Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells

BACKGROUND Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. OBJECTIVES In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. METHODS We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. FINDINGS It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. MAIN CONCLUSIONS Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis.

Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.

Modulatory effect of iron chelators on adenosine deaminase activity and gene expression in Trichomonas vaginalis

Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalisisolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival.

Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs

A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.

[In Vitro anti-Trichomonas activity of Freula assafoetida and garlic extracts]

Trichomoniasis is a disease caused by Trichomonas vaginalis and is the most common sexually transmitted disease after viral sexually transmitted ones. Trichomoniasis is usually treated with oral metronidazole and both patient and her partner should be treated. Considering the probable teratogenic effect of metronidazole and parasite drug resistance, it is necessary to find an alternative medication for treatment of trichomoniasis. In this study in vitro effect of garlic and Freula assafoetida extracts on Trichomonas vaginalis were evaluated. This In Vitro study was done in Yasuj Faculty of Medicine, Yasuj, Iran. Trichomonas vaginalis was cultured in TYI-S-33 medium. Effect of garlic and Freula assafoetida extracts in specified times and concentrations on Trichomonas vaginalis were assessed. Garlic extract was used in 0.1, 0,05 and 0.025 mg/ml while Freula assafoetida extract was used in 2, 1 and 0.5 mg/ml. The inhibitory effect of extract on Trichomonas was assessed by counting the alive parasites 1, 2 and 24 hours after exposure with extracts. Findings of this study showed that hydroalcoholic extract of Freula assafoetida at concentration of 0.5, 1 and 2 mg/ml killed 90% of the parasites in first hour of exposure and garlic extract at concentration of 0.1 mg/ml killed 95% of parasites after 2 hours. Moreover garlic extract killed 90% of parasites at concentration of 0.05, 0.025 and 0.0125 mg/ml after 24 hours of exposure even at low concentration. This study indicated that garlic and freula assafoetida have significat effect on Trichomonas vaginalis, therefore detecting the effective substances of these potent anti-parasitic herbs is recommended

Saponinas esteroidales de la planta Agave brittoniana (Agavaceae) con actividad contra el parásito Trichomona vaginalis

Steroidal saponins from the plant Agave brittoniana with activity against the parasite Trichomona vaginalis. The genus Agave (Agavaceae), includes more than 300 species; around 16 of them show an homogeneous distribution throughout Cuba. Agave brittoniana (ssp. brachypus), is an endemic subspecies that grows in the central region of the country and its leaves are traditionally used in the treatment of parasitic diseases. The parasite Trichomonas vaginalis causes the disease known as trichomoniasis, that infects the genital tract. To test in vitro the plant against Trichomona vaginalis, the dried and powdered leaves were extracted three times with ethanol-water (7 : 3) by maceration at room temperature. The solvent was removed under reduced pressure and the extract was suspended in distilled water, defatted with n-hexane, and extracted with water-saturated n-butanol. After solvent removal, a portion of the n-butanol extract was hydrolyzed. After extraction with ethyl acetate the hydrolysis products were compared with authentic sapogenins samples using thin layer chromatography (TLC). Most of the sapogenins (yuccagenin and diosgenin) were isolated and their structures were confirmed. using nuclear magnetic resonance (NMR) experiments. The n-butanol extract was subjected to a separation process through column chromatography to obtain five fractions. After multiple separation processes by reversed phase high performance liquid chromatography (HPLC), the most active one produced one refined fraction that contained two saponins with the same aglycone (diosgenin) and one yuccagenin based saponin. Best results of the activity were obtained with the yuccagenin derived glycoside. Rev. Biol. Trop. 56 (4): 16451652. Epub 2008 December 12.

El género Agave, familia Agavaceae, tiene más de 300 especies, con aproximadamente 16 distribuidas en toda Cuba. Una de ellas, el Agave brittoniana Trel. (ssp. brachypus), es una subespecie endémica y sus hojas son tradicionalmente utilizadas en el tratamiento de enfermedades parasitarias. Se realizaron estudios "in vitro" de la actividad de productos de esta planta frente a Trichomona vaginalis. Las hojas secas y pulverizadas fueron extraídas tres veces con una mezcla de etanol-agua (7: 3) mediante maceración a temperatura ambiente. El disolvente fue evaporado a presión reducida y el extracto fue suspendido en agua destilada, desengrasado con n-hexano, y extraído con n-butanol saturado con agua. Luego de una extracción con acetato de etilo, los productos de la hidrólisis fueron comparados con patrones de sapogeninas mediante la cromatografía de capa fina (CCD). Aislamos las sapogeninas mayoritarias (yuccagenina y diosgenina) y confirmamos sus estructuras utilizando técnicas de resonancia magnética nuclear. Por otra parte, el extracto n-butanólico fue sometido a un proceso de separación biodirigido mediante cromatografía de columna, obteniéndose cinco fracciones. Después de múltiples separaciones, la más activa rindió una fracción purificada con dos sapogeninas con el mismo aglicón (diosgenina) y un glicósido de yucagenina. Los mejores resultados de esta actividad fueron obtenidos con el glicósido derivado de la yucagenina.

In vitro sensitivity of Trichomonas vaginalis to DNA topoisomerase II inhibitors.

Vaginal trichomoniasis is a highly prevalent sexually transmitted disease caused by a microaerophilic protozoan Trichomonas vaginalis. The disease is one of the most common sexually transmitted disease and can augment the predisposition of individuals to human immunodeficiency virus (HIV) infection. Although the disease can be treated with metronidazole and related 5-nitroimidazole, cases of trichomonal vaginitis which are refractory to standard treatment seems to be increasing. Clearly, new antitrichomonad agents are needed and DNA topoisomerase II may acts as a new target for antitrichomonad agents. In this study, in vitro sensitivity of T. vaginalis to DNA topoisomerase II was investigated. Axenic culture of local strain of T. vaginalis was performed. Both eukaryotic and prokaryotic DNA topoisomerase II inhibitors such as ellipticine, amsacrine and fluoroquinolones were tested for effectiveness against T. vaginalis in vitro compared to metronidazole. T. vaginalis was sensitive to metronidazole under aerobic conditions. Minimal inhibitory concentrations (MICs) of eukaryotic DNA topoisomerase II inhibitors, ellipticine and amsacrine, were 6.4 mM and 64 mM, respectively. The MICs of prokaryotic DNA topoisomerase II or DNA gyrase inhibitors; ciprofloxacin, ofloxacin and norfloxacin were 64, 960 and 1,280 mM, respectively. Based on the results, among DNA topoisomerase II inhibitors ellipticine was the most effective drug against T. vaginalis in vitro whereas fluoroquinolones did not show high antitrichomonad activity.

Incidence of Trichomonas vaginalis among women having vaginal discharge, in Manisa, Turkey

In the present study, vaginal discharge samples obtained from 207 patients with vaginal discharge complaints were examined by utilizing wet mount preparations, Giemsa staining method and CPLM cultivation method. In 27 of them [13.1%], T. vaginalis was detected by both saline wet mount preparation and Giemsa staining method. But as a result of the cultivation in CPLM media of the vaginal discharge materials collected from infected samples, reproduction was observed in only 21 of them [10.2%]. A treatment of secnidazole by 2 g unique dose was applied to infected patients and their spouses and a complete recovery was followed up both microscopically and clinically

Pruebas fisico-quimicas y biologicas de laboratorio con preparaciones homeopaticas de Yumel / Physical chemical and biological tests of laboratory with homeopathic preparations of Yumel

Los autores desarrollaron un trabajo de investigacion en el laboratorio con preparaciones de Yumel (Guatteria gaumeri Greenman) para demostrar la accion de la dinamizacion con diferentes diluciones decimales empleando para tal analisis el espectrometro UV-Visivel y Fluorometria. Fueron realizadas tambien pruebas biologicas in vitro frente a cultivos de Trypanosoma cruzi y Trychomona vaginalis con resultados alentadores en relacion sl Trypanosoma estando en evaluacion la actividad frente a la Trychomona

Hemolytic activity of Trichomonas vaginalis and Titrichomonas foetus

The hemolytic activity of live isolates and clones of Trichomonas vaginalis and Tritrichomonas foetus was investigated. The isolates were tested against human erythrocytes. No hemolytic activity was detected by the isolates of T. foetus. Whereas the isolates of T. vaginalis lysed erythrocytes from all human blood groups. No hemolysin released by the parasites could be detected. Our preliminary results suggest than hemolysis depend on the susceptibility of red cell membranes to destabilization and the intervention of cell surface receptors as a mechanism of the hemolytic activity. The mechanism could be subject to strain-species-genera specific variation of trichomonads. The hemolytic activity of T. vaginalis is not due to a hemolysin or to a product of its metabolism. Pretreatment of trichomonads with concanavalin A reduced levels of hemolysis by 40 por cento.

Effect of estrogens on the adhesion of trichomonas vaginalis to epithelial cells in vitro

The feeding behavior of six triatomid species toward latex condoms filled with blood at 26 ñ 1 and 36 ñ 1 grade C observed for 4 h. The species studied were Triatoma infestans, Triatoma brasiliensis, Tritoma vitiiticeps, Triatoma pseudomaculata, Rhodnius and Panstrongylus, and 34 to 348 insects were studied in each group (average, 190). All the life stages of each species were used. Although most insects (80%, average for 6 species) preferred the warmer blood, 54% of the individuals of all species fed on blood kept at 26 grade C. For Triatoma pseudomaculata, a higher proportion of insects fed on blood at 26 grade C (92%) than on blood at 36 grade C (65%). These results suggest that bait containing insecticides which are effective through the digest tract may be useful in the control of these vectors even at room temperature

Murine pathogenicity of Trichomonas vaginalis & its susceptibility to metronidazole.

Eight isolates of T. vaginalis obtained from clinical specimens were tested for their pathogenicity in the murine model using the subcutaneous route of inoculation. In general, all isolates produced localised purulent abscesses in the mice. On comparison with a reference strain, all but two isolates were found to be significantly more pathogenic for mice than the reference strain, while two were comparable in their pathogenicity. All eight isolates were sensitive in vivo to metronidazole. In six of the eight isolates, metronidazole (30 mg/kg body wt) administered for four days to subcutaneously infected mice effected a 100 per cent cure. The ED50 values ranged from 3.4 to 23.94 mg/kg body weight. None of the isolates could be termed as resistant.

Rapid eradication of trichomonads by single-dosed nimorazole.

Previous in vitro studies demonstrated the rapidity of trichomonacidal action of nimorazole (Naxogin 500) which was twice that of metronidazole and many times that of tinidazole. Since rapid eradication of parasites can lead to a significant decrease in transmission rate, and hence, a lower prevalence of this sexually transmitted disease, a pilot study was designed to investigate the in vivo speed of action of nimorazole. Twenty females with positive wet smears for trichomonas vaginalis were treated with a single 2 gram-dose of nimorazole orally. Without any antiseptics, specimens of vaginal discharge were collected at 0 hour (before treatment), 3, 24 and 72 hours for parasite count and culture. After a single treatment with 2 g of nimorazole the cure rate was 65 per cent at 3 hours and 100 per cent at all points thereafter. The result of this pilot study supports previous in vitro findings that nimorazole rapidly eradicates vaginal parasites.

Tratamiento de la vaginitis causada por Trichomoas vaginalis, Candida albicans, Gardnerella vagilalis, solas o combinadas con la asociación de tinidazol/tioconazol / Treatment of vaginitis caused by Trichomonas vaginalis, Candida albicans, Gardnerella vaginallis, individually or combined, with a Tinidazole/Thioconazole association

Con el objeto de comprobar la eficacia y la tolerancia de la combinación de tinidazol con tioconazol, ambos fármacos derivdos del imidazol, en dosis de 150 y 100 mg respectivamente, para el tratamiento de las vaginitis causadas por Trichomonas vaginalis, Candida albicans y Gardnerella vaginalis ya sea como germen patógeno único o asocaidos entre sí, fueron estudiadas de manera avierta no comparativa, 27 pacientes adultas con leucorea, con diagnóstico clínico y bacteriológico de vaginitis por Trichomonas, Candida o Gardnella solas o en combinación. Descartadas las enfermas embarazadas, lactantes, alérgicas a los derivados imidazólicos y con otras enfermedades venéreas, entre otros excluyentes se revisaron clínica y bacteriológicamente al principio del estudio y una segunda vez en el 6o. y 15o. día después de haber aplicado por vía vaginal una tableta de tinidazol cada 12 hrs., durante tres días consecutivos. Se consideraron clínicamente duradas el 74% de las enfermas y mejoradas 26% sin falla alguna; y desde el punto de vista bacteriológico se consiguió 93% de curación en candidiasis, 100% en tricomoniasis y 50% de curación cuando el fermen fue Gardnerella, aún cuando se considera que el número de casos de este agente no permite una valoración correcta. Por otra parte, la tolerancia puede considerarse como una buena dada a la casi total ausencia de síntomas y signos adversos durante la aplicación del medicamento

Estudo aberto de Tioconazol/Tinidazol creme vaginal no tratamento de infecçöes vaginais causadas por Trichomonas vaginalis, Candida albicans e/ou Gardnerella vaginalis / Open non comparative study of Tioconazole/Tinidazole vaginal cream in the treatment of vaginal infections due to Trichomonas vaginalis, Candida albicans and/or Gardnerella vaginalis

No presente estudo, os autores analisaram a eficácia e tolerância da associaçäo tioconazol/tinidazol no tratamento tópico de 60 pacientes com infecçöes vaginais causadas por Trichomonas, Candida e Gardnerella. Durante os primeiros dez dias de tratamento, as pacientes interromperam a atividade sexual. Os resultados apresentados n 7§ dia após o início do tratamento mostraram uma intensa melhora de sinais e sintomas de vaginite, bem como cura microbiológica em 90% de pacientes com Trichomonas, 21% de pacientes com Candida e 38,1% de pacientes com Gardinerella. Aquelas recurrências foram atribuídas ao reinício da atividade sexual. Quanto a tolerabilidade, näo foram relatados eventos adversos relevantes

Effect of metronidazole on the cell surface charge of Trichomonas vaginalis and Tritrichomonas foetus

1. The effect of metronidazole, a drug used in the treatment of trichomoniasis, on the surface chage of both Trichomonas vaginalis and Tritrichomonas foetus was analyzed by cell electrophoresis. 2. Incubation of the parasites under anaerobic conditions for 2 or 120 min in the presence of 2.0 microng/ml metronidazole, a concentration which inhibits cell growth by about 50%, led to a marked decrease in the net surface charge of the parasites. 3. The metronidazole analogue, 1-hydroxyethyl-2-methyl-1-4-inhibited protozoan growth but had no effect on met surface charge. 4. These observations indicate that, in addition to its effect on intracellular structure, cuch as hydrogenosomes, metronidazole alters the surface of trichomonads

A comparison of various methods for the determination of viability of parasitic flagellates.

To find out a simple, rapid, accurate, and reliable viability assay for in vitro anti-protozoa drug testing, a comparison among the various methods for the determination of flagellate viability was made. Micro-colony forming efficiency up to 90% for three different flagellates was obtained by the agar plate method. Macro-colony forming efficiency obtained by the culture tube method was generally lower (20-45%). Viability determined by trypan blue exclusion correlated closely with viability determined by micro-colony count (r = 0.94). The growth of the remaining parasites survived from serum treatment was not inhibited while that from metronidazole pretreatment was inhibited. The standard regrowth assay based on the 48-hr cultivation could be applied only for those survival not inhibited. The inhibitory effect of metronidazole on parasites was further testified by the longer generation time for metronidazole-pretreated flagellates in comparison with that for control (p less than 0.001). If the inhibitory effect of a drug is involved in the mechanisms against parasites, only the trypan blue exclusion and the micro-colony count can give an accurate and reliable viability determination. Moreover, the simple and rapid diagnosis provided by trypan blue exclusion is especially useful for the in vitro anti-protozoa drug susceptibility test.

Evaluación del tratamiento local con Tioconazol-Tinidazol de las vaginitis producidas por Gardnerella vaginalis, Cándida albicans, Trichomonas vaginalis y sus asociaciones / Evaluation of local treatment with Thioconazole-Tinidazole in vaginitis produced by Gardnerella vaginalis, Candida albicans, Trichomonas vaginalis and their associations

Se presentan los resultados del tratamiento local con tioconazol-tinidazol, de las vaginitis producidas por Gardnerella vaginalis, Cándida albicans, Trichomonas vaginalis y sus asociaciones, en 73 pacientes con leucorrea, estudiadas clínica y bacteriológicamente, desde su ingreso a un protocolo especial y en los controles establecidos a los 7 y 30 días. Fueron sometidas a tratamiento, 24 pacientes con vaginitis por Cándida albicans,. 23 por Gardnerella vaginalis y 14 por Trichomonas vaginalis. En 12 casos se trataron asociaciones de estos agentes. La respuesta al tratamiento fue muy buena en los casos de candidiasis vulvovaginal, con un 95,8% de éxito. Un 78,3% de los casos de vaginitis por Gardnerella vaginalis y un 64,3% de las tricomoniasis vaginales, curaron clínica y bacteriológicamente. Los resultados obtenidos en las asociaciones microbianas fueron variables según los gérmenes comprometidos. El estudio permitió comprobar además, que sólo el 60% de los 230 casos de leucorreas analizados, tenían una vaginitis, el 40% restante presentó otras causas de leucorrea. En el total de las 138 vaginitis estudiadas, Gardnerella vaginalis se aisló en el 35,5%, Cándida albicans en el 31,9% y Trichomonas vaginalis en el 18,1%. Asociaciones de estos agentes se pesquisaron en un 14,5%. Finalmente, se destaca la importancia reactualizada del síntoma leucorrea como manifestación clínica de patologías más serias que las vaginitis, entre las cuales se describen las cervicitis, displasias y neoplasias tanto vaginales como cervicales

Avaliaçäo clínica de uma associaçäo ácido salicílico/ ácido bórico/alúmen amoniacal como coadjuvante no tratamento de vulvovaginites por Trichomonas vaginalis e Candida albicans / Clinical evaluation of a salicylic acid/boric acid/ammonium alum combination as coadjuvants in the treatment of Trichomonas vaginalis and Candida albicans vulvovaginitis

Estudou-se a eficácia e a tolerabilidade de uma associaçäo ácido salicílico/ácido bórico/alúmen amoniacal, em comparaçäo com vinagre, ambos em soluçäo para banhos de assento, como coajuvantes para acelerar a remissäo da sintomatologia de vulvovaginites causadas por Trichomonas vaginalis e/ou Candida albicans. A amostra estudada foi composta por 30 pacientes, com idades entre 24 e 41 anos, com duraçäo do quadro clínico entre 4 e 14 dias, as quais foram aleatoriamente distribuídas entre os dois grupos de tratamento, com 15 pacientes cada. Os banhos de assento, em ambos os grupos, foram feitos por 10 dias. Como tratamento de base para os microorganismos causadores das vulvovaginites, todas as pacientes, independentemente de grupo comparativo, usaram uma associaçäo nimorazol/clotrimazol/lisozima, na forma de creme vaginal, por seis noites consecutivas. A associaçäo ácido salicílico/ácido bórico/alúmen amoniacal foi superior ao vinagre, encurtando em vários dias a remissäo da sintomatologia das vulvovaginites (prurido, ardor, dor à micçäo, corrimento). A associaçäo nimorazol/clotrimazol/lisozima teve uma eficácia de 100% contra T. vaginalis e C. albicans. Nenhum destes dois tratamentos causou reaçöes adversas às pacientes

Tratamento das vulvovaginites por tricomoníase e candidíase com associaçäo nimorazol/clotrimazol/lisozima na forma de creme vaginal / Treatment of vulvovaginal trichomoniasis and candidiasis with a combination of nimorazole, clotrimazole, and lysozyme in the form of vaginal cream

Estudou-se a atividade da associaçäo nimorazol/clotrimazol/lisozima na forma de creme vaginal, por seis noites consecutivas, em 36 pacientes portadoras de vulvovaginites causadas por Trichomonas vaginalis e/ou Candida albicans. Tratavam-se concomitantemente os parceiros de pacientes portadoras de tricomoníase com associaçäo nimorazol/lisozima em dose única por via oral. A eficácia foi em 100% nas vulvovaginites por tricomoníase e 95,8% naquelas por candidíase, com rápida melhora dos sintomas e sinais. Houve também desaparecimento de bactérias associadas, Gram-positivas e negativas, em grande número de casos. Näo houve relato de efeitos adversos no uso das medicaçöes acima citadas