camiri, 184 in Boyuibe, age range 1-75 years), whereas in Javillo almost the entire population was enrolled (84 people, age range 4-70 years).
In the following studies, we surveyed only populations living in rural areas. In 1997, the survey concerned rural communities in the neighborhood of Camiri and Villa Montes (Gran Chaco Province, Department of Tarija), each having approximately 3,300 inhabitants. The sample size was adequate for an expected anti-T. cruzi antibodies prevalence of 80%, with a worst acceptable error of 5% and a confidence interval of 95%. The study populations consisted of 238 individuals from Camiri and 250 individuals from Villa Montes (age range 1-85 years).
In 2006, we surveyed the rural community of Bartolo (Hernando Siles Province, Department of the community populations (161 persons, age range 1-77 years).
populations; age range 1-83 years) were enrolled in the the community populations, age range 1-83 years) were enrolled in Ivamirapinta (Cordillera Province, Department of Santa Cruz).

Serologic assays
For all subjects, demographic data (sex, age) were recorded, and a sample of 5-10 ml venous blood was collected, stored at -20°C in Bolivia, then transported to Italy and stored at -70°C until tested. Serological tests were performed by using an indirect hemagglutination assay (IHA) in 1987 (Cellognost-Chagas, Behring Institute, Germany) and 1997 (Chagas Polychaco kit, Lemos Laboratories, Argentina), fixing the cutoff at 1:8 dilution, according to WHO recommendations [8]. In the surveys conducted in 2006 and 2013, two conventional tests based on different principles and detecting different antigens were used. In the case of ambiguous or discordant results, a third test has been used. In details, in 2006 samples were tested by a highperforming rapid diagnostic test (RDT) (Chagas Quick test, Cypress Diagnostic, Belgium), a based on a recombinant antigen, composed of nine different epitopes and by an immunoenzymatic assay (EIA) (Chagas Trypanosoma cruzi IgG, DRG Diagnostic, Germany), which used as antigens the Trypanosoma cruzi lysate.
cruzi recombinant and whole cell lysate antigens, respectively. A third test (BioELISA Chagas, Biokit S.A, Spain) based on recombinant antigens, was used to disclose discordance. Assays were performed and cutoff determined according to the respective manufacturer's instructions. Negative and positive controls provided in the kit were included in each serological run.

Ethics
The studies were programmed and conducted in agreement with the Ministry of Health of the Plurinational State of Bolivia (within the Convenio Ministerio de Salud y Deportes, Estado Plurinacional de Bolivia/Cátedra de Enfermedades Infecciosas, Universidad de Florencia, Italia), and with the support of the Guaraní political organization (Asamblea del Pueblo Guaraní). The study was approved by a local Ethics Committee (Colegio Médico de Santa Cruz, TDEM CITE No. 028/2017).

Statistical analysis
Statistical analysis of data was performed with STATA 11.0 (StataCorp, College Statio, TX, USA). Frequencies and percentages with 95% confidence intervals (CI) for categorical variables, medians, and interquartile ranges (IQR) for continuous variables were calculated. Chi-square test and logistic regression were used to investigate the association of positive tests with sex and age, respectively. Results were considered significant when the p-

Results
Demographic details and age-stratified T. cruzi seroreactivity of the populations studied in our crosssectional surveys are shown in detail in Table 1. In the Cordillera Province (Department of Santa Cruz), in 1987 we found a seroprevalence for T. cruzi of 73% (129/178) and 64% (117/184) in urban and semi-urban areas, respectively. At the same time, a significantly higher rate was found in the rural community of Javillo, where 98% (82/84) of the inhabitants presented anti-T. cruzi antibodies (p < 0.001, age-stratified Cochran-Mantel-Haenszel test). In the subsequent decades, we conducted two further surveys in similar rural settings of the Cordillera Province. In 1997, seroprevalence was 81% (193/238) in some rural communities near Camiri, while in 2013, we found 60% of positivity (76/126) in Ivamirapinta (municipality of Gutierrez).
As far as the Chaco territories belonging to the Department of Chuquisaca are concerned, in the two consecutive surveys we conducted in the rural community of Bartolo (municipality of Monteagudo, Hernando Siles Province) in 2006 and 2013, a not significant decline in T. cruzi seroprevalence was observed (70% and 63%, respectively, p = 0.106). Similarly, seropositivity rate among 1-10 year-old children showed a slight, not significant decline from 2006 to 2013 (25% and 20%, respectively, p = 0.666).
In the Department of Tarija, we found a seroprevalence of 74% (184/250) in rural communities around the city of Villa Montes (Gran Chaco Province) in 1997.
In all the surveyed populations, distribution of CD did not differ by sex, while it increased according to age (in all cases excluding Javillo -1987, where exposure resulted universal since the first years of life). Moreover, T. cruzi seroprevalence among women of reproductive age (15-45 years) has been persistently high in rural areas, ranging from 100% in 1987 to 74-79% of the most recent determinations in 2013 (Table  1).

Discussion
Relatively few studies reported data on CD epidemiology in the Bolivian Chaco, although it is acknowledged as one of the most affected areas in the planet. In Table 2, we report a summary of the main available studies concerning anti-T. cruzi antibodies seroprevalence in the human population of the Bolivian Chaco [9][10][11][12][13]. A detailed and accurate analysis of the epidemiological evolution is challenging, being hindered by several limitations. Above all, various serological techniques have been used in the different periods (IHA, EIA, ELISA, RDT), basically due to the sequential development and availability of newer assays along the years. However, as for the older IHA, whereas sensitivity widely varied from different manufacturers, specificity was considered very high (98-99%), suggesting that the high prevalence found in the first studies was not overestimated [14,15]. On the other hand, results from more recent studies are generally based on more than one serological test, being a definitive diagnosis of T. cruzi infection defined by at least two positive results, as recommended by the WHO [16]. Commercial ELISA used in our studies, have shown a pooled sensitivity of 99.3% (97.9%-99.9%) and a pooled specificity of 97.5% (88.5%-99.5%) in a meta-analysis [17]. Moreover, the surveys took place in different communities, and comprehensive information on entomological situation and insecticide-based interventions are sparse or not available for the great part of the surveyed communities. The Bolivian Chaco is a homogeneous ecological zone and the rural populations share the same living and health conditions. Here, the maintenance of active transmission is fostered by the reinvasion of the poor human dwellings by residual triatomine populations from areas not well treated in the domicile or in the peridomicile, or from neighboring untreated areas. This problem is enhanced by the recent emergence of foci of resistance to pyrethroid insecticides [10,18]. In the area of Gutierrez (Cordillera Province), systematic spray campaigns against domestic T. infestans, through blanket spraying Table 2. Available studies reporting anti-Trypanosoma cruzi antibodies seroprevalence in the human population of the Bolivian Chaco.

References
Year  [19]. The study included an entomological evaluation of the house infestation by T. infestans before the intervention, which showed an overall infestation of either intra and/or peridomestic infestation of 30.5%. Furthermore, an ecosystem approach for the control of CD in the Bolivia Chaco, based on the identification of the ecological, biological and social determinants of CD transmission was proposed in rural villages of the Cordillera Province to reduce the risks of house infestation [20]. Knowledge, attitudes and practices on the vector, CD and its prevention were deficient. Living conditions, which encompass wall conditions and cleanliness of the room, and the presence of domestic animals in rooms, in particular poultry, resulted the main risk factor for house infestation. Education activities and an integrated vector control approach, based on simple and low cost interventions on the domicile environment were realized through community participation, leading to a significant decrease T. infestans densities in the domiciles. Recently, the presence of multiple animal reservoirs, including canine population, was confirmed as a further challenge for disease control and prevention strategies in this area [21].
In fact, whilst burdened by some limitations, available data confirmed that T. cruzi transmission is still active throughout this area, both via vectorial and vertical route, albeit a partial reduction over the past 40 years may be assumed (Figure 2). This trend is especially accounted for by the youngest generations, reflecting a contained CD transmission in recent years, likely due to an improvement in housing conditions over time and the successful vector control programs. On the other hand, positivity rates in people >40 years old are still over 90%, reflecting the intense vectorborne T. cruzi exposure over the past decades, which resulted in a chronic, lifelong infection ( Figure 3). Subjects with chronic asymptomatic infection (indeterminate phase) had 30% to 40% of likelihood to  develop the chronic phase complications, with a cardiac, digestive, neurological, or mixed involvement, decades after the initial infection [22]. Cardiac disease could lead to progressive heart failure and sudden death, which are the main causes of death in patients with chronic Chagas heart disease [23]. In 2011, a high prevalence of ECG abnormalities and substantial evidence of Chagas cardiomyopathy (55/398, 13.8%), including bundle branch and/or atrioventricular blocks and rhythm disturbances associated with impaired left ventricular end-diastolic dimensions or ejection fraction, were found in a population-based survey among T. cruzi infected inhabitants living in a rural area of the Bolivian Chaco [24]. As expected, the prevalence of any abnormality rose proportionally with the age. The authors called for improving the access to basic cardiac care, such as annual ECG, antiarrhythmics, pacemakers, which could have an immediate impact on CD morbidity and mortality. By merging these findings, and the CD prevalence reported in the few available studies, the Working Group on Chagas Disease in Bolivia and Peru estimated that between 3,000 and 4,000 people in the Bolivian Chaco are in need of pacemakers [25].
The screening of pregnant women and the early treatment of infected newborns are priorities for the Bolivian NPCDC started in 2004. The program was based on serological testing during pregnancy, followup of children born from positive mothers until one year old, and treatment of positive children [26]. At national level, in 2016 the infection rate among pregnant women was 16.4%, and maternal transmission in Bolivia was estimated between 1.3% and 2.3% [6]. In the Bolivian Chaco both these rates are likely higher than the average of the country, especially in rural areas, where maternal seroprevalence stands at 60-70% and congenital transmission rate at 4-6%. Maternal risk factors for vertical transmission included higher circulating parasite loads, alterations in maternal immune response, younger age and twin births [27,28]. Of note, sustained domestic vector exposure, occurring in women living in infested houses, has been associated with decreased parasitemia and lower congenital transmission risk [28]. Furthermore, available data are still too scarce to elucidate whether maternal T. cruzi infection has an impact on fetal outcome. In the absence of effective vertical transmission, chronic maternal T. cruzi infection did not show effect on gestation outcome and fetal development [29]. However, early detection of congenital infection remains crucial. Benznidazole showed good efficacy and tolerance in the treatment of congenital Chagas disease [30]. In addition, the impact of treatment of women of childbearing age in preventing the vertical CD transmission have been recently highlighted [31].

Conclusion
Although positive steps have been taken in the last decades, CD remains a major public health concern throughout the Bolivian Chaco. Here, as well as in the neighbouring areas of Northwest Argentina, and Paraguay (known as Gran Chaco region in its entirety), an effective vector control is still challenging. The reduction to 0 of the high risk municipalities and the strengthening of the Program (Atención Integral de la Enfermedad de Chagas) are a specific target of the Developing Sectorial Plan 2016-2020 [6]. Long-term repeated insecticide-based campaigns need to be integrated with housing improvements and surveillance system in order to achieve a sustainable interruption of vector-borne CD transmission in this area.