SEARCH

SEARCH BY CITATION

Keywords:

  • Trypanosoma cruzi ;
  • mice;
  • treatment;
  • benznidazole;
  • Amazon
  • Trypanosoma cruzi;
  • souris;
  • traitement;
  • benznidazole;
  • Amazonie
  • Trypanosoma cruzi;
  • ratones;
  • tratamiento;
  • benznidazol;
  • Amazonía

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objective

To assess the susceptibility of Trypanosoma cruzi strains from Amazon to benznidazole.

Methods

We studied 23 strains of T. cruzi obtained from humans in the acute phase of Chagas disease, triatomines and marsupials in the state of Amazonas and from chronic patients and triatomines in the state of Paraná, Brazil. The strains were classified as TcI (6), TcII (4) and TcIV (13). For each strain, 20 Swiss mice were inoculated: 10 were treated orally with benznidazole 100 mg/kg/day (TBZ group) for 20 consecutive days and 10 comprised the untreated control group (NT). Fresh blood examination, haemoculture (HC), PCR, and ELISA were used to monitor the cure.

Results

The overall cure rate was 60.5% (109/180 mice) and varied widely among strains. The strains were classified as resistant, partially resistant or susceptible to benznidazole, irrespective of discrete typing units (DTUs), geographical origin or host. However, the TcI strains from Amazonas were significantly (= 0.028) more sensitive to benznidazole than the TcI strains from Paraná. The number of parasitological, molecular and serological parameters that were significantly reduced by benznidazole treatment also varied among the DTUs; the TBZ group of mice inoculated with TcIV strains showed more reductions (8/9) than those with TcI and TcII strains.

Conclusions

Benznidazole resistance was observed among natural populations of the parasite in the Amazon, even in those never exposed to the drug.

Objective

Evaluer la sensibilité de souches de Trypanosoma cruzi de l’Amazonie au benznidazole.

Méthodes

Nous avons étudié 23 souches de T. cruzi provenant de l'homme dans la phase aiguë de la maladie de Chagas, de triatomes et de marsupiaux dans l'Etat d'Amazonas, et de malades chroniques et triatomes de l’État du Paraná, au Brésil. Les souches ont été classées comme TcI (6), TcII (4) et TcIV (13). Pour chaque souche, 20 souris suisses ont été inoculées: 10 ont été traitées par voie orale avec du benznidazole à 100 mg/kg/jour (groupe TBZ) pendant 20 jours consécutifs et 10 formaient le groupe témoin non traité (NT). L'examen du sang frais, l'hémoculture, la PCR et l’ELISA ont été utilisés pour suivre le traitement.

Résultats

Le taux global de guérison était de 60,5% (109/180 souris) et variait considérablement entre les souches. Les souches ont été classées comme résistantes, partiellement résistantes ou sensibles au benznidazole, quelle que soit la DTU, la provenance géographique ou l'hôte. Cependant, les souches de TcI provenant d'Amazonas étaient significativement (P = 0,028) plus sensibles au benznidazole que les souches TcI de Paraná. Le nombre de paramètres parasitologiques, sérologiques et moléculaires qui ont été considérablement réduits par le traitement au benznidazole variait également selon les DTU; le groupe TBZ des souris inoculées avec des souches TcIV a révélé plus de réductions (8/9) que celui de souris inoculées avec des souches TcI et TcII.

Conclusions

La résistance au benznidazole a été observée dans les populations naturelles du parasite dans l'Amazonie, même dans celles qui n'ont jamais été exposées à ce médicament.

Objetivo

Evaluar la susceptibilidad de cepas de Trypanosoma cruzi del Amazonas al benznidazol.

Métodos

Hemos estudiado 23 cepas de T. cruzi obtenidas de humanos en la fase aguda de la enfermedad de Chagas, triatominos y marsupiales en el Estado del Amazonas, y de pacientes crónicos y triatominos en el Estado de Paraná, Brasil. Las cepas se clasificaron como TcI (6), TcII (4) y TcIV (13). Con cada cepa se inocularon 20 ratones Suizos: 10 fueron tratados oralmente con benznidazol 100 mg/kg/día (grupo TBZ) durante 20 días consecutivos y 10 conformaban el grupo control sin tratar (ST). Para monitorizar la curación, se utilizaron un examen de sangre fresca, hemocultivo, PCR y ELISA.

Resultados

La tasa total de curación fue del 60.5% (109/180 ratones) y varió ampliamente entre las cepas. Las cepas se clasificaron como resistentes, parcialmente resistentes o susceptibles al benznidazol, independientemente del grupo de tratamiento, origen geográfico u hospedero. Sin embargo, las cepas TcI del Amazonas eran significativamente (= 0.028) más susceptibles al benznidazol que las cepas TcI de Paraná. El número de parámetros parasitológicos, moleculares y serológicos que se reducían significativamente con el tratamiento con benznidazol también variaban entre los grupos de tratamiento; en el grupo TBZ los ratones inoculados con las cepas TcIV mostraban una mayor reducción (8/9) que aquellos inoculados con las cepas TcI y TcII.

Conclusiones

Se observó Resistencia al Benznidazol entre poblaciones naturales del parásito en el Amazonas, incluso entre aquellos que no habían estado previamente expuestos al medicamento.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chagas disease (ChD) or American trypanosomiasis is caused by the flagellate protozoan Trypanosoma cruzi. ChD, including vectors and reservoirs, extends from the southern United States to Argentina and Chile, affecting 21 countries [World Health Organization (WHO) 2002; Organización Panamericana de la Salud (OPAS) 2006]. Migration and urbanisation in Latin America have led to cases of the disease in non-endemic areas (Schmunis 2007).

In the Amazon region, ChD is an important emerging anthropozoonosis, with hundreds of cases reported in recent decades. Deforestation, substandard rural housing and harvesting in forests have increased human contact with peridomestic vectors and mammal reservoirs, increasing the cases of alleged sylvatic transmission of ChD (Brasil 2009). The Brazilian Ministry of Health reported that >90% of cases of acute ChD occurred in the Amazon, 7.9% of them in the state of Amazonas (Brasil 2012). In this region, ‘discrete typing units’ (DTUs) TcI and TcIV of T. cruzi predominate. TcI exists in a natural cycle involving vectors of the genus Rhodnius and wild and synanthropic didelphids (Marcili et al. 2009). TcI and TcIV are associated with acute human ChD cases in outbreaks triggered by oral transmission (Marcili et al. 2009; Valente et al. 2009; Monteiro et al. 2012).

Benznidazole (BZ) is the only drug available for specific treatment of ChD in Brazil (Anonymous 2005). However, natural resistance to BZ is common, and its effectiveness varies in different geographical areas (Andrade et al. 1985; Filardi & Brener 1987; Yun et al. 2009). Several studies have investigated the biological and clinical properties of T. cruzi, including the response to chemotherapeutic agents in mice inoculated with different strains (Andrade et al. 1985; Filardi & Brener 1987; Revollo et al. 1998; Toledo et al. 2003, 2004). Brener et al. (1976) first observed the heterogeneity of responses of T. cruzi strains to specific treatment. Andrade et al. (1985) demonstrated an association between resistance to treatment with BZ and nifurtimox (NFX) and the biological behaviour of the parasite. Filardi and Brener (1987) found that the susceptibility in vivo to these drugs of strains from different hosts and geographical origins varied widely, from 0% to 100% cure. Filardi and Brener (1987) and Toledo et al. (1997) reported natural resistance to drugs in strains isolated from both wild and domestic cycles.

Although these studies have made significant contributions, none used a representative set of T. cruzi strains from emerging ChD areas such as the Brazilian Amazon. Few studies have explored the genetic and biological framework of strains of T. cruzi from the western Brazilian Amazon, where ChD causes less morbidity and mortality than in the classic endemic areas, appearing mainly in the chronic latent form. The T. cruzi DTUs in this region that infect humans differ from T. cruzi DTUs in other regions of Brazil. Acute cases have been reported in the Amazon region since 1969 (Shaw et al. 1969), but to our knowledge, no data exist on in vivo susceptibility to chemotherapeutic agents for the strains circulating in this region. This study evaluated the BZ susceptibility of natural populations of T. cruzi from the state of Amazonas, belonging to DTUs TcI and TcIV, comparatively with strains TcI and TcII from the states of Paraná and Minas Gerais, traditional endemic areas.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Parasite strains

We evaluated 23 strains of T. cruzi recently obtained in Amazonas and Paraná from different hosts and belonging to three different DTUs (Figure 1, Table 1). Human-derived strains were isolated before the patients were treated. The Amazonas strains were classified as TcI and TcIV by using polymerase chain reaction (PCR) amplification of the mini-exon and rRNA genes and by sequencing the mitochondrial cytochrome C oxidase subunit II and glucose-6-phosphate isomerase genes (Monteiro et al. 2012). Strains obtained from patients living in Paraná, including two allochthonous strains from Minas Gerais, were also included in the study and were genotyped as TcI and TcII using randomly amplified polymorphic DNA, PCR of the rDNA gene and restriction fragment length polymorphism analysis of the mitochondrial cytochrome C oxidase subunit II gene (Spitzner et al. 2007; Abolis et al. 2011).

Table 1. Discrete typing units (DTUs), geographical origin, host, cure rates and profile of susceptibility to benznidazole in mice for the Trypanosoma cruzi strains studied
T. cruzi strainsDTUGeographical originHostCure rates (%)Susceptibility to benznidazole
  1. aParaná; bAmazonas; cMinas Gerais; dSanta Isabel do Rio Negro; ePCP, patient in the chronic phase of Chagas disease; fPAP, patient in the acute phase of Chagas disease; gR, resistant; hIS, intermediate sensitive; and iS, sensitive.

TPAN/BR/1995/PMARA38TcIArapongas/PRa Panstrongylus megistus 30.0 (3/10)Rg
MDID/BR/2007/AM30TcIManaus/AMb Didelphis marsupiais 55.6 (5/9)ISh
MHOM/BR/1994/PR150TcIJanuária/MGcPCPe57.1 (4/7)IS
MDID/BR/2007/AM39TcIManaus/AM D. marsupiais 57.1 (4/7)IS
TROB/BR/2009/AM56TcIApuí/AM Rhodnius robustus 83.3 (5/6)Si
TROB/BR/2007/AM37TcICoari/AM Rhodnius robustus 100.0 (9/9)S
MHOM/BR/2007/PR1219TcIIMaringá/PRPCP27.3 (3/11)R
MHOM/BR/2007/BS48TcIIColorado/PRPCP42.9 (3/7)IS
MHOM/BR/2009/PR2259TcIIVirgem da Lapa/MGPCP100.0 (6/6)S
MHOM/BR/2010/PR1226TcIIMaringá/PRPCP100.0 (8/8)S
MHOM/BR/2007/AM18TcIVCoari/AMPAPf28.6 (2/7)R
TROB/BR/2009/AM57TcIVApuí/AM Rhodnius robustus 28.6 (2/7)R
MHOM/BR/2007/AM14TcIVCoari/AMPAP30.0 (3/10)R
MHOM/BR/2010/AM67TcIVSIRNd/AMPAP42.9 (3/7)IS
MHOM/BR/2010/AM69TcIVSIRN/AMPAP45.5 (5/11)IS
MHOM/BR/2010/AM64TcIVSIRN/AMPAP57.1 (4/7)IS
MHOM/BR/2007/AM13TcIVCoari/AMPAP66.7 (4/6)IS
MHOM/BR/2007/AM05TcIVCoari/AMPAP66.7 (6/9)IS
MHOM/BR/2007/AM08TcIVCoari/AMPAP71.4 (5/7)S
MHOM/BR/2010/AM62TcIVSIRN/AMPAP71.4 (5/7)S
MHOM/BR/2007/AM15TcIVCoari/AMPAP77.8 (7/9)S
MHOM/BR/2007/AM04TcIVCoari/AMPAP83.3 (5/6)S
MHOM/BR/2010/AM68TcIVSIRN/AMPAP100.0 (7/7)S
image

Figure 1. Map of Brazil highlighting the states of Amazonas, Paraná and Minas Gerais and the hosts from which the Trypanosoma cruzi strains were isolated.

Download figure to PowerPoint

Inoculation of mice

For each T. cruzi strain, a group of 20 male Swiss mice aged 21–28 days was used. The animals were supplied by the UEM central animal facility and were kept in suitable temperature and humidity, with water and food ad libitum. Mice were inoculated intraperitoneally with 1.0 × 104 blood trypomastigotes per animal. For experiments with four strains (AM37, AM56, PR150 and PMARA38) presenting subpatent parasitaemia in Swiss mice, an inoculum of 200 metacyclic trypomastigotes (MT) from culture in LIT medium was used. For strain AM37, a second experiment using an inoculum of 2.0 × 106 MT was also performed.

Susceptibility to benznidazole

After inoculation, the mice were divided into two groups of 10 animals, in separate cages: treated (TBZ group) by gavage with daily doses of BZ (Lafepe, Brazil) of 100 mg/kg body weight for 20 consecutive days, starting on day 5 after inoculation (Filardi & Brener 1987); and untreated controls (NT group). From the 3rd day after inoculation (dai), they were subjected daily to fresh blood examination (FBE) to confirm the infection and parasitaemia record. At day 5, before starting treatment with BZ, they were weighed to ensure that the two groups showed no statistical differences in mean weight and to adjust the drug dose according to the weight of each animal. Some mice were treated without confirmation of infection, particularly those inoculated with TcI strains from Amazonas that present subpatent parasitaemia, because these strains show high infectivity to mice (Reis et al. 2012).

Both TBZ and NT groups were submitted to different diagnostic techniques for cure monitoring and determination of susceptibility to BZ. Mice with negative FBE, haemoculture (HC), PCR and immunoassay [Enzyme-linked immunosorbent assay (ELISA)] results after the treatment ended were considered cured (Toledo et al. 2003; Miyamoto et al. 2008). Those with at least one positive result in any test were considered not cured.

The cure rate in animals inoculated with each strain and treated with BZ was calculated from the ratio between the number of cured animals and the total number of treated animals X 100. To determine the in vivo susceptibility of T. cruzi strains to the chemotherapeutic agent, a criterion similar to Toledo et al. (2003) was adopted.

Fresh blood examination

Fresh blood examination was performed daily from day 3 until a negative result was obtained for at least 3 consecutive days in the case of patent parasitaemia or for 30 days in the case of subpatent parasitaemia, according to Brener (1962).

Parasitaemia curve

The parasitaemia curve was based on the search for trypomastigotes in the bloodstream by FBE. In addition to determining the percentage of animals presenting positive FBE (%+FBE), for the TBZ and NT groups, the mean patent period (PP), maximum peak of parasitaemia (Pmax) and day of maximum peak of parasitaemia (DPmax) were obtained for each strain (Toledo et al. 2002).

Haemoculture

Thirty days after treatment had ended, the mice were submitted to HC in LIT medium according to Filardi and Brener (1987). This technique allowed the determination of the percentage of mice presenting positive haemoculture (%+HC), for TBZ and NT groups, and was employed to confirm the infection in mice with subpatent parasitaemia.

Polymerase chain reaction (PCR)

Blood samples for PCR and HC were collected simultaneously. DNA was extracted using the Gomes et al. (1998) protocol as modified by Miyamoto et al. (2006). PCR amplification was performed using 121 and 122 primers to amplify a specific fragment of 330 base pairs (bp) of kinetoplast DNA (kDNA) of T. cruzi (Wincker et al. 1994). The reaction mixture was submitted to 35 amplification cycles with a Techne TC-512 thermocycler, UK. The PCR products were visualised in 4.5% polyacrylamide silver-stained gel (Santos et al. 1993). From the PCR results, the percentage of mice with positive PCR (%+PCR) was obtained for each strain and for the TBZ and NT groups.

Enzyme-linked immunosorbent assay

An ELISA modified according to Voller et al. (1980) with alkaline antigen of the T. cruzi Y (TcII) strain, peroxidase-labelled anti-mouse immunoglobulin G conjugated (Bethyl Laboratories, Montgomery, AL, USA), and samples of serum collected 90 and 180 days after the end of the treatment (Toledo et al. 2003) was used. From the ELISA results, the percentage of mice with positive ELISA (%+ELISA) was obtained for three and six months after the treatment and for TBZ and NT groups.

Mortality

Cumulative mortality (%MOR) was recorded daily during the infection course. This parameter was determined for each strain and for TBZ and NT groups.

Statistical analysis

Data analysis used BioEstat version 5.3 (Belém, Pará, Brazil). Chi-square or Fisher's exact tests were used to test differences in proportions, and Student's t-test was used to test differences in means. The cure rates were compared among DTUs (TcI, TcII, TcIV), between the states of Amazonas and Paraná, among hosts (human, vector, marsupial) and between TcI strains from Amazonas and Paraná. The comparison also included the TBZ and NT groups, for nine parameters: PP, Pmax, DPmax,%+FBE,%+HC,%+PCR,%+ELISA 3M,%+ELISA 6M and %MOR. A Kaplan–Meier survival analysis was performed using SPSS® version 16.0 for Windows (SPSS Inc.® Chicago, IL, USA) to detect differences in the time elapsed from the first day of treatment to the day when the FBE became negative. Log-rank test was used to test differences between TBZ and NT groups. Statistical significance was considered if P < 0.05.

Ethics

The use of human-derived stocks of T. cruzi obtained from Amazonas and Paraná was approved by the ethics committees of the respective institutions and the use of mice by the Ethics in Research on Animals Committee of the State University of Maringá.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Impact of the treatment on the curve of parasitaemia

The curves of mean parasitaemia of animals inoculated with all T. cruzi strains of each DTU, treated with BZ and untreated are shown in Figure 2a for the 6 TcI strains, Figure 2b for the four TcII strains, and Figure 2c for the 13 TcIV strains. Shortly after treatment with BZ began, the parasitaemia was suppressed in all mice inoculated with strains of the three DTUs studied.

image

Figure 2. Curve of mean parasitaemia of mice inoculated with strains of Trypanosoma cruzi belonging to discrete typing units (DTUs) TcI (a), TcII (b) and TcIV (c) treated with benznidazole (100 mg/kg/d – 20×) (TBZ) and untreated controls (NT).

Download figure to PowerPoint

Cure rates

Treatment with BZ of mice inoculated with the 23 strains of T. cruzi led to an overall cure rate of 60.5% (109/180 mice). Cure rates obtained for each strain ranged from 27.3% to 100% (Table 1). Cure rates for strain AM37 using inoculum of 2 × 102 and 2 × 106 MT/animal were 100% and 90%, respectively.

Mice inoculated with TcI strains showed a mean cure rate of 62.5% (30/48 mice), ranging from 30% to 100%; TcII 62.5% (20/32 mice), ranging from 27.3% to 100%; and TcIV 58% (58/100 mice), ranging from 28.6% to 100% (Table 1).

Considering only the mice inoculated with the 17 strains from Amazonas, the cure rate was 61.8% (81/131), ranging from 28.6% to 100% (Table 1). The cure rates by municipality were as follows: 53.8% (Apuí), ranging from 28.6% to 83.3%; 56.3% (Manaus), ranging from 55.6% to 57.1%; 61.5% (Santa Isabel do Rio Negro-SIRN), ranging from 42.9% to 100%; and 65.1% (Coari), ranging from 28.6% to 100%.

Mice inoculated with T. cruzi strains isolated in Paraná, both autochthonous and allochthonous, had an overall cure rate of 55.1% (27/49 mice); considering only the autochthonous strains, the cure rate was 47.2% (17/36 mice).

The cure rates of mice inoculated with strains of T. cruzi from different hosts were, in ascending order: 56.3% for D. marsupialis, 59.4% for triatomines, 60.2% for humans with ChD in the acute phase and 61.5% for humans in the chronic phase.

Susceptibility to BZ

Regardless of the DTU, geographical origin and host, 9/23 strains of T. cruzi were sensitive (S) to BZ, nine strains were intermediate in sensitivity (IS), and five were resistant (R) to BZ (Table 1). A wide cure gradient was recorded for each DTU, and each DTU set contained strains classified as S, IS and R to BZ (Figure 3).

image

Figure 3. Profile of in vivo susceptibility to benznidazole of Trypanosoma cruzi strains belonging to discrete typing units (DTUs) TcI (a), TcII (b) and TcIV (c) from the states of Amazonas and Paraná, Brazil, in mice treated by oral route, 100 mg/kg/day for 20 consecutive days.

Download figure to PowerPoint

Statistical comparisons

Cure rates for mice inoculated with strains from different DTUs, states and hosts did not differ significantly, except for the comparison between TcI strains from Amazonas and Paraná. TcI strains from Amazonas showed significantly higher (= 0.028) cure rates than TcI strains from Paraná (Figure 3a).

Irrespective of DTU, for mice inoculated with all 23 T. cruzi strains, treatment with BZ caused significant reductions in all nine parameters evaluated (Table 2). However, the number of parameters that showed significant reductions after treatment with BZ varied significantly among DTUs, and the animals inoculated with TcIV strains showed more parameters with significant reductions (8/9), except for %+PCR (Table 2).

Table 2. Statistical comparisons of the parasitological, molecular and serological parameters among mice treated with benznidazole (TBZ group) and untreated controls (NT group), inoculated with Trypanosoma cruzi strains belonging to different discrete typing units (DTUs)
ParameteraOverallTcITcIITcIV
TBZ × NT P TBZ × NT P TBZ × NT P TBZ × NT P
  1. a

    Statistical comparisons of the first three parameters were performed using the Student's t-test and of the other parameters with the chi-square or Fisher's exact tests.

  2. b

    Number of trypomastigotes/0.1 mL of blood at the peak of maximum parasitaemia.

  3. c

    Day of the peak of maximum parasitaemia; PA, parasitaemia absent.

  4. d

    Percentage of mice with positive fresh blood examination (FBE).

  5. e

    Percentage of mice with positive haemoculture (HC).

  6. f

    percentage of mice with positive polymerase chain reaction.

  7. g

    Percentage of mice with positive Enzyme-linked immunosorbent assay (ELISA) 3 months after the treatment.

  8. h

    Percentage of mice with positive ELISA 6 months after the treatment.

  9. i

    Cumulative mortality.

PP (in days)0.5 × 5.9<0.0010.0 × 0.32NS0.51 × 18.90.0110.73 × 4.8<0.001
Pmaxb (×103)1.46 × 16.020.0030.0 × 0.29NS0.42 × 57.40.0212.57 × 10.50.019
DPmaxc5.3 × 10.9<0.001PA × 19.5-6.0 × 18.20.0214.8 × 6.90.000
%+FBEd0.0 × 64.8<0.0010.0 × 17.10.0300.0 × 87.50.0000.0 × 79.30.049
%+HCe27.1 × 58.6<0.00112.5 × 55.60.00031.2 × 100.00.00034.0 × 49.40.034
%+PCRf37.3 × 62.2<0.00129.2 × 67.90.00037.5 × 96.60.00041.2 × 45.6NS
%+ELISA 3Mg41.8 × 80.4<0.00140.0 × 33.3NS63.6 × 100.0NS37.9 × 92.10.000
%+ELISA 6Mh61.4 × 96.70.00050.0 × 100.0NS71.4 × 100.0NS60.6 × 92.00.033
%MORi0.4 × 10.40.0000.0 × 8.50.0310.0 × 0.0NS0.8 × 14.60.003
Number of significant differences9/94/96/98/9 

Survival analysis showed a significant reduction (= 0.000) in the period from the first day of treatment until a negative FBE, for all the TBZ groups of mice (Figure 4), irrespective of the geographical origin, host or DTU of the strains.

image

Figure 4. Kaplan–Meier survival analysis showing the time elapsed from the beginning of the treatment with benznidazole until a negative fresh blood examination, in treated (TBZ) and untreated (NT) groups for discrete typing units (DTUs) TcI, TcII and TcIV.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The clonal evolution model postulated for T. cruzi (Tibayrenc & Ayala 1988) predicts a correlation between the phylogenetic divergence of parasite clonal genotypes and their biological and medical properties. Many researchers have studied the association between T. cruzi subspecific genetic diversity and the parasite's biological properties, including susceptibility in vitro and in vivo to various chemotherapeutic agents such as BZ, NFX and itraconazole (ITZ) (Andrade & Magalhães 1997; Revollo et al. 1998; Toledo et al. 2003, 2004). In general, different DTUs exhibit statistically different, although somewhat overlapping, biological properties.

Acute ChD caused by strains belonging to the DTU TcIV (formerly TcIIa; Brisse et al. 2000) has been described by Marcili et al. (2009). Monteiro et al. (2012) found that this DTU is the major aetiologic agent responsible for acute cases of the disease in the western Brazilian Amazon, including outbreaks of oral transmission. To our knowledge, ours is the first study focusing on the susceptibility of strains belonging to DTU TcIV derived from the Brazilian Amazon, to the specific chemotherapeutic agent BZ.

This in vivo study of the BZ susceptibility of strains of T. cruzi obtained from different sources in Amazonas and Paraná and belonging to DTUs TcI, TcII and TcIV showed a wide variation in cure rates, ranging from 27.3% to 100%. This result confirms previous studies that found rates varying from 0% to 100% in the in vivo susceptibility to BZ and NFX for strains of T. cruzi originating from different Brazilian states and Latin American countries (Andrade et al. 1985; Filardi & Brener 1987). Similar variation in susceptibility to BZ was observed among strains from different transmission cycles (Toledo et al. 1997) and clones of parasites belonging to different genotypes (Toledo et al. 2003).

In this study, we found that some Amazon strains were resistant to the specific chemotherapy, in agreement with Andrade et al. (1985) and Filardi and Brener (1987) who observed strains with natural resistance to the drug, even among people without previous exposure.

A correlation between susceptibility to chemotherapeutic agents and phenotypic and genotypic features was also described, respectively, by Andrade et al. (1985) and Toledo et al. (2003). However, the current study showed no clear association between susceptibility to BZ and the DTU of the strains, with no predominance of strains with a particular pattern of BZ resistance within the DTUs, despite the apparently greater BZ susceptibility of TcI strains from Amazonas than TcI strains from Paraná.

Unlike previous research, the use of PCR, a more sensitive technique for monitoring the cure of treated mice (Miyamoto et al. 2006, 2008), probably improved the efficiency in detecting therapeutic failure. This methodological difference may explain the divergent results concerning the correlation between genetic diversity of T. cruzi and susceptibility to BZ.

Evidence is increasing that T. cruzi DTUs are highly diverse, and this heterogeneity may be highly informative in epidemiological terms (Llewellyn et al. 2009). Although DTU TcI is the major cause of resurgent human disease in northern South America, it also occurs in sylvatic triatomine vectors and mammalian reservoirs throughout the continent. The great genetic variability of TcI isolates from different geographical regions in Colombia was sufficient to propose the existence of at least four haplotypes associated with distinct transmission cycles of the parasite (Herrera et al. 2007; Falla et al. 2009). The six TcI strains of this study were classified as R (1), SI (3) and S (2) to BZ, and mice inoculated with this DTU showed an overall cure rate of 62.5%, an intermediate level of resistance. These data are at variance with other studies that associated TcI with greater resistance to BZ (Andrade et al. 1985; Revollo et al.1998; Toledo et al. 2003). However, sylvatic TcI populations are extraordinarily genetically diverse and several genotypes exist within TcI (Llewellyn et al. 2009; Cura et al. 2010). Likewise, intra-DTU genetic variability was demonstrated for TcIV strains from Amazonas with those originating from SIRN Municipality containing several haplotypes (Monteiro et al. 2012). These genotypes/haplotypes may differ in susceptibility to drugs. Studying two TcI genotypes (genotypes 19 and 20; Tibayrenc & Ayala 1988), Toledo et al. (2003) found that all clones belonging to genotype 20 were resistant to ITZ and BZ, while genotype 19 clones varied in susceptibility to BZ.

The survival curves for TcI (Figure 4) showed that the most strains belonging to this DTU never developed patent parasitaemia, allowing us to conclude that FBE is an inadequate parameter for cure monitoring for Amazonian TcI strains and explaining the absence of a predominant susceptibility pattern.

Murta et al. (1998) also found no correlation between the response to specific chemotherapy with BZ and NFX and the genetics of the parasite for the DTUs TcI and TcII (formerly Z1 and Z2, respectively; Miles et al. 1977). However, the association between sensitivity to drugs and DTU TcVI (formerly zymodeme B; Romanha et al. 1979) was confirmed, and all TcVI strains evaluated to date are sensitive to treatment with BZ and NFX.

The different levels of susceptibility to BZ for strains from Paraná concord with previous findings in study that used a larger number of strains (Toledo et al. 1997). Two strains in the current study, PR2259 (TcII) and PR150 (TcI), were previously studied, and the BZ susceptibility of PR2259, re-isolated 10 years later from the same patient, was confirmed. However, PR150 was previously considered resistant (0% cure), but was assessed as IS (cure rate of 57.1%) in this study. In addition to its increased susceptibility to BZ, PR150 changed its biological behaviour in mice, displaying lower infectivity and parasitaemia from subpatent to patent, suggesting population selection due to manipulation of a polyclonal strain. Parasite subpopulations with different genotypes may be present in low percentages in the T. cruzi strain and could be selected after a long-term vertebrate host–T. cruzi interaction (Veloso et al. 2005), by successive blood passages or maintenance in acellular culture, influencing their susceptibility to BZ (Veloso et al. 2001; Caldas et al. 2008). The genetic heterogeneity of the DTU TcI suggests that the existence of correlations between genetic and biological properties should be investigated at the sub-DTU level (Revollo et al. 1998; Toledo et al. 2002, 2003; Cura et al. 2010).

In this study, there were no significant differences in cure rates when strains were grouped according to DTU, geographical origin or host, except that the TcI strains from Amazonas showed higher cure rates than TcI strains from Paraná. Specific treatment with BZ triggered a significant reduction in the parasitological, molecular or serological parameters for mice inoculated with all DTUs. However, a more significant reduction was recorded for TcIV, indicating that the treatment can be more or less beneficial for the host depending on the DTU. Parasitemia levels were up to 10 times lower in mice inoculated with T. cruzi strains from Amazonas in comparison with strains from Paraná (Reis et al. 2012), which may have affected these results, because the drug dose was the same for all animals. TcI and TcIV strains are prevalent in the Amazon, and these further findings in mice justify the use of BZ in the treatment of ChD patients in this region.

It is difficult to correlate the results for mice inoculated with strains from the Amazon region and patient outcomes, due to the scarcity of published data on the effectiveness of ChD treatment in this region. Although there is little evidence on the effect of specific treatment in Amazonian patients (Pinto et al. 2009, 2010; Valente et al. 2009), the literature suggests a correlation between treatment response in patients, and results of experimental chemotherapy in mice infected with the same strains (Filardi & Brener 1987; Andrade et al. 1992; Toledo et al. 2004).

In this study, TcIV strains isolated from acute Chagasic patients and from Rhodnius robustus in Amazonas varied in susceptibility to BZ in vivo, comprising sensitive, partially sensitive and resistant strains. Resistance to BZ was observed among T. cruzi strains from Amazonas, which were natural parasite populations without previous exposure to this drug. The cure rate of about 60%, the apparent greater sensitivity to BZ of TcI strains from Amazonas in relation to TcI strains from Paraná and the significant reductions in parameters of mice inoculated with Amazonian strains undergoing treatment with BZ indicate that this drug is appropriate to treat human patients from this region. However, more effective treatments for ChD need to be found.

In conclusion, the data presented here do not fit the predictions of the clonal evolution model of T. cruzi. The lack of association between the classification of the strains in DTUs and their in vivo susceptibility to BZ could be explained by the considerably higher genetic variability of T. cruzi strains from the sylvatic than from the domestic environment. Our results also warn of the necessity to take into account the lower phylogenetic subdivisions of this species (genotypes and haplotypes) in studies that seek to evaluate the correlation between genetic diversity and biological properties in T. cruzi.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors thank to the Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná and to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Abolis NG, Araújo SM, Toledo MJO, Fernandez MA & Gomes ML (2011) Trypanosoma cruzi I-III in southern Brazil causing individual and mixed infections in humans, sylvatic reservoirs and triatomines. Acta Tropica 120, 167172.
  • Andrade SG & Magalhães JB (1997) Biodemes and zymodemes of Trypanosoma cruzi strains: correlations with clinical data and experimental pathology. Revista da Sociedade Brasileira de Medicina Tropical 30, 2735.
  • Andrade SG, Magalhães JB & Pontes AL (1985) Evaluation of chemotherapy with benznidazole and nifurtimox in mice infected with Trypanosoma cruzi strains of different types. Bulletin of the World Health Organization 63, 721726.
  • Andrade SG, Rassi A, Magalhães JB, Ferriolli FF & Luquetti AO (1992) Specific chemotherapy of Chagas disease: a comparison between the response in patients and experimental animals inoculated with the same strains. Transactions of the Royal Society of Tropical Medicine and Hygiene 86, 624626.
  • Anonymous (2005) Consenso Brasileiro em Doença de Chagas. Secretaria de Vigilância em Saúde do Ministério da Saúde. Revista da Sociedade Brasileira de Medicina Tropical 38, 130.
  • Brasil (2009) [database on the Internet]. Ministério da Sáude. A Doença de Chagas no Brasil. Available at: http://portal.saude.gov.br/portal/arquivos/pdf/tabchagascasos0509. pdf. Accessed 23 February 2009.
  • Brasil (2012) [database on the Internet]. Ministério da Saúde. Epidemiologia da DCh. Available: http://portal.saude.gov.br/portal/saude/profissional/visualizartexto.cfm?idtext=31454. Accessed 23 February 2012.
  • Brener Z (1962) Therapeutic activity and criterion of cure on mice experimentally infected with Trypanosoma cruzi. Revista do Instituto de Medicina Tropical de São Paulo 4, 389396.
  • Brener Z, Costa CAG & Chiari C (1976) Differences in the susceptibility of Trypanosoma cruzi strains to active chemotherapeutic agents. Revista do Instituto de Medicina Tropical de São Paulo 18, 450455.
  • Brisse S, Barnabé C & Tibayrenc M (2000) Identification of six Trypanosoma cruzi phylogenetic lineages by random amplified polymorphic DNA and multilocus enzyme electrophoresis. International Journal for Parasitology 30, 3544.
  • Caldas S, Santos FM, Lana M et al. (2008) Trypanosoma cruzi: acute and long-term infection in the vertebrate host can modify the response to benznidazole. Experimental Parasitology 118, 315323.
  • Cura CI, Mejia-Jaramillo AM, Duffy T et al. (2010) Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced leader genes. International Journal for Parasitology 40, 15991607.
  • Falla A, Herrera C, Fajardo A, Montilla M, Vallejo GA & Guhl F (2009) Haplotype identification within Trypanosoma cruzi I in Colombian isolates from several reservoirs, vectors and humans. Acta Tropica 110, 1521.
  • Filardi LS & Brener Z (1987) Susceptibility and natural resistance of Trypanosoma cruzi strains to drugs used clinically in Chagas disease. Transactions of the Royal Society of Tropical Medicine and Hygiene 81, 755759.
  • Gomes ML, Macedo AM, Vago AR, Pena SD, Galvão LM & Chiari E (1998) Trypanosoma cruzi: optimization of polymerase chain reaction for detection in human blood. Experimental Parasitology 88, 2833.
  • Herrera C, Bargues MD, Fajardo A et al. (2007) Identifying four Trypanosoma cruzi I isolate haplotypes from different geographic regions in Colombia. Infection, Genetics and Evoution 7, 535539.
  • Llewellyn MS, Miles MA, Carrasco HJ et al. (2009) Genome-scale multilocus microsatellite typing of Trypanosoma cruzi discrete typing unit I reveals phylogeographic structure and specific genotypes linked to human infection. PLoS Pathogens 5, e1000410.
  • Marcili A, Valente VC, Valente SA et al. (2009) Trypanosoma cruzi in Brazilian Amazonia: lineages TCI and TCIIa in wild primates, Rhodnius spp. and in humans with Chagas disease associated with oral transmission. International Journal for Parasitology 39, 615623.
  • Miles MA, Toyé PJ, Oswaldo SC & Godfrey DC (1977) The identification by isoenzyme patterns if two distinct strain-groups of Trypanosoma cruzi, circulating independently in a rural area of Brazil. Transactions of the Royal Society of Tropical Medicine and Hygiene 71, 217225.
  • Miyamoto CT, Gomes ML, Marangon AV et al. (2006) Trypanosoma cruzi: sensitivity of the polymerase chain reaction for detecting the parasite in the blood of mice infected with different clonal genotypes. Experimental Parasitology 112, 198201.
  • Miyamoto CT, Gomes ML, Marangon AV et al. (2008) Usefulness of the polymerase chain reaction for monitoring cure of mice infected with different Trypanosoma cruzi clonal genotypes following treatment with benznidazole. Experimental Parasitology 120, 4549.
  • Monteiro WM, Magalhães LKC, Sá ARN et al. (2012) Trypanosoma cruzi IV causing outbreaks of acute Chagas disease and infections by different haplotypes in the Western Brazilian Amazonia. PLoS ONE 7(), e41284. doi:10.1371/journal.pone.0041284.
  • Murta SMF, Gazinelli RT, Brener Z & Romanha AJ (1998) Molecular characterization of susceptible and naturally resistant strains of Trypanosoma cruzi to benznidazole and nifurtimox. Molecular and Biochemical Parasitology 93, 203214.
  • Organización Panamericana de la Salud (OPAS) (2006) Estimación cuantitativa de la enfermedad de Chagas en las Americas Pan American Health Organization (PAHO), Rio de Janeiro, Brazil.
  • Pinto AYN, Ferreira AG, Valente VC, Harada GS & Valente SAS (2009) Urban outbreak of acute Chagas disease in Amazon region of Brazil: four-year follow-up after treatment with benznidazole. Revista Panamericana de Salud Pública 25, 7783.
  • Pinto AYN, Ferreira AG, Valente SAS et al. (2010) Alterações eletrocardiográficas durante e após tratamento com benzonidazol em fase aguda de doença de Chagas autóctone da Amazônia brasileira. Revista Pan-Amazônica de Saúde 1, 6776.
  • Reis D, Monteiro WM, Bossolani GDP et al. (2012) Biological behavior in mice of Trypanosoma cruzi isolates from Amazonas and Paraná, Brazil. Experimental Parasitology 130, 321329.
  • Revollo S, Oury B, Laurent JP et al. (1998) Trypanosoma cruzi: impact of clonal evolution of the parasite on its biological and medical properties. Experimental Parasitology 89, 3039.
  • Romanha AJ, Pereira AAS, Chiari E & Kilgour V (1979) Isoenzyme patterns of cultured Trypanosoma cruzi: changes after prolonged subculture. Comparative Biochemical and Physiology 62, 139142.
  • Santos FR, Pena SDJ & Epplen JT (1993) Genetic and population study of a Y-linked tetranucleotide repeat DNA polymorphism with a simple non-isotopic technique. Human Genetics 90, 655656.
  • Schmunis GA (2007) Epedimiology of Chagas' disease in non-endemic countries: the role of international migration. Memórias do Instituto Oswaldo Cruz 102, 7585.
  • Shaw J, Lainson R & Fraiha H (1969) Considerações sobre a epidemiologia dos primeiros casos autóctones de doença de Chagas registrados em Belém, Pará, Brasil. Revista de Saúde Pública 3, 153157.
  • Spitzner FL, Freitas JM, Macedo AM et al. (2007) Trypanosoma cruzi – triatomine associations and the presence of mixed infections in single triatomine bugs in Paraná state, Brazil. Acta Parasitologica 52, 7481.
  • Tibayrenc M & Ayala FJ (1988) Isozyme variability in Trypanosoma cruzi, the agent of Chagas disease: genetical, taxonomical, and epidemiological significance. Evolution 42, 277292.
  • Toledo MJO, Guilherme ALF, Silva JC et al. (1997) Trypanosoma cruzi: chemotherapy with Benznidazole in mice inoculated with strains from Paraná state and from different endemic areas of Brazil. Revista do Instituto de Medicina Tropical de São Paulo 39, 283290.
  • Toledo MJO, Lana M, Carneiro CM et al. (2002) Impact of Trypanosoma cruzi clonal evolution on its biological properties in mice. Experimental Parasitology 100, 161172.
  • Toledo MJO, Bahia MT, Carneiro CM et al. (2003) Chemotherapy with benznidazole and itraconazole for mice infected with different Trypanosoma cruzi clonal genotypes. Antimicrobial Agents and Chemotherapy 47, 223230.
  • Toledo MJO, Tafuri WL, Bahia MT, Tibayrenc M & Lana M (2004) Genetic diversity and drug resistence in Trypanosoma cruzi, the agent of Chagas disease. Research Advances in Antimicrobial Agents Chemotherapy 4, 1121.
  • Valente SAS, Valente VC, Pinto AYN et al. (2009) Analysis of an acute Chagas disease outbreak in the Brazilian Amazon: human cases, triatomines, reservoir mammals and parasites. Transactions of the Royal Society of Tropical Medicine and Hygiene 103, 291297.
  • Veloso VM, Carneiro CM, Toledo MJ et al. (2001) Variation in susceptibility to benznidazole in isolates derived from Trypanosoma cruzi parental strains. Memórias do Instituto Oswaldo Cruz 96, 10051011.
  • Veloso VM, Romanha AJ, Lana M et al. (2005) Influence of the long-term Trypanosoma cruzi infection in vertebrate host on the genetic and biological diversity of the parasite. Parasitology Research 96, 382389. doi: 10.1007/s00436-005-1373-z.
  • Voller A, Bidwell DE & Bartlett A (1980) Enzyme immunoassays in diagnostic medicine. Theory and practice. Bulletin of the World Health Organization 53, 5565.
  • Wincker P, Britto C, Pereira JB, Cardoso MA, Oelemann W & Morel CM (1994) Use of a simplified polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients in a rural endemic area. American Journal of Tropical Medicine and Hygiene 71, 771777.
  • World Health Organization (WHO) (2002) Control of Chagas Disease. Second Report of the WHO Experts Committee, Technical Report Series No. 995. World Health Organization, Geneva.
  • Yun O, Lima MA, Ellman T et al. (2009) Feasibility, drug safety, and effectiveness of etiological treatment programs for Chagas disease in Honduras, Guatemala and Bolivia: 10-year experience of Médecins Sans Frontières. Plos Neglected Tropical Diseases 3, 18.