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1.  Tolerability of inhaled N-chlorotaurine in the pig model 
Background
N-chlorotaurine, a long-lived oxidant produced by human leukocytes, can be applied in human medicine as an endogenous antiseptic. Its antimicrobial activity can be enhanced by ammonium chloride. This study was designed to evaluate the tolerability of inhaled N-chlorotaurine (NCT) in the pig model.
Methods
Anesthetized pigs inhaled test solutions of 1% (55 mM) NCT (n = 7), 5% NCT (n = 6), or 1% NCT plus 1% ammonium chloride (NH4Cl) (n = 6), and 0.9% saline solution as a control (n = 7), respectively. Applications with 5 ml each were performed hourly within four hours. Lung function, haemodynamics, and pharmacokinetics were monitored. Bronchial lavage samples for captive bubble surfactometry and lung samples for histology and electron microscopy were removed.
Results
Arterial pressure of oxygen (PaO2) decreased significantly over the observation period of 4 hours in all animals. Compared to saline, 1% NCT + 1% NH4Cl led to significantly lower PaO2 values at the endpoint after 4 hours (62 ± 9.6 mmHg vs. 76 ± 9.2 mmHg, p = 0.014) with a corresponding increase in alveolo-arterial difference of oxygen partial pressure (AaDO2) (p = 0.004). Interestingly, AaDO2 was lowest with 1% NCT, even lower than with saline (p = 0.016). The increase of pulmonary artery pressure (PAP) over the observation period was smallest with 1% NCT without difference to controls (p = 0.91), and higher with 5% NCT (p = 0.02), and NCT + NH4Cl (p = 0.05).
Histological and ultrastructural investigations revealed no differences between the test and control groups. The surfactant function remained intact. There was no systemic resorption of NCT detectable, and its local inactivation took place within 30 min. The concentration of NCT tolerated by A549 lung epithelial cells in vitro was similar to that known from other body cells (0.25–0.5 mM).
Conclusion
The endogenous antiseptic NCT was well tolerated at a concentration of 1% upon inhalation in the pig model. Addition of ammonium chloride in high concentration provokes a statistically significant impact on blood oxygenation.
doi:10.1186/1471-2466-9-33
PMCID: PMC2722574  PMID: 19602222
2.  In vitro effect of tinidazole and furazolidone on metronidazole-resistant Trichomonas vaginalis. 
Trichomonas vaginalis is a common sexually transmitted protozoan parasite. Although often considered simply a nuisance infection, T. vaginalis has been implicated in premature rupture of placental membranes and increases in the risk of acquiring human immunodeficiency virus. Metronidazole, a 5-nitroimidazole, is currently the drug of choice to treat T. vaginalis infection. Because some patients have severe reactions to metronidazole and others are infected with metronidazole-resistant T. vaginalis, we were prompted to investigate alternative therapies. Tinidazole, another 5-nitroimidazole used in other countries to treat T. vaginalis infections, and furazolidone, a nitrofuran presently used to treat giardiasis and infections with some anaerobic enteric bacteria, were investigated for effectiveness against 9 metronidazole-susceptible and 12 metronidazole-resistant T. vaginalis patient isolates. The in vitro aerobic and anaerobic minimum lethal concentrations (MLC) and the time for drug efficacy were determined. Tinidazole killed the metronidazole-susceptible isolates at a low MLC but was effective against only 4 of the 12 metronidazole-resistant isolates. In contrast, furazolidone was effective at a low MLC for all isolates. When tinidazole was effective, it required > 6 h to kill trichomonads. However, furazolidone killed both metronidazole-susceptible and resistant trichomonads within 2 to 3 h of exposure. These data suggest that furazolidone may be a good candidate for treating metronidazole-resistant trichomoniasis and that further investigation of this drug is warranted.
PMCID: PMC163276  PMID: 8723451
3.  Bactericidal Activity of N-Chlorotaurine against Biofilm-Forming Bacteria Grown on Metal Disks 
Many orthopedic surgeons consider surgical irrigation and debridement with prosthesis retention as a treatment option for postoperative infections. Usually, saline solution with no added antimicrobial agent is used for irrigation. We investigated the activity of N-chlorotaurine (NCT) against various biofilm-forming bacteria in vitro and thereby gained significant information on its usability as a soluble and well-tolerated active chlorine compound in orthopedic surgery. Biofilms of Staphylococcus aureus were grown on metal alloy disks and in polystyrene dishes for 48 h. Subsequently, they were incubated for 15 min to 7 h in buffered solutions containing therapeutically applicable concentrations of NCT (1%, 0.5%, and 0.1%; 5.5 to 55 mM) at 37°C. NCT inactivated the biofilm in a time- and dose-dependent manner. Scanning electron microscopy revealed disturbance of the biofilm architecture by rupture of the extracellular matrix. Assays with reduction of carboxanilide (XTT) showed inhibition of the metabolism of the bacteria in biofilms. Quantitative cultures confirmed killing of S. aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa biofilms on metal alloy disks by NCT. Clinical isolates were slightly more resistant than ATCC type strains, but counts of CFU were reduced at least 10-fold by 1% NCT within 15 min in all cases. NCT showed microbicidal activity against various bacterial strains in biofilms. Whether this can be transferred to the clinical situation should be the aim of future studies.
doi:10.1128/AAC.02700-13
PMCID: PMC4023792  PMID: 24492358
4.  Chemical Characterization and Biological Properties of NVC-422, a Novel, Stable N-Chlorotaurine Analog▿† 
During oxidative burst, neutrophils selectively generate HOCl to destroy invading microbial pathogens. Excess HOCl reacts with taurine, a semi-essential amino acid, resulting in the formation of the longer-lived biogenerated broad-spectrum antimicrobial agent, N-chlorotaurine (NCT). In the presence of an excess of HOCl or under moderately acidic conditions, NCT can be further chlorinated, or it can disproportionate to produce N,N-dichlorotaurine (NNDCT). In the present study, 2,2-dimethyltaurine was used to prepare a more stable N-chlorotaurine, namely, N,N-dichloro-2,2-dimethyltaurine (NVC-422). In addition, we report on the chemical characterization, in vitro antimicrobial properties, and cytotoxicity of this compound. NVC-422 was shown effectively to kill all 17 microbial strains tested, including antibiotic-resistant Staphylococcus aureus and Enterococcus faecium. The minimum bactericidal concentration of NVC-422 against Gram-negative and Gram-positive bacteria ranged from 0.12 to 4 μg/ml. The minimum fungicidal concentrations against Candida albicans and Candida glabrata were 32 and 16 μg/ml, respectively. NVC-422 has an in vitro cytotoxicity (50% cytotoxicity = 1,440 μg/ml) similar to that of NNDCT. Moreover, our data showed that this agent possesses rapid, pH-dependent antimicrobial activity. At pH 4, NVC-422 completely killed both Escherichia coli and S. aureus within 5 min at a concentration of 32 μg/ml. Finally, the effect of NVC-422 in the treatment of an E. coli-infected granulating wound rat model was evaluated. Treatment of the infected granulating wound with NVC-422 resulted in significant reduction of the bacterial tissue burden and faster wound healing compared to a saline-treated control. These findings suggest that NVC-422 could have potential application as a topical antimicrobial.
doi:10.1128/AAC.00158-11
PMCID: PMC3101424  PMID: 21422212
5.  Cytotoxic Activity of N-Chlorotaurine on Acanthamoeba spp.▿  
Acanthamoeba spp. are the causative agents of Acanthamoeba keratitis (AK), which mainly occurs in contact lens wearers, and of skin lesions, granulomatous amoebic encephalitis (GAE), and disseminating diseases in the immunocompromised host. AK therapy is complex and irritating for the eye, skin lesions are difficult to treat, and there is no effective treatment for GAE. Therefore, new anti-Acanthamoeba drugs are needed. We investigated the anti-Acanthamoeba activity of N-chlorotaurine (NCT), an endogenous mild antiseptic. It was shown that NCT has amoebicidal qualities, both in phosphate-buffered saline (PBS) and in amoebic culture medium. After 6 h of treatment with 10 mM NCT in PBS, the levels of trophozoites of all strains investigated already showed at least a 2-log reduction. When the trophozoites were treated with 20 mM NCT in culture medium, they showed a 2-log reduction after 24 h. The addition of NH4Cl to NCT led to a faster decrease in the numbers of living cells, if tests were carried out in PBS. A delay of excystation was observed when cysts were treated with 55 mM (1%) NCT in culture medium. A complete failure of excystment was the result of treatment with 1% NCT plus 1% NH4Cl in PBS. Altogether, NCT clearly demonstrated amoebicidal activity at concentrations well tolerated by human tissues and might be useful as a topical drug for the treatment of Acanthamoeba infections. The addition of ammonium chloride can be considered to enhance the activity.
doi:10.1128/AAC.00715-07
PMCID: PMC2224745  PMID: 18039920
6.  Novel Trichomonacidal Spermicides▿†‡ 
Metronidazole, the U.S. Food and Drug Administration-approved drug against trichomoniasis, is nonspermicidal and thus cannot offer pregnancy protection when used vaginally. Furthermore, increasing resistance of Trichomonas vaginalis to 5-nitro-imidazoles is a cause for serious concern. On the other hand, the vaginal spermicide nonoxynol-9 (N-9) does not protect against sexually transmitted diseases and HIV in clinical situations but may in fact increase their incidence due to its nonspecific, surfactant action. We therefore designed dually active, nonsurfactant molecules that were capable of killing Trichomonas vaginalis (both metronidazole-susceptible and -resistant strains) and irreversibly inactivating 100% human sperm at doses that were noncytotoxic to human cervical epithelial (HeLa) cells and vaginal microflora (lactobacilli) in vitro. Anaerobic energy metabolism, cell motility, and defense against reactive oxygen species, which are key to survival of both sperm and Trichomonas in the host after intravaginal inoculation, depend crucially on availability of free thiols. Consequently, molecules were designed with carbodithioic acid moiety as the major pharmacophore, and chemical variations were incorporated to provide high excess of reactive thiols for interacting with accessible thiols on sperm and Trichomonas. We report here the in vitro activities, structure-activity relationships, and safety profiles of these spermicidal antitrichomonas agents, the most promising of which was more effective than N-9 (the OTC spermicide) in inactivating human sperm and more efficacious than metronidazole in killing Trichomonas vaginalis (including metronidazole-resistant strain). It also significantly reduced the available free thiols on human sperm and inhibited the cytoadherence of Trichomonas on HeLa cells. Experimentally in vitro, the new compounds appeared to be safer than N-9 for vaginal use.
doi:10.1128/AAC.00199-11
PMCID: PMC3165359  PMID: 21709091
7.  Trichomonas vaginalis and trichomoniasis in the Republic of Korea 
Vaginal trichomoniasis, caused by Trichomonas vaginalis, is the most common sexually transmitted disease. More than 170 million people worldwide are annually infected by this protozoan. In the Republic of Korea, 10.4% of women complaining of vaginal symptoms and signs were found to be infected with T. vaginalis. However, despite its high prevalence, the pathogenesis of T. vaginalis infection has not been clearly characterized although neutrophil infiltration is considered to be primarily responsible for the cytologic changes associated with this infection. We hypothesized that trichomonads in the vagina sometime after an acute infection secrete proteins like excretory-secretory product that have a chemotactic effect on neutrophils, and that these neutrophils are further stimulated by T. vaginalis to produce chemokines like IL-8 and GRO-α, which further promote neutrophil recruitment and chemotaxis. Thus, neutrophil accumulation is believed to maintain or aggravate inflammation. However, enhanced neutrophil apoptosis induced by live T. vaginalis could contribute to resolution of inflammation. Macrophages may constitute an important component of host defense against T. vaginalis infection. For example, mouse macrophages alone and those activated by lymphokines or nitric oxide are known to be involved in the extracellular killing of T. vaginalis. In the host, T. vaginalis uses a capping phenomenon to cleave host immunoglobulins with proteinases and thus escape from host immune responses. Recently, we developed a highly sensitive and specific diagnostic polymerase chain reaction (PCR) technique using primers based on a repetitive sequence cloned from T. vaginalis (TV-E650), and found that the method enables the detection of T. vaginalis at concentrations as low as 1 cell per PCR mixture.
doi:10.3347/kjp.2006.44.2.101
PMCID: PMC2532633  PMID: 16809958
Trichomonas vaginalis; trichomoniasis; pathogenesis; PCR; neutrophil; macrophage
8.  In vitro inhibition of Giardia lamblia and Trichomonas vaginalis growth by bithionol, dichlorophene, and hexachlorophene. 
Bithionol, dichlorophene, and hexachlorophene, which are used in treating some helminthic infections, killed trophozoites of Giardia lamblia and Trichomonas vaginalis in modified BI-S-33 and Asami media, respectively. Virtually all G. lamblia and T. vaginalis cells were killed within 24 h with a 0.42 mM concentration of these compounds, except that 0.93 mM dichlorophene was required for sterilizing T. vaginalis in the same period. In modified BI-S-33 and Asami media from which bovine and human sera were omitted, respectively, the inhibitory actions of the compounds against in vitro growth of these protozoa were significantly enhanced. Trophozoites of G. lamblia and T. vaginalis could be killed in shorter than 10 min with 0.074 mM dichlorophene and 0.0025 mM hexachlorophene, respectively, in serum-free media. G. lamblia, which was incubated in the complete medium containing dichlorophene, showed a characteristic swelling of the ventral side which led to disruption of the parasite, whereas bithionol caused a thin crack in the cytoplasm of T. vaginalis incubated in Asami medium. The crack appeared to enlarge and result in vacuolization of T. vaginalis. These observations suggest that bithionol, dichlorophene, and hexachlorophene merit further evaluation to ascertain whether they are useful for treatment of giardiasis and trichomoniasis.
Images
PMCID: PMC176206  PMID: 3872626
9.  Hydrogenosome Metabolism Is the Key Target for Antiparasitic Activity of Resveratrol against Trichomonas vaginalis 
Metronidazole (MDZ) and related 5-nitroimidazoles are the recommended drugs for treatment of trichomoniasis, a sexually transmitted disease caused by the protozoan parasite Trichomonas vaginalis. However, novel treatment options are needed, as recent reports have claimed resistance to these drugs in T. vaginalis isolates. In this study, we analyzed for the first time the in vitro effects of the natural polyphenol resveratrol (RESV) on T. vaginalis. At concentrations of between 25 and 100 μM, RESV inhibited the in vitro growth of T. vaginalis trophozoites; doses of 25 μM exerted a cytostatic effect, and higher doses exerted a cytotoxic effect. At these concentrations, RESV caused inhibition of the specific activity of a 120-kDa [Fe]-hydrogenase (Tvhyd). RESV did not affect Tvhyd gene expression and upregulated pyruvate-ferredoxin oxidoreductase (a hydrogenosomal enzyme) gene expression only at a high dose (100 μM). At doses of 50 to 100 μM, RESV also caused overexpression of heat shock protein 70 (Hsp70), a protective protein found in the hydrogenosome of T. vaginalis. The results demonstrate the potential of RESV as an antiparasitic treatment for trichomoniasis and suggest that the mechanism of action involves induction of hydrogenosomal dysfunction. In view of the results, we propose hydrogenosomal metabolism as a key target in the design of novel antiparasitic drugs.
doi:10.1128/AAC.00009-13
PMCID: PMC3716124  PMID: 23478970
10.  Alternative Pathway of Metronidazole Activation in Trichomonas vaginalis Hydrogenosomes 
Antimicrobial Agents and Chemotherapy  2005;49(12):5033-5036.
Metronidazole and related 5-nitroimidazoles are the only available drugs in the treatment of human urogenital trichomoniasis caused by the protozoan parasite Trichomonas vaginalis. The drugs are activated to cytotoxic anion radicals by their reduction within the hydrogenosomes. It has been established that electrons required for metronidazole activation are released from pyruvate by the activity of pyruvate:ferredoxin oxidoreductase and transferred to the drug by a low-redox-potential carrier, ferredoxin. Here we describe a novel pathway involved in the drug activation within the hydrogenosome. The source of electrons is malate, another major hydrogenosomal substrate, which is oxidatively decarboxylated to pyruvate and CO2 by NAD-dependent malic enzyme. The electrons released during this reaction are transferred from NADH to ferredoxin by NADH dehydrogenase homologous to the catalytic module of mitochondrial complex I, which uses ferredoxin as electron acceptor. Trichomonads acquire high-level metronidazole resistance only after both pyruvate- and malate-dependent pathways of metronidazole activation are eliminated from the hydrogenosomes.
doi:10.1128/AAC.49.12.5033-5036.2005
PMCID: PMC1315937  PMID: 16304169
11.  Chemical composition of essential oil and anti trichomonas activity of leaf, stem, and flower of Rheum ribes L. extracts 
Objectives: Trichomoniasis is one of the most common sexually transmitted diseases in humans and is caused by the protozoan Trichomonas vaginalis. Nowadays, increasing resistance to drugs such as metronidazole resulted in many problem, so new effective remedies are needed. In this study, we evaluate constituents of essential oil and anti-trichomonas activity of Rheum ribes.
Materials and Methods: The essential oil from Rheum ribes L. flower growing wild in Iran was analyzed by GC/MS. The parasites were treated with different extract and fractions of the flower, stem, and leave of the plant. Anti-trichomonas activity was evaluated using an in vitro assay.
Results: In all, 19 compounds were identified; palmitic acid [27.08%], n-eicosane [9.9%], n-tetracosane [7.34%], linoleic acid [6.56%], and ethyl linoleate [4.76%] were the main components of the oil. Rheum ribes extracts and fractions concentration dependently inhibited the ability of parasites to growth. This was associated with polarity of solvent used for fractionation and plant parts used for extraction.
Conclusion: Findings demonstrate the potential of Rheum ribes extracts as an anti-trichomonas agent for human use. Further studies are required to evaluate its toxicity and safety.
PMCID: PMC4104631  PMID: 25050317
Essential oil; Rheum ribes; Trichomonas vaginalis
12.  Down-regulation of flavin reductase and alcohol dehydrogenase-1 (ADH1) in metronidazole-resistant isolates of Trichomonas vaginalis 
Molecular and Biochemical Parasitology  2012;183-222(2):177-183.
Graphical abstract
In isolates of Trichomonas vaginalis with reduced susceptibility metronidazole, flavin reductase and alcohol dehydrogenase-1 (ADH1) activities are down-regulated.
Highlights
► In clinical isolates of Trichomonas vaginalis with reduced metronidazole susceptibility flavin reductase is down-regulated. ► In clinical isolates of T. vaginalis with reduced metronidazole susceptibility alcohol dehydrogenase-1 (ADH1) is down-regulated. ► Thioredoxin reductase levels are not changed in metronidazole-resistant T. vaginalis clinical isolates.
The microaerophilic parasite Trichomonas vaginalis is a causative agent of painful vaginitis or urethritis, termed trichomoniasis, and can also cause preterm delivery or stillbirth. Treatment of trichomoniasis is almost exclusively based on the nitroimidazole drugs metronidazole and tinidazole. Metronidazole resistance in T. vaginalis does occur and is often associated with treatment failure. In most cases, metronidazole-resistant isolates remain susceptible to tinidazole, but cross resistance between the two closely related drugs can be a problem.
In this study we measured activities of thioredoxin reductase and flavin reductase in four metronidazole-susceptible and five metronidazole-resistant isolates. These enzyme activities had been previously found to be downregulated in T. vaginalis with high-level metronidazole resistance induced in the laboratory. Further, we aimed at identifying factors causing metronidazole resistance and compared the protein expression profiles of all nine isolates by application of two-dimensional gel electrophoresis (2DE).
Thioredoxin reductase activity was nearly equal in all strains assayed but flavin reductase activity was clearly down-regulated, or even absent, in metronidazole-resistant strains. Since flavin reductase has been shown to reduce oxygen to hydrogen peroxide, its down-regulation could significantly contribute to the impairment of oxygen scavenging as reported by others for metronidazole-resistant strains. Analysis by 2DE revealed down-regulation of alcohol dehydrogenase 1 (ADH1) in strains with reduced sensitivity to metronidazole, an enzyme that could be involved in detoxification of intracellular acetaldehyde.
doi:10.1016/j.molbiopara.2012.03.003
PMCID: PMC3341570  PMID: 22449940
Trichomonosis; Metronidazole resistance; Thiordoxin reductase; Flavin reductase; Alcohol dehydrogenase 1
13.  Murine Models of Vaginal Trichomonad Infections 
Trichomonas vaginalis and Tritrichomonas foetus cause common sexually transmitted infections in humans and cattle, respectively. Mouse models of trichomoniasis are important for pathogenic and therapeutic studies. Here, we compared murine genital infections with T. vaginalis and T. foetus. Persistent vaginal infection with T. foetus was established with 100 parasites but T. vaginalis infection required doses of 106, perhaps because of greater susceptibility to killing by mouse vaginal polymorphonuclear leukocytes. Infection with T. vaginalis persisted longest after combined treatment of mice with estrogen and dexamethasone, whereas infection was only short-lived when mice were given estrogen or dexamethasone alone, co-infected with Lactobacillus acidophilus, and/or pretreated with antibiotics. Infection rates were similar with metronidazole-resistant (MR) and metronidazole-sensitive (MS) T. vaginalis. High dose but not low dose metronidazole treatment controlled infection with MS better than MR T. vaginalis. These murine models will be valuable for investigating the pathogenesis and treatment of trichomoniasis.
doi:10.4269/ajtmh.2011.11-0123
PMCID: PMC3183775  PMID: 21976570
14.  Trichomonad invasion of the mucous layer requires adhesins, mucinases, and motility 
Sexually Transmitted Infections  1999;75(4):231-238.
BACKGROUND/OBJECTIVE: Trichomonas vaginalis, the causal agent of trichomonosis, is a flagellated parasitic protozoan that colonises the epithelial cells of the human urogenital tract. The ability of T vaginalis to colonise this site is in part a function of its ability to circumvent a series of non-specific host defences including the mucous layer covering epithelial cells at the site of infection. Mucin, the framework molecule of mucus, forms a lattice structure that serves as a formidable physical barrier to microbial invasion. The mechanism by which trichomonads traverse the mucous covering is unknown. Proteolytic degradation of mucin, however, may provide for a mechanism to penetrate this layer. The goal, therefore, was to determine how trichomonads cross through a mucous layer. METHODS: Secreted trichomonad proteinases were analysed for mucinase activity by mucin substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The importance of trichomonad mucinases for traversing the mucous layer was examined on an artificial mucin layer in invasion chambers. Adherence to mucin and tissue culture cells was measured using a microtitre plate assay. RESULTS: Trichomonad isolate 24402 secreted five proteinases when incubated in PBS. All five proteinases were shown to possess mucinase activity. These mucinases were able to degrade bovine submaxillary mucin and to a lesser extent porcine stomach mucin. These enzymes were active over a pH range of 4.5-7.0 and were inhibited with cysteine proteinase inhibitors. Furthermore, T vaginalis was shown to bind to mucin possibly via a lectin-like adhesin. Adherence to mucin was increased threefold when parasites were grown in iron deficient medium. Adherence to soluble mucin prevented attachment to HeLa cells. Proteinase activity, adherence, and motility were required for trichomonads to traverse a mucin layer in vitro. CONCLUSIONS: These results show that trichomonads can traverse the mucous barrier first by binding mucin followed by its proteolytic degradation. The data further underscore the importance of trichomonad proteinases in the pathogenesis of trichomonosis. Finally, this study suggests that interference with trichomonad mucin receptors and proteinases may be a strategy to prevent colonisation by this parasite. 



PMCID: PMC1758222  PMID: 10615308
15.  Analysis of human immunoglobulin-degrading cysteine proteinases of Trichomonas vaginalis. 
Infection and Immunity  1995;63(9):3388-3395.
Trichomonas vaginalis is a protozoan parasite that causes a widely distributed sexually transmitted disease (STD). Since immunoglobulin G (IgG) antibodies to specific trichomonad immunogens are found in serum and vaginal washes (VWs) from patients with trichomoniasis, a potential mechanism of immune evasion by this parasite might be the ability of T. vaginalis proteinases to degrade human immunoglobulins (Igs). Incubation of human IgG with lysates of T. vaginalis organisms resulted in time- and concentration-dependent degradation of the heavy chain. Secretory IgA was degraded similarly. Inhibitors of cysteine proteinases, when added to trichomonal lysates, abolished IgG and IgA degradation, while EDTA, a metalloproteinase inhibitor, did not. Substrate-gel electrophoresis with human IgG, IgM, or IgA copolymerized with acrylamide revealed several distinct cysteine proteinases in both lysates and culture supernatants from logarithmically growing parasites that degraded all classes of human antibodies. Trichomonal lysates and supernatants of numerous isolates tested all had Ig-degrading activity. Finally, proteolytic activity against IgG was detected in most (26 of 33; 78%) VWs from patients with trichomoniasis. In contrast, 18 of 28 (65%) VWs from women without trichomoniasis or from patients infected with other STDs had no detectable proteinases when tested in an identical manner. The other 10 of these 28 VWs (35%) had smaller amounts of detectable Ig-degrading proteinases. These differences in Ig-degrading proteinase activity between patients with and without trichomoniasis, regardless of coinfecting STDs, were statistically significant (P = 0.001). These results illustrate that T. vaginalis is capable of degrading human Igs.
PMCID: PMC173466  PMID: 7642267
16.  Production of Interleukin-8 by Human Neutrophils Stimulated with Trichomonas vaginalis  
Infection and Immunity  2004;72(3):1326-1332.
Neutrophils are the predominant inflammatory cells found in the vaginal discharges of patients infected with Trichomonas vaginalis. Although chemoattractants, such as leukotriene B4 and interleukin-8 (IL-8), are found in the vaginal discharges of symptomatic trichomoniasis patients, little is known about the mechanism of how neutrophils accumulate or mediate initial inflammatory response after acute T. vaginalis infection. We examined IL-8 production in neutrophils activated by T. vaginalis and evaluated the factors involved in T. vaginalis adherence that might affect IL-8 production. When human neutrophils were stimulated with live trophozoites, T. vaginalis lysate, or T. vaginalis excretory-secretory products, the live trichomonads induced higher levels of IL-8 production than the lysate or products did. When live trichomonads were pretreated with various inhibitors of proteinase, microtubule, microfilament, or adhesin (which are all known to participate in the adherence of T. vaginalis to vaginal epithelial cells), IL-8 production significantly decreased compared with the untreated controls. Furthermore, an NF-κB inhibitor (pyrrolidine dithiocarbamate), a mitogen-activated protein (MAP) kinase (MEK) inhibitor (PD98059), and a p38 MAP kinase inhibitor (SB203580) significantly suppressed IL-8 synthesis in neutrophils. These results suggest that live T. vaginalis, particularly adherent trophozoites, can induce IL-8 production in neutrophils and that this action may be mediated through the NF-κB and MAP kinase signaling pathways. In other words, T. vaginalis-induced neutrophil recruitment may be mediated via the IL-8 expressed by neutrophils in response to activation by live T. vaginalis.
doi:10.1128/IAI.72.3.1326-1332.2004
PMCID: PMC355987  PMID: 14977935
17.  Trichomonas vaginalis NYH286 phenotypic variation may be coordinated for a repertoire of trichomonad surface immunogens. 
Infection and Immunity  1987;55(9):1957-1962.
Trichomonas vaginalis isolate NYH286 was fractionated with immunoglobulin G of sera from patients with trichomoniasis. Subpopulations of trichomonads with phenotypes of either patient serum-immunoglobulin G reactive (PS+) or nonreactive (PS-) were obtained. Flow cytofluorometry of PS+ and PS- subpopulations with a monoclonal antibody called C20A3 which reacts with a high-molecular-weight immunogen of T. vaginalis gave corresponding fluorescent (positive) and nonfluorescent (negative) phenotypes. No relationship was seen between PS+ and PS- phenotypes and binding of soybean agglutinin, wheat germ agglutinin, and concanavalin A, showing that PS- organisms still possessed carbohydrate moieties on their surfaces based on lectin binding. Phenotypic variation among the PS+ and PS- trichomonads was observed during in vitro growth. A positive-to-negative phenotype shift was also recorded for parasites obtained from lesions of mice subcutaneously infected with PS+ trichomonads. The involvement of surface proteins in the differential PS+ and PS- reactions was supported by soluble antigen and whole cell radioimmunoprecipitation assays. Finally, enhanced parasitism and killing of HeLa cells in monolayer cultures were observed for PS- subpopulations as compared with PS+ counterparts. The data support the idea that phenotypic variation for T. vaginalis may be coordinated for a repertoire of trichomonad immunogens and that such membrane dynamics influence expression of virulence determinants for these sexually transmitted disease agents.
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PMCID: PMC260640  PMID: 3497876
18.  Strain of Trichomonas vaginalis Resistant to Metronidazole and Other 5-Nitroimidazoles 
A strain of Trichomonas vaginalis (IR-78), recently isolated from a patient afflicted with recurrent symptomatic trichomoniasis, showed resistance to metronidazole, tinidazole, and nimorazole in vitro as well as in vivo. In a serial dilution test using cysteine monohydrochloride–peptone–liver infusion–maltose medium, T. vaginalis IR-78 was only resistant under aerobic conditions. Under anaerobic conditions it was as susceptible as the normal reference strain. The minimal lethal concentrations of metronidazole, tinidazole, and nimorazole for IR-78 were 100, 50, and 50 μg/ml aerobically and 0.4, 0.4, and 0.2 μg/ml anaérobically, respectively. The efficacy of metronidazole, tinidazole, and nimorazole was assessed in vivo by oral administration to mice simultaneously infected with IR-78 both subcutaneously and intraperitoneally. The CD50 (dose needed to cure 50% of infections) of each compound was significantly higher for the subcutaneous than for the intraperitoneal infection. In contrast, there was little difference in CD50 for these infections in mice inoculated with a susceptible trichomonas strain. The CD50's for all three compounds against intraperitoneal and subcutaneous infections with IR-78 were 2 to >70 times higher than for susceptible strain E. Both forms of infection with IR-78 could always be cured with therapeutically acceptable doses of tinidazole and nimorazole; subcutaneous infections could not be cured with tolerated doses of metronidazole.
PMCID: PMC352642  PMID: 311617
19.  Extensive Genetic Diversity, Unique Population Structure and Evidence of Genetic Exchange in the Sexually Transmitted Parasite Trichomonas vaginalis 
Background
Trichomonas vaginalis is the causative agent of human trichomoniasis, the most common non-viral sexually transmitted infection world-wide. Despite its prevalence, little is known about the genetic diversity and population structure of this haploid parasite due to the lack of appropriate tools. The development of a panel of microsatellite makers and SNPs from mining the parasite's genome sequence has paved the way to a global analysis of the genetic structure of the pathogen and association with clinical phenotypes.
Methodology/Principal Findings
Here we utilize a panel of T. vaginalis-specific genetic markers to genotype 235 isolates from Mexico, Chile, India, Australia, Papua New Guinea, Italy, Africa and the United States, including 19 clinical isolates recently collected from 270 women attending New York City sexually transmitted disease clinics. Using population genetic analysis, we show that T. vaginalis is a genetically diverse parasite with a unique population structure consisting of two types present in equal proportions world-wide. Parasites belonging to the two types (type 1 and type 2) differ significantly in the rate at which they harbor the T. vaginalis virus, a dsRNA virus implicated in parasite pathogenesis, and in their sensitivity to the widely-used drug, metronidazole. We also uncover evidence of genetic exchange, indicating a sexual life-cycle of the parasite despite an absence of morphologically-distinct sexual stages.
Conclusions/Significance
Our study represents the first robust and comprehensive evaluation of global T. vaginalis genetic diversity and population structure. Our identification of a unique two-type structure, and the clinically relevant phenotypes associated with them, provides a new dimension for understanding T. vaginalis pathogenesis. In addition, our demonstration of the possibility of genetic exchange in the parasite has important implications for genetic research and control of the disease.
Author Summary
The human parasite Trichomonas vaginalis causes trichomoniasis, the world's most common non-viral sexually transmitted infection. Research on T. vaginalis genetic diversity has been limited by a lack of appropriate genotyping tools. To address this problem, we recently published a panel of T. vaginalis-specific genetic markers; here we use these markers to genotype isolates collected from ten regions around the globe. We detect high levels of genetic diversity, infer a two-type population structure, identify clinically relevant differences between the two types, and uncover evidence of genetic exchange in what was believed to be a clonal organism. Together, these results greatly improve our understanding of the population genetics of T. vaginalis and provide insights into the possibility of genetic exchange in the parasite, with implications for the epidemiology and control of the disease. By taking into account the existence of different types and their unique characteristics, we can improve understanding of the wide range of symptoms that patients manifest and better implement appropriate drug treatment. In addition, by recognizing the possibility of genetic exchange, we are more equipped to address the growing concern of drug resistance and the mechanisms by which it may spread within parasite populations.
doi:10.1371/journal.pntd.0001573
PMCID: PMC3313929  PMID: 22479659
20.  Pharmacodynamics of metronidazole determined by a time-kill assay for Trichomonas vaginalis. 
The pharmacodynamic effects of metronidazole on Trichomonas vaginalis have been poorly characterized. The present in vitro study was performed to characterize the relationship between killing of trichomonads and metronidazole exposure (metronidazole concentration and time of exposure). Five laboratory strains and five recent clinical isolates of T. vaginalis were studied. The minimum lethal concentrations (MLCs) of metronidazole for the strains ranged from 0.8 to 25 micrograms/ml under anaerobic conditions. Metronidazole exhibited concentration-dependent killing against T. vaginalis at concentrations ranging from 0.1 to > 10 times the MLC. The endpoint measurement, the kill rate constant, which was derived from the reduction in the logarithm of the colony count divided by exposure time, compared with the kill rate constant for the growth control was not affected by the time of assessment between 2 and 24 h. The kill rate constant-versus-metronidazole exposure curves were similar when concentration was expressed as a multiple of the MLC. There were no apparent differences between the clinical isolates and laboratory strains. These data suggest that peak metronidazole concentration and/or area under the plasma concentration-versus-time curve are the important pharmacodynamic parameters to be optimized.
PMCID: PMC162837  PMID: 7486930
21.  Modulation of dendritic cell function by Trichomonas vaginalis-derived secretory products 
BMB Reports  2015;48(2):103-108.
Trichomoniasis caused by the parasitic protozoan Trichomonas vaginalis is the most common sexually transmitted disease in the world. Dendritic cells are antigen presenting cells that initiate immune responses by directing the activation and differentiation of naïve T cells. In this study, we analyzed the effect of Trichomonas vaginalis-derived Secretory Products on the differentiation and function of dendritic cells. Differentiation of bone marrow-derived dendritic cells in the presence of T. vaginalis-derived Secretory Products resulted in inhibition of lipopolysaccharide-induced maturation of dendritic cells, down-regulation of IL-12, and up-regulation of IL-10. The protein components of T. vaginalis-derived Secretory Products were shown to be responsible for altered function of bone marrow-derived dendritic cells. Chromatin immunoprecipitation assay demonstrated that IL-12 expression was regulated at the chromatin level in T. vaginalis-derived Secretory Productstreated dendritic cells. Our results demonstrated that T. vaginalis-derived Secretory Products modulate the maturation and cytokine production of dendritic cells leading to immune tolerance. [BMB Reports 2015; 48(2): 103-108]
doi:10.5483/BMBRep.2015.48.2.116
PMCID: PMC4352611  PMID: 24965578
Chromatin; Cytokine; Dendritic cells; Tolerance; Trichomonas vaginalis
22.  Treatment of Infections Caused by Metronidazole-Resistant Trichomonas vaginalis 
Clinical Microbiology Reviews  2004;17(4):783-793.
Infections with the sexually transmitted protozoan Trichomonas vaginalis are usually treated with metronidazole, a 5-nitroimidazole drug derived from the antibiotic azomycin. Metronidazole treatment is generally efficient in eliminating T. vaginalis infection and has a low risk of serious side effects. However, studies have shown that at least 5% of clinical cases of trichomoniasis are caused by parasites resistant to the drug. The lack of approved alternative therapies for T. vaginalis treatment means that higher and sometimes toxic doses of metronidazole are the only option for patients with resistant disease. Clearly, studies of the treatment and prevention of refractory trichomoniasis are essential. This review describes the mechanisms of metronidazole resistance in T. vaginalis and provides a summary of trichomonicidal and vaccine candidate drugs.
doi:10.1128/CMR.17.4.783-793.2004
PMCID: PMC523556  PMID: 15489348
23.  Canine prostatic secretions kill Trichomonas vaginalis. 
Infection and Immunity  1982;37(1):77-81.
The zinc content of prostatic secretions is thought to be an important nonspecific defense against urinary tract infection in men. This investigation measured killing by prostatic fluid of Trichomonas vaginalis, a common sexually transmitted pathogen, and related this activity to zinc concentration. We used a canine model which closely resembles the human male genital tract. Prostatic secretions from all dogs killed all T. vaginalis isolates. There appear to be several mechanisms for killing of trichomonads by prostatic fluid. At prostatic fluid zinc concentrations comparable to those in normal men (greater than or equal to 3.2 mM), the rate of killing of trichomonads was proportional to the zinc concentration. At intermediate zinc levels, killing occurred by both zinc-dependent and zinc-independent mechanisms. A zinc-independent mechanism was responsible for antitrichomonal activity at relatively low zinc levels (less than 1.6 mM), comparable to those in the prostatic fluid of men with chronic prostatitis. This study suggests that the variable clinical spectrum of trichomoniasis in men may result from a balance between the zinc sensitivity of the T. vaginalis strains on one side and the content of both zinc and zinc-independent factors in prostatic fluid on the other.
PMCID: PMC347493  PMID: 6980837
24.  Clinical and Microbiological Aspects of Trichomonas vaginalis 
Clinical Microbiology Reviews  1998;11(2):300-317.
Trichomonas vaginalis, a parasitic protozoan, is the etiologic agent of trichomoniasis, a sexually transmitted disease (STD) of worldwide importance. Trichomoniasis is the most common nonviral STD, and it is associated with many perinatal complications, male and female genitourinary tract infections, and an increased incidence of HIV transmission. Diagnosis is difficult, since the symptoms of trichomoniasis mimic those of other STDs and detection methods lack precision. Although current treatment protocols involving nitroimidazoles are curative, metronidazole resistance is on the rise, outlining the need for research into alternative antibiotics. Vaccine development has been limited by a lack of understanding of the role of the host immune response to T. vaginalis infection. The lack of a good animal model has made it difficult to conduct standardized studies in drug and vaccine development and pathogenesis. Current work on pathogenesis has focused on the host-parasite relationship, in particular the initial events required to establish infection. These studies have illustrated that the pathogenesis of T. vaginalis is indeed very complex and involves adhesion, hemolysis, and soluble factors such as cysteine proteinases and cell-detaching factor. T. vaginalis interaction with the members of the resident vaginal flora, an advanced immune evasion strategy, and certain stress responses enable the organism to survive in its changing environment. Clearly, further research and collaboration will help elucidate these pathogenic mechanisms, and with better knowledge will come improved disease control.
PMCID: PMC106834  PMID: 9564565
25.  Early Repeated Infections with Trichomonas vaginalis among HIV-Positive and HIV-Negative Women 
Background
The purpose of the study was to examine whether early repeated infections due to Trichomonas vaginalis among human immunuodeficiency virus (HIV)–positive and HIV-negative women are reinfections, new infections, or cases of treatment failure.
Methods
Women attending an HIV outpatient clinic and a family planning clinic in New Orleans, Louisiana, who had culture results positive for T. vaginalis were treated with 2 g of metronidazole under directly observed therapy. At 1 month, detailed sexual exposure and sexual partner treatment information was collected. Isolates from women who had clinical resistance (i.e., who tested positive for a third time after treatment at a higher dose) were tested for metronidazole susceptibility in vitro.
Results
Of 60 HIV-positive women with trichomoniasis, 11 (18.3%) were T. vaginalis positive 1 month after treatment. The 11 recurrences were classified as 3 probable reinfections (27%), 2 probable infections from a new sexual partner (18%), and 6 probable treatment failures (55%); 2 of the 6 patients who experienced probable treatment failure had isolates with mild resistance to metronidazole. Of 301 HIV-negative women, 24 (8.0%) were T. vaginalis positive 1 month after treatment. The 24 recurrences were classified as 2 probable reinfections (8%) and 22 probable treatment failures (92%); of the 22 patients who experienced probable treatment failure, 2 had strains with moderate resistance to metronidazole, and 1 had a strain with mild resistance to metronidazole.
Conclusion
HIV-positive women were more likely to have sexual re-exposure than were HIV-negative women, although the rate of treatment failure was similar in both groups. High rates of treatment failure among both HIV-positive and HIV-negative women indicate that a 2-g dose of metronidazole may not be adequate for treatment of some women and that rescreening should be considered.
doi:10.1086/529149
PMCID: PMC3855851  PMID: 18444815

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