Two novel serum-free media for the culture of <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>

Definitive diagnosis of Trichomonas vaginalis, one of the common causes of sexually transmitted diseases in Turkey, relies on the microscopic examination of both fresh preparations and culture material of genital secretions and urine. The aim of this study was to compare the diagnostic efficacies of two culture media, serum-free TB1 and TB2, including iron and vitamin B12, respectively, with the well-known medium, TYM. Growth rate reached peak levels 48 h in TYM and 72 h in both TB1 and TB2 after inoculation. The highest amount of viable trophozoites has been obtained from TB1, almost equal to TYM but significantly higher than TB2. Fresh preparations obtained from the vaginal secretions of 119 patients have been examined and vaginal samples have been inoculated in TB1, TB2, and TYM. Viable T. vaginalis trophozoites have been detected in ten (8.4%) of fresh preparations and 11 (9.9%) of each medium. It is concluded that serum-free TB1 medium could be used effectively in both the isolation and maintenance of T. vaginalis culture in vitro.     

Inhibition of serum diamine oxidase discloses a constitutive putrescine release from cultured vascular smooth muscle cells

OBJECTIVE: To determine if putrescine and the higher polyamines spermidine and spermine are released from cultured vascular smooth muscle cells. MATERIAL: Vascular smooth muscle cell line A7r5. TREATMENT: Cells were treated with aminoguanidine (10 or 100 microM) for 1 to 24 h with or without fetal calf serum (10%) present in the culture medium. METHODS: Cellular and medium concentrations of polyamines were determined by liquid chromatography. Total cellular protein was determined by the Bradford procedure. Student's two-tailed t-test was used for statistical calculations. RESULTS: A constitutive release of putrescine was disclosed within 5 h if serum diamine oxidase was inhibited by 10 microM aminoguanidine. The release was linear with time for 24 h and specific for putrescine in the sense that the higher polyamines spermidine and spermine, despite similar cellular concentrations, were not released. Similar amounts of putrescine were released from the smooth muscle cells whether or not culture medium contained serum. Cells, that had been cultured in medium from which fetal calf serum had been omitted for last 48 h, contained less putrescine, spermidine and protein than those that persisted in medium that contained serum. CONCLUSION: A constitutive putrescine release from vascular smooth muscle cells is disclosed in the presence of aminoguanidine.     

Soluble Trichomonas vaginalis antigens in cell-free culture supernatants

Trichomonad proteins present in cell-free supernatants from logarithmic phase cultures of a pathogenic strain of Trichomonas vaginalis were detected with enzyme-linked immunosorbent assay employing antibody from mice and rabbit antisera. Numerous immunogenic membrane proteins of T. vaginalis were identified by immunoblotting of culture filtrate with rabbit antisera. Released membrane antigens ranging in molecular weight from 30 000 to 300 000 under reducing conditions were also detected in radioimmunoprecipitation assays using growth medium from mid-logarithmic phase organisms which were extrinsically labeled. Parallel experiments using [³H]thymidine-labeled T. vaginalis demonstrated that lysis was not solely responsible for the presence of parasite proteins in growth medium.     

Diagnosis of Trichomonas vaginalis Infection by PCR Using Vaginal Swab Samples

Trichomonas vaginalis infection is the most prevalent nonviral sexually transmitted disease (STD) in the world. A PCR test using vaginal swab samples for the detection of T. vaginalis was developed to add T. vaginalis infection to the growing list of STDs that can be detected by DNA amplification techniques. A primer set, BTUB 9/2, was designed to target a well-conserved region in the beta-tubulin genes of T. vaginalis. All strains (15 of 15) of T. vaginalis tested were successfully detected by PCR giving a single predicted product of 112 bp in gel electrophoresis. No such targeted product was amplified with DNA from Trichomonas tenax, Trichomonas gallinae, Chlamydia trachomatis, Neisseria gonorrhoeae, Giardia lamblia, Chilomastix sulcatus, Dientamoeba fragilis, and Entamoeba histolytica. An optimal analytical sensitivity of one T. vaginalis organism per PCR was achieved. Culture, performed with the Inpouch TV culture system, was examined daily with a light microscope to identify T. vaginalis. Twenty-three of 350 (6.6%) vaginal swab samples from women attending an army medical clinic were culture positive for T. vaginalis. Of these culture positive specimens, PCR detected 22 of 23 (96%) with primer set BTUB 9/2, and wet preparation detected only 12 of 23 (52%). Seventeen specimens were BTUB 9/2-PCR positive and culture negative. Ten of these discordant specimens were determined to be as true positive by PCR using primer sets TVA 5-1/6 and/or AP65 A/B, which target different regions in the T. vaginalis genome, and seven were determined to be false positive. The sensitivity of BTUB 9/2-PCR was 97% and the specificity was 98%. The sensitivities of culture and wet preparation were 70 and 36%, respectively. The diagnosis of T. vaginalis infection by PCR is a sensitive and specific method that could be incorporated into a joint strategy for the screening of multiple STDs by using molecular amplification methods.     

Putrescine — a potent endogenous anti-inflammatory substance in inflammatory exudates

Sponge-induced exudate from the rat and saline extracts of normal rat liver and rat liver damagedin situ by dimethylnitrosamine have been assayed for putrescine, spermidine and spermine levels. The three physiological oligoamines have been examined for anti-inflammatory activity by the carrageenan-induced oedema rat model and spermidine and putrescine were found to be anti-inflammatory with putrescine being about ten times as active as spermidine. Putrescine was also anti-inflammatory in the adjuvant arthritic rat model.Thioacetamide and theophylline were also anti-inflammatory and theophylline doubled the level of putrescine in rat liver. It was concluded that putrescine was a potent anti-inflammatory factor in the inflammatory exudates and extracts examined.     

Viability of Trichomonas vaginalis in Copan universal transport medium and eSwab transport medium

We compared the use of universal transport medium and eSwab transport medium held at room temperature or 37°C to bedside inoculation and immediate incubation of culture media for the detection of Trichomonas vaginalis. There were no significant culturable differences in the sensitivity of either of the transport media to that of bedside inoculation.     

Release of polyamines in cultures of rat parotid and liver cells

Rat parotid gland and liver cells were cultured for 6 and 24 h. The cells as well as their growth medium were analyzed on their content of the polyamines putrescine, spermidine and spermine. In control medium the content of polyamines was very low but already after 6 h substantial amounts of all three polyamines under study had been released into the medium from parotid as well as from liver cells. The release was much more pronounced from parotid compared to liver cells. Putrescine was accumulated in parotid cells as well as in the medium indicating that a new synthesis of this amine occurred in these cells.     

Release of polyamines in cultures of rat parotid and liver cells

Rat parotid gland and liver cells were cultured for 6 and 24 h. The cells as well as their growth medium were analyzed on their content of the polyamines putrescine, spermidine and spermine. In control medium the content of polyamines was very low but already after 6 h substantial amounts of all three polyamines under study had been released into the medium from parotid as well as from liver cells. The release was much more pronounced from parotid compared to liver cells. Putrescine was accumulated in parotid cells as well as in the medium indicating that a new synthesis of this amine occurred in these cells.     

Polyamine biosynthesis in trichomonads

Trichomonas vaginalis, Tritrichomonas foetus and Trichomitus batrachorum grown in modified Diamond's medium all had high concentrations of putrescine and lower concentrations of spermidine and spermine. Ornithine decarboxylase (ODC; EC 4.1.1.17) was detectable in all three species although at significantly different levels. Trichomonas vaginalis had the highest activity (typically around 1.85 nmol min-1 (mg protein)-1), Trichomitus batrachorum the lowest (0.11 nmol min-1 (mg protein)-1). The Trichomonas vaginalis ODC had an apparent Mr of 230 000 and was severely inhibited by alpha-difluoromethylornithine (DFMO). S-Adenosyl-methionine decarboxylase (EC 4.1.1.50) could not be detected in T. batrachorum but was present in the other two species. Arginine decarboxylase was apparently absent from all three. All three trichomonad species were able to accumulate spermidine and putrescine from the medium. When T. vaginalis was grown in the presence of DFMO (4 mM), which had little effect on parasite growth, ODC activity was reduced by over 99% and the polyamine content was altered; putrescine concentrations were decreased, those of spermidine and spermine remained the same or were raised. DFMO-treated cells accumulated more exogenous putrescine than untreated control cells. The results suggest that the lack of effect of DFMO on T. vaginalis in culture was due to the parasite being able to accumulate polyamines from the growth medium. It appears, therefore, that testing DFMO and similar compounds in axenic trichomonad cultures may well not give a true indication of their effectiveness in vivo where sources of exogenous polyamines may not be available.     

Regulation of polyamine content in cultured fibroblasts

The content of putrescine and of the polyamines (spermidine and spermine) and the activities of their biosynthetic enzymes were measured in 3T3 mouse fibroblasts and SV40-transformed mouse fibroblasts over the entire period from subculturing in fresh medium until confluence. The transformed cells had a substantially higher content of putrescine and spermidine than the 3T3 cells and higher activities of all of the biosynthetic enzymes. However, the ratio of spermine synthase to spermidine synthase was higher in the 3T3 cells, which correlated with their higher spermine-to-spermidine ratio. All of the biosynthetic enzymes increased in activity during cell growth. Ornithine decarboxylase increased 20-fold with a maximum at 24-36 h after culturing whereas S-adenosylmethionine decarboxylase increased 3-fold at the same time. Spermidine synthase increased 10- to 16-fold during the growth period whereas spermine synthase increased 2- to 3-fold. The relative enzyme activities and the changes in total polyamine content suggested that 1) the activity of S-adenosylmethionine decarboxylase limited the production of the polyamines and 2) the relative amounts of spermidine and spermine synthase determined the predominant polyamine that the available decarboxylated S-adenosylmethionine is used to synthesize. When 3T3 cells become quiescent at confluence, there was a substantial fall in the intracellular spermidine level because of a greatly increased excretion of spermidine into the medium. Spermine content also fell because there was an increased conversion of spermine into spermidine, which was then excreted. The specific excretion of spermidine did not occur with the transformed SV-3T3 cells.     

The use of a highly sensitive electrophoretic method to compare the proteinases of trichomonads

A highly sensitive electrophoretic method involving gelatin-containing polyacrylamide gels has been used to analyse trichomonad proteinases. Multiple forms, optimally active at pH 5–6, were present in all four species examined, but the species could be distinguished from one another by both quantitative and qualitative differences. The intestinal parasites, Trichomitus batrachorum and Pentatrichomonas hominis, had lower specific activities than the urogenital parasites, Trichomonas vaginalis and Trichomonas foetus, and, in the case of P. hominis, there were fewer enzyme forms. The high activity proteinases of Tritrichomonas foetus had low apparent molecular weights (<25 kDa), while the predominant enzymes of Trichomonas vaginalis were of high apparent molecular weight (68–110 kDa). Distinct differences were also observed between the proteinase patterns of various isolates of T. vaginalis. All of the enzymes were stimulated by dithiothreitol, suggesting that they were cysteine proteinases. This was confirmed for the T. vaginalis and Tritrichomonas foetus proteinases from their inhibition by antipain, leupeptin, TLCK and iodoacetic acid. The method allows the detection of proteinases in samples of Trichomonas vaginalis containing as few as 10⁴ cells or as little as 1 μg protein. It was also possible to detect proteinase activity released into the medium. For both T. vaginalis and Tritrichomonas foetus, the extracellular enzymes present during early log phase were qualitatively different from the intracellular proteinases, although the latter were present in samples of media obtained from later cultures (cell densities greater than 1 × 10⁵ parasites ml⁻¹). The results show the potential of this technique for detecting proteinases in trichomonad samples in studies aimed at determining proteinase function in pathogenesis and host-parasite relationships.     

Translation initiation factor eIF-5A, the hypusine-containing protein, is phosphorylated on serine and tyrosine and O-glycosylated in Trichomonas vaginalis

The eukaryotic translation factor eIF-5A is highly conserved throughout eukaryotes and undergoes an unusual polyamine-dependent post-translational modification called hypusination. Trichomonas vaginalis has two tveif-5a genes (tveif-5a1 and tveif-5a2), each encoding a 19-kDa protein. In this report, we describe the detection of two forms with different isoelectric points (5.2 and 5.5) that correspond to the precursor and mature TveIF-5A, respectively. In addition, we demonstrated that only the mature form of TveIF-5A is phosphorylated and glycosylated via two-dimensional gel electrophoresis-western blot (2DE-WB) assays using anti-phosphoserine and anti-phosphotyrosine antibodies and the SNA, ConA and MAA lectins. Interestingly, when the protozoa were grown in 1,4-diamino-2-butanone (DAB), an inhibitor of putrescine biosynthesis, and transferred to medium containing exogenous putrescine, a new spot with an isoelectric point of 5.3 was observed, presumably corresponding to a phosphorylated intermediate or deoxyhypusine form. Our data indicate that, in T. vaginalis, phosphorylations and glycosylations are necessary to obtain the mature TveIF-5A, and we confirm the identity of the precursor, intermediate and mature forms of TveIF-5A by mass spectrometry analysis.     

Evaluation of a Chromogenic Culture Medium for the Detection of Clostridium difficile

Purpose

Clostridium difficile (C. difficile) is an important cause of nosocomial diarrhea. Diagnostic methods for detection of C. difficile infection (CDI) are shifting to molecular techniques, which are faster and more sensitive than conventional methods. Although recent advances in these methods have been made in terms of their cost-benefit, ease of use, and turnaround time, anaerobic culture remains an important method for detection of CDI.

Materials and Methods

In efforts to evaluate a novel chromogenic medium for the detection of C. difficile (chromID CD agar), 289 fecal specimens were analyzed using two other culture media of blood agar and cycloserine-cefoxitin-fructose-egg yolk agar while enzyme immunosorbent assay and polymerase chain reaction-based assay were used for toxin detection.

Results

ChromID showed the highest detection rate among the three culture media. Both positive rate and sensitivity were higher from chromID than other culture media. ChromID was better at detecting toxin producing C. difficile at 24 h and showed the highest detection rate at both 24 h and 48 h.

Conclusion

Simultaneous use of toxin assay and anaerobic culture has been considered as the most accurate and sensitive diagnostic approach of CDI. Utilization of a more rapid and sensitive chromogenic medium will aid in the dianogsis of CDI.     

An electron microscope study of the interaction between Trichomonas vaginalis and epithelial cells of the human amnion membrane

The mechanism of cytopathogenicity of Trichomonas vaginalis is not well established. Adhesion of T. vaginalis to human epithelial cells is considered a prerequisite for parasitic infection and its pathogenic effect. To investigate cytopathological changes in the host caused by T. vaginalis infection, human amnion membrane was used as an in vitro model. T. vaginalis strain WAA38 from axenic culture was allowed to interact with the epithelial layer of the human amnion membrane for 6 and 9 h. Structural changes resulting from the interaction between parasite and host cells were studied with transmission (TEM) and scanning (SEM) electron microscopy. Analysis of the electron microscope data showed that T. vaginalis established contact with the host cells as early as after 6 h of incubation; however, a close attachment of parasites to the epithelial cells occurred only after 9 h. Amoeboid T. vaginalis formed numerous cytoplasmic extensions and adhered to the epithelial cells mostly through the portions of their body opposite the undulating membrane. A dense network of microfilaments was seen at the site of contact between T. vaginalis and epithelial cells. Damaged and desquamated epithelial cells were seen with TEM and SEM only in the areas where parasites were in direct contact with target cells.     

The effect of 3-(biphenyl-4-yl)-3-hydoxyquinuclidine (BPQ-OH) and metronidazole on Trichomonas vaginalis: a comparative study

Trichomonas vaginalis causes trichomoniasis in humans, a sexually transmitted disease commonly treated with metronidazole (MTZ), a drug that presents some toxicity, causing undesirable side effects. In addition, an increase in metronidazole-resistant parasites has been reported. Thus, the development of alternative treatment is recommended. To date, the search for antiparasitic drugs has been based on different approaches: identification of active natural products, identification of parasite targets, and the use of available compounds active against other pathogenic microorganisms. Here, we analyzed the in vitro antiproliferative and ultrastructural effects on T. vaginalis of BPQ-OH, a hydroxiquinuclidine derivative that inhibits squalene synthase and is active against several protozoa and fungi. We also compared the effects of BPQ-OH on T. vaginalis and mammalian cells with those of MTZ. We found that BPQ-OH inhibits in vitro proliferation of T. vaginalis, with an IC₅₀ of 46 μM after 24 h. Although this IC₅₀ is 16 times higher than that of MTZ (1.8 μM), BPQ-OH is less toxic for human cell lines than MTZ, with LC₅₀ values of 2,300 and 70 μM, and selective indexes of 50 and 39, respectively. Ultrastructural analyses demonstrated that BPQ-OH induced alterations in T. vaginalis, such as rounded and wrinkled cells, membrane blebbing and intense vacuolization, leading to cell death, whereas MTZ also caused significant changes, including a decrease in hydrogenosomes size and endoflagellar forms. Our observations identify BPQ-OH as a promising leading compound for the development of novel anti-T. vaginalis drugs and highlight the need for further testing this molecule using experimentally infected animals.     

Effect of DL-alpha-difluoromethylornithine on polyamine synthesis and interconversion in Trichomonas vaginalis grown in a semi-defined medium

Growth of Trichomonas vaginalis in a semi-defined medium was inhibited by 5 mM DL-alpha-difluoromethylornithine (DFMO). Using high pressure liquid chromatography (HPLC) analysis, putrescine and cadaverine levels were found to be 90 and 100% reduced, respectively after 120 h exposure, whilst spermidine and spermine levels were unchanged. Putrescine (40 microM) and cadaverine (6 microM) were detected in the spent media from control cultures. Neither of these diamines was detected in spent media from 72 h DFMO-treated cultures. Changes in intracellular levels of amine precursors were also determined by HPLC. There was a transient increase in ornithine to 39 nmol (mg protein)-1 at 48 h in the DFMO-treated cells while it remained undetectable in control cells throughout the experiment. Arginine and citrulline levels remained high, decreasing to control levels only after 72 h. Only spermine (1 mM) rescued DFMO-treated cells, and this is discussed with respect to the presence of a putative spermine-specific oxidase designated by its sensitivity to aminoguanidine. Aerobic incubation of growing (normal) cells with [14C]spermine resulted in the production of an unknown metabolite (19% of total label), whose content was reduced to 5% under anaerobic conditions. Decarboxylated S-adenosylmethionine remained undetectable in DFMO-treated cells, and the methylation index (ratio of S-adenosylmethionine to S-adenosylhomocysteine) did not change from the control value of 9.3. Ornithine decarboxylase, S-adenosylmethionine synthetase, S-adenosylmethionine:L-homocysteine methyltransferase, and S-adenosylhomocysteine hydrolase enzyme activities were detected. However, S-adenosylmethionine decarboxylase, spermidine synthase or spermine synthase were not detected. These findings are discussed with reference to the arginine dihydrolase pathway whose end products are putrescine and ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid Presumptive Identification of Gardnerella vaginalis (Haemophilus vaginalis) from Human Blood Agar Media

Presumptive identification of Gardnerella vaginalis from 48-h human blood agar cultures by using a Gram stain, hemolysis, and colonial morphology was highly accurate.     

Polyamine metabolism in filarial worms

The human and animal filarial parasites Onchocerca volvulus, Dirofilaria immitis, Brugia patei and Litomosoides carinii contained low levels of putrescine but much higher levels of spermidine and spermine as estimated by ion-pair high pressure liquid chromatography; N-acetylated polyamines were present only in minute amounts. Enzyme activities of ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19), respectively, were not detectable. Experiments carried out with O. volvulus and D. immitis demonstrated the uptake and bioconversion of labeled polyamines. There is evidence for the existence of a complete reverse pathway generating putrescine from spermidine and spermine, respectively, in both worms. N-Acetylating enzyme activities were detected in 100,000 X g preparations of homogenates from D. immitis which were capable to acetylate putrescine, spermidine and spermine. Long term incubation of the worms in the presence of labeled polyamines resulted in the excretion of putrescine and N-acetylputrescine.