[Downloaded free from http://www.jvbd.org on Thursday, October 29, 2020, IP: 177.254.211.171] J Vector Borne Dis 55, September 2018, pp. 222–229 Babesiosis prevalence in malaria-endemic regions of Colombia Juliana González1, Ignacio Echaide2, Adriana Pabón1, Juan Gabriel Piñeros J1, Silvia Blair1 & Alberto Tobón-Castaño1. 1 Malaria Group, Faculty of Medicine, University of Antioquia, Medellín, Colombia; 2Immunology and Parasitology Laboratory, Institute of Agricultural Technology (INTA), Rafaela, Argentina ABSTRACT Background & objectives: The presence of Babesia spp in humans, bovine cattle and ticks (the transmitting vector) has not been well characterized in Colombia. Babesia infection in humans can be overlooked due to similarity of the disease symptoms with malaria specially in the regions where malaria is endemic. The aim of the present work was to study the frequency of Babesia infection in humans, bovines and ticks in a malaria endemic region of Colombia, and explore the possible relationship of infection with host and the environmental factors. Methods: A cross-sectional study was carried out between August 2014 and March 2015 to determine the frequency of B. bovis and B. bigemina infection in a sample of 300 humans involved in cattle raising, in 202 bovines; and in 515 ticks obtained from these subjects, using molecular (PCR), microscopic and serological methods. In addition, the demographic, ecological and zootechnical factors associated with the presence of Babesia, were explored. Results: In the bovine population, the prevalence of infection was 14.4% (29/202); the highest risk of infection was found in cattle under nine months of age (OR = 23.9, CI 8.10–94.30, p = 0.0). In humans, a prevalence of 2% (6/300) was found; four of these six cases were positive for B. bovis. Self-report of fever in the last seven days in the positive cases was found to be associated with Babesia infection (Incidence rate ratio = 9.08; CI 1.34–61.10, p = 0.02). The frequency of B. bigemina infection in the collected ticks was 18.5% (30/162). Interpretation & conclusion: The study established the presence of Babesia spp in humans, bovines and ticks. The most prevalent species responsible for babesiosis in humans and bovines was B. bovis, while B. bigemina was the species most frequently found in the tick population. The results contribute to the knowledge of the epidemiology of babesiosis in the country and can provide guidelines for the epidemiological surveillance of this non-malarial febrile illness in humans as well as cattle. Key words Babesia bigemina; B. bovis; babesiosis; bovine; Colombia; human; tick INTRODUCTION Babesiosis is a parasitic disease caused by a group of Babesia species that parasitize various hosts such as bovine cattle, buffaloes and other animal species; and are considered zoonotic1–2. The infected tick bite is the main route of transmission of the Babesia3. Epidemiological studies in humans and cattle have used different diagnostic methods, including PCR and microscopy for identification of parasites, and indirect immunofluorescence assay (IFA) and ELISA for seroprevalence4–5. In tropical countries bovine babesiosis is highly prevalent and has a high economic impact; the main causative agents reported include Babesia bovis and B. bigemina. In Latin America, several studies have reported about the presence of bovine Babesia: (i) in Northern Brazil the prevalence of infection detected by PCR was 33.2% for B. bovis and 52% for B. bigemina; (ii) in Southern Brazil, the seroprevalence for B. bovis was 96.1% by PCR, whereas it was 68.8 and 52.5% by IFA for B. bovis and B. bigemina respectively6–7; (iii) in Mexico, an ELISA seroprevalence of 36% for B. bovis and 45% for B. bigemina was estimated8; and (iv) in Colombia, in the region of Valley of the Magdalena River, the frequency of infection was 22.4% by microscopy, while by PCR it was 63.3% (59.9% by B. bigemina and 3.4% by mixed infection). Seroprevalence by IFA was 65.6% (57, 1% by B. bovis and 25.9% by B. bigemina)9–10. Human babesiosis is an emerging tick-borne infectious disease having worldwide distribution. In most cases, it is associated with the population that works on cattle ranches or in moist wooded areas, where the vector is generally observed. The cases are commonly reported in Europe and North America, where the main causal organisms are B. microti, B. bovis, B. divergens and B. bigemina11–12. This disease causes an acute febrile syndrome like malaria and can be misdiagnosed due to morphological similarities of Babesia with Plasmodium parasite13. In Colombia two studies have described babesiosis in humans. The study carried out in the Magda- [Downloaded free from http://www.jvbd.org on Thursday, October 29, 2020, IP: 177.254.211.171] González et al: Babesiosis in Colombia lena Medio region reported 0.5% samples positive by microscopy and 3.6% seropositive by IFA for B. bovis or B. bigemina14; while the another study from the Department of Cordoba reported 30.6% positivity by IFA for B. microti15. The prevalence of bovine babesiosis in other regions of Colombia is not known, because this disease does not require mandatory notification to the health authorities9–10. Also the prevalence of infection is unknown in people living in malaria-endemic areas (where livestock farming is an important economic activity), due to its clinical and parasitological similarity with malaria that may confuse the diagnosis and ignore its existence. Undiagnosed cases or misdiagnosis might lead to serious consequences for the patient. This prevents the epidemiological surveillance of babesiosis and generate gaps in the epidemiological characterization of this infection. The objective of this study was to characterize and establish the magnitude of the Babesia infection in bovine cattle and humans, and to identify the presence of B. bovis and B. bigemina species in ticks, in two towns where farmers practice bovine ranching and which are endemic for malaria in the UrabáColombia region. 223 Colombia MATERIAL & METHODS Design and study site A cross-sectional descriptive study was carried out between February 2014 and March 2015 in two Urabá towns: Turbo (8° 05' 42" N; 76° 44' 23" W) and Necoclí (8° 25' 39" N; 76° 46' 58" W) (Fig. 1). In both towns, cattle ranching represents the second most important economy activity16; Turbo predominates in dual-purpose of rearing livestock, i.e. for milk and meat production (92%), while in Necoclí it is aimed at meat production (63%). It has been estimated that these municipalities have an average risk of malaria transmission with annual parasite rates of 2.7 and 2.8 per 1000 inhabitants for Turbo and Necoclí, respectively; without any report of human or bovine babesiosis17. Sample size Sample size (n) for bovine and human populations was estimated according to Lwanga et al18 and using the Epidat program (version 4.1), on the basis of the following data/criteria. For bovines: Total population = 280,767 (records of the Department of Agriculture of the Department of Antioquia in 2014 for both towns16), prevalence of babesiosis = 13.65% (the median of frequencies reported in Colombia9, 19; and sampling error = 5%. For humans : People related to livestock activity16 = 2559; prevalence Fig. 1: Location of sampling sites in the Urabá region (green area), Antioquia, Colombia— 1 Necocli; 2 Turbo. of babesiosis in exposed humans13 = 30.6%; and sampling error = 5%. The sample size calculated was 202 for bovines and 319 for humans. Sampling strategy and selection of the units for analysis The selection of the study units (bovines and humans) were made from each productive unit (PU) in total 18 localities from both towns. The PUs were chosen for their homogenous production characteristics (cattle farms) and health status (vaccination against brucellosis and aphtose fever), and for their proximity to the municipal head; in total 379 farms fulfilled these characteristics, 164 in Turbo and 215 in Necoclí. The PUs that had implemented vaccination against Babesia and applied tick control insecticides in the last eight days of the visit, were excluded. The selection was made by proportional fixation sampling proposed by Silva et al20 in 1993. Finally, 30 PUs located in 15 locations, eight in Turbo and seven in Necoclí, met the selection criteria (Fig. 1). [Downloaded free from http://www.jvbd.org on Thursday, October 29, 2020, IP: 177.254.211.171] 224 J Vector Borne Dis 55, September 2018 Selection of bovine and human subjects The bovine sample was divided according to their proportion reported in each town: 60% (n = 121) for Turbo and 40% (n = 81) Necoclí. Each sub-sample was distributed among the PUs from each town, and in each PU, the bovines were selected through a list of random numbers. The selected human subjects were town residents or working in a PU; when necessary, adjacent residents were included to complete the sampling of a PU. The selection of human subjects was based on the following inclusion criteria: Age over 18 yr, willingness to participate and sign the informed consent. Data collection Data recorded on a standardized form included information on: Production units: The productive and sanitary characteristics investigated were production orientation, type of pasture, use of tick control insecticides, deworming and quarantines. Cattle: Each animal was investigated for sex, race, age and presence of ticks; the clinical status (signs of infection) was evaluated by a veterinarian. The association between the presence of Babesia and factors of the herd, such as zootechnical orientation (dual purpose, meat, milk), pastures and availability of professional veterinarians were also explored. Humans: Sociodemographic characteristics (sex, age, ethnic group), labour activities (cattle farming, housewife, student), housing conditions (wall, floor, ceiling material), presence of disease symptoms in the last seven days (headache, fever, arthralgia) and presence of clinical signs at the time of the survey (pallor, jaundice, fever and haemorrhages). Ticks: The number of ticks was determined and registered for each bovine using the technique described by Álvarez et al21 in 2003. From each bovine, 1 to 5 ticks were captured and stored for seven days, guaranteeing the development of the vector parasitic cycle. Sampling and laboratory analysis DNA extraction and PCR: For the diagnosis of babesiosis in both, humans and cattle, 5 ml of venous blood was taken; 400 ml were distributed in two tubes with heparin and then stored at –20°C until analyzed for molecular diagnosis. DNA was extracted using the DNeasy Tissue and Blood kit, following the manufacturer's instructions22. The primers reported by Figueroa et al23 were used to amplify the 18S gene by nested PCR, modified by Terkawi et al24; the PCR products were examined on a 2% TAE agarose gel by electrophoresis at 100 volts for 40 min. The final prod- ucts were 291 bp for B. bovis and 178 bp for B. bigemina23. Quality control of the results was done with DNA samples sequenced by the Institute of Agricultural Technology of Argentina, INTA, Rafaela. Microscopic diagnosis: The presence of active infection and morphological identification of the species was performed by peripheral blood smears stained with Giemsa25 and read under a light microscope with a 100 × objective. A sample was considered negative when no parasites were identified in 300 fields. For quality control, two blind readings were performed on all the positive samples and on 10% of the negative samples. Serological diagnosis: Bovine and human sera were centrifuged at 2500 rpm. The presence of antibodies was determined by ELISA using a suspension of purified merozoites obtained in vitro from B. bovis or B. bigemina; a bovine IgG1 heavy chain anti-chain monoclonal antibody conjugate (M 23ADRI-Canada) and human IgA multispecies (Pierce Biotechnology, Rockford, IL, USA) samples were also used. The 10% of the samples were analyzed by immunofluorescence (IFA), a technique in which the parasites (B. bovis and B. bigemina) were first cultured in leukocyte free red blood cells with equine serum, until 5–6% parasitaemia. After thin blood smears preparation, 1/100 B. bovis and 1/120 B. bigemina sera dilutions were made. Fluorescent reactions were observed with a Leitz microscope equipped for epi-illumination using a 50 W mercury vapour lamp. The ELISA and IFA were performed as described by de Echaide et al26. Statistical analysis The analysis was carried out with the statistical program SPSS ver. 23 (IBM Corporation) licensed for the University of Antioquia. Descriptive analysis of quantitative variables was carried out using measures of central tendency (median or average) and dispersion [interquartile range–(IQR) or standard deviation (SD)]. Qualitative variables were analyzed by proportions; a bivariate analysis was performed for the bovine and human populations using as a dependent variable the PCR diagnosis of Babesia spp. Categorical variables were analyzed using the Chi-square test and the Fisher's exact test. Infection analysis in cattle was performed by logistic regression using the step-by-step method and infection in humans by Poisson regression. Applying the Hosmer Lemeshow (H–L) criterion (p ≤0.25) the variables entered into the models, and according epidemiological importance and biological plausibility. p-value <0.05 was considered as statistically significant. [Downloaded free from http://www.jvbd.org on Thursday, October 29, 2020, IP: 177.254.211.171] González et al: Babesiosis in Colombia Ethical statement The international ethical standards for biomedical research with human subjects established by the WHO and the ethical norms of the ministry of social protection of Colombia for human research (Resolution 8430 of 1993) and animal research (Law 84 of 1989) were followed. The collection of specimens were carried out in compliance with the regulations established by the Colombian government (National Environmental Licensing Authority Resolution ANLA 0524 of 2014). The procedures were approved by both the Bioethics Committee and the Ethical Committee for Animal Research of the University Research Headquarters of the University of Antioquia (Acts 13-32-436 of 2012 and 15-32-436 of 2015). RESULTS Babesia infection was diagnosed in bovine cattle, humans and ticks in five locations, namely El Tres, Alto Mulatos, Turbo, Mulatos, Las Changas, and Totumo) out of the 18 localities visited. Cattle characteristics and infection status Among the 202 bovines studied, majority (74.8%) were reared for dual purpose (meat and milk production), which were grazing on native pastures (63.9%) such as Brachiaria decumbens. The general characteristics of cattle are described in (Table 1). The majority were females (77.2%) corresponding to cross Cebu (Bos indicus); the median age was 48 months (IQR 9–84) with a high proportion of bovines over 48 months (44%). The bovines were mostly asymptomatic (83%) at the time of the study; 34 had a rectal temperature > 38.5°C without other clinical signs (Table 1). The prevalence of Babesia established by PCR in cattle was 14.4% (29/202); 19 infections were by B. bovis (65.5%), six by B. bigemina (20.7%) and four infections were due to both the species (13.8%). The prevalence of infection by microscopy was 4.5% (9/202); 77% for B. bovis (n = 7) and 33% for B. bigemina (n = 2) (Fig. 2). Antibodies against the Babesia species were found in 55.4% (112/202) population (by ELISA); 71.4% (80/112) for B. bovis and 73.2% (82/112) for B. bigemina. The age of the bovines was categorized according to the median and the age at greater risk for the presence of Babesia (<9 months); the frequency of babesiosis by molecular diagnosis was as follows: 41.5% between 0–9 months, 6.7% between 10–48 months; and 3.4% for animals older than 48 months. The frequencies of serum antibodies for Babesia in these groups were 77.4, 55 and 42.7%, respectively. 225 Table 1. Characteristics of the bovine and human population Characteristics Bovine variables Town Categories Turbo Necoclí Zootechnical orientation Dual purpose Meat Milk Pasture type Native Other Availability of veterinarians Yes No Sex Male Female Race Holstein × Cebu Simmental Gyr Holstein Fever Yes No Age <9 months 10–48 months >48 months Human variables Town Turbo Necoclí Sex Male Female Ethnic group Mestizo African descendant Indigenous Domestic animals in the Yes No house Primary activity Cattle farming Housewife Student Tick bites Yes No Fever Si No Shaking chills Yes No Headache Yes No Figures in parentheses indicate percentages. Number 76 126 151 40 11 129 73 177 25 46 156 103 12 8 3 35 167 53 60 89 (37.6) (62.4) (74.8) (19.8) (5.4) (63.9) (36.1) (87.6) (12.4) (22.8) (77.2) (51) (5.9) (4) (1.5) (17.3) (82.7) (26.2) (29.7) (44.1) 150 150 259 41 287 9 4 261 39 247 34 19 236 64 270 30 276 24 173 127 (50) (50) (86.3) (13.7) (95.7) (3) (1.3) (87) (13) (82.3) (11.3) (6.3) (68.3) (31.7) (90) (10) (92.0) (8) (57.7) (42.3) Fig. 2: Babesia bigemina in a Giemsa stained blood smear from a bovine (Urabá, Colombia). Pear-shaped B. bigemina inside a red blood cell (arrow). [Downloaded free from http://www.jvbd.org on Thursday, October 29, 2020, IP: 177.254.211.171] 226 J Vector Borne Dis 55, September 2018 Human subject characteristics and infection status The study was carried out in 300 residents. The median age was 35 yr (Range, 25–48); 95.7% were recognized as a mestizo population (people of mixed European and Amerindian ancestry). The houses were characterized by having wooden walls (53.7%), earthen floors (39%) and zinc roofs (62%). Of all the subjects studied 87% had domestic animals in the house. The most common clinical symptoms during the seven days prior to the study were headache (42.3%) and fever (30%); though joint pain and sore throat were also reported. Sociodemographic and clinical data are summarized in Table 1. The frequency of babesiosis in humans diagnosed by PCR was 2% (6/300); 66.6% (n = 4) for B. bovis and 33.3% (n = 2) for B. bigemina. By microscopy, Babesia spp was diagnosed in three cases (1%), two infections were due to B. bovis and one was due to B. bigemina. The agreement between both tests was 50%, with a Kappa index = 0.6. Seroprevalence in humans was 0.33% (1/300) with antibody titres in one subject for both species. Two positive subjects for Babesia presented fever and headache, one presented only headache and the other three were asymptomatic. The frequency of these symptoms does not differ with the subjects without infection (p >0.05, chisquare test). Vector characteristics and infection status Seventy percent (141 out of 202) of the bovines stud- ied were parasitized by ticks, from which 515 specimens were collected and then divided into 162 sets. These sets were classified according to species, stage and sex. The frequency of Babesia infection in the tick subsets was 18.5% (30/162); 73.3% due to B. bigemina infection (22/30), 16.7% due to B. bovis infection (5/30) and 10% due to infection by both species (3/30). Association between bovine and human babesiosis The logistic regression analysis for the bovine population, with a goodness of fit of 0.921, showed that bovines < 9 months of age presented the highest probability of infection by Babesia (Table 2). Poisson regression for humans indicates that babesiosis was associated with subjective fever in the last seven days [incidence rate ratio (IRR) = 9.08; CI = 1.34–61.10] with a goodness of fit for the model of 0.780 (Table 3). DISCUSSION Since the first case of babesiosis reported in humans in 1957 in Yugoslavia27, diverse studies have measured the frequency of Babesia in humans and cattle. To the best of our knowledge there are no studies investigating the presence of this infectious agent in the population of humans, bovines and vectors simultaneously. This study evaluated the prevalence of Babesia in these three populations, in a zone endemic for malaria, and favourable for Table 2. Bivariate and multivariate analysis of Babesia infection and livestock variables Variables Age (Months) <9 10–48 >48 Sex (Female) Town Adm. tick insecticide (spray) Pasture rotation (days) Presence of ticks Babesia Positive Babesia Negative Without ticks Crude OR 95% CI OR 20.3 2.1 5.7 0.4 72.5 0.5 0.3 2.1 2.6 1 0.1 0.8 0.8 0.9 0.6 5.1 9.1 1 0.7 0.5 0.2 0.2 2.5 1.3 p-value Adjusted* OR 95% CI OR 0.001 0.36 Ref 0.001 0.11 0.13 0.14 24 1.8 6.1 0.4 94.3 8.5 0.9 3.3 0.3 0.8 0.3 0.8 0.1 0.3 2.5 13.2 1.2 2.4 0.61 0.16 Ref 0.5 0.5 0.1 0.2 2.5 1.5 *p-value 0.001 0.48 Ref 0.845 0.1 0.08 0.72 0.41 0.21 Ref *Adjusted for all other livestock variables; OR: Odds ratio; CI: Confidence interval. Table 3. Bivariate and multivariate analysis of Babesia infection in humans and some individual variables Variables Fever informed by the participant Months dedicated to cattle ranching Bovine with ticks (PCR positive for Babesia spp) Working in cattle ranches Bovines PCR positive for Babesia Crude IRR 4.5 1 1.6 0.4 0.7 95% CI 0.8 0.9 0.8 0.1 0.2 IRR p-value 24.7 1 3.2 2.3 2.1 0.08 0.3 0.23 0.32 0.54 Adjusted IRR* 9 0.1 2.2 0.8 0.4 *Adjusted for all other variables; IRR: Relative risk index (Hosmer Lemeshow criteria, p <0.25); CI: Confidence interval. 95% CI 1.3 0.9 0.8 0.1 0.2 IRR p-value 61.1 1 5.9 7 1.9 0.024 0.97 0.123 0.79 0.54 [Downloaded free from http://www.jvbd.org on Thursday, October 29, 2020, IP: 177.254.211.171] González et al: Babesiosis in Colombia the presence of Babesia due to its eco-epidemiological conditions28. In the bovine population, this study found a higher frequency of infection for B. bovis (79.3%) compared to B. bigemina (34.5%), proportions that included coinfections. Although, both are transmitted by the same vectors, this could be explained by the fact that B. bovis infection can persist in hosts for 24 months or more compared to B. bigemina infection, which persists for 12 months29. Although, the frequency of bovine cases in this study is lower than that reported in other studies in Colombia, the species wise proportion coincides with that reported by Ríos et al10, 34, who identified a higher frequency of B. bovis (57.1%) than of B. bigemina (25.9%). The seroprevalence was higher than the presence of active infection, this can be supported by the fact that a large proportion of the bovines (73%) were from meat breeds that are more resistant to Babesia infection indicating high proportion of asymptomatic bovines. This resistance of cattle to clinical signs is an important factor in the maintenance of enzootic stability because it aids sporadic babesiosis outbreaks when new animals enter these areas. In cattle, a statistically significant association was found between the prevalence of Babesia infection by the PCR technique and the presence of antibodies by the ELISA diagnosis, with a higher risk in animals younger than nine months of age compared to adults. This is in agreement with earlier studies that reported greater susceptibility to infection at this age6–7. In the human subjects, infection prevalence of the disease was 2% by PCR and 1% by microscopy. Among the six positive subjects, three were positive by both methods and presented fever on the day of diagnosis, suggesting that they were in the acute phase of the disease and thus were potential transmitters of the infection32. By the serological technique (IFA), IgG antibodies were observed in one only person out of 300, which may be due to poor prior contact with the parasite or to the variability of these antibodies over time as evidenced by Gumber et al 29 who reported that in apes infected with B. microti, this immunoglobulin is detected in chronic phase of infection (56 days after contact with the parasite). Although, the frequency of Babesia infection in humans by microscopy and PCR was low for the both species studied, it predominated for B. bovis, similar to the findings reported by Ríos et al14, who found a seroprevalence of 2.1% for B. bovis and 1.5% for B. bigemina in a cattle zone endemic for malaria in Colombia. The exploration of sociodemographic and epidemiological variables in relation to the diagnosis of infection in humans showed no associations. Specifically, the sex 227 and age variables were not related to an increased risk of infection. This is similar to what Hong et al30 reported in their study, suggesting a similar exposure in women and men, though, an analysis was not performed according to their occupation. The frequency of infestation in tick groups was 18.5%. However, the history of tick bites in people during the last year was not associated with a risk of infection. This situation can be explained by a low frequency of tick infection, or this might be due to memory bias29. When analyzing clinical variables in people, fever was observed to be the most persistent and common symptom associated with Babesia infection similar to other infections31–33, and was present in up to 91% of the patients. Microscopic diagnosis is suitable as a diagnostic alternative, in cases of symptomatic animals and in acute stages of the disease, and is the most common procedure used by veterinarians to screen for possible clinical cases. However, its diagnostic capacity is much lower than the molecular diagnosis in asymptomatic animals28. In this study the identification of seropositive animals by microscopy was 4% as opposed to 14.4% by PCR. No association was found between Babesia infection in humans and bovines with the presence of the parasite in ticks. This may be due to the low prevalence of babesiosis in humans and the low parasitaemia in bovines, a condition that decreases the transmission capacity of the parasite34. The lower frequency of B. bovis in ticks corresponds to the fact that this species does not have vertical transmission, while B. bigemina has food, transovarial and vertical transmission35. This is in concordance with a study9 carried out in Puerto Berrío-Antioquia, Colombia that showed a greater presence of B. bigemina (79.2%) compared to B. bovis (9.4%). CONCLUSION The study established the presence of Babesia parasite in bovine cattle, humans and its vectors inhabiting a region endemic for malaria in Colombia. The prevalence was low (2%) for B. bovis and B. bigemina infection in humans; however, the frequency in bovines and ticks were 14.4 and 18.5%, respectively. Since, it is not mandatory to notify Babesiosis in cattle in Colombia, the epidemiology of this disease is not well known, and therefore, it is not suspected as a cause of disease in the human population. The presence of Babesiosis in humans, represents an important problem for diagnosis. The results contribute to the knowledge of the epidemiology of babesiosis in the country and can provide guidelines for the epidemiologi- [Downloaded free from http://www.jvbd.org on Thursday, October 29, 2020, IP: 177.254.211.171] 228 J Vector Borne Dis 55, September 2018 cal surveillance of non-malarial febrile illness in people and febrile pathologies in cattle. Conflict of interest The authors of the article have no conflict of interests to declare. ACKNOWLEDGEMENTS This work was funded by the University of Antioquia (Vice-Chancellor for Research), Colombia. The authorsthank the members of the Malaria Group, who collaborated on the project, namely Cecilia Giraldo-C and Luz Aida Gómez-R for the microscopic diagnosis; Juan Camilo Pérez for the molecular diagnosis, Andrés HolguínRocha for the taxonomic classification of the ticks; and Tatiana Lopera and Cesar Segura for their participation in the technical and academic meetings. Thanks are due to the Demography and Health Group of the National Faculty of Public Health (University of Antioquia), Colombia especially Professor Hugo Grisales for his contribution to the sampling design. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. Adl SM, Simpson AG, Farmer MA, Andersen RA, Anderson OR, Barta JR, et al. The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. 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