Detection of Trichomonas vaginalis by Allplex™ STI Essential Assay (Seegene) in clinical samples from the Brazilian public health system users.

The failures in Trichomonas vaginalis (TV) infection diagnosis leave more than half of cases unidentified. In this report, urine and vaginal discharge samples were analyzed by wet mount, culture examination, and real-time PCR by Allplex™ (Seegene®) kit, in a population assisted by the Brazilian Public Health System. From 747 samples, 2.81% were positive for TV in wet mount and culture, and 3.88% by Allplex™. Samples kept at - 80 ºC for 22 months did not impair the PCR technique. The sensitivity for wet mount, culture, and Allplex™ was 72, 100, and 100%, respectively. Allplex™ technique showed highest detection of TV.

Therapeutic potential of antimicrobial peptides against pathogenic protozoa.

Protozoal infections cause significant morbidity and mortality in humans and animals. The use of several antiprotozoal drugs is associated with serious adverse effects and resistance development, and drugs that are more effective are urgently needed. Microorganisms, mammalian cells and fluids, insects, and reptiles are sources of antimicrobial peptides (AMPs) that act against pathogenic microorganisms; these AMPs have been widely studied as a promising alternative therapeutic option to conventional antibiotics, aiming to treat infections caused by multidrug-resistant pathogens. One advantage of AMP molecules is their adaptability, as they can be easily fine-tuned for broad-spectrum or targeted activity by changing the amino acid residues in their sequence. Consequently, these variations in structural and physicochemical properties can alter the antimicrobial activities of AMPs and decrease resistance development. This article presents an overview of peptide activities against amebiasis, giardiasis, trichomoniasis, Chagas disease, leishmaniasis, malaria, and toxoplasmosis. AMPs and their analogs demonstrate great potential as therapeutics, with potent and selective activity, when compared with commercially available drugs, and hold the potential to act as new scaffolds for the development of novel anti-protozoal drugs.

Phenolic chalcones as agents against Trichomonas vaginalis.

Trichomonas vaginalis, a flagellated and anaerobic protozoan, is a causative agent of trichomoniasis. This disease is among the world's most common non-viral sexually transmitted infection. A single class drug, nitroimidazoles, is currently available for the trichomoniasis treatment. However, resistant isolates have been identified from unsuccessfully treated patients. Thus, there is a great challenge for a discovery of innovative anti-T. vaginalis agents. As part of our ongoing search for antiprotozoal chalcones, we designed and synthesized a series of 21 phenolic chalcones, which were evaluated against T. vaginalis trophozoites. Structure-activity relationship indicated hydroxyl group plays a role key in antiprotozoal activity. 4'-Hydroxychalcone (4HC) was the most active compound (IC50 = 27.5 µM) and selected for detailed bioassays. In vitro and in vivo evaluations demonstrated 4HC was not toxic against human erythrocytes and Galleria mellonella larvae. Trophozoites of T. vaginalis were treated with 4HC and did not present significant reactive oxygen species (ROS) accumulation. However, compound 4HC was able to increase ROS accumulation in neutrophils coincubated with T. vaginalis. qRT-PCR Experiments indicated that 4HC did not affect the expression of pyruvate:ferredoxin oxidoreductase (PFOR) and ß-tubulin genes. In silico simulations, using purine nucleoside phosphorylase of T. vaginalis (TvPNP), corroborated 4HC as a promising ligand. Compound 4HC was able to establish interactions with residues D21, G20, M180, R28, R87 and T90 through hydrophobic interactions, π-donor hydrogen bond and hydrogen bonds. Altogether, these results open new avenues for phenolic chalcones to combat trichomoniasis, a parasitic neglected infection.

Antimicrobial and Antibiofilm Activities of Copper(II)-1,10-phenanthroline-5,6-pione Against Commensal Bacteria and Fungi Responsible for Vaginal Microbiota Dysbiosis.

The disbalance of vaginal eubiotic microbiota can lead to overgrowth of Candida species and bacteria responsible for aerobic vaginitis, activating inflammatory pathways. The presence of Trichomonas vaginalis, a sexually transmitted protozoan pathogen, can be a predisposing factor for disordering the growth of bacterial/fungal pathogenic species due to the increase in pH and reduction of eubiotic microbiota. Herein, we evaluated the effects of the potent trichomonacidal compound, copper(II)-1,10-phenanthroline-5,6-dione (Cu-phendione), against pathogens responsible for candidiasis and aerobic vaginitis. Cu-phendione showed antimicrobial activity against Candida albicans, non-albicans Candida species (C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis) and Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus, Enterococcus faecalis, and Streptococcus agalactiae) bacteria. Moreover, Cu-phendione was able to interfere with the fungal biofilm formation. These results highlight the antimicrobial potential of Cu-phendione against bacterial and fungal strains of vaginitis-causing infectious agents.

Iron chelation and inhibition of metallopeptidases mediate anti-Trichomonas vaginalis activity by a novel 8-hydroxyquinoline derivative.

Trichomoniasis is a neglected parasitic infection, with no oral therapeutic alternatives to overcome the pitfalls of currently approved drugs. In this context, the search for new anti-Trichomonas vaginalis drugs is imperative. Here we report the selective anti-T. vaginalis activity of a substituted 8-hydroxyquinoline-5-sulfonamide derivative. Six different derivatives were evaluated for anti-T. vaginalis. In vitro and in vivo toxicity methods, association with metal ions, and investigation on the mechanism of action were performed with the most active derivative, PH 152. Cytotoxicity assays showed selectivity for the parasite and the low toxicity was confirmed in G. mellonella larvae model. The mode of action is related to iron chelation by disrupting Fe-S clusters-dependent enzyme activities in the parasite. Proteomic analysis indicated inhibition of metallopeptidases related to T. vaginalis virulence mechanisms and metabolic pathways. PH 152 presented selective trichomonacidal activity through multitarget action.

Anti-Trichomonas vaginalis activity and chemical analysis of metabolites produced by marine-associated fungi.

Trichomoniasis is the most common non-viral sexually transmitted infection worldwide and it may have serious consequences, especially for women. Currently, 5-nitroimidazole drugs are the treatment of choice for trichomoniasis, although presenting adverse effects and reported cases of drug resistance. Metabolites isolated from marine fungi have attracted considerable attention due to their unique chemical structures with diverse biological activities, including antiprotozoal activity. In this study, we showed the anti-Trichomonas vaginalis activity of fractions obtained from marine fungi and the chemical composition of the most active fraction was determined. Ethyl acetate fractions of the fungus Aspergillus niger (EAE03) and Trichoderma harzianum/Hypocrea lixii complex (EAE09) were active against T. vaginalis. These samples, EAE03 and EAE09, were also effective against the fresh clinical isolate metronidazole-resistant TV-LACM2R, presenting MIC values of 2.0 mg/mL and 1.0 mg/mL, respectively. The same MIC values were found against ATCC 30,236 T. vaginalis isolate. In vitro cytotoxicity revealed only the fraction named EAE03 with no cytotoxic effect; however, the active fractions did not promote a significant hemolytic effect after 1-h incubation. Already, the in vivo toxicity evaluation using Galleria mellonella larvae demonstrated that none of the tested samples caused a reduction in animal survival. The fraction EAE03 was followed for purification steps and analyzed by LC-DAD-MS. Eleven compounds were annotated, including butyrolactone, butanolide, and atromentin. Overall, the range of activities reported confirms the potential of marine fungi to produce bioactive molecules.

Optimization of Coumarins Extraction from Pterocaulon balansae by Box-Behnken Design and Anti-Trichomonas vaginalis Activity.

Trichomonas vaginalis causes trichomoniasis, a nonviral sexually transmitted infection with a high prevalence worldwide. Oral metronidazole is the drug of choice for the treatment of this disease, although high levels of T. vaginalis resistance to this agent are well documented in the literature. This study describes the anti-T. vaginalis activity of an optimized coumarin-rich extract from Pterocaulon balansae. Optimization was performed to maximize extraction of total coumarins by means of a 3-level Box-Behnken design, evaluating the effect of three factors: extraction time, plant : solvent ratio, and ethanol concentration. Optimum conditions were found to be 5 h extraction time and a plant : solvent ratio of 1% (w/v) and 60% (v/v) ethanol, which resulted in approximately 30 mg of total coumarins/g of dry plant. The coumarin-enriched extract exhibited a minimum inhibitory concentration of 30 µg/mL and an IC50 of 3.2 µg/mL against T. vaginalis, a low cytotoxicity, and a high selectivity index (18 for vaginal epithelial cells and 16 for erythrocytes). The coumarins permeation/retention profile through porcine vaginal mucosa was evaluated in Franz-type diffusion cells. After 8 h of kinetics, coumarins were detected in the tissue (4.93 µg/g) without detecting them in the receptor compartment. A significant increase of coumarins in the mucosa layers (8.18 µg/g) and receptor compartment (0.26 µg/g) was detected when a T. vaginalis suspension (2 × 105 trophozoites/mL) was previously added onto the mucosa. No alterations were visualized in the stratified squamous non-keratinized epithelium of the porcine vaginal mucosa after contact with the extract. Overall, these results suggest that the P. balansae coumarin-rich extract may have potential as a treatment for trichomoniasis.

Trichomonas vaginalis NTPDase inhibited by lycorine modulates the parasite-neutrophil interaction.

Lycorine is an Amaryllidaceae alkaloid that presents anti-Trichomonas vaginalis activity. T. vaginalis causes trichomoniasis, the most common non-viral sexually transmitted infection. The modulation of T. vaginalis purinergic signaling through the ectonucleotidases, nucleoside triphosphate diphosphohydrolase (NTPDase), and ecto-5'-nucleotidase represents new targets for combating the parasite. With this knowledge, the aim of this study was to investigate whether NTPDase and ecto-5'-nucleotidase inhibition by lycorine could lead to extracellular ATP accumulation. Moreover, the lycorine effect on the reactive oxygen species (ROS) production by neutrophils and parasites was evaluated as well as the alkaloid toxicity. The metabolism of purines was assessed by HPLC. ROS production was measured by flow cytometry. Cytotoxicity against epithelial vaginal cells and fibroblasts was tested, as well as the hemolytic effect of lycorine and its in vivo toxicity in Galleria mellonella larvae. Our findings showed that lycorine caused ATP accumulation due to NTPDase inhibition. The alkaloid did not affect the ROS production by T. vaginalis; however, it increased ROS levels in neutrophils incubated with lycorine-treated trophozoites. Lycorine was cytotoxic against vaginal epithelial cells and fibroblasts; conversely, it was not hemolytic neither exhibited toxicity against the in vivo model of G. mellonella larvae. Overall, besides having anti-T. vaginalis activity, lycorine modulates ectonucleotidases and stimulates neutrophils to secrete ROS. This mechanism of action exerted by the alkaloid could enhance the susceptibility of T. vaginalis to host immune cell, contributing to protozoan clearance.

Strongyloides stercoralis larvae or egg: Which came first?

Strongyloides stercoralis (SS) hyperinfection is a well-documented condition. However, SS eggs in stool samples are not commonly observed during routine analysis. Here, we report a case on SS hyperinfection where both larvae and eggs were observed in the stool sample of an immunossupressed liver allograft transplanted patient.

Anti-Trichomonas vaginalis activity of 1,10-phenanthroline-5,6-dione-based metallodrugs and synergistic effect with metronidazole.

Trichomonas vaginalis is responsible for the most common non-viral, sexually transmitted infection, human trichomoniasis, and is associated with an increased susceptibility to HIV. An escalation in resistance (2.5-10%) to the clinical drug, metronidazole (MTZ), has been detected and this compound also has adverse side-effects. Therefore, new treatment options are urgently required. Herein, we investigate the possible anti-T. vaginalis activity of 1,10-phenanthroline-5,6-dione (phendione) and its metal complexes, [Ag(phendione)2]ClO4 and [Cu(phendione)3](ClO4)2·4H2O. Minimum inhibitory concentration (MIC) against T. vaginalis ATCC 30236 and three fresh clinical isolates and mammalian cells were performed using serial dilution generating IC50 and CC50 values. Drugs combinations with MTZ were evaluated by chequerboard assay. A strong anti-T. vaginalis activity was found for all test compounds. IC50 values obtained for [Cu(phendione)3](ClO4)2·4H2O were similar or lower than those obtained for MTZ. In vitro assays with normal cells showed low cytotoxicity and [Cu(phendione)3](ClO4)2·4H2O presented a high selectivity index (SI) for fibroblasts (SI = 11.39) and erythrocytes (SI > 57.47). Chequerboard assay demonstrated that the combination of [Cu(phendione)3](ClO4)2·4H2O with MTZ leads to synergistic interaction, which suggests distinct mechanisms of action of the copper-phendione complex and avoiding the MTZ resistance pathways. Our results highlight the importance of phendione-based drugs as potential molecules of pharmaceutical interest.

Anti-Trichomonas vaginalis activity of chalcone and amino-analogues.

Trichomoniasis is the most common non-viral sexually transmitted disease worldwide and can lead to serious consequences in reproductive health, cancer, and HIV acquisition. The current approved treatment present adverse effects and drug resistance data on this neglected parasitic infection is underestimated. Chalcones are a family of molecules that present biological applications, such as activity against many pathogenic organisms including protozoan pathogens. Chalcone (1) and three amino-analogues (2-4) were synthesized by Claisen-Schmidt condensation reaction and had their activity evaluated against the parasitic protozoan Trichomonas vaginalis. This bioassay indicated the presence and position of the amino group on ring A was crucial for anti-T. vaginalis activity. Among these, 3'-aminochalcone (3) presented the most potent effect and showed high cytotoxicity against human vaginal cells. On the other hand, 3 was not able to exhibit toxicity against Galleria mellonella larvae, as well as the hemolytic effect on human erythrocytes. Trophozoites of T. vaginalis were treated with 3, and did not present significant reactive oxygen species (ROS) accumulation, but induced a significantly higher ROS accumulation in human neutrophils after co-incubation. T. vaginalis pyruvate:ferredoxin oxidoreductase (PFOR) and ß-tubulin gene expression was not affected by 3.

Anti-Trichomonas vaginalis activity of ursolic acid derivative: a promising alternative.

Trichomonas vaginalis is an extracellular parasite that binds to the epithelium of the human urogenital tract and causes the sexually transmitted infection, trichomoniasis. In view of increased resistance to drugs belonging to the 5-nitroimidazole class, new treatment alternatives are urgently needed. In this study, eight semisynthetized triterpene derivatives were evaluated for in vitro anti-T. vaginalis activity. Ursolic acid and its derivative, 3-oxime-urs-12-en-28-oic-ursolic acid (9), presented the best anti-T. vaginalis activity when compared to other derivatives, with minimum inhibitory concentration (MIC) at 25 µM. Moreover, 9 was active against several T. vaginalis fresh clinical isolates. Hemolysis assay demonstrated that 9 presented a low hemolytic effect. Importantly, 25 µM 9 was not cytotoxic against the Vero cell lineage. Finally, we demonstrated that compound 9 acts synergistically with metronidazole against a T. vaginalis metronidazole-resistant isolate. This report reveals the high potential of the triterpenoid derivative 9 as trichomonicidal agent.

Adenosine reduces reactive oxygen species and interleukin-8 production by Trichomonas vaginalis-stimulated neutrophils.

Trichomonas vaginalis is a flagellated protozoan that affects the human urogenital tract causing 276.4 million new infections a year. The parasite elicits a vaginal mucosal infiltration of immune cells, especially neutrophils which are considered to be primarily responsible for cytological change observed at the infection site as well as the major contributor in the inflammatory response against the parasite. Extracellular nucleotides and their nucleosides are signaling compounds involved in several biological processes, including inflammation and immune responses. Once in the extracellular space, the nucleotides and nucleosides can directly activate the purinergic receptors. Herein, we investigated the involvement of purinergic signaling on the production of reactive oxygen species (ROS) and cytokines by T. vaginalis-stimulated neutrophils. Parasites were able to induce an increase in ROS and IL-8 levels while they did not promote IL-6 secretion or neutrophil elastase activity. Adenine and guanine nucleotides or nucleosides were not able to modulate ROS and cytokine production; however, when T. vaginalis-stimulated neutrophils were incubated with adenosine and adenosine deaminase inhibitor, the levels of ROS and IL-8 were significantly reduced. These immunosuppressive effects were probably a response to the higher bioavailability of adenosine found in the supernatant as result of inhibition of enzyme activity. The involvement of P1 receptors was investigated by immunofluorescence and A1 receptor was the most abundant. Our data show that the influence of purinergic signaling, specifically those effects associated with adenosine accumulation, on the modulation of production of proinflammatory mediators by T. vaginalis-stimulated neutrophils contribute to the understanding of immunological aspects of trichomoniasis.

The Caatinga endemic Manilkara rufula possesses remarkable activity against Trichomonas vaginalis and Tritrichomonas foetus.

Tritrichomonas foetus infects the bovine urogenital tract, causing bovine trichomoniasis. Significant economic losses may occur due to infertility and abortion among cattle. Trichomonas vaginalis is the causative agent of trichomoniasis; the most common but overlooked non-viral sexually transmitted disease worldwide. Human and bovine trichomoniasis present treatment restrictions and efforts to identify new alternatives are essential. The present study evaluated the anti-trichomonads activities of seven fractions from northwest endemic plant Manilkara rufula. Flavonoids and condensed tannins were identified from these fractions by LC-DAD-MS/MS and MALDI-MS/MS. Altogether, the results demonstrated for the first time the structural description of tannins from leaves of M. rufula and the relation of these compounds with anti-T. vaginalis and anti-T. foetus activities. Overall, this report reveals the potential of M. rufula fractions against both parasites and shows new alternatives to treat the infection caused by trichomonads.

Trichomonas vaginalis clinical isolates: cytoadherence and adherence to polystyrene, intrauterine device, and vaginal ring.

The parasitism by Trichomonas vaginalis is complex and in part is mediated by cytoadherence accomplished via five surface proteins named adhesins and a glycoconjugate called lipophosphoglycan (TvLPG). In this study, we evaluated the ability of T. vaginalis isolates to adhere to cells, plastic (polystyrene microplates), intrauterine device (IUD), and vaginal ring. Of 32 T. vaginalis isolates, 4 (12.5%) were strong adherent. The T. vaginalis isolates TV-LACM6 and TV-LACM14 (strong polystyrene-adherent) were also able to adhere to IUD and vaginal ring. Following chemical treatments, results demonstrated that the T. vaginalis components, lipophosphoglycan, cytoskeletal proteins, and surface molecules, were involved in both adherence to polystyrene and cytoadherence. The gene expression level from four adhesion proteins was highest in trophozoites adhered to cells than trophozoites adhered to the abiotic surface (polystyrene microplate). Our data indicate the major involvement of TvLPG in adherence to polystyrene, and that adhesins are important for cytoadherence. Furthermore, to our knowledge, this is the first report showing the T. vaginalis adherence to contraceptive devices, reaffirming its importance as pathogen among women in reproductive age.

Caatinga plants: Natural and semi-synthetic compounds potentially active against Trichomonas vaginalis.

Trichomonas vaginalis causes trichomoniasis; the most common but overlooked non-viral sexually transmitted disease worldwide. The treatment is based at 5'-nitroimidazoles, however, failure are related to resistance of T. vaginalis to chemotherapy. Caatinga is a uniquely Brazilian region representing a biome with type desert vegetation and plants present diverse biological activity, however, with few studies. The aim of this study was to investigate the activity against T. vaginalis of different plants from Caatinga and identify the compounds responsible by the activity. A bioguided fractionation of Manilkara rufula was performed and four major compounds were identified: caproate of α-amyrin (1b), acetate of ß-amyrin (2a), caproate of ß-amyrin (2b), and acetate of lupeol (3a). In addition, six derivatives of α-amyrin (1), ß-amyrin (2) and lupeol (3) were synthesized and tested against the parasite. Ursolic acid (5) reduced about 98% of parasite viability after 2h of incubation and drastic ultrastructural alterations were observed by scanning electron microscopy. Moreover, 5 presented high cytotoxicity to HMVII and HeLa cell line and low cytotoxicity against Vero line at 50 µM (MIC against the parasite). Metronidazole effect against T. vaginalis resistant isolate was improved when in association with 5.

Amaurocine: Anti-Trichomonas vaginalis protein produced by the basidiomycete Amauroderma camerarium.

Trichomonas vaginalis is the causative agent of trichomoniasis, the most common nonviral STD worldwide. This infection can lead to severe health conditions, especially when women are affected. Metronidazole and tinidazole are the only choices of treatment. In this sense, natural bioactive compounds against T. vaginalis are an interesting approach in the search for more efficient therapies. Herein, amaurocine, a 12 kDa protein, produced by the mushroom Amauroderma camerarium was purified and tested against T. vaginalis, including two fresh clinical isolates. Amaurocine presented MIC values at 2.6 µM against the ATCC isolate 30236, and 5.2 µM against the fresh clinical isolates, TV-LACH1 and TV-LACM2. Furthermore, besides increasing human neutrophils nitric oxide release, amaurocine presented a low toxicity toward those cells, suggesting it exerts a proinflammatory character.

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. vaginalis isolates 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.

Iron from haemoglobin and haemin modulates nucleotide hydrolysis in Trichomonas vaginalis.

Extracellular ATP may act as a danger signalling molecule, inducing inflammation and immune responses in infection sites. The ectonucleotidases NTPDase and ecto-5'-nucleotidase are enzymes that modulate extracellular nucleotide levels; these enzymes have been previously characterised in Trichomonas vaginalis. Iron plays an important role in the complex trichomonal pathogenesis. Herein, the effects of iron on growth, nucleotide hydrolysis and NTPDase gene expression in T. vaginalis isolates from female and male patients were evaluated. Iron from different sources sustained T. vaginalis growth. Importantly, iron from haemoglobin (HB) and haemin (HM) enhanced NTPDase activity in isolates from female patients and conversely reduced the enzyme activity in isolates from male patients. Iron treatments could not alter the NTPDase transcript levels in T. vaginalis. Furthermore, our results reveal a distinct ATP, ADP and AMP hydrolysis profile between isolates from female and male patients influenced by iron from HB and HM. Our data indicate the participation of NTPDase and ecto-5'-nucleotidase in the establishment of trichomonas infection through ATP degradation and adenosine production influenced by iron.

Natural and synthetic compound anti-Trichomonas vaginalis: an update review.

Trichomonas vaginalis is a flagellate protozoan that causes trichomonosis, a sexually transmitted disease of worldwide importance. However, the infection has long received much less attention than other parasitic and sexually transmitted diseases. This negligence leads to poor diagnosis and underestimated prevalence values, and consequently, it has been associated to increasing acquisition and transmission of HIV, pregnancy outcomes, infertility, pelvic inflammatory disease, and cervical and prostate cancer. In view of increased resistance to drugs belonging to the nitroimidazole class, new treatment alternatives are urgently needed. Natural products provide an immeasurable wealth of active molecules, and a great number of new drugs have been originated from these compounds. In addition, new synthetic products or derivatives from old drugs also provide an alternative to treat trichomonosis. Albeit many studies have been performed with natural products against T. vaginalis, none of them progressed to clinical trials. Overall, inadequate financial investments are made, and no alternative treatment for trichomonosis has been discovered; meanwhile, hundreds of thousands of people will remain infected and suffering the serious consequences of this nonviral STD. Thus, it is highlighted that clinical trials for better understanding the potential in vitro are necessary and urgent in order to furnish a new therapeutic alternative for trichomonosis treatment. The current review attempts to give an overview on the potential of natural and synthetic products as antitrichomonal.