In vitro susceptibility to benznidazole, nifurtimox and posaconazole of Trypanosoma cruzi isolates from Paraguay / [Sensibilidad in vitro a benznidazol, nifurtimox y posaconazol de cepas de Trypanosoma cruzi de Paraguay].

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.

Sensibilidad in vitro a benznidazol, nifurtimox y posaconazol de cepas de Trypanosoma cruzi de Paraguay / In vitro susceptibility to benznidazole, nifurtimox and posaconazole of Trypanosoma cruzi isolates from Paraguay

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.

Adsorption capacity of different types of carbon nanotubes towards metronidazole and dimetridazole antibiotics from aqueous solutions: effect of morphology and surface chemistry.

The effect of surface chemistry and morphology of carbon nanotubes (CNTs) on their adsorption capacity towards dimetridazole (DTZ) and metronidazole (MNZ) antibiotics from water solutions was investigated in this work. The CNTs studied were single-walled carbon nanotubes (SWCNTs), CNTs doped with nitrogen (N-CNTs), multiwalled CNTs (MWCNTs), and MWCNTs functionalized with carboxylic groups (MWCNT-COOH). The experimental adsorption equilibrium data were best interpreted with the Redlich-Peterson (R-P) isotherm model. At T of 25 °C and pH of 7, the capacities of adsorption decreased as follows: SWCNT > MWCNT > N-CNT ≈ MWCNT-COOH, and the maximum capacities of SWCNT towards MNZ and DTZ were 101 mg/g and 84 mg/g, correspondingly. The SWCNT had the highest adsorption capacity because SWCNT presented the largest surface area, and was the only nanomaterial with a basic surface. The adsorption of both antibiotics on the CNTs was predominantly ascribed to the π-π stacking. The basic groups promoted the π-π stacking interactions and favored the adsorption capacity towards MNZ and DTZ. The capacity of SWCNT for adsorbing MNZ was lessening substantially by reducing the pH from 11 to 2, and the electrostatic interactions caused this trend. The Sheindorf-Rebuhn-Sheintuch adsorption model interpreted the data for the competitive adsorption of DTZ and MNZ on SWCNT adequately.

Novel nitroimidazole derivatives evaluated for their trypanocidal, cytotoxic, and genotoxic activities.

The current treatment of Chagas disease is based on the use of two drugs, nifurtimox (Nfx) and benznidazole (Bnz), both of which present limited efficacy in the chronic stage of the disease and toxic side effects. Thus, the discovery of novel compounds is urgently required. Herein, we report the successful synthesis of 4-nitroimidazole analogs of Bnz via nucleophilic aromatic substitution or cycloaddition reactions. The analogs were biologically evaluated, and compound 4 (4-cyclopropyl-1-(1-methyl-4-nitro-1H-imidazole-5-yl)-1H-1,2,3-triazole) was identified as the most potent against both the trypomastigote (IC50 = 5.4 µM) and amastigote (IC50 = 12.0 µM) forms of T. cruzi, showing activity in the same range as Bnz (IC50 = 8.8 and 8.7 µM, respectively). The cytotoxic and genotoxic activities of compounds 5, 4 and 11 were assessed. These three compounds were cytotoxic and genotoxic to RAW and HepG2 cells and mutagenic to Salmonella enterica strains. However, 4 exhibited toxic effects only at concentrations higher than those needed for trypanocidal activity. Molecular docking of 4 showed the importance of the size and π-π interactions between the nitroimidazole and the cofactor (flavin mononucleotide) of T.cruzi-nitroreductase (TcNTR). Moreover, the residues His503 and Tyr545 are relevant for binding to TcNTR. Our design strategy was capable of generating novel and active Bnz analogs.

Correlation between molecular charge densities and sensitivity of nitrogen-rich heterocyclic nitroazole derivative explosives.

Nitroazole derivatives are nitrogen-rich heterocyclic ring molecules with potential application as energetic materials. Thirty-three of them-nitroimidazoles, nitrotriazoles, and nitropyrazoles-were investigated. Computed density functional theory molecular charge densities were partitioned employing the accurate distributed multipole analysis (DMA) method. Based on the magnitude of the DMA atom-centered electric multipoles (monopole, dipole, and quadrupole values), mathematical models were developed to compute the impact sensitivity of the explosives composed of these molecules. Charge localization and delocalization of the ring nitrogen atoms as well as charges of the atoms of the nitro group affect the sensitivity of explosives composed of nitroazole derivatives. The sensitivity is strongly dependent on the ring position of the nitrogen atoms and the bonding site of the substituent groups. The N/C ratio and the repulsion of the non-bonding electron pairs of the vicinal nitrogen atoms of the ring also play an important role in the stability of nitroazoles. The influence of the withdrawing group (NO2) and the electron injector groups (NH2 and CH3) including their bonding position on the ring could be quantified.

HQSAR and random forest-based QSAR models for anti-T. vaginalis activities of nitroimidazoles derivatives.

Trichomonas vaginalis is the causative agent of trichomoniasis, a highly prevalent sexually transmitted infection worldwide. Nitroimidazole drugs, such as metronidazole and tinidazole, are the only recommended treatment, but cases of resistance represent at least 5%. In case of resistance or therapeutic failure, posology with higher doses is used, culminating in the increase of the toxic effects of the treatment. In this context, the development of new drugs becomes an eminent necessity. Hologram quantitative structure-activity relationship (HQSAR) models using nitroimidazole derivatives were generated to discover the relationship between the different chemical structures and the activity against cells and the selectivity against susceptible and resistant strains. One model of each strain was chosen for interpretation, both showed good internal coefficient (q2LOO values: 0.607 for susceptible strain and 0.646 for resistant strain subsets) and great values in other internal and external validations metrics. From the contribution of fragments to HQSAR models, several differences between the most and least potent compounds were found: 5-nitroimidazole contributes positively while 4-nitroimidazole negatively. QSAR models employing random forest (RF-QSAR) machine learning technique were also built and a robust model was obtained from resistant strain activity prediction (q2LOO equals to 0.618). The constructed HQSAR and RF-QSAR models were employed to predict the activity of three newly planned nitroimidazole derivatives in the design of new drugs candidates against T. vaginalis strains.

In vitro genotoxicity of nitroimidazoles as a tool in the search of new trypanocidal agents

BACKGROUND Only benznidazole (Bnz) (1) and nifurtimox (Nfx) (2) are licensed for the treatment of Chagas disease although their safety and efficacy profile are far from ideal. Farmanguinhos from Fiocruz has developed seven nitroimidazole compounds (4-10) analogs of megazol (3). OBJECTIVES To evaluate whether the genotoxic effect of 3 was abolished in the seven nitroimidazoles (4-10) analogs using the in vitro alkaline comet assay (CA) and the in vitro cytokinesis-block micronucleus assay (CBMN) in whole human blood cells (WHBC) and correlate this effect with their trypanocidal activity using bloodstream trypomastigote forms of Trypanosoma cruzi. METHODS The toxicity of 3-10 to WHBC in the in vitro CA was determined using the fluorescein diacetate/ethidium bromide assay. DNA damage in the in vitro CA was evaluated according to tail size in four classes (0-3) and methyl methane-sulfonate (MMS) was used as a positive control. The cytotoxicity of 3-10 to WHBC in the CBMN was measured using the cytokinesis-block proliferation index and the replication index. The number of the micronucleate cells in 2,000 binucleate cells by experimental group was determined. Mitomycin C and N-deacetyl-N-methylcolchicine were used as positive controls. FINDINGS Compound 3 showed a significant DNA strand break effect through the in vitro CA and highly significant clastogenic and/or aneugenic effect in the CBMN. Compounds 5, 6, 8, 9 and 10 showed negative results in the CBMN and positive results in the in vitro CA, while the inverse effect was observed for 4 and 7. MAIN CONCLUSIONS Compound 10 was the most promising to proceed with the development as a drug candidate in the treatment of Chagas disease showing absence of chromosomal cytogenetic damage and high activity against T. cruzi, about two times higher than 3 and the clinical drug 1.

In vitro anti-tuberculosis activity of azole drugs against Mycobacterium tuberculosis clinical isolates / Actividad in vitro de azoles contra aislamientos clínicos de Mycobacterium tuberculosis

Background: Latent tuberculosis has been associated with the persistence of dormant Mycobacterium tuberculosis in the organism of infected individuals, who are reservoirs of the bacilli and the source for spreading the disease in the community. New active anti-TB drugs exerting their metabolic action at different stages and on latent/dormant bacilli are urgently required to avoid endogenous reactivations and to be part of treatments of multi- and extensively-drug resistant tuberculosis (M/XDR-TB). It was previously reported that azole drugs are active against M. tuberculosis. For that reason, the aims of this study were to determine the in vitro activity of azole drugs, imidazole (clotrimazole, CLO and econazole, ECO) and nitroimidazole (metronidazole, MZ and ipronidazole, IPZ), against a collection of MDR M. tuberculosis clinical isolates; and to analyze their potential use in both the LTB and the active forms of M/XDR-TB treatments. Methods: A total of 55 MDR M. tuberculosis isolates and H37Rv were included. MZ and IPZ activity against M. tuberculosis isolates were tested using anaerobic culture conditions. The activity of ECO and CLO was measured by the minimal inhibitory concentration (MIC) using a microdilution colorimetric method. Results: MZ and IPZ showed bacteriostatic activity against M. tuberculosis strains. MIC5o and MIC90 to ECO was 4.0 µg/ml, while MIC50 to CLO was 4.0 µg/ml and MIC90 was 8.0 µg/ml respectively. Conclusion: All azole compounds tested in the study showed inhibitory activity against MDR M. tuberculosis clinical isolates.

Introducción: La tuberculosis (TB) latente ha sido asociada a la persistencia de Mycobacterium tuberculosis durmientes en el organismo de las personas infectadas, las cuales constituyen un reservorio del bacilo y una fuente de diseminación de la enfermedad en la comunidad. Urge la necesidad de contar con nuevos fármacos antituberculosos con acción sobre el bacilo en estado latente/durmiente, a fin de evitar reactivaciones endógenas y para ser incluidas en el tratamiento de la TB multirresistente y extensivamente resistente (M/XDR-TB). Se ha reportado que los azoles son activos contra M. tuberculosis. Por esta razón, los objetivos del presente estudio fueron determinar la actividad in vitro sobre aislamientos clínicos de M/XDR-TB de distintos azoles, incluyendo los imidazoles econazol (ECO) y clotrimazol (CLO) y los 5-nitro-imidazoles ipronidazol (IPZ) y metronidazol (MZ), así como analizar su potencial uso contra las formas latente y activa de esta enfermedad. Métodos: Fueron incluidos 55 aislamientos clínicos de M. tuberculosis MDR y la cepa de referencia H37Rv. Se evaluó la actividad del MZ y el IPZ sobre los aislamientos en condiciones de cultivo anaeróbico, mientras que la actividad del ECO y el CLO fue estimada determinando la concentración inhibitoria mínima (CIM) mediante el método colorimétrico de microdilución en placa. Resultados: El MZ y el IPZ presentaron actividad bacteriostática frente a las cepas de M. tuberculosis. La CIM50 y CIM90 del ECO fue de 4 µg/ml, mientras que el CLO presentó una CIM50 de 4 µg/ml y una CIM90 de 8 µg/ml. Conclusión: Todos los compuestos azólicos evaluados presentaron actividad inhibitoria frente a aislamientos clínicos de M. tuberculosis.

In vitro anti-tuberculosis activity of azole drugs against Mycobacterium tuberculosis clinical isolates.

BACKGROUND: Latent tuberculosis has been associated with the persistence of dormant Mycobacterium tuberculosis in the organism of infected individuals, who are reservoirs of the bacilli and the source for spreading the disease in the community. New active anti-TB drugs exerting their metabolic action at different stages and on latent/dormant bacilli are urgently required to avoid endogenous reactivations and to be part of treatments of multi- and extensively-drug resistant tuberculosis (M/XDR-TB). It was previously reported that azole drugs are active against M. tuberculosis. For that reason, the aims of this study were to determine the in vitro activity of azole drugs, imidazole (clotrimazole, CLO and econazole, ECO) and nitroimidazole (metronidazole, MZ and ipronidazole, IPZ), against a collection of MDR M. tuberculosis clinical isolates; and to analyze their potential use in both the LTB and the active forms of M/XDR-TB treatments. METHODS: A total of 55 MDR M. tuberculosis isolates and H37Rv were included. MZ and IPZ activity against M. tuberculosis isolates were tested using anaerobic culture conditions. The activity of ECO and CLO was measured by the minimal inhibitory concentration (MIC) using a microdilution colorimetric method. RESULTS: MZ and IPZ showed bacteriostatic activity against M. tuberculosis strains. MIC50 and MIC90 to ECO was 4.0µg/ml, while MIC50 to CLO was 4.0µg/ml and MIC90 was 8.0µg/ml respectively. CONCLUSION: All azole compounds tested in the study showed inhibitory activity against MDR M. tuberculosis clinical isolates.

Multiresidue method for identification and quantification of avermectins, benzimidazoles and nitroimidazoles residues in bovine muscle tissue by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) using a QuEChERS approach.

A quantitative and confirmatory multiresidue method for determining the presence of avermectins, benzimidazoles and nitroimidazoles in bovine muscle tissue by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was developed, optimized and validated, using a QuEChERS extraction. The evaluated performance parameters were linearity, selectivity, matrix effect, decision limits (CCα), detection capability (CCß), limits of detection (LOD), limits of quantification (LOQ), accuracy, precision and robustness. The validated method exhibited linearity with coefficient of determination (R2) higher than 0.90 in the working range from 0.5 to 2.0 times the maximum residue limit (MRL) or the minimum required performance level (MRPL) for the studied analytes, except for closantel, for which the linear study range was defined from 50 to 200µgkg-1. The method was selective in the presence of macrolides and lincosamides for all the studied analytes. The LOD varied from 0.007 to 66.715µgkg-1, whereas LOQ values ranging from 0.011 to 113.674µgkg-1 were found. The results of the evaluation of the accuracy and precision were satisfactory for all the studied analytes, and according to the assessment of the robustness, the method was not robust only for the analytes abamectin, moxidectin, doramectin fenbendazole sulfone, closantel, thiabendazole, hydroxyl-metronidazole and ronidazole. The performance parameters demonstrated total method adequacy for the detection and quantification of avermectins, benzimidazoles and nitroimidazoles residues in bovine muscle tissues.

Challenges and Persistent Questions in the Treatment of Trichomoniasis.

Trichomoniasis is a sexually transmitted disease (STD) caused by infection with the protozoan parasite Trichomonas vaginalis. It is considered the most prevalent non-viral sexually transmitted disease worldwide. Recently, the infection has been associated with adverse outcomes of pregnancy and increased risks of HIV acquisition and transmission, as well as an association with cervical and prostate cancers. The consequences of trichomoniasis are likely much greater than previously recognized, both at the individual and the community level. Since many cases are asymptomatic, and the most common approach used for diagnosis (wet mount) is also one of the least sensitive, millions of T. vaginalis infections remain undiagnosed and therefore untreated. The purpose of this review is to address what is known about the treatment of T. vaginalis infections and what additional approaches could be pursued. The increasing recognition of the potential public health implications of trichomoniasis has resulted in greater attention to improving effectiveness of the interventions for affected individuals. Currently, treatment relies almost solely on one class of drugs, the 5- nitroimidazoles, which causes concern should widespread drug resistance arise. There are also concerns regarding which 5-nitroimidazole to use as not all of them are active against T. vaginalis. Finally, new therapeutic targets and active compounds with treatment potential are considered.

Combination therapy in the management of giardiasis: What laboratory and clinical studies tell us, so far

Treatment failures in patients suffering from giardiasis are not uncommon feature. The most frequent approach in these cases is to treat these patients with longer repeated courses and/or higher doses of the primary therapy, or using drugs from a different class to avoid potential cross-resistance. However, a higher rate of adverse events may limit this strategy. In this context, combination therapy (CT) is emerging as a valuable option against refractory giardiasis. In the attempt to evaluate the benefits of CT, a number of experimental studies, clinical series, and randomized clinical trials (RCTs), as well as several veterinary studies have been performed, with varying results. Here, we present a critical analysis of the available information regarding CT for the treatment of Giardia infection, as well as the authors' opinion with respect to its use. RCTs of combination therapy are limited and the optimal combinations and administration strategies need yet to be clarified. Analyses of the cost-effectiveness and RCTs of CTs for Giardia infection are required to assess the role of these drugs for the control of giardiasis, mainly in the case of treatment failures linked to suspected drug tolerance are the case

Studies of genotoxicity and mutagenicity of nitroimidazoles: demystifying this critical relationship with the nitro group

Nitroimidazoles exhibit high microbicidal activity, but mutagenic, genotoxic and cytotoxic properties have been attributed to the presence of the nitro group. However, we synthesised nitroimidazoles with activity against the trypomastigotes of Trypanosoma cruzi, but that were not genotoxic. Herein, nitroimidazoles (11-19) bearing different substituent groups were investigated for their potential induction of genotoxicity (comet assay) and mutagenicity (Salmonella/Microsome assay) and the correlations of these effects with their trypanocidal effect and with megazol were investigated. The compounds were designed to analyse the role played by the position of the nitro group in the imidazole nucleus (C-4 or C-5) and the presence of oxidisable groups at N-1 as an anion receptor group and the role of a methyl group at C-2. Nitroimidazoles bearing NO2 at C-4 and CH3 at C-2 were not genotoxic compared to those bearing NO 2 at C-5. However, when there was a CH3 at C-2, the position of the NO2 group had no influence on the genotoxic activity. Fluorinated compounds exhibited higher genotoxicity regardless of the presence of CH3 at C-2 or NO2 at C-4 or C-5. However, in compounds 11 (2-CH3; 4-NO2; N-CH2OHCH2Cl) and 12 (2-CH3; 4-NO2; N-CH2OHCH2F), the fluorine atom had no influence on genotoxicity. This study contributes to the future search for new and safer prototypes and provide.

Management of chronic Giardia infection.

Advances in our understanding of chronic giardiasis (CG) may improve our care of patients in this stage of the disease. This review proposes a new concept of CG and highlights the recent advances in our understanding and management of this condition. According to this review, management requires, initially, an accurate diagnosis, which may exclude several conditions that can mimic CG. Optimal treatment requires a tailored approach which includes the recognition of the known modifiable causes of this health condition, assessment of symptoms and potential complications, their treatment utilizing, if necessary, a multidisciplinary team, and an ongoing monitoring for the effect of therapy - weighing the efficacy of individual drugs - all of these together may lead to a successful treatment of CG.