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.

Photodynamic therapy as a new approach to Trichomonas vaginalis inactivation.

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% ±â€¯7.18 and 80.21% ±â€¯7.11 of the sensitive strain, respectively, and 31.17% ±â€¯4.23 and 91.13% ±â€¯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.

Hematological profile in natural progression of giardiasis: kinetics of experimental infection in gerbils.

INTRODUCTION: The clinical manifestations of giardiasis and its impact are harmful to children, and may cause deficits in their physical and cognitive development. The pathogenic mechanisms are usually unknown and the available reports can be controversial. METHODOLOGY: The present study aimed to know, for the first time, the evolution of the hematological profile of the gerbils, experimentally infected with Giardia lamblia, up to the infection's resolution. Hematological variables have been tested. RESULTS: White blood cells have not presented meaningful alterations during the course of the infection. A significant reduction in the number of red blood cells (p = 0.021), in the concentration of hemoglobin (p = 0.029) and in the value of the hematocrit (p = 0.016) has been observed, starting from the second week of infection, ratifying an anemia related to giardiasis. Reduction in the level of serum iron starting from the third week of infection, despite not being significant, could suggest the participation of iron in the anemia. However, the weight of the animals was kept and the hematimetric parameters started to return to the basic values after the parasitological cure without iron reposition. CONCLUSIONS: The outcomes found suggest the idea that not only malabsorption but also other mechanisms such as chronic inflammation may be implicated in iron deficiency anemia in giardiasis and may explain how asymptomatic patients may have anemia without malabsorption. In this context, considering the highlighting character of the anemia in our study, we believe that anemia should be investigated in children with giardiasis. And in the cases of anemia without a definite etiology, giardiasis should also be investigated.

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.

Genotyping and Descriptive Proteomics of a Potential Zoonotic Canine Strain of Giardia duodenalis, Infective to Mice.

The zoonotic potential of giardiasis, as proposed by WHO since the late 70's, has been largely confirmed in this century. The genetic assemblages A and B of Giardia duodenalis are frequently isolated from human and canine hosts. Most of the assemblage A strains are not infective to adult mice, which can limit the range of studies regarding to biology of G. duodenalis, including virulence factors and the interaction with host immune system. This study aimed to determine the infectivity in mice of an assemblage A Giardia duodenalis strain (BHFC1) isolated from a dog and to classify the strain in sub-assemblages (AI, AII, AIII) through the phylogenetic analysis of beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. In addition, the proteomic profile of soluble and insoluble protein fractions of trophozoites was analyzed by 2D-electrophoresis. Accordingly, trophozoites of BHFC1 were highly infective to Swiss mice. The phylogenetic analysis of tpi and gdh revealed that BHFC1 clustered to sub-assemblage AI. The proteomic map of soluble and insoluble protein fractions led to the identification of 187 proteins of G. duodenalis, 27 of them corresponding to hypothetical proteins. Considering both soluble and soluble fractions, the vast majority of the identified proteins (n = 82) were classified as metabolic proteins, mainly associated with carbon and lipid metabolism, including 53 proteins with catalytic activity. Some of the identified proteins correspond to antigens while others can be correlated with virulence. Besides a significant complementation to the proteomic data of G. duodenalis, these data provide an important source of information for future studies on various aspects of the biology of this parasite, such as virulence factors and host and pathogen interactions.

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.

Prevalence of intestinal parasitism and associated symptomatology among hemodialysis patients.

Intestinal parasites are an important cause of morbidity and mortality. Immunocompromised individuals may develop more severe forms of these infections. Taking into account the immunity impairment in patients suffering from chronic renal failure (CRF), we will determine the prevalence and associated symptoms of intestinal parasites in these patients. Controls without CRF were used for comparison. Stool samples were collected and processed for microscopic identification of parasites using the Formalin-ether concentration method. For Cryptosporidium diagnosis, the ELISA technique was used. One hundred and ten fecal samples from hemodialysis patients were analyzed, as well as 86 from a community group used as control group. A result of 51.6% of intestinal parasites was observed in hemodialysis patients and 61.6% in the control group. Cryptosporidium and Blastocystis were the most common infections in patients with CRF (26.4% and 24.5%, respectively). Blastocystis was the most common infection in the control group (41.9%), however no individual was found positive for Cryptosporidium. Among the CRF patients, 73.6% were symptomatic, 54.3% of these tested positive for at least one parasite, in contrast to 44.8% in asymptomatic patients (p = 0.38). The most common symptoms in this group were flatulence (36.4%), asthenia (30.0%) and weight loss (30.0%). In the control group, 91.9% were symptomatic, 60.8% of these tested positive for at least one parasite, in contrast to 71.4% in asymptomatic patients (p = 0.703). A significant difference between the two groups was observed with regard to symptoms, with bloating, postprandial fullness, and abdominal pain being more frequent in the control group than in the hemodialysis group (all p < 0.05). Comparing symptomatic with asymptomatic, there was no association in either group between symptoms or the prevalence of parasitic infection, nor with the type of parasite or with multiple parasitic infections. Patients with chronic renal failure are frequent targets for renal transplantation, which as well as the inherent immunological impairment of the disease itself, results in immunosuppression by medication. For this reason, carriers of intestinal parasites with pathogenic potential can develop serious clinical complications influencing the success of transplantation. This fact, coupled with the high prevalence of intestinal parasites and the dissociation between symptoms and infection in CRF patients, suggests that the stool test should be incorporated in routine propedeutics. Furthermore, preventive measures for the acquisition of parasites through the fecal-oral contamination route should be introduced.

Prevalence of ittestinal parasitism and associated symptomatology among hemodialysis patients / Prevalência de parasitismo intestinal e sintomatologia associada em pacientes hemodialíticos

Intestinal parasites are an important cause of morbidity and mortality. Immunocompromised individuals may develop more severe forms of these infections. Taking into account the immunity impairment in patients suffering from chronic renal failure (CRF), we will determine the prevalence and associated symptoms of intestinal parasites in these patients. Controls without CRF were used for comparison. Stool samples were collected and processed for microscopic identification of parasites using the Formalin-ether concentration method. For Cryptosporidium diagnosis, the ELISA technique was used. One hundred and ten fecal samples from hemodialysis patients were analyzed, as well as 86 from a community group used as control group. A result of 51.6% of intestinal parasites was observed in hemodialysis patients and 61.6% in the control group. Cryptosporidium and Blastocystis were the most common infections in patients with CRF (26.4% and 24.5%, respectively). Blastocystis was the most common infection in the control group (41.9%), however no individual was found positive for Cryptosporidium. Among the CRF patients, 73.6% were symptomatic, 54.3% of these tested positive for at least one parasite, in contrast to 44.8% in asymptomatic patients (p = 0.38). The most common symptoms in this group were flatulence (36.4%), asthenia (30.0%) and weight loss (30.0%). In the control group, 91.9% were symptomatic, 60.8% of these tested positive for at least one parasite, in contrast to 71.4% in asymptomatic patients (p = 0.703). A significant difference between the two groups was observed with regard to symptoms, with bloating, postprandial fullness, and abdominal pain being more frequent in the control group than in the hemodialysis group (all p < 0.05). Comparing symptomatic with asymptomatic, there was no association in either group between symptoms or the prevalence of parasitic infection, nor with the type of parasite or with multiple parasitic infections. Patients with chronic renal failure are frequent targets for renal transplantation, which as well as the inherent immunological impairment of the disease itself, results in immunosuppression by medication. For this reason, carriers of intestinal parasites with pathogenic potential can develop serious clinical complications influencing the success of transplantation. This fact, coupled with the high prevalence of intestinal parasites and the dissociation between symptoms and infection in CRF patients, suggests that the stool test should be incorporated in routine propedeutics. Furthermore, preventive measures for the acquisition of parasites through the fecal-oral contamination route should be introduced.

Doenças parasitárias infectam grande número de indivíduos em todo o mundo. Manifestações clínicas mais severas podem se apresentar em pacientes imunocomprometidos. Considerando o importante comprometimento imunológico observado em pacientes com insuficiência renal crônica (IRC), foi determinada a prevalência e sintomas associados a parasitoses intestinais nesses pacientes em comparação a controles saudáveis. Foram coletadas amostras fecais de cada participante e processadas para identificação microscópica dos parasitas pelo método de concentração por formol-éter. Foi utilizada a técnica de ELISA para identificar coproantígenos de Cryptosporidium. Foram analisadas 110 amostras fecais de pacientes em hemodiálise e 86 de um grupo controle comunitário. Cryptosporidium e Blastocystis foram as infecções mais freqüentes nos pacientes em hemodiálise (26,4% e 24,5%, respectivamente). Blastocystis foi a infecção mais freqüente no grupo controle (41,9%), entretanto nenhum indivíduo positivo para Cryptosporidium foi identificado. Considerando os pacientes com IRC, 73,6% eram sintomáticos, sendo 54,3% positivos para algum parasita, contra 44,8% nos assintomáticos (p = 0,38). Os sintomas mais frequentes neste grupo foram flatulência (36,4%), adinamia (30,0%) e perda de peso (30,0%). No grupo controle, 91,9% eram sintomáticos, sendo 60,8% positivos para algum parasita, contra 71,4% nos assintomáticos (p = 0,703). Em relação aos sintomas, houve diferença significativa entre os dois grupos, sendo que flatulência, plenitude pós-prandial, e dor abdominal foram mais freqüentes no grupo controle que nos pacientes em hemodiálise (todos p < 0,05). Comparando-se sintomáticos com assintomáticos, não houve associação entre a sintomatologia e a prevalência de parasitose, nem com o tipo de parasita, e nem com o poliparasitismo, nos dois grupos. Considerando que pacientes com IRC são frequentes alvos de transplante renal, resultando em imunossupressão por medicamentos, que é somada à deficiência imunológica inerente à própria doença. Os portadores de parasitas intestinais com potencial patogênico podem desenvolver sérias complicações clínicas que influenciam o sucesso do transplante. Este fato, aliado a alta prevalência de parasitas intestinais e dissociação entre os sintomas e infecção nesses pacientes, sugerem a incorporação do exame de fezes na propedêutica de rotina dos mesmos, juntamente com medidas preventivas para a aquisição de parasitas com rota de contaminação fecal-oral.

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.