The Northern Bolivian Altiplano: a region highly endemic for human fascioliasis
Abstract
SummaryThe worldwide importance of human infection by Fasciola hepatica has been recognized in recent years. The endemic region between Lake Titicaca and the valley of La Paz, Bolivia, at 3800–4100 m altitude, presents the highest prevalences and intensities recorded. Large geographical studies involving Lymnaea truncatula snails (malacological, physico-chemical, and botanic studies of 59, 28 and 30 water bodies, respectively, inhabited by lymnaeids; environmental mean temperature studies covering a 40-year period), livestock (5491 cattle) and human coprological surveys (2723 subjects, 2521 of whom were school children) were conducted during 1991–97 to establish the boundaries and distributional characteristics of this endemic Northern Altiplano region. The endemic area covers part of the Los Andes, Ingavi, Omasuyos and Murillo provinces of the La Paz Department. The human endemic zone is stable, isolated and apparently fixed in its present outline, the boundaries being marked by geographical, climatic and soil-water chemical characteristics. The parasite distribution is irregular in the endemic area, the transmission foci being patchily distributed and linked to the presence of appropriate water bodies. Prevalences in school children are related to snail population distribution and extent. Altiplanic lymnaeids mainly inhabit permanent water bodies, which enables parasite transmission during the whole year. A confluence of several factors mitigates the negative effects of the high altitude.
Introduction
The worldwide importance of human infection by Fasciola hepatica has been recognized in recent years (Chen & Mott 1990; Mas-Coma et al. 1999a, b). Between 2.4 and 17 million people are presently affected by fascioliasis, according to recent estimations (Hopkins 1992; Rim et al. 1994). Among the 51 countries in which human cases have been reported (Chen & Mott 1990; WHO 1995; Esteban et al. 1998a), Bolivia presents the endemic region with the highest prevalences (Hillyer et al. 1992; Bjorland et al. 1995; Mas-Coma et al. 1995; Angles et al. 1997; Esteban et al. 1997a, b; Strauss et al. 1997; O'Neill et al. 1998) and intensities (Esteban et al. 1997a, b) recorded: prevalences of up to 72% by coprology in given communities; amounts of more than 1000 F. hepatica eggs per g of faeces are numerous in children. Moreover, clinical and pathological aspects, well known in human fascioliasis in general (Chen & Mott 1990; Mas-Coma et al. 1999a, b), become confusing in Bolivia owing to the association with other pathogenic parasites: up to 13 protozoan and 5 helminth species have been found concomitantly infecting F. hepatica-infected children; up to 8 parasite species associated with F. hepatica infection have been detected (Esteban et al. 1996; 1997a, 1998b, c).
All human reports in Bolivia concern localities and communities located in the Northern Bolivian Altiplano, between Lake Titicaca and the valley of the city of La Paz, at 3800–4100 m altitude. The parasite is well known to Aymara inhabitants of this region, who call it by the vernacular name of talpalako (Mas-Coma et al. 1995).
Previous studies (Ueno et al. 1975; Lobato Paraense 1982) described the existence of two lymnaeid snail-transmitting species in this region: Lymnaea viatrix and Lymnaea cubensis. However, recent morphological and anatomic (Oviedo et al. 1995), as well as molecular (Bargues & Mas-Coma 1997; Bargues et al. 1997) and isoenzymatic (Jabbour-Zahab et al. 1997) studies have proved that there is only one lymnaeid species instead of two and that this species is Lymnaea truncatula, imported from Europe.
The aim of this paper is to summarize the results obtained in the geographical studies conducted during a 7-year period to establish the boundaries and the distributional characteristics of this endemic region. This is the first time that a human fascioliasis-endemic zone has been analysed in its geographical entirety. So far studies on human fascioliasis elsewhere were always confined to mainly case or group reports, analyses of several epidemics, or a very few epidemiological surveys in given localities (Chen & Mott 1990).
Moreover, our studies aim to analyse whether a Geographical Information System (GIS) would be viable and useful in this area. Since GIS has proved to be useful for animal fascioliasis (Malone & Zukowski 1992), its development for human fascioliasis has recently been encouraged by several specialists (WHO 1995; Hillyer & Apt 1997).
Materials and methods
Geographical studies
A sufficiently large area was covered to assure that the whole endemic zone was included; the geographical boundaries covered were from 15°52′ to 17°50′ latitude and from 67°50′ to 68°57′ longitude (Figures 1 and 2). Hydrometeorological and climatic data were furnished by the Servicio Nacional de Meteorología e Hidrología (SENAMHI) of La Paz. For the establishment of the geographical distribution of F. hepatica and of human infection, studies performed from 1991 to 1997 involved lymnaeid snails, livestock analyses and human surveys.
Map of the Northern Bolivian Altiplano showing the area prospected from 1992 to 1997 and the localities where the parasite was found in humans ● localities with human fascioliasis; ○ localities in which the parasite was not detected in humans; &;squ localities not surveyed) and where lymnaeid-inhabited waters were detected (▴). Results obtained in previous surveys (1982–92) are also included. For the numbers and corresponding names of the human localities surveyed see Table 1.
Map of the Northern Bolivian Altiplano showing the area prospected from 1991 to 1994 and the localities where the parasite was found in cattle ● localities with fascioliasis in cattle; ○ localities in which the parasite was not detected in cattle). Results obtained in previous surveys on cattle and sheep (1972–93) are also included localities with fascioliasis in sheep; ▴ localities in which the parasite was not detected in sheep). For the numbers and corresponding names of cattle and sheep communities surveyed see Tables 2 and 3. □ human localities.
Lymnaeid snails
The geographical distribution of the intermediate snail host species L. truncatula was assessed by studying freshwater collections repeatedly in the different seasons of the year and throughout the 1992–97 period by malacological sampling methods (Malone 1987). The vegetation and the physical and chemical characteristics of the water bodies presenting lymnaeids were studied. Plant species were collected mainly in the immediate neighbourhood of the lymnaeids present in the water body. Analytical methods used for the determination of water characteristics were: American Public Health Association (APHA) methods for pH, electric conductivity and FNU (Formacine-Turbidity Units) turbidity (Greenberg et al. 1992); Hach methods for colour, FTU (Formacine-Nephelometric Units) turbidity, solids in suspension, phosphates, sulphates, nitrites, nitrates, and ammoniacal nitrogen; flame emission for sodium and potassium; atomic absorption for calcium and magnesium (Anonymous 1986).
Livestock analyses
Faecal samples from 5491 cattle aged 5 months to 12 years were collected from 107 communities of 11 zones in the Department of La Paz from 1991 to 1994 (Table 2). The number of samples collected from each community was a minimum of 10% of the cattle population. Faecal specimens were transported to the laboratory within 5 h. From each stool sample a quantity of 4 g was sedimented twice, first with 50 ml of detergent solution (1 ml per litre) after filtration and then with 50 ml water, and stained with methyl green before examination under the light microscope for F. hepatica eggs (Dennis et al. 1954).
Information on results obtained from sheep was also taken into account, although information furnished by cattle is more valuable to establish the geographical distribution of the parasite: the life-span of the adult liver fluke in sheep can be as long as 11 years (Dawes & Hughes 1964), while cattle are more resistant to reinfection and may self-cure, becoming egg-negative after 1 year of infection (De Leon & Quiñones 1981).
Human surveys
Human surveys involving 2723 subjects were performed from 1992 to 1997. Mainly school children (2521) were surveyed because children do not usually migrate, unlike Aymara adults, who sometimes move or travel both inside and outside the Northern Altiplano. The Bolivian educational system has given rise to small to mid-size schools strategically located according to the human population of the zones; each locality has its own school and in cases of dispersed communities and/or very small localities, a school receives the children from its whole neighbourhood.
Coprological surveys were made at random on a given day among all consenting persons; a clean 30-ml plastic, widemouthed, numerated container with snap-on lid was given to each person; subjects were then asked to try to fill the container with their own faeces and to return it immediately. Personal data were noted on delivery of the container. The low receptivity of Aymara adults made it impossible to obtain multiple stool specimens collected at two-or three-day intervals.
Stool samples were processed as described by Esteban et al. (1997b). A Kato-Katz slide was made from each stool sample and if possible (sufficient material) two aliquots of each one were preserved in Merthiolate-Iodine-Formalin (MIF) fixative (1 : 3) and 10% formalin solution (1 : 3). Samples fixed in MIF were processed by the direct MIF smear and MIF concentration techniques, and those fixed in 10% formalin were processed by a formol-ether concentration technique.
Institutional ethical review procedure
The surveys were carried out after informed consent was obtained from the local authorities of the community (among Aymaras, community authorities are responsible for transmitting parental consent after previous meetings), as well as from the Director and teachers of each school. The project was approved by the Secretaría Nacional de Salud del Ministerio de Desarrollo Humano (La Paz, Bolivia) and performed in collaboration with the INLASA Institute in La Paz, the official disease reference centre for Bolivia.
Results
Parasite geography
Human localities studied and prevalences obtained are noted in Table 1. Results obtained from cattle are noted in Table 2. F. hepatica infection has been studied in cattle and sheep from several Altiplanic localities (Table 3) (Ueno et al. 1975; Mas-Coma et al. 1995; Hillyer et al. 1996). The results of the geographical studies are shown in Figures 1 and 2. The distribution of the liver fluke shows clear overlapping in human and livestock hosts. Similarly, overlapping of the distribution of the parasite and the snail intermediate host was observed.
The endemic area covers the Northern Altiplano, also known as humid Altiplano, including part of Los Andes, Ingavi, Omasuyos and Murillo provinces of the Department of La Paz. Most of it concerns the two large corridors (planes separated by small hill chains) El Alto-Pucarani-Batallas and Tambillo-Aygachi-Huacullani, the plane of Laja in which both corridors reunite, and up to the route from El Alto to Oruro.
The human endemic zone appears isolated. The western boundary is marked by the coast of Lake Titicaca. The water of this lake is saline (the global contents of dissolved salts, measured by means of electric conductivity at 25 °C, is 1343–1521 μS/cm in the Small Lake depending on the season) (Iltis et al. 1991). Salinity has adverse effects for lymnaeids and studies on the malacofauna of Lake Titicaca have never found lymnaeids in its waters (Dejoux 1991).
The Achacachi zone represents the northernmost boundary. Studies carried out up to the locality of Ancoraimes suggest that the snail is absent from this zone, most probably due to its incapacity to survive the periodic floods by the saline waters of the Lake of the narrow land band between Lake Titicaca and the Eastern Andean chain. The rest of the septentrional boundaries, in the northern part of the El Alto-Batallas route, are constituted by the altitudinal increase of the slope foothills of the Eastern Andean chain, temperature decrease being the determining factor. The absence of the parasite in both humans and livestock and of the snail host in the numerous water collections of the corridor of Peñas-Kerani may be explained by the loss of the moderating influence of Lake Titicaca, the presence of a hill chain between the Lake and this corridor, the neighbourhood of the Eastern Andean chain and its influence on low temperatures, and a chemical composition of the water bodies adverse to lymnaeids.
In the eastern boundaries, the parasite extends into the valley of La Paz, although the disease there concerns mainly livestock; human subjects are only affected in the small subvalley of Achocalla. More to the south, the disease extends into the valley of the river Cala Jahuira, where it reaches only up to the mid-valley, temperature decrease related to altitudinal increase explaining this boundary.
The most meridional boundary is located in the southern part of the locality of Viacha. Studies showed two factors that explain the disease's incapacity to extend southward, in the direction of the Central Altiplano: first, a marked change of the climatic conditions (temperature decrease to a lower annual mean; night temperatures always near or below 0 °C; humidity decrease with annual mean rainfall of only 330 mm; strong winds in winter) in the Central Altiplano (Lorini & Liberman 1983; Ruthsatz 1983) as the consequence of the loss of the moderating influence of Lake Titicaca (Vacher et al. 1989; Roche et al. 1991); as known, in other regions the intramolluscan larval development of F. hepatica is arrested below 10 °C (Boray 1969); second, in the area south of Viacha, large extensions of land covered by a superficial salt layer in which water collections are always devoid of lymnaeids; large amounts of salts on the terrestrial surface are typical in the Central Altiplano (Salm & Gehler 1987). In the Tiwanaku-Guaqui corridor, the few isolated transmission foci of Yanarico and Chambi Grande are related to water bodies appearing as subsoil effluences; all other water bodies studied in this corridor correspond to rivers devoid of lymnaeids; this absence is probably related to their chemical composition, in turn a consequence of soil characteristics. Despite a report of L. viatrix in Guaqui (Lobato Paraense 1982), we were unable to find lymnaeids in Guaqui and its neighbourhood; most probably, the Bolivian material studied by Lobato Paraense (1982) came from the Yanarico-Chambi Grande area (or even from the Tambillo-Huacullani corridor), but Guaqui was mentioned to allow its location on large-scale maps. F. hepatica-infected cattle are rarely found in the surroundings of Guaqui, but in all cases animals are imported from Peru. Finally, studies carried out in the large southern corridor of Jesus de Machaca showed that it is too dry and cold; here also the loss of the moderating influence of Lake Titicaca becomes determinant.
Human infection
The endemic zone appears to be heterogeneous: there are subzones of greater or lesser prevalences in humans, as well as others even lacking the parasite (compare Figure 1 and Table 1). When analysing school children, prevalences show a relationship with the snail population distribution and extent. Schools located in areas where extensive water bodies inhabited by numerous lymnaeids exist (Pantini, Cutusuma, Chijipata Alto, Cullucachi, Calasaya, Huacullani, Lacaya Baja, Quiripujo) present higher prevalences. By contrast, in areas without freshwater collections (Caleria), with water bodies devoid of lymnaeids (Causaya) or with small water bodies with small snail populations (Achocalla, Belen Yayes, Cohana), prevalences are low or zero. Other areas present intermediate prevalences.
In large schools receiving children from different neighbouring areas with and without lymnaeid-inhabited water bodies, a concentration of infected subjects is detected among the children living or frequently visiting the transmission foci areas; for instance, in the school of Ancocagua a low prevalence was detected, although in a 6-child family 4 of the children presented very high intensities which could be explained by the fact that the children accompany the livestock to pastures.
The results obtained in the surveys carried out on adult subjects in Tauca, Chijipata Alto and El Alto (Mas-Coma et al. 1995; Esteban et al. 1997b; Angles et al. 1997) suggest that migration takes place within the endemic zone from areas of high prevalences to areas of low or null prevalences and vice versa. Similarly, infected adults can be diagnosed outside the transmission zone, as with several urban cases in hospitals of La Paz (Mas-Coma et al. 1995).
Distribution of transmission foci
Transmission foci are patchily distributed and determined by lymnaeid populations inhabiting local waters. Despite the extreme monomorphism detected in the Altiplanic L. truncatula populations (Jabbour-Zahab et al. 1997), they are able to adapt to different and very extreme physical and chemical conditions (Table 4). A similar observation can be made concerning the aquatic vegetation (Table 5), in which only a negative association has been observed with the plant called totora (Schoenoplectus californicus ssp. tatora), most probably because of the noxious secretions of its roots (Althaus 1966; Seidel 1978); in very few cases lymnaeids could be found in a water body in which totoras were present, although in these cases the snails were always far away from this plant. Among the numerous aquatic and semiaquatic plant species found in water collections presenting lymnaeids (Table 5), mainly Juncus ebracteatus and Mimulus glabratus and secondarily Nostoc sp. are contamination sources for human adults. Many other species appear to be involved in the transmission to children, such as Hydrocotyle ranunculoides, Eleocharis spp., Rorippa spp., other Juncaceae and Scrophulariaceae, Compositae, etc.
A specific characterization of the transmission foci becomes very difficult owing to the capacity of Altiplanic lymnaeids to inhabit different types of water bodies, such as small watercourses, natural and man-made canals, subsoil effluences from shallow phreatic layers, large and small rivers conducting waters coming from the perpetual snow amounts of the Eastern Andean chain, flooding areas (the so-called bofedales in Bolivian –Mas-Coma et al. 1995), shallow wells, pools, artificial fountains, overflowings, clean as well as markedly eutrophic waters, etc. Lymnaeids were usually found in stagnant or scarcely flowing water bodies, very rarely in running waters (such as after intense rainfall).
European populations of L. truncatula are markedly amphibious (Euzeby 1971). On the contrary, Altiplanic L. truncatula specimens are more aquatic and only rarely encountered in mud, out of water (a significant number of lymnaeids on mud, out of water, was only detected in 12% of the monthly collection visits performed to 6 different transmission foci studied throughout a year period). They mainly inhabit permanent water collections, never temporary water bodies (large or narrow streams, ponds in pastures, puddles besides the roads, etc.) such as those originating from rain during the rainy season (October–March). Temporary water bodies originating from rain do not persist for enough time to allow colonization by lymnaeids and even in rainy years the dry season is too long to allow the survival of buried and lethargic lymnaeids. This is related to the high evapotranspiration rates proper to the high altitude (in spite of the humidity of Lake Titicaca – see Roche et al. 1991), as well as to the long duration of the dry season coincident with the lowest temperatures of the year (April–September). Thus, lymnaeids scattered throughout patchily flooded pastures, which constitute the most important transmission foci in mid-Europe, are almost never found in the Altiplano. Similarly, the relationship of fascioliasis with rainy periods, well known in the northern hemisphere both in animals and humans (Chen & Mott 1990; Ripert et al. 1988), cannot be applied to the Northern Altiplano on the same terms. In the Altiplano, the existence of lymnaeid populations in the transmission foci throughout the whole year enables parasite transmission during all seasons.
In the Northern Bolivian Altiplano year, seasonal differences in mean temperature are very low, of only 5.0–6.5 °C. The great temperature differences, from 10.3 to 13.0 (on the coast of Lake Titicaca) up to 13.4–22.4 °C (far from the Lake) occur within a day (Table 6). Moreover, the Northern Altiplano was originally a forest of trees of altitude (Beck et al. 1988), but today few trees remain in the whole endemic zone (except willows and eucalyptus in a few given places, and a very few endemic kishuara Buddleya coriacea); the lack of trees and shrubs implies no shade and hence a direct intense sunshine (Vacher et al. 1989; Roche et al. 1991) which increases the temperature of water bodies at midday (daily temperature range in water bodies inhabited by lymnaeids: from 3.0 –10.0 °C at night to 15.0–28.0 °C during the day).
Discussion
Several conclusions can be drawn:
-
the human endemic zone appears isolated and apparently unable to extend from its present outline, its boundaries being marked by geographical, climatic and soil-water chemical characteristics;
-
the endemic zone shows remarkable stability, the transmission foci, marked by the presence of lymnaeid populations in water bodies, being maintained both during the year and throughout pluriannual periods;
-
the parasite distribution appears irregular in the endemic area, the transmission foci being patchily distributed and linked to the presence of appropriate water collections;
-
human prevalences in school children appear to be related to the distance to water bodies presenting lymnaeids and to snail population size and extent;
-
Altiplanic L. truncatula are less amphibious than European L. truncatula, inhabiting mainly permanent water bodies and appearing to be unable to colonize temporary water bodies; this enables parasite transmission during the whole year.
The absence of lymnaeids in Lake Titicaca waters is worth mentioning, since fascioliasis is traditionally linked to the Lake by the Aymara inhabitants of the endemic zone (Mas-Coma et al. 1995). The origin of this erroneous thought may be most probably found in the fact that the most human prevalent subzones (Pantini, Cutusuma, Chijipata Alto, Cullucachi, Calasaya; Huacullani, Chojasihui, Lacaya, Quiripujo) are located near the Lake. This is nothing more than the result of the confluence of numerous permanent water bodies (rivers coming from the Eastern Andean chain; subsoil effluences from shallow underground phreatic layers – see Liberman 1987) inhabited by important lymnaeid populations in these subzones, as the consequence of the slight slope of the plains, from about 4056 m altitude in the neighbourhood of El Alto to the 3809 m of Lake Titicaca.
Similarly, the observation of the negative association of totora with lymnaeids is contrary to the general local thought that fascioliasis transmission is related to this plant (Mas-Coma et al. 1995), the green stems and roots of which are used both for livestock and human consumption, respectively (Levieil & Orlove 1991). All other plant species present in Altiplanic water collections presenting lymnaeids may be considered potential human contamination sources: Altiplanic F. hepatica metacercariae attach to plant species belonging to different taxonomic groups (Ueno et al. 1975); according to our observations, Aymara children suck and/or chew all kinds of aquatic and semiaquatic vegetables they find when playing or accompanying livestock.
According to the boundaries of the endemic region, the human population at risk can be estimated at about 250000 Aymara people living in the rural area and more than 2 million people in the urban zones. The problem increases because of the situation of the endemic zone regarding the densely populated cities of La Paz and El Alto. The inhabitants of both cities cannot avoid crossing the endemic zone when going to Lake Titicaca (tourism, sport activities, week end trips, etc.), to Peru (the two closest frontiers are in Guaqui and Copacabana), to Laja and Tiwanaku (tourist and historical reasons) or to Oruro, Cochabamba and the rest of the country (the only route being that from El Alto). Moreover, both La Paz and El Alto are preferred destinations for emigrants from the endemic zone, who return frequently to their origins on holidays, at weekends, for family celebrations, etc. Related to all these movements, an increase of transport has taken place in recent years. This has favoured the carrying of Altiplanic vegetables from the endemic zone to El Alto and La Paz for human consumption, opening the possibility that plants carrying metacercariae can be sold in noncontrolled city markets, thus leading to urban infection.
Moreover, the endemic zone supports numerous populations of several potential definitive host species. Among the domestic livestock at risk, cattle and sheep are the most abundant, followed by pigs and donkeys. Studies on fascioliasis of Altiplanic sheep and cattle have already been done (Ueno & Morales 1973; Ueno et al. 1975; Mas-Coma et al. 1995; Hillyer et al. 1996). The participation of pigs and donkeys in parasite transmission in the Altiplano has recently been demonstrated, whereas horses, goats, llamas and alpacas do not seem to significantly participate in disease transmission (Mas-Coma et al. 1997). Domestic rabbits and domesticated guinea pigs, as well as wild hares and rodents were not found infected and may not be considered in the epidemiology of the disease because of their etoecological characteristics (Fuentes et al. 1997).
The paucity of the agricultural soil and the extreme hardness of farming at this altitude have led to the present situation, in which Aymara inhabitants largely depend on livestock. Following a custom of great social importance among Aymara Indians, each family traditionally sustains its own animals, usually more than one species, mainly sheep, cattle, pigs and donkeys. The increase of livestock has led to soil compaction by overpasturing, which in turn progressively decreases agricultural soil capacity. Moreover, this situation has deteriorated because of land laws introduced by the Bolivian government after the 1950s, by which direct legacy of land pieces from parents to sons has given rise to such property fragmentation that it has become impossible today for a family to survive from the production of such small land pieces (Mas-Coma et al. 1995).
Summing up, in the Northern Bolivian Altiplano a confluence of several factors takes place which mitigates the negative effects of the high altitude, mainly the cold temperatures and the high evapotranspiration rates. Among these factors are:
-
the nearness to the equator, with increasing temperature;
-
the neighbourhood of Lake Titicaca moderating temperatures and increasing humidity;
-
the existence of numerous fresh water bodies deriving from the thaw of the perpetual snow of the Eastern Andean Chain which, combined with the existence of shallow phreatic layers, assure the presence of permanent water collections for the survival of lymnaeids;
-
the absence of shade because of the lack of trees and shrubs permitting a marked daily increase of the temperature of the water bodies;
-
the scarcity of land offerings leading human inhabitants to a marked dependence on livestock, including various potential definitive host species.
But if all these factors may explain the adaptation of the parasite and the intermediate snail host to the Northern Altiplano, the reasons why the disease reaches such high human transmission rates still remains to be discovered. Prevalences in Altiplanic animal reservoir hosts are not high enough to understand the human prevalences detected.
Several characteristics make the Northern Bolivian Altiplano an ideal candidate for the application of GIS methods: the isolation and the relatively small size of the endemic area, the good visibility of the water collections owing to the absence of high or dense vegetation, the stability of the transmission foci, the lymnaeids being unable to colonize temporary waters, and the relation between human infection and the distance to the nearest water collections inhabited by lymnaeids. Only two factors can complicate it: the overlapping of the numerous populations of the different potential definitive host species and the exist-ence of water bodies lacking lymnaeids. The first can be monitored; the second will need special work in the field because vegetation species do not allow us to differentiate between water collections presenting and those lacking lymnaeids.
Acknowledgements
The authors acknowledge the facilities and collaboration received from the following institutions and centres in La Paz: Dirección Nacional de Epidemiología and Dirección Nacional de Relaciones Internacionales of the Ministerio de Previsión Social y Salud Pública; Comité Regional de Zoonosis and Centro Piloto La Paz of the Unidad Sanitaria La Paz; Dirección Nacional de Producción Pecuaria of the Ministerio de Asuntos Campesinos y Agropecuarios (M.A.C.A.); Office of the Pan American Health Organization; and Danish NGO Danchurchaid-Bolivia.
The study was supported by a Project (Contract No. TS3-CT94–0294) of the STD Programme of the European Commission (DG XII), Brussels, by the Programme of Scientific Cooperation with Latin America of the Instituto de Cooperación Iberoamericana of the Agencia Española de Cooperación Internacional (I.C.I.-A.E.C.I.), Madrid, and by Projects No. UE96–0001 and No. PM97–0099 of the Dirección General de Investigación Científica y Técnica (DGICYT), Spanish Ministry of Education and Culture, Madrid.
