Identification and characterization of NanH2 and NanH3, enzymes responsible for sialidase activity in the vaginal bacterium Gardnerella vaginalis.

Gardnerella vaginalis is abundant in bacterial vaginosis (BV), a condition associated with adverse reproductive health. Sialidase activity is a diagnostic feature of BV and is produced by a subset of G. vaginalis strains. Although its genetic basis has not been formally identified, sialidase activity is presumed to derive from the sialidase A gene, named here nanH1 In this study, BLAST searches predicted two additional G. vaginalis sialidases, NanH2 and NanH3. When expressed in Escherichia coli, NanH2 and NanH3 both displayed broad abilities to cleave sialic acids from α2-3- and α2-6-linked N- and O-linked sialoglycans, including relevant mucosal substrates. In contrast, recombinant NanH1 had limited activity against synthetic and mucosal substrates under the conditions tested. Recombinant NanH2 was much more effective than NanH3 in cleaving sialic acids bearing a 9-O-acetyl ester. Similarly, G. vaginalis strains encoding NanH2 cleaved and foraged significantly more Neu5,9Ac2 than strains encoding only NanH3. Among a collection of 34 G. vaginalis isolates, nanH2, nanH3, or both were present in all 15 sialidase-positive strains but absent from all 19 sialidase-negative isolates, including 16 strains that were nanH1-positive. We conclude that NanH2 and NanH3 are the primary sources of sialidase activity in G. vaginalis and that these two enzymes can account for the previously described substrate breadth cleaved by sialidases in human vaginal specimens of women with BV. Finally, PCRs of nanH2 or nanH3 from human vaginal specimens had 81% sensitivity and 78% specificity in distinguishing between Lactobacillus dominance and BV, as determined by Nugent scoring.

Enzymatic assay to test diamines produced by vaginal bacteria.

An enzymatic assay was developed to determine the concentration of diamines (DA) in clinical samples of vaginal fluids. Putrescine and cadaverine are DA produced by anaerobic bacteria and are typically present in the vaginal fluids of women with an abnormal microbiota, as occurs in bacterial vaginosis. The vaginal DA (VADA) assay is based on the enzyme diamine oxidase which reacts with putrescine and cadaverine to produce H2O2 in a quantitative manner. H2O2 concentration is measured spectrophotometrically by a chromogenic reaction catalyzed by horseradish peroxidase. The VADA assay proved to be capable of detecting DA concentrations as low as 4 mM and showed a dose-response relationship which was linear over DA concentrations ranging from 4 to 256 mM. Using clinical samples it was possible to show that the VADA assay can be performed on human vaginal swabs and that the mean DA concentration is significantly higher in samples positive for microbial pathogens.

Degradation, foraging, and depletion of mucus sialoglycans by the vagina-adapted Actinobacterium Gardnerella vaginalis.

Bacterial vaginosis (BV) is a polymicrobial imbalance of the vaginal microbiota associated with reproductive infections, preterm birth, and other adverse health outcomes. Sialidase activity in vaginal fluids is diagnostic of BV and sialic acid-rich components of mucus have protective and immunological roles. However, whereas mucus degradation is believed to be important in the etiology and complications associated with BV, the role(s) of sialidases and the participation of individual bacterial species in the degradation of mucus barriers in BV have not been investigated. Here we demonstrate that the BV-associated bacterium Gardnerella vaginalis uses sialidase to break down and deplete sialic acid-containing mucus components in the vagina. Biochemical evidence using purified sialoglycan substrates supports a model in which 1) G. vaginalis extracellular sialidase hydrolyzes mucosal sialoglycans, 2) liberated sialic acid (N-acetylneuraminic acid) is transported into the bacterium, a process inhibited by excess N-glycolylneuraminic acid, and 3) sialic acid catabolism is initiated by an intracellular aldolase/lyase mechanism. G. vaginalis engaged in sialoglycan foraging in vitro, in the presence of human vaginal mucus, and in vivo, in a murine vaginal model, in each case leading to depletion of sialic acids. Comparison of sialic acid levels in human vaginal specimens also demonstrated significant depletion of mucus sialic acids in women with BV compared with women with a "normal" lactobacilli-dominated microbiota. Taken together, these studies show that G. vaginalis utilizes sialidase to support the degradation, foraging, and depletion of protective host mucus barriers, and that this process of mucus barrier degradation and depletion also occurs in the clinical setting of BV.

Amylases in the Human Vagina.

Dominance of Lactobacillus species in vaginal communities is a hallmark of healthy conditions in the female genital tract. Key nutrients for lactobacilli include sugars produced when glycogen is degraded by α-amylase in the vagina. While α-amylase activity has been demonstrated in vaginal fluids, it is unclear whether α-amylases are produced solely by the host, bacteria in the vagina, or both. We screened cervicovaginal mucus from 23 reproductive-age women, characterized the species composition of vaginal communities, measured vaginal pH, and determined levels of amylase activity, glycogen, and lactic acid. Based on differences in these measured variables, one sample from each of four individual donors was selected for metagenomic and proteomic analyses. Of eight putative bacterial amylases identified in the assembled bacterial metagenomes, we detected four in vaginal fluids. These amylases were produced by various bacteria in different vaginal communities. Moreover, no two communities were the same in terms of which bacteria were producing amylases. Although we detected bacterial amylases in vaginal fluids, there was no clear association between the bacterial species that was dominant in a community and the level of amylase activity. This association was likely masked by the presence of human α-amylase, which was also detected in vaginal fluids. Finally, the levels of amylase activity and glycogen were only weakly associated. Our findings show, for the first time, that multiple amylases from both bacterial and human origins can be present simultaneously in the vagina. This work also suggests that the link between glycogen, amylase, and Lactobacillus in the vagina is complex.IMPORTANCE In this study, we show that multiple bacteria in the vaginal community produce amylases that hydrolyze glycogen into simpler sugars (i.e., maltose and maltotriose). These sugars serve as "common goods" that sustain bacterial populations in vaginal communities. Given the temporal changes that are observed in the human vaginal microbiome, we expect the kinds of bacterial amylases produced will also vary over time. These differences influence the pool of resources that are broadly shared and shape the species composition of the vaginal bacterial community.

Among pregnant women with bacterial vaginosis, the hydrolytic enzymes sialidase and prolidase are positively associated with interleukin-1beta.

OBJECTIVE: The objective of the study was to explore the mechanisms of local innate immunity induction and modulation in pregnant women with bacterial vaginosis (BV). STUDY DESIGN: A total of 200 singleton pregnant women in early gestation (12 +/- 4 weeks) with BV (Nugent 7-10) without concurrent vaginal infections with Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, syphilis, and yeast. Concentrations of vaginal interleukin (IL)-1beta and IL-8, the number of neutrophils, and the levels of sialidase and prolidase hydrolytic enzymes were determined in vaginal fluid. RESULTS: Concentrations of vaginal IL-1beta had a strong positive correlation with levels of sialidase (P < .001) and prolidase (P < .001). Conversely, such enzymes were negatively correlated with the ratio of IL-8/IL-1beta (both P < .001) and were not significantly associated with concentrations of IL-8. Notably, the number of vaginal neutrophils had a negative correlation with sialidase (P = .007). CONCLUSION: The strong induction of IL-1beta in BV-positive women appears to be associated with the production of the hydrolytic enzymes sialidase and prolidase by BV-associated bacteria. However, these 2 enzymes may inhibit the expected amplification of the proinflammatory IL-1beta cascade as evaluated by the down-regulation of the IL-8/IL-1beta ratio. A blunted response to IL-1beta signals may cause the poor rise of neutrophils, which is peculiar to BV. This impairment of local defense may contribute to increased susceptibility to adverse outcomes in BV-positive pregnant women.

Modulation of vaginal immune response among pregnant women with bacterial vaginosis by Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, and yeast.

OBJECTIVE: This study was undertaken to examine the influence of coinfections on vaginal innate and adaptive immunity, and microbial enzyme activities of pregnant women with bacterial vaginosis (BV). STUDY DESIGN: The population consisted of 265 singleton pregnant women in early gestation (<20 weeks) with BV (Nugent 7-10) who had vaginal fluid collected for measurement of interleukin-1beta (IL-1beta) and IL-8 concentrations, number of neutrophils, immunoglobulin A against Gardnerella vaginalis (anti-Gvh IgA), and activities of microbial sialidase and prolidase. RESULTS: Among women with BV, median levels of vaginal IL-1beta (4-fold, P = .005), IL-8 (4-fold, P < .001), and neutrophils (6-fold, P = .013) were greatly increased in women with T vaginalis with respect to women without any coinfection. Yeast increased the level of IL-8 (5-fold, P < .001), but not IL-1beta (P = .239) and neutrophils (P = .060). Chlamydia trachomatis and Neisseria gonorrhoeae had no effect on vaginal cytokines. None of the coinfections influenced vaginal anti-Gvh IgA, sialidase and prolidase activities. CONCLUSION: The strong proinflammatory cytokine induction by T. vaginalis may contribute to the observed increase in preterm birth among BV positive women coinfected with T. vaginalis treated with metronidazole.

The presence of the putative Gardnerella vaginalis sialidase A gene in vaginal specimens is associated with bacterial vaginosis biofilm.

Bacterial vaginosis (BV) is a difficult-to-treat recurrent condition in which health-associated lactobacilli are outnumbered by other anaerobic bacteria, such as Gardnerella vaginalis. Certain genotypes of G. vaginalis can produce sialidase, while others cannot. Sialidase is known to facilitate the destruction of the protective mucus layer on the vaginal epithelium by hydrolysis of sialic acid on the glycans of mucous membranes. This process possibly facilitates adhesion of bacterial cells on the epithelium since it has been linked with the development of biofilm in other pathogenic conditions. Although it has not been demonstrated yet, it is probable that G. vaginalis benefits from this mechanism by attaching to the vaginal epithelium to initiate biofilm development. In this study, using vaginal specimens of 120 women enrolled in the Ring Plus study, we assessed the association between the putative G. vaginalis sialidase A gene by quantitative polymerase chain reaction (qPCR), the diagnosis of BV according to Nugent score, and the occurrence of a BV-associated biofilm dominated by G. vaginalis by fluorescence in situ hybridisation (FISH). We detected the putative sialidase A gene in 75% of the G. vaginalis-positive vaginal specimens and found a strong association (p<0.001) between the presence of a G. vaginalis biofilm, the diagnosis of BV according to Nugent and the detection of high loads of the G. vaginalis sialidase A gene in the vaginal specimens. These results could redefine diagnosis of BV, and in addition might guide research for new treatment.

Variation in vaginal immune parameters and microbial hydrolytic enzymes in bacterial vaginosis positive pregnant women with and without Mobiluncus species.

OBJECTIVE: This study was undertaken to assess if levels of interleukin-1beta (IL-1beta), IL-8, sialidase, prolidase and immunoglobulin A against Gardenerella vaginalis hemolysin (anti-Gvh IgA) in vaginal secretions differ between BV+ women with (M+) and without (M-) Mobiluncus spp. STUDY DESIGN: Vaginal secretions were obtained from 265 women at their first prenatal care visit and assessed for all study parameters. Gram stain evaluation using Nugent criteria was performed and coinfection with sexually transmitted infections determined. Differences between BV+/M+ and BV+/M- women were evaluated using the chi2 statistic or Mann-Whitney test. RESULTS: Of the 265 BV+ women, 43% (n = 113) were M+ of which 97% (n = 110) had Nugent scores of 9 or 10 . BV+/M+ women had elevated levels of sialidase (median value: 4.11 nmol vs 1.91 nmol of converted substrate; P = .003) but no difference in prolidase, anti-Gvh IgA, IL-1beta, IL-8, levels were found between the two groups. BV+/M- women had significantly higher rates of coinfection with Trichomonas vaginalis. CONCLUSION: BV+/M+ women have higher vaginal concentrations of sialidase and lower rates of T. vaginalis compared with BV+/M- women. Further research is needed to assess the association of this, and other, microbiologic profiles to risk of adverse pregnancy outcome.

[Sialidase activity in women with bacterial vaginosis]. / Actividad sialidasa en mujeres con vaginosis bacteriana.

Bacterial vaginosis (VB) is a syndrome characterized by overgrowth of endogenous Gram negative bacterial flora and the lack of the normal flora. Within bacterial enzymes, sialidases have been considered a virulence factor of many pathogenic microorganisms colonizing the different mucous membranes. Their presence in vaginal discharges can be correlated with VB. The aim of this study was to detect the activity of this enzyme in women with this syndrome and without clinical evidence of genital infection. Out of a total 112 women studied, 51 were patients with VB and the other 61 women presented normal vaginal flora. For the quantification of enzyme activity, the technique based on the enzymatic hydrolysis of a derivative acid of the acetyl metoxifenil muramic acid was used. In the studied population both groups shared values from 0.5 to 5.1 nmoles of metoxifenol, whereas only 11 out of 52 patients with VB (21.17%), registered more than 5.1 nmoles. The presence of sialidase activity is not enough to confirm VB, except for values greater than 5.5 nmoles of the metoxifenol produced in the enzymatic reaction.

The Effects of Hormones and Vaginal Microflora on the Glycome of the Female Genital Tract: Cervical-Vaginal Fluid.

In this study, we characterized the glycome of cervical-vaginal fluid, collected with a Catamenial cup. We quantified: glycosidase levels; sialic acid and high mannose specific lectin binding; mucins, MUC1, MUC4, MUC5AC, MUC7; and albumin in the samples collected. These data were analyzed in the context of hormonal status (day of menstrual cycle, hormonal contraception use) and role, if any, of the type of the vaginal microflora present. When the Nugent score was used to stratify the subjects by microflora as normal, intermediate, or bacterial vaginosis, several important differences were observed. The activities of four of six glycosidases in the samples from women with bacterial vaginosis were significantly increased when compared to normal or intermediate women: sialidase, P = <0.001; α-galactosidase, P = 0.006; ß-galactosidase, P = 0.005; α-glucosidase, P = 0.056. Sialic acid binding sites as measured by two lectins, Maackia amurensis and Sambucus nigra binding, were significantly lower in women with BV compared to women with normal and intermediate scores (P = <0.0001 and 0.008 respectively). High mannose binding sites, a measure of innate immunity were also significantly lower in women with BV (P = <0.001). Additionally, we observed significant increases in MUC1, MUC4, MUC5AC, and MUC7 concentrations in women with BV (P = <0.001, 0.001, <0.001, 0.02 respectively). Among normal women we found that the membrane bound mucin MUC4 and the secreted MUC5AC were decreased in postmenopausal women (P = 0.02 and 0.07 respectively), while MUC7 (secreted) was decreased in women using levonorgestrel-containing IUDs (P = 0.02). The number of sialic acid binding sites was lower in the postmenopausal group (P = 0.04), but the number of high mannose binding sites, measured with Griffithsin, was not significantly different among the 6 hormonal groups. The glycosidase levels in the cervical-vaginal mucus were rather low in the groups, with exception of α-glucosidase activity that was much lower in the postmenopausal group (P<0.001). These studies present compelling evidence that the vaginal ecosystem responds to the presence of different vaginal microorganisms. These effects were so influential that it required us to remove subjects with BV for data interpretation of the impact of hormones. We also suggest that certain changes occurring in vaginal/cervical proteins are due to bacteria or their products. Therefore, the quantitation of vaginal mucins and lectin binding offers a new method to monitor bacteria-host interactions in the female reproductive tract. The data suggest that some of the changes in these components are the result of host processing, such as the increases in mucin content, while the microflora is responsible for the increases in glycosidases and the decreases in lectin binding. The methods should be considered a valid marker for insult to the female genital tract.

Diagnosis and microecological characteristics of aerobic vaginitis in outpatients based on preformed enzymes.

OBJECTIVE: Aerobic vaginitis (AV) is a recently proposed term for genital tract infection in women. The diagnosis of AV is mainly based on descriptive diagnostic criteria proposed by Donders and co-workers. The objective of this study is to report AV prevalence in southwest China using an objective assay kit based on preformed enzymes and also to determine its characteristics. MATERIALS AND METHODS: A total of 1948 outpatients were enrolled and tested by a commercial diagnostic kit to investigate the AV prevalence and characteristics in southwestern China. The study mainly examined the vaginal ecosystem, age distribution, Lactobacillus amount, and changes in pH. Differences within groups were analyzed by Wilcoxon two-sample test. RESULTS: The AV detection rate is 15.40%. The AV patients were usually seen in the sexually active age group of 20-30 years, followed by those in the age group of 30-40 years. The vaginal ecosystems of all the patients studied were absolutely abnormal, and diagnosed to have a combined infection [aerobic vaginitis (AV) + bacterial vaginitis (BV) 61.33%; 184/300]. Aerobic bacteria, especially Staphylococcus aureus and Escherichia coli, were predominantly found in the vaginal samples of these women. CONCLUSION: AV is a common type of genital infection in southwestern China and is characterized by sexually active age and combined infection predominated by the AV and BV type.

BVBLUE test for diagnosis of bacterial vaginosis in pregnant women attending antenatal care at Phramongkutklao Hospital.

BACKGROUND: Bacterial vaginosis (BV) is a disorder of the vaginal ecosystem characterized by a shift in the vaginal flora form the normally predominated lactobacillus to one dominated by sialidase enzyme-producing mixed flora. OBJECTIVES: To compare the sensitivity of BVBLUE test for diagnosis of bacterial vaginosis with Gram stain by using Nugent score as a gold standard. MATERIAL AND METHOD: From April to June, 2004, a total of 173 pregnant women who received antenatal care at Phramongkutklao Hospital had reached the study criteria. The speculum for this exam, used in the process of collecting vaginal secretions, must not be lubricated with any lubricants. The vaginal discharge was collected from the lower 1/3 of the vaginal wall. Gram stain score and BVBLUE test were conducted and compared. RESULTS: 173 patients were enrolled in the present study. BVBLUE test was compared to the standard method for the diagnosis of BV by Gram stain using Nugent score as a gold standard. The sensitivity, specificity, accuracy, positive and negative predictive value of BVBLUE test versus the Gram stain score for diagnosis of bacterial vaginosis were 94%, 96%, 96% 86%, and 98%, respectively. CONCLUSION: BVBLUE test for diagnosis of bacterial vaginosis had high sensitivity, specificity, accuracy, positive and negative predictive value (94%, 96%, 96%, 86%, and 98%, respectively).

α-Amylase in Vaginal Fluid: Association With Conditions Favorable to Dominance of Lactobacillus.

Vaginal glycogen is degraded by host α-amylase and then converted to lactic acid by Lactobacilli. This maintains the vaginal pH at ≤4.5 and prevents growth of other bacteria. Therefore, host α-amylase activity may promote dominance of Lactobacilli. We evaluated whether the α-amylase level in vaginal fluid is altered in women with bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) and whether its concentration was associated with levels of lactic acid isomers and host mediators. Vaginal fluid was obtained from 43 women with BV, 50 women with VVC, and 62 women with no vulvovaginal disorders. Vaginal fluid concentrations of α-amylase, secretory leukocyte protease inhibitor (SLPI), hyaluronan, hyaluronidase-1, ß-defensin, and elafin were measured by enzyme-linked immunosorbent assay (ELISA). Vaginal concentrations of neutrophil gelatinase-associated lipocalin (NGAL), matrix metalloproteinase (MMP) 8, and d- and l-lactic acid levels in these patients were previously reported. The median vaginal fluid α-amylase level was 1.83 mU/mL in control women, 1.45 mU/mL in women with VVC, and 1.07 mU/mL in women with BV. Vaginal levels of α-amylase were correlated with d-lactic acid (P = .003) but not with l-lactic acid (P > .05) and with SLPI (P < .001), hyaluronidase-1 (P < .001), NGAL (P = .001), and MMP-8 (P = .005). The exfoliation of glycogen-rich epithelial cells into the vaginal lumen by hyaluronidase-1 and MMP-8 may increase glycogen availability and promote α-amylase activity. The subsequent enhanced availability of glycogen breakdown products would favor proliferation of Lactobacilli, the primary producers of d-lactic acid in the vagina. Concomitant production of NGAL and SLPI would retard growth of BV-related bacteria.

Screen-printed amperometric microcell for proline iminopeptidase enzyme activity assay.

A microfabricated amperometric microcell was designed and used for the determination of proline iminopeptidase (PIP) enzyme activity in 2-10-microl samples. The measurements were made in the range of 10.3-841.5 mU/ml enzyme activities. The sensitivity of the determinations was between - 0.0195 and - 0.0203 microA ml/mU per min. The coefficient of variation of the determined values ranged between 2.8 (at 561.2 mU/ml) and 24.1% (at 10.3 mU/ml). The microcell was manufactured on an alumina substrate using screen-printed graphite working and Ag/AgCl reference electrodes. Elevated PIP activity in the vaginal fluid is a biochemical indicator of bacterial vaginosis. The method is appropriate to differentiate between normal (66+/-145 mU/ml) and elevated, diseased (704+/-145 mU/ml), values.

Lactate dehydrogenase and its isoenzymes in vaginal fluid in vaginitis/vaginosis cases and in healthy controls.

The objective of the study was to measure the lactate dehydrogenase (LD) activity in vaginal lavage fluid of women with vaginitis/vaginosis and in healthy pre- and post-menopausal controls. Also to analyse the LD isoenzyme patterns in such samples and compare the influence on the LD activity by different storage and sampling methods. Twenty of the women studied, who had no signs of inflammation as evidenced from vaginal wet smears, were pre-menopausal and 8 post-menopausal. Fifty-eight non-pregnant patients with vaginitis/vaginosis or non-inflammatory gynaecological conditions were analysed for LD isoenzyme patterns. The LD activity was correlated to vaginal pH. Furthermore, the LD activity was determined in another 100 women screened for Chlamydia trachomatis. Finally, the influence on the LD activity when sampling was made by a cytological brush vs vaginal lavage and analysed after different storage periods, as studied. The LD activity was elevated, i.e. >2 micro kat/L, in all but two of the women with leucorrhoea. Only women with bacterial vaginosis (BV) without leucorrhoea, had an increased LD activity. An increased vaginal pH correlated to the LD concentration. The LD activity was elevated in cases with vulvovaginal candidiasis, trichomoniasis, chlamydial cervicitis and senile colpitis. Storage of samples for up to six hours had no influence on the test outcome. Brush and lavage fluid samples did not differ with regard to the rate of positive LD tests. In healthy women, the LD activity is low and predominated by slow-migrating isoenzymes, i.e. LD 4 and 5. The LD activity is generally increased in cases of vaginitis and in women with BV and chlamydial cervicitis. In trichomoniasis, particularly high concentrations of LD 5, are found.

Mucinase and sialidase activity of the vaginal microflora: implications for the pathogenesis of preterm labour.

Evidence linking bacterial vaginosis (BV) to chorioamnionitis and spontaneous preterm birth is mounting. Successful treatment of BV could reduce the rate of late miscarriage and preterm birth. Mucinase and sialidase activity have been implicated in the pathogenesis of BV. This study extends the work of previous studies to investigate sialidase, other known mucin degrading enzymes and overall mucin degrading activity in samples of vaginal fluid from women with and without BV. Samples from 31 women were diagnosed for BV, and tested for enzyme activity using established assays. Activity was recorded in all samples. Significant increases in activity were detected in BV samples for sialidase using a mucin (BSM P<0.005) and serum type glycoprotein (AGP P<0.005) substrates, beta-galactosidase (P<0.001), and beta-N-acetylhexosaminidase (P<0.01). No significant increases in BV patients were detected in O-glycanase, proteinase, arylesterase, sulphatase or whole mucinase activities. These results support the hypothesis that certain BV-associated enzymes may detrimentally affect the mucosal barrier, permitting bacteria access to the uterus.

Application of a novel human cervical mucin-based assay demonstrates the absence of increased mucinase activity in bacterial vaginosis.

Enzymes produced in bacterial vaginosis (BV) have been proposed as possible mediators of pre-term birth. Most studies have concentrated on mid-trimester measurements of enzyme activity, and utilize synthetic substrates to measure enzyme activity, which may not accurately represent mucinase activity in vivo. We have developed a novel ELISA mucinase assay using biotinylated human cervical mucin as a substrate. The assay is rapid, sensitive and can be used to screen large numbers of samples. The new assay has been used to assess vaginal mucinase activities in 92 women <14 weeks gestational age with and without BV. No differences in mucinase activity were detected between normal and BV groups while significant elevation of sialidase and other glycosidases was confirmed as reported before. This study shows that significant mucinase activity is a normal event in the mucus barrier, but does not reflect changes identified for individual enzyme activities associated with BV.

Light microscopy observation of lytic enzymatic activity of the organisms associated with bacterial vaginosis.

In this study, cervico-vaginal smears taken from 500 patients were examined cytologically using the Papanicolaou technique. Seventeen of the 500 were classified as having bacterial vaginosis. Lytic enzymatic activity of the organisms on clue cells were determined at light microscopic level. The integrity of the cell and the smoothness of the cell membrane were disrupted. Small cavities on the cell membrane and hollows in the cytoplasm were observed. Due to the loss of cytoplasm, very narrow and thin tracks around the nucleus and in the cytoplasm resembling a cobweb were seen. It is suggested that these lytic cellular changes might be formed by the organisms on clue cells.

[Relationship between vaginal sialidase bacteria vaginosis and chorioammionitis].

OBJECTIVE: To study the relationship among vaginal sialidase, bacterial vaginosis (BV), chorioammionitis and subsequent adverse outcome of pregnancy. METHODS: Vaginal sialidase was measured by colorimetry in samples of vaginal discharges from 80 pregnant women with BV (study group) and 60 normal pregnant women at same gestation weeks (control group). Color turning blue means positive of sialidase activity, and no color changing means negative. The diagnosis of chorioammionitis was based on pathological examination. RESULTS: Ninety six dot three percent, 3.3% exhibited sialidase activities in study group and control group, respectively. There were significant differences between these two groups (P < 0.001). By measuring vaginal sialidase activity, the sensitivity, specificity, positive and negative predictive value in diagnosing BV were 96.3%, 96.7%, 97.5% and 95.1%, respectively. Chorioammionitis, premature rupture of membranes, premature delivery and puerperal infection in sialidase positive group were significantly higher than the negative group. Sensitivity, specialty of sialidase activity for chorioammionitis were 87.5%, 50.0%, respectively. CONCLUSION: Vaginal sialidase activity has strong relation with bacterial vaginosis. Measuring vaginal sialidase activity is a fast, easy, and useful method to diagnose bacterial vaginosis. It also has relation with chorioammionitis and subsequent adverse outcome of pregnancy.

Bacterial vaginosis and biomarkers of oxidative stress in amniotic fluid.

OBJECTIVE: In this study we tried to determine if the activities of the primary antioxidant enzymes are detectable in amniotic fluid and if they can be used as early biomarkers of complications in pregnancy connected with bacterial vaginosis. METHODS: This was a prospective study in which amniotic fluid was taken between 16 and 19 weeks of gestation. 161 pregnant women were divided into two groups: study group--patients with the treated local infection and control group--healthy pregnant women. Levels of reduced glutathione, and the activities of glutathione peroxidase, glutathione reductase, glutathione S-transferase, xanthine oxidase, superoxide dismutase and lipid peroxidation were determined spectrophotometrically in amniotic fluid samples. RESULTS: Concentration of malonyldialdehide (product of lipid peroxidation) varied greatly between investigated groups. Xanthine oxidase and superoxide dismutase activities, though very low, were present in amniotic fluid samples. Also, enzymes of glutathione cycle and reduced glutathione concentrations were detectable and showed certain variations. CONCLUSION: Although, biomarkers of antioxidant activity are present in the amniotic fluid, they are not different between women with and without bacterial vaginosis.