Diagnostico de laboratorio da trichomonose urogenital / Laboratory diagnostic of urogenital trichomonosis

O objetivo deste estudo e descrever as tecnicas usadas para o diagnostico de laboratorio do Trichomonas vaginalis. Foram avaliadas as possiveis diferenças na sensibilidade do exame direto convencional,no cultivo e nos testes sorologicas usados para o diagnostico da trichomonose.

[Bound amino acids in local strains of Trichomonas vaginalis]. / Izuchenie sviazannykh aminokislot mestnykh shtammov Trichomonas vaginalis.

Amino acid composition of water-soluble and water-insoluble proteins of 8 strains of Tr. vaginalis is studied. 17 amino acids are found in both protein hydrolyzates. Despite the complete coincidence of their qualitative compositions there are reliable differences in the quantitative contents of some amino acids. Differences in the contents of main amino acids of water-soluble proteins of different strains reflect the belonging of the latter to different sero-groups. No reliable differences in the quantitative contents of amino acids of both water-soluble and water-insoluble proteins in strains belonging to one sero-group are recognised.

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.

Candidiasis vaginal: diagnóstico y tratamiento en la práctica ginecológica / Candidiasis, vaginal: diagnosis and treatment in the practice

El tratamiento efectivo de las Candidiasis vaginal se ha convertido en un problema social y terapéutico, porque es una enfermedad muy molesta aunque no compromete la vida, compromete la felicidad de la vida conyugal. El uso de los conazoles en cualquiera de sus formas en el presente, es una solución. Pero el problema de las recurrencias frustran severamente los resultados, por lo que es necesario controlar, tratar los factores predisponentes para el fracaso o la recurrencia. Se debe tener en cuenta que el factor sexual en la que está envuelta la pareja, deberá ser considerado en la terapéutica paralela, así como todos los cuidados para evitar la infectación. Con el avance de la farmacología anti fungicida, deberemos considerar que mientras no consiga la forma de crear defensa en la vagina contra la Candida, ésta seguirá invadiéndola. Por lo tanto, es buena técnica de utilizar de manera profiláctica tratamiento local, de preferencia, o en el futuro, la dosis única oral para prevenir reinfecciones

Trichomonas vaginalis: preliminary characterization of a sperm motility inhibiting factor.

This study determined the effects of Trichomonas vaginalis trophozoites, subcellular fractions, and medium from axenic T. vaginalis cultures on human sperm motility and viability. Spent medium (pH 7.0) caused complete cessation of sperm motility after 15 minutes incubation. Trophozoite soluble fraction or formalin-killed trophozoites caused a 50 percent reduction in sperm motility, compared to 25 percent reduction caused by the trophozoite particulate fraction or the sterile medium and three percent by saline (control). Spent medium from T. vaginalis cultures reaching stationary growth phase produced the greatest reduction in sperm motility, suggesting that potency was related to time in culture and trophozoites per ml. The T. vaginalis spermicidal activity was heat-stable, trypsin-sensitive, and had a molecular weight of 12-15,000 by gel filtration. This proteinaceous substance was present in and secreted by T. vaginalis trophozoites during normal growth in axenic culture. Since this T. vaginalis byproduct rapidly killed sperm in vitro, its effects in humans may contribute to infertility in infected couples.

Presence of laminin-binding proteins in trichomonads and their role in adhesion.

Adhesion is regarded as an important feature in the pathogenesis of various microorganisms. Ability to recognize extracellular matrix proteins, such as laminin or fibronectin, has been correlated with invasiveness. We report that laminin enhances the adhesion of the parasitic protozoa Trichomonas vaginalis and Tritrichomonas foetus to a polystyrene substrate and to the surface of epithelial cells (Madin-Darby canine kidney cell line) in vitro. The enhancement was higher for T. vaginalis than for T. foetus. Addition of anti-laminin antibodies to medium significantly inhibited the adhesion of parasites to polystyrene substrate. Indirect immunofluorescence and transmission electron microscopy of replicas of the parasite's surface labeled with antibody-gold complexes showed laminin-binding sites distributed over the parasite surface. Iodinated P1 fragment of laminin, which retains the laminin-binding site, binds saturably to the parasite surface with a Kd of 19.5 nM, for about 3 X 10(5) binding sites per cell. Immunoblotting analysis of whole parasite extracts showed that a protein of 118 kDa is responsible for laminin binding.

Localization of acetylated alpha-tubulin in Tritrichomonas foetus and Trichomonas vaginalis.

We used monoclonal antibodies specific for acetylated and nonacetylated alpha-tubulin to detect and to localize microtubules containing acetylated alpha-tubulin (stable microtubules) in the pathogenic protozoa Tritrichomonas foetus and Trichomonas vaginalis. SDS-PAGE analysis showed that tubulin is a major protein of both parasites, being enriched in cytoskeletal preparations of whole cells extracted with Triton X-100. The monoclonal antibodies, which recognize all isoforms of alpha-tubulin (B-5-1-2) and only acetylated alpha-tubulin (6-11B-1), bind to the tubulin of T. foetus and T. vaginalis as seen by immunoblotting. Tubulin-containing structures were localized using immunofluorescence microscopy and transmission electron microscopy of the whole cytoskeleton previously incubated in the presence of the anti-tubulin antibodies and a second antibody-gold complex, and then processed using the negative staining or replica techniques. The results obtained indicate that, in addition to the flagellar microtubules, those which form the peltar-axostyle system represent stable microtubules containing acetylated alpha-tubulin.

Identification and properties of Trichomonas vaginalis proteins involved in cytadherence.

Trichomonas vaginalis NYH286 surface proteins which are candidates for mediating parasite cytadherence (adhesins) were identified. At least four trichomonad protein ligands ranging in relative molecular mass from 65 to less than or equal to 21 kilodaltons were found to selectively bind to chemically stabilized HeLa cells. The proteins were present on the surfaces of 10 different isolates of T. vaginalis examined; however, the nonpathogenic trichomonad T. tenax did not possess similar HeLa cell-binding proteins under identical experimental conditions, suggesting that these proteins are unique to the pathogenic human trichomonads. The surface nature of the candidate adhesins was confirmed by the ability of the proteins on intact, live organisms to be radioiodinated and to be removed with trypsin treatment. Rabbit antiserum (immunoglobulin G fraction) generated against adhesin proteins electroeluted from acrylamide preparations inhibited cytadherence compared with control immunoglobulin G. An adherence-negative subpopulation of T. vaginalis NYH286 organisms was also isolated. These nonadherent trichomonads did not synthesize the adhesin proteins. Interestingly, absence of adhesins from these parasites paralleled expression of a major immunogen known to undergo phenotypic variation. Revertant organisms derived from the adherence-minus subpopulation synthesized the adhesins and attached to HeLa cells. The emergence of revertant adherent T. vaginalis organisms also corresponded with the appearance of parasites which were without the major immunogen on their surface. Finally, it was determined that only those parasites lacking the major surface immunogen were capable of adherence and toxicity to HeLa cells.

Nitroimidazole and oxygen derived radicals detected by electron spin resonance in hydrogenosomal and cytosolic fractions from Trichomonas vaginalis.

Hydrogenosome-enriched fractions from Trichomonas vaginalis reduce a number of nitroimidazole derivatives to their respective electron spin resonance-detectable nitro-anion radicals. In the presence of of oxygen and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) a superoxide spin trapped adduct of DMPO was formed; the rate-determining step was the prior formation of the nitro-anion radical. Oxygen-derived radicals were detected with cytosolic fractions from a metronidazole-resistant isolate (CDC-85) when incubated with NADH or NADPH as respiratory substrate. The requirement for superoxide dismutase and catalase to completely abolish formation of these signals suggests contributions from both superoxide and peroxide. No oxygen-derived radicals were observed with cytosolic fractions from a metronidazole-susceptible strain (C1-NIH).

The dsRNA of Trichomonas vaginalis is associated with virus-like particles and does not correlate with metronidazole resistance.

Twelve metronidazole-resistant and twelve metronidazole-susceptible strains of Trichomonas vaginalis were tested for the presence of dsRNA. Three resistant and five susceptible strains were found to contain dsRNA which indicated that metronidazole resistance does not correlate with the absence of dsRNA. Electron microscopy showed the homogenates of all dsRNA-positive strains to contain virus-like particles 32-38 nm in diameter, while no such particles were found in the dsRNA-negative strains. A mutual relationship between the dsRNA and virus-like particles seems to exist.

A rapid method for isolation of double stranded RNA.

A rapid and simple method for the isolation and purification of dsRNA is presented. The crucial step of this method is the extraction of proteins and DNA with acid phenol. After the extraction, only RNA is left in the aqueous phase. ssRNA contamination of the RNA preparation can be greatly reduced when ammonium sulfate is present during the extraction.

S-adenosylmethionine and transmethylation reactions in trichomonads.

S-Adenosylmethionine (SAM) levels in trichomonads, a range of trypanosomatids and mouse liver were measured using HPLC techniques. The concentrations were found to be similar in each with the exception of Herpetomonas muscarum ingenoplastis, which contained approximately ten-fold more. Living trichomonads were found to incorporate exogenous L-methionine into intracellular SAM and its methyl carbon was also detected in lipids and nucleic acids, presumably through its involvement in transmethylation reactions. Norleucine and cycloleucine inhibited L-methionine uptake and incorporation into living Trichomonas vaginalis. Both the rates of incorporation of exogenous L-methionine into intracellular SAM and its involvement in transmethylation reactions were greater for Trichomonas vaginalis than for Tritrichomonas foetus. The results suggest that Trichomonas vaginalis and other trichomonads contain enzymes equivalent to SAM synthetase (EC 2.5.1.6) and SAM-dependent methyltransferases (EC 2.1.1).

The double-stranded RNA in Trichomonas vaginalis may originate from virus-like particles.

A linear 5.5-kilobase double-stranded RNA, identified in many strains and isolates of the parasitic protozoan Trichomonas vaginalis in a previous study, is found largely intact in ribonuclease-treated homogenates of the parasite. It can be pelleted with membranes from the homogenate at 12,500 X g and further purified in CsCl buoyant density-gradient centrifugations. The purified sample contains the double-stranded RNA as well as one major protein with an estimated molecular mass of 85 kDa in NaDodSO4/PAGE. Electron microscopic examinations indicated the presence of icosahedral virus-like particles of 33-nm diameter in the purified preparation. The exact location of the virus in T. vaginalis is not clear, except that it is not found in the nuclear fraction and is probably membrane-bound. No free virus can be recovered from the culture medium of T. vaginalis, and no successful infection of virus-free T. vaginalis strains by purified virus has yet been accomplished. There is no viral genomic sequence identifiable in host DNA. So far as we know, it is the first time a double-stranded RNA virus has been identified in a protozoan.

Resultados citológicos en pacientes con extendidos inflamatorio / Cytological results in patients with inflammatory smears

Se analiza el material de 300 pacientes que concurrieron al Dpto. de Ginecología y Citopatología de LALCEC, en los meses de marzo a mayo de 1986, se detectan 110 resultados colpocitológicos inflamatorios (36,6%), sus edades oscilan entre los 15 y 54 años con un promedio de 32. Se da la frecuencia de la flora de los extendidos cérvico-vaginales: Cocos (23,6%), Gardnerella (13,6%), Hongos (4,6%), Trichomonas (3,6%) y Flora Mixta (2,6%). Se analiza la flora de los extendidos cérvico-vaginales en relación a los antecedentes obstétricos: Parto, Aborto más Parto, Cesárea, Nuligestas, etc. Se realiza una comparación con los resultados de otros autores obtenidos de la bibliografía (AU)

Resultados citológicos en pacientes con extendidos inflamatorio / Cytological results in patients with inflammatory smears

Se analiza el material de 300 pacientes que concurrieron al Dpto. de Ginecología y Citopatología de LALCEC, en los meses de marzo a mayo de 1986, se detectan 110 resultados colpocitológicos inflamatorios (36,6%), sus edades oscilan entre los 15 y 54 años con un promedio de 32. Se da la frecuencia de la flora de los extendidos cérvico-vaginales: Cocos (23,6%), Gardnerella (13,6%), Hongos (4,6%), Trichomonas (3,6%) y Flora Mixta (2,6%). Se analiza la flora de los extendidos cérvico-vaginales en relación a los antecedentes obstétricos: Parto, Aborto más Parto, Cesárea, Nuligestas, etc. Se realiza una comparación con los resultados de otros autores obtenidos de la bibliografía

Isolation and characterization of DNA from Tritrichomonas foetus and Trichomonas vaginalis.

High molecular weight DNA samples free of contaminating proteins or RNA were obtained from Tritrichomonas foetus or Trichomonas vaginalis by lysing the cells in 4 M guanidinium thiocyanate before centrifuging in CsCl density gradient and then purifying the DNA band by NACS-37 column chromatography. The bulk DNA from either organism acted as a single component in ion-exchange chromatography, agarose gel electrophoresis, CsCl density gradient centrifugation and thermal denaturation. T. foetus DNA showed a melting temperature (Tm) of 82 degrees C corresponding to a 31% GC content whereas T. vaginalis DNA melted at 84 degrees C to suggest 36% GC. Both DNA samples demonstrated 35 to 42% hyperchromicity when fully melted. Cot analysis revealed the presence of repetitive sequences in both DNAs: approximately 46.7% in T. foetus DNA and 53.3% in T. vaginalis DNA. The unique sequences of these two protozoan DNAs are of a similar size of about 2.5 X 10(7) base pairs. Agarose gel electrophoresis of restriction fragments of the two purified DNA samples gave distinct banding patterns that were characteristic of the two species of protozoan parasites.

Further studies on the surface saccharides in Trichomonas vaginalis strains by fluorescein-conjugated lectins.

Fluorescence emitted by individual cells of several Trichomonas vaginalis strains, nearly all of which were cloned, incubated with fluorescein-conjugated lectins in the absence (experimental) or presence (control) of inhibitory sugars, or else in phosphate-buffered saline alone (autofluorescence) was measured with a Leitz MPV Compact microfluorometer. Irrespective of whether the organisms were postfixed in formalin or glutaraldehyde, the relative fluorescence emitted by the cells was closely comparable, provided that appropriate neutral density filters were employed. However, autofluorescence was much higher for glutaraldehyde-fixed trichomonads. Therefore, although better preserved and more amenable to subsequent manipulations, such organisms were found unsuitable for use in "qualitative" titration of the fluorescence emitted by various strains. Provided that the necessary precautions were taken, comparable fluorescence readings were obtained with trichomonads affixed to glass slides by heat (41 degrees C, on a section spreader) or by a cytologic centrifuge (Cytospin 2). Large numbers of concanavalin A (Con A)-binding sites were present on organisms of all strains, irrespective of their virulence for human patients and as estimated by the subcutaneous mouse assay; these sites were shown with the aid of D-mannose to be mannose or mannose-related residues. More binding sites for soybean agglutinin (SBA) were found on the virulent than on avirulent strains. On the basis of inhibition experiments, the sugar residues mainly responsible for these differences appeared to be D-lactose residues. Similar differences were observed with Ricinus communis agglutinin Type I (RCA I), for which D-galactose was employed as the competing sugar. However, with two cloned strains the situation with regard to RCA I binding was reversed - more of the lectin bound to a mild than to a virulent strain. The results obtained with Ricinus communis Type II agglutinin (RCA II) were often similar to those noted for RCA I; however, in most instances the inhibition with N-acetyl-D-galactosamine (GalNAc) was lower. Furthermore, the results noted with the GalNAc-specific agglutinins from Dolichus biflorus and Helix pomatia suggested that only very few GalNAc residues were available for binding on the surfaces of all T. vaginalis strains examined in the course of this study. Statistical analyses of fluorescence emitted by four clones of each, Balt 42 (virulent) and JH31A (avirulent) T. vaginalis strain upon incubation with Con A and SBA revealed homogeneity of these strains with regard to the number of the specific surface saccharide residues, D-mannose and D-lactose.(ABSTRACT TRUNCATED AT 400 WORDS)

Thiol groups on the surface of anaerobic parasitic protozoa.

Evidence is presented that Giardia lamblia and Entamoeba histolytica, phylogenetically unrelated aerotolerant anaerobes, have crucial thiol groups on or easily accessible to their external surface. Both parasites were killed by three structurally unrelated thiol-blocking reagents which penetrate intact cells poorly or not at all. The parasites were protected from p-chloromercuribenzenesulfonic acid (10-100 microM) by cysteine or by reduced glutathione. Killing was arrested with identical kinetics by addition of either cysteine (which quickly penetrates the cells) or bovine serum albumin (which does not penetrate intact cells) at various times after p-chloromercuribenzenesulfonic acid, indicating that the reactive site may be on the outer surface of the cell. Proteins lacking cysteine did not protect. Sensitivity of three other protozoa to p-chloromercuribenzenesulfonic acid was also tested. Trichomonas vaginalis (anaerobic) was at least as sensitive as E. histolytica and G. lamblia, while Crithidia fasciculata and Paramecium tetraurelia (both aerobic) were less sensitive. Thiol groups on the G. lamblia surface were demonstrated directly by fluorescence-activated cell sorter analysis of trophozoites which had been modified with a thiol-specific hapten, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl)ethylenediamine and reacted with fluorescent antibody to this hapten.