Comparison of enzymatic activities in different Candida species isolated from women with vulvovaginitis.

INTRODUCTION: Comparing the activities of secreted enzymes in different fungal species can improve our understanding of their pathogenic role. Secretion of various enzymes by Candida species has been considered for determination of their virulence in different Candida infections including vulvovaginitis. AIMS: The aim of this study was to determine and compare the activity of secreted enzymes in Candidia strains isolated from women suspected to vulvovaginal candidiasis (VVC) and referred to some health centers in Khuzestan, Southwestern Iran. PATIENTS AND METHODS: The vaginal secretion samples were taken by swap from 250 suspected women with symptoms of vulvovaginal candidiasis and cultured on CHROMagar Candida medium. Identification of the isolated Candida from culture positive samples performed by the color of colonies and some standard mycological procedures. Activities of phospholipase, hemolysin-α, hemolysin-ß, esterase and proteinase were measured in vitro by standard laboratory protocols. The enzymatic activity index (EAI) was calculated for each enzyme in accordance to relevant protocols. RESULTS: Totally in eighty cases (32%), a Candida strain was isolated which found to be as 52 (65%) Candida albicans; 12 (15%) C. glabrata; 10 (12.5%) C. dubliniensis; 4 (5%) C. krusei, C. tropicalis and C. parapsilosis species (each=1; 1.3%). Among C. albicans strains, 89.1% produced all studied enzymes, while 86% of C. glabrata strains failed to produce proteinase and phospholipase. The EAIs in decreasing order were as hemolysin-ß=0.2895, hemolysin-α=0.5420, esterase=0.5753, proteinase=0.7413, and phospholipase=0.7446, respectively. Activity of phospholipase, esterase and proteinase secreted by C. albicans and C. dubliniensis were significantly more than those released by C. glabrata and C. krusei, while 86% of C. glabrata strains did not show esterase activity. On the other hand, the activity rates of hemolysin α and ß among all studied isolates were almost similar. CONCLUSION: In the present study, the prevalence of VVC among investigated women was higher than the previous report from Khuzestan but C. albicans has yet remained the predominant agent of VVC in this area. Given to the EAI, the virulence of C. albicans in VVC can be mediated by phospholipase, esterase and proteinases.

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.

[Protease and phospholipase activities of Candida albicans isolated from vaginal secretions with different pH values]. / Actividades proteinasa y fosfolipasa de aislamientos de Candida albicans provenientes de secreciones vaginales con distintos valores de pH.

Even though vulvovaginal candidiasis and bacterial vaginosis are seldom simultaneously found, we have detected this association at an above average frequency. Thus, we set out to study the activity of proteinases and phospholipases, virulence factors of Candida albicans, to assess their role in the above mentioned association. Of a total of 70 Candida isolates were retrieved from samples of vaginal secretions analyzed at our Diagnostic Service, 65 were identified as C. albicans (a group of n=26 obtained from clinical samples of pH>4.5 and a group of n=39 from clinical samples of pH=or<4.5). The evaluation of phospholipases activity was performed on malt agar and Sabouraud dextrose agar with the addition of egg yolk as substrate. The proteolytic activity was detected on plates of agar base medium with the addition of bovine albumin serum as substrate as sole nitrogen source. Phospholipases activity was essentially the same in both groups of samples (p=0.2003). Proteolytic activity was detected in 61.5% of the isolates from the group with pH=or<4.5 and in 96.2% in the group with pH>4.5; being the former much higher than the latter (p=0.0001). Based on these results we postulate that the simultaneous occurrence of bacterial vaginosis and vulvovaginal candidiasis could be related to the proteolytic activity but unrelated to phospholipases activity.

Increased susceptibility of secretor factor gene Fut2-null mice to experimental vaginal candidiasis.

Fut2-LacZ-null mice, which are a model of the human ABO and Lewis nonsecretor group, display increased susceptibility to experimental yeast vaginitis, indicating a role for alpha(1,2)fucosylated cervical glycans in mucosal defense. However, the lack of significant effect of competitive inhibition by exogenous neoglycoproteins in this study emphasizes the complexity of Candida-epithelial cell adhesion events.

Leukocyte esterase activity in vaginal fluid of pregnant and non-pregnant women with vaginitis/vaginosis and in controls.

OBJECTIVES: To determine the leukocyte esterase (LE) activity in vaginal lavage fluid of women with acute and recurrent vulvovaginal candidosis (VVC and RVVC respectively), bacterial vaginosis (BV), and in pregnant and non-pregnant women without evidence of the three conditions. Also to compare the result of LE tests in women consulting at different weeks in the cycle and trimesters of pregnancy. The LE activity was correlated to vaginal pH, number of inflammatory cells in stained vaginal smears, type of predominating vaginal bacteria and presence of yeast morphotypes. METHODS: One hundred and thirteen women with a history of RVVC, i.e. with at least four attacks of the condition during the previous year and who had consulted with an assumed new attack of the condition, were studied. Furthermore, we studied 16 women with VVC, 15 women with BV, and 27 women attending for control of cytological abnormalities, who all presented without evidence of either vaginitis or vaginosis. Finally, 73 pregnant women were investigated. The LE activity in vaginal fluid during different weeks in the cycle of 53 of the women was measured. RESULTS: In the non-pregnant women, an increased LE activity was found in 96, 88, 73 and 56% of those with RVVC, VVC and BV and in the non-VVC/BV cases, respectively. In 73% of pregnant women in the second trimester, and 76% of those in the third, the LE test was positive. In all groups of non-pregnant women tested, the LE activity correlated with the number of leukocytes in vaginal smears, but it did not in those who were pregnant. There was no correlation between LE activity and week in cycle. The vaginal pH showed no correlation to LE activity in any of the groups studied. CONCLUSIONS: The use of commercial LE dipsticks has a limited value in the differential diagnosis of RVVC, VVC and BV. There is no correlation between the LE activity in vaginal secretion on one hand and vaginal pH, week in the menstrual cycle and trimester in pregnancy on the other. Women with BV often have signs of inflammation as evidenced by a positive LE test and inflammatory cells in genital smears.

High aspartyl proteinase production and vaginitis in human immunodeficiency virus-infected women.

Vaginal isolates of Candida albicans from human immunodeficiency virus-positive (HIV+) and HIV- women with or without candidal vaginitis were examined for secretory aspartyl proteinase (Sap) production in vitro and in vivo and for the possible correlation of Sap production with pathology and antimycotic susceptibility in vitro. HIV+ women with candidal vaginitis were infected by strains of C. albicans showing significantly higher levels of Sap, a virulence enzyme, than strains isolated from HIV+, C. albicans carrier subjects and HIV- subjects with vaginitis. The greater production of Sap in vitro was paralleled by greater amounts of Sap in the vaginal fluids of infected subjects. In an estrogen-dependent, rat vaginitis model, a strain of C. albicans producing a high level of Sap that was isolated from an HIV+ woman with vaginitis was more pathogenic than a strain of C. albicans that was isolated primarily from an HIV-, Candida carrier. In the same model, pepstatin A, a strong Sap inhibitor, exerted a strong curative effect on experimental vaginitis. No correlation was found between Sap production and antimycotic susceptibility, as most of the isolates were fully susceptible to fluconazole, itraconazole, and other antimycotics, regardless of their source (subjects infected with strains producing high or low levels of Sap, subjects with vaginitis or carrier subjects, or subjects with or without HIV). Thus, high Sap production is associated with virulence of C. albicans but not with fungal resistance to fluconazole in HIV-infected subjects, and Sap is a potentially new therapeutic target in candidal vaginitis.

Chronic vaginal candidiasis responsive to biotin therapy in a carrier of biotinidase deficiency.

BACKGROUND: Many patients experience recurrent or persistent episodes of vaginal candidiasis. Some of these women might be carriers of an inborn error of biotin metabolism (either biotinidase deficiency or holocarboxylase synthetase activity). These women might benefit from administration of pharmacologic amounts of biotin. CASE: A 38-year-old gravida 2, para 2 carrier of biotinidase deficiency presented with a 14-month history of persistent vaginal candidiasis, despite appropriate therapy. After 3 months of pharmacologic doses of biotin, her symptoms resolved completely. CONCLUSION: Given that 1 in every 123 individuals is predicted to be a carrier of biotinidase deficiency, there might be other women with chronic vaginal candidiasis who will respond to biotin administration.

Ultrastructural localization of the secretory aspartyl proteinase in Candida albicans cell wall in vitro and in experimentally infected rat vagina.

Detection and ultrastructural localization of aspartyl proteinase (Sap) in Candida albicans experimentally infecting rat vagina were studied. Two Sap-positive (Sap+) and one Sap-negative (Sap-) strains of the fungus, endowed with high and low experimental vaginopathic potential, respectively, were used. Both Sap+ strains produced consistent Sap levels in the rat vagina, while the Sap- strain did not produce any measurable Sap. Electron microscopy of thin sections of chemically-fixed vaginal scrapings showed clear evidence of hyphae of proteolitic strains of C. albicans invading the keratinized epithelial cell layer of the vagina. The fungal cells exhibited a pronounced fibrillar layer on the cell wall with a marked intermixing of fungal and vaginal materials especially pronounced at the hyphal tip. Post-embedding immunogold techniques with the use of anti-Sap polyclonal and the specifically generated monoclonal antibody GF1 showed that Sap was essentially localized in the cell wall of C. albicans early during infection, in a cytological pattern mirroring Sap localization in C. albicans cells grown in Sap-inductive media in vitro. In summary, the data offer a new biochemical and ultrastructural evidence that Sap is actively secreted during experimental rat vaginitis by C. albicans. Cell wall localization of Sap is probably inherent to this active secretion process.

Evidence for a role for secreted aspartate proteinase of Candida albicans in vulvovaginal candidiasis.

The presence of the secretory aspartate (acid) proteinase in the vaginal fluid of candidal vaginitis patients and controls was studied by ELISA and immunoblot (Western blot). In addition, a proteinase-deficient mutant strain of Candida albicans (IR24) was compared with the wild-type parent strain (10261) for ability to infect the vagina of pseudoestrus rats under estradiol treatment. Among the 67 women examined, proteinase was detected only in 22 harboring C. albicans (range, 42-233 ng/ml of vaginal fluid), at concentrations significantly higher in the 14 vaginitis patients than in the 8 asymptomatic fungal carriers. Western blots confirmed the presence of only one protein band of approximately 43 kDa, corresponding to that of the purified proteinase, in the ELISA-positive vaginal fluids. Experimental vaginal infection was significantly more extensive and persistent in rats infected with the proteinase-producer strain than in those challenged with the proteinase-deficient mutant, and the enzyme was detected in the vaginas of the former but not of the latter animals. Both strains 10261 and IR24 developed hyphal forms to a roughly similar extent during infection, and both showed a comparable adherence in vitro to vaginal and buccal epithelial cells. The clinical and experimental evidence support a role for secretory proteinase as a virulence factor in the pathogenesis of candidal vaginitis.