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Table of Contents
RESEARCH ARTICLE
Year : 2019  |  Volume : 56  |  Issue : 3  |  Page : 252-262

Molecular characterization of Trypanosoma cruzi and Leishmania spp. coinfection in mammals of Venezuelan coendemic areas


1 Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso” (BIOMED); Departamento de Clinico Integral, Facultad de Ciencias de la Salud, Universidad de Carabobo Sede Aragua, Maracay, Venezuela
2 Instituto de Zoología y Ecología Tropical (IZET), Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas, Venezuela
3 Centro de Investigaciones en Enfermedades Tropicales (CIET-UC), Facultad de Ciencias de la Salud, Universidad de Carabobo, San Carlos, Cojedes, Venezuela
4 Centro de Medicina Tropical de Oriente, Universidad de Oriente (UDO) Núcleo Anzoátegui, Barcelona, estado Anzoátegui, Venezuela
5 Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso” (BIOMED), Maracay, Venezuela
6 Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso” (BIOMED); Centro Nacional de Referencia de Flebótomos, BIOMED, Facultad de Ciencias de la Salud, Universidad de Carabobo, Maracay, Venezuela
7 Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso” (BIOMED); Departamento de Parasitología, Facultad de Ciencias de la Salud, Universidad de Carabobo Sede Aragua, Maracay, estado Aragua, Venezuela

Date of Submission28-Feb-2019
Date of Acceptance15-May-2019
Date of Web Publication09-Jul-2020

Correspondence Address:
Dr E Ferrer
Instituto de Investigaciones Biomédicas “Dr Francisco J. Triana Alonso” (BIOMED), Universidad de Carabobo Sede Aragua, Maracay
Venezuela
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.289394

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  Abstract 

Background & objectives: Trypanosoma cruzi and Leishmania spp. are protozoans that cause American trypanosomiasis and leishmaniasis, respectively. In endemic foci where both diseases coincide, coinfection can occur. The objective of this work was the characterization of the parasites involved in coinfection in several endemic areas of Venezuela.
Methods: Molecular characterization was done in 30 samples of several species of mammals (Didelphis marsupialis, Equus mulus, Rattus rattus, Canis familiaris, Felis catus, and Sciurus granatensis) from the states of Anzoategui, Cojedes and Capital District diagnosed with T. cruzi and Leishmania spp. coinfections. For the typing of T. cruzi DTUs, the markers of miniexon, 24Sa rDNA, 18Sa rDNA, and hsp60-PCR-RFLP (EcoRV) were used. Infection by Leishmania spp. was characterized by miniexon multiplex PCR for complexes of Leishmania and ITS1-PCR-RFLP (HaeIII, HhaI, and RsaI) for the identification of the species.
Results: The T. cruzi TcI was present in 100% of the coinfected mammals, which included 76.7% of triple infection by T. cruzi TcI-complex–L. (L) mexicana–L. infantum/chagasi, 13.3% of double infection by T. cruzi TcI-L. mexicana and 10% of double infection by T. cruzi Tcl—L. infantum/chagasi.
Interpretation & conclusion: These results suggest that the double or triple infection is a phenomenon existing in almost all the coendemics areas and mammals studied, which might influence the mechanisms of adaptation and pathogenicity of these parasites.

Keywords: Coinfection; Leishmania; mammals; molecular characterization; reservoirs; Trypanosoma cruzi


How to cite this article:
Viettri M, Herrera L, Aguilar C M, Morocoima A, Reyes J, Lares M, Lozano-Arias D, García-Alzate R, Chacón T, Feliciangeli M D, Ferrer E. Molecular characterization of Trypanosoma cruzi and Leishmania spp. coinfection in mammals of Venezuelan coendemic areas. J Vector Borne Dis 2019;56:252-62

How to cite this URL:
Viettri M, Herrera L, Aguilar C M, Morocoima A, Reyes J, Lares M, Lozano-Arias D, García-Alzate R, Chacón T, Feliciangeli M D, Ferrer E. Molecular characterization of Trypanosoma cruzi and Leishmania spp. coinfection in mammals of Venezuelan coendemic areas. J Vector Borne Dis [serial online] 2019 [cited 2020 Oct 25];56:252-62. Available from: https://www.jvbd.org/text.asp?2019/56/3/252/289394




  Introduction Top


Leishmaniasis and American trypanosomiasis are diseases caused by protozoans under the Trypanosomatidae family. Leishmaniasis is caused by Leishmania parasites which are transmitted by the bite of infected phlebotomine sandflies. American trypanosomiasis or Chagas disease is caused by Trypanosoma cruzi and mostly transmitted to humans by contact with faeces or urine of triatomine bugs or foods contaminated with these fluids[1],[2].

Chagas disease is a potentially life-threatening illness with cardiovascular, digestive, and neurological complications. About 6 to 7 million people worldwide are estimated to be infected, principally in Latin America. However, it has been increasingly detected in other countries mainly due to population mobility[1]. Several outbreaks have been reported in Venezuela, an endemic country with increasing prevalence of the disease[3],[4],[5],[6],[7]. On the other hand, there are three main forms of leishmaniasis; cutaneous, mucocutaneous, and visceral that depend on the species and the host immune response. An estimated 700,000 to 1 million new cases and 20,000 to 30,000 deaths occur annually across the world[2]. In Venezuela, cutaneous leishmaniasis (CL) is distributed throughout the country while visceral leishmaniasis (VL) is mostly limited to northwestern and northern areas[8],[9].

Trypanosoma cruzi constitutes a group of six heterogeneous sub-populations (lineages) or discrete typing units (DTUs) with high genetic variability, namely TcI, TcII, TcIII, TcIV, TcV and TcVI[10],[11]. This genetic variability has been associated with differential distribution of T. cruzi DTUs in geographic areas and host tissues, with an influence on the pathogenesis of the disease[10],[12]. Similarly, the genus Leishmania is divided into subgenres, mainly Leishmania and Viannia. These subgenres are divided into complexes, which are represented by several species with equal morphological characteristics, but which differ in their geographic distribution, biological and immunological behaviour[13].

Many molecular markers have been used for the characterization of T. cruzi[10],[11]. The methodologies for identification of DTUs are centred mainly on the markers described by Brisse et al[14], and Lewis et a/[15], Sullivan et al[16] and Sturm et al[11]. For Leishmania spp. the identification of the complexes is mainly based on Leishmania miniexon multiplex PCR described by Harris et al[18], while ITS1-nested-PCR–RFLP described by Schönian et al[19] and Cruz et al[20] is mainly used for species identification.

Epidemiological changes produce a close relationship between the enzootic cycle and the human, favouring the presence of these parasites in rural and urban areas[7],[21],[22],[23],[24],[25],[26],[27]. The T. cruzi and Leishmania spp. can share mammal reservoirs, including man; and geographical areas, suggesting the possibility of coinfection. There are limited studies of coinfection between T. cruzi and Leishmania spp. in animal reservoirs worldwide[28],[29],[30]; and even less that characterize the coinfection. Hence, the objective of the present study was to characterise the American Trypanosomiasis–Leishmaniasis coinfection in mammals from Venezuelan coendemic areas.


  Material & Methods Top


Samples

Control samples: The reference strains of Trypanosoma cruzi, DTU TcI–isolates MDID/VE/1984/Dm28c, TMAC/VE/2007/LH5; DTU TcIII–MDID/VE/2001/LH45 and DTU TcV– MHUM/PA/2007/LH31; and the reference strains of Leishmania spp.–isolates L.(V) braziliensis MHOM/BR/1975/M2903, L.(L) mexicana MHOM/BZ/1982/BEL21, and L.(L) donovani MHOM/ IN1980/DD8 were used as controls for the study. All the strains were cultured in vitro in the liver infusion-tryptose (LIT) medium according to Viettri et al[30].

Experimental samples: A descriptive approach was used for the collection of samples (intentional sampling) from mammals (Didelphis marsupialis, Equus mulus, Rattus rattus, Canis familiaris, Felis catus, and Sciurus granatensis) living in the co-endemic areas of American Trypanosomiasis and Leishmaniasis, previously diagnosed with T. cruzi–Leishmania spp. coinfection[30]. Samples were taken from Cojedes, Anzoategui, and Capital District states[30] [Figure 1].
Figure 1: Map of Venezuela highlighting the studied areas.

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DNA extraction

The DNA extraction from parasites in culture and blood on filter paper were performed according to Viettri et al[30]. Briefly, to parasitic pellets (controls) and blood-impregnated filter paper (samples) were added 1 ml of distilled water and incubated for 30 min at room temperature. Subsequently, these were centrifuged for 3 min at 14,000 rpm, the supernatant was discarded leaving a volume of 50 ml; 200 ml of the 5% Chelex®-100 resin was added and incubated for 30 min at 56 °C. They were then shaken for 10 sec and incubated at 100 °C for 10 min, shaken again for 10 sec, centrifuged for 3 min at 14,000 rpm, and finally, 200 μL of the DNA containing supernatant was extracted. It was transferred to a microtube and stored at -20 °C until use.

Identification of T. cruzi DTUs

The molecular characterization of T. cruzi was carried out using the methodologies/protocol described by: (a) Brisse et al[14] using the following molecular markers –(i) intergenic region of the miniexon; (ii) D7 divergent domain of the 24Sa rDNA; and (iii) size-variable domain of the 18S rDNA. Amplification cycles were performed according to Brisse et al[14] and adapted by Rivera et al[26] [Table 1]; (b) Lewis et al[15] using 24Sa rDNA; (c) Sullivan et al[16] using hsp60-PCR-RFLP (EcoRV); and (d) Sturm et al[11]. The PCR products were digested with the enzyme EcoRV, according to the manufacturer’s protocols. Negative controls were the reaction mixture and water and positive controls were the reference strains of the most common DTU of T. cruzi circulating in the area [Table 1].
Table 1: Primers, PCR conditions and amplification programs employed

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Identification of Leishmania species

The identification of Leishmania species was performed using the molecular markers by Leishmania miniexon multiplex PCR and ITS1-nested-PCR–RFLP. The reaction of Leishmania miniexon multiplex PCR was carried out according to the protocols described by Harris et al[18], and adapted by Reyes et al[31] [Table 1]. The ITS1- nested-PCR–RFLP was performed according to the protocols described by Schönian et al[19] and Cruz et al[20] [Table 1]. The PCR products were digested with the enzymes HaeIII, HhaI, and RsaI according to the manufacturer’s protocols. Negative controls were the reaction mixture and water and positive controls were the reference strains of the most common species of Leishmania spp. circulating in the area.

Agarose gel electrophoresis

The amplification products of all the PCR and RFLP reactions were evaluated by 2% agarose gel electrophoresis, stained with 0.5 (μg/ml ethidium bromide. The DNA was separated in a horizontal electrophoresis chamber (Minicell™ EC370M BioRad®, Philadelphia, USA), with a constant voltage between 60–100 V and TAE (Tris base, 40 mM acetic acid, 0.5 M EDTA; pH 8) as a buffer. The DNA bands were visualized on a photodocumentation system, Gel Doc 1000 (BioRad®, Philadelphia, USA) using the multi-analyst program, and the size of the amplification bands was compared with a marker size of 100 base pairs (bp) (BenchTop 100 bp DNA Ladder, Promega, Madison, USA).

Purification and sequencing of miniexon PCR product

The amplification products of the miniexon of the T. cruzi and Leishmania spp. were purified from agarose gels with the commercial Wizard® SV Gel kit and PCR CleanUp System (Promega, Madison, USA), according to the manufacturer’s protocol and were sequenced in the facility of DNA sequencing at the Unit of Genetic and Forensic Studies (UEGF) of the Venezuelan Institute of Scientific Research (IVIC), Venezuela. The sequences obtained were compared with the sequences deposited in the GenBank.

Ethical statement

The study was approved (Reference No. TDR/PRD/ ETHICS/2000.1) by the Committee of Bioethics of the Institute of Biomedical Researches “Dr Francisco J. Triana Alonso” of the University of Carabobo (BIOMED-UC), Venezuela; and conducted in accordance with the guidelines for humans and animals care by the Commission of Bioethics of the Ministry of Science and Technology, Venezuela and the “Operational guidelines for ethics committees” that review biomedical research. Signed informed consent was obtained by the owners of animals for obtaining blood samples according to ethical rules.


  Results Top


Determination of T. cruzi DTUs

Miniexon PCR: All the 30 DNA samples from the co-infected specimens showed the amplification of a band of 350 bp corresponding to T. cruzi TcI, according to the algorithms proposed by Brisse et al[14] and Zingales et al[10] [Table 2], [Figure 2]a.
Table 2: Results of the T. cruzi molecular markers (amplification bands) and the identified DTUs in mammalian samples positive for coinfection (T. cruzi/Leishmania spp.) from different endemic areas of Venezuela

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Figure 2: Amplification products of T. cruzi markers in agarose gel electrophoresis: (a) Miniexon-intergenic region (1–16); (b) 24Sα rDNA: Pattern-I (1–9), Not amplified (NA, 10–17), Pattern-II (18–19); and (c) 24Sα rDNA: Pattern-III (1–2), Pattern-IV (3–5), Pattern-V (6–9) and Pattern-VI (10–11). M: Molecular weight marker (100 bp); TcI: Positive control; TcV: Positive control; and (C–): Negative control.

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24Sα rDNA PCR: A total of 22 DNA samples from the coinfected specimens showed the presence of a band of 110 bp corresponding to T. cruzi TcI [Table 2]; however, they also showed amplification of atypical bands that do not coincide with the algorithms proposed by Brisse et al[14] and Zingales et al[10]. In [Figure 2]b and [Figure 2]c, six amplification patterns are shown with various bands and a group of eight DNA samples that did not amplify. Of the six amplification patterns, the pattern-I (9/30) corresponds to the DNA samples from Anzoátegui and Cojedes states (C. familiaris, D. marsupialis), pattern-II (2/30) corresponds to the samples from Capital District (R. rattus) [Figure 2]b, pattern-III (2/30) corresponds to the samples from Anzoátegui (F. ca- tus and C. familiaris) while the amplification patterns-IV, V and VI corresponds (9/30) to the samples of DNA from the Cojedes state (C. familiaris and E. mulus) [Figure 2]c.

18S rDNA PCR: The amplification products obtained with this molecular marker showed the typical 175 bp band corresponding to T. cruzi TcI, and additional atypical bands that did not coincide with the algorithms proposed by Brisse et al[14] and Zingales et al[10]. The [Figure 3]a and [Figure 3]b, show seven different amplification patterns observed. The amplification pattern-I (2/30) corresponds samples from the Anzoátegui state (C. familiaris, D. marsupialis), the pattern-II and VII (5/30) corresponds samples from the Cojedes state (C. familiaris, S. granatensis), the pattern- III (2/30) corresponds samples from the Capital District (R. rattus), while the patterns-IV, V and VI corresponds samples (21/30) from the states of Anzoátegui and Cojedes (C. familiaris, D. marsupialis, E. mulus, and F. catus).
Figure 3: Amplification products of T. cruzi markers in agarose gel electrophoresis: (a) 18 S rDNA Pattern-I (1–2), Pattern-II (3–6), Pattern-III (7–8), Pattern-IV (9–17); (b) 18 S rDNA Pattern-V (1–10), Pattern-VI (11–12), Pattern-VII (13); and (c) PCR-RFLP HSP60/EcoRV samples (1–30). M: Molecular weight marker (100 bp); TcI: Positive control; TcV: Positive control; and (C–): Negative control.

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hsp60 PCR-RFLP(EcoRV): The PCR amplification of the T. cruzi hsp60, was performed as described earlier[16] using the reference strains of T. cruzi. The optimal reaction conditions included 1.5 mM MgCl2, 0.2 mM dNTPs, 0.4 μM for each primer (hsp60-F and hsp60-R) and DNA polymerase 1.0 U. The analyzed DNA samples showed the amplification of the expected diagnostic band of 432–462 bp [Figure 3]c, which when incubated with the enzyme EcoRV, showed no digestion for the amplification products of TcI, TcII, and TcIV (as expected), while for the TcV, three fragments (462, 314 and 148 bp approximately) were observed (as expected, due to the partial digestion of the product of 462 bp). Analysis of the results obtained with all the markers, confirms the evidence of the presence of the TcI in 100% of the coinfected samples.

Identification of Leishmania species

Miniexon multiplex PCR: [Table 3] shows the sizes of the products of the miniexon from the states of Anzoategui, Cojedes and the Capital District, wherein 23 of the 30 samples showed the presence of two diagnostic bands, one between 218–240 bp and another between 351–397 bp, indicating the presence of DNA from the complexes L. (L) mexicana and L. (L) donovani, respectively, [Figure 4]a. While in 4 of the 30 samples (one from the Capital District and three from the Cojedes), a single band of approximately 240 bp was observed compatible with the complex L. (L) mexicana [Figure 4]b. Evaluation of the remaining three samples from Anzoátegui state showed a unique amplification product of approximately 397 bp, which corresponds to what is expected in the complex L. (L) donovani [Figure 4]b [Table 3].
Figure 4: Amplification products of Leishmania markers in agarose gel electrophoresis: (a) Miniexon multiplex PCR, samples with double amplification (1–13); and (b) Miniexon multiplex PCR, samples with simple amplification (1–7). M: Molecular weight marker (100 bp); (C+1): Positive control L. braziliensis; (C+2): Positive control L. Mexicana; (C+3): Positive control L. donovani; (C–): Negative control.

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Table 3: Results of the Leishmania molecular markers (amplification bands) in mammalian samples positive for coinfection (T. cruzi/Leishmania spp.) from different endemic areas of Venezuela

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ITS1-nested PCR-RFLP (HaeIII): All the DNA samples analysed showed the amplification of a band of approximately 300 bp (280–330 bp), which when digested with the enzyme HaeIII, yielded four positive samples for L. mexicana (186-88-59 bp) and three samples positive for L. infantum/chagasi (184-72-55 bp) [Figure 5]a [Table 3]. Two other groups of samples were also detected; one confirmed by atypical restriction pattern-I (12/30) and another by atypical restriction pattern-II (11/30) whose restriction patterns were 186-72-55 bp and 164-90-59 bp, respectively [Table 3].
Figure 5: Amplification products of Leishmania markers in agarose gel electrophoresis: (a) PCR-RFLP ITS1/HaeIII—(C+1): L. mexicana; (C+2): L. donovani; (C+3): L. braziliensis; (b) PCRRFLP ITS1/HhaI; (C+1): L. donovani; (C+2): L. mexicana, (C+3): L. braziliensis; (c) PCR-RFLP ITS1/RsaI–(C+1): L. braziliensis; (C+2): L. mexicana; (C+3): L. donovani. Lanes 1–7: Samples with typical restriction pattern in all cases (a, b, and c); M: Molecular weight marker (100 bp).

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RFLP ITS1/HhaI: The digestion of PCR product with HhaI, yielded four samples positive for L. mexicana (168-87–78 bp) and three samples positive for L. infantum/chagasi [Figure 5]b. The rest of the samples were separated into two other groups, based on the atypical restriction patterns I and II, consisting 12 (approximate band size 280 bp) and 11 samples (approximate restriction pattern 200–100 bp), respectively [Table 3].

RFLP ITS1/RsaI: By the digestion of PCR product with RsaI, four samples positive for L. mexicana (221–112 bp) and three samples positive for L. infantum/chagasi (210–101 bp) were identified [Figure 5]c. In addition, two other groups comprising 12 (approximate band size 280 bp ) and 11 samples (restriction pattern 210-112 bp) were identified based on atypical restriction patterns I and II, respectively [Table 3]. Analysis of the results obtained with the markers for Leishmania identification, revealed that the species L. (L) mexicana and L. (L) infantum/chagasi were present in four and three samples, respectively (of the 30 samples). Single Leishmania species could not be identified from the rest 23 samples; however, Leishmania complexes, L. (L). mexicana and L. (L). donovani, were identified [Table 3] and [Table 4].
Table 4: Characterisation of T. cruzi DTU and Leishmania complex/species in mammalian samples positive for coinfection T. cruzi/Leishmania spp. from different endemic areas of Venezuela

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Sequencing and analysis of the intergenic region of miniexon

In order to investigate the possible intra-DTU genetic variations of T. cruzi and intra complexes genetic variations of the Leishmania spp., the products of the miniexon of T. cruzi and Leishmania spp. were sequenced. The analysis of the chromatograms did not allow to edit the sequences for the phylogenetic analysis, due to the presence of two or three different types of DNA in the same sample, which caused interference during the sequencing process. However, the partial sequences of the 350 bp product from the intergenic miniexon region of T. cruzi of six sequenced samples could be compared with the sequences deposited in the GenBank, showing a high percentage of similarity with Venezuelan isolates of T. cruzi TcI.


  Discussion Top


There are limited studies of coinfection between T. cruzi and Leishmania spp. in mammals worldwide and even less that characterize the coinfection. In Venezuela, although several endemic foci of both diseases coincide, our previous work[30] was the first study of coinfection on mammals; however, in that work, neither the T. cruzi DTUs nor the Leishmania species involved in the coinfection were specified/characterised. The objective of this research was the molecular characterization of the sub-populations of T. cruzi and the Leishmania species that coexist in common mammals (possible reservoir).

The study observed the presence of T. cruzi TcI DNA in all (100%) of the coinfected mammals. The amplification of the 24Sa rDNA and 18Sa rDNA subunits generated amplification products characteristic to the TcI. However, additional amplification bands were generated that did not coincide with the characterization algorithms proposed by Brisse et al[14] and Zingales et al[10]. This is probably due to the presence of Leishmania spp. DNA in the characterized samples. Even though several works have demonstrated the specificity of the primers of the 24Sα rDNA and 18Sα rDNA[14], they have never been used for mixed infections.

For this reason, it is possible that the primers for T. cruzi hybridize with some DNA sequences of Leishmania spp., considering that ribosomal genes have fairly conserved sequences in the kinetoplastid protozoa genome[32].

The T. cruzi PCR and RFLP results were confirmed with the sequencing of the products of the miniexon-in-tergenic region. The nucleotide sequences obtained showing a high percentage of identity (98%) with sequences of Venezuelan strains, characterized as TcI were deposited in the GenBank[26]. The study results agree with the studies of Añez et al[3], Carrasco et al[24] and Rivera et al[26], reporting similarity of96, 94.1, and 95.5%, respectively, for the isolates obtained from triatomines, mammals reservoirs and humans in Venezuela, corresponding to TcI. Some authors have reported that TcI strain circulates in mammals from almost 17 states of Venezuela, except Barinas, Anzoátegui, and Nueva Esparta where in addition to TcI small proportion of TcIII also circulates[23],[24].

TcI predominates in the countries of Central America, Mexico, Venezuela, Colombia and northern Brazil where the clinical form of the disease is chagasic myocardiopathy, while the rest of the DTUs are distributed to southern Brazil and southern countries from South America, where the digestive (megaesophagus and megacolon) and neurological forms are common, apart from chagasic myocardiopathy[10],[12].

On the other hand, the atypical amplification patterns of the 24Sa rDNA and 18Sa rDNA subunits are distributed in most of the samples, regardless of the species and geographical location; except for the two samples of the species R. rattus, from the Capital District, (urban area), which showed unique amplification patterns, incompatible-with the rest samples. These results show some similarities with the research of Rivera et al[26] which reported that TcI isolates from the Capital District are intrinsically homogeneous, but in turn, show differences with the sequences of rural isolates from the Anzoátegui, Cojedes and Guárico states, which could be due to genetic selection in domestic corridors. Studies conducted in Bolivia, Mexico, Brazil, Colombia, Argentina and Venezuela, report variations in the sequences of the miniexon-intergenic region of TcI isolates from various vectors and reservoirs including man[26],[33],[34],[35]. Genetic heterogeneity in this sub-population of T. cruzi could be related to the geographical distribution of some isolates and certain mechanisms of adaptation to different conditions, such as a coinfection.

Regarding Leishmania, the complexes L. (L) mexicana and L. (L) donovani were detected in the samples analyzed, of which 76.7% had mixed infection with both complexes. The samples coinfected with the complexes L. (L) mexicana–L. (L) donovani, showed atypical restriction patterns by ITS1-PCR-RFLP, not compatible with any of the reference species; perhaps the presence of two species belonging to the same sub-genus (non-common feature) in the same sample of DNA, could interfere with the enzymatic digestion. Only the L. mexicana species was identified in four positive samples for the L. (L) mexicana complex. Although the digestion of the amplification product ITS1/HaeIII, HhaI and RsaI to did not allow the identification of species in the positive samples for the complex L. (L) donovani, it can be deduced, that the samples with DNA of the complex L. (L) donovani corresponds to the species L. infantum/chagasi since it is the only species of this complex present in the country.

In Venezuela, several studies reveal the presence of the complexes L. (L) donovani, L. (L) mexicana and L. (V) braziliensis, circulating in sandfly vectors, animal reservoirs (C. familiaris, E. asinus, D. marsupialis, and R. rattus) and man, causing the various clinical forms of the disease[21],[22],[25],[27],[36],[37],[38],[39],[40]. The epidemiological complexity of some endemic areas, with a very heterogeneous distribution of these complexes, raises the possibility of overlapping the transmission cycles of different species, allowing many of the foci of visceral and tegumentary leishmaniasis to coincide. This is related to the displacement of human populations that contribute to the dispersion of Leishmania spp. beyond its traditional ecological distribution[40].

In Brazil, some studies reveal the presence of double and triple infection caused by several Leishmania species in mammals[41],[42],[43],[44],[45] and humans[46]. However, in Venezuela, it is the first time that the presence of the complexes L. (L) mexicana–L. (L) donovani, causing mixed infection in animal reservoirs is reported. Moreover, the study also identified L. infantum/chagasi in traditional areas of CL These results have important implications in the epidemiological control of VL and CL in Venezuela, as the studied animals/mammals represent a considerable source of infection, due to their close relationship with humans in rural and urban areas.

The results showed in this study represent the first finding of triple infection in domestic and synanthropic reservoirs of trypanosomatids of public health importance (T. cruzi–L. (L) mexicana–L. infantum/chagasi) in Venezuela. This study provides important data, not only in the form of diagnosis and characterization of these parasites,-but also in the form of their epidemiology; since reservoirs, vectors and man converge in a common ecotope, like some of these infected wild mammals, e.g. squirrels, incorporated as non-traditional pets, represents a potential risk in the active transmission of Chagas disease, CL and VL. Other studies have also indicated the presence of these protozoans coexisting in several species of mammals including man[28],[29],[47],[48],[49],[50],[51],[52]. These results invite the design of new schemes future programmes to control these parasites.

Further, the study also demonstrated the interference caused in the characterization techniques due to the double (T. cruzi–Leishmania sp.) or triple infections [T cruzi–L. (L) Mexicana–L. infantum/chagasi]. This experience could serve as an example to others investigations with doubtful results, and knowledge on the possibility of coinfection. On the other hand, it is often thought that non-specific bands or atypical patterns in molecular diagnostic techniques are due to genetic variability, and may be the effect of interference by another similar DNA, in case of coinfection. Although, we cannot rule out that the non-specific bands or atypical patterns found in this work may be due to genetic variability of circulating parasites in these areas with respect to the reference strains, that precisely, because the interference of several DNAs in the same sample could not be clarified with the sequencing. Diagnostic problems may occur when different parasites are present in the same sample, especially when the similarities between phylogenetically close parasites affect the accuracy of diagnostic tests. Coinfection can lead to diagnostic errors and delays, and it can influence the effectiveness and safety of treatment[53]. Despite being a common trait among wild mammals, mixed infections and their consequences for the host’s health and parasite transmission are still a poorly known phenomenon[50].

The studied parasites share a set of similarities: belong to the same order and family, common antigenic and genetic characteristics, a complex relationship with their invertebrate vectors and a similar evolutionary cycle, being the cells of the phagocyte mononuclear system capable of being infected by both protozoa. A study has shown that T. cruzi can differentiate and multiply within parasitophorous vacuoles in macrophages coinfected with L. amazonensis[54]. These similarities raise the possibility of genetic recombination in the cells of the reservoirs, with the production of hybrid clones or different genotypes with genetic and antigenic characteristics that may or may not influence the mechanisms of adaptation and pathogenicity of these parasites. The results of the study necessitates further research, since they only show the tip of the iceberg in the complex dynamics of the coinfection between T. cruzi and the species of the genus Leishmania.


  Conclusion Top


Based on the results it can be inferred that T. cruzi TcI strain is present in all of the coinfected mammals residing in the co-endemic areas of Venezuela. Triple infection by T. cruzi TcI and complex L. (L) mexicana–L. infantum/chagasi is more frequent than the double infection by T. cruzi TcI–L. mexicana or T. cruzi TcI–L. infantum/chagasi. Use of multiple techniques for diagnosis and molecular characterization of the species is essential since coinfection can cause interference in diagnostic techniques. Coinfection might influence the mechanisms of adaptation and pathogenicity of these parasites and the effectiveness of treatment.

Conflict of interest

The authors declare no conflict of interest.


  Acknowledgements Top


Funding was provided by the Proyects: Misión Cien- cia 2008000911-2 and 2008000911-6, FONACIT, MPPS and Proyecto Estratégico UCV-UC-UDO, FONACIT, MPPS, 2011000470.



 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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