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Background: For indigenous people in Colombia, high infection rates with Chagas disease (CD) are known. Methods: In 2018 and 2020, nine villages were screened for CD. CD-positive patients could enter a drug observed treatment. While, in 2018, Benznidazole (BNZ) was provided as the first-line drug by the government, nifurtimox (NFX) was administered in 2020. Results: Of 121 individuals treated with BNZ, 79 (65%) suffered from at least one adverse event (AE). Of 115 treated with NFX, at least one AE occurred in 96 (84%) patients. In 69% of BNZ cases, the side effects did not last longer than one day; this applied to 31% of NFX cases. Excluding extreme outlier values, average duration of AEs differed highly significantly: BNZ (M = 0.7, SD = 1.4) and NFX (M = 1.7, SD = 1.5, p < 0.001). Using an intensity scale, AEs were highly significantly more severe for NFX (M = 2.1, SD = 0.58) compared to BZN (M = 1.1, SD = 0.38), p < 0.001. When analyzing the duration in relation to the intensity, the burden of AEs caused by NFX was significantly more pronounced. Dropouts (n = 2) due to AEs were in the NFX-group only. Conclusions: Side effects caused by BNZ were significantly fewer, as well as milder, shorter in duration, and more easily treatable, compared to NFX.

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Se presenta un caso de enfermedad de Chagas en fase aguda en un paciente masculino de 40 años, proce-dente de la Vereda Buenos Aires, Municipio de Miraflores, Departamento del Guaviare. El paciente acudió a urgencias con fiebre, cefalea, astenia, adinamia y disuria. Se realizó un frotis de sangre y un análisis de orina, con resultados positivos para infección urinaria sintomática, pero negativos para malaria. Cinco días más tarde se confirmó el diagnóstico de enfermedad de Chagas en fase aguda tras recibir un resultado positivo para Trypanosoma cruzi. El paciente recibió tratamiento con nifurtimox y benznidazol, y se llevó a cabo una investigación de contactos y zonas de riesgo, búsqueda activa comunitaria e institucional, entomológica y de reservorios, y una vigilancia de laboratorio para detectar posibles casos de infección en la comunidad. Se identificaron cinco casos con síntomas similares, pero las pruebas parasitológicas fueron negativas. Se aplic-aron medidas de educación sanitaria para prevenir la propagación de la enfermedad.

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Cyclophilins (CyPs) are a family of enzymes involved in protein folding. Trypanosoma cruzi, the causative agent of Chagas disease, has a 19-kDa cyclophilin, TcCyP19, that was found to be secreted in parasite stages of the CL Brener clone and recognized by sera from T. cruzi-infected mice and patients. The levels of specific antibodies against TcCyP19 in T. cruzi-infected mice and subjects before and after drug treatment were measured by an in-house enzyme linked immunosorbent assay (ELISA). Mice in the acute and chronic phase of infection, with successful trypanocidal treatments, showed significantly lower anti-TcCyP19 antibody levels than untreated mice. In children and adults chronically infected with T. cruzi, a significant decrease in the anti-TcCyP19 titers was observed after 12 months of etiological treatment. This decrease was maintained in adult chronic patients followed-up 30-38 months post-treatment. These results encourage further studies on TcCyP19 as an early biomarker of trypanocidal treatment efficiency.

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Nifurtimox is recommended for the treatment of Chagas disease; however, long-term follow-up data are scarce. This prolonged follow-up phase of the prospective, historically controlled, CHICO clinical trial evaluated seronegative conversion in pediatric patients aged <18 years with Chagas disease who were followed for 4 years after nifurtimox treatment. Patients were randomly assigned 2:1 to nifurtimox 60-day or 30-day regimens comprising 10 to 20 mg/kg/day for patients aged <12 years and body weight <40 kg, and 8 to 10 mg/kg/day for those aged ≥12 years and body weight ≥40 kg. Anti-Trypanosoma cruzi antibodies decreased during the study period, achieving seronegative conversion in 16 (8.12%) and 8 (8.16%) patients in the 60-day and 30-day nifurtimox regimens, respectively, with corresponding incidence rates per 100 patients/year of seronegative conversion of 2.12 (95% confidence interval [CI]: 1.21 to 3.45) and 2.11 (95% CI: 0.91 to 4.16). Superiority of the 60-day nifurtimox regimen was confirmed by the lower limit of the 95% CI being higher than that (0%) in a historical placebo control group. Children aged <2 years at baseline were more likely to reach seronegative conversion during the 4-year follow-up than older children. At any annual follow-up visit, >90% of evaluable patients had persistently negative quantitative PCR results for T. cruzi DNA. No adverse events potentially related to treatment or caused by protocol-required procedures were documented for either treatment regimen. This study confirms the effectiveness and safety of a pediatric formulation of nifurtimox administered in an age- and weight-adjusted regimen for 60 days to treat children with Chagas disease.

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Nifurtimox is a nitroheterocyclic drug employed for treatment of trypanosomiases (Chagas disease and West African sleeping sickness); its use for certain cancers has also been assessed. Despite having been in the market for over 50 years, knowledge of nifurtimox is still fragmentary and incomplete. Relevant aspects of the chemistry and biology of nifurtimox are reviewed to summarize the current knowledge of this drug. These comprise its chemical synthesis and the preparation of some analogues, as well as its chemical degradation. Selected physical data and physicochemical properties are also listed, along with different approaches toward the analytical characterization of the drug, including electrochemical (polarography, cyclic voltammetry), spectroscopic (ultraviolet-visible, nuclear magnetic resonance, electron spin resonance), and single crystal X-ray diffractometry. The array of polarographic, ultraviolet-visible spectroscopic, and chromatographic methods available for the analytical determination of nifurtimox (in bulk drug, pharmaceutical formulations, and biological samples), are also presented and discussed, along with chiral chromatographic and electrophoretic alternatives for the separation of the enantiomers of the drug. Aspects of the drug likeliness of nifurtimox, its classification in the Biopharmaceutical Classification System, and available pharmaceutical formulations are detailed, whereas pharmacological, chemical, and biological aspects of its metabolism and disposition are discussed.

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Nifurtimox (NFX) is a nitrofuran derivative used to treat Chagas disease, a neglected disease caused by the protozoan Trypanosoma cruzi. The drug is very sparingly soluble in aqueous media and no other solid phases of NFX have been reported to date. The preparation of the amorphous mode of NFX is reported, as well as its characterization by hot stage microscopy, thermal (differential scanning calorimetry and thermogravimetric analysis), spectroscopic (solid state nuclear magnetic resonance, mid-infrared, and near-infrared), diffractometric and functional (powder dissolution rate) means. The stability of the new phase was investigated. This was characterized using thermal, spectroscopic, and diffractometric methods, finding out its spontaneous reversion to the crystalline state, as sign of instability. In addition, the amorphous material proved to be sensitive to temperature, pressure, and mechanical stress, all of which accelerated phase conversion. However, it was able to remain stable in a model polymeric amorphous solid dispersion with PEG 4000 for more than one month. An approach for monitoring the conversion of the amorphous phase to its crystalline counterpart under thermal stress by chemometric analysis of mid-infrared spectra at different temperatures is also disclosed.

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Chagas disease is a zoonotic infectious disease caused by the protozoan parasite Trypanosoma cruzi. It is distributed worldwide, affecting around 7 million people; there is no effective treatment, and it constitutes a leading cause of disability and premature death in the Americas. Only two drugs are currently approved for the treatment, Benznidazole and Nifurtimox, and both have to be activated by reducing the nitro-group. The T. cruzi aldo-keto reductase (TcAKR) has been related to the metabolism of benznidazole. TcAKR has been extensively studied, being most efforts focused on characterizing its implication in trypanocidal drug metabolism; however, little is known regarding its biological role. Here, we found that TcAKR is confined, throughout the entire life cycle, into the parasite mitochondria providing new insights into its biological function. In particular, in epimastigotes, TcAKR is associated with the kinetoplast, which suggests additional roles of the protein. The upregulation of TcAKR, which does not affect TcOYE expression, was correlated with an increase in PGF2α, suggesting that this enzyme is related to PGF2α synthesis in T. cruzi. Structural analysis showed that TcAKR contains a catalytic tetrad conserved in the AKR superfamily. Finally, we found that TcAKR is also involved in Nfx metabolization.

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Chagas disease (CD) is a parasitic zoonosis endemic in Central and South America affecting nearly 10 million people, with 100 million people at high risk of contracting the disease. Treatment is only effective when received at the early stages of the disease and it involved two drugs (nifurtimox (NFX) and benznidazole (BNZ)). Both treatments require multiple daily administrations of high doses, suffer from variable efficacy and insufficient efficacy in chronic CD, many side effects, and a very long duration of treatment that results in poor compliance, while combined available therapies that lead to reduced duration of treatment are not available and polypharmacy reduces compliance and increases the cost further. Here we present self-nanoemulsified drug delivery systems (SNEDDS) able to produce easily scalable combined formulations of NFX and BNZ that can allow for tailoring of the dose and can be easily converted to oral solid dosage form by impregnation on mesoporous silica particles. SNEDDS demonstrated an enhanced solubilisation capacity for both drugs as demonstrated by flow-through studies and in vitro lipolysis studies. High loading of SNEDDS to Syloid 244 and 3050 silicas (2:1 w/w) allowed clinically translatable amounts of both NFX and BNZ to be loaded. Tablets prepared from NFX-BNZ combined SNEDDS loaded on Syloid 3050 silicas demonstration near complete dissolution in the flow through cell apparatus compared to NFX and BNZ commercial tablets respectively (Lampit® and Rochagan®). NFX-BNZ-SNEDDS demonstrated nanomolar efficacy in epimastigotes and amastigotes of T. cruzi with acceptable selectivity indexes and demonstrated enhanced survival and reduced parasitaemia in acute murine experimental models of CD. Thus, the results presented here illustrate the ability for an easily scalable and personalised combination oral therapy prepared from GRAS excipients, enabling treatment access worldwide for the treatment of CD.

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PURPOSE: The objective of this study was to molecularly characterize Mexican isolates of T. cruzi obtained from infected triatomine bugs (the vectors of T. cruzi) and to evaluate their susceptibility to Nifurtimox (NFX). METHODS: Three isolates obtained from Triatoma dimidiata (collected in the State of Veracruz) and one isolate obtained from Triatoma bassolsae (collected in the State of Puebla) were molecularly characterized and the expression of genes associated with natural resistance to NFX was analyzed by qPCR. RESULTS: Molecular characterization by PCR showed that isolates Zn3, Zn5, and SRB1 belong to the DTU TcI, while isolate Sum3 belongs to TcIV. The latter was also confirmed by sequencing of mitochondrial genes. Isolate Zn5 was the most sensitive to treatment with NFX (IC50, 6.8 µM), isolates SRB1 and Zn3 were partially resistant (IC50, 12.8 µM and 12.7 µM) and isolate Sum3 showed a high degree of resistance to NFX (IC50, 21.4 µM). We also found an association between decreased NTR1 or OYE gene expression with NFX resistance. CONCLUSION: Our results also evidenced a high variability in the susceptibility to NFX of these T. cruzi isolates Central and Southeastern Mexico, suggesting the presence of naturally resistant isolates circulating in the country. These results have important implications for defining treatment policies for patients with Chagas disease.

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Chagas disease (ChD), caused by Trypanosoma cruzi, has a global prevalence due to patient migration. However, despite its worldwide distribution, long-term follow-up efficacy studies with nifurtimox (NF) are scarce and have been conducted with only small numbers of patients. A retrospective study of a large cohort of ChD treated children and adults with NF. Treatment response was evaluated by clinical, parasitological, and serological after-treatment evaluation. A total of 289 patients were enrolled, of which 199 were children and 90 adults. At diagnosis, 89.6% of patients were asymptomatic. Overall, all symptomatic patients showed clinical improvement. At baseline, parasitemia was positive in 130 of 260 (50%) patients. All but one adult patient had cleared their parasitemia by the end of treatment. That patient was considered a treatment failure. Median follow-up time for children was 37.7 months, with an interquartile range of (IQR25-75 12.2 to 85.3), and for adults was 14.2 months (IQR25-75, 1.9 to 33.8). After treatment, a decrease of T. cruzi antibodies and seroconversion were observed in 34.6% of patients. The seroconversion profile showed that, the younger the patient, the higher the rate of seroconversion (log rank test; P value, <0.01). At least 20% seroreduction at 1 year follow-up was observed in 33.2% of patients. Nifurtimox was highly effective for ChD treatment. Patients had excellent treatment responses with fully resolved symptoms related to acute T. cruzi infection. Clearance of parasitemia and a decrease in T. cruzi antibodies were observed as markers of treatment response. This study reinforces the importance of treating patients during childhood since the treatment response was more marked in younger subjects. (This protocol was registered at ClinicalTrials.gov under registration number NCT04274101).

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Trypanosoma cruzi is the causative agent of Chagas disease which is currently treated by nifurtimox (NFX) and benznidazole (BZ). Nevertheless, the mechanism of action of NFX is not completely established. Herein, we show the protective effects of T. cruzi mitochondrial peroxiredoxin (MPX) in macrophage infections and in response to NFX toxicity. After a 3-day treatment of epimastigotes with NFX, MPX content increased (2.5-fold) with respect to control, and interestingly, an MPX-overexpressing strain was more resistant to the drug. The generation of mitochondrial reactive species and the redox status of the low molecular weight thiols of the parasite were not affected by NFX treatment indicating the absence of oxidative stress in this condition. Since MPX was shown to be protective and overexpressed in drug-challenged parasites, non-classical peroxiredoxin activity was studied. We found that recombinant MPX exhibits holdase activity independently of its redox state and that its overexpression was also observed in temperature-challenged parasites. Moreover, increased holdase activity (2-fold) together with an augmented protease activity (proteasome-related) and an enhancement in ubiquitinylated proteins was found in NFX-treated parasites. These results suggest a protective role of MPX holdase activity toward NFX toxicity. Trypanosoma cruzi has a complex life cycle, part of which involves the invasion of mammalian cells, where parasite replication inside the host occurs. In the early stages of the infection, macrophages recognize and engulf T. cruzi with the generation of reactive oxygen and nitrogen species toward the internalized parasite. Parasites overexpressing MPX produced higher macrophage infection yield compared with wild-type parasites. The relevance of peroxidase vs. holdase activity of MPX during macrophage infections was assessed using conoidin A (CA), a covalent, cell-permeable inhibitor of peroxiredoxin peroxidase activity. Covalent adducts of MPX were detected in CA-treated parasites, which proves its action in vivo. The pretreatment of parasites with CA led to a reduced infection index in macrophages revealing that the peroxidase activity of peroxiredoxin is crucial during this infection process. Our results confirm the importance of peroxidase activity during macrophage infection and provide insights for the relevance of MPX holdase activity in NFX resistance.

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Chagas disease (CD) is a worldwide problem, with over 8 million people infected in both rural and urban areas. CD was first described over a century ago, but only two drugs are currently available for CD treatment: benznidazole (BZN) and nifurtimox (NF). Treating CD-infected patients, especially children and women of reproductive age, is vital in order to prevent long-term sequelae, such as heart and gastrointestinal dysfunction, but this aim is still far from being accomplished. Currently, the strongest data to support benefit-risk considerations come from trials in children. Treatment response biomarkers need further development as serology is being questioned as the best method to assess treatment response. This article is a narrative review on the pharmacology of drugs for CD, particularly BZN and NF. Data on drug biopharmaceutical characteristics, safety and efficacy of both drugs are summarized from a clinical perspective. Current data on alternative compounds under evaluation for CD treatment, and new possible treatment response biomarkers are also discussed. Early diagnosis and treatment of CD, especially in paediatric patients, is vital for an effective and safe use of the available drugs (i.e. BZN and NF). New biomarkers for CD are urgently needed for the diagnosis and evaluation of treatment efficacy, and to guide efforts from academia and pharmaceutical companies to accelerate the process of new drug development.

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As a result of globalization and constant migratory flows, Chagas disease is now present in almost all continents. The management and treatment of the disease is often influenced by the economic and social context of the societies that host patients. In this manuscript, we aim to provide a comparative review of approaches to patients with Chagas disease in the Americas and Europe.

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OBJECTIVES: This study aimed to describe the electrocardiographic and echocardiographic status of chronic Chagas disease (cChD) patients treated with nifurtimox. METHODS: An observational study was performed in 146 cChD patients followed over a mean of 7.9 years. RESULTS: Of the 146 patients, 41 (28.1%) with normal electrocardiogram (ECG) at baseline maintained this condition, 34 (23.3%) with altered ECG at baseline normalised the alterations, and 46 (31.5%) with ECG abnormalities at baseline maintained this condition [23 (15.8%) with small alterations]. Finally, 25 cases (17.1%) in indeterminate phase altered the ECG. Differences before and after follow-up (P < 0.001) were found. The percentage of beneficial treatment was different than expected by chance (Z = 4.8; P < 0.001) and the annual percentage of cases that developed ECG alterations was lower than that of a historical cohort of untreated patients (P < 0.001). An echocardiogram was performed in 68 patients with baseline ECG alterations. The ejection fraction (EF) was normal in 57 (83.8%) and abnormal in 11 (16.2%). In 38 patients with ECG abnormalities that did not progress after treatment, EF and segmental motility (SM) were normal in 31 (81.6%) and 26 (68.4%), respectively. In 17 patients with ECG abnormalities, EF and SM were normal in 15 (88.2%) and 14 (82.4%) cases, respectively. CONCLUSION: Less progression to cardiomyopathy compared with a historical untreated cohort as well as the EF/SM results in patients with abnormal ECG that did not progress and in indeterminate cChD that altered the ECG suggests a beneficial effect of nifurtimox.

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Drug combinations have been evaluated for Chagas disease in an attempt to improve efficacy and safety. In this line, the objective of this work is to assess the effects of treatment with nitro drugs combinations using benznidazole (BZ) or nifurtimox (NFX) plus the sulfone metabolite of fexinidazole (fex-SFN) in vitro and in vivo on Trypanosoma cruzi infection. The in vitro interaction of fex-SFN and BZ or NFX against infected H9c2 cells by the Y strain was classified as an additive (0.5⩾ΣFIC<4), suggesting the possibility of a dose reduction in the in vivo T. cruzi infection. Next, the effect of combining suboptimal doses was assessed in an acute model of murine T. cruzi infection. Drug combinations led to a faster suppression of parasitemia than monotherapies. Also, the associations led to higher cure levels than those in the reference treatment BZ 100 mg day−1 (57.1%) (i.e. 83.3% with BZ/fex-SFN and 75% with NFX/fex-SFN). Importantly, toxic effects resulting from the associations were not observed, according to weight gain and hepatic enzyme levels in the serum of experimental animals. Taken together, this study is a starting point to explore the potential effects of nitro drugs combinations in preclinical models of kinetoplastid-related infections.

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Nifurtimox (NF) is one of the only two drugs currently available for Chagas disease (ChD) treatment. However, data on NF safety are scarce, and many physicians defer or refuse NF treatment because of concerns about drug tolerance. In a retrospective study of adverse drug reactions (ADRs) associated with NF treatment of ChD, children received NF doses of 10 to 15 mg/kg/day for 60 to 90 days, and adults received 8 to 10 mg/kg/day for 30 days. A total of 215 children (median age, 2.6 years; range, 0 to 17 years) and 105 adults (median age, 34 years; range, 18 to 57 years) were enrolled. Overall, 127/320 (39.7%) patients developed ADRs, with an incidence of 64/105 adults and 63/215 children (odds ratio [OR] = 3.7; 95% confidence interval [CI], 2.2 to 6.3). We observed 215 ADRs, 131 in adults (median, 2 events/patient; interquartile range for the 25th to 75th percentiles [IQR25-75], 1 to 3) and 84 in children (median, 1 event/patient; IQR25-75 = 1 to 1.5) (Padjusted < 0.001). ADRs were mainly mild and moderate. Severe ADRs were infrequent (1.2% in children and 0.9% in adults). Nutritional, central nervous, and digestive systems were the most frequently affected, without differences between groups. Treatment was discontinued in 31/320 (9.7%) patients without differences between groups. However, ADR-related discontinuations occurred more frequently in adults than in children (OR = 5.5, 95% CI = 1.5 to 24). Our study supports the safety of NF for ChD treatment. Delaying NF treatment due to safety concerns does not seem to be supported by the evidence. (This study has been registered in ClinicalTrials.gov under identifier NCT04274101.).

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Introduction: Trypanosoma cruzi, the causative agent of Chagas disease, shows substantial phenotypic and genotypic heterogeneity, which can influence the epidemiological and clinical variations of the disease and the sensitivity to the drugs used in the treatment. Objective: To assess the in vitro susceptibility to benznidazole, nifurtimox, and posaconazole of 40 cloned strains of T. cruzi isolated in Paraguay belonging to different genotypes, hosts, and localities. Materials and methods: We incubated the parasites in their epimastigote stage in LIT culture medium with different concentrations of each drug in triplicate assays. The degree of susceptibility was estimated by the inhibitory concentrations of 50 and 90% (IC50 and IC90) to obtain the average values and the standard deviation for each strain and drug. We determined the statistical significance between groups by analysis of variances with the Wilcoxon/Kruskal-Wallis non-parametric test and values of p<0.05. Results: A wide range of drug response was observed. Two groups of parasites (A and B) were identified as having significant differences in susceptibility to benznidazole (p<0.0001), and three groups (A, B, C) to nifurtimox and posaconazole (p<0.0001). Conclusions: Overall, the isolates were more susceptible to nifurtimox than benznidazole and posaconazole. Such differences highlight the heterogeneity of T. cruzi populations circulating in Paraguay, an aspect to consider in the treatment and follow up of patients.

Introducción. Trypanosoma cruzi, agente causal de la enfermedad de Chagas, exhibe una sustancial heterogeneidad fenotípica y genotípica que puede influir en las variaciones epidemiológicas y clínicas de la enfermedad, así como en la sensibilidad a los fármacos utilizados en el tratamiento. Objetivo. Evaluar la sensibilidad in vitro al benznidazol, el nifurtimox y el posaconazol de 40 cepas clonadas de T. cruzi de Paraguay, con distintos genotipos, huéspedes y localidades de origen. Materiales y métodos. En su estado epimastigote, los parásitos se incubaron en medio de cultivo LIT (Liver Infusion Tryptose) con diferentes concentraciones de cada fármaco en ensayos por triplicado. El grado de sensibilidad se estimó a partir de las concentraciones inhibitorias del 50 y el 90% (IC50 e IC90) y se obtuvieron los valores promedio y la desviación estándar de cada cepa y fármaco. La significación estadística entre grupos se determinó mediante análisis de varianzas con el test no paramétrico de Wilcoxon/Kruskal-Wallis y valores de p<0,05. Resultados. Se observó un amplio rango de respuesta a los fármacos. Se identificaron dos grupos de parásitos (A y B) con diferencias significativas en la sensibilidad al benznidazol (p<0,0001), y tres grupos (A, B, C) en cuanto a la sensibilidad al nifurtimox y el posaconazol (p<0,0001). Conclusiones. En general, las cepas fueron más sensibles al nifurtimox que al benznidazol y el posaconazol. Estas diferencias evidencian la heterogeneidad de las poblaciones de T. cruzi que circulan en Paraguay, lo que debe considerarse en el tratamiento y el seguimiento de las personas afectadas.

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Resumen: Introducción. Trypanosoma cruzi, agente causal de la enfermedad de Chagas, exhibe una sustancial heterogeneidad fenotípica y genotípica que puede influir en las variaciones epidemiológicas y clínicas de la enfermedad, así como en la sensibilidad a los fármacos utilizados en el tratamiento. Objetivo. Evaluar la sensibilidad in vitro al benznidazol, el nifurtimox y el posaconazol de 40 cepas clonadas de T. cruzi de Paraguay, con distintos genotipos, huéspedes y localidades de origen. Materiales y métodos. En su estado epimastigote, los parásitos se incubaron en medio de cultivo LIT (Liver Infusion Tryptose) con diferentes concentraciones de cada fármaco en ensayos por triplicado. El grado de sensibilidad se estimó a partir de las concentraciones inhibitorias del 50 y el 90% (IC50 e IC90) y se obtuvieron los valores promedio y la desviación estándar de cada cepa y fármaco. La significación estadística entre grupos se determinó mediante análisis de varianzas con el test no paramétrico de Wilcoxon/Kruskal-Wallis y valores de p<0,05. Resultados. Se observó un amplio rango de respuesta a los fármacos. Se identificaron dos grupos de parásitos (A y B) con diferencias significativas en la sensibilidad al benznidazol (p<0,0001), y tres grupos (A, B, C) en cuanto a la sensibilidad al nifurtimox y el posaconazol (p<0,0001). Conclusiones. En general, las cepas fueron más sensibles al nifurtimox que al benznidazol y el posaconazol. Estas diferencias evidencian la heterogeneidad de las poblaciones de T. cruzi que circulan en Paraguay, lo que debe considerarse en el tratamiento y el seguimiento de las personas afectadas.

Abstract: Introduction: Trypanosoma cruzi, the causative agent of Chagas disease, shows substantial phenotypic and genotypic heterogeneity, which can influence the epidemiological and clinical variations of the disease and the sensitivity to the drugs used in the treatment. Objective: To assess the in vitro susceptibility to benznidazole, nifurtimox, and posaconazole of 40 cloned strains of T. cruzi isolated in Paraguay belonging to different genotypes, hosts, and localities. Materials and methods: We incubated the parasites in their epimastigote stage in LIT culture medium with different concentrations of each drug in triplicate assays. The degree of susceptibility was estimated by the inhibitory concentrations of 50 and 90% (IC50 and IC90) to obtain the average values and the standard deviation for each strain and drug. We determined the statistical significance between groups by analysis of variances with the Wilcoxon/Kruskal-Wallis non-parametric test and values of p<0.05. Results: A wide range of drug response was observed. Two groups of parasites (A and B) were identified as having significant differences in susceptibility to benznidazole (p<0.0001), and three groups (A, B, C) to nifurtimox and posaconazole (p<0.0001). Conclusions: Overall, the isolates were more susceptible to nifurtimox than benznidazole and posaconazole. Such differences highlight the heterogeneity of T. cruzi populations circulating in Paraguay, an aspect to consider in the treatment and follow up of patients.

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We ascertain the in vitro Benznidazole (BZN) and Nifurtimox (NFX) susceptibility pattern of epimastigotes, trypomastigotes, and amastigotes of 21 T. cruzi strains, from patients, reservoir, and triatomine bugs of various geographic origins. Using this panel of isolates, we compute the Epidemiological cut off value (COwt). Then, the frequency of the susceptible phenotype (Wild type) towards benznidazole (BZN) and nifurtimox (NFX) within this set of strains belonging to three discrete typing units (DTUs), TcI, TcII, and TcV, was deduced. We observed that the susceptibility status of individual T. cruzi isolates toward BZN and NFX is related to the genetic background and underlying factors that are probably related to the individual life trait history of each strain. Analyzing drug susceptibility in this conceptual framework would offer the possibility to evidence a link between isolates expressing a low susceptibility level (not wild-type) as defined by the COwt value and none-curative treatment. It will also permit us to track drug-resistant parasites in the T. cruzi population.

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Neglected tropical diseases (NTDs) constitute a group of infectious diseases prevalent in countries with tropical and subtropical climate that affect the poorest individuals and produce high chronic disability associated with serious problems for the health system and socioeconomic development. Chagas disease or American trypanosomiasis is included on the NTDs list. However, even though this disease affects more than 10 million people, mostly in Latin America, causing the death of over 10,000 people every year, only two drugs are approved for its treatment, benznidazole and nifurtimox. These antiparasitic agents were developed almost half a century ago and present several biopharmaceutical disadvantages such as low aqueous solubility and permeability limiting their bioavailability. In addition, both therapeutic agents are available only as tablets and a liquid pediatric formulation is still lacking. Therefore, novel pharmaceutical strategies to optimize the pharmacotherapy of Chagas disease are urgently required. In this regard, nanotechnological approaches may be a crucial alternative for the delivery of both drugs ensuring an effective pharmacotherapy although the successful bench-to-bedside translation remains a major challenge. The present work reviews in detail the formulation and in-vitro/in-vivo analysis of different nanoformulations of nifurtimox and benznidazole in order to enhance their solubility, dissolution, bioavailability and trypanocidal activity.

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