Geographical Distribution of Trypanosoma cruzi Genotypes in Venezuela Herna ´ n J. Carrasco 1 *, Maikell Segovia 1 , Martin S. Llewellyn 2 , Antonio Morocoima 3 , Servio Urdaneta-Morales 4 , Cinda Martı´nez 5 , Clara E. Martı´nez 1 , Carlos Garcia 1 , Marlenes Rodrı ´guez 1 , Raul Espinosa 6 , Belkisyole ´ A. de Noya 7 , Zoraida Dı´az-Bello 7 , Leidi Herrera 4 , Sinead Fitzpatrick 2 , Matthew Yeo 2 , Michael A. Miles 2 , M. Dora Feliciangeli 8 1 Laboratorio de Biologı ´a Molecular de Protozoarios, Instituto de Medicina Tropical, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela, 2 Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom, 3 Centro de Medicina Tropical, Facultad de Medicina, Universidad de Oriente, Puerto la Cruz, Venezuela, 4 Laboratorio de Biologı ´a de Vectores y Para ´sitos, Instituto de Zoologı ´a y Ecologı ´a Tropical, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela, 5 Direccio ´ n General de Salud Ambiental, Ministerio del Poder Popular para la Salud, Maracay, Venezuela, 6 Hospital Miguel Pe ´rez Carren ˜ o, Instituto Venezolano de los Seguros Sociales, Caracas, Venezuela, 7 Seccio ´ n de Inmunologı ´a, Instituto de Medicina Tropical, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela, 8 BIOMED, Universidad de Carabobo, Maracay, Venezuela Abstract Chagas disease is an endemic zoonosis native to the Americas and is caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The parasite is also highly genetically diverse, with six discrete typing units (DTUs) reported TcI – TcVI. These DTUs broadly correlate with several epidemiogical, ecological and pathological features of Chagas disease. In this manuscript we report the most comprehensive evaluation to date of the genetic diversity of T. cruzi in Venezuela. The dataset includes 778 samples collected and genotyped over the last twelve years from multiple hosts and vectors, including nine wild and domestic mammalian host species, and seven species of triatomine bug, as well as from human sources. Most isolates (732) can be assigned to the TcI clade (94.1%); 24 to the TcIV group (3.1%) and 22 to TcIII (2.8%). Importantly, among the 95 isolates genotyped from human disease cases, 79% belonged to TcI - a DTU common in the Americas, however, 21% belonged to TcIV- a little known genotype previously thought to be rare in humans. Furthermore, were able to assign multiple oral Chagas diseases cases to TcI in the area around the capital, Caracas. We discuss our findings in the context of T. cruzi DTU distributions elsewhere in the Americas, and evaluate the impact they have on the future of Chagas disease control in Venezuela. Citation: Carrasco HJ, Segovia M, Llewellyn MS, Morocoima A, Urdaneta-Morales S, et al. (2012) Geographical Distribution of Trypanosoma cruzi Genotypes in Venezuela. PLoS Negl Trop Dis 6(6): e1707. doi:10.1371/journal.pntd.0001707 Editor: Philippe Bu ¨ scher, Institute of Tropical Medicine, Belgium Received March 9, 2012; Accepted May 10, 2012; Published June 26, 2012 Copyright: ß 2012 Carrasco et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by FONACIT G-2005000827; FONACIT S1-98000388; European Union Seventh Framework grant number 223034; and the Wellcome Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]Introduction Trypanosoma cruzi, the etiological agent of Chagas disease, infects approximately 8 million people in Latin America [1]. A further 20 million people are at risk of infection. Chagas disease is widely dispersed across 21 countries in the Americas, with a natural distribution (in wild transmission cycles) from the Southern States of the USA [2] to Central Argentina [3]. Chagas disease is a vector-borne zoonosis, and transmission is generally achieved via the infected faeces of various triatomine bug species, evacuated during a blood meal. Infection is maintained in wild transmission cycles by numerous mamma- lian reservoir hosts, especially opossums (Didelphis sp.) and armadillos (Dasypus sp.) [4]. Human infection occurs at foci throughout the natural distribution of T. cruzi where triatomines have adapted to exploit the domestic setting, but also orally (via ingestion of triatomine contaminated foodstuffs) in endemic countries, as well as via blood transfusion, organ transplantation and congenital infection in and outside of areas of traditional endemicity [1]. T. cruzi is likely to be ancient and indigenous to the Americas [5,6]. Indeed, the parasite demonstrates considerable genetic diversity as initially revealed by multilocus enzyme electrophoresis (MLEE) [7–9]. These early studies supported the typing of the T. cruzi into three main groups or zymodemes, called Z1, Z2 and Z3. The implementation of further molecular techniques in combina- tion with MLEE, allow the division of the T. cruzi species in six groups or discrete typing units (DTU), denoted TcI, TcIIa, TcIIb, TcIIc, TcIId and TcIIe [10]. More recently, in a meeting of experts held in Brazil [11], a new nomenclature was recom- mended for the intraspecific classification of T. cruzi discrete typing units (DTUs) into TcI, TcII, TcIII, TcIV, TcV and TcVI. However, while the T. cruzi DTUs are relatively genetically stable in space and time, their evolutionary, ecological and epidemiolgi- cal significance is far from clear [4,12]. Some limited patterns emerge (reviewed in [13]), TcII, TcV and TcVI seem largely restricted to domestic transmission cycles south of the Amazon basin, where they cause considerable human disease. TcIII is infrequent from domestic sources, strongly associated with Dasypus novemcinctus in terrestrial transmission cycles, and found throughout www.plosntds.org 1 June 2012 | Volume 6 | Issue 6 | e1707
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Geographical Distribution of Trypanosoma cruziGenotypes in VenezuelaHernan J. Carrasco1*, Maikell Segovia1, Martin S. Llewellyn2, Antonio Morocoima3,
Servio Urdaneta-Morales4, Cinda Martınez5, Clara E. Martınez1, Carlos Garcia1,
Marlenes Rodrıguez1, Raul Espinosa6, Belkisyole A. de Noya7, Zoraida Dıaz-Bello7, Leidi Herrera4,
Sinead Fitzpatrick2, Matthew Yeo2, Michael A. Miles2, M. Dora Feliciangeli8
1 Laboratorio de Biologıa Molecular de Protozoarios, Instituto de Medicina Tropical, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela,
2 Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom,
3 Centro de Medicina Tropical, Facultad de Medicina, Universidad de Oriente, Puerto la Cruz, Venezuela, 4 Laboratorio de Biologıa de Vectores y Parasitos, Instituto de
Zoologıa y Ecologıa Tropical, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela, 5 Direccion General de Salud Ambiental, Ministerio del Poder
Popular para la Salud, Maracay, Venezuela, 6 Hospital Miguel Perez Carreno, Instituto Venezolano de los Seguros Sociales, Caracas, Venezuela, 7 Seccion de Inmunologıa,
Instituto de Medicina Tropical, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela, 8 BIOMED, Universidad de Carabobo, Maracay, Venezuela
Abstract
Chagas disease is an endemic zoonosis native to the Americas and is caused by the kinetoplastid protozoan parasiteTrypanosoma cruzi. The parasite is also highly genetically diverse, with six discrete typing units (DTUs) reported TcI – TcVI.These DTUs broadly correlate with several epidemiogical, ecological and pathological features of Chagas disease. In thismanuscript we report the most comprehensive evaluation to date of the genetic diversity of T. cruzi in Venezuela. Thedataset includes 778 samples collected and genotyped over the last twelve years from multiple hosts and vectors, includingnine wild and domestic mammalian host species, and seven species of triatomine bug, as well as from human sources. Mostisolates (732) can be assigned to the TcI clade (94.1%); 24 to the TcIV group (3.1%) and 22 to TcIII (2.8%). Importantly, amongthe 95 isolates genotyped from human disease cases, 79% belonged to TcI - a DTU common in the Americas, however, 21%belonged to TcIV- a little known genotype previously thought to be rare in humans. Furthermore, were able to assignmultiple oral Chagas diseases cases to TcI in the area around the capital, Caracas. We discuss our findings in the context of T.cruzi DTU distributions elsewhere in the Americas, and evaluate the impact they have on the future of Chagas diseasecontrol in Venezuela.
Citation: Carrasco HJ, Segovia M, Llewellyn MS, Morocoima A, Urdaneta-Morales S, et al. (2012) Geographical Distribution of Trypanosoma cruzi Genotypes inVenezuela. PLoS Negl Trop Dis 6(6): e1707. doi:10.1371/journal.pntd.0001707
Editor: Philippe Buscher, Institute of Tropical Medicine, Belgium
Received March 9, 2012; Accepted May 10, 2012; Published June 26, 2012
Copyright: � 2012 Carrasco et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by FONACIT G-2005000827; FONACIT S1-98000388; European Union Seventh Framework grant number 223034; and theWellcome Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Parasite isolationT. cruzi isolates were obtained from chagasic outpatients from
different geographical areas of Venezuela attending the Instituto
de Medicina Tropical (IMT) of the Universidad Central de
Venezuela (UCV) as well as chagasic patients living in rural areas
of Venezuela where Chagas disease is endemic. Another group of
patients were from urban areas of Caracas, the Capital city, and
the neighbor State Vargas (see Table 1), all of them in the acute
phase of the disease, presumably infected via oral transmission. All
patient isolates were collected under informed consent following
the ethical permissions of the Research Ethics Commission of the
Institute of Tropical Medicine, Faculty of Medicine, Universidad
Central de Venezuela. The second group of T. cruzi isolates was
obtained from seven different species of triatomine bug and
originated from insects brought to the IMT by members of the
public and those obtained during fieldwork. Triatomines were
identified to species level according to Lent and Wygodzinksy,
1979 [23] and in some case via molecular methods (as part of
Fitzpatrick et al., 2008 [24]) as detailed in Table 1. A third group of
T. cruzi isolates was found infecting nine species of wild and
domestic mammal (see Table 2), captured during multiple field
expeditions to endemic and urban regions.
Parasite culture and DNA isolationParasites were isolated via several different techniques. Briefly:
parasites from chagasic patients were obtained by indirect
xenodiagnosis, by hemoculture of peripheral blood, or by i.p.
inoculation of Balb/c mice with peripheral blood. From wild and
domestic mammals, parasites were isolated by direct xenodiagno-
sis, by hemoculture from peripheral or cardiac blood, or by i.p.
inoculation of Balb/c mice with cardiac blood. From triatomine
bugs, naturally infected or used in the xenodiagnosis, the parasites
were isolated by direct culture of feces in blood agar or by i.p.
inoculation of Balb/c mice with bug faeces. To achieve
xenodiagnosis, we used 12 to 15 instar nymphs of Rhodnius prolixus,
3rd or 4th stage, reared in the laboratory. Initially the parasites
were grown in biphasic medium blood-agar followed by culture in
Author Summary
Chagas disease is caused by a protozoan parasite calledTrypanosoma cruzi. T. cruzi infects a wide variety ofmammal species in Latin America as well as man, and isspread by multiple species of blood sucking triatomineinsect vectors. The presence of genetic diversity in T. cruziin the Americas is well established, with six different majorgenetic types in circulation. The genetic diversity of T. cruziin Venezuela is relatively poorly understood. In this workwe present the results from the genotyping of over sevenhundred isolates from 17 of the 24 states. Our datasetcomprises strains isolated from wild and domestic animals,several species of triatomine vector, as well as from humanChagas disease cases, including those associated with oraltransmission of T. cruzi. Amongst other findings, our datareveal a surprisingly high frequency of atypical genotypesin humans, particularly TcIV, which has rarely beenreported. We evaluate our findings in the context of T.cruzi diversity elsewhere in the Americas, and assess theimpact they have on the future of Chagas disease controlin Venezuela.
Figure 4. Distribution of Trypanosoma cruzi Discrete Typing Units (DTUs) in Venezuela. The three maps display samples collected fromhumans (top left), vectors (top right), and mammal reservoirs (bottom left). Pie chart area is proportional to sampling size. Pie segment colourrepresents DTU identity. Inset on each map shows the capital (Caracas) and surrounding states in greater detail.doi:10.1371/journal.pntd.0001707.g004
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