Investigación de Trichomonas vaginalis durante el embarazo mediante diferentes metodologías / Investigation of Trichomonas vaginalis through different methodologies during pregnancy

Los objetivos del trabajo fueron conocer la prevalencia de tricomonosis en pacientes embarazadas y evaluar la utilidad de diferentes metodologías para su diagnóstico. Se estudiaron prospectivamente 223 mujeres embarazadas. Trichomonas vaginalis se investigó utilizando distintos exámenes microscópicos, cultivo en medio sólido y medio líquido. Se evaluó la sensibilidad y especificidad de la microscopía considerando a los cultivos en ambos medios como método de referencia. La prevalencia del parásito obtenida por cultivo (medio líquido más medio sólido) fue de 4,5% (10/223) siendo la detección por examen en fresco, coloración de May-Grunwald Giemsa, fresco con solución acética formolada (SAF)/azul de metileno y por cultivo en medio sólido y líquido de 1,3%, 1,8%, 1,8% y 4,5% respectivamente. La sensibilidad del examen en fresco fue 30%, para el May-Grunwald Giemsa y el SAF/azul de metileno fue 40%. Utilizando conjuntamente los tres exámenes microscópicos, la sensibilidad se elevó al 50% y la especificidad fue 100% para todos los exámenes microscópicos. El cultivo en medio líquido detectó el 100% de los casos positivos , mientras que el medio sólido sólo el 50%. Por la baja sensibilidad de la microscopía para T. vaginalis, en embarazadas asintomáticas recomendamos la utilización del cultivo en medio líquido durante el embarazo, para instaurar un tratamiento precoz.

The aim of this study was to conduct a survey regarding the prevalence of trichomoniasis in pregnant patients and to evaluate the utility of different diagnostic methods. Two hundred and twenty three vaginal swab specimens from pregnant women were prospectively examined. Trichomonas vaginalis was investigated by various microscopic examinations, solid culture medium and liquid culture medium. The sensitivity and specificity of microscopy were evaluated by considering both culture media as the "gold standards". The prevalence of T. vaginalis obtained by both culture media (liquid plus solid media) was 4.5% (10/223). The prevalence of T. vaginalis obtained by direct smear, May-Grunwald Giemsa staining, sodium acetate-acetic acid-formalin (SAF)/Methylene blue staining-fixing technique, solid medium and liquid medium was 1.3%, 1.8%, 1.8% and 4.5%, respectively. The sensitivity of the direct smear was 30 %, but for the May- Grunwald Giemsa staining and the SAF/Methylene blue staining-fixing technique was 40%. Considering the three microscopic examinations altogether, the sensitivity rose to 50% and the specificity was 100% for all of them. The solid medium detected only 50% of the positive cases; the liquid medium detected 100%. Due to the low sensitivity obtained with microscopy in asymptomatic pregnant patients, we recommend the use of the liquid medium during pregnancy, in order to provide an early treatment.

Prevalencia de candidiasis vaginal en embarazadas: Identificación de levaduras y sensibilidad a los antifúngicos / Prevalence of vaginal candidiasis in pregnant women: Identification of yeasts and susceptibility to antifungal agents

La mujer embarazada es más susceptible tanto a la colonización como a la infección vaginal por levaduras. El objetivo de este trabajo fue determinar la prevalencia de levaduras aisladas de exudados vaginales de mujeres embarazadas y evaluar la sensibilidad a los antifúngicos de uso frecuente. Se estudiaron 493 pacientes en el período comprendido desde diciembre de 1998 hasta febrero de 2000. La prevalencia de Candida spp. fue 28% (Candida albicans 90,4%, Candida glabrata 6,3%, Candida parapsilosis 1,1%, Candida kefyr 1,1%, especies no identificadas 1,1%). Se determinó la sensibilidad a fluconazol, ketoconazol, itraconazol y nistatina por el método de difusión en agar Shadomy. Todos los aislamientos de C. albicans, C. kefyr y C. parapsilosis fueron sensibles in vitro a los antifúngicos probados, mientras que 1 de 6 aislamientos de C. glabrata presentó resistencia extendida a todos los azoles, pero sensibilidad a nistatina. En mujeres embarazadas C. albicans fue la levadura más frecuentemente aislada de exudados vaginales y continúa siendo ampliamente sensible a los antifúngicos; sólo en C. glabrata se observó resistencia a los azoles. Se recomienda la identificación de la levadura a nivel de especie particularmente en el caso de falla terapéutica y en infecciones recidivantes o crónicas.

Pregnant women are more susceptible to both vaginal colonization and infection by yeast. Our objectives were to determine the prevalence in pregnant women of yeasts isolated from vaginal exudates and their susceptibility to current antifungal drugs. A total of 493 patients was studied between December 1998 and February 2000. The prevalence of Candida spp. was 28% (Candida albicans 90.4%; Candida glabrata 6.3%; Candida parapsilosis 1.1%, Candida kefyr 1.1%; unidentified species 1.1%). The diffusion test in Shadomy agar was employed to determine the susceptibility to fluconazole, ketoconazole, itraconazole and nistatine. All C. albicans, C. kefyr and C. parapsilosis isolates were susceptible in vitro to the antifungal agents tested, while 1 in 6 C. glabrata isolates showed resistance to azole drugs; all strains were susceptible to nistatine. In pregnant women, C. albicans was the yeast most frequently isolated from vaginal exudates; it continues to be highly susceptible to antifungal drugs. Azole resistance was detected only among C. glabrata isolates. Identification to the species level is recommended, specially in cases of treatment failure and recurrent or chronic infection.

Toxocara canis infections in a pig model: immunological, haematological and blood biochemistry responses.

The immunological, haematological and enzymatic responses to the inoculation in pigs of 100,000 embryonated eggs of Toxocara canis were studied. Fifteen females were inoculated and three remained as controls. Haematological values were analysed from day 7 p.i. until day 126 p.i. In the inoculated group, white blood cells were raised on day 14 p.i. and eosinophil values on days 7, 14, 21, 35 and 49 p.i. showing significant differences compared with controls (P < 0.05). Absolute eosinophil counts (per ml) presented two rises, the first on days 7, 14 and 21 p.i. and the second on days 35 and 49 p.i. Blood biochemistry was maintained within normal values. Serological examination by ELISA to determine antibody levels against Toxocara canis L2/L3 excretory-secretory (ES) antigens showed values higher than the positive cut-off (1:32) from day 7 p.i. and until the end of the study on day 126 p.i., presenting two peaks: one on day 28 p.i. and the second covering days 49 to 56 p.i. Western blots of sera of inoculated animals presented, from day 7 p.i., two polypeptide bands of 55 and 70 kDa MW and, from day 56 p.i., an additional band of 120 kDa MW, all of which persisted until the end of the study. Immunological responses were sustained over time. No direct correlation was observed between the rise in eosinophils and antibody titres. To validate the conclusions, more studies are required on the polypeptide bands.

Detección de la actividad de desoxiribonucleasa (DNasa) en cepas de Clostridium chauvoei / [Deoxyribonuclease activity detection in Clostridium chauvoei strains]

Beta toxin of C. chauvoei has desoxiribonuclease (DNase) activity which is regarded as one of its virulence factors. The production of DNase was detected in strains isolated from bovines, using as controls C. chauvoei ATCC 10092, and C. perfringens Type A and C. septicum, both laboratory isolates. The enzyme activity was made evident on a DNA substrate observing the macroscopic degradation. A simple methodology was developed using a commercial medium for DNase test, with the incorporation of sterile horse serum. Each strain was streaked on the surface of the medium, incubated in anaerobic atmosphere at 37 degrees C for 48 hours. The plates were revealed with HCI 1 N. The appearance of a clear and transparent zone around and under the microbial growing was considered a positive reaction. Enzyme activity was detected in 10 of 12 strains and also in the controls. The serum addition to the commercial basal medium allows the optimum development of the microorganism showing the enzymatic digestion zone.

Toxocara canis in experimentally infected pigs: migratory pattern and tissue lesions.

Fifteen Yorkshire female pigs were inoculated with 100,000 infective T. canis eggs. Three animals were used as uninfected controls. Groups of three infected pigs were euthanized by accepted methods on days 7, 14, 21, 28 and 126 p.i., respectively. Larvae were recovered from all animals included in each group slaughtered on days 7 and 14 p.i.; on day 21 p.i. from two pigs, on day 28 p.i. from one, and no larvae were found on day 126 p.i. Differences in the mean number of larvae per gram in lymph nodes, liver and lungs between slaughter days, were significant for livers on day 7 p.i. and for lungs on day 14 p.i. (P < 0.10). The decrease over time was significant in all the organs that previously had larvae. Larvae were not found in the other organs and tissues analysed. Macroscopical lesions were found in the liver, lungs and lymph nodes on days 7, 14, 21, and 28 p.i. The entire surface of the liver was covered with small white spots on day 7 p.i., on days 14 and 21 p.i. the spots were distinctly nodular and, in some places, individual lesions were confluent. Lesions had apparently started to heal on days 28 and 126 p.i. appearance was normal. Lymph nodes were enlarged and oedematous during the first 4 weeks and the lungs had small areas of consolidation visible all over the surface, but by day 126 p.i., no visible lesions could be seen. Microscopical lesions were observed in the liver on day 7 p.i., with a largely periportal hepatitis. Numerous eosinophils and lymphocytes were present. The typical granulomatous reaction was observed on days 14 and 21 p.i. with a central necrotic core and a narrow region of fibroblastic tissue. By day 28 p.i. lesions had almost disappeared and the number of eosinophils was fewer. There were fewer leukocytes and the fibrous tissue had disappeared from the liver on day 126 p.i. For the first 3 weeks, pictures of the lymph nodes and the lungs were characterised by the formation of a granuloma. In the center of the granuloma larvae were observed. The majority of the lesions had healed by day 126 p.i.

Portación de estreptococo grupo B en mujeres embarazadas / [Group B Streptococcus carriers among pregnant women]

Streptococcus agalactiae--group B streptococci (GBS)--is a main cause of severe neonatal infections with a high mortality rate. The detection of pregnant GBS carriers (5-35

) allows intrapartum administration of antibiotic prophylaxis to these women and prevents perinatal infection. We studied the prevalence of GBS in 259 patients between 28 and 37 weeks gestation from April 2000 to March 2002. The anorectum (AR) and vaginal introitus swabs (VI) were cultured in selective Todd-Hewitt broth containing colistin (10 micrograms/ml) and nalidixic acid (15 micrograms/ml) while vaginal swabs (VFS) were cultured following conventional methods. A total of 47 strains of EGB were isolated from 259 patients (18.15

). The prevalence in different samples were: 5.40

in AR and 17.76

in VI + AR (reference method). The isolates were tested against penicillin, ceftriaxone, erythromycin, clindamycin, vancomycin, gentamicin and streptomycin to determine the minimum inhibitory concentration. The resistance phenotypes of erythromycin-resistant GBS were determined by the double-disk test. All strains were susceptible to penicillin, ceftriaxone and vancomycin, only one strain was erythromycin and clindamycin resistant by IMLSB mechanism. None of the isolated strains had a high resistant level to aminoglycosides. The sensitivity of cultures increased when selective broths were used as the primary detection method.

Antiviral activity of enterocin CRL35 against herpesviruses.

Enterocin CRL35 is an antibacterial polypeptide of 3.5 x 10(3) Da produced by Enterococcus faecium CRL35. A series of experiments are described that show the enterocin also had antiviral activity against thymidine-kinase positive (tk+) and deficient (tk-) strains of herpes simplex (HSV) type 1 and 2 in Vero and BHK-21 cells. This activity was observed at 100 microg/ml, 15-fold lower than the cytotoxic concentration. In both cell lines there was a 2 log inhibition of infectivity. The compound inhibited viral multiplication in a dose-dependent manner and had no virucidal effect. Enterocin CRL35 also inhibited the virion-associated host shutoff in infected Vero cells showing that intracellular viral multiplication was affected.

Flagellin, a major protein present in SDS-PAGE profiles of Sarkosyl-OMP-enriched fractions from Bordetella bronchiseptica Bvg- or modulated Bvg+ strains.

The bvg or vir locus positively regulates the expression of many Bordetella virulence-associated determinants (encoded by vag genes), including cell envelope proteins, in response to environmental stimuli. On the other hand, several genes named vrg genes are negatively controlled by the bvg regulon (Knapp and Mekalanos, 1988). Flagellin is encoded by a vrg gene, which is expressed when the principal virulence factors are eliminated during antigenic modulation or in phase variants (Akerley et al., 1992). We have previously analyzed SDS-PAGE profiles of Sarkosyl-outer membrane protein (OMP)-enriched fractions from B. bronchiseptica Bvg- and modulated Bvg+ strains and reported a major band associated with the avirulent phenotype (Passerini de Rossi et al., 1995). In order to characterize this band we have purified flagellar filaments from Bvg- and modulated Bvg+ strains, and analyzed them by SDS-PAGE. These profiles revealed a single major band of 40 or 45 kDa depending on the strain. The N-terminal amino acid sequence of the putative flagellin expressed by BB7200a was identical over the first 21 residues analyzed to that of the flagellin from the modulated strain BB7865 reported by Akerley et al. (1992). Comparison of the SDS-PAGE profile of flagellar filaments with that of the OMP-enriched fraction of the corresponding strain showed that the flagellum-associated polypeptide had the same electrophoretic mobility as that of the characteristic band of the avirulent phenotype. Furthermore, this band was absent in the OMP-enriched fraction profile from a Bvg- strain subjected to a treatment that removes flagella. Our results indicate that the major protein observed in SDS-PAGE profiles of Sarkosyl-OMP-enriched fractions from B. bronchiseptica Bvg- and modulated Bvg+ strains corresponds to flagellin.

Meliacine, an antiviral compound from Melia azedarach L., inhibits interferon production.

A glycopeptide isolated from the high plant Melia azedarach L. (meliacine) inhibits the in vitro replication of several RNA and DNA animal viruses. Interferon (IFN) production was depressed greatly in meliacine treated L929 cells and primary mouse embryo fibroblast cultures (MEF) induced with Newcastle disease virus (NDV) or poly(rI).poly(rC). This action was observed when meliacine was added before, simultaneously or early after induction with poly(rI).poly(rC) or NDV. In addition, accumulation of acid-resistant IFN was strongly diminished in adult mice treated intraperitoneally with meliacine. Though meliacine causes a strong inhibition of IFN both in vitro and in vivo, we do not know how selectively it affects the IFN system.

Induction of a refractory state to viral infection in mammalian cells by a plant inhibitor isolated from leaves of Melia azedarach L.

A partially purified plant inhibitor (Meliacin) isolated from Melia azedarach L induced in cells a refractory state to virus infection. Meliacin was active in a large variety of continuous and/or primary cell cultures. A state of maximum virus resistance was achieved after 2 h of incubation and was maintained for at least 15 h; later on it declined but it was fully regained after a second pulse of Meliacin. Interferon was not detected in the supernatant of cells treated with Meliacin and a measurable increase in ds-RNA dependent protein kinase activity was not observed in extracts of Meliacin-treated cells. The antiviral state was not transferred by either extracellular fluid or direct cell-to-cell contact. An active cell metabolism was required for Meliacin action, which was partially reversed in the presence of actinomycin D. It appears that Meliacin is not an interferon-like substance, which induces an antiviral state based on a still unexplained mechanism.

Influence of the metabolic environment on oxygen uptake and isometric developed tension of isolated rat portal vein.

Oxygen uptake and isometric developed tension (IDT) of isolated rat portal vein were simultaneously measured after glucose removal, following the readmission of metabolic substrates and addition of 2-deoxy-D-glucose (2-DG) or insulin. In control conditions, basal O2 uptake was 0.128 +/- 0.004 microliter O2/mg dry weight/min (n = 50). After 40 min of incubation in a glucose-free medium, IDT and O2 uptake decayed to a similar degree. Addition of 11 mM glucose, 5 mM pyruvate, acetate, lactate or butyrate to hypodynamic veins, elicited differential recoveries in the mechanical and metabolic parameters, pyruvate and acetate being the most effective. Insulin (0.1 U/ml) added 5 min prior to readmission of 11 mM glucose, increased the restoration of IDT without any effect on oxygen uptake. 2-DG 5.5 mM, when added in glucose-free medium, abolished IDT but only depressed the cellular respiration by 38% at 20 min; these effects were decreased in presence of glucose (11 mM). These results suggest that the oxygen uptake of this vessel depends, among other factors, on the exogenous substrate offered. They show also the relationship between peak of developed isometric force and metabolic activity as well as the relevance of glycolytic and oxidative pathways in this tissue.

Ensayos de citotoxicidad y actividad antiviral de extractos crudos y semipurificados de hojas verdes de Melia azedarach L / [Assays of cytotoxicity and antiviral activity of crude and semipurified extracts of green leaves of Melia azedarach L.]

Crude extracts from fresh green leaves of Melia azedarach L contain an antiviral factor (FAV) able to inhibit the replication of several animal viruses, e.g. Polio, VSV, HSV, FMDV, Sindbis, Junín, Pichinde and Tacaribe in Vero or BHK-21 cells. Crude preparations were subjected to different steps of purification like chromatography on Sephadex G-100 and DEAE-Sephadex. The antiviral activity of G-100 and DEAE fractions was fully conserved, whereas contaminating proteins were lost. Two types of cytotoxicity tests were performed with the different fractions. Two-fold serial dilutions of each of them were added to preformed monolayers of Vero or BHK-21 cells and cellular viability was tested. While crude extracts were toxic at low dilutions (less than or equal to 1:10), G-100 and DEAE fractions were not. The other cytotoxicity assay consisted in seeding the cells in the presence of different concentrations of each fraction. G-100 fraction affected cell growth at low dilutions (less than or equal to 1:5), while DEAE fraction did not. It should be remarked that the purification procedure rendered a partial purified DEAE fraction with an increased specific activity (antiviral activity/mg of protein). It is concluded that an antiviral factor devoid of toxicity exists in M. azedarach L extracts, which exhibited a broad spectrum of antiviral activity.

Effects of oxamate, an inhibitor of lactate dahydrogenase, upon the spontaneous or oxytocin-induced motility and over pyruvate levels of uterine horns isolated from ovariectomized or estrus rats.

The stability with time of the spontaneous as well as the oxytocin-triggered functional activity of uterine borns isolated from induced estrus rats and immersed in a medium with lactate as the substrate was not affected by the presence of oxomate (an inhibitor of lactate dehydrogenase) at 10 or 20mM. On the contrary, the oxytocin-driven and the spontaneous motility of preparations obtained from 15 days castrated rats diminished significantly following in addition of the enzyme inhibitor. Furthermore, uterine borns from ovariectomized animals injected with 17-beta estradiol, regain the lack of functional sensivity towards oxamate. In addition, determinations of pyruvate levels in homogenates of uterine tissue, previously suspended in lactate medium (as in the functional experiments), demonstrate to be significantly reduced, following oxamate. Several possible reasons, attempting to explain these findings, are discussed.

PIP: The pharmacological effects of oxamate on the spontaneous or the oxytocin-induced motility of isolated rat uterus in a solution with lactate as the sole external substrate were investigated during estrus, following ovariectomy, and after castration accompanied with estradiol replacement therapy. Uterine preparations were tested for mechanical activity (isometric-developed tension and the frequency of contractions). Initial oxytocin-induced activity of the uterus of rats in induced estrus was significantly greater than spontaneous activity (p.01). Oxamate added to the suspension was ineffective in modifying this activity. In castrate rats, however, the addition of oxamate to the medium significantly reduced oxytocin-driven and spontaneous motility. Those ovariectomized rats receiving replacement therapy were also resistant to oxamate effects. The effects of oxamate upon pyruvate levels of oxytocin-exposed uterine tissue were also determined. In the presence of oxamate, levels of uterine pyruvate in all the hormonal conditions were significantly lower than in controls (p.001).

Simian virus 40 deoxyribonucleic acid replication. I. Effect of cycloheximide on the replication of SV40 deoxyribonucleic acid in monkey kidney cells and in heterokaryons of SV40-transformed and susceptible cells.

Infectious deoxyribonucleic acid (DNA) was extracted from green monkey kidney (CV-1) cultures at various times after the cultures were infected with simian virus 40 (SV40) at input multiplicities of 0.01 and 0.1 plaque-forming unit (PFU) per cell. A pronounced decrease in infectious DNA was observed from 3 to 16 hr after virus infection, suggesting that structurally altered intracellular forms may have been generated early in infection. Evidence is also presented that SV40 DNA synthesis requires concurrent protein synthesis. DNA replication was studied in the presence and absence of cycloheximide in: (i) SV40-infected and uninfected cultures of CV-1 cells; (ii) cultures synchronized with 1-beta-d-arabinofuranosylcytosine (ara-C) for 24 to 30 hr prior to the addition of cycloheximide; and (iii) in heterokaryons of SV40-transformed hamster and susceptible monkey kidney cells. DNA synthesis was determined by pulse-labeling the cultures with (3)H-thymidine at various times from 24 to 46 hr after infection. In addition, the total infectious SV40 DNA was measured. Addition of cycloheximide, even after early proteins had been induced, grossly inhibited both SV40 and cellular DNA syntheses. The activities of thymidine kinase, DNA polymerase, deoxycytidylate deaminase, and thymidylate kinase were measured; these enzyme activities remained high for at least 9 hr in the presence of cycloheximide. SV40 DNA prelabeled with (3)H-thymidine before the addition of cycloheximide was also relatively stable during the time required for cycloheximide to inhibit further DNA replication.