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The emergence of nitroimidazole resistant isolates has been an aggravating factor in the treatment of trichomoniasis, the most common non-viral sexually transmitted disease in the world. This highlights the importance of new technologies that are safe, effective, and have minor side effects or resistance. Hence, we evaluated the effectiveness of photodynamic therapy on the inactivation of Trichomonas vaginalis in vitro. We used methylene blue as a photosensitizing substance, and a light-emitting diode (LED) for irradiation of metronidazole sensitive and resistant strains. Our results showed that only the presence of light did not interfere with parasite growth; however, methylene blue isolated or associated with light inhibited 31.78% ± 7.18 and 80.21% ± 7.11 of the sensitive strain, respectively, and 31.17% ± 4.23 and 91.13% ± 2.31 of the resistant strain, respectively. The high trichomonicidal activity of the photodynamic therapy, associated with low cost and ease of application, signalize its great therapeutic potential not only when conventional treatment fails, but also routinely in women with trichomoniasis.

Trichomoniasis is the most common non-viral, sexually transmitted infection affecting humans worldwide. The main treatment for trichomoniasis is metronidazole (MTZ). However, adverse effects and reports of resistance have stimulated the development of therapeutic alternatives. The ease of manipulation of the side chains of MTZ coupled with its safety makes this molecule attractive for the development of new drugs. In this context, we evaluated the activity of the chlorinated MTZ derivative, MTZ-Cl, on sensitive and resistant strains of Trichomonas vaginalis. MTZ-Cl presented a remarkable activity against both sensitive and resistant strains. In vitro and in vivo toxicity assays indicated that the new molecule is safe for future clinical trials. Furthermore, we noticed different rates of free radical production between the sensitive and resistant strains. MTZ-Cl induced a higher release of nitric oxide (NO, ~ 9000 a.u.) by both sensitive and resistant strains. However, the sensitive strain produced a greater amount of H2O2 (~ 1,800,000 a.u.) and superoxide radicals (~ 350,000 a.u.) in the presence of MTZ. In the resistant strain, production of these radicals was more prominent when MTZ-Cl was used. Collectively, these results suggest that NO is an important molecule in the trichomonacidal activity against resistant and sensitive strains, suggesting an alternative pathway for MTZ-Cl activation. We highlight the high trichomonacidal potential of MTZ-Cl, improving the effectiveness of treatment and reducing side effects. In addition, MTZ-Cl is derived from a well-established drug on the world market that presents low toxicity to human cells, suggesting its safety to proceed with future clinical trials.

Trichomonas vaginalis is an amitochondrial parasite that causes human trichomoniasis. Despite metronidazole effectiveness, resistant cases are becoming more frequent. This scenario reveals the need to develop new therapeutic options. Photodynamic Therapy (PDT) is an experimental treatment that involves the activation of photosensitive substances and the generation of cytotoxic oxygen species and free radicals to promote the selective destruction of target tissues. In previous work, we identified an excellent in vitro PDT activity using methylene blue and light emitting diode against metronidazole sensitive and resistant strains of T. vaginalis. Here, we evaluated the efficacy of PDT in vivo and its high trichomonicidal activity was assessed through transmission electron microscopy. Female Balb/c mice were infected intravaginally with T. vaginalis trophozoites. On the third day of infection, methylene blue was introduced into the vaginal canal, which then received 68.1 J/cm2 of radiation for 35.6 s. Twenty-four hours after treatment the vaginal canal of the animals was scraped and the samples processed by the immunocytochemistry technique. Besides that, in vitro photodynamic treatment was performed and T. vaginalis trophozoites were processed by transmission electron microscopy. PDT significantly reduced infection in animals treated, compared to control groups, being as efficient as metronidazole. Morphological changes observed have suggested that PDT activity on T. vaginalis was due to necrosis. These results, added to the high trichomonicidal activity of PDT confirm its feasibility for trichomoniasis treatment.

BackgroundTrichomonas vaginalis is an amitochondrial parasitic that causes human trichomoniasis, the most common non-viral sexually transmitted infection in the world. The therapy of choice is metronidazole (MTZ). Despite MTZ effectiveness, resistant cases are becoming more frequent. Another point to emphasize are the side effects that may result in treatment discontinuation, leading to further spread of infection and emergence of resistant strains. This scenario reveals the need to develop new therapeutic options. Photodynamic therapy (PDT) is an experimental treatment that involves the activation of photosensitive substances and the generation of cytotoxic oxygen species and free radicals to promote the selective destruction of target tissues. A previous study, from our group, identified an excellent in vitro PDT activity using methylene blue and light emitting diode against MTZ sensitive and resistant strains of T. vaginalis. The aim of this study was to evaluate the efficacy of PDT in vivo and clarify its high trichomonicidal potential by evaluating its action upon T. vaginalis trophozoites through transmission electron microscopy (TEM).MethodologySeven-week-old female Balb/c mice were infected intravaginally with T. vaginalis trophozoites. On the third day of infection, methylene blue was introduced into the vaginal canal of the animals, which then received 68.1 J / cm2 of radiation for 35.6 sec. Control groups without infection and infected, treated with metronidazole were also included for comparison. Twenty-four hours after treatment the vaginal canal of the animals was scraped and the samples processed by the immunocytochemistry technique. After in vitro photodynamic treatment, T. vaginalis trophozoites were processed for TEM. Ultrathin sections were collected in 400-mesh copper grids, contrasted with 5% uranyl acetate and 3% lead citrate, in aqueous solutions for 20 and 5 min., respectively and observed in a Jeol JEM 230 transmission electron microscope.ResultsTEM showed morphological changes such as centripetal displacement of organelles, cannibalism, hydrogenosomal damage, intense cytoplasmic vacuolization, dilated endoplasmic reticulum cisternae and membrane discontinuity, in both resistant and sensitive strains, suggesting that trichomonicidal activity is mainly due to necrosis.PDT significantly reduced infection in animals treated with a single therapy session, compared to control groups, being statistically as efficient as MTZ.ConclusionsOur results demonstrated high trichomonicidal activity of PDT with morphological alterations compatible with necrosis. Therefore these results indicate that PDT represents not only an alternative therapy for refractory trichomoniasis, but also routinely for this important neglected parasitic disease.

The aim of this study was to evaluate the immunocytochemistry (ICC) to diagnose trichomoniasis, particularly asymptomatic infections. By culture serial dilutions, ICC was able to detect 1 trophozoite/mL, while the culture was positive up to 100 trophozoites/mL. The ICC in vivo detection capability was assessed in vaginal secretions of mice experimentally infected and in vaginal swabs from asymptomatic HIV-positive pregnant women compared with culture. All vaginal secretion samples from mice were positive according to both methods. Swabs from fifty-five asymptomatic women were positive in four (7.27%) of them by culture. Beyond these four, another ten (25.45%) women were positive by immunocytochemistry, proving their higher sensitivity (p=0.002), noticing 3.5 times more positives. ICC had better performance in both successive dilutions as in asymptomatic women, showing higher sensitivity and specificity. In this way, its facility of execution and cost-effectiveness support its practicality, as a routine procedure to diagnose trichomoniasis not only when the parasite load is lower but probably in all clinical scenarios.

The phytochemical study of hexane extract from leaves of Salacia crassifolia resulted in the isolation of 3β-palmitoxy-urs-12-ene, 3-oxofriedelane, 3β-hydroxyfriedelane, 3-oxo-28-hydroxyfriedelane, 3-oxo-29-hydroxyfriedelane, 28,29-dihydroxyfriedelan-3-one, 3,4-seco-friedelan-3-oic acid, 3β-hydroxy-olean-9(11):12-diene and the mixture of α-amirin and β-amirin. β-sitosterol, the polymer gutta-percha, squalene and eicosanoic acid were also isolated. The chemical structures of these constituents were established by IR, 1H and 13C NMR spectral data. Crude extracts and the triterpenes were tested against Entamoeba histolytica, Giardia lamblia and Trichomonas vaginalis and no activity was observed under the in vitro assay conditions. The hexane, chloroform, ethyl acetate and ethanol crude extracts, and the constituent 3,4-seco-friedelan-3-oic acid and 28,29-dihydroxyfriedelan-3-one showed in vitro antimicrobial activity against Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Streptococcus sanguinis and Candida albicans.

A amebíase é uma doença humana causada pelo protozoário Entamoeba histolytica, um parasito patogênico e invasivo que causa morte de cerca de 100 mil indivíduos por ano em todo mundo. Algumas espécies do gênero Maytenus têm sido utilizadas na medicina popular brasileira para tratamento da diarreia. Estas plantas são promissoras fontes de triterpenos bioativos de interesse medicinal. Doze extratos brutos e quatro triterpenos, previamente conhecidos, isolados a partir de galhos e raízes de Maytenus gonoclada, foram investigados in vitro em relação ao potencial amebicida em diferentes intervalos de tempo. Extratos brutos, lupeol, 3-30-di-hidroxi-lup-20(29)-eno e 3-oxo-11-hidroxilup-20 (29)-eno não exibiram atividade contra E. histolytica, contudo, o triterpeno tingenona foi ativo. A citotoxicidade da tingenona foi avaliada utilizando células mononucleares do sangue periférico humano. A IC50 encontrada foi de 8,9 µM. Neste caso a dose citotóxica está muito próxima da dose amebicida, inviabilizando o prosseguimento dos estudos com a substância in natura. A citotoxicidade da tingenona sobre células humanas poderia constituir restrição para seu emprego em tratamentos antiparasitários. Contudo a facilidade de obtenção e grandes quantidades, sinaliza sua utilização para modificações na estrutura da molécula visando obter derivados com potente atividade e menor toxicidade. Amebiasis is a human disease caused by the protozoan Entamoeba histolytica, a parasite that causes invasive pathogenic and death of about 100.000 individuals per year worldwide. Some species of the genus Maytenus have been used in Brazilian folk medicine to treat diarrhea. These plants are promising sources of bioactive triterpenes of medicinal interest. Twelve extracts and four triterpenes, previously known, isolated from the branches and roots of Maytenus gonoclada were investigated "in vitro" against potential amoebicide at different time intervals. Crude extracts, lupeol, 3-30-dihydroxy-lup-20 (29)-ene and 3-oxo-11-hydroxylup-20 (29)-ene exhibited no activity against E. histolytica, however, the triterpene tingenone was active. The cytotoxicity was evaluated using the tingenone mononuclear cells human peripheral blood. The IC50 of 8.9 M was found. In this case the cytotoxic dose is very close to the dose amoebicide, precluding further studies with substance "in nature". The cytotoxicity of tingenone on human cells could be restricted to its use in parasite treatments. However the ease of obtaining large amounts and, pointing their use for changes in the structure of the molecule to obtain derivatives with potent activity and lower toxicity.

By reacting maleic anhydride with amines, we synthesized the derivatives N -ethyl, N -(2-ethylamine), N -piperidinyl, N -phenyl, and N -phenylhydrazinyl maleamic acids. The purity of these products was initially verified by melting range and the presence of only one spot observed by thin layer chromatography. The chemical structures of the obtained N -alkyl maleamic acids were confirmed through infrared (IR) and hydrogen and carbon nuclear magnetic resonance ( 1 H and 13 C NMR) spectrometry. Due to the already proven pharmacological activity of maleimides, maleic anhydride and its N -alkyl maleamic acids were subjected to in vitro assays to observe antiviral (SA-11 rotavirus), antibacterial ( Escherichia coli, Staphylococcus aureus, and Bacillus cereus ), antifungal ( Colletotrichum musae, Fusarium solani f. sp. phaseoli, Fusarium solani f. sp. piperis Alb., and Penicillium sp.), and antiprotozoal ( Trichomonas vaginalis , Giardia lamblia , and Entamoeba histolytica ) effects. To study the anti-rotavirus properties, firstly the 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) method was used to establish the median cytotoxicity concentration (CC 50 ) of the compounds, using MA-104 cell line. Under the experimental conditions used, cytotoxic, anti-rotavirus, antibacterial, and antifungal properties were not observed for these compounds.

The influence of inflammation on the number of trophozoites and on the murine amoebic liver abscess area following infection with Entamoeba histolytica and E. dispar was evaluated. Immunohistochemistry and digital morphometry were used to identify and quantify the trophozoites, neutrophils, macrophages, and lesions. Positive correlation was observed between the number of trophozoites and inflammatory cells. A significant decrease in parasitism and inflammation in groups treated with dexamethasone was observed. The scarceness or absence of trophozoites in the treated groups suggest the importance of the inflammatory response in the production of amoebic hepatic abscesses in spite of the inherent virulence of the parasite being decisive in the establishment of the lesion.