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PubMed1986

Growth and cytopathogenicity of Trichomonas vaginalis in tissue cultures.

The primary purpose of this study was to identify the mammalian tissue cultures which were most suitable for investigations of the cytopathogenicity of Trichomonas vaginalis. A recently isolated strain of the organism was inoculated into 15 different tissue cultures which were maintained in an appropriately modified growth medium. Proliferation of the protozoon was accompanied by the progressive disintegration of cell culture monolayers. Initial focal lesions consisting of detached cells and an accumulation of trichomonads gradually enlarged until the entire monolayer was disrupted. When judged by the size of the inoculum required to obtain this effect, differences among the tissue cultures were noted. An inoculum of approximately 10(3) viable trichomonads was sufficient to completely disrupt monolayers of HeLa 229, HeLa, McCoy, HEp-2, and RK-13 cells. To obtain a comparable effect with other cells, 10- to 100-fold higher levels of inoculum were required. Polyethylene glycol concentrates from culture filtrates contained a cell-detaching factor (CDF) which caused detachment and clumping of susceptible cells. Freshly seeded cells in growth medium containing CDF failed to form a monolayer. Aggregates of cells maintained for up to 1 week in the presence of CDF remained viable and formed a monolayer after being washed and suspended in normal growth medium. The activity of the CDF was not lost during 1 week of contact with the cells. The CDF may contribute to the pathogenicity mechanisms of T. vaginalis.

Cell Adhesion↗
PubMed1989

Isolation of a cell-detaching factor of Trichomonas vaginalis.

The pathogenetic role of soluble products of Trichomonas vaginalis growth in culture is controversial. To evaluate this role, T. vaginalis was grown in broth and cell culture and the cell-free filtrate was applied to fresh cell culture monolayers. When adjusted to pH 6.5, filtrates obtained from 22-h culture growth totally disrupted McCoy, HEp-2, human foreskin fibroblast, and Chinese hamster ovary cell monolayers within 6 h. These detached cells remained greater than 90% viable. This cell-detaching factor (CDF) was heat and acid labile, with a pH optimum of 6.5. CDF has trypsinlike activity which disrupts monolayer cells, but cells do not die if the pH is controlled. CDF was purified by ethanol precipitation, ammonium sulfate fractionation, and ion-exchange and gel filtration column chromatography. A 200,000-molecular-weight glycoprotein which was also immunogenic by immunoblot with human sera reactive to T. vaginalis was isolated in this manner. This confirms the presence of a specific soluble CDF derived from T. vaginalis whose application may be important as a diagnostic tool and in further studies of pathogenesis.

Ammonium Sulfate↗
PubMed1996

Modified thioglycolate medium: a simple and reliable means for detection of Trichomonas vaginalis.

Despite the declining rate of sexually transmitted diseases in developed countries, trichomoniasis is still one of the most common venereal infections. While diagnosis of this condition is commonly based on the microscopic wet-mount method, culture remains the most accurate single procedure for detecting the presence of Trichomonas vaginalis in clinical samples. In the present study, the efficacy of a modified formula of the commonly available thioglycolate medium was compared with that of the standard Diamond's medium for detection of T. vaginalis in samples from 176 women with vaginal symptoms. Thioglycolate medium supplemented with yeast extract, horse serum, and antimicrobial agents was as reliable as Diamond's medium for detection of T. vaginalis in vaginal fluid samples. Modified thioglycolate medium may be used as a readily available, low-cost substitute for the standard medium for culturing T. vaginalis.

Animals↗
PubMed2003

Validation of a urine-based PCR-enzyme-linked immunosorbent assay for use in clinical research settings to detect Trichomonas vaginalis in men.

Trichomonas vaginalis infection is highly prevalent worldwide and is associated with urethritis, prostatitis, and urethral strictures in men. However, the natural history and importance of T. vaginalis in men are poorly understood, in part because of difficulties in diagnosing infection. Traditional detection methods rely on culture and wet-mount microscopy, which can be insensitive and time consuming. Urethral swabs are commonly used to detect T. vaginalis in men, but discomfort from specimen collection is a barrier to large studies. One thousand two hundred twenty-five Malawian men attending sexually transmitted disease and dermatology clinics were enrolled in this cross-sectional study to validate detection by urine-based PCR-enzyme-linked immunosorbent assay (ELISA) with urine and urethral swab culture as the reference standard. This assay for detection of amplified T. vaginalis DNA in first-catch urine (< or = 30 ml) performed with a sensitivity of 92.7%, a specificity of 88.6%, and an adjusted specificity of 95.2% compared to culture of urethral swabs or urine sediment. For clinical research settings in which urethral swabs are not available and culture is not feasible, the urine-based PCR-ELISA may be useful for detection of trichomoniasis in men.

Adolescent↗
PubMed2004

Performance of a new, rapid assay for detection of Trichomonas vaginalis.

Trichomonas vaginalis infection is highly prevalent, may have serious health consequence, and is readily treatable. However, screening has been limited by currently available tests, which tend to be insensitive, expensive, or require a delay before results are reported. The XenoStrip-Tv (Xenotope Diagnostics, Inc., San Antonio, Tex.) was evaluated on vaginal swab specimens from 936 women attending sexually transmitted disease clinics in Seattle, Wash. (n = 497), and Birmingham, Ala. (n = 439). T. vaginalis prevalence by culture (InPouch; Biomed) was 8.7% in Seattle and 21.0% in Birmingham. Compared to culture, the XenoStrip assay in Seattle was 76.7% (95% confidence interval [95% CI] = 61.4 to 88.2) sensitive and 99.8% (95% CI = 98.8 to 99.9) specific, and in Birmingham it was 79.4% (95% CI = 69.6 to 87.1) sensitive and 97.1% (95% CI = 94.8 to 98.6) specific. The positive predictive values were 97.1% in Seattle and 87.9% in Birmingham; the negative predictive values were 97.8 and 94.7%, respectively. Rapid test performance did not vary by vaginal symptoms or by the presence of other vaginal or cervical syndromes or infections. The sensitivity did vary by day of culture-positive result, with a 71% decline in XenoStrip sensitivity for every additional day delay until T. vaginalis was first detected in cultures (odds ratio = 0.29, 95% CI = 0.18 to 0.49). The rapid assay was more sensitive than wet preparation microscopy (78.5% versus 72.4% [P = 0.04]) but was less specific (98.6% versus 100% [P = 0.001]). The XenoStrip rapid assay is well suited for use in settings with a moderately high prevalence of T. vaginalis infection, particularly when microscopy is not practical.

Adult↗
PubMed1993

Multiple double-stranded RNA segments are associated with virus particles infecting Trichomonas vaginalis.

Previous studies demonstrated that some isolates of the sexually transmitted protozoan Trichomonas vaginalis are infected with a nonsegmented, double-stranded RNA (dsRNA) virus. A reexamination of the total dsRNA extracted from several virus-harboring isolates indicated the presence of at least three dsRNAs with sizes ranging from 4.8 to 4.3 kbp. The double-stranded nature of each of the three segments was determined by hybridization experiments using riboprobes of opposite polarities obtained from cDNA generated to each of the segments. All three segments were present in agar clones originating from single organisms of T. vaginalis isolates, suggesting that the three segments were not the result of a mixed population of trichomonads harboring different sizes of dsRNA. The three segments were associated with CsCl-purified virus particles, as evidenced by electron microscopy, and RNAse treatment of the preparation containing virus particles did not destroy the dsRNAs. Finally, the individual dsRNA segments were purified for use as probes to determine whether the three dsRNAs shared any sequence homology. Each end-labeled dsRNA segment did not cross-hybridize to any of the other two segments, a finding consistent with the hybridization of labeled cDNAs to only the segments from which they were derived. These results show that the coding capacity of the dsRNA virus may be at least three times greater than that estimated earlier and illustrates further the complexity of this virus-parasite interrelationship.

Animals↗
PubMed1994

Unique double-stranded RNAs associated with the Trichomonas vaginalis virus are synthesized by viral RNA-dependent RNA polymerase.

Most Trichomonas vaginalis isolates are carriers of the multisegmented double-stranded RNA (dsRNA) virus. In vitro polymerase assays were performed to demonstrate the RNA-dependent RNA polymerase (RDRP) activity of purified particles. Transcripts which comigrated with the dsRNAs of the virus were readily detected as synthesized products, indicating viral RDRP activity. In addition, smaller-sized dsRNA species, possibly two of approximately 700 bp (s1) and one of 500 bp (s2), were synthesized by purified virus particles of the CsCl gradient surrounding the virus peak. No cross-hybridization with either s1 or s2 and the dsRNA segments occurred, suggesting that s1 and s2 were synthesized from different templates. An RNase A protection assay revealed that the synthesized s1 and s2 polymerase products were double stranded. Furthermore, hybridization of products with strand-specific RNA of s1 generated from cDNA indicated that only one strand was synthesized in vitro. s1 and s2 were not visualized in ethidium bromide-stained agarose gels of dsRNA of infected trichomonads grown in batch cultures. However, dsRNA profiles of the same infected organisms cultivated under defined continuous-flow conditions contained readily detectable levels of s1 and s2, indicating that amplification of s1 and s2 occurred under specific environmental conditions. These newly discovered dsRNAs were not detected in all of the virus-carrying isolates. Finally, it is noteworthy that the s1 and s2 dsRNAs and the RDRP activity were not detected in trichomonal isolates without virus or in virus-negative progeny derived from virus-positive parental isolates. These data indicate the possibility of variations in the number of dsRNAs and/or of the presence of satellites in trichomonads infected with the multisegmented virus.

Animals↗
PubMed1994

Trichomonas vaginalis with a double-stranded RNA virus has upregulated levels of phenotypically variable immunogen mRNA.

Some isolates of Trichomonas vaginalis carry a double-stranded RNA virus (TVV) and undergo phenotypic variation between surface expression and cytoplasmic expression of a prominent immunogen (P270). Only trichomonads with TVV express P270 on the surface. Northern (RNA) blots using a specific cDNA encoding the repeat element of the phenotypically varying P270 immunogen as a probe showed accumulation of P270 transcript only in isolates with TVV (V+) in contrast to isolates without the virus (V-). To test further the influence of virus infection on P270 mRNA expression, V- progeny, derived from the parental V+ isolates, were tested. Trichomonads of V- progeny, like the fresh clinical V- isolates, also showed no accumulation of P270 mRNA. By immunoblotting with a monoclonal antibody to the key repeated epitope of P270, it was found that V- organisms had quantitatively less immunoreactive protein than the parental V+ isolates. Although V+ and V- isolates contained proteins immunoreactive with the monoclonal antibody to P270, it was necessary to test for the presence of the P270 gene among all the isolates. As expected, Southern blots demonstrated that V+ and V- trichomonads possessed the gene encoding P270. These data suggest that the double-stranded RNA virus of T. vaginalis is involved in the regulation of P270 mRNA accumulation.

Animals↗
PubMed1995

Characterization of double-stranded RNA satellites associated with the Trichomonas vaginalis virus.

Three small and distinct satellite double-stranded RNAs (dsRNAs) denoted s1, s1', and s2 were recently described for a Trichomonas vaginalis isolate harboring a dsRNA virus. Since characterization of these satellite dsRNAs might provide insight into the virus replication cycle and virus-host interactions, full-length cDNAs to s1 and s1' dsRNAs were synthesized and sequenced. s1 dsRNA has 688 bp, and s1' dsRNA has 616 bp. A 228-bp open reading frame that begins at nucleotide 37 was detected on a putative sense strand of s1. All satellite RNAs were found associated with RNA-dependent RNA polymerase (RDRP) activity that banded on CsCl gradients. Within carrier trichomonads, satellite RNAs synthesized single-stranded replicative intermediates. An in vitro assay was established to assess replication of satellite RNAs. Transcripts generated from s1 cDNA, for example, served as a template for the viral RDRP. These templates had a polarity similar to that of the replicative intermediate found in the satellite-harboring parasites. Importantly, the recognition of s1 RNA was shown to be specific, since unrelated RNAs did not serve as templates for RDRP under the same experimental conditions. The data indicate that the cDNA of s1 has a specific and essential sequence needed for recognition by the viral RDRP and for subsequent RNA synthesis. Both s1 and s1' have conserved domains, albeit of unproven function, but which may be required for replication.

Animals↗
PubMed2001

Initiator recognition in a primitive eukaryote: IBP39, an initiator-binding protein from Trichomonas vaginalis.

While considerable progress has been made in understanding the mechanisms of transcription in higher eukaryotes, transcription in single-celled, primitive eukaryotes remains poorly understood. Promoters of protein-encoding genes in the parasitic protist Trichomonas vaginalis, which represents one of the deepest-branching eukaryotic lineages, have a bipartite structure with gene-specific regulatory elements and a conserved core promoter encompassing the transcription start site. Core promoters in T. vaginalis appear to consist solely of a highly conserved initiator (Inr) element that is both a structural and a functional homologue of its metazoan counterpart. Using DNA affinity chromatography, we have isolated an Inr-binding protein from T. vaginalis. Cloning of the gene encoding the Inr binding protein identified a novel 39-kDa protein (IBP39). We show that IBP39 binds to both double and single Inr motifs found in T. vaginalis genes and that binding requires the conserved nucleotides necessary for Inr function in vivo. Analyses of the cloned IBP39 gene revealed no homology at the protein sequence level with identified proteins in other organisms or the presence of known DNA-binding domains. The relationship between IBP39 and Inr-binding proteins in metazoa presents interesting evolutionary questions.

Amino Acid Sequence↗
PubMed1988

New rapid latex agglutination test for diagnosing Trichomonas vaginalis infection.

A newly developed latex agglutination test for Trichomonas vaginalis infection was compared for sensitivity, specificity, efficiency, and positive and negative predictive values with microscopy, culture, and an enzyme linked immunosorbent assay (ELISA) in the diagnosis of 395 women attending a genitourinary medicine clinic. T vaginalis infection was diagnosed in 42 (11%) women. The sensitivities of both the latex agglutination test and the ELISA were 95% compared with 74% for microscopy and 76% for culture. The latex test was specific and showed no cross reaction with a wide range of other genital tract infections. The latex agglutination test can detect antigen in both soluble and insoluble forms, and as it is simple to perform, can be undertaken during routine examination without recourse to special equipment or training. Further evaluation is required.

Enzyme-Linked Immunosorbent Assay↗
PubMed2003

Double stranded RNA virus in South African Trichomonas vaginalis isolates.

AIMS: To screen Trichomonas vaginalis isolates from South Africa for the presence of a small double stranded RNA virus designated T vaginalis virus (TVV). METHODS: TVV was detected by simultaneous extraction of DNA and RNA, and its presence confirmed by electron microscopy and nuclease digestions. RESULTS: TVV was detected in 59 of 72 (81.9%) isolates. CONCLUSIONS: These results indicate a possible higher infection rate of South African T vaginalis isolates by the double stranded RNA virus than has been reported for isolates elsewhere.

Animals↗
PubMed1976

Sensitivity of Trichomonas vaginalis to metronidazole, tinidazole, and nifuratel in vitro.

Prompted by the sensitivity of trichomonads to metronidazole and nifuratel in clinical practice, a study was conducted in 1971-1972 of 63 consecutive strains of Trichomonas vaginalis isolated from women with clinically refractory vaginal discharge. Their susceptibility to metronidazole, tinidazole, and nifuratel was tested, using a serial tube dilution technique. The minimum concentrations which in 48 hrs caused immobilization and lysis of trichomonads cultured in Diamond's medium was assessed. No differences in drug potency could be determined. The median trichomonistatic and trichomonicidal concentrations were 0-1 and 0-6 mug/ml. respectively when using an inoculum of 10,000 organisms per ml. An inoculum of 100,000 per ml. resulted in inhibitory concentrations of 1-0 and killing concentrations of 3-3 mug./ml. These levels are readily attained in blood and vaginal tissue after oral ingestion of the two imidazole derivatives. Thus, metronidazole has maintained its efficacy since it was first introduced more than a decade ago. The few therapeutic failures with metronidazole and tinidazole are considered to have been caused by pharmacokinetic deficiencies in the patients, or by re-infection.

Drug Resistance, Microbial↗
PubMed1977

Ornidazole: a new antiprotozoal compound for treatment of Trichomonas vaginalis infection.

A new anti-protozoal compound, ornidazole, a derivative of nitroimidazole, was given in a single 2 g dose to 20 women with Trichomonas vaginalis infection. All the women were cured, but they suffered some side-effects. Plasma levels of ornidazole reached a peak five to eight times higher than minimum inhibitory concentrations and exceeded this level for at least 36 hours. It is therefore possible that a smaller dose might have had an adequate trichomonicidal effect and fewer side-effects. Further studies are in progress.

Adolescent↗
PubMed1978

Studies on strain sensitivity of Trichomonas vaginalis to metronidazole.

Ninety-four strains of Trichomonas vaginalis isolated from unselected patients in Vienna during the period from November 1975 to November 1975 were subjected to sensitivity tests against metronidazole in vitro and in vivo. Nearly 90% of the isolates examined showed in vitro growth inhibition at concentrations of 0.4 to 1.6 microgram metronidazole/ml and only about 10% at concentrations of less than 0.4 microgram/ml. All isolates showed some inhibition of growth after incubation for 48 hours with 3.2 microgram metroidazole/ml. Tests on treatment carried out on experimentally infected mice revealed that all strains of T. vaginalis examined were sensitive to metronidazole (MDE: less than 3 x 35 mg/kg peroral).

Animals↗
PubMed1978

Abnormal forms of Trichomonas vaginalis.

Abnormal forms of Trichomonas vaginalis have been demonstrated by both conventional and scanning electron microscopy after inoculation of media with clinical material from cases of trichomonal vaginitis. Twenty-six cases of vaginitis have been studied; 10 of them showed the abnormal forms of trichomonads after growth in a modification of the medium described by Bushby and Copp (1955), while 16 showed only normal forms.

Atypical Bacterial Forms↗
PubMed1978

Absence of detectable local antibody in genitourinary tract secretions of male contacts of women infected with Trichomonas vaginalis.

Samples of semen and urine were obtained from 37 male contacts of women with proved Trichomonas vaginalis infection; on culture, eight (22%) of the men were shown to harbour the parasite. However, significant amounts of antitrichomonal antibody were found in only two of these samples, and the amounts present were very small. A further 10 samples were tested but none was found to contain antibody. The asymptomatic nature and low parasite numbers commonly described in infections in men is thus unlikely to be due to a vigorous local immune response.

Animals↗
PubMed1981

Behaviour and pathogenicity of Trichomonas vaginalis in epithelial cell cultures: a study by light and scanning electron microscopy.

The behaviour and pathogenic effects of Trichomonas vaginalis in mammalian cell cultures were studied using light microscopy and scanning electron microscopy. Six hours after inoculation of the parasites into the cell cultures about 10% of the epithelial monolayer was destroyed. The parasites adhered to the epithelial cells, developed an amoeboid morphology, and crawled over and under the monolayer of cells. These observations suggest that the adhesiveness, amoeboid morphology, and motility of T vaginalis may be important mechanisms in the injury caused to the vaginal epithelium.

Animals↗