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PubMed1989

Cervical intraepithelial neoplasia among women with papillomavirus infection compared to women with Trichomonas infection.

This study was undertaken to determine if women with papillomavirus infection were more likely to develop cervical intraepithelial neoplasia (CIN) than women with other sexually transmitted infections. Women with cytologic evidence of papillomavirus infection and a comparison group of women with cytologic evidence of Trichomonas infection were identified from a reexamination of cervicovaginal smears initially evaluated between 1973 and 1981 in a private pathology laboratory. To identify subsequent CIN among these women the authors reviewed records of private obstetric and gynecology practices and public clinics through 1983. After accounting for possible confounding variables, including age, marital status, frequency of cervical cytologic examination, and selected characteristics of pregnancy history, a 2.7-fold increase in the rate of CIN was found among the 613 women with prior papillomavirus infection compared to the 410 women with previous Trichomomas infection. The rate ratio was higher among women with more severe cervical neoplasia than for those with less severe neoplasia and for those followed for greater than 12 months than for those followed 7 to 12 months. The results of this study provide further evidence of the role of papillomavirus in the etiology of cervical cancer.

Adult↗
PubMed1994

Determination of the viability of Trichomonas vaginalis using flow cytometry.

In clinical laboratories, viability of Trichomonas vaginalis is determined by using light microscopy (differential count of motile to nonmotile organisms). Alternative methods are proposed that utilise flow cytometry. Under an epifluorescence microscope, live organisms fluorescence intensely green with fluorescein diacetate (FDA), whereas dead cells fluoresce orange with propidium iodide (PI). Flow cytometric histograms of green versus red fluorescence reveal distinct populations for live and dead cells. The anionic oxonal probe DiBAC4(3) is a membrane potential sensitive dye that distributes between the inside of the cell and the medium. Live organisms are less fluorescent than dead organisms when stained with the oxonol probe. Valinomycin, dicyclohexylcarbodiimide, and vanadate all give significant changes in the fluorescence intensities of cultures stained with the oxonol probe compared with control cultures, indicating that this probe is detecting changes in plasma membrane potential. Both FDA/PI and oxonol staining protocols allow good discrimination between populations and permit counts that are more statistically significant than those obtained by light microscopy. These methods remove the subjectiveness of microscopic counts and would increase the accuracy of susceptibility assays.

Animals↗
PubMed2005

Trichomonas vaginalis P16 Immunoreactivity in cervicovaginal Pap tests: a diagnostic pitfall.

Detection of p16 is emerging as a useful biomarker for Human Papillomavirus (HPV)-related dysplastic and malignant lesions of the cervix, and as such has potential application to cervicovaginal Papanicolaou (Pap) specimens. While evaluating p16 immunocytochemistry in our laboratory we observed one Pap test in which Trichomonas vaginalis stained positively for p16. We therefore proceeded to determine the frequency of T. vaginalis immunoreactivity for p16 in 10 consecutive, satisfactory, liquid-based Pap tests diagnosed as negative for intraepithelial lesion or malignancy in which T. vaginalis was present. For each case, a ThinPrep slide prepared from residual vial material was immunostained with p16. In an additional case, a prepared cell block was stained with p16. T. vaginalis were consistently p16 positive. We document, for the first time, p16 immunoreactivity of T. vaginalis that, on a Pap slide and cell block, may be morphologically misinterpreted as small dysplastic or malignant epithelial cells. The presence of this parasite in Pap tests may also potentially hinder the use of p16 as an adjunct to liquid-based cervical screening cytology.

Adolescent↗
PubMedDate not supplied

Gonorrhea, Trichomonas and Candida among gravid and nongravid women in cameroon.

A case-control study of 1358 pregnant and nonpregnant women with and without symptoms of a lower genital tract infection were investigated for evidence of venereal disease. The prevalence of gonorrhea, Trichomonas and Candida in pregnancy was 15.0%, 20.6% and 41.3%, respectively, while the equivalent percentages in the nonpregnant women varied from 16.5% to 21.6%, 2.4% to 22.8% and 33.3% to 39.3%. However, these differences are not statistically significant. The implications of the findings are discussed and a screening program suggested.

Cameroon↗
PubMed1981

In vitro susceptibility of Trichomonas vaginalis to zinc.

The prostate has long been considered to be a site of sequestration of Trichomonas vaginalis. From this reservoir, organisms are thought to be shed during intercourse and produce repeated infections in the female genitourinary tract. Bacteria known to cause prostatitis are inhibited by concentrations of zinc found in uninfected prostatic secretions. The present study demonstrates a similar inhibitory effect of zinc on the growth of T. vaginalis. This finding would suggest that, in otherwise healthy males, the prostate is not likely to be a reservoir for latent infection by these organisms.

In Vitro Techniques↗
PubMed2002

A form of cell death with some features resembling apoptosis in the amitochondrial unicellular organism Trichomonas vaginalis.

One of hallmarks of apoptosis is the degradation and concomitant compaction of chromatin. It is assumed that caspases and caspase-independent pathways are rate limiting for the development of nuclear apoptosis. The caspase-independent pathway involves apoptosis-inducing factor (AIF) and leads to DNA fragmentation and peripheral chromatin condensation. Both pathways are the result of activation of death signals that the mitochondrion receives, integrates, and responds to with the release of various molecules (e.g., cytochrome c and AIF). In fact, both pathways have in common the final point of the DNA fragmentation and the mitochondrial origin of molecules that initiate the apoptotic events. Here, we examine the question of whether apoptosis or apoptotic-like processes exist in a unicellular organism that lacks mitochondria. We herein show that a form of cell death with some features resembling apoptosis is indeed present in Trichomonas vaginalis. Characterization of morphological aspects implicated in this event together with the preliminary biochemical data provided may lead to new insight about the evolutionary relationships between the different forms of programmed cell death identified so far.

Animals↗
PubMed1994

The intracellular amino acid pools of Giardia intestinalis, Trichomonas vaginalis, and Crithidia luciliae.

The total intracellular amino acid profiles of Giardia intestinalis trophozoites, Trichomonas vaginalis, and Crithidia luciliae were determined by sensitive amino acid analysis. The three protozoan parasites exhibited distinctively different amino acid profiles, but all three were dominated by high concentrations of intracellular alanine. This common feature suggests that alanine synthesis is a major aspect of intermediary metabolism in these protozoan parasites. There were also distinctively different aspects, particularly those related to arginine metabolism. Ornithine, citrulline, and ammonia were found in G. intestinalis trophozoites, but no intracellular arginine was detected. This pattern is consistent with the high activity of giardial arginine deiminase and the arginine dihydrolase pathway. However, in contrast, both T. vaginalis and C. luciliae contained considerable intracellular pools of arginine. When the G. intestinalis trophozoites were divided into the two populations existing in in vitro culture--attached and nonattached--there were no significant differences between the amino acid profiles of the two populations, with the exception of citrulline, which was found in lower concentrations in the nonattached cells. The T. vaginalis profile was characterised by high concentrations of valine and leucine, whereas the C. luciliae profile was dominated by high levels of glutamate and proline. Overall, the analysis of the total amino acid pool provides a valuable technique to rapidly highlight those amino acids of potential metabolic significance and to provide a rapid technique for defining the nature of amino acid metabolic interactions in situ.

Amino Acids↗
PubMed1998

Trichomonas vaginalis and Tritrichomonas foetus: expression of chitin at the cell surface.

The expression of chitin as a structural component of Trichomonas vaginalis and Tritrichomonas foetus was demonstrated by using enzymatic hydrolysis by recombinant (rec-) chitinase, chemical analysis, lectin, fluorescent Calcofluor and antibody binding, glycosidases of known specificity, high-performance liquid chromatography (HPLC), and flow cytometry. Chitinous structures were characterized by their insolubility in hot alkali and by releasing glucosamine on hydrolysis with 6 N HCl. N,N'-Diacetylchitobiose and N,N,'N''-triacetylchitotriose were identified by HPLC as enzymatic hydrolysis products of the alkali-resistant polysaccharide. The location of chitin on the surface of T. vaginalis and T. foetus was inferred from the decreased reactivity with whole parasites of ligands such as Lycopersicon esculentum (TOL) and Solanum tuberosum lectins, fluorescent Calcofluor, and anti-chitin antibody, after cell treatment with rec-chitinase. Binding of [125I]TOL showed that, in T. vaginalis and T. foetus, the numbers of lectin receptors per cell were 4.2 x 10(5) and 3.0 x 10(5), respectively. Binding of the lectin to the trichomonad surface was markedly decreased by treatment with rec-chitinase. TOL interaction with the parasites was not affected by N-acetyl-beta-D-glucosaminidase treatment, showing that the lectin receptors consisted of beta-linked GlcNAc polymers and not of terminal beta-linked GlcNAc residues.

Animals↗
PubMed1998

Trichomonas vaginalis: expression and characterisation of recombinant S-adenosylhomocysteinase.

The gene encoding S-adenosylhomocysteinase activity (S-adenosylhomocysteine hydrolase, SAHH; EC 3.3.1.1) in Trichomonas vaginalis has been expressed in Escherichia coli to facilitate the characterisation of the enzyme. Expression of this gene using the pQE-30 (6xHis N-terminal tag) expression system (QIAGEN) has enabled the one-step purification of 6 mg of active recombinant enzyme from a 100-ml bacterial culture by affinity chromatography using a nickel-NTA matrix. The recombinant enzyme has a molecular weight of approximately 56,000 and identification of tryptic peptides by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry has shown that the purified recombinant protein is identical in primary structure to the predicted sequence. The presence of the N-terminal 6xHis tag in the recombinant enzyme did not appear to affect its kinetic and other properties, which are similar to those exhibited by the "native" enzyme present in cell-free extracts of T. vaginalis. These properties include a similar apparent Km for adenosine (20-25 microM for the recombinant and 5-10 microM for the native enzymes, respectively) and similar inhibition/inactivation patterns exhibited by adenosine analogues such as arabinosyl adenine (ara-A).

Adenosylhomocysteinase↗
PubMed2000

Trichomonas vaginalis: characterization, expression, and phylogenetic analysis of a carbamate kinase gene sequence.

The gene encoding carbamate kinase (CBK, ATP:carbamate phosphotransferase, EC 2.7.2.2) from Trichomonas vaginalis has been sequenced and its expression in this protozoon has been verified using reverse-transcription polymerase chain reaction. The codon usage and percentage nucleotide composition in the coding and noncoding regions are consistent with other genes isolated from this parasite. Phylogenetic analysis of this gene has suggested possible speciation events that are congruent with other studies, with suggestions of lateral gene transfer. The gene was expressed in Escherichia coli using the pQE-30 expression system, and the recombinant protein was purified using affinity chromatography. The expression of the recombinant protein was identified via Western blotting and matrix-assisted laser desorption ionization mass spectrometry of tryptic peptides. Preliminary kinetic assays have revealed that the recombinant enzyme has a K(m) similar to that of the native enzyme and size-exclusion chromatography has shown that the enzyme is active as the homodimer.

Amino Acid Sequence↗
PubMed2000

Trichomonas vaginalis: chromatin and mitotic spindle during mitosis.

The mitotic phases and the changes that the chromatin and mitotic microtubules undergo during mitosis in the sexually transmitted parasite Trichomonas vaginalis are described. Parasites arrested in the gap 2 phase of the cell cycle by nutrient starvation were induced to mitosis by addition of fresh whole medium. [(3)H] Thymidine labeling of trichomonad parasites for 24 h showed that parasites have at least four synchronic duplications after mitosis induction. Fixed or live and acridine orange (AO)-stained trichomonads analyzed at different times during mitosis by epifluorescence microscopy showed that mitosis took about 45 min and is divided into five stages: prophase, metaphase, early and late anaphase, early and late telophase, and cytokinesis. The AO-stained nucleus of live trichomonads showed green (DNA) and orange (RNA) fluorescence, and the nucleic acid nature was confirmed by DNase and RNase treatment, respectively. The chromatin appeared partially condensed during interphase. At metaphase, it appeared as six condensed chromosomes, as recently reported, which decondensed at anaphase and migrated to the nuclear poles at telophase. In addition, small bundles of microtubules (as hemispindles) were detected only in metaphase with the polyclonal antibody anti-Entamoeba histolytica alpha-tubulin. This antibody showed that the hemispindle and an atractophore-like structure seem to duplicate and polarize during metaphase. In conclusion, T. vaginalis mitosis involves five mitotic phases in which the chromatin undergoes different degrees of condensation, from chromosomes to decondensed chromatin, and two hemispindles that are observed only in the metaphase stage.

Acridine Orange↗
PubMed1995

Iron mediates Trichomonas vaginalis resistance to complement lysis.

Trichomonas vaginalis, a sexually transmitted disease agent in humans, is readily lysed by activation of the alternative complement pathway. The parasite became resistant following growth in medium supplemented by iron compared to parasites grown in medium depleted of iron, which were readily killed by complement. The resistance to complement was dependent on iron concentration while divalent cations other than iron were ineffective, showing specific regulation of this property by iron. Lactoferrin, but not transferrin, rendered low-iron-parasites resistant to complement lysis, reinforcing the in vivo modulation by a known source of iron for this parasite. Pretreatment of high-iron, complement-resistant parasites with proteinase inhibitors resulted in lysis by complement, indicating that resistance was likely due to proteinase degradation of C3 on the trichomonal surface.

Animals↗
PubMed1996

Trichomonas vaginalis haemolysis: pH regulates a contact-independent mechanism based on pore-forming proteins.

There is a controversy in literature about involvement of secreted factors in the pathogenetic mechanisms of Trichomonas vaginalis, described mostly as contact-dependent. We found that the protozoan, under triggering conditions, is able to release molecules that lead to lysis without direct contact between parasite and target cells as a prerequisite. In this paper we characterize contact-independent cytotoxicity using the red blood cell as a cellular model. Contact-independent haemolysis is a phenomenon were pH exerts a key role, triggering the secretion of a lytic molecule and regulating its activity. A partial physicochemical characterization of the haemolytic factor suggests that a protein of M(r) > 30 kDa could be the effector responsible for damage. Furthermore, the parasite-induced membrane permeabilization, detected by measuring potassium escape from the target cell, and an effective osmotic protection by carbohydrates allowed us to relate the previously described pore-forming mechanism involved in contact-dependent cytotoxicity with the contact-independent lysis.

Animals↗
PubMed1996

Molecular characterization of a third malic enzyme-like AP65 adhesin gene of Trichomonas vaginalis.

Adherence to the vaginal epithelium by the sexually transmitted parasite Trichomonas vaginalis is mediated by four trichomonad surface proteins (AP65, AP51, AP33 and AP23). We recently showed that the 65-kDa adhesin is a member of a multigene family comprised of two similar but distinct proteins, AP65-1 and AP65-2, encoded by the genes ap65-1 and ap65-2, respectively. An additional immuno-crossreactive clone, the 1.2 kb F11.1 cDNA, was isolated from a phagemid expression library and encoded a fusion protein of approximately 46,000 daltons (46 kDa) that bound to HeLa cell surfaces. A significant portion of the 5' end was missing which, using the 5'-RACE method, was obtained and combined with the F11.1 clone to give a full-length cDNA. The ap65-3 gene encoded for a protein of 567 amino acids with a molecular mass of 63.1 kDa. The gene showed 88% and 96% identity at the DNA level with ap65-1 and ap65-2, respectively. Restriction mapping confirmed that the three AP65 genes are different. Southern analysis revealed that the ap65-3 gene is present in the T. vaginalis genome in multiple copies. Experiments with agar clones of trichomonads showed that each gene of the multigene family is present in all parasites, and Northern analysis showed that ap65-3 is expressed and transcriptionally regulated by iron. The ap65-3 gene had a leader sequence and, as with ap65-1 and ap65-2, showed significant homology to malic enzyme. Finally, analysis of the 3'-untranslated regions revealed that the transcript of ap65-3 had a long poly (A) tail in comparison to ap65-1 and ap65-2. Even more intriguing, sequences were found that may relate to differential degradation of select AP65 transcripts, such as the sequence motifs AUUUA for ap65-1 mRNA and UUAUUUAU for the ap65-2 mRNA, which were not found for ap65-3.

Amino Acid Sequence↗
PubMed1998

Only two of the Trichomonas vaginalis triplet AP51 adhesins are regulated by iron.

The sexually transmitted parasite Trichomonas vaginalis cytoadheres to the vaginal epithelium, and four candidate trichomonad adhesins have been identified. One such protein, termed AP51, was characterized further. To do this, we studied a 1 kb cDNA clone (AP51.2) isolated from a phagemid expression library, which encoded a fusion protein of approximately 38 kDa that was immuno-crossreactive with anti-AP51 serum and retained functional adhesive properties. We performed 5'-PCR amplification to recover the missing 5' end in order to provide the complete cDNA sequence for the gene encoded by AP51.2 (ap51-2). Other PCR products revealed almost complete sequences for two additional ap51 genes, making AP51 a member of a multigene family of at least three distinct proteins and genes. The ap51-1 and ap51-3 genes each encoded for 407 amino acids while ap51-2 encoded 408 amino acids, and not unexpectedly, these genes had a high percent identity at the DNA and amino acid levels. Mapping confirmed the sequence distinctions and uniqueness of the three ap51 genes. Southern analysis using gene-specific probes revealed the single copy nature of each of the ap51 genes, all of which were present among the numerous agar clones of single trichomonads of the isolates tested. Importantly, Northern analysis showed transcriptional regulation by iron of only the ap51-1 and ap51-3 genes but not ap51-2, perhaps indicating the presence of two bona fide isoforms of the ap51 genes. The 3'-untranslated region of ap51-3 had a short poly (A) tail as well as the sequence motif AUUUA, which may relate to differential degradation of ap51-3 transcripts, in comparison to ap51-1 and ap51-2. Finally, the ap51 genes had partial homology to the beta-subunit of succinyl-CoA synthetase, reinforcing the idea that molecular mimicry may play a role in host parasitism by T. vaginalis.

Adhesins, Bacterial↗
PubMed1998

Molecular analysis of the gene encoding the immunodominant phenotypically varying P270 protein of Trichomonas vaginalis.

Trichomonas vaginalisis a flagellated protozoan responsible for the most common non-viral sexually transmitted disease. The immunogen P270 was previously found to be up-regulated in expression and to undergo phenotypic variation between surface versus cytoplasmic localization in trichoImonads harbouring a dsRNA virus. In this report, we characterize the entire p270 open reading frame (ORF) and the unknown flanking 5;- and 3;-unique, non-repeat coding sequences of the gene in addition to untranslated regions. Consistent with an earlier report (Dailey & Alderete, 1991, Infect. Immun. 59: 2083-88), a significant portion of the gene consists of a tandemly repeated 333 bp element that contains the sequence coding for the epitope DREGRD detected by murine monoclonal antibody and antibody from the sera of patients. The non-repeat coding regions for the 5;- and 3;-ends were 69 nucleotides (23 amino acids) and 1183 nucleotides (395 amino acids), respectively. Sequencing of repeat elements showed them to be identical, affirming the highly-conserved nature of this element throughout the gene. The start codon was immediately preceded by the 12 nucleotide consensus sequence (TCATTTTTAATA) found in other trichomonad protein-coding genes. A very AT-rich, non-coding region was identified upstream of the p270 ORF. P270 appears to contain a leader sequence at the amino-terminus and transmembrane domain at the carboxy-terminus. No significant homology was found with any reported proteins at either the nucleotide or amino acid level.

Amino Acid Sequence↗
PubMed1995

Identification of a satellite double-stranded RNA in the parasitic protozoan Trichomonas vaginalis infected with T. vaginalis virus T1.

Co-infection by a 0.5-kb small double-stranded (ds) RNA together with Trichomonas vaginalis virus (TVV) genomic 4.6-kb dsRNA is commonly observed in a number of T. vaginalis isolates. By molecular cloning and primer extension experiments, the 497-bp cDNA sequence of a 0.5-kb dsRNA co-infecting with TVV-T1 in T vagina/is T1 isolate was elucidated. Consistent with the replication cycle of a typical dsRNA virus, a plus-strand viral RNA beginning at +1 of the 0.5-kb dsRNA was identified in infected T. vaginalis T1 cells by primer extension and Northern hybridization studies. The 0.5-kb dsRNA was separately encased in TVV capsids from the viral genomic dsRNA, as shown by protein analysis and electron microscopic examination of viral particles purified by multiple rounds of CsCl gradient centrifugation. The riboprobes transcribed from a cloned cDNA of the 0.5-kb dsRNA exhibited strong hybridization to a small dsRNA in a T vaginalis T9 isolate, which harbors a TVV-T9 distantly related to TVV-T1, but the same probes showed very little hybridization to the viral genomic dsRNA of both TVV-T1 and TVV-T9. Very little sequence homology between the 0.5-kb dsRNA and the 4.6-kb dsRNA in TVV-T1 was found by computer-assisted analysis, suggesting that the small dsRNA in T. vaginalis T1 is not derived from the genome of TVV-T1 or other distantly related T. vaginalis viruses. These results suggest that the small dsRNAs in T vaginalis are satellite RNAs of T. vaginalis virus.

Animals↗
PubMed1996

Genomic organization and sequence conservation in type I Trichomonas vaginalis viruses.

To reveal the genetic conservation of type I Trichomonas vaginalis viruses (TVV) we cloned and sequenced the 4.6-kb ds RNA of a TVV-T5 isolate for comparison with the cDNA sequence of a related TVV-T1 ds RNA. Analogous to TVV-T1, the TVV-T5 ds RNA also contains an upstream capsid protein gene overlapped with a downstream RNA-dependent RNA polymerase (RDRP) gene by a +1 reading frame shift. A conserved ribosomal slippage heptamer (C CUU UUU) was found within the consensus 14-nt overlap, and the context of the sequence surrounding the heptamer suggests a potential ribosomal frameshifting in the biosynthesis of RDRP from the initiation of capsid protein either through two consecutive -1 shifts or a +1 shift.

Animals↗