Identification of a surface antigen of Trichomonas vaginalis.

A major surface antigen of Trichomonas vaginalis was purified by using three independently derived monoclonal antibodies (two immunoglobulin M and one immunoglobulin G1) prepared against T. vaginalis PHS-2J. A 115,000-molecular-weight antigen and one or more components with a molecular weight of 58,000 to 64,000 were recovered when any of the three antibodies was used as an immunoadsorbent. The purified antigen reacted with all three monoclonal antibodies in an enzyme-linked immunosorbent assay, indicating that the antibodies recognized the same antigen but not necessarily the same determinant. The purified antigen was sensitive to both pronase digestion and periodate oxidation. The antigen was shown to be on the external surface of some but not all T. vaginalis isolates by agglutination of live organisms with the monoclonal antibodies.     

Characterization of Trichomonas vaginalis virus proteins in the pathogenic protozoan T. vaginalis

Summary.  The 4.6-kb double-stranded (ds) RNA of Trichomonas vaginalis virus (TVV)-T1 has been shown to encode two overlapping genes, cap and pol. In this study, a serum for specifically detecting viral cap gene product was raised against a recombinant protein, and sera for specifically detecting pol gene product were raised against synthetic oligopeptides. A 75-kDa major protein and a 160-kDa minor protein were detected by anti-CAP serum in a TVV-T1 sample, indicating that the 75-kDa protein is the viral capsid protein. The 160-kDa protein alone was also detected by two distinct anti-POL sera, indicating that the pol gene is expressed as a CAP-POL fusion protein. These results suggest that the TVV-T1 genome is arranged into a cap-pol organization in a manner similar to that of viruses in family Totiviridae. Accepted December 12, 1997 Received September 8, 1997     

Antigen-specific proliferation responses of peripheral blood lymphocytes to Trichomonas vaginalis antigen in patients with Trichomonas vaginitis.

This report describes the development of an assay system which overcomes the difficulty of detecting immune responses of patients with Trichomonas vaginitis by making use of peripheral blood leukocytes obtained from such patients. When peripheral blood leukocytes from the patient were stimulated in microcultures with the soluble antigen extracted from Trichomonas vaginalis, significant degrees of proliferation ensued, as measured by the incorporation of [methyl-3H]thymidine 4 to 5 days after initiation. The antigen-induced proliferation response of peripheral blood leukocytes is specific for T. vaginalis antigen. The T. vaginalis-specific [methyl-3H]thymidine incorporation is mediated by Leu-1-positive cells, namely, T lymphocytes, in the peripheral blood leukocyte population. This assay system should prove useful for the analysis of the immune response to the protozoa in patients with Trichomonas vaginitis.     

Detection of Trichomonas vaginalis antigen in women by enzyme immunoassay.

An enzyme immunoassay (EIA) was developed for the detection of Trichomonas vaginalis antigen in vaginal swabs. Four hundred and eighty two women attending a sexually transmitted disease (STD) clinic were tested; 44 (9.1%) were positive by culture, 32 (6.6%) were positive by wet film examination, and 54 (11.2%) were considered to be positive for trichomonal antigen by EIA. Taking culture as the reference method, the EIA had a sensitivity of 93.2% and a specificity of 97.5%. The predictive value of a positive test was 82% and that of a negative test was 99.3%.     

Generation of interleukin-8 from human monocytes in response to Trichomonas vaginalis stimulation.

Neutrophils are the predominant inflammatory cells found in the vaginal discharges of patients with Trichomonas vaginalis infection. We have investigated the possible role of interleukin-8 (IL-8) in the inflammatory response elicited by T. vaginalis infection. This study has shown that T. vaginalis induces blood monocytes to produce large amounts of bioactive IL-8, mainly by membrane components of T. vaginalis (MTV). Monocyte-derived IL-8 induced by MTV was dose and time dependent. The peak level of IL-8 was 102 +/- 11 ng/ml of conditioned media (mean +/- standard error; n = 5) obtained from MTV-stimulated monocytes (MTVCM) at 36 h of cultivation. With a multichamber chemotactic assay, we found an optimal neutrophil chemotaxis (177 +/- 14 migrated cells) induced by MTVCM collected at 16 h of cultivation when the level of IL-8 was 42 +/- 8 ng/ml. A neutralizing monoclonal antibody directed against IL-8, but not the irrelevant antibodies, significantly blocked the neutrophil chemotactic activity (decreased from 153 +/- 6 to 23 +/- 3 migrated cells; n = 3 [P < 0.001]) induced by MTVCM. Moreover, the maximum increase of the IL-8 mRNA level from MTV-treated monocytes was observed after a 5-h cultivation and decreased thereafter. Monocytes cocultured with MTV in the presence of a neutralizing monoclonal antibody directed against tumor necrosis factor alpha, but not against IL-1 beta, decreased IL-8 production by 25% (P < 0.05), indicating that the release of IL-8 in MTV-stimulated monocytes is partially dependent on tumor necrosis factor alpha. The capacity of MTV-induced monocytes to synthesize IL-8 suggests that these cells can contribute to the induction of the acute inflammatory response seen in T. vaginalis infection.     

Comparison of four methods to detect Trichomonas vaginalis.

Four methods for the detection of Trichomonas vaginalis in vaginal secretions from 88 symptomatic patients were compared: wet-mount examination, Kupferberg liquid medium, Hirsch charcoal agar, and the Papanicolaou smear. Hirsch diphasic slants and Kupferberg medium did not significantly differ in sensitivity from direct examination of wet mounts. The Papanicolaou smear identification of trichomonads was found to be the least sensitive method evaluated.     

Inorganic pyrophosphatase of Trichomonas vaginalis.

Trichomonas vaginalis homogenates were found to have an acid inorganic pyrophosphatase activity with a specific activity at pH 4.8 of about 7 nmol min-1 (mg protein)-1. This activity was localized predominantly in hydrolase containing particles, showed structure-bound latency and was tightly membrane-bound. The activity showed no magnesium dependence, a Km of about 2 mM inorganic pyrophosphate, a pH optimum of 5.2 and was inhibited by fluoride at millimolar levels. No evidence was obtained for the existence of a cytosolic magnesium-dependent activity but the existence of a low level of magnesium-independent cytosolic activity cannot be excluded. These observations correlate with the importance of cytosolic inorganic pyrophosphate in the carbohydrate catabolism in T. vaginalis.     

Immune response to hepatitis B surface antigen.

A total of 69 persons were investigated for assessment of cell-mediated and humoral immunity to hepatitis B surface antigen (HBsAg). Three groups, each consisting of 20 normal persons, 20HBsAg carriers, and 20 convalescent hepatitis B patients, were studied for HBsAg, anti-HBs, and leukocyte migration inhibition with purified HBsAg. Sequential sampling if an additional group of nine acute hepatitis B patients defined the cellular and humoral immune response to HBsAg. The antigen was eliminated rapidly by mounting of cell-mediated immune response detectable for a limited period, followed by antibody response in relatively few patients moore than 3 months after clearance of circulating HBsAg.     

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.     

Comparative immunoelectrophoretic studies of total water-soluble extracts of Trichomonas vaginalis, T. tenax and T. hominis

An antigen characterization was carried out by the method of two-dimensional immunoelectrophoresis, and on this basis the antigen community and the antigenic differences between the 3 Trichomonas species parasitic in man were investigated. In the homologous antigen-antibody-systems a maximum number of precipitation curves is formed--21 in T. vaginalis and 20 each in T. tenax and T. hominis. According to our setting of the experiment T. vaginalis has 5 specific antigens in regard to T. tenax and 3 in regard to T. hominis. T. tenax has 2 specific antigens in regard to T. vaginalis and 7 in regard to T. hominis, T. hominis has 2 specific antigens in regard to T. vaginalis and 3 in regard to T. tenax. The presence of antigenic differences is important for the immunological characterization of the 3 species and demonstrates their validity.     

Antibody to Trichomonas vaginalis in human cervicovaginal secretions.

Human cervicovaginal secretions were obtained from patients at the Gynecology and Obstetrics Clinics at National Taiwan University Hospital and Cathay General Hospital, Republic of China. Among the 500 patients examined, 33 (6.6%) were infected with Trichomonas vaginalis as determined by the culture method. Secretions from 24 of the infected patients and 30 noninfected women were assayed for anti-T. vaginalis immunoglobulins by the indirect immunofluorescent antibody technique. A few serum samples from both infected and noninfected persons were also included in this study. Immunoglobulin G (IgG) antibody against T. vaginalis was detected in 17 (70.8%) secretions from the infected women. Among the 17 positive secretions, anti-parasite IgA was found in two specimens, IgE was found in three, and IgM was found in one. Of the 30 secretions, 7 (23.3%) from noninfected women also contained anti-parasite IgG. Low levels of natural anti-trichomonad IgG and IgM were detected in the sera of normal persons. Infection with T. vaginalis caused an increase in the serum IgG antibody titer. Cross-reaction between T. vaginalis and Pentatrichomonas hominis was also observed.     

Contact-dependent cytopathogenic mechanisms of Trichomonas vaginalis.

The cytopathogenic mechanisms of Trichomonas vaginalis have been debated since the 1940s. We examined the following three proposed pathogenic mechanisms: contact-dependent extracellular killing, cytophagocytosis, and extracellular cytotoxins. Serial observations of Chinese hamster ovary (CHO) cell monolayers exposed to trichomonads revealed that (i) trichomonads form clumps, (ii) the clumps adhere to cells in culture, and (iii) monolayer destruction occurs only in areas of contact with T. vaginalis. Kinetic analysis of target cell killing by trichomonads revealed that the probability of CHO cell death was related to the probability of contact with T. vaginalis, supporting the observation by microscopy that trichomonads kill cells only by direct contact. Simultaneous studies of 111indium oxine label release from CHO cells and trypan blue dye exclusion demonstrated that T. vaginalis kills target cells without phagocytosis. Filtrates of trichomonad cultures or from media in which trichomonads were killing CHO cells had no effect on CHO cell monolayers, indicating that trichomonads do not kill cells by a cell-free or secreted cytotoxin. The microfilament inhibitor cytochalasin D (10 micrograms/ml) inhibited trichomonad killing of CHO cell monolayers by 80% (P less than 0.0001). In contrast, the microtubule inhibitor vinblastine (10(-6) M) caused only 17% inhibition of trichomonad destruction of CHO cell monolayers (P less than 0.020), whereas colchicine (10(-6) M) had no effect. T. vaginalis kills target cells by direct contact without phagocytosis. This event requires intact trichomonad microfilament function; microtubule function appears not to be essential.     

Immune response to synthetic peptides of hepatitis delta antigen.

Hepatitis delta antigen (HDAg) is the only viral protein known to be expressed during hepatitis delta virus (HDV) infection. Detection of antibody to HDAg (anti-HD) is the usual method for diagnosis of HDV infection since viremia lasts only a few weeks. In an effort to identify the major epitopes recognized by humans during natural infection, four oligopeptides including residues 2 to 17 (SP1), 155 to 172 (SP2), 168 to 182 (SP3), and 189 to 211 (SP4) of the HDAg molecule were synthesized and probed by enzyme-linked immunosorbent assay with a panel of 80 serum specimens from 45 patients suffering from either HDV-hepatitis B virus coinfections (n = 17) or HDV superinfections (n = 28). Sera from infected patients recognized these relatively short peptides. Peptide SP2 was the most antigenic; 71% of serum specimens reacted. Antibody to SP2 was also the commonest in sera taken early in the course of the disease. Peptide SP2 corresponds to one of the two regions which is highly conserved between different isolates. Among the 63 serum specimens which scored anti-HD positive by a commercial assay, all but 3 reacted to at least one of the peptides (95% agreement). Peptide assays appeared to be significantly more sensitive than the commercial assay with native HDAg early in the course of HDV infection since 14 of 17 (82%) serum specimens which scored anti-HD negative in the commercial assay reacted to one or more peptides. All serum specimens giving one or more positive results with the various peptides were confirmed as being HDV positive by an inhibition assay with free peptide in solution. The immune response to HDAg peptides vared greatly between individuals. No specific reactivity profile could be assigned to those with either HDV-hepatitis B virus coinfections or HDV superinfections. Overall, HDAg peptides appeared to be convenient reagents in addition to native antigen for the development of new and improved diagnostic tests for HDV infection.     

Evaluation of affirm VP Microbial Identification Test for Gardnerella vaginalis and Trichomonas vaginalis.

A commercial system (Affirm VP Microbial Identification Test; MicroProbe Corp.) for detection of vaginal pathogens was evaluated with 176 consecutive women attending a sexually transmitted disease clinic for genital complaints. Vaginal swab specimens were used for culture of Gardnerella vaginalis and Trichomonas vaginalis, preparation of a vaginal smear for Gram stain interpretation, and wet mount evaluation. An additional swab was used to evaluate the 30-min nonisotopic oligonucleotide probe test. The automated probe system detected G. vaginalis in 69 (95%) of 73 women having > 5 x 10(5) CFU of G. vaginalis per ml by culture, and 20 (43%) of 47 specimens with < or = 5 x 10(5) CFU of G. vaginalis per ml. There were three false positives and four false negatives for the Affirm VP test compared with > 5 x 10(5) CFU of G. vaginalis per ml. The probe system detected G. vaginalis in 57 (90%) of 63 vaginal specimens from women having clue cells on wet mount examination, and in only 3 (3%) of 113 women without clue cells, suggesting that the Affirm probe for G. vaginalis could be used as a surrogate for wet mount examination for clue cells. The T. vaginalis probe was positive for 12 of 12 specimens positive by wet mount and 12 of 15 specimens positive by culture. There were no false positives and three false negatives for the Affirm VP test compared with culture and/or wet mount for T. vaginalis. The Affirm VP Microbial Identification System is a rapid, objective, and automated test for the detection of T. vaginalis and clinically significant levels of G. vaginalis that is comparable to wet mount examination for clue cells and is superior to wet mount examination for the detection of trichomonads.     

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.     

Rapid assay for immunological detection of Trichomonas vaginalis.

Trichomoniasis is a common sexually transmitted disease with an estimated incidence of 4 million to 8 million cases a year in the United States. The most commonly used method of diagnosis is a direct microscopic observation (wet mount) of vaginal secretions and, although both rapid and inexpensive, the sensitivity of this technique is generally 50 to 70%. We developed an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Trichomonas vaginalis which is both rapid and sensitive (detection limit of approximately 100 trichomonads per ml). This assay, which employs affinity-purified rabbit anti-T. vaginalis antibodies in a "sandwich" configuration, is simple to perform and is neither interfered with nor appears to cross-react with other microorganisms which are common inhabitants of the urogenital tract. One hundred and seventy-seven consecutive unselected patients attending a clinic for sexually transmitted diseases were evaluated for trichomoniasis by a broth culture technique monitored for up to 7 days (and considered here to be the standard for positivity), the conventional wet mount method, a solid culture procedure, and the ELISA. Of these, 84 were positive by culture; 33 were positive by the wet mount; and despite the fact that the vaginal specimens were diluted 20-fold during the culture procedures prior to testing in the ELISA, 65 were positive by ELISA. In addition to exhibiting a sensitivity of 77%, the specificity of the ELISA was 100%. These results demonstrate that the ELISA is a significant improvement over the wet mount method for the diagnosis of trichomoniasis.     

Clinical and Microbiological Aspects of Trichomonas vaginalis

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.     

Molecular probe for identification of Trichomonas vaginalis DNA.

Trichomoniasis is one of the most widespread sexually transmitted diseases in the world. Diagnosis can be achieved by several methods, such as direct microscopic observation of vaginal discharge, cell culture, and immunological techniques. A 2.3-kb Trichomonas vaginalis DNA fragment present in strains from diverse geographic areas was cloned and used as a probe to detect T. vaginalis DNA in vaginal discharge by a dot blot hybridization technique. This probe was specific for T. vaginalis DNA. It recognized strains from two regions in Italy (Sardinia, Piemonte) and from Mozambique (Africa). In addition, our probe did not cross-react with bacterial (Escherichia coli, Enterococcus spp., group B streptococci, Gardnerella vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and Lactobacillus spp.), viral (herpes simplex virus type 2), fungal (Candida albicans), protozoan (Entamoeba histolytica, Giardia lamblia, Plasmodium falciparum, Leishmania major, and Leishmania infantum), or human nucleic acids. The probe reacted with Pentatrichomonas hominis and Trichomonas foetus. The limit signal recognized by our probe corresponded to the DNA of 200 T. vaginalis isolates. The 2.3-kb probe was used in a clinical analysis of 98 samples. Of these, 20 samples were found to be positive both with the probe and by cell culture, and only 14 of these were positive by a standard wet mount method.