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Am. J. Trop. Med. Hyg., 80(2), 2009, pp. 239-241
Copyright © 2009 by The American Society of Tropical Medicine and Hygiene

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SHORT REPORT


Concurrent Detection of Trypanosoma cruzi Lineages I and II in Domestic Triatoma dimidiata from Guatemala

Pamela M. Pennington*, Claudia Paiz, Laura M. Grajeda, AND Celia Cordón-Rosales
Center for Health Studies, Universidad del Valle de Guatemala, Guatemala City, Guatemala

 

ABSTRACT

The agent of Chagas disease, Trypanosoma cruzi, is phylogenetically divided into two lineages, T. cruzi I (TCI) and II (TCII). TCI is found in sylvatic and domestic habitats across South America. Despite a high prevalence of TCII in domestic habitats in South America, it has been rarely found in domestic habitats in Central America and Mexico. This may be caused by limitations in detection tests previously used. A modified hemi-nested polymerase chain reaction assay was developed to improve detection of TCI and TCII mini-exon genes. This method detected TCI and II concurrently in 84% of 44 cultured isolates from Triatoma dimidiata specimens collected inside homes across the disease-endemic area in Guatemala.

Trypanosoma cruzi, the agent of Chagas disease, is divided into two genetically and biologically distinct lineages, T. cruzi I (TCI) and T. cruzi II (TCII).1–6 TCII is subdivided into five discrete typing units, IIa–e. 7,8 TCIIa and IIc were derived from an ancestral hybridization between TCI and TCIIb. TCIId and TCIIe were derived from a hybridization between TCIIb and TCIIc.9 TCI is associated with sylvatic and domestic environments across South America, having a strong association with arboreal marsupial reservoirs.3–5,10,11 TCII is associated with domestic environments in South America, with sylvatic raccoons in North America and sylvatic armadillos in South America. 3,12–19

Studies on T. cruzi diversity in Central America and Mexico suggest that TCI is the primary lineage circulating in domestic and sylvatic environments. 15,20–26 In most of these studies, parasites were isolated from human blood or triatomine vectors through passage in mice and/or culture. 20,22–25 Some studies in Mexico typed parasites directly in triatomine blood meals by polymerase chain reaction (PCR). 21,26 Cultured isolates were typed by isoenzyme analysis and molecular markers such as the mini-exon gene. 15,20,22–25 Among these studies, those that used the mini-exon marker to type TCI and TCII used a multiplex PCR procedure designed to amplify in one tube different size products representing each lineage, in which the mini-exon for TCI is 200 basepairs (bp) and the mini-exon for TCII is 250 bp. 5,20–24,26–28

Using the published mini-exon multiplex assay, only the TCII product was obtained with the CL Brener strain, a TCIIb/TCIIc hybrid. 23 Having a TCIIc ancestor, this strain has retained a TCI mini-exon sequence in its genome. 29 This finding suggests that the multiplex assay is not efficient at detecting both genes simultaneously under certain conditions, possibly because of different amplification efficiencies. Recently, the mini-exon PCR procedure was adapted to amplify the TCI and II mini-exon genes in two tubes and with a hemi-nested format to increase sensitivity. 18 For the present study, a hemi-nested PCR was designed to increase the sensitivity for both genes in cultured specimens. To verify the presence of TCII in the samples, a TCII-specific PCR was repeated in a separate reaction from TCI to minimize competition between the two targets in hybrid strains or mixed infections.

The modified PCR was tested on T. cruzi isolates cultured from the intestines of Triatoma dimidiata. Triatomines were collected in domestic environments across the disease-endemic area of Guatemala during a baseline entomologic survey (2000–2005). Houses were randomly selected (n = 7,271) in 572 randomly chosen villages across the departments of Zacapa, Jutiapa, Santa Rosa, and Chiquimula. The house sample size was estimated on the basis of the 1994 National Census and on parameters of an estimated 30% infestation level (5% precision, 95% confidence interval, 80% statistical power). Two entomology technicians searched each house for 15–30 minutes. Collected triatomines were microscopically screened for T. cruzi metacyclic infection and selected positive live specimens were asceptically dissected. The intestinal contents were homogenized in 1 mL of penicillin/streptomycin (3,000 U/mL), incubated overnight at 4°C, and cultured at 28°C in 3 mL of liver infusion tryptose liquid medium supplemented with 10% fetal bovine serum. DNA was extracted from positive cultures with the IsoQuick kit according to manufacturer’s instructions (Orca Research Inc., Bothwell, WA).

A hemi-nested PCR was designed as an adaptation of previously published primer sequences (Figure 1AGo). The external PCR was performed in a final volume of 20 µ L with 1 µ L of DNA, 0.2 µ M of each primer KMe1F (5'-TTCTGTACT ATATTGGTA-3') and KMe1R (5'-CAATATAGTACAGAA ACTG-3'), 0.15 mM of each dNTP, 1.5 mM MgCl2, 0.04 U/µ L of Taq polymerase, 75 mM Tris-HCl, pH 8.5, 20 mM (NH4)2SO4, and 0.1% Tween 20 (CLP, San Diego, CA). 30 An initial 5-minute denaturation at 94°C was followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 48°C for 30 seconds, extension at 72°C for 30 seconds, and a final extension of 72°C for 10 minutes. For the second reaction, 1 µ L of the first reaction was added to a master mixture containing the same reagent conditions as before, except for a different combination of primers, each at 0.15 µ M. 28 This multiplex detects TCI (200 basepairs bp), TCII (250 basepairs), Zymodeme 3 (150 basepairs) and T. rangeli (100 basepairs) using the primers: TC1F (5'-ACACTTTCTGTGGCGCTGATCG-3'), TC2F (5'-TTGCTCGCACACTCGGCTGCAT-3'), TC3F (5'-CCGCGAACAACCCCTAATAAAAATG-3'), TRR (5'-CCTATT GTGATCCCCATCTTCG-3'), and MER (5'-TACCAATAT AGTACAGAAACTG-3'). The PCR conditions were the same as those for the external reaction except for an annealing at 55°C. The external and internal reactions were performed in different laminar flow hoods and mineral oil was used to prevent product aerosols. Negative reagent controls were included with all reactions. Products were separated by electrophoresis on 10% polyacrylamide gels (24:1) in 0.5x Tris-borate-EDTA buffer using a Mini Protean II vertical gel system (Bio-Rad, Hercules, CA) and visualized with silver staining or ethidium bromide staining under ultraviolet light.


Figure 1
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  		FIGURE 1. Hemi-nested multiplex polymerase chain reaction (PCR) for the mini-exon genes for Trypanosoma cruzi lineages I (TCI) and II (TCII). A , Schematic representation of hemi-nested PCR design. Arrows show the 5' to 3' direction of the primers. Gray boxes indicate mini-exon repeats. External primers (KMe1F, KMe1R), internal primers for TCI and TCII (TC1F, TC2F, MER) (TC3 and TR not shown). B, Polyacrylamide gel electrophoresis of amplified TCI (200 basepairs) and TCII (250 basepairs) mini-exon genes in cultures from Triatoma dimidiata intestinal contents. DNA was extracted from T. cruzi strains cultured from intestinal contents and the hemi-nested multiplex PCR was performed. Tula = Tulahuen, TCII strain from Chile; H7 = TCI strain from Guatemala; 27, 29, 30, 33, 34, 35, and 36 = T. cruzi strains isolated from T. dimidiata; C = negative reagent control; - = unloaded lane; Tr = T. rangeli control; MM = 25-basepair molecular mass marker (Promega, Madison, WI).

 
A total of 44 isolates were analyzed from across Guatemala: Jutiapa (n = 24), Zacapa (n = 1), Chiquimula (n = 3), and Santa Rosa (n = 16). This technique detected TCI and TCII concurrently in 84% (37 of 44) of T. dimidiata specimens collected inside homes across 29 different municipios in four departments of Guatemala. Single TCI infections were detected in 16% (7 of 44) of the samples. TCI and TCII were detected concurrently in specimens from all four departments. Figure 1BGo shows an example of amplification results of both genes in some cultured samples and the controls used. The H7 strain was recently typed with the mini-exon gene as TCI but our PCR conditions showed the presence of TCI and TCII in this strain. 23 The product observed above the 300-basepair marker in Tula and H7 is probably the 600-basepair product expected from the external primers.

Although these findings contradict previous results for the region, several nonexclusive scenarios could explain them. TCI may grow better in culture and/or may be found in higher concentrations in triatomines, making it difficult to detect TCII with the published multiplex. 19 TCI may be the primary lineage infecting humans, and TCII may be maintained by a domestic reservoir. Finally, there may be a high prevalence of hybrid strains circulating in domestic environments. These results provide a new perspective on the transmission dynamics of Chagas disease in Central America.

Molecular evolution studies estimate that both lineages may have diverged 3–88 million years ago. 4,31 One hypothesis proposes that an ancestral lineage was distributed in North and South America 84.4 million years ago, when both continents were still connected.2 After the Cenozoic Era, South America separated from North America and TCI developed in South America, associated with marsupials.2 TCII would have developed in North America in association with placentals, subsequently entering South America during the Oligocene Epoch (38 million years ago) with the island hoppers (primates and rodents) or during the Pliocene-Pleistocene Boundary (5 million years ago) through the great faunal exchange by the Panama isthmus.

Another hypothesis suggests that both lineages evolved in South America after the Cretacic Period and Cenozoic Era.2 In this case, TCI would have been associated with arboreal marsupials and TCII with cave-dwelling edentates such as armadillos. 10,32 Cave-dwelling placentals that migrated to South America after the Oligocene Epoch would have become infected with TCII upon entry into these niches. We propose that, regardless of the origin of these lineages, the great faunal exchange would have enabled both lineages to enter Mesoamerica.

Thus, the high proportion of TCI/TCII strains could have important epidemiologic and evolutionary implications. Given that TCI and TCII show different susceptibilities to drugs, the resistance profiles of Central American strains should be evaluated and compared with those of South American strains. 33 Isoenzyme analysis of Central American strains previously showed some evidence of genetic exchange, with lack of deviation from Hardy-Weinberg equilibrium at one of three loci. 34 This finding may be explained by the presence of mixed infections. The characterization of cloned strains and the use of microsatellites are currently underway and should help elucidate whether these strains are mixed or hybrid strains. Future work will include subtyping of the TCII samples. On the basis of these results, we propose that the isthmus provided an environment that enabled interaction between hosts carrying both lineages during faunal exchanges, leading to mixed infections and/or hybrid formation in cave-dwelling triatomine species such as T. dimidiata.

Received July 8, 2008. Accepted for publication October 20, 2008.

Acknowledgments: We thank Ben Beard and Ellen Dotson for their support during these research projects, and Sandra Juarez, Ben Beard, and Henry Bishop for providing control strains.

Financial support: This study was partially supported by grants from the Gorgas Memorial Research Institute Award and Tropical Disease Research grant A30460 to Pamela M. Pennington, and project 085–2006, "Estudio del Ciclo de Transmisión del Trypanosoma cruzi Mediante la Tipificación Genética de Este Parásito," funded by Fondo para el Desarrollo de la Ciencia y Tecnología within Consejo Nacional de Ciencia y Tecnología to Laura M. Grajeda.

* Address correspondence to Pamela M. Pennington, Center for Health Studies, Research Institute, Universidad del Valle de Guatemala, 18 Avenida 11-95 Zona 15 VHIII, Guatemala City, Guatemala, 01015. E-mail: pamelap{at}uvg.edu.gt Back

Authors’ addresses: Pamela M. Pennington, Claudia Paiz, Laura M. Grajeda, and Celia Cordón-Rosales, Center for Health Studies, Research Institute, Universidad del Valle de Guatemala, 18 Avenida 11-95 Zona 15 VHIII, Guatemala City, Guatemala, 01015, E-mails: pamelap{at}uvg.edu.gt and ccordon{at}gt.cdc.gov.

 

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