Photodynamic therapy effect on the ultrastructure of Trichomonas vaginalis trophozoites and their effectiveness in experimentally infected animals

Background Trichomonas 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). Methodology Seven-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. Results TEM 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. Conclusions Our 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.


47
Author summary 48 Trichomoniasis is the most prevalent non-viral Sexually Transmitted Infection (STI) 49 worldwide. Although not causing death, it is associated with other potentially fatal STIs 50 such as HIV. The success of their control is impaired by promiscuity, directing the 51 efforts to restrain the transmission of disease to treatment. MTZ is the drug of choice.

52
Despite its effectiveness, cases of resistance are frequent. It adds up to the resistance, 53 the side effects that can interrupt the treatment culminating in more resistance and 54 transmission of the disease. In this context, therapeutic alternatives may represent a 55 solution not only to solve the increasing resistance, but also for trichomoniasis control. 56 We evaluated the efficacy of photodynamic therapy (PDT) as trichomonicide in 57 experimentally infected animals and its action on the parasite was evaluated by     The pre-estrogenization procedure followed the protocol standardized by [13]. 155 Then, on day 0 and day 1 all animals were infected intravaginally with 1 x 10 7 T. 156 vaginalis trophozoites, strain JT (sensitive to MTZ). In a previous study [15], this strain 157 was shown to be significantly more resistant to PDT than the CDC 085 strain (resistant 158 to MTZ), so we employed it for the in vivo assays. processed for the immunocytochemistry technique, as previously described [14]. For 167 each experimental group, the trophozoites were quantified in ten random microscopic 9 9 168 fields the intensity of the infection was considered intense (above 12 trophozoites per 169 field), moderate (5 to 11 trophozoites per field) and low (1 to 4 trophozoites per field).

185
Microscopic analysis showed that the parasites were blue MB-stained, indicating that 186 the 30 minutes exposure was sufficient for the photosensitizer to react with the 187 protozoan compartments (Fig 1).   (Fig 2A and 2B).

205
In the presence of only MB, the trophozoites exhibited several changes in their 206 ultrastructure. Some parasites displayed centripetal relocation of the organelles to the 207 inner (more anaerobic) portions of the cytoplasm (Fig 2C), accumulation of numerous 208 glycogen granule aggregates, dilated hydrogenosomal peripheral vesicles, moderate 209 vacuolation and slightly reduced cytoplasmic electron density (Fig 2D).

210
In the PDT groups, process of phagocytosis of hydrogenosomes from the 211 extracellular medium (Fig 2E) associated with a cytoplasmic exclusion zone 212 characterizing the cytoskeleton participation in the parasite cannibalism. In addition, the 213 presence of large and numerous autophagosomes with high cytoplasm-derived content, 214 highly extracted cytoplasmic matrix, small expansion in the Golgi cis region with many 11 11 215 budding vesicles, low hydrogenosome numbers, and dilated endoplasmic reticulum 216 cisternae are prominent (Fig 2F).

217
Many trophozoites presented membrane-containing autophagosomes located at 218 the periphery of the cells and fusing with the plasma membrane (Fig 2G). The advanced 219 PDT effects included intense cellular damage, with structural disorganization and 220 intense vacuolization, membrane discontinuity, extensive cytoplasmic extraction, and 221 large debris assemblies (Fig 2J).  The resistant trophozoites CTR and LC groups, presented typical hydrogenosomes, but 239 with reduced dimensions as compared to the sensitive strain, normal cytoskeleton and 240 intact organelles including nuclei, which were eventually multiple (Fig 3 A and 3B).

243
The early effects of PDT are marked by granular hydrogenosome matrix and 244 high vacuolization, followed by membrane discontinuity leading to cytoplasmic 245 extraction ( Fig 3D). We also observed the formation of large myelin-like Figs 246 containing cytoplasmic material with glycogen granules (Fig 3E).  whereas the other half had a moderate one (Fig 4). In addition, 25% of the animals in 277 the MB group had intense, 50% moderate and 25% mild type infections. The animals in 278 the PDT group showed 37.5% mild infection and 62.5% were absent. These data 279 demonstrate that PDT significantly (p < 0.05) reduced infection in animals treated with 280 a single treatment session, being statistically as efficient as the MTZ group (Fig 4).

304
In previous work, our group confirmed the efficacy and safety of photodynamic 305 treatment with MB on susceptible and resistant strains of T. vaginalis [11]. Among the 306 photosensitizers currently used, MB, a phenothiazine compound, has proven PDT-307 associated activity [18,19]. In this work, we used LED technology that, although less 308 common in PDT, proved to be a good alternative in the treatment of cutaneous and 309 mucosal wounds [20].   In addition, parasites of the sensitive strain exposed to PDT presented intense