JAC Advance Access originally published online on April 21, 2004
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Journal of Antimicrobial Chemotherapy (2004) 53, 1045-1053
© 2004 The British Society for Antimicrobial Chemotherapy
Effects of specific treatment on parasitological and histopathological parameters in mice infected with different Trypanosoma cruzi clonal genotypes
1 Departamento de Análises Clínicas, Centro de Ciências da Saúde, Bloco I-90, Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900, Maringá, PR; 2 Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Campus Pampulha, 31270-901, Belo Horizonte, MG; 3 Departamento de Ciências Biológicas, Instituto de Ciências Exatas e Biológicas, Campus Universitário Morro do Cruzeiro, Universidade Federal de Ouro Preto (UFOP), 35400-000, Ouro Preto, MG; 4 Departamento de Análises Clínicas and 5 Departamento de Farmácia, Escola de Farmácia, Rua Costa Sena, 171, UFOP, 35400-000, Ouro Preto, MG, Brazil
Received 13 December 2002; returned 7 March 2003; revised 11 December 2003; accepted 3 March 2004
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Abstract |
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The goal of this study was to verify the effect of specific treatment on parasitological and histopathological parameters in mice experimentally infected with different Trypanosoma cruzi clonal genotypes. Twenty cloned stocks were selected, representative of the whole phylogenetic diversity of the protozoan and belonging to the clonal genotypes 19 and 20 (T. cruzi I) and 39 and 32 (T. cruzi II). The stocks were inoculated in 40 BALB/c mice divided into four groups: (i) treated with benznidazole, (ii) treated with itraconazole and (iii and iv) untreated control groups (NT) for each drug, respectively. Seven parameters related to parasitaemia curves and histopathological lesions were analysed. Four during the acute phase (AP) and three during both the AP and chronic phase (CP) of infection. Statistical comparison between benznidazole-treated and NT groups for the biological parameters showed significant differences for all genotypes. Benznidazole treatment led to lower patent period, maximum of parasitaemia, day of maximum parasitaemia and area under the parasitaemia curve for all genotypes analysed. Percentage of positive haemoculture during AP and CP was lower for genotypes 19 and 32. Tissue parasitism (TP) and inflammatory process (IP) during AP were lower for genotypes 19 and 32, respectively. In general, itraconazole treatment induced a smaller reduction in these same parameters between treated and NT animals in relation to benznidazole treatment. Our results indicate that phylogenetic divergence among T. cruzi clonal genotypes must be taken in account in chemotherapy and studies dealing with all aspects of the parasite and the disease.
Keywords: experimental chemotherapy, benznidazole, itraconazole, genetic groups
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Introduction |
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Recent studies have shown the importance of parasite persistence in the maintenance and clinical evolution of Chagas’ disease, especially concerning chagasic cardiopathy.1,2 Moreover, the effects of specific treatment on its clinical evolution3,4 and in lesions observed in experimental infections5–8 have been shown to be beneficial. Although the results of these studies were controversial, particularly when related to treated patients, nowadays, more importance has been given to Chagas’ disease chemotherapy and clinical assays with alternative compounds.
Andrade9 correlated specific arrays of morphobiological and behavioural characters with particular types within the aetiological agent Trypanosoma cruzi. Variability in the drug susceptibility of different T. cruzi strains has been described by several authors10,11 using especially benznidazole and nifurtimox, usually employed in the treatment of human Chagas’ disease. Based on these facts, the strains of T. cruzi were classified9 as types I, II and III equivalent to biodemes I, II and III described later.12
Several studies have demonstrated that T. cruzi shows great biological and genetic variability. The characterization of T. cruzi populations from different geographical areas of Brazil by multilocus enzyme electrophoresis (MLEE) demonstrated the existence of six different zymodemes (Z): Z1, Z2 (or ZA), Z3, ZB, ZC and ZD.13,14 Tibayrenc et al.15 considering the isoenzymic profiles of T. cruzi stocks isolated from different hosts (vertebrate and invertebrate) of the domestic, peridomestic and sylvatic cycles, proposed a complex multiclonal structure for this species distributed among 43 distinct genetic groups, zymodemes, natural clones or clonets. According to the nomenclature recently proposed, T. cruzi is subdivided into two major phylogenetic subdivisions: T. cruzi I and T. cruzi II.16 Stocks of T. cruzi II have been shown to be more susceptible to benznidazole and itraconazole treatment than stocks of T. cruzi I.17
Correlation between the phylogenetic divergence of the parasite (clonal genotypes) and its biological and medical properties has been demonstrated.18–20 More recently, statistical association between parameters related to virulence, pathogenicity and susceptibility to chemotherapeutic agents benznidazole and itraconazole, on the one hand, and genetic distance of the parasite stocks on the other was demonstrated by Toledo and colleagues17,21 in mice inoculated with the same standard set of stocks studied above.
This study aims to evaluate the effects of specific chemotherapy with benznidazole and itraconazole on parasitological and histopathological parameters in BALB/c mice experimentally infected with T. cruzi cloned stocks from genotypes 19 and 20 (T. cruzi I), and 39 and 32 (T. cruzi II) during the acute and chronic phases of infection.
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Materials and methods |
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Parasites
The same standardized sample of 20 T. cruzi stocks used in previous studies was used.17–21 The stocks were cloned using micromanipulation, with visual verification under a microscope. They were fully characterized by MLEE with 22 different loci22 and randomly amplified polymorphic DNA (RAPD) with 10 primers.23 They are representative of major clonets (widespread clonal genotypes) 19, 20, 39 and 32,15,23 and were obtained from different ecogeographical areas of Latin America and from several hosts of the domestic and sylvatic transmission cycles. Ten stocks belonged to genotypes 19 (SP104 cl1, Cutia cl1, Gamba cl1, 13379 cl7 and OPS21 cl11) and 20 (SO34 cl4, Cuíca cl1, P209 cl1, Esquilo cl1 and P11 cl3), classified as T. cruzi I, and 10 stocks belonged to genotypes 39 (SC43 cl1, Bug2148 cl1, Bug2149 cl11, MN cl2 and SO3 cl5) and 32 (MAS cl1, CBB cl3, TU18 cl2, IVV cl4 and MVB cl8), classified as T. cruzi II. Clonal genotypes 19, 20, 39 and 32 illustrate different phylogenetic relationships. Genotypes 32 and 39 are more closely related to each other, whereas genotype 19/20 is more distantly related to both 32 and 39. Lastly, 19 and 20 are very close to each other.
Infection of mice
Groups of 40 female isogenic BALB/c mice (from Instituto de Ciências Biológicas, UFMG), 28–30 days old, were inoculated through an intraperitoneal route with 10 000 blood trypomastigotes/animal of each stock. The number of parasites in the inoculum was determined according to Brener.24 Inocula were obtained from breast-fed Swiss mice previously inoculated with a large number of metacyclic trypomastigotes obtained from late stationary culture in LIT (liver-infusion tryptose) medium, after treatment with guinea pig serum.25 The period from 2 to 3 months after the infection during which the parasitaemia was detectable for fresh blood examination was considered as the acute phase (AP), and the period after 90 days of infection when the mice presented parasitaemia sub-patent was considered as the chronic phase (CP).
Treatment schemes
After detection of infection, the animals were divided into two groups: 20 treated (T) and 20 untreated controls (NT). Ten mice from the first group were treated orally with daily doses of benznidazole (Roche) 100 mg/kg body weight for 20 consecutive days:26 five mice during the AP of infection starting the treatment approximately 10 days post-infection (d.p.i.) and five mice during the CP starting the treatment on the 90th d.p.i. The other 10 mice were treated orally with daily doses of itraconazole (Biolab) 100 mg/kg body weight for 60 consecutive days:27 five during the AP and five during the CP. Both compounds were suspended in distilled water using gum arabic and each mouse received 0.2 mL of this suspension, by gavage.
Parameters evaluated
Five microlitres of blood was collected daily from the tail of T and NT mice, examined microscopically for living trypomastigotes and the parasites were counted according to Brener.24 The parasitaemia curve, patent period (PP), maximum of parasitaemia (MP), day of maximum parasitaemia (DMP) and the area under the parasitaemia curve (PAR) were determined.
Haemoculture
Thirty to 35 days after the end of benznidazole or itraconazole treatment, the surviving mice, either from the AP or the CP, were submitted to haemoculture in 3 mL of LIT liquid medium according to Filardi & Brener.26 The results were expressed as percentages of positive haemoculture (%+HC).
Histopathological analysis
Histopathological analysis was carried out only on animals treated with benznidazole and in NT groups. For each T. cruzi stock, eight mice were necropsied for histopathological studies: four mice benznidazole-treated (two on the 30th d.p.i. and two on the 110th d.p.i.); the same number of animals from the NT groups was evaluated. The following organs and tissues were collected: (1) heart, (2) skeletal muscle, (3) gastrointestinal tract including stomach, small and large intestine, (4) genitourinary tract including bladder, kidney and uterine tubes, (5) brain, (6) liver, (7) pancreas and (8) fat tissue. This material was routinely processed and embedded in paraffin. Five-micrometre sections of three blocks containing every organ of each mouse were obtained for further evaluation. These preparations were stained with haematoxylin–eosin (HE) for microscopic examination. The tissue parasitism (TP) and inflammatory process (IP) displayed by different organs or tissues were classified as absent (–), mild (+), moderate (++) and severe (+++) during the AP and CP of infection. The percentages of organs or tissues with TP and IP were compared in T and NT animals.
Statistical analysis
Statistical analysis of the virulence parameters was completed as follows: PP, MP and DMP were compared using the non-parametric Mann–Whitney test and the %+HC-AP and %+HC-CP were compared using the 
2 test. The area under the parasitaemia curve was compared using the non-parametric Kolmogorov-Smirnov test. Histopathological data (TP and IP) were analysed using the non-parametric Kruskal–Wallis test.28
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Results |
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Patent period (PP)
Benznidazole treatment. Animals inoculated with stocks from genotypes 19, 20, 39 and 32, and benznidazole-treated, displayed highly significant (P < 0.001) reductions in PP (Table 1).
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Itraconazole treatment. In animals inoculated with stocks from the four genotypes, itraconazole treatment promoted a lower reduction in PP than benznidazole treatment (Table 2). Differences in PP between itraconazole-treated and NT animals were significant (P < 0.05) only for mice inoculated with stocks from genotype 19.
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Maximum parasitaemia (MP)
Benznidazole treatment. Animals infected with T. cruzi stocks from the four genotypes and benznidazole-treated showed a major reduction (P < 0.001) in the MP in relation to NT animals (Table 1 and Figure 1).
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Itraconazole treatment. Itraconazole treatment reduced the parasitaemia in mice infected with the four genotypes (Table 2 and Figure 2). However, a significant reduction (P < 0.05) in the MP was observed only in mice infected with genotype 20.
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Day of maximum parasitaemia (DMP)
Benznidazole treatment. The DMP was significantly lower (P < 0.005) in mice inoculated with T. cruzi stocks belonging to genotypes 19, 20, 39 and 32, and benznidazole-treated, in relation to NT animals (Table 1 and Figure 1).
Itraconazole treatment. There were no significant differences in relation to DMP between itraconazole-treated and NT animals inoculated with stocks from the four genotypes (Table 2).
Area under the parasitaemia curves (PAR)
Benznidazole treatment. All animals infected with all T. cruzi genotypes and benznidazole-treated showed sub-patent parasitaemia immediately after beginning treatment (Figure 1). The PAR from benznidazole-treated animals was statistically smaller than that observed in NT animals (P < 0.001) for the four genotypes.
Itraconazole treatment. Animals infected with stocks that showed patent parasitaemia displayed reduction in PAR when itraconazole-treated (Figure 2). However, significant differences in PAR (P < 0.001) were observed between T and NT animals, only in mice inoculated with stocks from genotype 20. In general, itraconazole-treated animals remained with patent parasitaemia during treatment. Some animals infected with stocks from genotype 20 showed an increase in PAR after the end of treatment, between the 75th and the 95th d.p.i. (Figure 2).
Percentages of positive haemoculture (%+HC)
Benznidazole treatment during the acute phase. The %+HC in animals infected with T. cruzi stocks from genotypes 19, 20, 39 and 32, benznidazole-treated during the AP, and in NT controls are shown in Table 1. A significant decrease (P < 0.001) in the %+HC was observed in animals benznidazole-treated and infected with stocks from genotypes 19 and 32 in relation to the NT animals.
Benznidazole treatment during the chronic phase. The %+HC in animals infected with T. cruzi stocks from genotypes 19, 20, 39 and 32, benznidazole-treated during the CP, and in NT animals are shown in Table 1. Differences in the %+HC between benznidazole-treated and NT animals were also significant (P < 0.001) only in animals infected with stocks from genotypes 19 and 32 (Table 1).
Itraconazole treatment during the acute phase. In animals infected with T. cruzi stocks from all genetic groups, the differences in the %+HC between itraconazole-treated and NT groups were not significant (Table 2).
Itraconazole treatment during the chronic phase. Only animals inoculated with stocks from genotype 32 displayed a significant reduction (P < 0.05) in the %+HC when itraconazole-treated during the CP (in relation to the NT groups) (Table 2).
Tissue parasitism (TP)
In NT animals infected with stocks belonging to the four genotypes, TP was detected in mice infected with genotypes 19, 20 (Figure 3b) and 32 during the AP, and in animals infected with genotype 39 during the CP (Table 3).
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Benznidazole treatment during the acute phase. Parasites were not observed in mice infected with T. cruzi stocks from genotype 19 and benznidazole-treated during the AP and the reduction in parasitism was significant (P < 0.05) in relation to the NT group, as shown in Table 3.
No significant reduction (P = 0.06) in TP was observed between animals (benznidazole-treated and NT) infected with T. cruzi stocks from genotype 20 (Table 3). Mild parasitism was still found in two organs/tissues of benznidazole-treated animals infected with stocks belonging to genotype 20.
Mice infected with stocks belonging to genotype 39 displayed a significantly higher TP (P = 0.02) after benznidazole treatment in the AP than the NT group, which curiously did not display parasitism in this phase of infection (Table 3). Amastigotes were found in the gastrointestinal tract (Bug2149 cl10 and MN cl2 stocks), in skeletal muscle (Bug2149 cl10 and MN cl2 stocks) and in the genitourinary tract (MN cl2 stock), although few in number.
In animals infected with stocks from genotype 32, the reduction in TP with benznidazole treatment during the AP was not significant (Table 3).
Benznidazole treatment during the chronic phase. TP in animals infected with all T. cruzi genotypes was not found after treatment in the CP (Table 3). In mice infected with genotype 39 and benznidazole-treated (different from what was observed during the AP), a significant reduction (P < 0.05) in parasitism was observed during the CP (Table 3). Only NT mice infected with genotype 39 displayed parasites in tissues during the chronic phase of infection.
Inflammatory process (IP)
Benznidazole treatment during the acute phase. The percentages of organs/tissues of animals (benznidazole-treated and NT) infected with stocks from genotypes 19, 20, 39 and 32 during the AP with IP are shown in Table 3. Animals treated during the acute phase (T-AP) displayed IP that ranged from moderate to mild. Severe IP was observed only in NT animals infected with genotype 20 (Figure 3a). Moderate IP was found only five times in animals treated during the AP. Mild inflammation was found 50 times in the T-AP groups: 11, 15, 12 and 12 times for mice infected with genotypes 19, 20 (Figure 3c), 39 and 32, respectively.
Considering the mice infected with stocks from all genotypes, benznidazole treatment during the AP of infection reduced the percentage of organs/tissues with IP from 51.3% (NT animals) to 34.4% (T-AP). Nevertheless, a significant reduction was observed only in animals infected with genotypes 20 (P < 0.05) and 32 (P < 0.005).
When inflammation of a particular organ/tissue was compared between T-AP animals and NT groups, it was observed that on the one hand, AP treatment promoted a significant decrease in IP in the genitourinary tract from animals infected with genotype 19, in skeletal muscle from those infected with genotype 20 and in fat tissue from those infected with genotype 32. On the other hand, a significant increase in the inflammatory reaction in skeletal muscle from animals infected with genotype 39 (data not shown) was observed.
Benznidazole treatment during the chronic phase. The percentage of organs/tissues from animals (benznidazole-treated and NT) infected with stocks from genotypes 19, 20, 39 and 32 during the CP is shown in Table 3.
The frequencies of moderate inflammation were 5, 1, 5 and 3 in mice infected with genotypes 19, 20, 39 (Figure 4c) and 32, respectively. Mild inflammation was more frequent and detected 46 times: 9, 13, 11 and 13 times in organs/tissues from animals infected with genotypes 19, 20, 39 and 32 (Figure 4a), respectively.
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A significant reduction in the percentage of organs/tissues with IP was not observed in animals infected with genotypes 19, 20 and 32 and benznidazole-treated. A significant increase (P < 0.05) in IP frequency was observed in mice infected with genotype 39 and benznidazole-treated (Table 3 and Figure 4c). Benznidazole-treated and NT mice infected with this genotype showed 50.0% and 27.5%, respectively, of the organs/tissues with IP during the CP.
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Discussion |
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The in vivo susceptibility to benznidazole and itraconazole, for the same set of cloned stocks studied here, was previously determined by Toledo et al.,17 which demonstrated in treated mice that genotype 19 was partially susceptible to benznidazole and resistant to itraconazole in both phases of infection, whereas genotype 20 was resistant to both chemotherapeutic agents during the AP and CP. On the other hand, genotype 39 was considered partially susceptible to benznidazole in both phases of infection, to itraconazole only in the CP and resistant to itraconazole during the AP. Genotype 32 was considered susceptible to benznidazole in both phases of infection and to itraconazole in the CP. This genotype was also considered partially susceptible to itraconazole during the AP of infection.
Mice experimentally infected with the four T. cruzi genotypes, and benznidazole-treated, presented a significant reduction in four parameters related to virulence (PP, MP, DMP and area under the parasitaemia curve) in animals infected with all genotypes and in %+HC of those infected with genotypes 19 (partially susceptible to benznidazole) and 32 (susceptible to benznidazole) in both phases of infection. This demonstrates the powerful suppressor effect of benznidazole on parasitaemia, even in mice infected with T. cruzi genotypes (19 and 20) that display low susceptibility to drugs. However, the extension of benefits obtained with treatment seems to be related to the genotype of the parasite involved in the infection.
Different from what was observed with benznidazole treatment, itraconazole treatment, on the other hand, promoted reduction in a small number of virulence parameters. There was a significant decrease in the PP of animals infected with genotype 19 and in the MP of mice infected with genotype 20 and treated during the AP. A significant reduction in %+HC was verified only in animals infected with genotype 32 after treatment during the CP. This demonstrates that itraconazole has a smaller and slower suppressor effect on parasitaemia than benznidazole, which may be due, in part, to different action mechanisms of the two chemotherapeutic agents.29,30
The evaluation of the effects of benznidazole treatment on histopathological data in animals infected with the four T. cruzi genotypes demonstrated that, after AP treatment of the infection, this chemotherapeutic agent promoted a significant reduction in TP in animals infected with genotype 19 (P < 0.05) and in IP in mice infected with genotypes 20 (P < 0.05) and 32 (P < 0.005). A significant reduction in TP with benznidazole treatment was also observed in animals infected with genotype 39 only after CP treatment. These findings are similar to the results of Andrade et al.,5,6 Segura et al.7 and Toledo et al.,8 which demonstrated the useful effect of the chemotherapeutic agent on histopathological parameters (parasitism and inflammation), apparently independent of the degree of drug susceptibility of the T. cruzi strain studied. A good example of this is the benznidazole treatment response of mice infected with genotype 20. Although reportedly resistant to both chemotherapeutic agents (benznidazole and itraconazole),17 mice infected with this genotype displayed a significant reduction in five of the parameters studied: PP, MP, DMP, PAR and IP-AP.
Stocks from genotype 39 again displayed a particular behaviour. Animals infected with this genotype and benznidazole-treated during the AP displayed a significantly higher parasitism than the NT group, demonstrating a negative effect of the treatment on the course of infection. Lana et al.31 and Toledo et al.21 suggested a higher susceptibility of this genotype to the host immune response in relation to genotypes 19/20 and 32. Data previously obtained by Brisse et al.32 with stocks from genetic group 39, suggested that it is a hybrid clonet harbouring a combination of the genes of the two putative parental genotypes. This genotype showed contradictory results between TP during the AP (TP absent) and CP of infection (TP present). Only mice infected with stocks of this clonal genotype showed no association between the levels of TP and IP.21 Moreover, Toledo et al.17 verified that from five stocks of genotype 39, MN cl2 and Bug2149 cl10 were considered, respectively, partially resistant and resistant to benznidazole, which could justify, in part, the detection of amastigotes in tissues of mice infected with these two stocks and benznidazole-treated. The fact that there was no parasitism in animals from the NT group deserves special attention.
These results indicate that, at least for genotype 39, AP treatment appears to have a negative influence on the host–parasite relationship. In addition, it is possible that during the AP, animals infected with genotype 39 modulate an immune response able to maintain TP scarce or absent and that the introduction of treatment interfered with this process. It was observed that treatment in the CP, although having significantly reduced the TP in animals infected with genotype 39, increased (P < 0.05) the percentage of organs/tissues with IP.
Results obtained from the other three genotypes corroborate data from other groups of investigators, who observed beneficial effects of specific treatment with benznidazole in the reduction in histopathological lesions in experimental murine infections6–8 as well as in the clinical evolution of human Chagas’ disease.3,4 However, the curious results observed after benznidazole treatment, only in mice infected with genotype 39, show the influence of parasite genetics in chemotherapy results. This fact has to be seriously considered in drug design and especially by the clinician in any decision about individual treatment. The literature has progressively demonstrated the importance of parasite genetics in drug susceptibility/resistance.17,33
These results also show the necessity of implementing studies that establish the effect of specific chemotherapy on the immunological state as well as on cell populations in the peripheral blood of the patient as suggested by Gontijo et al.34 Some authors have observed experimentally that benznidazole treatment may modify the synthesis of macrophage mediators such as nitrite, IL-6, IL-10 and TNF-
.35 Besides its trypanocidal activity, benznidazole may change the balance between pro- and anti-inflammatory mediators, with important consequences for the course of the T. cruzi infection. Moreover, it was demonstrated that host immune response could influence the results of specific chemotherapy for experimental Chagas’ disease. Benznidazole treatment efficacy was significantly decreased when mice infected with different T. cruzi strains were immunosuppressed with cyclophosphamide.36
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Acknowledgements |
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We thank Dr Michel Tibayrenc for the stocks of T. cruzi studied here and Maria Chaves from the Federal University of Ouro Preto for technical support in obtaining and preparing the histopathological sections. This work was supported by grants from Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG), Brazil.
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Footnotes |
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* Corresponding author. Tel: +55-44-2614518; Fax: +55-44-2614490; E-mail: mjotoledo{at}uem.br
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References |
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