Transmission electron microscopy revealing the mechanism of action of photodynamic therapy on Trichomonas vaginalis.

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.

Immunocytochemistry Improving the Diagnosis of Trichomonas vaginalis Infections.

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.

High prevalence of enteroparasitosis in urban slums of Belo Horizonte-Brazil. Presence of enteroparasites as a risk factor in the family group.

The present study evaluates the prevalence of enteroparasitosis in the urban slums of Belo Horizonte, Brazil and the risk of transmitting enteroparasites to the family members of infected individuals. Stool samples were collected and examined at clinical laboratories near each slum. Individuals were identified and classified as positive for parasitosis (IP(+)), and individuals with negative stool tests were classified as negative for parasitosis (IP(-)) and enrolled as control patients. We collected samples from 594 patients, of which 20·2% and 79·8% were classified as IP(+) and IP(-), respectively. In addition, 744 family members (FIPs) effectively participated in the study by providing fecal samples. In total, 1338 participants were evaluated. Of these, 34·6% were tested positive for parasitosis. Blastocystis was the most prevalent parasite, infecting 22·4% of individuals. Among FIPs, the overall prevalence was 46·1%. Of these, 50·6% and 44·7% were classified as FIPs(+) and FIPs(-), respectively. These results showed that IP(+) did not impact the prevalence of infection within the studied communities, not constituting index cases of specific risk behaviors, suggesting that, in fact, these communities are exposed to similar oral-fecal routes of contamination.

Effects of metronidazole analogues on Giardia lamblia: experimental infection and cell organization.

The chemotherapeutic agents used for the treatment of giardiasis are often associated with adverse side effects and are refractory cases, due to the development of resistant parasites. Therefore the search for new drugs is required. We have previously reported the giardicidal effects of metronidazole (MTZ) and its analogues (MTZ-Ms, MTZ-Br, MTZ-N(3), and MTZ-I) on the trophozoites of Giardia lamblia. Now we evaluated the activity of some giardicidal MTZ analogues in experimental infections in gerbils and its effects on the morphology and ultrastructural organization of Giardia. The giardicidal activity in experimental infections showed ED(50) values significantly lower for MTZ-I and MTZ-Br when compared to MTZ. Transmission electron microscopy was employed to approach the mechanism(s) of action of MTZ analogues upon the protozoan. MTZ analogues were more active than MTZ in changing significantly the morphology and ultrastructure of the parasite. The analogues affected parasite cell vesicle trafficking, autophagy, and triggered differentiation into cysts. These results coupled with the excellent giardicidal activity and lower toxicity demonstrate that these nitroimidazole derivates may be important therapeutic alternatives for combating giardiasis. In addition, our results suggest a therapeutic advantage in obtaining synthetic metronidazole analogues for screening of activities against other infectious agents.

The old and new therapeutic approaches to the treatment of giardiasis: where are we?

Giardia lamblia is the causative agent of giardiasis, one of the most common parasitic infections of the human intestinal tract. This disease most frequently affects children causing abdominal pain, nausea, vomiting, acute or chronic diarrhea, and malabsorption syndrome. In undernourished children, giardiasis is a determining factor in retarded physical and mental development. Antigiardial chemotherapy focuses on the trophozoite stage. Metronidazole and other nitroimidazoles have been used for decades as the therapy of choice against giardiasis. In recent years many other drugs have been proposed for the treatment of giardiasis. Therefore, several synthetic and natural substances have been tested in search of new giardicidal compounds. This study is a review of drugs used in in vitro and in vivo tests, and also drugs tested in clinical trials (nonrandomized and randomized).

Effect of metronidazole analogues on Giardia lamblia cultures.

We comparatively evaluate the effect of metronidazole (MTZ) and its five analogues on trophozoites of Giardia lamblia axenically growing. The compounds MTZ-Ms, MTZ-I, MTZ-Br, MTZ-N(3), and MTZ-NH(3)Cl were obtained by molecular modification of the side chain of MTZ. Four of them presented higher giardicidal activity when compared with MTZ. Among them, MTZ-Br and MTZ-I were the most active, without cytotoxic effects against mitogen-activated human peripheral blood mononuclear cells (PBMC). The alteration of MTZ side chain constitutes a fruitful field to develop new drugs for the treatment not only of giardiasis but also of other diseases and signalize that metronidazole analogues are promising candidates as giardicidal and should be further evaluated.

A simple colourimetric method to determine anti-giardial activity of drugs.

A new colourimetric method to be used for the screening of compounds with giardicidal activity was developed. After the end of drug incubation, the remaining viable cells were fixed with methanol and stained by methylene blue. The inclusion of methylene blue by Guardia lamblia trophozoites was measured spectrophotometrically to determine the anti-giardial activity of metronidazole. The 50% inhibitory concentration (IC(50)) was 1.96 +/- 0.13 microM. The reliability of this method was assessed by comparison with the IC(50) obtained using a haemocytometer to get the number of viable cells (1.82 +/- 0.43 microM). This method provides a feasible, cheap and reliable method to determine the anti-giardial activity of drugs.