Chagas Disease Vectors of Paraguay: Entomoepidemiological Aspects of Triatoma sordida (Stål, 1859) and Development of an Identification Key for Paraguayan Triatomines Based on Cytogenetics Data

Abstract.

Approximately 150,000 people are living with Chagas disease in Paraguay. Although the country has been since 2008 considered as one of the countries that succeeded in interrupted the vector transmission of Chagas by Triatoma infestans in houses of the eastern region, there are nine other species notified in the country that are potential vectors and also deserve attention from vector control programs. Thus, we carried out an entomoepidemiological study of T. sordida in the eastern and western regions of the country and we developed an identification key for Paraguay's triatomines based on cytogenetic data. Between the years 2003 to 2004, 271 specimens of T. sordida were captured in domestic, peridomestic, and wild ecotopes, with 131 insects caught in the eastern (Alto Paraguay, Boquerón and Pte. Hayes) and 140 in the western region of Paraguay (Guairá and Paraguarí). High rates of peridomicillary infestation were observed for both regions. Besides that, the natural infection of the captured insects was detected by optical microscopy in 12% and 10%, and by PCR in 21% and 20% in the eastern and western regions, respectively. Based on cytogenetic data from nine of ten species notified in Paraguay, an identification key was developed to differentiate all taxa. Thus, given the vectorial importance of T. sordida, we highlight the need for continued attention from Paraguay's vector control programs for this species. Further, we provide a taxonomic key that assists in the correct classification of Paraguayan triatomines.

INTRODUCTION

Chagas disease is a potentially life-threatening neglected tropical disease caused by the protozoan Trypanosoma cruzi (Chagas, 1909) (Kinetoplastida, Trypanosomatidae).¹ It is found mainly in 21 Latin American countries, where it is mostly vector-borne, more specifically by triatomines (Hemiptera, Triatominae).¹ An estimated 8 million people are infected worldwide, and more than 25 million people at risk of acquiring the disease, which causes more than 10,000 deaths per year, being the vector control remains the most useful method to prevent new infections.¹

Approximately 150,000 people are living with Chagas disease in Paraguay. Although the country has been since 2008 considered as one of the countries that succeeded in interrupted the vector transmission of Chagas by Triatoma infestans (Klug, 1834) in houses of the eastern region (which represents a significant public health achievement in the country),² there are nine other species notified in the country (grouped in the Psammolestes Bergroth, 1911, Panstrongylus Berg, 1879 and Triatoma Laporte, 1832 genera)³ that are potential vectors and also deserve attention from vector control programs.

Triatoma sordida (Stål, 1859), for example, is a species of secondary importance for the transmission of the etiologic agent of Chagas disease that, after controlling the T. infestans domiciliary populations,² has been gaining epidemiological importance,^4–6 once control activities passed to notify T. sordida in regions where T. infestans had been eliminated.⁷ This vector, abundant in peridomiciliary regions,^7,8 has the adaptive capacity to inhabit houses,⁷ as well as an estimated 87% risk of domiciliary transmission of T. cruzi in the Chaco Paraguay region.⁵

Thus, to assist in the entomological monitoring of Chagas disease vectors in Paraguay, we carried out an entomoepidemiological study of T. sordida in the eastern and western regions of the country, developing an identification key for Paraguay's triatomines based on cytogenetic data.

MATERIALS AND METHODS

Between the years 2003 to 2004, 271 specimens of T. sordida were captured in domestic, peridomestic and wild ecotopes, being 131 insects caught in the eastern (Alto Paraguay, Boquerón and Pte. Hayes) and 140 in the western region of Paraguay (Guairá and Paraguarí) to calculate the infestation levels. Triatomines were classified based on the taxonomic key of Lent and Wygodzinsky.⁹ The analysis of the infection rate by optical microscopy (OM) was performed based on Gonzalez-Britez et al.,¹⁰ and the intestinal content from all adult specimens (131 from the eastern and 140 from the western region) was extracted and examined. Also, based on Sánchez et al.,¹¹ the infection rate analysis was also performed by polymerase chain reaction (PCR) in 77 specimens from the eastern and 60 from the western region that were randomly selected from the samples analyzed by MO. Additionally, from cytogenetic data of triatomines present in Paraguay [chromosome number analyzed with lacto-acetic orcein, constitutive heterochromatin pattern analyzed with C-banding, as well as localization of 45S rDNA probe analyzed with fluorescent in situ hybridization (FISH) and composition of AT-rich and CG-rich DNA analyzed with CMA₃/DAPI],^12–18 an identification key was developed based on Borsatto et al.^19,20 Still, from the literature data, we have grouped all notifications of T. sordida in the different ecotopes of Paraguay, as well as information related to infection rates and food sources.

RESULTS AND DISCUSSION

As reported by Moncayo and Silveira,⁸ high rates of peridomiciliary infestation were observed in the eastern and western regions of Paraguay (Table 1). For the eastern region, although no specimens have been collected in the domiciliary environment of Alto Paraguay (Table 1), Sánchez et al.⁵ have already reported infected T. sordida in houses from that department (Table 2). In addition, despite Sánchez et al.⁵ also have notified this species infected with T. cruzi in areas of peridomicile and intradomicile of Boquerón and Pte. Hayes (Table 2), we present for the first time the collection record of specimens in wild environments of these departments (Table 1), which may represent an important link in the epidemiological chain to maintain the vectorial transmission of Chagas disease.²¹ Triatoma sordida has also been notified infected in the departments of San Pedro and Cordillera (peridomicile and intradomicile) and Conception (intradomicile), as well as without infection in the Central department (intradomicile) (Table 2).^8,11,22

Table 1.

Number of specimens and infestation rate of T. sordida collected in the eastern and western regions of Paraguay

Paraguay		T. sordida
Region	Department	D	P	W
	Alto Paraguay	0	27	15
Eastern	Boquerón	6	31	5
	Pte. Hayes	16	28	3
	Infestation	16.8%	65.6%	17.6%
Western	Guairá	11	53	0
	Paraguarí	17	59	0
	Infestation	20%	80%	0%

D = domicily; P = peridomocily; W = wild.

Table 2.

Review of ecotopes, infection, and food source of T. sordida from Paraguay

Region	Department	D	P	W	I	II	IP	FS
	Alto Paraguay	+5	+*5	+*	+*5	+5	+5	–
	Boquerón	+*5	+*5	+*	+*5	+5	+5	–
	Pte. Hayes	+*5,8	+*5,8	+*	+*5	+5	+5	Human,6,8 bird,6,8 rodent,6 cat,6 dog,6,8 goat6
Eastern	Concepción	+3	–	–	+3	+3	–	Human,3 chicken3
	San Pedro	+1,4	+4	–	+4	+4	+4	Human,1,4 bird,4 marsupial,4 rodent4
	Cordillera	+1,2,4	+4	–	+4	+4	+4	Human,1,4 Bird4
	Central	+2	–	–	–	–	–	–
	Guairá	+*	+*	–	+*	–	–	–
Western	Misiones	+2	–	–	–	–	–	–
	Paraguarí	+*1, 2,4	+*4	–	+*4	+4	+4	Human,1,4–7 Bird,4–7 Marsupial,4 Rodent,4–7 Cat,5–7 Dog,4–7 Goat6,7

⁺= with notification; – = without notification; * = notified by the authors; ¹Sánchez et al.⁶; ²Moncayo and Silveira⁸; ³Sánchez et al.²³; ⁴Sánches et al.¹¹; ⁵Sánches et al.⁵; ⁶Gonzalez et al.⁶; ⁷Gonzalez-Britez et al.¹⁰; ⁸Fraenkel et al.²⁴; D = domiciliary; FS = food source; IP = natural infection with T. cruzi in the region peridomiciliary; P = peridomiciliary; W = wild; I = natural infection with T. cruzi; II = natural infection with T. cruzi in the intradomiciliary region.

As for the western region, the observed results for Paraguarí (Table 1) resemble those already reported in the literature (Table 2), and, for the first time, T. sordida was reported in peridomiciliary and intradomiciliary ecotopes in the department of Guairá (Table 1 and 2). Besides, this vector has already been reported in the domiciliary region of the Misiones department (Table 2). It is worth noting that no specimens were collected in the wild environment of the western of Paraguay (Table 1).

Sánchez et al.⁵ highlight that vector control in the Paraguayan Chaco (western region) was limited by the isolation of the houses, through the persistence of domestic infestations, as well as by cultural customs of the inhabitants (indigenous tribes), being the detection of natural infection and the identification of the food source of triatomines important to assess the factors associated with the species' capacity and vectorial competence.

As observed in the present study (Table 1), other entomoepidemiological studies carried out in the 21st century in the western region also point to the same rates shown in Table 1 (for example, infestation rates from 18–20% in domicile and 81% in peridomicile).⁶ Besides, Sánchez et al.⁶ observed T. cruzi infection in 17.3% of the analyzed samples [similar to those of the western region (Table 3)], confirming the vectorial capacity of T. sordida.

Table 3.

Infection rate of specimens and infestation rate of T. sordida collected in the eastern and western regions of Paraguay.

T. sordida		MO		PCR
Region	Department	n	P	n	P
	Alto Paraguay	42	3 (7.1%)	24	4 (16.6%)
Eastern	Boquerón	42	5 (11.9%)	24	4 (16.6%)
	Pte. Hayes	47	8 (17%)	29	8 (27.6%)
Western	Guairá	64	5 (7.8%)	26	4 (15.4%)
	Paraguarí	76	9 (11.8%)	34	8 (23.5%)

MO = optica microscopy; n = sample; P = positive for T. cruzi; PCR = polymerase chain reaction.

Food sources were evaluated in T. sordida from four departments in the eastern and one department in the western region (Table 2), being mammals and birds the main blood source (Table 2). It is worth mentioning that human blood was detected in specimens from all evaluated departments (Table 2). In the eastern region, for example, the rates of human blood among the samples already analyzed were: Concepción (72%),²² San Pedro (10%),¹¹ Pte. Hayes (24%)¹⁰ and Cordillera (82%).¹¹ In the western region, the rate was 7.5% in T. sordida of Paraguarí.¹¹

Of the 131 T. sordida from the eastern and 140 from the western region, analyzed by OM, the natural infection was detected in approximately 12% and 10%, respectively (Table 3). As for the 77 specimens from the eastern and 60 from the western region analyzed by PCR, about 21% and 20%, respectively, were infected (Table 3) (confirming the importance of molecular analyses to assess the infection rate of triatomines). In general, infection rates followed the decreasing pattern: Pte. Hayes > Boquerón > Alto Paraguay in the eastern and Paraguarí > Guairá in the western region (Table 3).

The infection rates observed by Sánchez et al.¹¹ in Paraguarí were similar to those observed in Table 3 by PCR. This department, along with Cordillera, is characterized as endemic areas in the western region, with a human infection rate ranging from 10–12% in the 15 to 45 age group.⁵ In addition, the infection rates analyzed by PCR for the Chaco region (20%) are similar to that observed by Sánchez et al.⁵ These data, when combined to the domiciliary colonization of T. sordida infected with T. cruzi⁶ and the high rate of infection in indigenous communities (over 50%)⁶ highlight one more time the vectorial importance of T. sordida in the western region.

Based on karyotype, constitutive heterochromatin pattern in chromatin and chromosomes, as well as localization of 45S rDNA probe and CMA3/DAPI pattern in chromosomes for nine of ten species notified in Paraguay (with the exception of P. guentheri Berg, 1879 that does not present cytogenetic data available in the literature), an identification key was developed and allowed to differentiate all taxa (Table 4). As all species of triatomines are potential vectors of Chagas disease,⁹ developing alternatives for the correct identification of these vectors (such as the taxonomic key proposed here) can assist in the activities of the Paraguayan health professionals working with vector control.

Table 4.

Identification key for Paraguayan triatomines based on cytogenetic data

Identification key
1. Karyotype with 2n = 21 chromosomes (18A + X1X2Y)	Panstrongylus megistus
2. Karyotype with 2n = 22 chromosomes (20A + XY)	3
3a. Prophase without heterochromatin blocks dispersed inside the nucleus	4
3b. Prophase with heterochromatic blocks dispersed inside the nucleus	6
4a. 45S rDNA probe located in X and Y chromosomes	Psammolestes coreodes
4b. 45S rDNA probe located in an autosome pair	5
5a. An autosome pair with dot CMA3 in one end	Triatoma guazu
5b. Without dot CMA3 in autosomes	Triatoma guasayana
6a. Heterochromatin in 3–4 large pairs of autosomes	7
6b. Heterochromatin in all autosomes	8
7a. X chromosome with heterochromatin	Triatoma infestans
7b. X chromosome without heterochromatin	Triatoma platensis
8a. 45S rDNA probe located in X chromosome	Triatoma sordida
8b. 45S rDNA probe located in a large autosome pair and X chromosome	Triatoma delpontei
9. Karyotype with 2n = 23 chromosomes (20A + X1X2Y)	Panstrongylus geniculatus

Thus, given the vectorial importance of T. sordida, we highlight the need for continued attention from Paraguay's vector control programs for this species. Besides, we contribute to the development of a taxonomic key that assists in the correct classification of notified triatomines until the moment in Paraguay.