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Antimicrobial Agents and Chemotherapy, September 2007, p. 3282-3289, Vol. 51, No. 9
0066-4804/07/$08.00+0     doi:10.1128/AAC.01590-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Impact of Dual Infections on Chemotherapeutic Efficacy in BALB/c Mice Infected with Major Genotypes of Trypanosoma cruzi{triangledown}

H. R. Martins,1* R. Moreira Silva,2 H. M. S. Valadares,3 M. J. O. Toledo,4 V. M. Veloso,5 D. M. Vitelli-Avelar,7 C. M. Carneiro,1,7 G. L. L. Machado-Coelho,1,2 M. T. Bahia,1,5 O. A. Martins-Filho,6 A. M. Macedo,3 and M. Lana1,7

Núcleo de Pesquisas em Ciências Biológicas, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto,1 Departamento de Farmácia, Escola de Farmácia, Universidade Federal de Ouro Preto,2 Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais,3 Departamento de Análises Clínicas, Universidade Estadual de Maringá, Pr,4 Departamento de Ciências Biológicas, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto,5 Centro de Pesquisas René Rachou, FIOCRUZ,6 Departamento de Análises Clínicas, Escola de Farmácia, Universidade Federal de Ouro Preto, MG, Brazil7

Received 20 December 2006/ Returned for modification 17 March 2007/ Accepted 21 June 2007


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ABSTRACT
 
The aim of this work was to investigate the impact of dual infections with stocks of Trypanosoma cruzi major genotypes on benznidazole (BZ) treatment efficacy. For this purpose, T. cruzi stocks representative of the genetic T. cruzi lineages, displaying different susceptibilities to BZ, belonging to the major T. cruzi genotypes broadly dispersed in North and South America and important in Chagas’ disease epidemiology were used. Therapeutic efficacy was observed in 27.8% of the animals treated. Following BZ susceptibility classification, significant differences were observed in dual infections on the major genotype level, demonstrating that combinations of genotypes 19+39 and genotypes 19+32 led to a shift in the expected BZ susceptibility profile toward the resistance pattern. Analysis on the T. cruzi stock level demonstrated that 9 out of 24 dual infections shifted the expected BZ susceptibility profile compared with the respective single infections, including shifts toward lower and higher BZ susceptibilities. Microsatellite identification was able to identify a mixture of T. cruzi stocks in 7.7% of the T. cruzi isolates from infected and untreated mice (6.9%) and infected and treated but not cured mice (9.0%), revealing in some mixtures of BZ-susceptible and -resistant stocks that the T. cruzi stock identified after BZ treatment was previously susceptible in single infections. Considering the clonal structure and evolution of T. cruzi, an unexpected result was the identification of parasite subpopulations with distinct microsatellite alleles in relation to the original stocks observed in 12.2% of the isolates. Taken together, the data suggest that mixed infections, already verified in nature, may have an important impact on the efficacy of chemotherapy.


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INTRODUCTION
 
Chagas’ disease, which is caused by the protozoan parasite Trypanosoma cruzi, is widespread in North and South America, from Mexico to southern Argentina and Chile. Although great advances have been reached in vectorial and transfusional transmission, reducing the incidence of Chagas’ disease by more than 70% (28), it is estimated that 13 million individuals still remain infected in Central and Latin America and 200,000 new cases occur per year in areas where the disease is endemic (47). The infection is characterized by a brief acute phase, followed by a chronic phase in which most patients remain asymptomatic. Approximately 30% of these people develop a silent and debilitating disease with complex and varied clinical pictures (14). This reality justifies the studies of specific Chagas’ disease chemotherapy that currently have shown unsatisfactory and controversial results. Therefore, the treatment of individuals with Chagas’ disease remains a great challenge. In this context, several studies have pointed out the relevance of parasite-related features to the genesis and perpetuation of tissue lesions (21, 29), as well as the beneficial effect of effective treatment, even for uncured patients, on the prognosis and clinical evolution of the disease (5, 16, 43).

Despite intensive research to develop new drugs that are safer and efficient in the chemotherapy of Chagas’ disease (42), only two drugs are available for human disease chemotherapy, including benznidazole (BZ; Rochagan, Rodanil [Roche]) and nifurtimox (Lampit; Bayer). These nitro derivatives are used in prolonged treatment schedules and cause important adverse reactions that hamper treatment continuity. The results of chemotherapy are more successful during the acute phase of the disease, and an approximately 40 to 76% parasitological cure rate could be observed. The efficacy of treatment in the chronic phase is low or zero, and a parasitological cure is obtained in 0 to 20% of the patients treated (19, 34).

It has been proposed that parasite genetic aspects are involved and make important contributions to explain the differences observed after treatment when screening results in distinct geographic areas are compared. Also, the existence of strains naturally resistant or susceptible to nitro derivatives has been described (4, 15, 18, 39). Considering that T. cruzi undergoes predominantly clonal evolution (36), a correlation among phylogenetic divergence and parasite biological and medical properties is expected. Several in vitro and in vivo studies (3, 33, 39, 41) with parasites from different T. cruzi genotypes, including the major genotypes broadly dispersed in North and South America and observed in different hosts (humans, several animal species, and vectors), support this hypothesis. It is natural to suppose that these genotypes that are so prevalent in nature should have an important role in the epidemiology of Chagas’ disease. These studies have demonstrated that T. cruzi I isolates are more drug resistant than T. cruzi II isolates (39, 41).

The existence of mixed infections in vertebrate and invertebrate hosts has been verified (8, 35) and raises questions about their relevance to the epidemiology of Chagas’ disease. At least in experimental mixed infections, important interactions between subpopulations of the mixtures resulted in changes in parasite biological properties (12, 13, 25, 32). This suggests that mixed infections might have important consequences for morbidity, the dynamics of parasite transmission, and the response to chemotherapy, since individuals in areas where the disease is endemic may have undergone several reinfections (10).

Thus, the goal of the present study was to evaluate in BALB/c mice the impact of dual T. cruzi infections on chemotherapy effectiveness during the acute phase, compared with the respective single infections, with distinct stocks of the major T. cruzi genotypes.


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MATERIALS AND METHODS
 
Parasites. Eight standard T. cruzi stocks representative of four distinct major genotypes (36) were used, including Cuica cl1 and P209 cl1 of genotype 20; Gamba cl1 and OPS21 cl1 of genotype 19, both of which genotypes belong to the T. cruzi I lineage; Bug2148 cl1 and SO3 cl5 of hybrid genotype 39, classified as belonging to the T. cruzi lineage; and IVV cl4 and MAS cl1 of genotype 32, belonging to the T. cruzi II lineage (6). These stocks were isolated from different ecogeographical areas of Latin America (38) and previously characterized through multilocus enzyme electrophoresis and random polymorphic DNA analysis as described by Tibayrenc et al. (37).

The choice of the parasite stocks used was based on their sensitivity to BZ and virulence in BALB/c mice; the polar stocks combining these properties in each genotype were selected as described by Toledo et al. (39). The majority of the isolates (six of eight) showed the same BZ susceptibility profile in the acute and chronic phases of infection, except stocks Gamba cl1 (40% and 25% cure rates, respectively) and IVV cl4 (40% and 80% cure rates, respectively). Stocks OPS21 cl1, SO3 cl5, and MAS cl1 (genotypes 19, 39, and 32, respectively) represented the most BZ-susceptible isolates (100% cure rate); Gamba cl1 and IVV cl4 (genotypes 19 and 32) were partially susceptible to BZ in the acute phase (40% cure rate); and P209 cl1, Cuica cl1, and Bug2148 cl1 (genotypes 20, 20, and 39, respectively) were the most resistant isolates (0% cure rate).

Experimental design and infections. Groups of 12 28- to 30-day-old BALB/c female mice from the Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, were intraperitoneally inoculated with 10,000 T. cruzi blood trypomastigotes/animal in single infections or 5,000 blood trypomastigotes of each T. cruzi stock in dual infections. The number of parasites was determined as described by Brener (9). Twenty-four different parasite combinations were compared by using single infections with each of the eight T. cruzi stocks evaluated (see Table 2). Replicate batches of experiments representative of each genotype combination were performed.


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  		TABLE 2. Microsatellite identification of T. cruzi isolates from BALB/c mice infected with different major genotypesa

Treatment schedule. BZ treatment was performed in the acute phase of infection, starting at day 10 following inoculation, considering only those situations where infection was confirmed by fresh-blood examination (FBE) or hemoculture. Groups of six animals were treated with 100 mg of BZ (Rochagan, Rodanil; Roche) per kg of body weight for 20 consecutive days. The drug was suspended in gum arabic and administered by gavage. A group of six infected untreated animals was always included in each experimental batch.

Cure criteria. Drug susceptibility and resistance were defined by cure criteria based on parasitological (FBE, hemoculture, and PCR) and serological methods, including conventional serology by enzyme-linked immunosorbent assay (ELISA) and nonconventional serology by flow cytometric detection of anti-live trypomastigote antibodies (FC-ALTA).

Animals with negative parasitological and serological test results were considered cured. Those animals with negative parasitological test results that were positive by conventional serology but negative by nonconventional serology were classified as dissociated and also considered cured as described by Kretteli and Brener (22). Animals with positive parasitological and serological test results were considered not cured.

In this work, cure rates were analyzed by considering the following three levels of T. cruzi genetic subdivisions: (i) T. cruzi lineages (T. cruzi I, T. cruzi, and T. cruzi II), (ii) major genotypes (19, 20, 39, and 32), and (iii) T. cruzi stocks (the T. cruzi stocks used for dual infections). Two variables named the observed and expected cure rates were measured. The observed cure rate was calculated for each group of animals as follows: number of cured animals/total number of infected treated animals x 100. Taking BZ susceptibility as an intrinsic feature of T. cruzi populations, the expected cure rate was estimated by considering the null hypothesis that during dual T. cruzi infection, stocks do not interfere with one another's BZ susceptibility. Therefore, the expected cure rate for a dual infection always reflects the cure rate of the most resistant stock present in the mixture. As an example, when considering infection with two T. cruzi stocks, one BZ susceptible and the other BZ resistant, the expected cure rate for the dual infection was considered to be the cure rate of the resistant stock in the mixture. However, when T. cruzi genetic subdivisions were compared at the lineage or genotype level, the global expected cure rates were reported as the average of the expected cure rates of the T. cruzi combinations within each genetic subdivision.

Following the expected cure rate determination, the BZ susceptibility profile of each single or dual infection was classified according to Filardi and Brener (15) as resistant (cure rates of ≤33%), partially susceptible (cure rates of 33% to 66%), or susceptible (cure rates of >66%).

(i) FBE. Blood samples from mouse tail veins were collected daily and exhaustively examined by optical light microscopy for detection of living trypomastigotes up to day 60 after infection.

(ii) Hemoculture. Hemocultures were carried out 30 days after treatment according to Filardi and Brener (15). Blood collected from the orbital sinus vein was inoculated into 3 ml of liver infusion tryptose medium and maintained at 28°C. Each tube was examined for detection of parasites at days 30, 60, 90, and 120 following hemoculture. The isolated parasites were further characterized by microsatellite assay to characterize the T. cruzi stocks present in infected and untreated animals and infected and treated but not cured animals.

(iii) PCR. Blood samples were collected from the orbital sinus vein at day 30 after treatment with a 1:2 mixture of 6 M guanidine-0.2 M EDTA (pH 8.0) and stored at room temperature (7). DNA extraction was carried out as described by Wincker et al. (46) with slight modifications introduced by Gomes et al. (17). PCR amplifications were carried out with 9-µl reaction mixtures containing 10 mM Tris-HCl (pH 9,0), 0.1% Triton X-100, 75 mM KCl, 3.5 mM MgCl2, each deoxynucleoside triphosphate (dATP, dCTP, dGTP, and dTTP; Sigma Company) at 0.2 mM, 0.5 U of Platinum Taq DNA polymerase (Invitrogen), and 10 pmol of each oligonucleotide primer (S35 and S36, described by Ávila et al. [7] and provided by Invitrogen). To the reaction mixture, we added 2 µl of the DNA sample and overlaid the mixture with 30 µl of mineral oil to avoid evaporation. After an initial denaturation step of 5 min at 94°C, 35 cycles of amplification were performed in a thermocycler (PTC-150; MJ Research), each one consisting of 1 min at 95°C for DNA denaturation, 1 min at 65°C for primer annealing, and 1 min at 72°C for primer extension, followed by a final extension step of 10 min at 72°C. Amplified DNA was visualized by 6% polyacrylamide gel electrophoresis and silver staining. Positive, negative, and reagent controls were processed in parallel with each assay.

(iv) Conventional serology (ELISA). ELISAs were performed according to the methodology of Voller et al. (44). Samples of mouse serum were collected 2 and 3 months after treatment and stored at –20°C. Sera were tested at a 1:80 dilution in phosphate-buffered saline by using the T. cruzi antigen prepared by alkaline extraction of parasites of strain Y obtained at exponential growth in LIT medium. Antibody binding was detected by using peroxidase-labeled anti-mouse immunoglobulin G (IgG; Sigma Immunochemical Reagents, St. Louis, MO). Absorbance (optical density) was read in a spectrophotometer with a 490-nm filter (model 3550; Bio-Rad). Positive and negative controls were processed in parallel with each assay. The cutoff value calculated for each plate was the mean absorbance of 10 negative control serum samples plus 2 standard deviations.

(v) Nonconventional serology (FC-ALTA). The flow cytometric technique used to detect anti-live trypomastigote antibodies (FC-ALTA) was carried out with serum samples collected 3 months after treatment as described by Martins-Filho et al. (26) and adapted to microplates by Cordeiro et al. (11). Serum samples from experimental animals were assayed at a 1:1,500 dilution by using goat anti-mouse IgG (Sigma Immunochemical Reagents, St. Louis, MO) labeled with fluorescein isothiocyanate to assess IgG reactivity as described by Toledo et al. (39). The results are expressed as the percentage of positive fluorescent parasites (PPFP) based on the internal control of nonspecific binding of the fluorescein isothiocyanate-conjugated second-step reagent. Positive and negative controls were included in all experimental batches. Flow cytometric measurements were performed on a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). Samples were considered negative when the PPFP was ≤20% and positive when the PPFP was >20%, as described by Martins-Filho et al. (26).

Microsatellite assay. Microsatellite analyses initially performed with DNA obtained from pure cultivated T. cruzi stocks by using eight microsatellite loci (TcAAT8, TcATT14, TcCAA10, TcGAG10, TcTAC15, TcTAT20, TcAAAT6, and SCLE10) allowed the selection of four loci (TcAAT8, TcAAT14, TcAAAT6, and SCLE10) able to distinguish each T. cruzi stock in a dual infection. The primers used to identify the selected loci included TcAAT8 (fluorescein 5'-ACCTCATCGGTGTGCATGTC-3' and 5'-TATTGTCGCCGTGCAATTTC-3'), TcTAT20 (fluorescein 5'-GATCCTTGAGCAGCCACCAA-3' and 5'-CAAATTCCCAACGCAGCAGC-3', TCAAAT6 (fluorescein 5'-GCCGTGTCCTAAAGAGCAAG-3' and 5'-GGTTTTAGGGCCTTTAGGTG-3'), and SCLE10 (fluorescein 5'-GATCCGCAATAGGAAAC-3' and 5'-GTGCATGTTCCATGGCTT-3'). The majority of the stocks had only one or two different allele sizes, as expected for cloned populations. An exception was the Cuica cl1 stock, which had three allele sizes for the SCLE10 locus, suggesting a mixture of subpopulations.

Parasites were cultivated in LIT medium, and DNA extractions were performed as described by Tibayrenc et al. (37). PCR assays were performed in a total volume of 15 µl containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100 (buffer B; Promega, Madison, WI), 2.5 mM MgCl2 (Promega), 0.5 U of Taq DNA polymerase (Promega), each deoxynucleoside triphosphate at 250 µM, each primer at 0.3 µM, and 3 µl of template DNA (1 ng/µl). The mixture was covered with mineral oil to avoid evaporation.

Amplification was performed with a PT100 thermocycler (MJ Research) by using the step-down protocol (20) modified for amplification of T. cruzi DNA and comprising an initial denaturation step of 94°C for 5 min, annealing at 58°C for 30 s, primer extension at 72°C for 1 min, and denaturation at 94°C for 30 s. After each five cycles, the annealing temperature was decreased to 55, 53, 51, and 48°C. At the last temperature, the number of cycles was increased to 15, followed by a final extension at 72°C for 10 min.

(i) Allele sizes. To determinate allele sizes, a volume of 1 to 3 µl of the PCR fluorescent products was analyzed in a 6% denaturing polyacrylamide gel (8 M urea) with an ALF sequencer (GE Healthcare, Milwaukee, WI) in comparison to fluorescent DNA fragments of 50 to 500 bp by using Allelelocator software (GE Healthcare).

Statistical analysis. The percentages of positivity obtained by several methods for each group were compared by the {chi}2 test. Fisher's exact test was used to compare the observed cure rates of the different T. cruzi subdivisions and to compare the expected and observed cure rates. Tests were performed with SPSS 12.0 software for Windows. Differences were considered statistically significant at P < 0.05.


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RESULTS
 
Profile of T. cruzi susceptibility to BZ. In order to ascertain an accurate cure control of the animals treated, we validated the cure criteria to be applied to biological samples from infected mice subjected to BZ treatment or not treated. The results demonstrated that the cure criteria adopted were appropriate for the evaluation of therapeutic efficacy (Table 1). On the basis of the proposed cure criteria, therapeutic effectiveness was observed in 27.8% of the animals treated, classified as treated and cured (19.1%) or dissociated (8.7%). Therapeutic failure was detected in 72.2% of the animals treated, which were considered infected and treated but not cured. The PCR was the more sensitive parasitological test and was positive for 27 out of 29 treated and not cured animals with negative hemoculture results. The serological methods were equally sensitive, except for dissociated animals, for which, by definition, there is a discordance between the ELISA and FC-ALTA results. The PCR and FC-ALTA (nonconventional serological method) results showed a strong correlation in the dissociated group and reinforce its classification as the treated and cured group.


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  		TABLE 1. Panoramic overview of parasitological and immunological cure criteria applied to BALB/c mice infected with T. cruzi stocks of different major genotypes

(i) BZ susceptibility profile of T. cruzi lineages. Figure 1 (left panel) illustrates the cure rates of BALB/c mice for T. cruzi phylogenetic lineages. Mice singly infected with the T. cruzi I and T. cruzi lineages showed cure rates of 35.4% and 54.4%, respectively, which characterize them as partially susceptible to BZ. On the other hand, animals singly infected with the T. cruzi II lineage showed a cure rate of 70.0%, typifying them as susceptible to BZ treatment.


Figure 1
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  		FIG. 1. Profile of T. cruzi susceptibility to BZ. The left panel shows the cure rates for single and dual infections with T. cruzi lineages (T. cruzi I, T. cruzi, and T. cruzi II). The right panel shows the cure rates for single and dual infections with genotypes 20, 19, 39, and 32. Different letters represent significant differences at P < 0.05 between the observed cure rates of the T. cruzi genetic groups studied. An asterisk represents a shift in the BZ susceptibility profile based on the observed and expected cure rates for dual infections regarding the susceptibility pattern proposed by Filardi and Brener (15). {blacksquare}, resistant (cure rates of ≤33%); {square}, partially susceptible (cure rates of 33% to ≤66%); {square}, susceptible (cure rates of >66%).

The observed cure rates of dual infections ranged from 0% for the T. cruzi I mixture to 59.1% for the T. cruzi + T. cruzi II combination. Despite the distinct cure rates observed for the T. cruzi I + T. cruzi (24%) and T. cruzi I + T. cruzi II (17.4%) combinations in comparison to the other groups of animals, both of these dual infections led to a resistant BZ susceptibility profile (<33% cure rates) similar to that of the T. cruzi I + T. cruzi I combination (Fig. 1, left panel).

The global expected cure rates for combinations of T. cruzi lineages were calculated as the mean cure rate based on the expected cure rates of the stocks used for each mixture.

As the T. cruzi I + T. cruzi I dual infection always included BZ-resistant stocks, the global expected cure rate was 0%. As the T. cruzi I + T. cruzi dual infection still displayed a predominance of combinations including BZ-resistant stocks, the global expected cure rate was 17.7%. However, as the T. cruzi I + T. cruzi II dual infection included combinations of BZ-resistant and partially BZ-susceptible or BZ-susceptible stocks, the global expected cure rate was 26.7%. As the T. cruzi + T. cruzi II dual infection displayed a predominance of partially BZ-susceptible stocks, the expected cure rate was 35%.

Comparative analysis of the observed and expected cure rates did not demonstrate any significant shift in the BZ susceptibility profile, despite the slight change in the cure rate observed for T. cruzi + T. cruzi II (59.1%) in comparison to the expected value (35.0%) (Fig. 1, left panel).

(ii) BZ susceptibility profile of the major T. cruzi genotypes. Figure 1 (right panel) illustrates the cure rates of BALB/c mice for the major T. cruzi genotypes. Comparison of the major T. cruzi genotypes demonstrated significant differences in the observed cure rates. While mice infected with T. cruzi genotype 20 displayed a cure rate of 0%, animals infected with genotype 19 or 32 showed susceptibility to BZ treatment (cure rate of 70%) (Fig. 2, right panel). Animals infected with genotype 39 were partially susceptible to BZ (cure rate of 50.0%).


Figure 2
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  		FIG. 2. BZ susceptibility profile of single and dual infections with T. cruzi stocks of different genotypes. T. cruzi genotypes and stocks in bold are resistant to BZ (genotypes 20 and 39), those in gray are partially susceptibility to BZ (genotypes 19 and 32), and those in normal type are susceptible to BZ (genotypes 19, 39, and 32). Results are expressed as observed cure rates (dual infections) and expected cure rates (based on a theoretical dual infection, expressed as the susceptibility profile of the T. cruzi stock more resistant in the respective single infection). An asterisk represents a shift in the BZ susceptibility profile based on differences between the observed and expected cure rates for dual infections and the observed and expected cure rates regarding the susceptibility profile proposed by Filardi and Brener (15). {blacksquare}, resistant (cure rates of ≤33%); {square}, partially susceptible (cure rates of 33% to ≤66%); {square}, susceptible (cure rates of >66%). Connecting lines highlight dual infections that show a shift between the observed and expected cure rates.

The cure rates of dual infections with major T. cruzi genotypes ranged from 0% for the mixture of genotypes 20+19 to 59.1% for genotypes 39+32. Despite the significant differences observed for the combination of genotype 32 or 39 with genotype 20 or 19, those changes were not able to change the pattern of susceptibility to BZ treatment, which was confined to the region of resistance (Fig. 1, right panel).

For all combinations including genotype 20, which comprise BZ-resistant stocks, the global expected cure rate was 0%. As the dual infections including genotype 19 comprised a distinct predominance of BZ-susceptible (genotypes 39 and 32) or BZ-resistant (genotype 39) stocks, the global expected cure rates ranged from 35% to 55%. However, as the dual infection with genotype 32 and genotype 39 included combinations with partially BZ-susceptible and BZ-susceptible stocks, the global expected cure rate was 35%.

Despite slight changes in the observed cure rates of the other genotype combinations, those changes did not alter the expected BZ susceptibility profile (Fig. 1, right panel). Comparative analysis of the observed and expected values demonstrated that the combinations of genotypes 19+39 and genotypes 19+32 led to a shift in the expected BZ susceptibility profile toward the resistant pattern and no significant changes were observed for the other dual-genotype infections. Indeed, the observed cure rates of the dual infections with genotypes 19 and 39 (25.9%) and genotypes 19 and 32 (22.7%) were lower than the expected cure rates of 35% and 55.0%, respectively, and also in comparison to those observed for single infections with genotypes 19 (70%), 32 (70%), and 39 (50%).

(iii) BZ susceptibility profile of single and dual infections with different T. cruzi stocks. Figure 2 shows a comparison of the cure rates of BALB/c mice infected with single and dual T. cruzi stocks. Comparison of the observed cure rates of dual infections with the corresponding expected cure rates, based on the cure rate of the T. cruzi stock more resistant in the respective single infection, is presented in Fig. 2. Data analysis demonstrates that 9 out of the 24 dual infections studied (combinations highlighted with asterisks in Fig. 2) shifted the expected BZ susceptibility profile compared with the respective single infections (connecting lines highlight shifts in the BZ susceptibility profile). It is important to note that in three cases the differences between the observed and expected cure rates were especially remarkable, i.e., for Bug2148 cl1 + MAS cl1 (P = 0.015), Bug2148 cl1 + OPS21 cl11 (P = 0.030), and OPS21 cl11 + MAS cl1(P = 0.015). The OPS21 cl11 + MAS cl1 combination of two T. cruzi stocks 100% susceptible to BZ proved to be resistant to BZ treatment, with the dual infection showing only a 14.3% cure rate. On the other hand, the mixture of Bug2148 cl1 + MAS cl1 including one clone 100% resistant and other 100% susceptible to BZ proved to be susceptible to BZ treatment, with the dual infection displaying a cure rate of 83.3% (Fig. 2).

Shifts in the profiles of BZ susceptibility were observed in all of the genotype combinations, including 20+39 (n = 2), 20+32 (n = 1), 19+39 (n = 3), 19+32 (n = 2), and 39+32 (n = 1) but not 20+19. Four combinations led to increased resistance to BZ compared to the expected values (OPS21 cl11 + MAS cl1, OPS21 cl11 + SO3 cl5, Gamba cl1 + IVV cl4, and Gamba cl1 + SO3 cl5). On the other hand, five combinations yield increased BZ susceptibility (Bug2148 cl1 + MAS cl1, Cuica cl1 + SO3 cl5, P209 cl1 + SO3 cl5, Bug2148 cl1 + OPS21 cl11, and P209 cl1 + IVV cl4).

Microsatellite identification of T. cruzi isolates. The microsatellite allele sizes were determined for all T. cruzi samples isolated from infected and untreated mice and infected and treated but not cured mice, as shown in Table 2. T. cruzi characterization was performed for 180 stocks isolated from 102 dually infected and untreated mice and 78 dually infected and treated but not cured mice. A mixture of T. cruzi stocks from the same animal was observed in a low percentage of the isolates (7/102 [6.9%] from infected and untreated mice and 7/78 [9.0%] from infected and treated but not cured mice, for a total of 14/180 [7.7%]).

It is important to mention that stocks of genotype 32 were not detected in any of the combinations, while stocks of genotype 39 were only observed in the combination of genotypes 39 and 32 (Table 2).

In combinations of genotypes 19 and 20, stocks of both genotypes were isolated from the infected and untreated group and the infected and treated but not cured group. Curiously, in animals infected with a mixture of Cuica cl1 and Gamba cl1 (genotype 20 [100% resistant to BZ in single infections] and genotype 19 [40.0% susceptible to BZ in single infections]), Gamba cl1 was detected in all except one of the isolates from infected and untreated mice (four of five) and in all of those from infected and treated but not cured mice (six of six). Moreover, in animals infected with a mixture of P209 cl1 and OPS21 cl1 (genotype 20 [100% resistant to BZ in single infections] and genotype 19 [100% susceptible to BZ in single infections]), although both stocks were observed in the same proportion (three of six) in isolates from infected and untreated mice, only OPS21 cl11 (100% susceptible to BZ) was observed in the infected and treated but not cured group (five of five) (Table 2).

In animals infected with mixtures of genotypes 19 and 39, the stocks belonging to genotype 19 (Gamba cl1 [40% susceptible to BZ in single infections] and OPS21 cl11 [100% susceptible to BZ in single infections]) were preferentially isolated when combined with Bug2148 cl1, the most resistant stock of genotype 39, with a cure rate of 0% (Table 2).

An unexpected result was the identification of a parasite subpopulation with distinct microsatellite alleles in relation to the original stocks, named unknown T. cruzi stocks, observed in 22 (12.2%) of the total of 180 stocks isolated. These unknown T. cruzi stocks were observed more often in infected and treated but not cured animals (13/22 [59.1%]) than in infected and untreated animals (9/22 [40.9%]) and were similarly isolated from animals infected with different combinations of genotypes 32 (12/22) and/or 39 (12/22).

The majority of the unknown T. cruzi stock isolates (14/22) showed alleles sizes of 263/263 bp for the TcAAAT6 locus, except for the mixtures Bug2148 cl1 + Cuica cl1 and Bug2148 cl1 + Gamba cl1, where different allele sizes of 271/275 bp were detected in six isolates. The two isolates obtained from mice infected with mixtures of genotypes 39+32 (Bug2148 cl1 + IVV Cl4 and IVV cl4 + SO3 cl5) and untreated displayed an allele size of 292/292 bp for the TcAAT8 locus but not the heterozygote profile of 250/292 bp expected for Bug2148 cl1 or SO3 cl5.


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DISCUSSION
 
Several studies have demonstrated that parasite-related features play an important role in the efficacy of chemotherapy in Chagas’ disease and, more recently, that genetic aspects of parasites may have a close relationship to the therapeutic response (4, 15, 39, 41). Our group verified that in mixed infections, interactions between populations of T. cruzi may occur, resulting in important changes in the biological characteristics of the parasite and the evolution of the infection (12, 25, 32). Moreover, the importance of mixed infections in the epidemiology of Chagas’ disease (8, 35) and the evidence that they may result in significant alterations in the host-parasite relationship motivated the studies on the efficacy of chemotherapy in dual infections. Therefore, the goal of the present investigation was to evaluate in BALB/c mice the impact of dual T. cruzi infections on chemotherapy effectiveness, compared with the respective single infections with stocks from distinct major T. cruzi genotypes.

The results of the present investigation corroborate this hypothesis and demonstrate clearly that mixed infections show responses to BZ treatment distinct from the expected response based on single-infection analyses. In addition, they suggest that the establishment of a correlation between the parasite's genetics and the treatment response may be difficult in dual infections.

As previously demonstrated by Toledo et al. (39, 41), T. cruzi I isolates and their combinations in dual infections were more resistant to BZ than T. cruzi II isolates. Hybrid T. cruzi genotype 39 showed a resistance pattern intermediate between those of the first two groups. When the upper T. cruzi divisions were considered, shifts in the BZ susceptibility profile in dual infections were not detected. On the other hand, changes in the BZ susceptibility profile were evident when the lesser T. cruzi subdivisions (mixtures of major T. cruzi genotypes or stocks) were considered. These results support the observation of Toledo et al. (39) that it is necessary to take into account the lesser phylogenetic subdivisions because of the heterogeneous behavior of stocks from the same great genetic subdivisions. For instance, shifts in the BZ susceptibility profile were observed only in combinations of genotypes 19+39 and 19+32. When infections were considered at the T. cruzi stock level, shifts were also detected in combinations of stocks of several other genotypes. For instance, we observed an increase in the cure rate of six dual infections involving almost all of the genotype combinations tested (except 19+32 and 20+19) and a reduction in two, including genotypes 19+39 and 19+32. Moreover, when the isolated subpopulations were identified by microsatellite profile, interesting and curious results were verified in some mixtures. In four cases (Cuica cl1 + Gamba cl1, Bug2148cl + Gamba cl1, Bug2148 cl1 + OPS21 cl11, and P209 cl1 + OPS21 cl11), the stock identified was the one more susceptible to BZ, to the detriment of the resistant one. Indeed, these data suggest that the expected correlation between susceptibility to treatment and genetic combination is difficult to establish for dual infections.

Moreover, 23 out of 180 isolates showed microsatellite profiles distinct from those expected for the original clones in the mixtures. This occurred in 62.5% of the animals from the infected and treated but not cured group (14/23) and 33.5% (9/23) of those in the infected and untreated group. In two cases, concomitant changes in the microsatellite profile and susceptibility to BZ were observed. Unexpectedly, the supposed clone Cuica cl1 revealed three microsatellites locus profiles with three or four picks, suggestive of polyclonal populations. Possibly, this result was due to the high resolution of the microsatellite assay, which allows T. cruzi detection in biological samples with low parasite contents (23). These microsatellite profile changes were unexpected because we used clonal T. cruzi stocks predicted to remain genetically stable (38), even considering the possibility of genetic exchange (24). However, genetic changes have already been observed in cultures of clonal protozoan parasites as a result of drug resistance selection or even spontaneously or by modification of the culture conditions used (1, 2, 27, 30, 31, 45). Furthermore, we cannot exclude the possibility that these original stocks possessed different subpopulations present at levels too low for detection by conventional molecular techniques and/or that the passage of these stocks in laboratory animals or culture propitiated the preferential growth of such subpopulations.

Although identification by microsatellite analysis verified the presence of only one stock in most isolates, our findings demonstrated that stocks isolated from mice not cured showed a BZ susceptibility profile different from that expected on the basis of single infections. This may suggest too the coexistence of two T. cruzi stocks, at least at the beginning of the infection, which was sufficient to promote these changes. These findings may also represent a multifactorial phenomenon, and we cannot discount the possibility of parasite selection at the beginning of the infection, prior to the establishment of BZ treatment. Moreover, there is also the possibility of parasite selection during the isolation procedures and during in vitro growth before microsatellites characterization (8, 13) or even the unavailability of blood to specific T. cruzi stocks sequestered in tissue sites (40).

Interactions between T. cruzi clonal stocks of different genotypes suggesting reciprocal stimulation or inhibition were already demonstrated in experimental infections of Triatoma infestans (12, 32) and mice (25). Those authors observed that clones with a low ability to develop in invertebrate or vertebrate hosts in monoclonal infections were able to multiply and to remain in mixed infections (19 or 20 + 32) at levels even higher than that of the most virulent clone. These data corroborated the results obtained here and emphasize the importance of studies of the effect of mixed infections on the evolution of Chagas’ disease.

Recent studies have revealed a high incidence of mixed infections in humans (35) and vectors (8), confirming the relevance of mixed infections on the evolution of Chagas’ disease. Moreover, the dynamic of their circulation in nature through vertebrate and invertebrate hosts needs to be studied further. The contradictory results obtained in human treatment by different authors are probably influenced by the occurrence of mixed infection, since individuals in areas where the disease is endemic can be subjected to several reinfections (10). In conclusion, data presented here show that the treatment of mixed infections is more complex than the treatment of single infections because of the possibility of interactions between the populations involved, resulting in new characteristics and opening new perspectives to drive further investigations in the field of Chagas’ disease treatment.


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ACKNOWLEDGMENTS
 
We thank Michel Tibayrenc, IDR, Montpellier, France, for providing the T. cruzi stocks studied here.

We thank the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPEMIG (Fundação de Amparo a Pesquisa do Estado de Minas Gerais), Brazil, for financial support.


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FOOTNOTES
 
* Corresponding author. Mailing address: Núcleo de Pesquisas em Ciências Biológicas, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Campus Universitário, Morro do Cruzeiro, 35400-000 Ouro Preto, MG, Brazil. Phone: 55-31-3559-1691. Fax: 55-31-3559-1800. E-mail: helen{at}nupeb.ufop.br Back

{triangledown} Published ahead of print on 16 July 2007. Back


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Antimicrobial Agents and Chemotherapy, September 2007, p. 3282-3289, Vol. 51, No. 9
0066-4804/07/$08.00+0     doi:10.1128/AAC.01590-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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