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    Hybridization patterns of blood and T. infestans used in xenodiagnosis. (A) Ethidium bromide staining from blood and T. infestans. (B) Hybridization with the universal probe. (C–F) Patterns of hybridization with probes TcIIb, TcIId, TcI, and TcIIe, respectively.

  • 1

    OPS/OMS, 1999. Informe de la VII Reunión de la Comisión Intergubernamental de la Iniciativa del Cono Sur. Washington, DC. Organización Panamericana de la Salud.

  • 2

    Andrade SG, 1999. Trypanosoma cruzi: clonal structure of parasite strains and the importance of principal clones. Mem Inst Oswaldo Cruz 94 :185–187.

    • Search Google Scholar
    • Export Citation
  • 3

    Tibayrenc M, Ayala FJ, 1988. Isozyme variability in Trypanosoma cruzi, the agent of Chagas disease: genetical, taxonomical, and epidemiological significance. Evolution 42 :277–292.

    • Search Google Scholar
    • Export Citation
  • 4

    Machado C, Ayala FJ, 2001. Nucleotide sequences provide evidence of genetic exchange among distantly related lineages of Trypanosoma cruzi. Proc Natl Acad Sci USA 98: 7396–7401.

    • Search Google Scholar
    • Export Citation
  • 5

    Gaunt MC, Yeo M, Frame IA, Russell Stethard J, Carrasco HJ, Taylor M, Solis S, Veazey P, Miles GAJ, Acosta N, Rojas de Arias A, Miles MA, 2003. Mechanism of genetic exchange in American trypanosomes. Nature 421 :936–939.

    • Search Google Scholar
    • Export Citation
  • 6

    Chiari E, 1999. Chagas disease diagnosis using polymerase chain reaction, hemoculture and serologic methods. Mem Inst Oswaldo Cruz 94 :299–300.

    • Search Google Scholar
    • Export Citation
  • 7

    Solari A, Campillay R, Ortiz S, Wallace A, 2001. Identification of Trypanosoma cruzi genotypes circulating in Chilean chagasic patients. Exp Parasitol 97 :226–233.

    • Search Google Scholar
    • Export Citation
  • 8

    Miles MA, Apt W, Widmer G, Povoa MM, Schofield CJ, 1984. Isozyme heterogeneity and numerical taxonomy of Trypanosoma cruzi stocks from Chile. Trans R Soc Trop Med Hyg 78 :526–535.

    • Search Google Scholar
    • Export Citation
  • 9

    Devera R, Fernandez O, Coura JR, 2003. Should Trypanosoma cruzi be called “cruzi” complex? A review of the parasite diversity and the potential of selecting population after in vitro culturing and mice infection. Mem Inst Oswaldo Cruz 98 :1–12.

    • Search Google Scholar
    • Export Citation
  • 10

    Tibayrenc M, Ayala F, 1987. Forte corrélation entre classification isoenzymatique et variabilité de l’ADN kinétoplastique chez Trypanosoma cruzi. CR Acad Sci III 304 :89–92.

    • Search Google Scholar
    • Export Citation
  • 11

    Carreño H, Rojas C, Aguilera X, Apt W, Miles MA, Solari A, 1987. Schizodeme analyses of Trypanosoma cruzi zymodemes from Chile. Exp Parasitol 64 :252–260.

    • Search Google Scholar
    • Export Citation
  • 12

    Barnabé C, Brisse S, Tibayrenc M, 2000. Population structure and genetic typing of Trypanosoma cruzi, the agent of Chagas disease: a multilocus enzyme electrophoresis approach. Parasitology 120 :513–526.

    • Search Google Scholar
    • Export Citation
  • 13

    Montamat E, Durand S, Bocco JL, De Luca GM, Blanco A, 1999. Identification of Trypanosoma cruzi zymodemes by kinetoplast DNA probes. J Eukaryot Microbiol 46 :155–159.

    • Search Google Scholar
    • Export Citation
  • 14

    Martins-Filho OA, Eloi-Santos SM, Teixeira Carvalho A, Corrêa Oliveira R, Rassi A, 2002. Double-Blind study to evaluate flow cytometry analysis of anti-live trypomastigote antibodies for monitoring treatment efficacy in cases of human Chagas disease. Clin Diagn Lab Immunol 9 :1107–1113.

    • Search Google Scholar
    • Export Citation
  • 15

    Schenone H, 1999. Xenodiagnosis. Mem Inst Oswaldo Cruz 94 :289–294.

  • 16

    Zulantay I, Honores P, Solari A, Apt W, Ortiz S, Osuna A, Rojas A, Lopez B, Sanchez G, 2004. Use of polymerase chain reaction (PCR) and hybridization assays to detect Trypanosoma cruzi in chronic chagasic patients treated with itraconazole or allopurinol. Diagn Microbiol Infect Dis 48 :253–257.

    • Search Google Scholar
    • Export Citation
  • 17

    Torres JP, Ortiz S, Muñoz S, Solari A, 2004. Trypanosoma cruzi isolates from Chile are heterogeneous and composed of mixed populations when characterized by schizodeme and Southern analyses. Parasitology 128 :161–168.

    • Search Google Scholar
    • Export Citation
  • 18

    Revollo S, Oury B, Laurent JP, Barnabé C, Quesney V, Carriere V, Noel S, Tibayrenc M, 1998. Trypanosoma cruzi: impact of clonal evolution of the parasite on its biological and medical properties. Exp Parasitol 89 :30–39.

    • Search Google Scholar
    • Export Citation
  • 19

    Breniere SF, Bosseno MF, Telleria J, Bastrenta B, Yaksic N, Noireau F, Alcazar JL, Barnabé C, Wincker P, Tibayrenc M, 1998. Different behavior of two Trypanosoma cruzi major clones: transmission and circulation in young Bolivian patients. Exp Parasitol 89 :285–295.

    • Search Google Scholar
    • Export Citation
  • 20

    Bosseno MF, Espinoza B, Sanchez B, Breniere SF, 2000. Mexican Trypanosoma cruzi stocks: analysis of minicircles kDNA homologies by cross-hybridization. Mem Inst Oswaldo Cruz 95 :473–476.

    • Search Google Scholar
    • Export Citation
  • 21

    Sturn NR, Vargas NS, Westenberger SJ, Zingales B, Campell DA, 2003. Evidence for multiple hybrid groups in Trypanosoma cruzi. Int J Parasitol 33 :269–279.

    • Search Google Scholar
    • Export Citation
  • 22

    Apt W, Aguilera X, Arribada A, Gomez L, Miles MA, Widmer G, 1987. Epidemiology of Chagas disease in Northern Chile: Isozyme profiles of Trypanosoma cruzi from domestic and sylvatic transmission cycles and their association with cardiopathy. Am J Trop Med Hyg 37 :302–307.

    • Search Google Scholar
    • Export Citation
  • 23

    Oliveira RP, Broude NE, Macedo AM, Cantor CR, Smith CL, Pena SDJ, 1998. Probing the genetic population structure of Trypanosoma cruzi with polymorphic microsatellites. Proc Natl Acad Sci USA 95 :3776–3780.

    • Search Google Scholar
    • Export Citation
  • 24

    Brener Z, Andrade Z, Barral-Netto M, 2000. Trypanosoma cruzi e doença de Chagas. Editora Guanabara Koogan. Rio de Janeiro: S.A. Segunda Edição, 89–125.

  • 25

    Castro AM, Luquetti AO, Rassi A, Rassi GG, Chiari E, Galvao LMC, 2002. Blood culture and polymerase chain reaction for the diagnosis of the chronic phase of human infection with Trypanosoma cruzi. Parasitol Res 88 :894–900.

    • Search Google Scholar
    • Export Citation
  • 26

    Sanchez G, Wallace A, Olivares M, Aguilera X, Apt W, Solari A, 1990. Biological characterization of Trypanosoma cruzi zymodemes: in vitro differentiation of epimastigotes and infectivity of culture metacyclic trypomastigotes into mice. Exp Parasitol 71 :125–133.

    • Search Google Scholar
    • Export Citation
  • 27

    Contreras VT, Araque W, Delgado VS, 1994. Trypanosoma cruzi: metacyclogenesis in vitro. Changes in the properties of metacyclic trypomastigotes maintained in the laboratory by different methods. Mem Inst Oswaldo Cruz 89 :253–259.

    • Search Google Scholar
    • Export Citation
  • 28

    Silveira PA, Lana M, Britto C, Bastrenta B, Tibayrenc M, 2000. Experimental Trypanosoma cruzi biclonal infection in Triatoma infestans: detection of distinct clonal genotypes using kinetoplast DNA probes. Int J Parasitol 30 :843–848.

    • Search Google Scholar
    • Export Citation
  • 29

    Bosseno MF, Telleria J, Vargas F, Yaksic N, Noireau F, Morin A, Breniere SF, 1996. Trypanosoma cruzi: Study of the distribution of two widespread clonal genotypes in Bolivian Triatoma infestans vectors shows a high frequency of mixed infections. Exp Parasitol 83: 275–282.

    • Search Google Scholar
    • Export Citation
  • 30

    Tibayrenc M, 1999. Toward an integrated genetic epidemiology of parasitic protozoa and other pathogens. Annu Rev Genet 33 :449–477.

 
 
 
 

 

 

 

 

 

 

 

 

 

VARIATION IN TRYPANOSOMA CRUZI CLONAL COMPOSITION DETECTED IN BLOOD PATIENTS AND XENODIAGNOSIS TRIATOMINES: IMPLICATIONS IN THE MOLECULAR EPIDEMIOLOGY OF CHILE

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  • 1 Program of Cellular and Molecular Biology, Biomedical Sciences Institute, Faculty of Medicine, University of Chile, Santiago, Chile; Public Health School, Faculty of Medicine, University of Chile, Santiago, Chile

To identify Trypanosoma cruzi clones from chronically infected individuals, they were transferred to triatomines by the xenodiagnosis test (XD) with Triatoma infestans. Polymerase chain reaction (PCR) and hybridization assays were performed to detect minicircle DNA in human blood samples and triatomine feces, using probes to determine the T. cruzi clones present. T. cruzi clone 19 (TcI) resulted the most prevalent in humans, with a frequency of 0.70 compared with a frequency of 0.53 in triatomines. T. cruzi clone 39 (TcIId) was the most prevalent in T. infestans, with a frequency of 0.65 compared with 0.33 in humans. The T. cruzi clone 43 (TcIIe) was not detected in blood samples; nevertheless, it was present at a rate of 0.17 in T. infestans feces. In conclusion, the T. cruzi clones are associated to each host, suggesting that selective amplification of clones occurs in human and triatomines.

INTRODUCTION

Trypanosoma cruzi, the etiologic agent of Chagas disease, is widely spread over Latin America and is transmitted by triatomine bugs. At least 10 million people are at risk of infection.1 T. cruzi isolates are complex multiclonal populations that differ in their genetic, biologic, biochemical, and molecular characteristics and in their behavior in the vertebrate host. T. cruzi isolates differ in intrinsic characters such as antigenic composition, susceptibility to chemotherapy, isoenzyme patterns, and genomic profiles of DNA.2 However, a clonal structure has been suggested for T. cruzi populations; a nucleotide sequence provides evidence of genetic exchange among lineages of T. cruzi, and T. cruzi hybrids can be experimentally generated.35 Great efforts have been made in detecting T. cruzi in biologic samples, and polymerase chain reaction (PCR) has shown a variable degree of efficiency. The sensitivity of the methods ranged from 96–100% compared with serologic diagnosis.6,7 Studies of T. cruzi strains to determine genetic diversity of this parasite based on a few isoenzymes described three main parasite groups or “zymodemes” (Z1, Z2, and Z3).8 Later, with the analysis of isoenzymatic profiles at 15 gene loci and T. cruzi samples isolated from a variety of vertebrate and invertebrate hosts, it was possible to identify at least 43 distinct natural isoenzyme profiles or clones.3 T. cruzi contains nuclear and kinetoplast DNA (nDNA and kDNA), both of which contain many repetitive sequences that are highly suitable for PCR detection. Characterization of different T. cruzi isolates has also been achieved by studying restriction fragment length polymorphism (RFLP) of kDNA. The parasites exhibiting the same RFLP patterns belong to a “schizodeme.”9 Different groups of investigators have shown a close correlation between zymodemes and schizodemes, suggesting that nuclear structural genes coding for enzymes and extranuclear (kDNA) have had a parallel evolution.10,11 Currently, the taxon T. cruzi is divided into two divisions (I and II) on the basis of isoenzyme phenotypes, DNA profiles, ribosomal, and mini-exon DNA sequence polymorphisms. T. cruzi I corresponds to zymodeme Z1, and it has been proposed that T. cruzi II consists of five sub-lineages, one corresponding to Z2 and another to Z3.12 The clinical evolution of T. cruzi infection varies widely in different patients and geographic regions. It is possible that the variability in the clinical picture of Chagas disease could be related in part to T. cruzi clones, harboring different pathogenicity.13 T. cruzi kDNA contains two components: minicircles and maxicircles. The minicircles have two kinds of sequence elements: a conserved region repeated four times per molecule and four variable and divergent regions intercalated among the repeats.

The aim of this study was to determine the different T. cruzi clones circulating in the blood of chronically infected individuals and those detected in Triatoma infestans after xenodiagnosis (XD). This evaluation is important to determine the distribution of T. cruzi clones in the two hosts (vertebrate and invertebrate) of the Chagas disease transmission cycle. These results support the selection of specific clones by the different hosts implicated in Chagas disease transmission cycle.

MATERIALS AND METHODS

Patients and blood samples.

We tested 57 chronically infected individuals (average, 40 years old) of the IV and V regions of Chile who were positive by conventional serology enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IFI). Also, flow cytometry was performed for detection of active infection.14 Two milliliters of blood was mixed with a similar volume (6 mol/L) of the buffer Guanidine HCl and 0.2 mol/L EDTA for PCR assays. XD was performed simultaneously. The samples were incubated at 98°C for 15 minutes, and DNA was extracted by phenol extraction and frozen at −20°C until used.

Informed consent.

Informed consent was obtained from all human adult participants. The project was approved by the Ethical Committee of the Medicine Faculty, University of Chile.

XD and triatomine samples.

This test was carried out using two cylindrical wooden boxes with seven uninfected third-instar nymphs of T. infestans each, as described previously.15 Microscopic examination of insect feces was performed 30, 60, and 90 days after feeding, and a pool of these samples was collected to be used in PCR. The pool of triatomine feces for the PCR test consisted of all the samples obtained at 30, 60, and 90 days from the triatomines fed with peripheral blood of the infected individuals, no matter whether the XD was positive or negative at these time. The material was placed in Diamond medium and incubated at 98°C for 15 minutes and centrifuged at 4,000 rpm for 3 minutes, and the supernatant was passed through a P6 column to remove low-molecular-weight DNA polymerase inhibitors. Finally, the wash through was collected and frozen at −20°C until used.

DNA extraction and PCR amplification.

PCR assays were performed in triplicate from blood and triatomines feces, according to conditions described.7 Essentially, 800 μL of the blood mixture was phenol extracted. The final aqueous phase was mixed with 1/10 volume of 3 mol/L ammonium acetate and 2 volumes of cold absolute ethanol and left overnight at −20°C to precipitate the DNA. The pellet obtained was centrifuged (12,000 rpm for 15 minutes at 4°C) and washed in 70% ethanol. The pellet was dried to 4°C and resuspended in 50 μL of sterile distilled water. PCR amplification of DNA was performed three times. The reaction mixture was composed of 5 μL of the DNA sample in a volume of 50 μL and conditions already described using primers 121 (5′-AAA-TAATGTACGGG (T/G) GAGATGCATGA-3′), and 122 (5′-GGTTCGATTGGGGTTGGTGTAATATA-3′). PCR assays are performed in a DNA clean chamber to avoid contamination. Every 22 samples, a single amplification of a DNA preparation from a confirmed infected individual was included as positive control, and a tube from a confirmed non-infected individual was included as a negative control of PCR. The same was done for triatomine samples. A similar protocol was used as a PCR internal control for amplification of the human β-globin gene sequences. Finally a fraction of the reaction mixture was electrophoresed on 2% agarose gels, denatured, transferred to a nylon membrane, and cross-linked by UV irradiation.

Hybridization with universal and specific radioactive probes.

Hybridization was performed using total kDNA as a universal probe16 and exposed in a Personal Molecular Imager-FX (Bio-Rad Laboratories, Hercules, CA). The hybridization assays were performed at least three times with the universal probe and specific probes.

Specific probes.

The probes used in this study were from T. cruzi clones: CBBcl3, NRcl3, sp104cl1, and v195cl1. They correspond to clones 32 (TcIIb), 39 (TcIId), 19 (TcI), and 43 (TcIIe), respectively, and were prepared as described.17 Clone sp104 was first isolated from Mepraia spinolai (silvatic cycle). Meantime the CBB and NR clones were isolated from chronically infected individuals, and clone v195 from T. infestans (domestic cycle). Probes were hybridized against a panel containing minicircle amplicons of T. cruzi clones as control.

RESULTS

Samples of blood and triatomines fed from all infected individuals were evaluated by PCR and hybridization tests with the universal probe and then with a panel of four specific probes. Positives were considered those cases that PCR and hybridization with the universal probe showed the 330 bp corresponding to minicircle DNA. Controls with non-infected humans and triatomines used were performed in parallel and resulted negative (data not shown). Figure 1A shows a representative experiment with PCR results from blood and T. infestans of a selected group of studied infected individuals. Figure 1B shows the Southern analysis with the universal probe that confirmed PCR results and increased the detection sensitivity obtained by the PCR assay. Figure 1, C–F, also shows the hybridization patterns obtained after Southern analysis with the four specific probes. Probes revealed all possible patterns of hybridization; there were samples positive with zero, one, two, three, and four probes. The hybridization results for the 57 chronically infected individuals are summarized in Table 1. Subsequently, we analyzed 1) infection rates of blood samples and triatomine bugs infected with each T. cruzi clone and 2) infection rates of mixed infections in each host. Most of the individuals were infected with T. cruzi clones 19 (TcI) or 32 (TcIIb) (P = 0.0001) or a mixture of both clones in their blood. However, when this analysis was performed with triatomines fed from the same infected individuals, the most prevalent T. cruzi clones detected were 39 (TcIId) and 19 (TcI). Generally, frequencies of T. cruzi clones found in peripheral blood and triatomine feces were different. For example: in blood, the most frequent clone was 19 (TcI) (0.70); however, in triatomines, the most frequent was 39 (TcIId) (0.65). Percentages of infected individuals in which a single T. cruzi clone was detected in blood were 26.3%, 17.5%, and 8.8% for the T. cruzi clones 19 (TcI), 32 (TcIIb), and 39 (TcIId), respectively. These values contrast with those found in triatomines, which were 7.0%, 0%, and 19.3%, respectively. Statistical analysis indicated significant differences in detection of clones 32 (TcIIb) and 39 (TcIId) among hosts. Interestingly, clone 43 (TcIIe) was not detected in blood; nevertheless, this clone was detected at a frequency of 0.17 in T. infestans (P = 0.0001). Table 1 also shows that in 45.5% and 54.2% of the cases, mixed infections were detected in blood and triatomine samples, respectively. Analyzing peripheral blood, 1.7% of the samples did not hybridize with any specific probe tested; this increased to 17.5% in triatomine samples. These remaining samples were probably infected by T. cruzi clones different from 19 (TcI), 32 (TcIIb), 39 (TcIId), or 43 (TcIIe). The results of the analysis of representative cases are shown in Table 2. In case 19, a single clone, clone 19 (TcI), was detected in blood, whereas in feces, the following clones were detected: 32 (TcIIb), 39 (TcIId), and 43 (TcIIe). In case 99, clone 32 (TcIIb) was detected in blood and clone 43 (TcIIe) in triatomines. Finally, in case 48, several T. cruzi clones were detected in blood, but none were found in T. infestans, and the converse was seen in case 46 (Table 2). Similar cases were found in other samples shown in Table 2. These results show the importance of analyzing T. cruzi genotypes in both systems in parallel. It is important to notice that we were using four well-selected probes that have a high percentage of detection (98% in blood samples and 82% in triatomines) of the infecting T. cruzi clones circulating in chronically infected individuals. Identical profiles of T. cruzi with a single clone in both samples were only detected in three infected individuals (5% of cases; data not shown), suggesting that most infected individuals were infected with mixed T. cruzi clones.

DISCUSSION

The biologic behavior of T. cruzi natural clones is linked to their phylogenetic diversity. Those T. cruzi clones that are phylogenetically distant tend to show very distinct properties for various biologic parameters such as in vitro sensitivity to antichagasic drugs and infectivity in cell cultures among others.18 These results confirm to a large extent previous data of the distribution of T. cruzi clones reported in Bolivia. Indeed, these authors provided evidence that the distribution of the two major T. cruzi clones in humans was significantly different from that observed in T. infestans of the same area.19 In this study, we analyzed for the first time the distribution of different T. cruzi clones in human subjects and T. infestans fed with peripheral blood of the same human subjects (XD). As a whole, the frequencies of each T. cruzi clone found in the two hosts were different. Therefore, they have different adaptations in vertebrate and invertebrate hosts. Minicircle DNA was detected in blood and triatomine samples by PCR; however, when this assay was complemented with a further hybridization test and a universal probe, detection in blood samples increased from 87.8% to 100% of the cases and in triatomines from 86.6% to 96.4% of the cases. This can be explained by the higher sensitivity of the radioactive signal than ethidium bromide staining. The clone 19 (TcI) was the most frequent in humans and clone 39 (TcIId) in triatomines. Interestingly, 39 (TcIId) and 43 (TcIIe) clones are genetically very related and more adapted to the invertebrate host than human hosts. These results contrast with those obtained from blood of 0- to 10-year-old chagasic patients of Chile. Frequencies obtained with the same method for similar T. cruzi clones differ in prevalence. The difference can be explained by temporal fluctuation of T. cruzi clones during the natural course of Chagas disease and/or by different geographical distribution of these clones in Chile.7 Others different T. cruzi clones are probably circulating. They can be genetically slightly different from those studied here because they do not cross-hybridize with the probes used, same as that reported in Mexico where genetically related T. cruzi clones exhibit an absence of cross-hybridization between minicircles kDNA.20 Most probably, these T. cruzi clones are TcIIa or TcIIc, which have been described in other endemic areas.21 These unknown T. cruzi clones may be present mixed with the others or alone, as determined here in 1 blood sample and in 10 triatomine samples. Moreover, these other T. cruzi clones seem to be even more adapted to the invertebrate host because they are more abundant than in the vertebrate host. However, with the panel of probes used, we are confident that the most important T. cruzi clones circulating in humans were detected. The genotyping studies of infecting T. cruzi clones in chronically infected individuals show that the systems are complementary, because different T. cruzi clones are usually found in blood and triatomines. Using both samples, we think that is possible to obtain a complete profile of T. cruzi clones circulating in a single infected individual because each one shows a differential behavior to a particular host. This represents the real molecular epidemiology picture of Chagas disease in an endemic area of Chile, because this methodology directly detects T. cruzi clones in a host, in contrast to previous studies that used parasite cultures, bypassing the final interpretation of epidemiologic data.8,22 The selection of specific T. cruzi clones in humans has been explained by probable differences in infectivity and/or parasitemia control by the immune system.19,23 It is a frequent observation that, in chronic chagasic patients, a T. cruzi clone may be selected during the long-term interaction and differential tissue tropism that may interfere with T. cruzi sub-population distribution.24 There is clear evidence that supports the selective role of the vertebrate host.25 These “filters” might select those populations or clones that are more apt in the new environment. Also development and metacyclogenesis (i.e., the passage of epimastigotes to trypomastigotes) is different in T. cruzi clones, which depends on the genetic nature of both parasite and vector.26,27 It has been shown that the infection of T. cruzi is possibly regulated by biochemical factors induced in different parts of the insect, for example, peptide hemolytic with lytic activity, which destroys several T. cruzi clones with different kinetics of lysis or lectins with different specificities in stomach, intestine, and hemolymph in R. prolixus, which represents a regulatory mechanism of interactions between the parasite and the vector.28 In an effort to show that T. cruzi clones have different adaptations in the diverse hosts, groups of investigators analyzing biclonal experimental infections of T. cruzi in T. infestans observed that almost one half of the biclonal infections were not detectable after the conclusion of the T. cruzi cycle, with important differences in the detection of these double infections according to the T. cruzi clones used. The elimination of a clonal genotype seems to be a frequent but not constant pattern in the biclonal infections of T. cruzi. This selection could be independent of the involved T. cruzi clone; nevertheless, it is possible that T. infestans would determine the persistence of certain clones and not of others. As a whole, the finding described here of one T. cruzi clone in one host and a different clone in the other allows us to conclude that almost all circulating parasites are differentially adapted to each host and represent mixtures of T. cruzi clones in ratios not possible to determinate by the standard PCR used here that is not quantitative. The expected number of chronically infected individuals presenting mixed infections in the studied areas should be very important in epidemiologic studies because mixed infections can be acquired in two ways: 1) infection by a vector presenting mixed infections and 2) re-infection with different T. cruzi clonal genotypes. Mixed infections reported here were in 45.5% of human and 54.2% of triatomine samples, which are the same figures reported in young Chilean patients or Bolivian patients and triatomines,7,19,29 suggesting that humans are early infected with T. cruzi mixture. It has been proposed that the biologic relevance of these mixtures may be great, for example, the phenomena of clonal cooperation and clonal hitch-hiking.30 Moreover, a genotype that does not proliferate in invertebrate hosts has the possibility of proliferating in the vertebrate after transmission, allowing the constant in the nature of a large diversity of T. cruzi clones.

Table 1

Detection of T. cruzi clones TcI, TcIIb, TcIId, and TcIIe in blood and T. infestans

ClonesBloodT. infestans
Only TcIIb10 (17.5%)0
Only TcIId5 (8.8%)11 (19.3%)
Only TcI15 (26.3%)4 (7.0%)
Only TcIIe01 (1.7%)
TcIIb + TcIId1 (1.7%)2 (3.5%)
TcIIb + TcI12 (21.0%)2 (3.5%)
TcIIb + TcIIe01 (1.7%)
TcIId + TcI5 (8.8%)15 (26.3%)
TcIId + TcIIe00
TcI + TcIIe01 (1.7%)
TcIIb + TcIId + TcI8 (14.0%)3 (5.3%)
TcIIb + TcIId + TcIIe02 (3.5%)
TcIIb + TcI + TcIIe01 (1.7%)
TcIIb + TcIId + TcI + TcIIe04 (7.0%)
Others1 (1.7%)10 (17.5%)
Total5757
Frequency of TcIIb0.540.26
Frequency of TcIId0.330.65
Frequency of TcI0.700.53
Frequency of TcIIe00.17
Two or more clones45.5%54.2%
Table 2

Different profiles of T. cruzi clones in human and triatomine samples

Patient no.T. cruzi clones in human bloodT. cruzi clones in T. infestansTotal number of identified clones
Representative cases of all studied.
10TcITcI1
19TcITcIIb, TcIId, TcIIe4
26TcIIb, TcIId, TcITcIIb, TcIId, TcI, TcIIe4
36TcIIb, TcI2
38TcIIdTcIId, TcI, TcIIe3
46TcIId, TcI, TcIIe3
48TcIIb, TcIId, TcI3
94TcITcIIb, TcIIe3
99TcIIbTcIIe2
Figure 1.
Figure 1.

Hybridization patterns of blood and T. infestans used in xenodiagnosis. (A) Ethidium bromide staining from blood and T. infestans. (B) Hybridization with the universal probe. (C–F) Patterns of hybridization with probes TcIIb, TcIId, TcI, and TcIIe, respectively.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 6; 10.4269/ajtmh.2006.74.1008

*

Address correspondence to Aldo Solari, Program of Cellular and Molecular Biology, Faculty of Medicine, University of Chile, 70086 Santiago 7. Chile, E-mail: asolari@med.uchile.cl

Authors’ addresses: Ximena Coronado, Marlene Rozas, Sylvia Ortiz, Gittith Sanchez, Helena Albrecht, and Aldo Solari, Program of Cellular and Molecular Biology, Faculty of Medicine, University of Chile, 70086 Santiago 7, Chile, E-mail: asolari@med.uchile.cl. Ines Zulantay and Werner Apt, Program of Cellular and Molecular Biology, Faculty of Medicine, University of Chile, 9183 Santiago, Chile, E-mail: wapt@med.uchile.cl. Jorge Rodriguez. Public Health School, Faculty of Medicine, University of Chile, 9183 Santiago 7, Chile, E-mail: jrodrigu@med.uchile.cl.

Acknowledgments: The authors thank Carezza Botto-Mahan and M. Leonor Bustamante for valuable comments on this manuscript.

Financial support: This work was supported by Grants DI-Sal 03/06-2, Fondecyt 1040731, and Fondecyt 1040762. Additional support was obtained from the following grants Postgraduate Department fellowship PG/83/2003, University of Chile.

REFERENCES

  • 1

    OPS/OMS, 1999. Informe de la VII Reunión de la Comisión Intergubernamental de la Iniciativa del Cono Sur. Washington, DC. Organización Panamericana de la Salud.

  • 2

    Andrade SG, 1999. Trypanosoma cruzi: clonal structure of parasite strains and the importance of principal clones. Mem Inst Oswaldo Cruz 94 :185–187.

    • Search Google Scholar
    • Export Citation
  • 3

    Tibayrenc M, Ayala FJ, 1988. Isozyme variability in Trypanosoma cruzi, the agent of Chagas disease: genetical, taxonomical, and epidemiological significance. Evolution 42 :277–292.

    • Search Google Scholar
    • Export Citation
  • 4

    Machado C, Ayala FJ, 2001. Nucleotide sequences provide evidence of genetic exchange among distantly related lineages of Trypanosoma cruzi. Proc Natl Acad Sci USA 98: 7396–7401.

    • Search Google Scholar
    • Export Citation
  • 5

    Gaunt MC, Yeo M, Frame IA, Russell Stethard J, Carrasco HJ, Taylor M, Solis S, Veazey P, Miles GAJ, Acosta N, Rojas de Arias A, Miles MA, 2003. Mechanism of genetic exchange in American trypanosomes. Nature 421 :936–939.

    • Search Google Scholar
    • Export Citation
  • 6

    Chiari E, 1999. Chagas disease diagnosis using polymerase chain reaction, hemoculture and serologic methods. Mem Inst Oswaldo Cruz 94 :299–300.

    • Search Google Scholar
    • Export Citation
  • 7

    Solari A, Campillay R, Ortiz S, Wallace A, 2001. Identification of Trypanosoma cruzi genotypes circulating in Chilean chagasic patients. Exp Parasitol 97 :226–233.

    • Search Google Scholar
    • Export Citation
  • 8

    Miles MA, Apt W, Widmer G, Povoa MM, Schofield CJ, 1984. Isozyme heterogeneity and numerical taxonomy of Trypanosoma cruzi stocks from Chile. Trans R Soc Trop Med Hyg 78 :526–535.

    • Search Google Scholar
    • Export Citation
  • 9

    Devera R, Fernandez O, Coura JR, 2003. Should Trypanosoma cruzi be called “cruzi” complex? A review of the parasite diversity and the potential of selecting population after in vitro culturing and mice infection. Mem Inst Oswaldo Cruz 98 :1–12.

    • Search Google Scholar
    • Export Citation
  • 10

    Tibayrenc M, Ayala F, 1987. Forte corrélation entre classification isoenzymatique et variabilité de l’ADN kinétoplastique chez Trypanosoma cruzi. CR Acad Sci III 304 :89–92.

    • Search Google Scholar
    • Export Citation
  • 11

    Carreño H, Rojas C, Aguilera X, Apt W, Miles MA, Solari A, 1987. Schizodeme analyses of Trypanosoma cruzi zymodemes from Chile. Exp Parasitol 64 :252–260.

    • Search Google Scholar
    • Export Citation
  • 12

    Barnabé C, Brisse S, Tibayrenc M, 2000. Population structure and genetic typing of Trypanosoma cruzi, the agent of Chagas disease: a multilocus enzyme electrophoresis approach. Parasitology 120 :513–526.

    • Search Google Scholar
    • Export Citation
  • 13

    Montamat E, Durand S, Bocco JL, De Luca GM, Blanco A, 1999. Identification of Trypanosoma cruzi zymodemes by kinetoplast DNA probes. J Eukaryot Microbiol 46 :155–159.

    • Search Google Scholar
    • Export Citation
  • 14

    Martins-Filho OA, Eloi-Santos SM, Teixeira Carvalho A, Corrêa Oliveira R, Rassi A, 2002. Double-Blind study to evaluate flow cytometry analysis of anti-live trypomastigote antibodies for monitoring treatment efficacy in cases of human Chagas disease. Clin Diagn Lab Immunol 9 :1107–1113.

    • Search Google Scholar
    • Export Citation
  • 15

    Schenone H, 1999. Xenodiagnosis. Mem Inst Oswaldo Cruz 94 :289–294.

  • 16

    Zulantay I, Honores P, Solari A, Apt W, Ortiz S, Osuna A, Rojas A, Lopez B, Sanchez G, 2004. Use of polymerase chain reaction (PCR) and hybridization assays to detect Trypanosoma cruzi in chronic chagasic patients treated with itraconazole or allopurinol. Diagn Microbiol Infect Dis 48 :253–257.

    • Search Google Scholar
    • Export Citation
  • 17

    Torres JP, Ortiz S, Muñoz S, Solari A, 2004. Trypanosoma cruzi isolates from Chile are heterogeneous and composed of mixed populations when characterized by schizodeme and Southern analyses. Parasitology 128 :161–168.

    • Search Google Scholar
    • Export Citation
  • 18

    Revollo S, Oury B, Laurent JP, Barnabé C, Quesney V, Carriere V, Noel S, Tibayrenc M, 1998. Trypanosoma cruzi: impact of clonal evolution of the parasite on its biological and medical properties. Exp Parasitol 89 :30–39.

    • Search Google Scholar
    • Export Citation
  • 19

    Breniere SF, Bosseno MF, Telleria J, Bastrenta B, Yaksic N, Noireau F, Alcazar JL, Barnabé C, Wincker P, Tibayrenc M, 1998. Different behavior of two Trypanosoma cruzi major clones: transmission and circulation in young Bolivian patients. Exp Parasitol 89 :285–295.

    • Search Google Scholar
    • Export Citation
  • 20

    Bosseno MF, Espinoza B, Sanchez B, Breniere SF, 2000. Mexican Trypanosoma cruzi stocks: analysis of minicircles kDNA homologies by cross-hybridization. Mem Inst Oswaldo Cruz 95 :473–476.

    • Search Google Scholar
    • Export Citation
  • 21

    Sturn NR, Vargas NS, Westenberger SJ, Zingales B, Campell DA, 2003. Evidence for multiple hybrid groups in Trypanosoma cruzi. Int J Parasitol 33 :269–279.

    • Search Google Scholar
    • Export Citation
  • 22

    Apt W, Aguilera X, Arribada A, Gomez L, Miles MA, Widmer G, 1987. Epidemiology of Chagas disease in Northern Chile: Isozyme profiles of Trypanosoma cruzi from domestic and sylvatic transmission cycles and their association with cardiopathy. Am J Trop Med Hyg 37 :302–307.

    • Search Google Scholar
    • Export Citation
  • 23

    Oliveira RP, Broude NE, Macedo AM, Cantor CR, Smith CL, Pena SDJ, 1998. Probing the genetic population structure of Trypanosoma cruzi with polymorphic microsatellites. Proc Natl Acad Sci USA 95 :3776–3780.

    • Search Google Scholar
    • Export Citation
  • 24

    Brener Z, Andrade Z, Barral-Netto M, 2000. Trypanosoma cruzi e doença de Chagas. Editora Guanabara Koogan. Rio de Janeiro: S.A. Segunda Edição, 89–125.

  • 25

    Castro AM, Luquetti AO, Rassi A, Rassi GG, Chiari E, Galvao LMC, 2002. Blood culture and polymerase chain reaction for the diagnosis of the chronic phase of human infection with Trypanosoma cruzi. Parasitol Res 88 :894–900.

    • Search Google Scholar
    • Export Citation
  • 26

    Sanchez G, Wallace A, Olivares M, Aguilera X, Apt W, Solari A, 1990. Biological characterization of Trypanosoma cruzi zymodemes: in vitro differentiation of epimastigotes and infectivity of culture metacyclic trypomastigotes into mice. Exp Parasitol 71 :125–133.

    • Search Google Scholar
    • Export Citation
  • 27

    Contreras VT, Araque W, Delgado VS, 1994. Trypanosoma cruzi: metacyclogenesis in vitro. Changes in the properties of metacyclic trypomastigotes maintained in the laboratory by different methods. Mem Inst Oswaldo Cruz 89 :253–259.

    • Search Google Scholar
    • Export Citation
  • 28

    Silveira PA, Lana M, Britto C, Bastrenta B, Tibayrenc M, 2000. Experimental Trypanosoma cruzi biclonal infection in Triatoma infestans: detection of distinct clonal genotypes using kinetoplast DNA probes. Int J Parasitol 30 :843–848.

    • Search Google Scholar
    • Export Citation
  • 29

    Bosseno MF, Telleria J, Vargas F, Yaksic N, Noireau F, Morin A, Breniere SF, 1996. Trypanosoma cruzi: Study of the distribution of two widespread clonal genotypes in Bolivian Triatoma infestans vectors shows a high frequency of mixed infections. Exp Parasitol 83: 275–282.

    • Search Google Scholar
    • Export Citation
  • 30

    Tibayrenc M, 1999. Toward an integrated genetic epidemiology of parasitic protozoa and other pathogens. Annu Rev Genet 33 :449–477.

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