5-Nitroindazole-based compounds: further studies for activity optimization as anti-Trypanosoma cruzi agents.

In this study, a new series of eleven 5-nitroindazole derivatives (10-20) and a related 6-nitroquinazoline (21) was synthesized and tested in vitro against different forms of the kinetoplastid parasite Trypanosoma cruzi, etiological agent of Chagas disease. Among these compounds, derivatives 11-14 and 17 showed trypanocidal profiles on epimastigotes (IC50 = 1.00-8.75 µM) considerably better than that of the reference drug benznidazole, BZ (IC50 = 25.22 µM). Furthermore, the lack of cytotoxicity observed for compounds 11, 12, 14, 17 and 18 over L929 fibroblasts, led to a notable selectivity (SI) on the extracellular replicative form of the parasite: SIEPI > 12.41 to > 256 µM. Since these five derivatives overpassed the cut-off value established by BZ (SIEPI ≥ 10), they were moved to a more specific assay against the intracellular and replicative form of T. cruzi, i.e, amastigotes. These molecules were not as active as BZ (IC50 = 0.57 µM) against this parasite form; however, all of them showed remarkable IC50 values lower than 7 µM. Special mention deserve compounds 12 and 17, whose SIAMA were > 246.15 and > 188.23, respectively. The results compiled in the present work, point to a positive impact over the trypanocidal activity of the electron withdrawing substituents introduced at position 2 of the N-2 benzyl moiety of these compounds, especially fluorine, i.e., derivatives 12 and 17. These outcomes, supported by the in silico prediction of good oral bioavailability and suitable risk profile, reinforce the 5-nitroindazole scaffold as an adequate template for preparing potential antichagasic agents.

Activity profile of two 5-nitroindazole derivatives over the moderately drug-resistant Trypanosoma cruzi Y strain (DTU TcII): in vitro and in vivo studies.

In previous studies, we have identified several families of 5-nitroindazole derivatives as promising antichagasic prototypes. Among them, 1-(2-aminoethyl)-2-benzyl-5-nitro-1,2-dihydro-3H-indazol-3-one, (hydrochloride) and 1-(2-acetoxyethyl)-2-benzyl-5-nitro-1,2-dihydro-3H-indazol-3-one (compounds 16 and 24, respectively) have recently shown outstanding activity in vitro over the drug-sensitive Trypanosoma cruzi CL strain (DTU TcVI). Here, we explored the activity of these derivatives against the moderately drug-resistant Y strain (DTU TcII), in vitro and in vivo. The outcomes confirmed their activity over replicative forms, showing IC50 values of 0.49 (16) and 5.75 µm (24) towards epimastigotes, 0.41 (16) and 1.17 µm (24) against intracellular amastigotes. These results, supported by the lack of toxicity on cardiac cells, led to better selectivities than benznidazole (BZ). Otherwise, they were not as active as BZ in vitro against the non-replicative form of the parasite, i.e. bloodstream trypomastigotes. In vivo, acute toxicity assays revealed the absence of toxic events when administered to mice. Moreover, different therapeutic schemes pointed to their capability for decreasing the parasitaemia of T. cruzi Y acute infected mice, reaching up to 60% of reduction at the peak day as monotherapy (16), 79.24 and 91.11% when 16 and 24 were co-administered with BZ. These combined therapies had also a positive impact over the mortality, yielding survivals of 83.33 and 66.67%, respectively, while untreated animals reached a cumulative mortality of 100%. These findings confirm the 5-nitroindazole scaffold as a putative prototype for developing novel drugs potentially applicable to the treatment of Chagas disease and introduce their suitability to act in combination with the reference drug.

Experimental models in Chagas disease: a review of the methodologies applied for screening compounds against Trypanosoma cruzi.

One of the main problems of Chagas disease (CD), the parasitic infection caused by Trypanosoma cruzi, is the lack of a completely satisfactory treatment, which is currently based on two old nitroheterocyclic drugs (i.e., nifurtimox and benznidazole) that show important limitations for treating patients. In this context, many laboratories look for alternative therapies potentially applicable to the treatment, and therefore, research in CD chemotherapy works in the design of experimental protocols for detecting molecules with activity against T. cruzi. Phenotypic assays are considered the most valuable strategy for screening these antiparasitic compounds. Among them, in vitro experiments are the first step to test potential anti-T. cruzi drugs directly on the different parasite forms (i.e., epimastigotes, trypomastigotes, and amastigotes) and to detect cytotoxicity. Once the putative trypanocidal drug has been identified in vitro, it must be moved to in vivo models of T. cruzi infection, to explore (i) acute toxicity, (ii) efficacy during the acute infection, and (iii) efficacy in the chronic disease. Moreover, in silico approaches for predicting activity have emerged as a supporting tool for drug screening procedures. Accordingly, this work reviews those in vitro, in vivo, and in silico methods that have been routinely applied during the last decades, aiming to discover trypanocidal compounds that contribute to developing more effective CD treatments.

Drug repositioning for novel antitrichomonas from known antiprotozoan drugs using hierarchical screening.

AIM: Metronidazole is the most widely used drug in trichomoniasis therapy. However, the emergence of metronidazole-resistant Trichomonas vaginalis isolates calls for the search for new drugs to counter the pathogenicity of these parasites. RESULTS: Classification models for predicting the antitrichomonas activity of molecules were built. These models were employed to screen antiprotozoal drugs, from which 20 were classified as active. The in vitro experiments showed moderate to high activity for 19 of the molecules at 10 µg/ml, while 3 compounds yielded higher activity than the reference at 1 µg/ml. The 11 most active chemicals were evaluated in vivo using Naval Medical Research Institute (NMRI) mice. CONCLUSION: Benznidazole showed similar results as metronidazole, and can thus be considered as a potential candidate in antitrichomonas therapy.

Novel Imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxides as antiproliferative trypanosoma cruzi drugs: Computational screening from neural network, synthesis and in vivo biological properties.

A new family of imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide with antiproliferative Trypanosoma cruzi properties was identified from a neural network model published by our group. The synthesis and evaluation of this new class of trypanocidal agents are described. These compounds inhibit the growth of Trypanosoma cruzi, comparable with benznidazole or nifurtimox. In vitro assays were performed to study their effects on the growth of the epimastigote form of the Tulahuen 2 strain, as well as the epimastigote and amastigote forms of CL clone B5 of Trypanosoma cruzi. To verify selectivity towards parasite cells, the non-specific cytotoxicity of the most relevant compounds was studied in mammalian cells, i.e. J774 murine macrophages and NCTC clone 929 fibroblasts. Furthermore, these compounds were assayed regarding the inhibition of cruzipain. In vivo studies revealed that one of the compounds, 19, showed interesting trypanocidal activity, and could be a very promising candidate for the treatment of Chagas disease.

Biological approaches to characterize the mode of action of two 5-nitroindazolinone prototypes on Trypanosoma cruzi bloodstream trypomastigotes.

The phenotypic activity of two 5-nitroindazolinones, i.e. 2-benzyl-1-propyl (22) and 2-benzyl-1-butyl (24) derivatives, previously proposed as anti-Trypanosoma cruzi prototypes, was presently assayed on bloodstream trypomastigotes (BT) of the moderately drug-resistant Y strain. Further exploration of putative targets and cellular mechanisms involved in their activity was also carried out. Therefore, transmission electron microscopy, high-resolution respirometry and flow cytometry procedures were performed on BT treated for up to 24 h with the respective EC50 value of each derivative. Results demonstrated that although 22 and 24 were not as active as benznidazole in this in vitro assay on BT, both compounds triggered important damages in T. cruzi that lead to the parasite death. Ultrastructural alterations included shedding events, detachment of plasma membrane and nuclear envelope, loss of mitochondrial integrity, besides the occurrence of a large number of intracellular vesicles and profiles of endoplasmic reticulum surrounding cytoplasmic organelles such as mitochondrion. Moreover, both derivatives affected mitochondrion leading to this organelle dysfunction, as reflected by the inhibition in oxygen consumption and the loss of mitochondrial membrane potential. Altogether, the findings exposed in the present study propose autophagic processes and mitochondrial machinery as part of the mode of action of both 5-nitroindazolinones 22 and 24 on T. cruzi trypomastigotes.

Bond-based bilinear indices for computational discovery of novel trypanosomicidal drug-like compounds through virtual screening.

Two-dimensional bond-based bilinear indices and linear discriminant analysis are used in this report to perform a quantitative structure-activity relationship study to identify new trypanosomicidal compounds. A data set of 440 organic chemicals, 143 with antitrypanosomal activity and 297 having other clinical uses, is used to develop the theoretical models. Two discriminant models, computed using bond-based bilinear indices, are developed and both show accuracies higher than 86% for training and test sets. The stochastic model correctly indentifies nine out of eleven compounds of a set of organic chemicals obtained from our synthetic collaborators. The in vitro antitrypanosomal activity of this set against epimastigote forms of Trypanosoma cruzi is assayed. Both models show a good agreement between theoretical predictions and experimental results. Three compounds showed IC50 values for epimastigote elimination (AE) lower than 50 µM, while for the benznidazole the IC50 = 54.7 µM which was used as reference compound. The value of IC50 for cytotoxicity of these compounds is at least 5 times greater than their value of IC50 for AE. Finally, we can say that, the present algorithm constitutes a step forward in the search for efficient ways of discovering new antitrypanosomal compounds.

Novel solid dispersions of benznidazole: preparation, dissolution profile and biological evaluation as alternative antichagasic drug delivery system.

Solid dispersions (SD) of benznidazole (BNZ) in sodium deoxycholate (NaDC) or low-substituted hydroxypropylcellulose (L-HPC) were developed by freeze-drying process to improve the solubility of this low water-soluble drug and consequently, its trypanocidal activity. Although the dissolution studies showed a progressive decrease in the release rate of BNZ when formulated in the presence of NaDC, the increase in the surfactant concentration resulted in a better trypanocidal profile on epimastigotes, as well as in an enhancement of the unspecific cytotoxicity. However, such an effect was not so evident on amastigotes and in vivo (blood-trypomastigotes), where high concentrations of surfactant (BNZ:NaDC ≥ 1:6) experimented a loss of activity, correlating this fact with the minor cession of BNZ these formulations accomplished in acidic locations (i.e., dissolution test medium). According to the in vitro results, we reformulated the promising SD-1:3 (IC50 epimastigotes = 33.92 ± 6.41 µM, IC50 amastigotes = 0.40 ± 0.05 µM and LC50 = 183.87 ± 12.30 µM) replacing NaDC by L-HPC, which achieved the fastest dissolution profile. This fact, together with the safety this carrier ensures (LC50 > 256 µM), prompted us to evaluate the cellulose SD in vivo, improving the effectiveness of its NaDC equivalent (%AUPC = 96.65% and 91.93%, respectively). The results compiled in the present work suggest these solid dispersions as alternative drug delivery systems to improve the limited chemotherapy of Chagas disease.

Development of novel benznidazole formulations: physicochemical characterization and in vivo evaluation on parasitemia reduction in Chagas disease.

This work aims to develop novel benznidazole (BZN) solid dispersions (SD) to improve its solubility and bioavailability properties. Low-substituted hydroxypropylcellulose (L-HPC) and sodium deoxycholate (NaDC) were evaluated as carriers. BZN solid dispersions containing different ratios of carrier were prepared by a freeze-drying process and characterized by SEM, powder X-ray diffraction (XRD), differential scanning calorimetry (DSC) and dissolution studies. The reduced BNZ crystallinity in the new formulations was confirmed by XRD, and supported by DSC. BNZ:L-HPC solid dispersion at a 1:3 ratio (w/w) (SD-1:3 L-HPC) improved the BNZ dissolution rate (85% at 5 min) in comparison with BNZ raw material (23% at 5 min). However, NaDC formulations showed a prolonged release (24% at 30 min for SD-1:3 NaDC), due to the formation of a sustained release matrix in acidic medium. In vivo studies performed in a murine model of Chagas disease showed that the formulation achieving the highest parasitemia suppression at a low dose of 25mg/kg/day after five days of treatment was SD-1:3 L-HPC (60% of parasitemia suppression versus 33% of suppression exerted by BNZ), suggesting that BNZ:L-HPC systems enhance the bioavailability of the drug.

Antiprotozoan lead discovery by aligning dry and wet screening: prediction, synthesis, and biological assay of novel quinoxalinones.

Protozoan parasites have been one of the most significant public health problems for centuries and several human infections caused by them have massive global impact. Most of the current drugs used to treat these illnesses have been used for decades and have many limitations such as the emergence of drug resistance, severe side-effects, low-to-medium drug efficacy, administration routes, cost, etc. These drugs have been largely neglected as models for drug development because they are majorly used in countries with limited resources and as a consequence with scarce marketing possibilities. Nowadays, there is a pressing need to identify and develop new drug-based antiprotozoan therapies. In an effort to overcome this problem, the main purpose of this study is to develop a QSARs-based ensemble classifier for antiprotozoan drug-like entities from a heterogeneous compounds collection. Here, we use some of the TOMOCOMD-CARDD molecular descriptors and linear discriminant analysis (LDA) to derive individual linear classification functions in order to discriminate between antiprotozoan and non-antiprotozoan compounds as a way to enable the computational screening of virtual combinatorial datasets and/or drugs already approved. Firstly, we construct a wide-spectrum benchmark database comprising of 680 organic chemicals with great structural variability (254 of them antiprotozoan agents and 426 to drugs having other clinical uses). This series of compounds was processed by a k-means cluster analysis in order to design training and predicting sets. In total, seven discriminant functions were obtained, by using the whole set of atom-based linear indices. All the LDA-based QSAR models show accuracies above 85% in the training set and values of Matthews correlation coefficients (C) vary from 0.70 to 0.86. The external validation set shows rather-good global classifications of around 80% (92.05% for best equation). Later, we developed a multi-agent QSAR classification system, in which the individual QSAR outputs are the inputs of the aforementioned fusion approach. Finally, the fusion model was used for the identification of a novel generation of lead-like antiprotozoan compounds by using ligand-based virtual screening of 'available' small molecules (with synthetic feasibility) in our 'in-house' library. A new molecular subsystem (quinoxalinones) was then theoretically selected as a promising lead series, and its derivatives subsequently synthesized, structurally characterized, and experimentally assayed by using in vitro screening that took into consideration a battery of five parasite-based assays. The chemicals 11(12) and 16 are the most active (hits) against apicomplexa (sporozoa) and mastigophora (flagellata) subphylum parasites, respectively. Both compounds depicted good activity in every protozoan in vitro panel and they did not show unspecific cytotoxicity on the host cells. The described technical framework seems to be a promising QSAR-classifier tool for the molecular discovery and development of novel classes of broad-antiprotozoan-spectrum drugs, which may meet the dual challenges posed by drug-resistant parasites and the rapid progression of protozoan illnesses.

In vitro phenotypic screening of 7-chloro-4-amino(oxy)quinoline derivatives as putative anti-Trypanosoma cruzi agents.

In this study, a series of 22 pre-synthesized 7-chloro-4-amino(oxy)quinoline derivatives was assayed in vitro as potential antichagasic agents. A primary screening against Trypanosoma cruzi epimastigotes and a non-specific cytotoxicity assay on murine fibroblasts were simultaneously performed, resulting quinolines 3, 7 and 12 with great selectivity (SI) on the extracellular parasite (SI7, SI3, SI12 and SIBZ >9.44). Therefore, the activity of these derivatives was evaluated on intracellular amastigotes, achieving derivative 7 the best SI (SI=12.73). These results, supported by the in silico prediction of a good oral bioavailability and a suitable risk profile, propose the 4-amino-7-chloroquinoline scaffold as a potential template for designing trypanocidal prototypes.

Selective activity of 2,4-diaryl-1,2,3,4-tetrahydroquinolines on Trypanosoma cruzi epimastigotes and amastigotes expressing ß-galactosidase.

The growth inhibitory effect on Trypanosoma cruzi epimastigotes and the unspecific cytotoxicity over NCTC-929 fibroblasts of two series of previously synthesized 2,4-diaryl-1,2,3,4-tetrahydroquinolines (THQ), have been studied in vitro and compared with those of benznidazole (BZ). Derivatives AR39, AR40, AR41, AR91 and DM15 achieved outstanding selectivity indexes (SI) on the extracellular form (SITHQ>SIBZ>9.44) and thus, were tested in a more specific in vitro assay against amastigotes, showing less effectiveness than the reference drug (SIBZ>320) but also accomplishing great selectivity on the intracellular stage (SITHQ>25). These promising results, supported by the in silico prediction of high bioavailability and less potential risk than benznidazole, reveal several tetrahydroquinolines as prototypes of potential antichagasic drugs.

Identification in silico and in vitro of novel trypanosomicidal drug-like compounds.

Atom-based bilinear indices and linear discriminant analysis are used to discover novel trypanosomicidal compounds. The obtained linear discriminant analysis-based quantitative structure-activity relationship models, using non-stochastic and stochastic indices, provide accuracies of 89.02% (85.11%) and 89.60% (88.30%) of the chemicals in the training (test) sets, respectively. Later, both models were applied to the virtual screening of 18 in-house synthesized compounds to find new pro-lead antitrypanosomal agents. The in vitro antitrypanosomal activity of this set against epimastigote forms of Trypanosoma cruzi is assayed. Predictions agree with experimental results to a great extent (16/18) of the chemicals. Sixteen compounds show more than 70% of epimastigote inhibition at a concentration 100 µg/mL. In addition, three compounds (CRIS 112, CRIS 140 and CRIS 147) present more than 70% of epimastigote inhibition at a concentration of 10 µg/mL (79.95%, 73.97% and 78.13%, respectively) with low values of cytotoxicity (19.7%, 7.44% and 20.63%, correspondingly).Taking into account all these results, we could say that these three compounds could be optimized in forthcoming works. Even though none of them resulted more active than nifurtimox, the current results constitute a step forward in the search for efficient ways to discover new lead antitrypanosomals.

Ligand-based discovery of novel trypanosomicidal drug-like compounds: in silico identification and experimental support.

Two-dimensional bond-based linear indices and linear discriminant analysis are used in this report to perform a quantitative structure-activity relationship study to identify new trypanosomicidal compounds. A database with 143 anti-trypanosomal and 297 compounds having other clinical uses, are utilized to develop the theoretical models. The best discriminant models computed using bond-based linear indices provides accuracies greater than 90 for both training and test sets. Our models identify as anti-trypanosomals five out of nine compounds of a set of already-synthesized substances. The in vitro anti-trypanosomal activity of this set against epimastigote forms of Trypanosoma cruzi is assayed. Both models show a perfect agreement between theoretical predictions and experimental results. The compounds identified as active ones show more than 98% of anti-epimastigote elimination (AE) at a concentration of 100 µg/mL. Besides, three compounds show more than 70% of AE at a concentration of 10 µg/mL. Finally, compounds with the best "activity against epimastigote forms/unspecific cytotoxicity" ratio are evaluated using an amastigote susceptibility assay. It should be noticed that, compound Va7-71 exhibit a 100% of intracellular amastigote elimination and shows similar activity when compared to a standard trypanosomicidal as nifurtimox. Finally, we can emphasize that, the present algorithm constitutes a step forward in the search for efficient ways of discovering new anti-trypanosomal compounds.

Saponinas esteroidales de la planta Agave brittoniana (Agavaceae) con actividad contra el parásito Trichomona vaginalis

Steroidal saponins from the plant Agave brittoniana with activity against the parasite Trichomona vaginalis. The genus Agave (Agavaceae), includes more than 300 species; around 16 of them show an homogeneous distribution throughout Cuba. Agave brittoniana (ssp. brachypus), is an endemic subspecies that grows in the central region of the country and its leaves are traditionally used in the treatment of parasitic diseases. The parasite Trichomonas vaginalis causes the disease known as trichomoniasis, that infects the genital tract. To test in vitro the plant against Trichomona vaginalis, the dried and powdered leaves were extracted three times with ethanol-water (7 : 3) by maceration at room temperature. The solvent was removed under reduced pressure and the extract was suspended in distilled water, defatted with n-hexane, and extracted with water-saturated n-butanol. After solvent removal, a portion of the n-butanol extract was hydrolyzed. After extraction with ethyl acetate the hydrolysis products were compared with authentic sapogenins samples using thin layer chromatography (TLC). Most of the sapogenins (yuccagenin and diosgenin) were isolated and their structures were confirmed. using nuclear magnetic resonance (NMR) experiments. The n-butanol extract was subjected to a separation process through column chromatography to obtain five fractions. After multiple separation processes by reversed phase high performance liquid chromatography (HPLC), the most active one produced one refined fraction that contained two saponins with the same aglycone (diosgenin) and one yuccagenin based saponin. Best results of the activity were obtained with the yuccagenin derived glycoside. Rev. Biol. Trop. 56 (4): 16451652. Epub 2008 December 12.

El género Agave, familia Agavaceae, tiene más de 300 especies, con aproximadamente 16 distribuidas en toda Cuba. Una de ellas, el Agave brittoniana Trel. (ssp. brachypus), es una subespecie endémica y sus hojas son tradicionalmente utilizadas en el tratamiento de enfermedades parasitarias. Se realizaron estudios "in vitro" de la actividad de productos de esta planta frente a Trichomona vaginalis. Las hojas secas y pulverizadas fueron extraídas tres veces con una mezcla de etanol-agua (7: 3) mediante maceración a temperatura ambiente. El disolvente fue evaporado a presión reducida y el extracto fue suspendido en agua destilada, desengrasado con n-hexano, y extraído con n-butanol saturado con agua. Luego de una extracción con acetato de etilo, los productos de la hidrólisis fueron comparados con patrones de sapogeninas mediante la cromatografía de capa fina (CCD). Aislamos las sapogeninas mayoritarias (yuccagenina y diosgenina) y confirmamos sus estructuras utilizando técnicas de resonancia magnética nuclear. Por otra parte, el extracto n-butanólico fue sometido a un proceso de separación biodirigido mediante cromatografía de columna, obteniéndose cinco fracciones. Después de múltiples separaciones, la más activa rindió una fracción purificada con dos sapogeninas con el mismo aglicón (diosgenina) y un glicósido de yucagenina. Los mejores resultados de esta actividad fueron obtenidos con el glicósido derivado de la yucagenina.

[Steroidal saponins from the plant Agave brittoniana with activity against the parasite Trichomona vaginalis]. / Saponinas esteroidales de la planta Agave brittoniana (Agavaceae) con actividad contra el parásito Trichomona vaginalis.

The genus Agave (Agavaceae), includes more than 300 species; around 16 of them show an homogeneous distribution throughout Cuba. Agave brittoniana (ssp. brachypus), is an endemic subspecies that grows in the central region of the country and its leaves are traditionally used in the treatment of parasitic diseases. The parasite Trichomonas vaginalis causes the disease known as trichomoniasis, that infects the genital tract. To test in vitro the plant against Trichomona vaginalis, the dried and powdered leaves were extracted three times with ethanol-water (7:3) by maceration at room temperature. The solvent was removed under reduced pressure and the extract was suspended in distilled water, defatted with n-hexane, and extracted with water-saturated n-butanol. After solvent removal, a portion of the n-butanol extract was hydrolyzed. After extraction with ethyl acetate the hydrolysis products were compared with authentic sapogenins samples using thin layer chromatography (TLC). Most of the sapogenins (yuccagenin and diosgenin) were isolated and their structures were confirmed. using nuclear magnetic resonance (NMR) experiments. The n-butanol extract was subjected to a separation process through column chromatography to obtain five fractions. After multiple separation processes by reversed phase high performance liquid chromatography (HPLC), the most active one produced one refined fraction that contained two saponins with the same aglycone (diosgenin) and one yuccagenin based saponin. Best results of the activity were obtained with the yuccagenin derived glycoside.

Selective activity of polyene macrolides produced by genetically modified Streptomyces on Trypanosoma cruzi.

The growth inhibitory effects on Trypanosoma cruzi of several natural tetraene macrolides and their derivatives were studied and compared with that of amphotericin B. All tetraenes strongly inhibited in vitro multiplication. Proliferation of epimastigotes was arrested by all these drugs at < or =3.6 microM, which were also active on amastigotes proliferating in fibroblasts. Compared with amphotericin B, the compounds were less effective but also less toxic, showing no effect on the proliferation of J774 and NCTC 929 mammalian cells at concentrations active against the parasites. CE-108B (a polyene amide) appeared to be an especially potent trypanocidal compound, with strong in vivo trypanocidal activity and very low or no toxic side effects, and thus should be considered for further studies.

Development of resazurin microtiter assay for drug sensibility testing of Trypanosoma cruzi epimastigotes.

A quantitative colorimetric assay using the oxidation-reduction indicator resazurin was developed to measure cytotoxicity of compounds against the protozoan parasite Trypanosoma cruzi. This method is based on the detection of colorimetric changes caused by the oxidation (blue) and reduction (pink) capabilities of resazurin dye, an indicator for metabolic cell function. To validate the assay, the experimental conditions were adjusted, such as number of parasites, dye concentration, and time of incubation, with respect to linearity and lower limit of detection. We found that absorbances increased linearly, with the plating density of parasites as low as 5-100 x 10(4)/well (r=0.99; p<0.001) when they were incubated for 5 h at 28 degrees C in the presence of 10% resazurin solution (3 mM). When the cytotoxicity of the reference drugs nifurtimox and benznidazole was measured with this assay and compared to the microscopic counting method, the same range was obtained, demonstrating that the resazurin microtiter assay is valid for the screening of new trypanocidal compounds. This test is very simple, fast, sensitive, and cheap.

Biological variability in clinical isolates of Trichomonas vaginalis.

Eighteen clinical isolates of Trichomonas vaginalis were obtained from women who attended health centers of the Government of Madrid. A total of 1,848 vaginal specimens recovered during the gynaecological examination were seeded in culture tubes containing liquid Diamond medium. Pathogenicity to mice was determined after intraperitoneal inoculation of mice by quantification of mortality and gross damage to abdominal organs. As could be expected, a broad variability was obtained, being some of the isolates more virulent than the reference strain. Regarding to metronidazole susceptibility, none resistant isolate was found but different degrees of susceptibility were determined.

Biological variability in clinical isolates of Trichomonas vaginalis

Eighteen clinical isolates of Trichomonas vaginalis were obtained from women who attended health centers of the Goverment of Madrid. A total of 1,848 vaginal specimens recovered during the gynaecological examination were seeded in culture tubes containing liquid Diamond medium. Pathogenicity to mice was determined after intraperitoneal inoculation of mice by quantification of mortality and gross damage to abdominal organs. As could be expected, a broad variability was obtained, being some of the isolates more virulent than the reference strain. Regarding to metronidazole susceptibility, none resistant isolate was found but different degrees of susceptibility were determined