Human Trichomoniasis Research Articles

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.

The Role of Iron Status in the Early Progression of Metronidazole Resistance in Trichomonas vaginalis Under Microaerophilic Conditions

Trichomonas vaginalis is the etiological agent of human trichomoniasis. Metronidazole has high treatment success rate among trichomoniasis patients. However, metronidazole-resistant T.vaginalis has been reported, contributing in an increasing number of refractory cases. The mechanism of metronidazole resistance in this parasite is still unclear. In the vaginal environment, where the microaerophilic conditions prevail but the iron concentration is constantly fluctuating, the metronidazole resistance profile of T.vaginalis could be altered. In this study, we developed metronidazole-resistant strains of T.vaginalis and evaluate if iron availability is important to the action of the drug. The modulation of iron levels and iron chelation affected the actions of metronidazole both in susceptible and resistant strains. Interestingly, the early resistant strains exhibited minor iron content. The results of transcription analysis in the early resistant strains showed dysregulation in the expression of genes that codified proteins involved in iron transporter, iron-sulfur cluster assemblage, and oxidative stress response, which could not be observed in the late resistant and susceptible strains. Our results indicate that iron content plays an important role in the metronidazole action in T.vaginalis and likely to be related to iron-sulfur proteins involved in metronidazole activation and oxidative stress via Fenton reaction.

Potato Peels and Their Bioactive Glycoalkaloids and Phenolic Compounds Inhibit the Growth of Pathogenic Trichomonads.

Potato peel, a waste product of the potato processing industry, is high in bioactive compounds. We investigated the in vitro antitrichomonad activity of potato peel powders prepared from commercial Russet, red, purple, and fingerling varieties as well as several known potato components, alkaloids and phenolic compounds, against three pathogenic strains of trichomonads. Trichomonas vaginalis is a sexually transmitted protozoan parasite that causes the human disease trichomoniasis. Two distinct strains of the related Tritrichomonas fetus infect cattle and cats. The glycoalkaloids α-chaconine and α-solanine were highly active against all parasite lines, while their common aglycone solanidine was only mildly inhibitory. α-Solanine was several times more active than α-chaconine. The phenolic compounds caffeic and chlorogenic acids and quercetin were mildly active against the parasites. Most of the potato peel samples were at least somewhat active against all three trichomonad species, but their activities were wide-ranging and did not correspond to their glycoalkaloid and phenolic content determined by HPLC. The two Russet samples were the most active against all three parasites. The purple potato peel sample was highly active against bovine and mostly inactive against feline trichomonads. None of the test substances were inhibitory toward several normal microflora species, suggesting the potential use of the peels for targeted therapeutic treatments against trichomonads.

Characterization of a novel endogenous cysteine proteinase inhibitor, trichocystatin-3 (TC-3), localized on the surface of Trichomonas vaginalis.

Trichomonas vaginalis is a flagellated protist responsible for human trichomoniasis. T. vaginalis has three genes encoding for endogenous cysteine proteinase (CP) inhibitors, known as trichocystatin-1 through trichocystatin-3 (TC-1, TC-2, and TC-3). These inhibitors belong to the cystatin family. In this study, we characterized trichocystatin-3 (TC-3), an endogenous cysteine proteinase (CP) inhibitor of T. vaginalis. TC-3 possesses a signal peptide in the N-terminus and two putative glycosylation sites (typical of family 2, cystatins) but lacks the PW motif and cysteine residues (typical of family 1, stefins). Native TC-3 was recognized as an ∼18 kDa protein band in a T. vaginalis protein extract. By confocal microscopy, endogenous TC-3 was found in the Golgi complex, cytoplasm, large vesicles, and the plasma membrane. These localizations are consistent with an in silico prediction. In addition, the purified recombinant protein (TC-3r) functions as an inhibitor of cathepsin L CPs, such as human liver cathepsin L and trichomonad CPs, present in a proteinase-resistant extract (PRE). Via a pull-down assay using TC-3r as bait and PRE, we identified several trichomonad CPs targeted by TC-3, primarily TvCP3. These CP-TC-3 interactions occur in vesicles, in the cytoplasm, and on the parasite surface. In addition, TC-3r showed a protective effect on HeLa cell monolayers by inhibiting trichomonad surface CPs involved in cellular damage. Our results show that the endogenous inhibitor TC-3 plays a key role in the regulation of endogenous CP proteolytic activity.

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.

Phytochemical-rich foods inhibit the growth of pathogenic trichomonads

BackgroundPlants produce secondary metabolites that often possess widespread bioactivity, and are then known as phytochemicals. We previously determined that several phytochemical-rich food-derived preparations were active against pathogenic foodborne bacteria. Trichomonads produce disease (trichomoniasis) in humans and in certain animals. Trichomonads are increasingly becoming resistant to conventional modes of treatment. It is of interest to test bioactive, natural compounds for efficacy against these pathogens.MethodsUsing a cell assay, black tea, green tea, grape, pomegranate, and jujube extracts, as well as whole dried jujube were tested against three trichomonads: Trichomonas vaginalis strain G3 (found in humans), Tritrichomonas foetus strain D1 (found in cattle), and Tritrichomonas foetus-like organism strain C1 (found in cats). The most effective of the test substances was subsequently tested against two metronidazole-resistant Trichomonas vaginalis strains, and on normal mucosal flora.ResultsBlack tea extract inhibited all the tested trichomonads, but was most effective against the T. vaginalis organisms. Inhibition by black tea was correlated with the total and individual theaflavin content of the two tea extracts determined by HPLC. Metronidazole-resistant Trichomonas vaginalis strains were also inhibited by the black tea extract. The response of the organisms to the remaining preparations was variable and unique. We observed no effect of the black tea extract on common normal flora bacteria.ConclusionsThe results suggest that the black tea, and to a lesser degree green tea, grape seed, and pomegranate extracts might present possible natural alternative therapeutic agents to treat Trichomonas vaginalis infections in humans and the related trichomonad infections in animals, without negatively affecting the normal flora.

Diamine derivative anti-Trichomonas vaginalis and anti-Tritrichomonas foetus activities by effect on polyamine metabolism

Human and bovine trichomoniasis are sexually transmitted diseases (STD) caused by Trichomonas vaginalis and Tritrichomonas foetus, respectively. Human trichomoniasis is the most common non-viral STD in the world and bovine trichomoniasis causes significant economic losses to breeders. Considering the significant impact of the infections caused by these protozoa and the treatment failures, the search for new therapeutic alternatives becomes crucial. In this study the effect of diamines and amino alcohols in the in vitro viability of trichomonads was evaluated. Screening demonstrated the high activity of diamine 4 against these protozoa. Although cytotoxicity against HMVII cell line and slight hemolysis were observed in vitro, the compound showed no toxic effect on the Galleria mellonella in vivo model. Importantly, diamine 4 was active against both trichomonads species at 6h and 24h of incubation, and these effects was reverted by putrescine, a polyamine, suggesting competition for the same metabolic pathway. These findings indicate that the mechanism of action of diamine 4 is through the polyamine metabolism, a pathway distinct from that presented by metronidazole, the drug usually used to treat trichomoniasis and to which resistance is widely reported. These data demonstrate the importance of diamines as potential novel candidates as anti-T. vaginalis and anti-T. foetus agents.

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.

Characterization of cyclin-dependent kinases and Cdc2/Cdc28 kinase subunits in Trichomonas vaginalis.

Cyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.

Antiprotozoal Effects of the Tomato Tetrasaccharide Glycoalkaloid Tomatine and the Aglycone Tomatidine on Mucosal Trichomonads

The present study investigated the inhibitory effects of the commercial tetrasaccharide tomato glycoalkaloid tomatine and the aglycone tomatidine on three mucosal pathogenic protozoa that are reported to infect humans, cattle, and cats, respectively: Trichomonas vaginalis strain G3, Tritrichomonas foetus strain D1, and Tritrichomonas foetus strain C1. A preliminary screen showed that tomatine at 100 μM concentration completely inhibited the growth of all three trichomonads. In contrast, the inhibition of all three pathogens by tomatidine was much lower, suggesting the involvement of the lycotetraose carbohydrate side chain in the mechanism of inhibition. Midpoints of concentration-response sigmoid plots of tomatine on the three strains correspond to IC50 values, the concentration that inhibits 50% of growth of the pathogenic protozoa. The concentration data were used to calculate the IC50 values for G3, D1, and C1 of 7.9, 1.9, and 2.2 μM, respectively. The results show an approximately 4-fold variation from the lowest to the highest value (lowest activity). Although the inhibition by tomatine was not as effective as that of the medicinal drug metronidazole, the relatively low IC50 values for both T. vaginalis and T. foetus indicated tomatine as a possible natural alternative therapeutic for trichomoniasis in humans and food-producing (cattle and pigs) and domestic (cats) animals. Because tomatine has the potential to serve as a new antiprotozoan functional (medical) food, the distribution of this glycoalkaloid in tomatoes and suggestions for further research are discussed.

Evaluation of DNA Based Techniques for the Diagnosis of Human Vaginal Trichomoniasis in North Indian Population

Evaluation of DNA Based Techniques for the Diagnosis of Human Vaginal Trichomoniasis in North Indian Population

Comparative aspects of immunity and vaccination in human and bovine trichomoniasis: a review.

Trichomonas vaginalis and Tritrichomonas foetus are important extracellular protozoans that cause, respectively, human and bovine venereal diseases. Trichomonads are extracellular parasites that primarily inhabit the genital tracts of the mammalian hosts where they overcome the mucus barrier and parasitize mucosa by contact-dependent or contact-independent cytotoxicity. Transient immunity is usually achieved by the host after clinical infection. At present, vaccination in cattle reduces infection rates and reproductive wastage in affected herds. After vaccination, immunoglobulin G (IgG) levels increase in systemic circulation while immunoglobulin A (IgA) levels rise in the vagina. Only moderate protection is conferred by means of vaccination. Future vaccine development strategies are needed for cattle to enhance the antigenic component or use adjuvant that strongly activates the innate immune response to produce safe and potent vaccines. This paper reviews the current knowledge of the immunology of trichomoniasis infection and the challenges and potential of vaccines in the control of the infection in human and bovine trichomoniasis.

Nitric oxide maintains cell survival of Trichomonas vaginalis upon iron depletion.

BackgroundIron plays a pivotal role in the pathogenesis of Trichomonas vaginalis, the causative agent of highly prevalent human trichomoniasis. T. vaginalis resides in the vaginal region, where the iron concentration is constantly changing. Hence, T. vaginalis must adapt to variations in iron availability to establish and maintain an infection. The free radical signaling molecules reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been proven to participate in iron deficiency in eukaryotes. However, little is known about the roles of these molecules in iron-deficient T. vaginalis.MethodsT. vaginalis cultured in iron-rich and -deficient conditions were collected for all experiments in this study. Next generation RNA sequencing was conducted to investigate the impact of iron on transcriptome of T. vaginalis. The cell viabilities were monitored after the trophozoites treated with the inhibitors of nitric oxide (NO) synthase (L-NG-monomethyl arginine, L-NMMA) and proteasome (MG132). Hydrogenosomal membrane potential was measured using JC-1 staining.ResultsWe demonstrated that NO rather than ROS accumulates in iron-deficient T. vaginalis. The level of NO was blocked by MG132 and L-NMMA, indicating that NO production is through a proteasome and arginine dependent pathway. We found that the inhibition of proteasome activity shortened the survival of iron-deficient cells compared with untreated iron-deficient cells. Surprisingly, the addition of arginine restored both NO level and the survival of proteasome-inhibited cells, suggesting that proteasome-derived NO is crucial for cell survival under iron-limited conditions. Additionally, NO maintains the hydrogenosomal membrane potential, a determinant for cell survival, emphasizing the cytoprotective effect of NO on iron-deficient T. vaginalis. Collectively, we determined that NO produced by the proteasome prolonged the survival of iron-deficient T. vaginalis via maintenance of the hydrogenosomal functions.ConclusionThe findings in this study provide a novel role of NO in adaptation to iron-deficient stress in T. vaginalis and shed light on a potential therapeutic strategy for trichomoniasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1000-5) contains supplementary material, which is available to authorized users.

Immunity in urogenital protozoa

Innate and adaptive immunity play a significant role in urogenital infections. Innate immunity is provided by the epithelial cells and mucus lining along with acidic pH, which forms a strong physical barrier against the pathogens in female reproductive tract. Cells of innate immune system, antimicrobial peptides, cytokines, chemokines and adaptive immunity in the reproductive tract are evolved during infection, and a pro-inflammatory response is generated to fight against the invading pathogen Trichomonas vaginalis, a primary urogenital protozoa, the etiological agent of human trichomoniasis, a curable sexually transmitted infection. The involvement of the urogenital tract by other protozoal infections such as P. falciparum, Trypanosoma, Leishmania, Toxoplasma, Entamoeba histolytica and Acanthamoeba infection is rarely reported. Trichomonas induce pro-inflammatory and immunosuppressive responses in infected subjects. Multifactorial pathogenic mechanisms including parasite adherence, cysteine proteases, lipophosphoglycan, free radical, cytokine generation and Toll-like receptors appear to interplay with the induction of local and systemic immune responses that ultimately determine the outcome of the infection. However, the involvement of urogenital pathogen-specific immune mechanisms and effect of normal local resident flora on the outcome (symptomatic vs. asymptomatic) of infection are poorly understood. Moreover, immune interactions in trichomoniasis subjects co-infected with bacterial and viral pathogens need to be elucidated.

Are the fatty acids responsible for the higher effect of oil and alcoholic extract of Nigella sativa over its aqueous extract on Trichomonas vaginalis trophozoites?

Trichomoniasis, the disease caused by the flagellate protozoan Trichomonas vaginalis is the sexually transmitted infection with the largest annual incidence. Metronidazole is the drug of choice recommended for the treatment of human trichomoniasis but it can lead to drug resistance and many other adverse effects. So, it is necessary for new alternatives for the treatment of this infection. Medicinal plants or herbs could be good alternative regimens to be inexpensive, effective and safe to use. In the present study, the therapeutic potential of Nigella sativa aqueous and alcoholic extracts as well as seeds oil was examined. Different concentrations of these plant preparations were incubated in vitro with cultivated T. vaginalis trophozoites and its effect on growth was compared with metronidazole under the same conditions. Both the alcoholic extract and oil proved to be valuable agents as efficient as metronidazole in treating T. vaginalis infection. The remarkable effect of N. sativa oil may be attributed to the fact that the active principles extracted from N. sativa seeds are mostly from its essential oil (omega 3, 6, 9 as well as 7 fatty acids). However, further experimental and clinical investigations are needed to evaluate and standardize the doses of these natural products to be safe and efficient.

Molecular and morphologic identification of Pentatrichomonas hominis in swine

A marasmic pig with watery diarrhea was identified to harbor the human-pathogenic protist Pentatrichomonas hominis by PCR and sequence analysis of three genetic loci (ITS1-5.8S rRNA-ITS2, 18S rRNA, and EF-1α), electron microscopy, and infection experiments. DNA sequencing and phylogenetic analysis indicated the organism isolated in this study was most closely related to P. hominis. SEM and TEM observation of the ultrastructure demonstrated that it had a morphology identical to P. hominis. The result of experimental infections with P. hominis exhibited that the cells had the ability to propagate in the cecum of piglets and fecal–oral route might be the major way in which pigs became infected. The present study confirmed that swine could be a host for P. hominis and might serve as a reservoir for human trichomoniasis.

In Vitro Effect of Pomegranate Extract on Trichomonas Tenax

The incidence of Trichomonas tenax (T. tenax) in patients with acute ulcerative gingivitis has been demonstrated in several published reports. Metronidazole was known as the most effective drug for human trichomoniasis, however, drug resistance and toxicity appeared. This study was designed in vitro to investigate the inhibitory activity of Punica granatum (P. granatun) ethanol extract on the growth and motility of T. tenax in comparison to metronidazole. Pomegranate ethanol extract group was treated with concentrations of 12.5, 25, 50, and 100 µg/ml. Metronidazole group and blank control were included. At 12 h, 24 h 48 h and 72 hr after drug treatment, the anti-T. tenax effect of pomegranate ethanol extract was tested by microscope counting method. The results showed 60% motility of T.tenax trophozoite after treatment with 12.5µg/100 ml of pomegranate ethanol extract group and 25 µg/ml showed higher anti-T. tenax (P<0.01). The ethanol extract of pomegranate peel has a remarkable effect on T. tenax, and among the groups, 60% ethanol extract shows the best anti-T. tenax activity. (Gehad T. El-Sherbini and Nahla M. Shoukry In Vitro effect of pomegranate peel extract on Trichomonas tenax) Life Science Journal 2012; 9(3):791-797). (ISSN: 1097-8135). http://www.lifesciencesite.com. 113

Trichomonas stableri n. sp., an agent of trichomonosis in Pacific Coast band-tailed pigeons (Patagioenas fasciata monilis).

Trichomonas gallinae is a ubiquitous flagellated protozoan parasite, and the most common etiologic agent of epidemic trichomonosis in columbid and passerine species. In this study, free-ranging Pacific Coast band-tailed pigeons (Patagioenas fasciata monilis) in California (USA) were found to be infected with trichomonad protozoa that were genetically and morphologically distinct from T. gallinae. In microscopic analysis, protozoa were significantly smaller in length and width than T. gallinae and were bimodal in morphology. Phylogenetic analysis of the ITS1/5.8S/ITS2, rpb1, and hydrogenosomal Fe-hydrogenase regions revealed that the protozoan shares an ancestor with Trichomonas vaginalis, the sexually-transmitted agent of trichomoniasis in humans. Clinical and pathologic features of infected birds were similar to infections with T. gallinae. Evidence presented here strongly support taxonomical distinction of this parasite, which we hereby name Trichomonas stableri n. sp. This work contributes to a growing body of evidence that T. gallinae is not the sole etiologic agent of avian trichomonosis, and that the incorporation of molecular tools is critical in the investigation of infectious causes of mortality in birds.

Evaluation of the effect of miltefosine on Trichomonas vaginalis

Trichomonas vaginalis causes trichomoniasis in humans, a sexually transmitted disease commonly treated with metronidazole (MTZ). MTZ is known to cause undesirable side effects, and MTZ-resistant parasites have been reported. Thus, the development of an alternative treatment is desirable. Miltefosine (MLT) is an alkylphosphocholine synthetic lipid analogue that displays antiparasitic activity against Leishmania, Trypanosoma cruzi, Entamoeba histolytica, Acanthamoeba spp., Giardia lamblia, T. vaginalis and some fungi. Moreover, it has been used for oral treatment of visceral leishmaniosis in several countries. Here, we analysed the MLT-induced antiproliferative effect on T. vaginalis as well its effect on the fine structure and viability of the parasite. We observed a dose-dependent effect with an IC50 of 14.5 and 20 μM after 24 and 48 h, respectively. Furthermore, reversibility assays demonstrated that new incubations were necessary in order to maintain the antiproliferative effect. Ultrastructural analyses demonstrated that MLT induced several alterations, including the appearance of wrinkled and rounded cells, membrane blebbing, intense vacuolization and nuclear condensation, all indicative of cell death by apoptosis. In addition, the quantitative analyses of the viability assays using combined markers of live and dead cells demonstrated that treatment with the IC50 concentration of MLT significantly reduced the number of viable parasites compared with untreated cells. Taken together, these observations suggest that MLT is a promising compound for the treatment of trichomoniasis.

Opportunistic Human Infections Caused by Tritrichomonas species: A Mini-Review

The main purpose of this mini-review is to provide information on opportunistic human infections caused by Tritrichomonas spp. Rare cases of human trichomoniasis, including fatal meningoencephalitis and peritonitis, have been reported exclusively in immunosuppressed or immunocompromised individuals, suggesting the protozoa are human opportunistic pathogens. These infections are readily diagnosed by the characteristic morphology of the protozoa. Species identification is augmented by testing with molecular methods, such as PCR and DNA sequencing. Risk factors for infection and treatment are also discussed.