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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.

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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 HO (~ 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.

2 Background 3 Trichomonas vaginalis is an amitochondrial parasitic that causes human trichomoniasis, 4 the most common non-viral sexually transmitted infection in the world. The therapy of 5 choice is metronidazole (MTZ). Despite MTZ effectiveness, resistant cases are 6 becoming more frequent. Another point to emphasize are the side effects that may result 7 in treatment discontinuation, leading to further spread of infection and emergence of 8 resistant strains. This scenario reveals the need to develop new therapeutic options. 9 Photodynamic therapy (PDT) is an experimental treatment that involves the activation 10 of photosensitive substances and the generation of cytotoxic oxygen species and free 11 radicals to promote the selective destruction of target tissues. A previous study, from 12 our group, identified an excellent in vitro PDT activity using methylene blue and light 13 emitting diode against MTZ sensitive and resistant strains of T. vaginalis. The aim of 14 this study was to evaluate the efficacy of PDT in vivo and clarify its high 15 trichomonicidal potential by evaluating its action upon T. vaginalis trophozoites through 16 transmission electron microscopy (TEM). 17 18 Methodology 19 Seven-week-old female Balb/c mice were infected intravaginally with T. vaginalis 20 trophozoites. On the third day of infection, methylene blue was introduced into the 21 vaginal canal of the animals, which then received 68.1 J / cm 2 of radiation for 35.6 sec. 22 Control groups without infection and infected, treated with metronidazole were also 23 included for comparison. Twenty-four hours after treatment the vaginal canal of the 24 animals was scraped and the samples processed by the immunocytochemistry technique.3 3 25 After in vitro photodynamic treatment, T. vaginalis trophozoites were processed for 26 TEM. Ultrathin sections were collected in 400-mesh copper grids, contrasted with 5% 27 uranyl acetate and 3% lead citrate, in aqueous solutions for 20 and 5 min., respectively 28 and observed in a Jeol JEM 230 transmission electron microscope. 29 30 Results 31 TEM showed morphological changes such as centripetal displacement of organelles, 32 cannibalism, hydrogenosomal damage, intense cytoplasmic vacuolization, dilated 33 endoplasmic reticulum cisternae and membrane discontinuity, in both resistant and 34 sensitive strains, suggesting that trichomonicidal activity is mainly due to necrosis. 35 PDT significantly reduced infection in animals treated with a single therapy session, 36 compared to control groups, being statistically as efficient as MTZ. 37 38 Conclusions 39 Our results demonstrated high trichomonicidal activity of PDT with morphological 40 alterations compatible with necrosis. Therefore these results indicate that PDT 41 represents not only an alternative therapy for refractory trichomoniasis, but also 42 routinely for this important neglected parasitic disease.43 44