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    Geographical distribution and location of the municipalities and dwellings in study area (Aguazul and Mani, Casanare, Colombia).

  • 1.

    World Health Organization, 2015. Chagas disease in Latin America: an epidemiological update based on 2010 estimates. Wkly Epidemiol Rec 6: 3344.

    • Search Google Scholar
    • Export Citation
  • 2.

    Dias JCP, Silveira AC, Schofield CJ, 2002. The impact of Chagas disease control in Latin America: a review. Mem Inst Oswaldo Cruz 97: 603612.

    • Search Google Scholar
    • Export Citation
  • 3.

    Abad-Franch F, Monteiro F, 2007. Biogeography and evolution of Amazonian triatomines (Heteroptera: Reduviidae): implications for Chagas disease surveillance in humid forest ecoregions. Mem Inst Oswaldo Cruz 102: 5769.

    • Search Google Scholar
    • Export Citation
  • 4.

    Corredor A, Santacruz M, Páez S, Guatame LA, 1990. Distribución de los triatominos domiciliarios en Colombia. Bogotá, Colombia: Ministerio de Salud, Instituto Nacional de Salud, 44.

    • Search Google Scholar
    • Export Citation
  • 5.

    Sandoval CM, Gutiérrez R, Luna S, Amaya M, Esteban L, Ariza H, Angulo VM, 2000. High density of Rhodnius prolixus in rural house in Colombia. Trans R Soc Trop Med Hyg 94: 372373.

    • Search Google Scholar
    • Export Citation
  • 6.

    Angulo VM, Esteban L, Luna KP, 2012. Attalea butyracea próximas a las viviendas como posible fuente de infestación domiciliaria por Rhodnius prolixus (Hemíptera: Reduviidae) en los Llanos Orientales de Colombia. Biomedica 32: 277285.

    • Search Google Scholar
    • Export Citation
  • 7.

    Moncayo A, 2003. Chagas disease: current epidemiological trends after the interruption of vectorial and transfusional transmission in the Southern Cone countries. Mem Inst Oswaldo Cruz 98: 577591.

    • Search Google Scholar
    • Export Citation
  • 8.

    Farfán-García AE, Castellanos-Domínguez YZ, Luna-Marín KP, Angulo-Silva VM, 2013. Concordance of two serological tests for the diagnosis of Chagas disease. Rev Salud Publica (Bogota) 15: 208219.

    • Search Google Scholar
    • Export Citation
  • 9.

    Wisnivesky-Colli C, Paulone I, Pérez A, Chuit R, Gualtieri JM, Solarz N, Smith A, Segura EL, 1987. A new tool for continuous detection of the presence of triatomine bugs, vectors of Chagas' disease, in rural households. Medicina (B Aires) 47: 4550.

    • Search Google Scholar
    • Export Citation
  • 10.

    Angulo-Silva VM, Esteban L, Urbano P, Hincapié E, Núñez LA, 2013. Comparación de métodos para la captura de triatomínos (Hemíptera: Reduviidae) en palmas Attalea butyracea en los Llanos Orientales de Colombia. Biomedica 33: 653659.

    • Search Google Scholar
    • Export Citation
  • 11.

    Farfán AE, Gutiérrez R, Angulo VM, 2007. ELISA para la identificación de los patrones alimentarios de Triatominae en Colombia. Rev Salud Publica (Bogota) 9: 602608.

    • Search Google Scholar
    • Export Citation
  • 12.

    Fernandes O, Souto RP, Castro JA, Pereira JB, Fernandes NC, Junqueira AC, Naiff RD, Barrett TV, Degrave W, Zingales B, Campbell DA, Coura JR, 1998. Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences. Am J Trop Med Hyg 58: 807811.

    • Search Google Scholar
    • Export Citation
  • 13.

    Fitzpatrick S, Feliciangeli MD, Sanchez-Martin MJ, Monteiro FA, Miles MA, 2008. Molecular genetics reveal that silvatic Rhodnius prolixus do colonise rural houses. PLoS Negl Trop Dis 2: e210.

    • Search Google Scholar
    • Export Citation
  • 14.

    Sanchez-Martin MJ, Feliciangelli MD, Campbell-Lendrun D, Davies RC, 2006. Could the Chagas disease elimination programme in Venezuela be compromised by reinvasion of houses by sylvatic Rhodnius prolixus bug populations? Trop Med Int Health 11: 15851593.

    • Search Google Scholar
    • Export Citation
  • 15.

    Salazar-Gutiérrez FR, Trujillo-Guiza ML, Escobar-Martinez MC, 2013. Prevalence of Trypanosoma cruzi infection among people aged 15 to 89 years inhabiting the department of Casanare (Colombia). PLoS Negl Trop Dis 7: e2113.

    • Search Google Scholar
    • Export Citation
  • 16.

    Feliciangeli MD, Sanchez-Martin MJ, Suárez B, Marrero R, Torrellas A, Bravo A, Medina M, Martínez C, Hernández M, Duque N, Toyo J, Angel R, 2007. Risk factors for Trypanosoma cruzi infection in Barinas State, Venezuela. Am J Trop Med Hyg 76: 915921.

    • Search Google Scholar
    • Export Citation
  • 17.

    Crisante G, Rojas A, Teixeira MGM, Añez N, 2006. Infected dogs as a risk factor in the transmission of human Trypanosoma cruzi infection in western Venezuela. Acta Trop 98: 247254.

    • Search Google Scholar
    • Export Citation
  • 18.

    Pineda V, Saldava A, Monfante I, Santamaría A, Gottdenker NL, Yabley MJ, Rapoport G, Calzada JE, 2011. Prevalence of trypanosome infection in dogs from Chagas disease endemic regions in Panama, Central America. Vet Parasitol 178: 360363.

    • Search Google Scholar
    • Export Citation
  • 19.

    Turriago BC, Vallejo G, Guhl F, 2008. Seroprevalencia de Trypanosoma cruzi en perros de dos áreas endémicas en Colombia. Rev Fac Med 16: 1118.

    • Search Google Scholar
    • Export Citation
  • 20.

    Ramírez JD, Turriago B, Tapia-Calle G, Guhl F, 2013. Understanding the role of dogs (Canis lupus familiaris) in the transmission dynamics of Trypanosoma cruzi genotypes in Colombia. Vet Parasitol 196: 216219.

    • Search Google Scholar
    • Export Citation
  • 21.

    Guhl F, Ramírez JD, 2013. Retrospective molecular integrated epidemiology of Chagas disease in Colombia. Infect Genet Evol 20: 148154.

 

 

 

 

 

Human Trypanosomiasis in the Eastern Plains of Colombia: New Transmission Scenario

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  • Centro de Investigaciones en Enfermedades Tropicales, Universidad Industrial de Santander, Santander, Colombia

Characteristics of Trypanosoma cruzi infection were studied in a rural area of the eastern plains of Colombia. Using enzyme-linked immunosorbent assay and indirect fluorescent-antibody tests, the infection was determined in 11.6% of the inhabitants of 142 dwellings. During 6 months of community surveillance, in 42.3% dwellings, 609 triatomines were collected (597 Rhodnius prolixus and seven, three, one, and one of Panstrongylus geniculatus, Psammolestes arturi, Eratyrus mucronatus, and Triatoma maculata, respectively). Rhodnius prolixus was found in 80% peridomiciliary Attalea butyracea palms examined with baited traps, and its infection with T. cruzi was 30% and 38.5% in dwellings and palms, respectively. Trypanosoma cruzi was isolated in five of 35 triatomines and in one of 24 dogs. The blood of domestic and wild animals was identified in triatomines collected in the intradomicile and in palms. These results support the extension of the wild cycle of T. cruzi to human dwellings and the characterization of a new scenario for transmission in Colombia.

Trypanosoma cruzi infection continues to affect nearly 6 million people in 21 countries of the Americas1 despite the success of control programs against domestic triatomines.2 There are zones where dwellings are infested by other species of Triatominae with peridomiciliary and wild ecotopes. In the Amazon, some of the more than 27 wild species described, principally of the genus Rhodnius, have made incursions into dwellings, with the risk of infecting human populations.3

In Colombia, the Andean region in the eastern zone below 2,000 m above sea level has been considered endemic for Chagas disease because of the high degree of domiciliary infestation, wide dispersion, and the high densities of Rhodnius prolixus.4,5 A decade after the adoption of a government program to control domiciliary triatomines, in our observation, this species has been controlled. However, another transmission scenario has begun to be known with checking of the wild cycle in peridomiciliary palms as a transmission risk factor in the departments of the eastern plains6 and the reporting of high prevalence rates of human infection with T. cruzi.7 This study was conducted for the purpose of clarifying the epidemiological characteristics of this scenario in this region.

In December 2008, the prevalence of T. cruzi infection was evaluated in the inhabitants of 142 dwellings in the department of Casanare, dispersed in rural areas of the municipalities of Maní (04°49′02″N/72°17′19″W) and Aguazul (05°10′23″N/72°33′17″W), which were fumigated by the program of Vector-Transmitted Diseases (Enfermedades Transmitidas por Vectores [ETV], in Spanish) in that department. This territory corresponds to the alluvial and eolic plains of the Orinoquía, composed of flooded savannas with herbaceous and graminaceous vegetation in the humid warm thermal floor, with an average temperature of 25–27°C and an annual rainfall of 2,400–2,600 mm (Figure 1).

Figure 1.
Figure 1.

Geographical distribution and location of the municipalities and dwellings in study area (Aguazul and Mani, Casanare, Colombia).

Citation: The American Society of Tropical Medicine and Hygiene 94, 2; 10.4269/ajtmh.15-0406

The dwellings are constructed with brick walls, cement, tile or earth floors, and zinc tile roofs, with open spaces between the roof and the walls and no protection against the entrance of insects or other animals. Their peridomiciliary surroundings are not clearly demarcated and have rustic constructions for the shelter of domestic animals, small areas of subsistence crops, pastures with scattered palms, and also pockets of forests, mainly “Royal Palm” (Attalea butyracea), where domesticated and wild animals roam freely.

In visits to their homes, information about Chagas disease, as well as the purpose of the study, was offered to raise awareness for their participation. Those who agreed to participate were asked to provide written informed consent.

Of 492 inhabitants, 50% were women and 50% were men, with a mean age of 28.5 years and a range from 0 to 88 years. A blood sample was taken from adults, or minors authorized by their parents, on Whatman No. 3 filter paper (Sigma-Aldrich, St. Louis, MO) to determine T. cruzi infection by means of the tests enzyme-linked immunosorbent assay (ELISA) and immuno fluorescent-assay (IFA), using the filter paper technique.8

During 6 months in the dry season, the inhabitants, ETV program technicians, and researchers conducted entomological surveillance of domiciliary infestation by triatomines using three methods: community surveillance, actively searching (hours/man) without vacating, and “Maria” biosensors.9 In dwellings with palms located within 100 m of the peridomicile, individuals of A. butyracea and Cocus nucífera were examined using live bait traps6,9; the degree of infestation and colonization and the clustering (number of collected triatomines/number of palms or dwellings infested) and density (number of collected triatomines/number of palms or dwellings examined) in the two habitats (dwellings and palms) were established.10 The intestinal content of the triatomines collected alive was used for identifying their food sources by ELISA technique11 and determining the natural infection by direct examination and polymerase chain reaction (PCR). Using primers S35 and S36,6 a 330-bp fragment corresponding to the kinetoplast minicircle DNA sequence specific to T. cruzi was amplified. Trypanosoma cruzi in triatomines and domestic mammals was isolated and classified as T. cruzi, T. cruzi I, or T. cruzi II using the TC/TC1/TC2 primers for the intergenic spacer of mini-exon genes.12

The study was conducted within the framework of a research project approved by the ethics committee of the Industrial University of Santander.

Serological tests revealed infection in 57 of 492 (11.6%) inhabitants, 33 men and 24 women, and a range between 7 and 76 years, including four children under 15 years of age. In 60 of 142 dwellings, 609 triatomines (597 specimens of R. prolixus and seven, three, one, and one adult Panstrongylus geniculatus, Psammolestes arturi, Eratyrus mucronatus, and Triatoma maculata, respectively) were collected by community surveillance, resulting in a degree of infestation of 42.3%, a degree of colonization of 1.7%, a clustering of 10.9 triatomines per infested dwelling, and a density of 4.3 triatomines per dwelling.

In 24 of 28 A. butyracea examined, 336 specimens of R. prolixus were collected, and there were none in two C. nucífera individuals examined, resulting in a degree of infestation of 80% and colonization of 75% and a density of 11.2 and a clustering of 14.

The presence of flagellates was observed by the direct examination of feces in 20 of 85 triatomines collected in dwellings (23.5%), in 56 of 170 collected in palms (33%), and in two of 24 (8.3%) triatomines used for xenodiagnosis in dogs. Infection with T. cruzi was determined by PCR in nine of 30 triatomines collected from dwellings (30%) and in 10 of 26 triatomines collected in palms (38.5%).

Trypanosoma cruzi was isolated in five of 35 triatomines, three collected in dwellings and two in palms, and in one of 24 dogs. All were determined to be T. cruzi I. None were isolated in any of 11 cats, three pigs, one horse, and one cow on which xenodiagnoses was performed. Domestic animals and wild animals were identified as the blood food source in six of 34 triatomines collected in the intradomicile and in 31 of 224 triatomines collected in A. butyracea (Table 1).

Table 1

Number of individuals according to place of collection, natural infection, and food sources of Rhodnius prolixus

Place of collectionAdultsNymphsTotal% Infection (PCR)Food source identified (no. of triatomines) (n = 224)
FemalesMalesIIIIIIIVV
Dwellings4021940000159730.0Chicken (1)
Sheep (1)
Iguana (3)
Primate (1)
Peridomiciliary palms (Attalea butyracea)5738154656626233638.5Chicken (11)
Sheep (6)
Equine (5)
Iguana (3)
Bat (4)
Rat (1)
Iguana and chicken (1)

PCR = polymerase chain reaction.

The presence of residents infected with T. cruzi, which included children, indicates the active transmission of the parasite to the human population in the Colombian Orinoquía, where no intradomiciliary triatomine colonies were found. The elevated domiciliary infestation by adult R. prolixus insects in recently fumigated dwellings, a large proportion of which were infected with T. cruzi, can explain this transmission and reveal the ineffectiveness of fumigation for controlling intrusions by wild triatomines. The low physical protection of the dwelling and the discovery of infestation by large colonies of R. prolixus in the majority of the peridomiciliary A. butyracea examined suggest that these colonies are the source of the domiciliary infestation.

The identification of the blood of domestic animals in the intestinal contents of the triatomines collected in palms and the blood of wild animals in the adult insects collected in the intradomicile indicates a constant flow of the populations of these insects from the palms toward the domiciles and the return of these to their wild habitats. This pattern may signify the extension of the wild transmission cycle to the human environment, a situation that highlights the contact risk of its inhabitants with these populations of infected triatomines, as was suggested earlier for this region.6 This flow of triatomines from wild ecotopes to dwellings has been previously documented in Colombia (K. P. Luna, Thesis Dissertation, Universidad Industrial de Santander, 2009) and Venezuela.13,14

The infection of the human inhabitants with T. cruzi in this study agrees with that found in school-age children, in pregnant women, and in the general population in rural areas of this region,7,15 and it is similar to that found in the state of Barinas in Venezuela's Orinoquía.16

The observation of T. cruzi in 8.3% of the dogs examined, although much lower than the prevalence found in Venezuela and Panama,17,18 is similar to that found in dogs inhabiting dwellings with domiciliary triatomines in endemic areas of the Andes zone in Colombia.19,20 Trypanosoma cruzi I isolated in triatomines and mammals in this study is the discrete typing unit most frequently found in Colombia and in R. prolixus in this region and is associated with severe cardiopathy in Colombia and Argentina.21

The presence of infected people, the high domiciliary infestation by permanent intrusion of infected adult specimens of R. prolixus from highly infested peridomiciliary palms, and the identification of domestic and wild hosts as a common food source in palm and intradomiciliary triatomines and in infected dogs in the dwellings support the extension of the wild cycle of T. cruzi to human domiciles.

These findings are characterizing a new scenario of T. cruzi transmission to the residents in this region that is unlike what traditionally has been known in the Colombian Andes zone, where there are high densities and wide dispersion of mainly domestic R. prolixus.

The increasing emergence of human settlements by population migration to a territory recently incorporated into petroleum exploitation and the agroindustrial production of African palms as well as the inefficiency of traditional chemical measures for controlling the infestation by R. prolixus from palm species that are part of the natural landscape of the region require the implementation of a monitoring program for the detection and prompt treatment of acute infections.

Similarly, it is necessary to implement actions for health education and monitoring of the infestation and testing of new alternatives for the control of the permanent intrusion of triatomines and wild mammals to avoid the risk of vectorial transmission and oral transmission by food contamination.

  • 1.

    World Health Organization, 2015. Chagas disease in Latin America: an epidemiological update based on 2010 estimates. Wkly Epidemiol Rec 6: 3344.

    • Search Google Scholar
    • Export Citation
  • 2.

    Dias JCP, Silveira AC, Schofield CJ, 2002. The impact of Chagas disease control in Latin America: a review. Mem Inst Oswaldo Cruz 97: 603612.

    • Search Google Scholar
    • Export Citation
  • 3.

    Abad-Franch F, Monteiro F, 2007. Biogeography and evolution of Amazonian triatomines (Heteroptera: Reduviidae): implications for Chagas disease surveillance in humid forest ecoregions. Mem Inst Oswaldo Cruz 102: 5769.

    • Search Google Scholar
    • Export Citation
  • 4.

    Corredor A, Santacruz M, Páez S, Guatame LA, 1990. Distribución de los triatominos domiciliarios en Colombia. Bogotá, Colombia: Ministerio de Salud, Instituto Nacional de Salud, 44.

    • Search Google Scholar
    • Export Citation
  • 5.

    Sandoval CM, Gutiérrez R, Luna S, Amaya M, Esteban L, Ariza H, Angulo VM, 2000. High density of Rhodnius prolixus in rural house in Colombia. Trans R Soc Trop Med Hyg 94: 372373.

    • Search Google Scholar
    • Export Citation
  • 6.

    Angulo VM, Esteban L, Luna KP, 2012. Attalea butyracea próximas a las viviendas como posible fuente de infestación domiciliaria por Rhodnius prolixus (Hemíptera: Reduviidae) en los Llanos Orientales de Colombia. Biomedica 32: 277285.

    • Search Google Scholar
    • Export Citation
  • 7.

    Moncayo A, 2003. Chagas disease: current epidemiological trends after the interruption of vectorial and transfusional transmission in the Southern Cone countries. Mem Inst Oswaldo Cruz 98: 577591.

    • Search Google Scholar
    • Export Citation
  • 8.

    Farfán-García AE, Castellanos-Domínguez YZ, Luna-Marín KP, Angulo-Silva VM, 2013. Concordance of two serological tests for the diagnosis of Chagas disease. Rev Salud Publica (Bogota) 15: 208219.

    • Search Google Scholar
    • Export Citation
  • 9.

    Wisnivesky-Colli C, Paulone I, Pérez A, Chuit R, Gualtieri JM, Solarz N, Smith A, Segura EL, 1987. A new tool for continuous detection of the presence of triatomine bugs, vectors of Chagas' disease, in rural households. Medicina (B Aires) 47: 4550.

    • Search Google Scholar
    • Export Citation
  • 10.

    Angulo-Silva VM, Esteban L, Urbano P, Hincapié E, Núñez LA, 2013. Comparación de métodos para la captura de triatomínos (Hemíptera: Reduviidae) en palmas Attalea butyracea en los Llanos Orientales de Colombia. Biomedica 33: 653659.

    • Search Google Scholar
    • Export Citation
  • 11.

    Farfán AE, Gutiérrez R, Angulo VM, 2007. ELISA para la identificación de los patrones alimentarios de Triatominae en Colombia. Rev Salud Publica (Bogota) 9: 602608.

    • Search Google Scholar
    • Export Citation
  • 12.

    Fernandes O, Souto RP, Castro JA, Pereira JB, Fernandes NC, Junqueira AC, Naiff RD, Barrett TV, Degrave W, Zingales B, Campbell DA, Coura JR, 1998. Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences. Am J Trop Med Hyg 58: 807811.

    • Search Google Scholar
    • Export Citation
  • 13.

    Fitzpatrick S, Feliciangeli MD, Sanchez-Martin MJ, Monteiro FA, Miles MA, 2008. Molecular genetics reveal that silvatic Rhodnius prolixus do colonise rural houses. PLoS Negl Trop Dis 2: e210.

    • Search Google Scholar
    • Export Citation
  • 14.

    Sanchez-Martin MJ, Feliciangelli MD, Campbell-Lendrun D, Davies RC, 2006. Could the Chagas disease elimination programme in Venezuela be compromised by reinvasion of houses by sylvatic Rhodnius prolixus bug populations? Trop Med Int Health 11: 15851593.

    • Search Google Scholar
    • Export Citation
  • 15.

    Salazar-Gutiérrez FR, Trujillo-Guiza ML, Escobar-Martinez MC, 2013. Prevalence of Trypanosoma cruzi infection among people aged 15 to 89 years inhabiting the department of Casanare (Colombia). PLoS Negl Trop Dis 7: e2113.

    • Search Google Scholar
    • Export Citation
  • 16.

    Feliciangeli MD, Sanchez-Martin MJ, Suárez B, Marrero R, Torrellas A, Bravo A, Medina M, Martínez C, Hernández M, Duque N, Toyo J, Angel R, 2007. Risk factors for Trypanosoma cruzi infection in Barinas State, Venezuela. Am J Trop Med Hyg 76: 915921.

    • Search Google Scholar
    • Export Citation
  • 17.

    Crisante G, Rojas A, Teixeira MGM, Añez N, 2006. Infected dogs as a risk factor in the transmission of human Trypanosoma cruzi infection in western Venezuela. Acta Trop 98: 247254.

    • Search Google Scholar
    • Export Citation
  • 18.

    Pineda V, Saldava A, Monfante I, Santamaría A, Gottdenker NL, Yabley MJ, Rapoport G, Calzada JE, 2011. Prevalence of trypanosome infection in dogs from Chagas disease endemic regions in Panama, Central America. Vet Parasitol 178: 360363.

    • Search Google Scholar
    • Export Citation
  • 19.

    Turriago BC, Vallejo G, Guhl F, 2008. Seroprevalencia de Trypanosoma cruzi en perros de dos áreas endémicas en Colombia. Rev Fac Med 16: 1118.

    • Search Google Scholar
    • Export Citation
  • 20.

    Ramírez JD, Turriago B, Tapia-Calle G, Guhl F, 2013. Understanding the role of dogs (Canis lupus familiaris) in the transmission dynamics of Trypanosoma cruzi genotypes in Colombia. Vet Parasitol 196: 216219.

    • Search Google Scholar
    • Export Citation
  • 21.

    Guhl F, Ramírez JD, 2013. Retrospective molecular integrated epidemiology of Chagas disease in Colombia. Infect Genet Evol 20: 148154.

Author Notes

* Address correspondence to Victor Manuel Angulo-Silva, Centro de Investigaciones en Enfermedades Tropicales, Universidad Industrial de Santander, Parque Tecnológico de Guatiguará, Sede UIS Guatiguará, Km 2 Vía Refugio, Piedecuesta, Santander, Colombia 681011. E-mail: pitorio@hotmail.com

Financial support: This article was developed by the research group of the Center for Research in Tropical Diseases (Centro de Investigaciones en Enfermedades Tropicales, CINTROP, in Spanish) of the Industrial University of Santander (Universidad Industrial de Santander, in Spanish), in collaboration with the community of the study region and funded by the National Administrative Department of Science and Technology of Colombia (Departamento Administrativo Nacional de Ciencia y Tecnología de Colombia, COLCIENCIAS, in Spanish) (RC: 288-2007), the Industrial University of Santander, the University of the American Tropics (Universitaria del Trópico Americano, UNITROPICO, in Spanish), and the program of Vector-Transmitted Diseases of the Health Secretary of the department of Casanare. Support for publication was received from the Financing Contract, RC-380 2011, entered into between COLCIENCIAS and the Temporary Union National Research Program for the prevention, control, and comprehensive treatment of Chagas disease in Colombia (Unión Temporal Programa Nacional de Investigación para la Prevención, Control y Tratamiento Integral de la Enfermedad de Chagas en Colombia, in Spanish).

Authors' addresses: Victor Manuel Angulo-Silva, Yeny Zulay Castellanos-Domínguez, Mónica Flórez-Martínez, Lyda Esteban-Adarme, William Pérez-Mancipe, Ana Elvira Farfán-García, and Katherine Paola Luna-Marín, Center for Research in Tropical Diseases, Industrial University of Santander, Piedecuesta, Santander, Colombia, E-mails: pitorio@hotmail.com, yenyzu1@hotmail.com, mflorezm@gmail.com, lestebanar@hotmail.com, williamperezuis@yahoo.com, anaelvira05@gmail.com, and plunamar23@gmail.com.

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