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Characteristics of Triatomine infestation and natural Trypanosoma cruzi infection in the State of Rio Grande do Norte, Brazil

Abstract:

INTRODUCTION

Natural and artificial ecotope infestation by the kissing bug triatomines and their colonization and infection by Trypanosoma cruzi , the Chagas disease agent, were evaluated in nine municipalities of the State of Rio Grande do Norte, Brazil.

METHODS

Following identification, triatomine intestinal contents were analyzed by direct microscopic examination, xenoculture, and polymerase chain reaction (PCR) for parasite detection. Trypanosoma cruzi isolates were genotyped using three different markers.

RESULTS

Of 842 triatomines captured, 65% were Triatoma brasiliensis , 17.8% Triatoma pseudomaculata , 12.5% Panstrongylus lutzi , and 4.7% Rhodnius nasutus . Triatoma brasiliensis and P. lutzi adults were found in the intradomicile. T. brasiliensis, T. pseudomaculata , and R. nasutus nymphs and adults were found in the peridomicile and wild environment. Intradomiciliary and peridomiciliary infestation indexes were 5.6% and 33.7%, respectively. In the peridomicile, chicken coops were the most infested ecotope. The T. cruzi triatomine infection rate was 30.2%, of which PCR detected 29%. P . lutzi (78.1%), T . brasiliensis (24.5%), and T . pseudomaculata (22.7%) were the most infected species. TcII and III genotypes were detected in T. brasiliensis and TcIII in P. lutzi .

CONCLUSIONS

T. brasiliensis was found in all environments and most ecotopes with high T. cruzi infection rates. High infection rates were also detected in T . pseudomaculata and P. lutzi , suggesting their role in the interchange between the wild and peridomestic transmission cycles. The combination of PCR, microscopic examination, and xenoculture contributed to improving T. cruzi infection evaluation in triatomine bugs. The TcII and TcIII genotypes were predominant in the study area.

Keywords:
Trypanosoma cruzi; Triatominae natural infection; PCR; Xenoculture. Direct microscopic examination.

INTRODUCTION

Trypanosoma cruzi is the etiological agent of Chagas disease, and its main vectors belong to the genera Panstrongylus, Rhodnius , and Triatoma . Among these blood-sucking reduviid bugs of the subfamily Triatominae, 70 of the over 148 Triatominae species described 11. Galvão C, Paula AS. Sistemática e evolução dos vetores. : Galvão C, editor. Vetores da doença de Chagas. Curitiba (PR): Sociedade Brasileira de Zoologia 2015. p. 26-31.) are naturally infected by T. cruzi22. Zeledón R, Rabinovich JE. Chagas' disease: an ecological appraisal with special emphasis on its insect vectors. Annu Rev Entomol 1981; 26:101-133.. Infection is maintained primarily within three overlapping cycles: domiciliary, peridomiciliary, and sylvatic 33. Deane LM. Animal reservoirs of Trypanosoma cruzi in Brazil. Rev Bras Malariol D Trop 1964; 16:27-48.. Vector transmission remains the most important route of parasite to human transmission owing to the natural distribution of T. cruzi in the triatomine species adapted to domestic or peridomestic environments; this adaptation to human dwellings strongly determines human infection rates 44. Coura JR, Borges-Pereira J. Chagas disease: 100 years after its discovery. A systemic review. Acta Trop 2010; 115:5-13..

The Brazilian Northeast, one of the poorest and most underdeveloped regions within Brazil, is considered the most important region therein for American trypanosomiasis, where native species like Triatoma brasiliensis Neiva, 1911, Triatoma pseudomaculata Corrêa and Espínola, 1964, Panstrongylus lutzi Neiva & Pinto, 1926, and Rhodnius nasutus Stal, 1859 are widespread 55. Dias JCP, Machado EMM, Fernandes AL, Vinhaes MC General situation and perspectives of Chagas disease in Northeastern region, Brazil. Cad Saude Publica 2000; 16:13-34.) (66. Gurgel-Gonçalves R, Galvão C, Costa J, Peterson AT. Geographic distribution of Chagas disease vectors in Brazil based on ecological niche modeling. J Trop Med 2012; 2012:705326.. Triatoma brasiliensis is distributed in nine Northeast states, Tocantins, and Minas Gerais 77. Silveira AC, Feitosa VR, Borges R. Distribuição de triatomíneos capturados no ambiente domiciliar, no período de 1975/1983, no Brasil. Rev Bras Malariol D Trop1984; 36:15-312.. Considered the main vector of T. cruzi in the Northeastern semiarid regions, it colonizes both sylvatic and domestic environments but is more frequently captured in peridomestic areas 55. Dias JCP, Machado EMM, Fernandes AL, Vinhaes MC General situation and perspectives of Chagas disease in Northeastern region, Brazil. Cad Saude Publica 2000; 16:13-34.) (88. Costa J, Almeida CE, Dotson EM, Lins A, Vinhaes M, Silveira AC et al. The epidemiologic importance of Triatoma brasiliensis as a Chagas disease vector in Brazil: a revision of domiciliary captures during 1993-1999. Mem Inst Oswaldo Cruz 2003; 98:443-449.) (99. Sarquis O, Borges-Pereira J, Mac Cord JR, Gomes TF, Cabello PH, Lima MM. Epidemiology of Chagas disease in Jaguaruana, Ceará, Brazil. I. Presence of triatomines and index of Trypanosoma cruzi infection in four localities of a rural area. Mem Inst Oswaldo Cruz2004; 99:263-270.) (1010. Almeida CE, Pacheco RS, Haag K, Dupas S, Dotson EM, Costa J. Inferring from the Cyt B gene the Triatoma brasiliensis Neiva, 1911 (Hemiptera: Reduviidae: Triatominae) genetic structure and domiciliary infestation in the state of Paraíba, Brazil. Am J Trop Med Hyg 2008; 78:791-802.) (1111. Carbajal de la Fuente AL, Dias-Lima A, Lopes CM, Emperaire L, Walter A, Ferreira A, et al. Behavioral plasticity of Triatominae related to habitat selection in Northeast Brazil. J Med Entomol 2008; 45:14-19.. Triatomine control remains problematic as this region is the center of dispersion and has higher T. brasiliensis concentrations 1212. Forattini OP. Biogeography, origin, and distribution of triatominae domicile dispersal in Brazil. Rev Saude Publica 1980; 14:265-299.) (1313. Alencar JE. História natural da doença de Chagas no Estado do Ceará, Imprensa Universitária, Fortaleza, CE, p. 341, 1987.) (1414. Silveira AC, Vinhaes MC. Chagas disease: the epidemiological and control aspects. Rev Soc Bras Med Trop 1998; 31:15-60.; it is further aggravated by local vector control activity discontinuation and wild and domestic environmental overlap 1515. Câmara ACJ, Varela-Freire AA, Valadares HMS, Macedo AM, D'Ávila DA, Machado CR et alGenetic analyses of Trypanosoma cruzi isolates from naturally infected triatomines and humans in northeastern Brazil. Acta Trop 2010; 115:205-211.) (1616. Câmara ACJ, Lages-Silva E, Sampaio GHF, D'Ávila DA, Chiari E, Galvão LMC. Homogeneity of Trypanosoma cruzi I, II, and III populations and the overlap of wild and domestic transmission cycles by Triatoma brasiliensis in northeastern Brazil. Parasitol Res 2013; 112:1543-1550.) (1717. Martins K, Andrade CM, Barbosa-Silva AN, Nascimento GB, Chiari E, Galvão LMC et al. Trypanosoma cruzi III causing the indeterminate form of Chagas disease in a semi-arid region of Brazil. Int J Infect Dis 2015; 39:68-75.Triatoma pseudomaculata also demonstrates high domiciliation capacity, thereby increasing its colonization rate in several states 55. Dias JCP, Machado EMM, Fernandes AL, Vinhaes MC General situation and perspectives of Chagas disease in Northeastern region, Brazil. Cad Saude Publica 2000; 16:13-34.) (1818. Silveira AC. New challenges and the future of control. Rev Soc Bras Med Trop2011; 44 (supl 2):122-124.; such native triatomines that sporadically invade or reinvade human dwellings further complicate vector control consolidation 1818. Silveira AC. New challenges and the future of control. Rev Soc Bras Med Trop2011; 44 (supl 2):122-124..

Triatomines can be infected with several flagellates and T. cruzi infection level evaluation in wild, peridomiciliary, and domiciliary environments has relevance for control programs aimed at reducing human infections 1919. Shikanai-Yasuda MA, Ochs DE, Tolezano JE, Kirchhoff LV. Use of the polymerase chain reaction for detecting Trypanosoma cruzi in triatomine vectors. Trans R Soc Trop Med Hyg 1996; 90:649-651.) (2020. Botto-Mahan C, Ortiz S, Rozas M, Cattan PE, Solari A. DNA evidence of Trypanosoma cruzi in Chilean wild vector Mepraia spinolai (Hemiptera: Reduviidae). Mem Inst Oswaldo Cruz2005; 100:237-239.. Trypanosoma cruzi has been detected in the excreta or intestinal contents of triatomines by direct microscopic examination (DME) 1919. Shikanai-Yasuda MA, Ochs DE, Tolezano JE, Kirchhoff LV. Use of the polymerase chain reaction for detecting Trypanosoma cruzi in triatomine vectors. Trans R Soc Trop Med Hyg 1996; 90:649-651.) (2121. Moser DR, Kirchhoff LV, Donelson JE. Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction. J Clin Microbiol 1989; 27:1477-1482., necessitating the ability to distinguish this protozoan from other trypanosomatids. Although DME is reliable and relatively inexpensive, disadvantages related to sensitivity, specificity 2222. Russomando G, Rojas AA, Almiron M, Figueredo A, Ferreira ME, Morita K. Trypanosoma cruzi : polymerase chain reaction-based detection in dried feces of Triatoma infestans . Exp Parasitol 1996; 83:62-66.) (2323. Braz LMA, Raiz-Jr R, Amato-Neto V, Alárcon RS, Gakyia E, Okay TS. The detection of Trypanosoma cruzi in Triatoma infestans : comparison of a PCR-based assay with microscopical examination. Ann Trop Med Parasitol 2007; 101:461-465.) (2424. Pizarro JC, Lucero DE, Stevens L. PCR reveals significantly higher rates of Trypanosoma cruzi infection than microscopy in the Chagas vector, Triatoma infestans : High rates found in Chuquisaca, Bolivia. BMC Infect Dis 2007; 7:66-73., and poor performance on dead insects 2525. Dorn PL, Engelke D, Rodas A, Rosales R, Melgar S, Brahney B et alUtility of the polymerase chain reaction in detection of Trypanosoma cruzi in Guatemalan Chagas' disease vectors. Am J Trop Med Hyg 1999; 60:740-745.exist. Xenoculture is used for quality control to confirm negative intestinal content results ascertained by DME, whereas positive cultures allow for T. cruzi strain isolation 2626. Bronfen E, Rocha FSA, Machado GBN, Perillo MM, Romanha AJ, Chiari E. Isolation of Trypanosoma cruzi samples by xenodiagnosis and hemoculture from patients with chronic Chagas' disease. Mem Inst Oswaldo Cruz1989; 84:237-240.pursuant to e.g. genetic studies. For detecting T. cruzi in reduviid bug feces or urine and mammalian blood samples, polymerase chain reaction (PCR) is generally superior 2121. Moser DR, Kirchhoff LV, Donelson JE. Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction. J Clin Microbiol 1989; 27:1477-1482.) (2222. Russomando G, Rojas AA, Almiron M, Figueredo A, Ferreira ME, Morita K. Trypanosoma cruzi : polymerase chain reaction-based detection in dried feces of Triatoma infestans . Exp Parasitol 1996; 83:62-66.) (2727. Breniére SF, Bosseno MF, Revollo S, Rivera MT, Carlier Y, Tibayrenc M. Direct identification of Trypanosoma cruzi natural clones in vectors and mammalian hosts by polymerase chain reaction amplification. Am J Trop Med Hyg 1992; 46:335-341.and can also genotype T. cruzi using different targets.

Such methodology has increased the rate of positivity of infection in field studies, which is especially important in areas where there is scarce information on vector infection following control and surveillance programs. A recent seroepidemiological survey showed high seroprevalence for municipalities in the west and central mesoregions of the State of Rio Grande do Norte (RN) 2828. Brito CRN, Sampaio GHF, Câmara ACJ, Nunes DF, Azevedo PRM, Chiari E et al. Seroepidemiology of Trypanosoma cruzi infection in the semiarid rural zone of the State of Rio Grande do Norte, Brazil. Rev Soc Bras Med Trop2012; 45:346-352., and genotyping studies identified T . cruzi I (TcI) in T. brasiliensis and TcIII (formerly called TcIIc) in armadillos 2929. Marcili A, Lima L, Valente VC, Valente SA, Batista JS, Junqueira ACV et al. Comparative phylogeography of Trypanosoma cruzi TCIIc: New hosts, association with terrestrial ecotopes, and spatial clustering. Infect Genet Evol 2009; 9:1265-1274., TcI and TcII in humans, TcII and TcIII in T. brasiliensis , and TcIII in P . lutzi1515. Câmara ACJ, Varela-Freire AA, Valadares HMS, Macedo AM, D'Ávila DA, Machado CR et alGenetic analyses of Trypanosoma cruzi isolates from naturally infected triatomines and humans in northeastern Brazil. Acta Trop 2010; 115:205-211..

The purpose of this study was to evaluate the occurrence of triatomine infestation of natural and artificial ecotopes, and the colonization and T. cruzi infection in triatomines collected from different environments in the west and central mesoregions of the State RN, Brazil. Diagnostic method efficacy and reproducibility were evaluated and the T. cruzi populations isolated from positive triatomines were genotyped to establish their genetic groups.

METHODS

Study area

State of Rio Grande do Norte, located in northeastern Brazil, is divided into 167 municipalities distributed in four mesoregions: West, Central, Agreste, and East. About 90% of the territory represents arid and semiarid climates, where the predominant biome is the Caatinga, characterized as containing thorny shrubs, irregular structure, and partially uncovered soil. This study was conducted in the municipalities of Apodi, Caraúbas, Governador Dix-Sept Rosado, Lucrécia, Mossoró, Severiano Melo, São Miguel, Caicó, and Serra Negra do Norte in the West and Central mesoregions ( Figure 1), which were selected in reference to the seroepidemiological survey conducted between 2007 and 2009 2828. Brito CRN, Sampaio GHF, Câmara ACJ, Nunes DF, Azevedo PRM, Chiari E et al. Seroepidemiology of Trypanosoma cruzi infection in the semiarid rural zone of the State of Rio Grande do Norte, Brazil. Rev Soc Bras Med Trop2012; 45:346-352..

Figure 1:
Map of the State of Rio Grande do Norte highlighting the West and Central mesoregions. The study area showing the surveyed municipalities is shown in dark grey.

Triatomine collection and identification

Triatomines were captured in rural areas of the nine municipalities in intradomicile, peridomicile, and wild environments from March 2009 to August 2012. The intradomiciliary environment was surveyed in 250 domiciliary units (DU) together with 187 artificial structures in the peridomicile such as chicken coops (n = 110), corrals (n = 25), pigsties (n = 2), piles of tiles (n = 44), old stone fence structures (n = 2), and dry carnauba palm tree ( Copernicia prunifera ) straw (n = 4). Entomological indicators were used to calculate the colonization and species peridomiciliary and domiciliary infestation indices 3030. World Health Organization (WHO). Control of Chagas' Disease. Technical Report Series 811. Genebra: WHO; 1991..

Wild environment captures were performed in Apodi, Caraúbas, and Serra Negra do Norte in rock outcrops (n = 9), stone fences (n = 2), bird's nests (n = 5), and carnauba palm trees (n = 16), the latter as previously described 3131. Diotaiuti L, Dias JCP. Occurrence and biology of Rhodnius neglectus Lent, 1954 in palm trees of suburban areas of Belo Horizonte, Minas Gerais. Mem Inst Oswaldo Cruz1984; 79:293-301..

In Serra Negra do Norte , wild triatomines capture was conducted at the Seridó Ecological Station (ESEC-Seridó)/ Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio), created by Decree 87222 of 05/31/1982, Law 6902 04.27.1982 as a Conservation Unit. Some adult insects were captured in station lodgings and were classified as an undefined ecotope. Insects were captured both day and night by the principal author with the assistance of the respective Municipal Health Secretariat technicians via manual searches using tweezers and a flashlight in all environments and without the use of insect dislodging substances, and were individually identified as described 3232. Carcavallo RU, Rodríguez MEF, Salvatella R, Curto de Casas SI, Sherlock IS, Galvão C, et alHabitats and related fauna. : Carcavallo RU, Galindéz-Girón I, Jurberg J, Lent H, editors. Atlas of Chagas Disease Vectors in the Americas. Vol II Rio de Janeiro (RJ): FIOCRUZ; 1998. p. 561-600..

Natural infection of triatomine bugs

Direct microscopic examination . Collected triatomines were examined individually and their intestinal content was removed under aseptic conditions and placed in a well of a 24-well plate containing 500µL sterile saline solution. After homogenization, 5µL suspension was used to identify trypanosomatid forms via DME at 400× magnification 2626. Bronfen E, Rocha FSA, Machado GBN, Perillo MM, Romanha AJ, Chiari E. Isolation of Trypanosoma cruzi samples by xenodiagnosis and hemoculture from patients with chronic Chagas' disease. Mem Inst Oswaldo Cruz1989; 84:237-240.and smear stained by Giemsa (1,000).

Xenoculture . Approximately 250µL intestinal content suspension was seeded in 15mL tubes containing liver infusion tryptose culture medium 3333. Camargo EP. Growth and differentiation in Trypanosoma cruzi . I - Origin of metacyclic trypanosomes in liquid media. Rev Inst Med Trop 1964; 6:93-100.plus McNeal Novy Niccole or blood agar and incubated at 28C. Aliquots were examined after 15, 30, and 60 days by DME (400×) 2626. Bronfen E, Rocha FSA, Machado GBN, Perillo MM, Romanha AJ, Chiari E. Isolation of Trypanosoma cruzi samples by xenodiagnosis and hemoculture from patients with chronic Chagas' disease. Mem Inst Oswaldo Cruz1989; 84:237-240..

PCR with species-specific primers. Deoxyribonucleic acid (DNA) was extracted via phenol-chloroform 3434. Gomes ML, Macedo AM, Vago AR, Pena SDJ, Galvão LMC, Chiari E Trypanosoma cruzi : optimization of polymerase chain reaction for detection in human blood. Exp Parasitol1998; 88:28-33.) using 200µL diluted insect intestinal content solution (v/v) in 0.2M guanidine-HCl 6M/ethylenediaminetetraacetic acid (EDTA) 3535. Ávila HA, Sigman DS, Cohen LM, Millikan RC, Simpson L. Polymerase chain reaction amplification of Trypanosoma cruzi kinetoplast minicircle DNA isolated from whole blood lysates: diagnosis of chronic Chagas' disease. Mol Biochem Parasitol 1991; 48:211-222.) maintained for 5-7 days at room temperature and stored at 4ºC until DNA extraction. PCR amplifications were performed in duplicate, as described 3434. Gomes ML, Macedo AM, Vago AR, Pena SDJ, Galvão LMC, Chiari E Trypanosoma cruzi : optimization of polymerase chain reaction for detection in human blood. Exp Parasitol1998; 88:28-33., using specific primers to identify T. cruzi3636. Degrave W, Fragoso SP, Britto C, Van Heuverswyn H, Kidane GZ, Cardoso MAB et al. Peculiar sequence organization of kinetoplast DNA minicircles from Trypanosoma cruzi . Mol Biochem Parasitol1988; 27:63-70.. PCR assays were performed in a DNA clean chamber to avoid contamination and positive and negative controls were used to monitor each step.

For Trypanosoma cruzi and Trypanosoma rangeli Tejera, 1920 differential diagnosis, multiplex PCR was performed as described 3737. Souto RP, Vargas N, Zingales B. Trypanosoma rangeli : discrimination from Trypanosoma cruzi based on a variable domain from the large subunit ribosomal RNA gene. Exp Parasitol1999; 91:306-314.. As positive controls, Trypanosoma cruzi cell cultures of the Colombian strains ( T. cruzi I), JG ( T. cruzi II), and T. rangeli were used.

Trypanosoma cruzi isolates for genotyping

Cultured parasite genomic DNA obtained using phenol-chloroform was used as PCR assay templates 3838. Macedo AM, Martins MS, Chiari E, Pena SDJ. DNA fingerprinting of Trypanosoma cruzi : a new tool for characterization of strains and clones. Mol Biochem Parasitol1992; 55:147-153.. Trypanosoma cruzi isolates were typed using three different parasite genomic sequences as reported 3939. D'Ávila DA, Macedo AM, Valadares HMS, Gontijo ED, Castro AM, Machado CR et al. Probing population dynamics of Trypanosoma cruzi during progression of the chronic phase in chagasic patients. J Clin Microbiol2009; 47:1718-1725., with the 24Sα ribosomal (rRNA) gene D7 domain 4040. Souto RP, Zingales B. Sensitive detection and strain classification of Trypanosoma cruzi by amplification of a ribosomal RNA sequence. Mol Biochem Parasitol1993; 62:45-52., mitochondrial cytochrome oxidase subunit 2 gene (COII) 4141. Freitas JM, Augusto-Pinto L, Pimenta JR, Bastos-Rodrigues L, Gonçalves VF, Teixeira SMR et al. Ancestral genomes, sex, and the population structure of Trypanosoma cruzi . PLOS Pathog 2006; 2:e24., and the spliced leader genes intergenic region 4242. Burgos JM, Altcheh J, Bisio M, Duffy T, Valadares HMS, Seidenstein ME et al. Direct molecular profiling of minicircle signatures and lineages of Trypanosoma cruzi bloodstream populations causing congenital Chagas disease. Int J Parasitol 2007; 37:1319-1327.) as markers for six discrete typing units (DTUs) 4343. Zingales B, Andrade SG, Briones MRS, Campbell DA, Chiari E, Fernandes O et al. A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz2009; 104:1051-1054., using T. cruzi reference strains 1515. Câmara ACJ, Varela-Freire AA, Valadares HMS, Macedo AM, D'Ávila DA, Machado CR et alGenetic analyses of Trypanosoma cruzi isolates from naturally infected triatomines and humans in northeastern Brazil. Acta Trop 2010; 115:205-211.) (4444. Lages-Silva E, Crema E, Ramírez LE, Macedo AM, Pena SDJ, Chiari E. Relationship between Trypanosoma cruzi and human chagasic megaesophagus: blood and tissue parasitism. Am J Trop Med Hyg 2001; 65:435-441.) (4545. Monteiro WM, Magalhães LKC, de-Sá ARN, Gomes ML, Toledo MJO, Borges L et al. Trypanosoma cruzi IV causing outbreaks of acute Chagas disease and infections by different haplotypes in the Western Brazilian Amazonia. PloS One 2012; 7:e41284.and clones 4646. Zingales B, Pereira MES, Oliveira RP, Almeida KA, Umezawa ES, Souto RP et al. Trypanosoma cruzi genome project: biological characteristics and molecular typing of clone CL Brener. Acta Trop1997; 68:159-173.) (4747. Federici EE, Abelmann WH, Neva FA. Chronic and progressive myocarditis and myositis in C3H mice infected with Trypanosoma cruzi . Am J Trop Med Hyg 1964; 13:272-280.as DTU controls.

All PCR products were analyzed by 6% polyacrylamide gel electrophoresis and visualized by silver staining 4848. Santos FR, Pena SDJ, Epplen JT. Genetic and population study of a Y-linked tetranucleotide repeat DNA polymorphism with a simple non-isotopic technique. Hum Gene 1993; 90:655-656..

Statistical analysis

To quantify the concordance between the results of different methods, generalized Kappa ( hat k ) coefficients were estimated and an approximate 95% confidence interval (CI95%) for k [lower confidence limit (LCL) and upper confidence limit (UCL)] 4949. Fleiss JL, Levin B, Paik MC. Statistical Methods for Rates and Proportions. 3rd ed. Hoboken: Wiley; 2003.was calculated and classified accordingly 5050. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics 1977; 33:159-174..

RESULTS

Table 1shows that were captured 842 triatomines including T . brasiliensis (65%), T . pseudomaculata (17.8%), P . lutzi (12.5%), and R . nasutus (4.7%). T. brasiliensis was predominant in intradomicile (n = 12), peridomicile (n = 323), and wild environments (n = 212). Table 1shows specimen numbers per species, environment, and municipality.

Table 1:
Number of triatomine bugs captured in different environments in the west and central mesoregions in nine municipalities in the State of Rio Grande do Norte, Brazil (2009-2012).

In the wild, 48.5% (16/33) of the ecotopes were infested by triatomines. High infestation was observed in stone fences (100%) and rock outcrops (75%) in Serra Negra do Norte in comparison with other ecotopes. In the peridomicile, the infestation index was 33.7% (63/187) with T. brasiliensis and T. pseudomaculata at indices at 26.2% (49/187) and 5.9% (11/187), respectively, primarily in chicken coops. The intradomicile infestation index was 5.6% (14/250). Table 2shows the colonization index of each ecotope by triatomine species.

Table 2:
Entomological indicators observed in the ecotopes of different environments in the municipalities of the State of Rio Grande do Norte.

All the insects were examined; 30.2% exhibited T. cruzi infection, with P. lutzi demonstrating the highest rates (78.1%). The highest infection rate was detected by PCR (29%), detecting T. cruzi infection in most P . lutzi (69.5%) and T . brasiliensis (24.3%) specimens ( Figure 2A). Kappa evinced no agreement between T. cruzi detection methods for T. brasiliensis [ hat k = −0.0112; CI95% = (−0.0596; 0.0372)], T. pseudomaculata [ hat k = 0.0036; CI95% = (−0.0888; 0.0960)], P. lutzi [ hat k = 0.0690; CI95% = (−0.0414; 0.1794)], and R. nasutus ( hat k = 0.1652; CI95% = (−0.0137; 0.3441)]. The global infection index was 0.12 [CI95% = (0.0773; 0.1553)] indicating slight methodological agreement, but significantly different from zero as excluded by the CI.

Figure 2B shows that the highest infected specimen rate (53.3%) occurred in the intradomicile [primarily P. lutzi (66.7%)], followed by the wild environment (40.5%) and peridomicile (20.1%) [primarily P. lutzi and T. pseudomaculata (78.4% and 27.2%, respectively)].

Figure 2:
Rate of natural infection of triatomine species by Trypanosoma cruzi as determined using different methods and in three distinct environments. A) Infection rate of triatomine species by detection method and global infection index. B) Infection rate of triatomines in different capture environments and total number of infected specimens. T. brasiliensis: Triatoma brasiliensis ; T. pseudomaculata: Triatoma pseudomaculata; P. lutzi: Panstrongylus lutzi; R. nasutus: Rhodnius nasutus; PCR: polymerase chain reaction.

In the wild, T. brasiliensis presented high infection indices in Serra Negra do Norte (31.4%) and Caraúbas (26.1%) rock outcrops. T. cruzi infection was also detected in R. nasutus in carnauba palm trees. In the peridomicile, T. brasiliensis presented high infection indexes in piles of tiles (38.1%) and corrals (37.5%) in Governador Dix Sept Rosado and Caraúbas, respectively, and T. pseudomaculata in chicken coops (30.8%) in Caraúbas ( Table 2).

Intestinal contents of all infected triatomines and 100 uninfected specimens were submitted to multiplex PCR using primers for T. rangeli and produced no overlapping data, thus confirming that T. cruzi- specific amplification.

Trypanosoma cruzi DTUs were identified in 15 samples from three municipalities. We isolated 3 TcII and 3 TcIII stocks from T . brasiliensis captured in Caicó and Serra Negra do Norte , respectively,and 9 TcIII T . cruzi were isolated from P . lutzi in Caraúbas ( Table 3).

Table 3:
Geographical origin, development stage, environment, and genetic typing of Trypanosoma cruzi stocks from triatomine species.

DISCUSSION

This general assessment of triatomine occurrence demonstrated that the species T . brasiliensis, T. pseudomaculata, P . lutzi , and R . nasutus continue to exist in artificial environments in RN State municipalities, where they have been registered since the 1950s 77. Silveira AC, Feitosa VR, Borges R. Distribuição de triatomíneos capturados no ambiente domiciliar, no período de 1975/1983, no Brasil. Rev Bras Malariol D Trop1984; 36:15-312.) (88. Costa J, Almeida CE, Dotson EM, Lins A, Vinhaes M, Silveira AC et al. The epidemiologic importance of Triatoma brasiliensis as a Chagas disease vector in Brazil: a revision of domiciliary captures during 1993-1999. Mem Inst Oswaldo Cruz 2003; 98:443-449.) (1414. Silveira AC, Vinhaes MC. Chagas disease: the epidemiological and control aspects. Rev Soc Bras Med Trop 1998; 31:15-60.) (1515. Câmara ACJ, Varela-Freire AA, Valadares HMS, Macedo AM, D'Ávila DA, Machado CR et alGenetic analyses of Trypanosoma cruzi isolates from naturally infected triatomines and humans in northeastern Brazil. Acta Trop 2010; 115:205-211.) (5151. Lucena DT. Doença de Chagas no NordesteRev Bras Malariol D Trop1959; 11:675-696.) (5252. Castro-Filho J, Silveira AC. Distribuição da Doença de Chagas no Brasil. Rev Bras Malariol D Trop1979; 31:85-89.and where high seroprevalence of human T . cruzi infection has been estimated 2828. Brito CRN, Sampaio GHF, Câmara ACJ, Nunes DF, Azevedo PRM, Chiari E et al. Seroepidemiology of Trypanosoma cruzi infection in the semiarid rural zone of the State of Rio Grande do Norte, Brazil. Rev Soc Bras Med Trop2012; 45:346-352.. The presence of other triatomine species has also been reported 55. Dias JCP, Machado EMM, Fernandes AL, Vinhaes MC General situation and perspectives of Chagas disease in Northeastern region, Brazil. Cad Saude Publica 2000; 16:13-34.) (77. Silveira AC, Feitosa VR, Borges R. Distribuição de triatomíneos capturados no ambiente domiciliar, no período de 1975/1983, no Brasil. Rev Bras Malariol D Trop1984; 36:15-312.) (88. Costa J, Almeida CE, Dotson EM, Lins A, Vinhaes M, Silveira AC et al. The epidemiologic importance of Triatoma brasiliensis as a Chagas disease vector in Brazil: a revision of domiciliary captures during 1993-1999. Mem Inst Oswaldo Cruz 2003; 98:443-449.) (5252. Castro-Filho J, Silveira AC. Distribuição da Doença de Chagas no Brasil. Rev Bras Malariol D Trop1979; 31:85-89., but these were not found in this study.

Triatoma brasiliensis and Triatoma pseudomaculata were found in most municipalities as expected, being the main species caught in the semiarid RN region. R . nasutus was captured only in Apodi, Caraúbas, and Caicó municipalities and P. lutzi was found only in Serra Negra do Norte and in Caraúbas, as described 5151. Lucena DT. Doença de Chagas no NordesteRev Bras Malariol D Trop1959; 11:675-696.. In agreement with our studies, T. brasiliensis was the most frequently identified species, followed by T. pseudomaculata and R. nasutus , as observed in several studies 99. Sarquis O, Borges-Pereira J, Mac Cord JR, Gomes TF, Cabello PH, Lima MM. Epidemiology of Chagas disease in Jaguaruana, Ceará, Brazil. I. Presence of triatomines and index of Trypanosoma cruzi infection in four localities of a rural area. Mem Inst Oswaldo Cruz2004; 99:263-270.) (5353. Coutinho CFS, Souza-Santos R, Teixeira NFD, Georg I, Gomes TF, Boia MN et al. An entomoepidemiological investigation of Chagas disease in the state of Ceará, Northeast Region of Brazil., Cad Saude Publica 2014; 30:785-793..

We identified T. brasiliensis in the wild environment, peridomicile, and intradomicile with high T. cruzi infection rates. This species has been reported as semi-domestic, since it is an autochthonous species capable of colonizing domiciles and the peridomicile 1212. Forattini OP. Biogeography, origin, and distribution of triatominae domicile dispersal in Brazil. Rev Saude Publica 1980; 14:265-299.. It is highly dispersed and frequent owing to its eurytopic characteristics, is not hygrophilous, is able to withstand very high temperatures, and thus is acclimatized to the vast expanse of the Northeastern region 1313. Alencar JE. História natural da doença de Chagas no Estado do Ceará, Imprensa Universitária, Fortaleza, CE, p. 341, 1987..

Despite the absence of intradomicile colonies, frequent adult T. brasiliensis invasions occur because of the proximity of the wild environment with DUs. Residential lighting attracts these insects 5454. Freitas SPC, Freitas ALC, Prazeres SM, Gonçalves TCM. Influence of anthropic habits in the dispersion of Triatoma pseudomaculata Corrêa & Espínola, 1964 through Mimosa tenuiflora (Willdenow) (Mimosaceae) in the State of Ceará, Brazil. Cad Saude Publica2004; 20:333-336., potentially explaining their localized capture. Peridomicile colonization was observed by T. brasiliensis and T. pseudomaculata nymphs, most frequently in chicken coops where they develop dense colonies, facilitating intradomiciliary invasion. T. brasiliensis was also found in piles of tiles, corrals, and associated with domestic and synanthropic animals, according to municipality. This species strongly associates with native rodents, especially Galea spp., which thrive around rural dwellings in this state 5555. Valença-Barbosa C, Lima MM, Sarquis O, Bezerra CM, Franch-Abad F. Modeling disease vector occurrence when detection is imperfect II: Drivers of site-occupancy by synanthropic Triatoma brasiliensis in the Brazilian Northeast. PLoS Negl Trop Dis 2014a; 8:e2861.. The epidemiological profile similarities among the municipalities studied suggest the need for interventions to prevent parasite transmission in this environment to domestic animals and humans.

Degradation of the natural wild vector habitat allows the bugs to move close to human habitations; such coexistence increases the probability of human infection 5656. Toledo MJO, Kühl JB, Silva SV, Gasperi V, Araújo SM. Biogeography, origin, and distribution of triatominae domiciliarity in Brazil. Rev Saude Publica1997; 3:197-203.. In the wild, T . brasiliensis was mainly found colonizing rocky outcrops and stone fences. Its occurrence in Northeastern Brazil is associated with rock formation distribution 66. Gurgel-Gonçalves R, Galvão C, Costa J, Peterson AT. Geographic distribution of Chagas disease vectors in Brazil based on ecological niche modeling. J Trop Med 2012; 2012:705326.. However, this species can also be found in shrubby cacti co-occupied by native rodents in Ceará 5757. Valença-Barbosa C, Lima MM, Sarquis O, Bezerra CM, Abad-Franch F. Short report: A common Caatinga cactus, Pilosocereus gounellei , is an important ecotope of wild Triatoma brasiliensis population in the Jaguaribe Valley of Northeastern Brazil. Am J Trop Med Hyg 2014b; 90:1059-1062..

Triatoma pseudomaculata is widely distributed in the wild and is considered to be difficult to control 55. Dias JCP, Machado EMM, Fernandes AL, Vinhaes MC General situation and perspectives of Chagas disease in Northeastern region, Brazil. Cad Saude Publica 2000; 16:13-34.. T. pseudomaculata is primarily associated with Caatinga and areas of the Cerrado in Brazil 5858. Carbajal de la Fuente AL, Porcasi X, Noireau F, Diotaiuti L, Gorla DE. The association between the geographic distribution of Triatoma pseudomaculata and Triatoma wygodzinskyi (Hemiptera: Reduviidae) with environmental variables recorded by remote sensors. Infect Genet Evol2009; 9:54-61., and is an arboricolous species 5959. Dias-Lima AG, Menezes D, Sherlock I, Noireau F. Wild habitat and related fauna of Panstrongylus lutzi (Reduviidae, Triatominae). J Med Entomol2003; 40:989-990.. Here, the natural habitat of T . pseudomaculata was identified as bird's nests, corroborating previous findings 3232. Carcavallo RU, Rodríguez MEF, Salvatella R, Curto de Casas SI, Sherlock IS, Galvão C, et alHabitats and related fauna. : Carcavallo RU, Galindéz-Girón I, Jurberg J, Lent H, editors. Atlas of Chagas Disease Vectors in the Americas. Vol II Rio de Janeiro (RJ): FIOCRUZ; 1998. p. 561-600..

In the study area, the natural R. nasutus habitat was the Carnauba palm, considered the major ecotope of this species 99. Sarquis O, Borges-Pereira J, Mac Cord JR, Gomes TF, Cabello PH, Lima MM. Epidemiology of Chagas disease in Jaguaruana, Ceará, Brazil. I. Presence of triatomines and index of Trypanosoma cruzi infection in four localities of a rural area. Mem Inst Oswaldo Cruz2004; 99:263-270.) (6060. Lima MM, Coutinho CFS, Gomes TF, Oliveira TG, Duarte R, Borges-Pereira J, et al. Risk presented by Copernicia prunifera palm trees in the Rhodnius nasutus distribution in a Chagas disease-endemic area of the Brazilian northeast. Am J Trop Med Hyg 2008; 79:750-754..Colonization foci were observed in the peridomicile in dry carnauba straw used by the rural population to manufacture household items in Apodi. This can be explained by the proximity of palm trees with DUs that attract insects as previously described or by passive insect transport by residents living in carnauba extraction areas, where storing dry leaves in the peridomicile is common. In the State of Ceará, R . nasutus has been frequently found colonizing the peridomicile owing to the use of carnauba straw for chicken coop roof construction and household goods manufacture 99. Sarquis O, Borges-Pereira J, Mac Cord JR, Gomes TF, Cabello PH, Lima MM. Epidemiology of Chagas disease in Jaguaruana, Ceará, Brazil. I. Presence of triatomines and index of Trypanosoma cruzi infection in four localities of a rural area. Mem Inst Oswaldo Cruz2004; 99:263-270.) (6161. Sarquis O, Sposina R, Oliveira TG, Mac Cord JR, Cabello PH, Borges-Pereira J et alAspects of peridomiciliary ecotopes in rural areas of Northeastern Brazil associated to triatomine (Hemiptera, Reduviidae) infestation, vectors of Chagas disease. Mem Inst Oswaldo Cruz2006; 101:143-147..

Panstrongylus lutzi was the most infected species in the intradomicile and wild environment. The former is worrying because of the T. cruzi transmission risk to domestic animals and humans and potential introduction of a new parasite genetic group into the transmission cycles, since this area is currently exclusively infected with DTU III 1515. Câmara ACJ, Varela-Freire AA, Valadares HMS, Macedo AM, D'Ávila DA, Machado CR et alGenetic analyses of Trypanosoma cruzi isolates from naturally infected triatomines and humans in northeastern Brazil. Acta Trop 2010; 115:205-211.. P. lutzi has restricted distribution in areas of the semiarid northeast although it has wide geographical distribution and a high T. cruzi infection rate in the State of Pernambuco. Its domiciliation has occurred in the States of Ceará and Pernambuco 6262. Silveira AC, Dias JCP. The control of vectorial transmission. Rev Soc Bras Med Trop2011; 44:52-63.) (6363. Silva MBA, Barreto AVMS, Silva HA, Galvão C, Rocha D, Jurberg J et al Synanthropic triatomines (Hemiptera, Reduviidae) in the State of Pernambuco, Brazil: geographical distribution and natural Trypanosoma infection rates between 2006 and 2007. Rev Soc Bras Med Trop2012; 45:60-65.. P. lutzi is found in hollow Auxemma oncocalyx trunks in Ceará 6464. Garcia MHHM, Souza L, Souza RCM, Paula AS, Borges EC, Barbosa SE et al. Occurrence and variability of Panstrongylus lutzi in the State of Ceará, Brazil. Rev Soc Bras Med Trop2005; 38:410-415.and in armadillo burrows in the semiarid Caatinga in Bahia 5959. Dias-Lima AG, Menezes D, Sherlock I, Noireau F. Wild habitat and related fauna of Panstrongylus lutzi (Reduviidae, Triatominae). J Med Entomol2003; 40:989-990.. However, the P . lutzi natural habitat was not identified despite extensive searching in the wild environment of the Seridó Ecological Station where most specimens were captured; thus, further studies are required 1515. Câmara ACJ, Varela-Freire AA, Valadares HMS, Macedo AM, D'Ávila DA, Machado CR et alGenetic analyses of Trypanosoma cruzi isolates from naturally infected triatomines and humans in northeastern Brazil. Acta Trop 2010; 115:205-211.) (5151. Lucena DT. Doença de Chagas no NordesteRev Bras Malariol D Trop1959; 11:675-696..

Currently, vector transmission is considered residual by a few native and peridomestic species such as T. brasiliensis and T. pseudomaculata . There is also a risk of progressive domiciliation of certain species previously considered sylvatic such as P. lutzi and the possibility of human infection directly related to the parasite enzootic cycle 6262. Silveira AC, Dias JCP. The control of vectorial transmission. Rev Soc Bras Med Trop2011; 44:52-63.. P. lutzi holds relevance toward maintaining the peridomestic and domestic T. cruzi transmission cycles, the risk of invasion and eventual colonization therein, and the consequent parasite transmission to domestic animals and humans. These results highlight the increasing epidemiological importance of P. lutzi and indicate the continuing necessity of maintaining epidemiological surveillance against T. cruzi transmission in the study area.

Here, we reported a slight agreement among T. cruzi detection methods used to evaluate infection in triatomines, and the importance of its association to field studies. T . cruzi infection varied among triatomine species according to method with PCR showing high positivity whereas detection by DME in intestinal contents was low. However, higher infection rates were reported with flagellates morphologically similar to T . cruzi in Pernambuco 6363. Silva MBA, Barreto AVMS, Silva HA, Galvão C, Rocha D, Jurberg J et al Synanthropic triatomines (Hemiptera, Reduviidae) in the State of Pernambuco, Brazil: geographical distribution and natural Trypanosoma infection rates between 2006 and 2007. Rev Soc Bras Med Trop2012; 45:60-65., and similar or higher triatomine infection rates have been reported between methods 1919. Shikanai-Yasuda MA, Ochs DE, Tolezano JE, Kirchhoff LV. Use of the polymerase chain reaction for detecting Trypanosoma cruzi in triatomine vectors. Trans R Soc Trop Med Hyg 1996; 90:649-651.) (2323. Braz LMA, Raiz-Jr R, Amato-Neto V, Alárcon RS, Gakyia E, Okay TS. The detection of Trypanosoma cruzi in Triatoma infestans : comparison of a PCR-based assay with microscopical examination. Ann Trop Med Parasitol 2007; 101:461-465.) (2424. Pizarro JC, Lucero DE, Stevens L. PCR reveals significantly higher rates of Trypanosoma cruzi infection than microscopy in the Chagas vector, Triatoma infestans : High rates found in Chuquisaca, Bolivia. BMC Infect Dis 2007; 7:66-73.) (2525. Dorn PL, Engelke D, Rodas A, Rosales R, Melgar S, Brahney B et alUtility of the polymerase chain reaction in detection of Trypanosoma cruzi in Guatemalan Chagas' disease vectors. Am J Trop Med Hyg 1999; 60:740-745.. High PCR positivity in relation to DME has been previously observed, indicating its utility for epidemiological studies 1919. Shikanai-Yasuda MA, Ochs DE, Tolezano JE, Kirchhoff LV. Use of the polymerase chain reaction for detecting Trypanosoma cruzi in triatomine vectors. Trans R Soc Trop Med Hyg 1996; 90:649-651.) (2323. Braz LMA, Raiz-Jr R, Amato-Neto V, Alárcon RS, Gakyia E, Okay TS. The detection of Trypanosoma cruzi in Triatoma infestans : comparison of a PCR-based assay with microscopical examination. Ann Trop Med Parasitol 2007; 101:461-465.) (2424. Pizarro JC, Lucero DE, Stevens L. PCR reveals significantly higher rates of Trypanosoma cruzi infection than microscopy in the Chagas vector, Triatoma infestans : High rates found in Chuquisaca, Bolivia. BMC Infect Dis 2007; 7:66-73.) (6565. Cominetti MC, Almeida RFC, Gonçalves GMA, Andreotti R. Monitoring Trypanosoma cruzi infection in triatomines using PCR in Mato Grosso do Sul, Brazil. Rev Soc Bras Med Trop2013; 46:277-280.. PCR can also directly detect T. cruzi vector infection thus improving triatomine evaluation and should be used to assess infection rates in dead insects 2525. Dorn PL, Engelke D, Rodas A, Rosales R, Melgar S, Brahney B et alUtility of the polymerase chain reaction in detection of Trypanosoma cruzi in Guatemalan Chagas' disease vectors. Am J Trop Med Hyg 1999; 60:740-745.owing to its higher relative degree of precision. Thus, PCR represents the best tool for parasite detection, confirming the majority of infections ascertained by DME and xenoculture. However, these remain useful as the combination of methods can contribute to monitoring T. cruzi in triatomines and enhance confidence regarding triatomine positivity.

The Trypanosoma cruzi DTUs identified in 15 isolates from triatomine bugs were TcII and TcIII in T . brasiliensis and TcIII in P . lutzi . Previous findings showed these DTUs circulating in these species in the semiarid zone of RN 1515. Câmara ACJ, Varela-Freire AA, Valadares HMS, Macedo AM, D'Ávila DA, Machado CR et alGenetic analyses of Trypanosoma cruzi isolates from naturally infected triatomines and humans in northeastern Brazil. Acta Trop 2010; 115:205-211.) (1717. Martins K, Andrade CM, Barbosa-Silva AN, Nascimento GB, Chiari E, Galvão LMC et al. Trypanosoma cruzi III causing the indeterminate form of Chagas disease in a semi-arid region of Brazil. Int J Infect Dis 2015; 39:68-75.and highlight the need to understand T . cruzi population distribution in this area. Naturally heterogeneous T . cruzi populations involving TcI, TcII, and TcIII circulate among humans and triatomines in three different municipalities without domicile colonization 1717. Martins K, Andrade CM, Barbosa-Silva AN, Nascimento GB, Chiari E, Galvão LMC et al. Trypanosoma cruzi III causing the indeterminate form of Chagas disease in a semi-arid region of Brazil. Int J Infect Dis 2015; 39:68-75., and high genetic similarities exist among T . cruzi populations circulating in different hosts, localities, and environments 1616. Câmara ACJ, Lages-Silva E, Sampaio GHF, D'Ávila DA, Chiari E, Galvão LMC. Homogeneity of Trypanosoma cruzi I, II, and III populations and the overlap of wild and domestic transmission cycles by Triatoma brasiliensis in northeastern Brazil. Parasitol Res 2013; 112:1543-1550.. TcII was detected in the peridomicile in Caicó and in the wild in Serra Negra do Norte , indicating its participation in sylvatic and peridomestic cycles. The original primary host of TcII appears to be primates. Whereas its ecological niche has yet to be determined, isolates have been described from opossums in the Atlantic forest and from sylvatic primates, suggesting that such primates might be the primary original mammalian host 6666. Lisboa CV, Mangia RH, De Lima NRC, Martins A, Dietz J, Baker AJ et al. Distinct patterns of Trypanosoma cruzi infection in Leontopithecus rosalia in distinct Atlantic coastal rainforest fragments in Rio de Janeiro - Brazil. Parasitology 2004; 129:703-711.. TcII has also been isolated from the armadillo Euphractus sexcintus in the Paraguayan Chaco 6767. Miles MA, Llewellyn MS, Lewis MD, Yeo M, Baleela R, Fitzpatrick S, et al. The molecular epidemiology and phylogeography of Trypanosoma cruzi and parallel research on Leishmania : looking back and to the future. Parasitology 2009; 136:1509-1528.. This DTU has been shown as the primary cause of severe acute and chronic Chagas disease in the Atlantic Forest and central region of Brazil and represents the etiological disease agent in São Felipe in the State of Bahia, where the domestic vector is P. megistus6767. Miles MA, Llewellyn MS, Lewis MD, Yeo M, Baleela R, Fitzpatrick S, et al. The molecular epidemiology and phylogeography of Trypanosoma cruzi and parallel research on Leishmania : looking back and to the future. Parasitology 2009; 136:1509-1528..

TcIII was isolated from T . brasiliensis in the wild environment and from P. lutzi captured in the domiciliary and wild environments of two different localities. TcIII is a poorly understood T . cruzi genetic group predominantly identified among wild cycles of parasite transmission infecting terrestrial mammals and triatomine vectors, but is also a potentially important emergent human disease agent 6868. Llewellyn MS, Lewis MD, Acosta N, Yeo M, Carrasco HJ, Segovia M et al. Trypanosoma cruzi IIc: phylogenetic and phylogeographic insights from sequence and microsatellite analysis and potential impact on emergent Chagas'disease. PLoS Negl Trop Dis2009; 3:e510.. A few triatomine species have been described in sylvatic TcIII transmission such as Panstrongylus geniculatus Latreille, 1811, and Triatoma rubrovaria Blanchard, 1843, both mainly sylvatic vectors frequently associated with terrestrial ecotopes 6969. Gaunt M, Miles M. The ecotopes and evolution of triatomine bugs (Triatominae) and their associated trypanosomes. Mem Inst Oswaldo Cruz2000; 95:557-565.. TcIII is found in a broad range of terrestrial mammals and its transmission may occur inside triatomine-infested burrows by both vectorial and oral routes 2929. Marcili A, Lima L, Valente VC, Valente SA, Batista JS, Junqueira ACV et al. Comparative phylogeography of Trypanosoma cruzi TCIIc: New hosts, association with terrestrial ecotopes, and spatial clustering. Infect Genet Evol 2009; 9:1265-1274.) (7070. Yeo M, Acosta N, Llewellyn M, Sánchez H, Adamson S, Miles GA et al. Origins of Chagas disease: Didelphis species are natural host of Trypanosoma cruzi I and armadillos hosts of Trypanosoma cruzi II, including hybrids. Int J Parasitol2005; 35:225-233.. Overlapping geographic areas of TcIII and TcI isolates occur across South America, with shared wild mammals and vectors in terrestrial ecotopes 2929. Marcili A, Lima L, Valente VC, Valente SA, Batista JS, Junqueira ACV et al. Comparative phylogeography of Trypanosoma cruzi TCIIc: New hosts, association with terrestrial ecotopes, and spatial clustering. Infect Genet Evol 2009; 9:1265-1274.. TcIII in domestic transmission cycles, while intermittent 6868. Llewellyn MS, Lewis MD, Acosta N, Yeo M, Carrasco HJ, Segovia M et al. Trypanosoma cruzi IIc: phylogenetic and phylogeographic insights from sequence and microsatellite analysis and potential impact on emergent Chagas'disease. PLoS Negl Trop Dis2009; 3:e510., implies a role as a human disease agent. Furthermore, TcIII may be under-reported in both domestic and sylvatic transmission cycles because some typing methodologies fail to distinguish between TcIV and TcIII 7171. Fernandes O, Souto RP, Castro JA, Pereira JB, Fernandes NC, Junqueira ACV et al. 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 1998; 58:807-811.. The TcII and TcIII identified in this study corroborate previous findings and reinforce the need for constant epidemiological surveillance of T . brasiliensis and P. lutzi to prevent TcIII spread to the domestic cycle 1515. Câmara ACJ, Varela-Freire AA, Valadares HMS, Macedo AM, D'Ávila DA, Machado CR et alGenetic analyses of Trypanosoma cruzi isolates from naturally infected triatomines and humans in northeastern Brazil. Acta Trop 2010; 115:205-211..

TcI was not detected in triatomines in the studied area. However, most studies conducted in northeastern Brazil have isolated this DTU from T . brasiliensis and T . pseudomaculata , with most naturally infected T. brasiliensis isolates being TcI 7272. Pacheco RS, Brito CMM, Sarquis O, Pires MQ, Borges-Pereira J, Lima MM. Genetic heterogeneity in Trypanosoma cruzi strains from naturally infected triatomine vectors in northeastern Brazil: epidemiological implications. Biochem Genet 2005; 43:519-530.. TcI has also been identified in T . pseudomaculata and R. nasutus in the peridomicile and natural environments of five State of Ceará peri-urban and urban localities 7373. Brito CMM, Lima MM, Sarquis O, Pires MQ, Coutinho CFS, Duarte R et al. Genetic polymorphism in Trypanosoma cruzi I isolated from Brazilian Northeast triatomines revealed by low-stringency single specific primer-polymerase chain reaction. Parasitol Res2008; 103:1111-1117.. Therein, high TcI and TcII infection rates were detected in peridomestic T . brasiliensis and T . pseudomaculata , respectively 7474. Gumiel M, da Mota FF, Rizzo VS, Sarquis O, de Castro DP, Lima MM et al. Characterization of the microbiota in the guts of Triatoma brasiliensis and Triatoma pseudomaculata infected by Trypanosoma cruzi in natural conditions using culture independent methods. Parasit Vectors 2015; 8:245.. TcI has been most frequently identified in triatomines of the State of Mato Grosso do Sul 7575. Cominetti MC, Csordas BG, Cunha RC, Andreotti R. Geographical distribution of Trypanosoma cruzi in triatomine vectors in the State of Mato Grosso do Sul, Brazil. Rev Soc Bras Med Trop2014; 47:747-755.. The majority of isolates from humans, reservoirs, and vectors from Amazonia correspond to TcI 2929. Marcili A, Lima L, Valente VC, Valente SA, Batista JS, Junqueira ACV et al. Comparative phylogeography of Trypanosoma cruzi TCIIc: New hosts, association with terrestrial ecotopes, and spatial clustering. Infect Genet Evol 2009; 9:1265-1274.and it has been detected in humans from State of RN 1515. Câmara ACJ, Varela-Freire AA, Valadares HMS, Macedo AM, D'Ávila DA, Machado CR et alGenetic analyses of Trypanosoma cruzi isolates from naturally infected triatomines and humans in northeastern Brazil. Acta Trop 2010; 115:205-211.) (1616. Câmara ACJ, Lages-Silva E, Sampaio GHF, D'Ávila DA, Chiari E, Galvão LMC. Homogeneity of Trypanosoma cruzi I, II, and III populations and the overlap of wild and domestic transmission cycles by Triatoma brasiliensis in northeastern Brazil. Parasitol Res 2013; 112:1543-1550., with recent isolates from patients with various clinical forms of Chagas disease 1717. Martins K, Andrade CM, Barbosa-Silva AN, Nascimento GB, Chiari E, Galvão LMC et al. Trypanosoma cruzi III causing the indeterminate form of Chagas disease in a semi-arid region of Brazil. Int J Infect Dis 2015; 39:68-75.. T. cruzi genotypes isolated from triatomines can vary according to vector-specific physiology and ecological habitat 4343. Zingales B, Andrade SG, Briones MRS, Campbell DA, Chiari E, Fernandes O et al. A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz2009; 104:1051-1054., highlighting the necessary to accurately identify TcI in triatomines.

Our results suggest that combining methodologies contributed to increased parasite detection and to identifying infection foci to precisely determine triatomine distribution. Despite low parasite detection via xenoculture and DME, these methods allow the isolation of T. cruzi for further study. Owing to high peridomestic ecotope infestation and high T . brasiliensis and T . pseudomaculata T . cruzi infection, we recommend efficient entomological surveillance programs to detect possible colonization. This study contributes to our knowledge of T. cruzi diagnosis and identification in field-collected triatomines, further demonstrating that T. cruzi II and III predominate in the study area and, with future research, are essential for vector control and human infection prevention.

ACKNOWLEDGMENTS

The authors would like to thank the Secretariat of State for Public Health of Rio Grande do Norte, represented by the health authorities and agents of the Municipal Secretaries of the West and Central mesoregions for their indispensable support of the survey field activities. The authors are also grateful to Adalberto Antonio Varela-Freire MSc ( in memoriam ) and Philip S.P. Badiz for critical reading and revision of the manuscript.

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  • This work was supported by research grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico MCT/CNPq number 472251/2010-4 (EC); MCTI/CNPq/MS-SCTIE-Decit number 404056/2012-1 (LMCG); Programa Nacional de Incentivo à Parasitologia Básica/CAPES number 23038.005288/2011-48 (ACJC); research fellowships from the CNPq (LMCG and EC), and a scholarship from CAPES (ANBS).

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    Jan-Feb 2016
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