Benznidazole Treatment: Time- and Dose-Dependence Varies with the Trypanosoma Cruzi Strain

pathogens

Article Benznidazole Treatment: Time- and Dose-Dependence Varies with the Trypanosoma cruzi Strain

Kátia da Silva Fonseca 1,†, Luísa Perin 1,†,Nívia Carolina Nogueira de Paiva 1, Beatriz Cristiane da Silva 1, Thays Helena Chaves Duarte 2, Flávia de Souza Marques 2, Guilherme de Paula Costa 1, Israel Molina 1,3, Rodrigo Correa-Oliveira 1,4, Paula Melo de Abreu Vieira 1,2 and Cláudia Martins Carneiro 1,5,*

1 Laboratory of Immunopathology, Nucleus of Biological Sciences Research, Federal University of Ouro Preto, Ouro Preto 35400-000, Brazil; [email protected] (K.d.S.F.); [email protected] (L.P.); [email protected] (N.C.N.d.P.); [email protected] (B.C.d.S.); [email protected] (G.d.P.C.); [email protected] (I.M.); correa@cpqrr.fiocruz.br (R.C.-O.); [email protected] (P.M.d.A.V.) 2 Laboratory of Morphopathology, Department of Biological Sciences, Nucleus of Biological Sciences Research, Institute of Exact and Biological Sciences, Federal University of Ouro Preto, Ouro Preto 35400-000, Brazil; [email protected] (T.H.C.D.); fl[email protected] (F.d.S.M.) 3 Tropical Medicine and International Health Unit, Department of Infectious Diseases, Vall d’Hebron University Hospital, Universitat Autònoma de Barcelona, PROSICS Barcelona, 08035 Barcelona, Spain 4 Laboratory of Cellular and Molecular Immunology, René Rachou Research Center, Oswaldo Cruz Foundation, Belo Horizonte 30190-002, Brazil 5 Department of Clinical Analysis, School of Pharmacy, Federal University of Ouro Preto, Ouro Preto 35400-000, Brazil Citation: Fonseca, K.d.S.; Perin, L.; * Correspondence: [email protected]; Tel.: +55-31-3559-16-94; Fax: +55-31-3559-16-80 de Paiva, N.C.N.; da Silva, B.C.; †Kátia da Silva Fonseca and Luísa Perin contributed equally to this work. Duarte, T.H.C.; Marques, F.d.S.; Costa, G.d.P.; Molina, I.; Correa-Oliveira, R.; Abstract: As the development of new drugs for Chagas disease is not a priority due to its neglected Vieira, P.M.d.A.; et al. Benznidazole disease status, an option for increasing treatment adherence is to explore alternative treatment Treatment: Time- and Dose- regimens, which may decrease the incidence of side effects. Therefore, we evaluated the efficacy Dependence Varies with the of different therapeutic schemes with benznidazole (BNZ) on the acute and chronic phases of the Trypanosoma cruzi Strain. Pathogens disease, using mice infected with strains that have different BNZ susceptibilities. Our results show 2021, 10, 729. https://doi.org/ that the groups of animals infected by VL-10 strain, when treated in the chronic phase with a lower 10.3390/pathogens10060729 dose of BNZ for a longer period of time (40 mg/kg/day for 40 days) presented better treatment efficacy than with the standard protocol (100 mg/kg/day for 20 days) although the best result in the Academic Editor: treatment of the animals infected by the VL-10 strain was with100 mg/kg/day for 40 days. In the Vyacheslav Yurchenko acute infection by the Y and VL-10 strains of T. cruzi, the treatment with a standard dose, but with a

Received: 10 April 2021 longer time of treatment (100 mg/kg/day for 40 days) presented the best results. Given these data, Accepted: 13 May 2021 our results indicate that for BNZ, the theory of dose and time proportionality does not apply to the Published: 9 June 2021 phases of infection.

Publisher’s Note: MDPI stays neutral Keywords: chagas disease; Trypanosoma cruzi; benznidazole; therapeutic strategies; mice with regard to jurisdictional claims in published maps and institutional afﬁl- iations. 1. Introduction Chagas disease is a neglected protozoan disease that affects more than 6 million people worldwide and is endemic in Latin America [1]. It is caused by the hemoﬂagellate Copyright: © 2021 by the authors. parasite Trypanosoma cruzi (T. cruzi), belonging to the order Kinetoplastida and family Licensee MDPI, Basel, Switzerland. Tripanosomatidae, whose main invertebrate hosts and vectors of Chagas disease, are This article is an open access article hematophagous insects of the subfamily Triatominae [2,3]. T. cruzi has a high genetic distributed under the terms and heterogeneity and using different molecular markers, genotyping of the strains of the conditions of the Creative Commons parasite was carried out, in order to classify it in several subgroups (or discrete typing Attribution (CC BY) license (https:// units, DTUs) termed TcI–TcVI. However, studies have suggested that the DTUs would creativecommons.org/licenses/by/ have the status of subspecies [3]. 4.0/).

Pathogens 2021, 10, 729. https://doi.org/10.3390/pathogens10060729 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 729 2 of 10

Benznidazole (BNZ), the reference drug in the treatment of Chagas disease, shows satisfactory results in the acute and recent chronic phases of infection, but causes numerous side effects that lead to treatment interruption by significant numbers of patients [4–10]. There are few efforts to discover new drugs for Chagas disease, therefore, an option for increasing treatment adherence is to explore alternative treatment regimens with BNZ, which could decrease the incidence of side effects. A number of studies have suggested that it is possible to reduce the dose of BNZ currently used for treatment of Chagas disease. First, researchers reported that 20% of adults who prematurely abandoned BNZ treatment due to adverse effects, displayed negative serology for T. cruzi [11]. Furthermore, although plasma concentrations of BNZ in children subjected to the treatment are lower than in adults, the treatment efficacy is proven, suggesting that the BNZ dose used in the treatment of adults could be decreased and still generate similar exposure to that observed in children, maintaining efficacy and reducing side effects [12]. A prospective study of population pharmacokinetics showed, through dose simulations, that it is possible to decrease the dose of BNZ in relation to the conventional dose while keeping the plasma concentration within the recommended target range, indicating the need to evaluate whether lower doses could be as efficient as the conventional dose [13]. Moreover, animals subjected to intermittent treatment that reduces the total dose of BNZ can be cured of T. cruzi infection [14]. However, a recent study showed that to obtain parasitological cure in mice chronically infected with resistant T. cruzi strains it is necessary to increase the dose of BNZ and extend the length of treatment [15]. Lastly, mice in the acute phase of T. cruzi infection, when treated with BNZ, have a dependent dose-time response profile [16]. It still remains necessary to evaluate other therapeutic regimens by altering dose and time of treatment simultaneously and, mainly, to perform the evaluation in the chronic period of the infection, the phase when most patients are treated. Therefore, the objective of this work was to evaluate the influence of the dose and time treatment in curing mice in the acute and chronic phases of the infection against strains with different susceptibilities to BNZ treatment.

2. Materials and Methods 2.1. Animals and Ethics One hundred and twenty Swiss mice, 30 days old, weighing 18–25 g, and provided by the Animal Science Center of the Federal University of Ouro Preto (CCA, UFOP), were kept under control conditions with regular cycles of light, temperature of 23 ± 2 ◦C, and availability of food and water ad libitum. All procedures were performed according to ethical principles recommended by the National Council for Control of Animal Experimen- tation (CONCEA) and approved by the ethics committee of Federal University of Ouro Preto (Protocol CEUA-UFOP number: 2016/33). The mice were monitored daily by the researchers, who have extensive experience, so that visible physical changes were communicated to the veterinarian responsible for the sector, in order to determine the appropriate handling of the animal. We emphasize that some physical changes are characteristic of infection by Trypanosoma cruzi, such as weight loss and bristly fur, and in none of the evaluations carried out by the veterinarian throughout the project, were the animals euthanized.

2.2. Therapeutic Schemes Mice were inoculated with 5 × 103 of the blood trypomastigote forms of T. cruzi, all trypomastigotes were obtained from the blood samples of infected albino Swiss mice at the peak of parasitemia. These animals were divided into three groups: infected with Y strain (DTU II) and treated in the acute phase (n = 40); infected with VL-10 strain (DTU II) and treated in the acute phase (n = 40); and infected with VL-10 strain and treated in the chronic phase (n = 40). To exclude variables, the inoculum was the same for all phases/strains. Y Pathogens 2021, 10, 729 3 of 10

strain was not observed in chronic phase because the animals are not able to survive until chronic phase with this inoculum. In the acute phase and in the chronic phase of this experiment, groups of eight animals were analyzed: (1) infected and untreated; (2) infected and treated with BNZ 100 mg/kg/day for 20 days; (3) infected and treated with BNZ 100 mg/kg/day for 40 days; (4) infected and treated with BNZ 40 mg/kg/day for 20 days; and (5) infected and treated with BNZ 40 mg/kg/day for 40 days. BNZ was prepared by pulverization of 100 mg tablets (Pharmaceutical Laboratory of Pernambuco State, Recife, Brazil), suspended in an aqueous solution of methylcellulose 0.5% (Sigma-Aldrich, St. Louis, MO, USA), and each animal received, according to body weight, the suspension of the drug by gavage. The treatment was initiated in the acute phase after conﬁrmation of infection. The mice in the untreated group received oral gavage with water and the treated groups received BNZ suspended in a solution of water and methylcellulose. As this drug has low solubility in water [17], this strategy is necessary to enable greater homogeneity of the suspended substance, being the methylcellulose the suspending agent most used in pharmaceutical preparations, including in studies carried out by Chagas disease research groups [16,18,19]. Animals were evaluated in relation to survival, fresh blood examination (FBE), parasitemia, real-time polymerase chain reaction (qPCR) of blood ﬁve days after immunosuppression with cyclophosphamide (CyI), necropsy and qPCR of heart and colon 30 days after CyI. Treatment by gavage of animals in the chronic phase was initiated 120 days after infection. The same parameters evaluated in the acute phase were used in the chronic phase. The experiments lasted from 78 days (acute phase—20 days) to 213 days (chronic phase—40 days).

2.3. Cure Criteria For therapeutic evaluation, the analysis included: survival, parasitemia, and cure through FBE and qPCR after CyI. Only animals that were negative in both tests were considered cured [20].

2.3.1. Survival Animals were followed daily after inoculum up to 30 days after the end of the treatment and mortality was recorded [20].

2.3.2. Parasitemia Parasitemia was evaluated daily from the 4th day after infection until examination was negative for ﬁve consecutive days, following the adapted methodology [21].

2.3.3. Immunosuppression and Fresh Blood Examination Thirty days after the end of treatment all mice were subjected to CyI. Parasitemia was evaluated daily during this process and until ﬁve days after CyI, adapted methodology [22].

2.3.4. Real-Time Polymerase Chain Reaction (qPCR) qPCR in Total Blood Peripheral blood of mice was collected ﬁve days after the end of CyI, stored in Eppendorf tubes containing EDTA (anticoagulant), and kept in ice until extraction. Samples were subjected to procedures of DNA extraction using DNeasy® Blood and Tissue Kit (Qiagen®, Hilden, Germany), following the manufacturer’s recommendations.

qPCR in Tissue Fragments of heart and colon were collected during necropsy and then subjected to procedures of DNA extraction using WizardTM Genomic DNA Puriﬁcation Kit (Promega, Madison, WI, USA), following the manufacturer’s recommendations. Pathogens 2021, 10, x FOR PEER REVIEW 4 of 10

qPCR in Tissue Fragments of heart and colon were collected during necropsy and then subjected to procedures of DNA extraction using WizardTM Genomic DNA Purification Kit Pathogens 2021, 10, 729 4 of 10 (Promega, Madison, WI, USA), following the manufacturer’s recommendations.

2.4. Statistical Analyses 2.4. StatisticalStatistical Analyses analyses of the data were performed using GraphPad Prisma software (GraphPadStatistical Software, analyses San of Diego, the data CA, were USA). performed The parasitemia using GraphPad was expressed Prisma as software mean ± (GraphPadstandard error. Software, To compare San Diego, the area CA, under USA). the The curve parasitemia (AUC), Kruskal-Wallis was expressed test/ as mean Dunn’s± standardmultiple error.comparisons To compare test the was area underused, theand curve Kruskal-Wallis (AUC), Kruskal-Wallis test/Dunn’s test/Dunn’s multiple multiplecomparisons comparisons test were test used was to used, compare and Kruskal-Wallismaximum parasitemia test/Dunn’s peak. multiple comparisons test wereThe useddata towere compare evaluated maximum by contingency parasitemia tabl peak.e, Fisher exact test, and odds ratio to compareThe datathe percentage were evaluated of cure by among contingency the different table, Fishergroups. exact Differences test, and in odds mean ratio values to comparewere considered the percentage significant of cure at pamong < 0.05. the different groups. Differences in mean values were considered signiﬁcant at p < 0.05. 3. Results 3. Results Mice infected with Y strain and treated with all protocols showed a reduction in the parasitemiaMice infected peak and with patent Y strain period and treatedcompared with to alluntreated protocols animals showed (Figure a reduction 1). in the parasitemia peak and patent period compared to untreated animals (Figure1).

Figure 1. Effect of different benznidazole (BNZ) therapeutic protocols on parasitemia by Trypanosoma cruzi strains with Figure 1. Effect of different benznidazole (BNZ) therapeutic protocols on parasitemia by Trypanosoma cruzi strains with different susceptibilities to BNZ. (A) Y: partially susceptible, and (B) VL-10: resistant. Standard treatment for mice: BNZ different susceptibilities to BNZ. (A) Y: partially susceptible, and (B) VL-10: resistant. Standard treatment for mice: BNZ 100 mg/kg/day for 20 days. Parasitemia was measured by an adapted version of the Brener method. 100 mg/kg/day for 20 days. Parasitemia was measured by an adapted version of the Brener method. Animals infected with Y strain presented a survival rate of 63% when untreated, and 0% cure rate, Table1. Treatment with 100 mg/kg/day of BNZ improved the survival rate to 100%, with a cure rate of 75% for the 20-day treatment, which increased to an 87% cure rate when the treatment duration was extended to 40 days. However, for this strain, the lower dose of BNZ (40 mg/kg/day) resulted in no cure for either 20-day or 40-day treatments. Moreover, survival rates with 40 mg/kg/day of BNZ for 40 days decreased Pathogens 2021, 10, x FOR PEER REVIEW 5 of 10 Pathogens 2021, 10, x FOR PEER REVIEW 5 of 10 Pathogens 2021, 10, x FOR PEER REVIEW 5 of 10 Pathogens 2021, 10, x FOR PEER REVIEW 5 of 10

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Table 1. Time- and dose-dependencewith increase of BNZ intreatment treatment on different duration Trypanosoma (100% survival cruzi strains for 20 in days the acute of treatment phase of Chagas and 88% for Table 1. Time- and dose-dependence of BNZ treatment on different Trypanosoma cruzi strains in the acute phase of Chagas disease.Table 1. Time- and dose-dependence40 days of of treatment).BNZ treatment on different Trypanosoma cruzi strains in the acute phase of Chagas disease.Table 1. Time- and dose-dependence of BNZ treatment on different Trypanosoma cruzi strains in the acute phase of Chagas disease. disease. Positive qPCR Table 1. Time- and dose-dependence of BNZ treatment on different TrypanosomaPositive cruzi qPCRstrains in the acute phase of Chagas Survival Positive Positiveɸ qPCR Total Positive disease. Positive qPCR Survival Positive ɸ Total Positive Strain £ Group Survival Positive ɸ Total Positive Cure # § Survival Positive ɸ Total Positive £ Positive Positive qPCR Total Positive # Strain Group FBE Cure # StrainStrain £ GroupGroup Survival § Cure # Strain £ Group § FBEFBE¶ Blood HeartHeart ColonColon MiceMice Cure # § FBE ¶ Blood Heart Colon Mice Untreated 5/8 (63%) 5/5 (100%) ¶ FBE *-- 5/5 (100%) 0 Blood Heart Colon Mice a,d,e BNZ 100 mg/kg/day—20 days 8/8 (100%) 0/8 (0%) ¶ 1/8 (13%)Blood 1/8 (13%)Heart 2/8 (25%)Colon 2/8 (25%) Mice 75 Y BNZ 100Untreated mg/kg/day—40 days 8/8 5/8 (100%) (63%) 1/8 5/5 (22%)(100%) 0/7 (0%)* 0/8 - (0%) 0/8 - (0%) 5/5 1/8 (100%) (13%) 87 0a,b,d,e BNZBNZ 100 40 mg/kg/day—20 mg/kg/day—20Untreated days days 8/8 8/8 5/8(100%) (100%) (63%) 7/8 5/5 0/8 (88%) (100%) (0%) 1/11/8 (100%)(13%) * 1/8 1/7 (13%) (14%)- 2/8 7/7 (25%) - (100%) 5/52/8 8/8 (100%)(25%) (100%) 75 0a,d,e0 BNZ 40 mg/kg/day—40Untreated days 7/8 (88%) 5/8 (63%) 7/7 5/5 (100%) (100%) - * 0/7 (0%) - 7/7 (100%) - 5/5 7/7 (100%) 0 0b BNZ 100 mg/kg/day—20Untreated days 8/8 (100%) 5/8 (63%) 0/8 (0%) 5/5 (100%) 1/8 (13%) * 1/8 (13%) - 2/8 (25%) - 2/8 (25%) 5/5 (100%) 75 a,b,d,e a,d,e 0 Y BNZBNZ 100 100 mg/kg/day—40 mg/kg/day—20 days days 8/8 8/8 (100%) (100%) 1/8 0/8 (22%) (0%) 0/7 1/8 (0%) (13%) 0/8 1/8 (0%) (13%) 0/8 2/8 (0%) (25%) 1/8 2/8 (13%) (25%) 87 75 a,d,e Y BNZ 100BNZ mg/kg/day—40 Untreated100 mg/kg/day—20 days 8/8days 8/8 (100%) (100%) 8/8 (100%) 8/8 1/8 (100%) (22%) 0/8 (0%) 0/7 - (0%) 1/8 (13%)8/8 0/8 (100%)(0%) 1/8 (13%) 0/8 8/8 (0%) (100%) 2/8 (25%) 1/8 8/8 (13%) (100%) 2/8 (25%) 87 a,b,d,e0 75 a,d,e Y BNZBNZBNZ 40 100 100 mg/kg/day—20 mg/kg/day—20mg/kg/day—40 days days days 7/8 8/8 8/8(88%) (100%) (100%) 6/7 7/8 1/8 (88%) (88%) (22%) 1/1 0/1 0/7 (100%) (0%) (0%) 1/7 5/7 0/8 (14%) (71%) (0%) 7/7 0/87/7 (100%) (100%)(0%) 8/8 7/71/8 (100%) (100%)(13%) 87 00 a,b,d,e VL-10 Y BNZ BNZ40 mg/kg/day—20 100 mg/kg/day—40 days days 8/8 (100%) 8/8 (100%) 7/8 (88%) 1/8 (22%) 1/1 (100%) 0/7 (0%) 1/7 (14%) 0/8 (0%) 7/7 (100%) 0/8 (0%) 8/8 (100%) 1/8 (13%) a,b,d,e 0b 87 a,b,d,e BNZBNZBNZ 40 10040 mg/kg/day—40 mg/kg/day—40mg/kg/day—20 days days days 8/8 7/8 (100%) 8/8 (88%) (100%) 5/8 7/7 7/8 (63%)(100%) (88%) 0/31/1 (0%) -(100%) 0/7 1/74/8 (0%) (50%)(14%) 7/7 7/7 7/8(100%) (100%) (88%) 7/7 8/8 7/8 (100%) (100%) (88%) 12 0 0 BNZBNZ 40BNZ 40 mg/kg/day—40 mg/kg/day—20 40 mg/kg/day—20 days days days 8/8 7/8(100%) (88%) 8/8 (100%) 8/8 7/7 (100%) (100%) 7/8 (88%) - - 1/1 (100%)8/8 0/7 (100%)(0%) 1/7 (14%) 7/7 8/8 (100%) (100%) 7/7 (100%) 7/7 8/8 (100%) (100%) 8/8 (100%) 00 b 0 BNZBNZ 40 mg/kg/day—40mg/kg/day—40 days days 7/8 (88%) 7/8 (88%) 7/7 7/7 (100%) (100%) - - 6/6 0/7 ** (100%)(0%) 7/7 6/6 (100%) (100%) 7/7 7/7 (100%) 0 b BNZ 40 mg/kg/day—40 days 7/8 (88%) 7/7 (100%) - 0/7 (0%) 7/7 (100%) 7/7 (100%) 0 b (*) All mice diedUntreated with positive FBE; (**) 1 8/8 mouse (100%) died after 8/8 (100%) presenting positive - FBE. 8/8 Swiss(100%) female 8/8 mice(100%) were inoculated 8/8 (100%) with 5 × 0 103 trypomastigotesBNZ 100 mg/kg/day—20Untreated of the Y, and days VL-10 strains. 8/87/8 (100%)(88%)Up to 8/86/730 days (100%)(88%) after treatment. 0/1 (0%) - Fresh 8/85/7 (100%)(71%) blood exam 7/7 8/8 (100%)(100%) (FBE) after 7/7 cyclophosphamide8/8 (100%)(100%) 0 0 VL-10 Untreated 8/8 (100%) 8/8 (100%) - 8/8 (100%) 8/8 (100%) 8/8 (100%) 0 BNZ 100 mg/kg/day—20Untreated days 7/8 (88%) 8/8 (100%) 6/7 (88%) 8/8 (100%) 0/1 (0%) - 5/7 (71%) 8/8 (100%) 7/7 (100%) 8/8 (100%) 7/7 (100%) 8/8 (100%) a,b,d,e 0 0 VL-10immunosuppression. BNZBNZ 100 100 mg/kg/day—40 mg/kg/day—20Quantitative days days real-time 8/8 7/8 (100%) polymerase (88%) 5/8 chain6/7 (63%) (88%) reaction 0/3 0/1(qPCR) (0%) (0%) after 4/8 cyclophosphamide5/7 (50%) (71%) 7/8 7/7 (88%) (100%) immunosuppression. 7/8 7/7 (88%) (100%) Blood 12 0 VL-10qPCR BNZ was 10040 performedBNZ mg/kg/day—20 mg/kg/day—40 100 mg/kg/day—20 5 days after days days immunosuppression, days 8/8 8/8 (100%)(100%) 7/8 (88%) 8/8 5/8only (100%) (63%) for 6/7 negative (88%) 0/3 -(0%) FBE 0/1 assays. (0%) 8/8 4/8 (100%) (50%) Tissue 5/7 (71%) qPCR 8/8 7/8 (100%) (88%) was 7/7 performed(100%) 8/8 7/8 (100%) (88%) 30 7/7 days (100%) after 12 0a,b,d,e 0 VL-10 # a,b,d,e immunosuppressionBNZ 100 mg/kg/day—40 for all mice. daysCured: when 8/8 (100%) all tests (FBE 5/8 and (63%) qPCR) were 0/3 (0%) negative. 4/8 Letters (50%) represent 7/8 a(88%) significant 7/8difference (88%) in cure 12 a,b,d,e BNZ BNZ40 mg/kg/day—20 100 mg/kg/day—40 days days 8/8 (100%) 8/8 (100%) 8/8 (100%) 5/8 (63%) - 0/3 (0%) 8/86/6 (100%) ** 4/8 (50%) 8/8 (100%) 7/8 (88%) 8/8 (100%) 7/8 (88%) 0 12 rates whenBNZ compared 40 mg/kg/day—20 to: “a” Untreated days group; 8/8 (100%) “b” BNZ 100 8/8 mg/kg/day—20 (100%) - days; “d” 8/8 BNZ(100%) 40 mg/kg/day—20 8/8 (100%) days 8/8 (100%) and “e” BNZ 0 BNZ 40BNZ mg/kg/day—40 40 mg/kg/day—20 days days 7/8 (88%) 8/8 (100%) 7/7 (100%) 8/8 (100%) - - 6/6 8/8** (100%)6/6 (100%) 8/8 (100%) 7/7 (100%) 8/8 (100%) 0 0 40 mg/kg/day—40 BNZ 40 mg/kg/day—40 days. days 7/8 (88%) 7/7 (100%) - (100%)6/6 ** 6/6 (100%) 7/7 (100%) 0 BNZ 40 mg/kg/day—40 days 7/8 (88%) 7/7 (100%) - (100%)£ 6/6 **6/6 (100%) 7/7 (100%) 0 (*) All mice died BNZ with 40 mg/kg/day—40 positive FBE; (**)days 1 mouse 7/8 died (88%) after 7/7presenting (100%) positive - FBE.(100%) Swiss female6/6 mice (100%) were inoculated 7/7 (100%) 0 with(*) All 5 ×mice 103 trypomastigotesdied with positive of FBE;the Y, (**) and 1 mouseVL-10 diedstrains. after § Up presenting to 30 days positive after treatment. FBE. £ (100%)Swiss ¶ Fresh female blood mice exam were (FBE) inoculated after (*) All mice died with positive FBE;Untreated (**) 1 mouse animals died infectedafter presenting with VL-10 positive strain FBE. presented £ Swiss female similar mice features were inoculated when the fol- with 5(*) × All103 trypomastigotesmice died with positive of the Y, FBE; and (**) VL-10ɸ 1 mouse strains. died § Up after to 30presenting days after positive treatment. FBE. ¶£ FreshSwiss bloodfemale exam mice (FBE)were inoculatedafter cyclophosphamidewith 5 × 103 trypomastigotes immunosuppression. of the Y, and VL-10 Quantitative strains. § Up real-time to 30 days polymeraseafter treatment. chain ¶ Fresh reaction blood exam(qPCR) (FBE) after after cyclophosphamidewith 5 × 103 trypomastigotes immunosuppression.lowing of treatments the Y, andɸ QuantitativeVL-10 were compared:strains. real-time§ Up BNZ to 30 100dayspolymerase mg/kg/day after treatment. chain for 20¶reaction Fresh days, blood BNZ(qPCR) exam 40 mg/kg/day (FBE)after after cyclophosphamidecyclophosphamide immunosuppression. immunosuppression. Blood ɸ qPCRQuantitative was perfor real-timemed 5 days polymerase after immunosuppression, chain reaction only (qPCR) for negative after cyclophosphamidecyclophosphamide immunosuppression. immunosuppression.for 20 days, Blood and BNZ qPCR ɸ 40 wasQuantitative mg/kg/day performed real-time 5 for days 40 after days polymerase immunosuppression, (0% cure),# chain Table 1.reaction By only changing for negative(qPCR) the timeafter FBEcyclophosphamide assays. Tissue qPCR immunosuppression. was performed 30 Blood days qPCRafter immunosuppression was performed 5 days for after all mice. immunosuppression, Cured: when all only tests for (FBE negative and FBE assays.cyclophosphamide Tissue qPCR immunosuppression.wasof performed the standard 30 days protocol Blood after qPCR immunosuppression (BNZ was 100 perfor mg/kg/daymed 5for days all foraftermice. 20 immunosuppression, # days) Cured: to when BNZ all 100 tests mg/kg/dayonly (FBE for and negative for qPCR)FBE assays. were negative. Tissue qPCR Letters was represent performed a significant 30 days after difference immunosuppression in cure rates when for all compared mice. # Cured: to: “a” when Untreated all tests group; (FBE “b” and qPCR)FBE were assays. negative. Tissue Letters qPCR40 represent was days, performed there a significant is an30 improvementdays difference after immunosuppression in cure in therapeutic rates when efficacycomparedfor all mice. (12%). to: # “a”Cured: These Untreated when results all group; tests indicate “b”(FBE thatand BNZqPCR) 100 were mg/kg/day—20 negative. Letters days; represent“d” BNZ 40a significant mg/kg/day difference—20 days in and cure “e” rates BNZ when 40 mg/kg/day—40 compared to: “a” days. Untreated group; “b” BNZ 100qPCR) mg/kg/day—20 were negative. days; Lettersfor “d” both BNZrepresent strains, 40 mg/kg/day a Ysignificant and VL-10,—20 difference days the and therapeutic in “e” cure BNZ rates 40 efficacy mg/kg/day—40when compared is time- anddays. to: “a” dose-dependent Untreated group; in “b” the BNZ 100 mg/kg/day—20 days; “d” BNZ 40 mg/kg/day—20 days and “e” BNZ 40 mg/kg/day—40 days. BNZ 100 mg/kg/day—20acute days;When phase, “d” infected BNZ i.e., 40 the withmg/kg/day efficacy the VL-10—20 increases days strain, and proportionally all “e” the BNZ mice 40 mg/kg/day—40 totreated the increase in the days. chronic in dose phase or length with of When infected with the VL-10 strain, all the mice treated in the chronic phase with 100treatment. mg/kg/dayWhen infected for 40 withdays thesurvived VL-10 and strain, 62% all were the cured,mice treated Table 2,in although the chronic when phase treated with 100 mg/kg/dayWhenWhen infected for infected 40 withdays with thesurvived VL-10the VL-10 and strain, 62%strain, all were the all micecured,the mice treated Table treated in2, although the in chronicthe chronic when phase treated phase with with with100 40mg/kg/day mg/kg/day for for 40 days40 days, survived these animalsand 62% had were already cured, shownTable 2, cure although of 25%, when which treated is a with100 mg/kg/day40100 mg/kg/day mg/kg/day for for 40for 40 days40 days, days survived thesesurvived animals and and 62% had62% were alreadywere cured, cured, shown Table Table2 ,cure although 2, of although 25%, when which when treated is treateda promisingwith 40 mg/kg/day result considering for 40 days, the resistancethese animals of the had evaluated already strain.shown All cure of theof 25%, mice which infected is a promisingwithwith 40 mg/kg/day 40result mg/kg/day considering for 40for days,40 the days, resistance these these animals animalsof the had evaluated had already already strain. shown shown All cure of cure ofthe 25%, miceof 25%, which infected which is a is a withpromising the VL-10 result strain considering and untreated the resistance survived, of butthe noevaluated cure was strain. observed. All of the mice infected withpromising thepromising VL-10 result strain result considering and considering untreated the resistance the survived, resistance of thebut of evaluated nothe cure evaluated was strain. observed. strain. All of All the of mice the mice infected infected with the VL-10 strain and untreated survived, but no cure was observed. with with the VL-10 the VL-10 strain strain and untreatedand untreated survived, survived, but no but cure no cure was observed.was observed.

Table 2. Time- and dose-dependence of BNZ treatment on VL-10 Trypanosoma cruzi strain in the chronic phase of Chagas disease.

Positive Positive qPCR Total Cure # Strain Group Survival FBE Blood Heart Colon Positive mice (%)

Untreated 8/8 (100%) 5/8 (63%) 2/3 (67%) 4/5 * (80%) 2/5 (40%) 8/8 (100%) 0 BNZ 100 mg/kg/day—20 days 8/8 (100%) 5/8 (63%) 2/3 (67%) 2/7 * (29%) 4/7 (57%) 8/8 (100%) 0 VL-10 BNZ 100 mg/kg/day—40 days 8/8 (100%) 2/8 (25%) 0/6 (0%) 0/8 (0%) 1/8 (13%) 3/8 (38%) 62 a,b BNZ 40 mg/kg/day—20 days 8/8 (100%) 4/8 (50%) 3/4 (75%) 1/7 * (14%) 1/7 (14%) 8/8 (100%) 0 c BNZ 40 mg/kg/day—40 days 8/8 (100%) 6/8 (75%) 0/2 (0%) 0/5 * (0%) 0/5 (0%) 6/8 (75%) 25 a,b,c,d (*) Mice died with positive FBE. Swiss female mice were inoculated with 5 × 103 trypomastigotes of the VL-10 strains and were treated 120 days after infection. Up to 30 days after treatment. Fresh blood exam (FBE) after cyclophosphamide immunosuppression. Quantitative real-time polymerase chain reaction (qPCR) after cyclophosphamide immunosuppression. Blood qPCR was performed 5 days after immunosuppression, only for negative FBE assays. Tissue qPCR was performed 30 days after immunosuppression for all mice. # Cured: when all tests (FBE and qPCR) were negative. Letters represent a signiﬁcant difference in cure rates when compared to: “a” Untreated group; “b” BNZ 100 mg/kg/day—20 days; “c” BNZ 100 mg/kg/day—40 days; “d” BNZ 40 mg/kg/day—20 days.

When comparing the different strains submitted to the same treatment during the acute phase of infection, the proﬁle of susceptibility to benznidazole found in the literature [23] is maintained: animals infected with the Y strain presented higher percentages of cure, and those infected with the VL-10 strain showed less therapeutic success (Figure2). Pathogens 2021, 10, x FOR PEER REVIEW 6 of 10

Pathogens 2021, 10, x FOR PEER REVIEW 6 of 10

Table 2. Time- and dose-dependence of BNZ treatment on VL-10 Trypanosoma cruzi strain in the chronic phase of Chagas disease.Table 2. Time- and dose-dependence of BNZ treatment on VL-10 Trypanosoma cruzi strain in the chronic phase of Chagas disease. Positive Positive qPCR ɸ Total Cure # Strain £ Group Survival § FBEPositive ¶ Blood PositiveHeart qPCR ɸColon PositiveTotal mice (%)Cure # Strain £ Group Survival § Untreated 8/8 (100%) 5/8 FBE(63%) ¶ 2/3 Blood(67%) 4/5 *Heart (80%) 2/5 Colon(40%) Positive 8/8 (100%) mice 0(%) BNZ 100 mg/kg/day—20Untreated days 8/8 8/8 (100%) (100%) 5/8 5/8 (63%) (63%) 2/3 2/3 (67%) (67%) 2/7 4/5 * (29%)* (80%) 4/7 2/5 (57%) (40%) 8/8 8/8 (100%) (100%) 0 0 VL-10 BNZBNZ 100 100 mg/kg/day—40 mg/kg/day—20 days days 8/8 8/8 (100%) (100%) 2/8 5/8 (25%) (63%) 0/6 2/3 (0%) (67%) 0/82/7 (0%)* (29%) 1/8 4/7 (13%) (57%) 3/8 8/8 (38%) (100%) 62 a,b 0 VL-10 BNZBNZ 40 100 mg/kg/day—20 mg/kg/day—40 days days 8/8 8/8 (100%) (100%) 4/8 2/8 (50%) (25%) 3/4 0/6 (75%) (0%) 1/7 0/8* (14%) (0%) 1/7 1/8 (14%) (13%) 8/8 3/8 (100%) (38%) 0 62 c a,b BNZBNZ 40 40 mg/kg/day—40 mg/kg/day—20 days days 8/8 8/8 (100%) (100%) 6/8 4/8 (75%) (50%) 0/2 3/4 (0%) (75%) 0/5 1/7 * *(0%) (14%) 0/5 1/7 (0%) (14%) 6/8 8/8 (75%) (100%) 25 a,b,c,d 0 c (*) Mice diedBNZ with 40 mg/kg/day—40 positive FBE. £ Swissdays female 8/8 (100%)mice were 6/8 inoculated (75%) with 0/2 (0%)5 × 10 3 trypomastigotes 0/5 * (0%) 0/5 of(0%) the VL-10 6/8 strains(75%) and 25 a,b,c,d § ¶ were(*) Mice treated died 120 with days positive after infection.FBE. £ Swiss Up female to 30 micedays wereafter inoculatedtreatment. with Fresh 5 × blood 103 trypomastigotes exam (FBE) after of the cyclophosphamide VL-10 strains and ɸ immunosuppression.were treated 120 days after Quantitative infection. § Upreal-time to 30 dayspolymerase after treatment. chain ¶ Freshreaction blood (qPCR)exam (FBE) after after cyclophosphamide cyclophosphamide immunosuppression.immunosuppression. Blood ɸ qPCRQuantitative was perfor real-timemed 5 days polymerase after immunosuppression, chain reaction on(qPCR)ly for negative after FBEcyclophosphamide assays. Tissue # qPCRimmunosuppression. was performed 30 Blood days afterqPCR immunosuppression was performed 5 days for afterall mice. immunosuppression, Cured: when all tests only (FBE for negative and qPCR) FBE were assays. negative. Tissue LettersqPCR representwas performed a significant 30 days differenc after immunosuppressione in cure rates when for compared all mice. #to: Cured: “a” Untreated when all tests group; (FBE “b” and BNZ qPCR) 100 mg/kg/day were negative.— 20Letters days; “c”represent BNZ 100 a significant mg/kg/day differenc—40 days;e in “d”cure BNZ rates 40 when mg/kg/day compared—20 to: days. “a” Untreated group; “b” BNZ 100 mg/kg/day— 20 days; “c” BNZ 100 mg/kg/day—40 days; “d” BNZ 40 mg/kg/day—20 days. When comparing the different strains submitted to the same treatment during the acute phaseWhen of comparing infection, thethe profiledifferent of susceptibilitystrains subm ittedto benznidazole to the same found treatment in the duringliterature the [23]acute is maintained:phase of infection, animals the infected profile of with susceptibility the Y strain to benznidazole presented higher found percentages in the literature of Pathogens 2021, 10, 729 cure,[23] andis maintained: those infected animals with theinfected VL-10 with strain the showed Y strain less presented therapeutic higher success percentages (Figure6 2). of of 10 cure, and those infected with the VL-10 strain showed less therapeutic success (Figure 2).

75 87

75 62

25 a 12 25 a a 000 0 0 120 0 0 0 0 a 000 0 0 0 0 0 0 0

FigureFigure 2. 2.EfficacyEfficacy of oftreatment treatment compared compared between between strains strains and and stages stages of ofinfection. infection. “a” “a” represents represents significant significant difference difference in in [ relationFigurerelation to2. to AcuteEfficacy Acute phase phaseof treatment Y. Y. “ “ compared “” represents between aa significantsignificant strains difference difference and stages among among of infection. the the different different “a” represents phases phases of infection,of significant infection, but differencebut for for the the same in samerelationtreatment treatment to andAcute and strain phase strain (p

infection in animals infected by Y and VL-10 strains followed the theory of proportionality of BNZ dose and time. These data corroborate with preclinical studies that suggest that treatment in the acute phase of infection by strains partially susceptible or resistant to BNZ is dose- and time- dependent [16]. Mazzeti and collaborators evaluated the standard dose of BNZ (100 mg/kg) with treatment periods varying between 10, 20 or 40 days; and the standard BNZ treatment period (20 days) with doses ranging between 25, 50, 75, 100, and 300 mg/kg/day. However, the study did not evaluate the chronic phase of experimental Chagas disease and the PCR was limited to blood. In addition, we evaluated a lower dose (40 mg/kg/day) during the standard treatment period (20 days) and a longer period (40 days), whereas they evaluated a lower dose (50 mg/kg/day) only using the standard treatment time. Mice were considered cured only when they presented negative FBE after being subjected to immunosuppression, as well negative blood, colon, and heart qPCR. In the chronic phase of the disease, T. cruzi persists in the gastrointestinal tract, mainly in the stomach and colon [30]. When we analyzed fragments of colon of all the mice infected by VL-10 strain, treated or untreated, they were positive in 97% and 25% of the tests in the acute and chronic phases, respectively. Lastly, the acute phase of infection by Y strain showed 53% positivity. Our results demonstrate that independently of the T. cruzi strain, the colon is an important place of parasite residence in mice, thus this organ is a crucial place of analysis in post-treatment evaluation. Previous study reports the importance of including colon PCR (combined with blood PCR and FBE after immunosuppression) as one more of the cure criteria for evaluating the effectiveness of compounds in the experimental Chagas disease treatment [31]. Corroborating with our results, the colon presents important tissue parasitism independent of the parasite inoculation pathway (oral or intraperitoneal); in addition, this parasitism is greater in the acute phase when compared to the chronic phase [32]. In relation to qPCR analysis in cardiac tissue of animals infected by VL-10 strain, we found, respectively 84% and 22% positivity in the acute and chronic phases of infection, and for Y strain the results showed 7% positivity in the acute phase of infection. These results were consistent once VL-10 strain is myotropic, and Y strain is macrophagotropic [33–35]. In addition, these results are similar to those reported in another study where it was observed better therapeutic report for animals treated with nitroheterocyclics in the chronic phase [36]. This present study concluded, as well as Francisco et al. that, in mice, a chronic T. cruzi infection is easier to cure when compared to the acute infection. Francisco et al. using highly sensitive bioluminescence imaging concluded that a possible explanation for this finding would be the fact that the parasite burden in the chronic phase is lower than in the acute phase and restricted to some locations. Furthermore, using BNZ for a longer period of time can be important for exposing dormant forms to BNZ. In this way, parasites that are actively replicating would be affected at the beginning of the treatment regimen, while the remaining dormant forms would be killed until the end of the treatment [15]. In summary, for experimental Chagas disease: (1) infections by Y strain should be treated with the standard treatment and, where possible, with the standard dose for a longer period (better results); and (2) VL-10 strain infections treated in the acute and chronic phase respond better with the standard dose for a longer time, although a dose reduction with time increased is also an alternative in chronic phase. It is also noted that the treatment of infection by VL-10 strain obtained better results in the chronic phase when compared to the acute phase of the disease. In relation to methodology the present study used 8 animals per group and this number was determined by sample calculation. Furthermore, in the literature it is common the sample size to range from 6 to 10 animals per group [16,19,37]. In Chagas disease the evaluation of the effectiveness of a treatment is very difficult due to the absence of well- defined cure criteria and in general a combination of different methods is necessary [38,39]. Pathogens 2021, 10, 729 8 of 10

In this study, we opted to use an immunosuppression protocol and subsequent analysis with ESF and blood qPCR [19,20,22]. Similar to other studies, we opted to extend the qPCR assessment to tissues, in order to increase the robustness of the analysis [31,40,41]. The blood PCR is a suitable method to detect therapeutic failure in murine T. cruzi infection. However, PCR tests performed on tissues from animals treated with BNZ and considered cured, still detected T. cruzi DNA [38]. Therefore, we also perform qPCR in the heart and colon, organs known to be affected by Chagas disease [30,31,42,43]. Taken together, our data indicate that treatment for patients with Chagas disease remains a challenge, as the strains have different results when treated with BNZ in the different phases of infection. Thus, the development of new protocols seems to be a promising route of investigation, and new approaches for therapeutic effectiveness could include strain characterization, potentiating discovery of biomarkers of resistance that will allow individualized treatment, as well as the discovery of new drugs.

Author Contributions: Conceived and designed the experiments: K.d.S.F., L.P., P.M.d.A.V., I.M., C.M.C. Performed the experiments: K.d.S.F., L.P., B.C.d.S., F.d.S.M., G.d.P.C., N.C.N.d.P., T.H.C.D. Analyzed the data: K.d.S.F. and L.P. Contributed with reagents/materials/analysis tools: C.M.C. and R.C.-O. Wrote the paper: K.d.S.F., L.P., F.d.S.M. and T.H.C.D. All authors have read and agreed to the published version of the manuscript. Funding: Funding was provided by the Universidade Federal de Ouro Preto (UFOP), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), research fellowships from CNPq (Carneiro CM, Correa-Oliveira R), CAPES—Science With- out Borders and Senior Research Visitor (Molina I, Correa-Oliveira R), and BERENICE (Collaborative Project supported by the European Commission under the Health Innovation Work Programme of the 7th Framework Programme). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Institutional Review Board Statement: All procedures were performed according to ethical principles recommended by the National Council for Control of Animal Experimentation (CONCEA) and approved by the ethics committee of Federal University of Ouro Preto (Protocol CEUA-UFOP number: 2016/33). Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We thank the Animal Facility at Ouro Preto Federal University (UFOP), Minas Gerais State, Brazil (CCA/UFOP) for providing the animals. Conflicts of Interest: We have no conflict of interest to declare.

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