Estrogen-dependent induction of persistent vaginal candidosis in naïve mice.

The capacity of estrogen to induce vaginal candidosis (VC) in the absence of previous or concurrent Candida albicans infections was examined. Adult female Balb/c mice were evaluated for vaginal C. albicans burden, C. albicans-specific delayed type hypersensitivity (DTH) responses and vaginal lymphocyte population kinetics at several time-points while receiving weekly injections of 0.5 mg estrogen. Estrogen treatment resulted in the appearance of significant levels of C. albicans vaginal colonization, which persisted for a period of 4 weeks. A marked suppression of DTH responses mounted against subsequent C. albicans challenge was observed. Absolute number of vaginal T lymphocytes gradually increased by several folds especially at weeks 5-6 following the start of estrogen treatment. These results clearly indicate that estrogen, independent of other predisposing factors, is capable of perturbing the commensal relationship between the host and the fungus, which results in the induction of persistent VC.

Vaginal T lymphocyte population kinetics during experimental vaginal candidosis: evidence for a possible role of CD8+ T cells in protection against vaginal candidosis.

Vaginal candidosis represents a significant health problem to women of childbearing age worldwide. It has been postulated that localized T cells play a role in protection against vaginal candidosis. In an attempt to evaluate the role of vaginal T cells in protection against vaginal candidosis, T cell population kinetics was evaluated using an oestrogen-dependent vaginal candidosis murine model. Vaginal T lymphocytes were isolated at different time points post C. albicans inoculation, viable cells were enumerated, phenotypically analysed for the expression of CD3, CD4 and CD8 T cell markers and absolute numbers of T cell subsets were calculated. Oestrogen-induced persistence of vaginal candidosis resulted in a significant increase in the total number of vaginal lymphocytes within 24-48 h post infection; increased vaginal lymphocyte numbers persisted throughout the infection period. The number of CD3+ T cells dramatically increased following C. albicans administration and was maintained at high levels throughout the infection period. The majority of CD3+ T cells were of the CD8+ type; however, considerable numbers of both CD4+ T cells and CD4+CD8+ T cells were also observed throughout the infection period. The considerable and persistent increase in vaginal T cell numbers in general and that of CD8+ T cells in particular are evidence of the possible role played by localized T cells in protection against vaginal candidosis.

Persistent colonization and transient suppression of DTH responses in an estrogen-dependent vaginal candidosis murine model.

Recurrence of vaginal candidosis in women of childbearing age has been attributed to several predisposing factors including the presence of significant amounts of estrogen in the reproductive tract. In this study, the effect of estrogen on the level of C. albicans colonization, persistence of infection and suppression of DTH responses was investigated in an estrogen-dependent vaginal candidosis murine model. Mice were first injected subcutaneously with 0.5 mg of estradiol valerate 72 hours prior to C. albicans intravaginal inoculation and at weekly intervals thereafter for a period of up to 4 weeks; the inoculum consisted of 2 x 10(7) stationary-phase C. albicans blastoconidia in a volume of 20 microl. C. albicans colonization was evaluated in the spleen, liver, kidney, small intestine and reproductive tract of estrogen-treated and control mice 72 hours following inoculation, DTH responses were evaluated 2 and 5 weeks following primary inoculation and persistence of infection was evaluated at days 2, 3, 4, 8, 12, 19 and 26 post inoculation. Estrogen-treated mice exhibited higher levels of C. albicans colonization compared with control mice; this was most evident in the small intestine and reproductive tract. Estrogen treatment resulted in pronounced suppression of C. albicans-specific DTH responses; in that average footpad swelling was 4.7 mm in untreated mice compared with 2.3 mm in estrogen-treated mice. Long-term estrogen treatment resulted in the persistence of infection; in contrast, C. albicans infection resolved by day 8 post inoculation in untreated mice. DTH responses assayed 5 weeks post primary inoculation in treated mice were on average 4.1 mm, this was similar to that observed in untreated mice tested for DTH response 2 and 5 weeks post primary inoculation. These results suggest that, on the one hand, estrogen has an enhancing effect on C. albicans colonization and persistence of infection. On the other, estrogen seems to suppress DTH responses within the first 2 weeks post infection; persistence of infection under the influence of estrogen, however, seems to coexist with detectable systemic cell-mediated immunity.

Increased incidence of vulvovaginal candidiasis caused by Candida glabrata in Jordan.

Due to the rising importance of Candida glabrata and other non-albicans Candida as principle human opportunistic pathogens, 356 women with abnormal vaginal discharge who attended a private obstetrics and gynecology clinic in Amman, Jordan, between January 1999 and February 2001 were examined. The isolation rate of Candida spp. from high-vaginal swabs was 44.9%. CHROMagar Candida and conventional mycological methods identified six isolated Candida spp., including C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, and C. kefyr. The percentages of C. albicans, C. glabrata, and C. tropicalis isolates were 43.1%, 32.5%, and 8.1%, respectively. C. albicans was isolated in combination with C. tropicalis and C. krusei in two patients. Statistical analysis of the present results clearly show an increase in the incidence of vulvovaginal candidiasis caused by C. glabrata in the two study periods, 1994-1996 and 1999-2001 (P = 0.0186). In contrast, comparing the proportions of vulvovaginal candidiasis caused by C. albicans in the two periods, there was no significant difference. These results may have significant clinical implications, as C. glabrata are innately less susceptible to most antifungal agents than C. albicans; these findings support viewing this organism as a major pathogen.

Characterization of phenotype-based pathogenic determinants of various Candida albicans strains in Jordan.

Sixty-six clinical isolates of Candida albicans representing 14 different strain types were tested for their phospholipase and proteinase activities in correlation with adherence to buccal epithelial cells (BECs) and lethality to mice. Variations in phospholipase and proteinase production as well as adherence to BECs were observed both among isolates of the same strain type and between isolates of different strain types. All isolates tested, irrespective of strain type, produced low levels of phopholipase (0.5 mm for strain -BCD- and 2.7 mm for strain ABC--) and acid proteinase (0.6 mm for strain A---E and 2.2 mm for strain --C--). A correlation was noted between adherence, phospholipase and proteinase production, and lethality to mice. C. albicans isolates, which adhered most strongly to BECs, exhibited higher levels of phospholipase and proteinase activities as well as higher pathogenicity. This was most evident in strain type --C--, which exhibited higher adherence ability (mean 717 +/- 21 yeasts/100 BEC), and proteinase activity (mean 2.2 mm), and relatively higher phospholipase activity (mean 2.4 mm) compared with those of other strains. Additionally, this type was more prevalent and showed significantly higher levels of tissue colonization in the liver, kidneys, and spleen compared with most other strain types in both subjects with healthy dentates and complete denture wearers. These results clearly demonstrate the significant role of phospholipase and proteinase activities on the adherence of C. albicans and their overall influence on the pathogenesis of Candida species.

Candida albicans strain differentiation in complete denture wearers.

Strain differentiation of 66 clinical isolates of Candida albicans obtained from healthy dentate and complete denture wearers was performed. Resistogram method based on differences in the resistance of C. albicans isolates to sodium selenite, boric acid, cetrimide, sodium periodate and silver nitrate was used for strain differentiation. Of the 32 potential strains that can be distinguished, 14 different resistogram strains of C. albicans were found among the 66 isolates tested. Strain-C--was the most predominant (24.3% of total isolates), while strain A-CDE was the least predominant (1.5%). The results showed no particular association of certain strains with Candida infections in complete denture wearers. Sensitivity to antifungal agents showed that isolates from different strains were most sensitive to amphotericin B and nystatin and least sensitive to miconazole.

Effects of date extract on adhesion of Candida species to human buccal epithelial cells in vitro.

The adherence of three Candida species to human buccal epithelial cells (BEC) following treatment with different concentrations of date extract was investigated in vitro, as well as the effect of a mouth rinse with date extract on the adhesion of yeast to BEC. Adhesion of C. albicans, C. tropicalis and C. kefyr to BEC was significantly reduced after both short- and long-term periods of yeast exposure to various concentrations of date extract (reduction between 25% and 52% of the control value). A similar inhibition of adherence was observed upon pre-incubation of BEC with date extract. There was a significant reduction (P<0.001) in the adherence of yeast to BEC collected immediately or 5-20 min after an oral rinse with 10% date extract. No statistically significant difference was observed in the adhesion of BEC collected 30 min after an oral rinse with date extract and control BEC. In addition, pre-treatment of either Candida or BEC, or both, with date extract resulted in reduced adherence, the magnitude of which was largest when both types of cells were pre-treated. Date extract also inhibited germ-tube formation of C. albicans (56-85% inhibition), which might contribute to the effects on adherence.

Incidence and distribution of Candida species isolated from human skin in Jordan.

In the period between December 1992 and September 1993 a total of 1060 clinically suspected cases of dermatomycoses were examined for causative fungi. Of these 330 (31%) were suspected as cutaneous candidosis. A higher incidence was observed in adults (62.9%) than in children (36.2%) and infants (19.5%). The infected patients that were adults were divided into four categories: diabetics; those receiving steroid; those receiving antibiotic therapy; and a non-risk control group. Analysis of the data revealed that the incidence rate appears to be approximately doubled in diabetes (49.3%), steroid therapy (33.8%) and antibiotic therapy (27.4%) than in the control group (15.7%). Candida albicans was the most frequent isolate (57.6%) followed by Candida tropicalis (16.5%).

Prevalence of dermatophytoses in the Zarqa district of Jordan.

A total of 350 clinically suspected cases of dermatomycoses were examined for causative fungi during July 1997 to September 1998. Mycotic infection was demonstrable by microscopy and culture in 199 (56.8%) cases. The most common superficial mycotic infections were tinea pedis (35.2%) followed by tinea capitis (23.1%), tinea unguium (21.6%) and tinea corporis (10.6%). Most of the infected patients were 1-9, 20-29 and 30-39 years old. Men were mainly infected with tinea cruris and tinea pedis, while women were infected with tinea pedis, tinea unguium and tinea capitis. The frequencies of etiological agents isolated from patients were as follows: Trichophyton mentagrophytes var. interdigitale (32.7%), T. rubrum (28.6%), Epidermophyton floccosum (20.1%), Microsporum canis (11.1%), T. schoenleinii (4%), T. verrucosum (2%), T. violaceum (1%), and M. gypseum (0.5%). The number of infections varied with the seasons. The highest number of cases of tinea pedis and tinea cruris occurred in the summer months, while tinea capitis, tinea corporis and tinea unguium occurred in the spring and winter months.

The prevalence of Candida albicans populations in the mouths of complete denture wearers.

Using imprint cultures, the prevalence of oral candidosis and the frequency of isolation of Candida albicans and its density in oral mucosal sites and denture surfaces was determined in 190 healthy dentate subjects and 230 complete denture wearers. Candida colonization was 36.8% and 78.3% in healthy dentate and complete denture wearers patients, respectively. In the healthy dentate subjects the tongue, palate and cheeks, and in complete denture wearers additionally the upper and lower dentures, were the most frequently and densely colonized oral sites. Oral carrier rate and density of C. albicans were both higher in the denture wearers diabetic group than in the control non-diabetic group. Smoking was associated with an increase in the frequency and density of the yeast in denture wearers. Attention to these predisposing factors could reduce the incidence of oral candidosis particularly in immunocompromised patients.

Ultrastructural effects of date extract on Candida albicans.

The effect of Berhi date extract on the ultrastructure of Candida albicans was studied by scanning and transmission electron microscopy. Exposure of yeast to 5% (w/v) date extract showed evidence of weakening in the cell wall with indications of cell distortion and partial collapse in some cases as seen by scanning electron microscopy. Increasing the concentration of date extract (20%, w/v) led to more drastic damage to the yeast with cell lysis and concurrent leakage of cytoplasmic material with eventual cell death. Ultrastructural investigation showed irregular shapes of cells treated with date extract, with prominent effects on cell wall layers. Cell membranes lost their integrity, aggregation of the cytoplasmic contents and large detachment of plasmalemma from cell wall was observed in the treated cells. These results suggest that date extract may have multiple effects on Candida with an increasing potential of using it for prophylaxis purposes.

Effect of sub-inhibitory concentration of chlorhexidine on lipid and sterol composition of Candida albicans.

The effect of a sub-inhibitory concentration of chlorhexidine on lipid and sterol composition of Candida albicans was investigated. The total lipid content of this yeast grown in the presence of chlorhexidine was reduced whilst the total sterol content was increased compared with control-grown cells. Lipids and sterol analyses of this yeast grown in the presence and absence of chlorhexidine are presented. Chlorhexidine-grown yeast had a higher level of phosphatidylethanolamine, phosphatidylcholine and monogalactosyldiacylglycerol. Lower proportions of phosphatidylinositol plus phosphatidylserine, phosphatidic acid and cardiolipin were found in C. albicans grown in the presence of the drug when compared with control-grown yeast. The major fatty acids in control-grown cells were C16 and C18. Drug grown-cells had higher proportions of palmitic acid (16:0) and stearic acid (18:0), but lower proportions of palmitoleic acid (16:1) and oleic acid (18:1). Chlorhexidine also decreased the unsaturated-to-saturated fatty acid ratio, while the C16/C18 ratios increased compared to control-grown cells. Differences in the fatty acid composition of major phospholipids and neutral lipids between drug and control-grown yeast were also detected. Sterol analysis of control-grown cells showed that the major sterol present was ergosterol (55.4% wt). A significant increase in ergosterol and obtusifoliol was observed in chlorhexidine-treated cells and a significant decrease in squalene and lanosterol. Our results suggested that chlorhexidine affected the lipid and sterol composition of C. albicans.

Isolation of Candida species from domestic sewage in Jordan.

Saccharomyces cerevisiae and various species of Candida were isolated and identified from the Al-Baqa'a sewage treatment station. Potentially pathogenic yeasts were detected in sewage samples and Candida krusei was found in the treated effluent. There was a 90-100% reduction in the number of yeast found in treated sewage effluent compared with raw sewage. Seasonal variations of total yeast counts are also reported.

Prevalence and susceptibility of vaginal yeast isolates in Jordan.

The prevalence of vaginal yeast species has been studied in 140 women (41 pregnant, 66 infertile and 33 healthy controls) attending a gynaecological private clinic in Amman, Jordan. Yeast species were isolated from pregnant (68.2%), infertile (51.5%) and healthy control (48.4%) women. Patients manifesting one, two or three symptoms of vulvovaginitis were 22.1%, 26.8% or 24.2% respectively. Asymptomatic cases and cases with more than three symptoms were 22.4% and 4.5% respectively. Candida albicans was the dominant species (in 51.3% of the patients) followed by C. glabrata (17.9%). The percentage occurrence as well as the pattern of Candida species differed among the different groups of patients. Candida kefyr was found to be significantly higher in the infertile women. In vitro sensitivity tests using amphotericin B, nystatin, miconazole nitrate and chlorhexidine were carried out; amphotericin B was the most effective and miconazole nitrate the least.

Subinhibitory concentration of octenidine and pirtenidine: influence on the lipid and sterol contents of Candida albicans.

The effect of subinhibitory concentrations of octenidine and pirtenidine on the lipid and sterol composition of Candida albicans was investigated. The total lipid and sterol contents of C. albicans grown in the presence of either octenidine or pirtenidine were reduced compared with control-grown cells. The major differences in the lipid composition of drug-grown and control cells were phosphatidylglycerol, phosphatidylcholine and monogalactosyldiacylglycerol, which increased in the presence of octenidine and pirtenidine. Lower proportions of phosphatidic acid were found in yeasts grown in the presence of the drugs when compared with control C. albicans. Fatty acid analysis of control-grown cells showed that the major fatty acids were C16 and C18. Drug-grown cells had higher proportions of palmitic and linolenic acids but lower proportion of oleic acid. The C16/C18 ratios were higher for octenidine- and pirtenidine-grown cells than control cells. Differences in the fatty acid composition of major phospholipids and neutral lipids between drug-grown and control yeasts were also observed. Sterol analysis of control-grown cells showed that the major sterol present was ergosterol (65.9%). A significant increase in squalene and 4,14-dimethylzymosterol was observed in pirtenidine-treated cells, while octenidine-treated cells showed an increase in zymosterol and obtusifoliol contents. Our results suggest that octenidine and pirtenidine affect the lipids and sterol of C. albicans in different ways. The implications of these findings on the mode of action of these two drugs is discussed.

Pathogenicity determinants of Candida.

The incidence of infections due to Candida albicans and other related species has increased in recent years. A number of factors have contributed to this, e.g. the use of a wide range of potent antibacterial and immunosuppressive therapeutic agents and the increased incidence of immune-deficiency diseases such as AIDS. Pathogenicity determinants which confer virulence on C. albicans, and other Candida species to a lesser extent, have been reviewed. These include factors related to species and strains, adherence, dimorphism, toxin and enzyme production and cell surface composition. This review clearly shows that C. albicans virulence is a function of a multiplicity of factors working jointly to overcome the host defences. A lack or debility in any of these parameters will reflect negatively on its infectivity and make it difficult for Candida to establish itself, particularly in a healthy individual.

Combinations of antifungal and antineoplastic drugs with interactive effects on inhibition of yeast growth.

Interactive effects among antifungal and antineoplastic drugs contributed to toxicities when combinations of these drugs were used to inhibit the growth of five Candida spp. Drug interactions were measured by growth inhibition in both liquid and solid media, by viable cell counts and by examination using scanning electron microscopy. Large cooperative effects on toxicity were demonstrated between some antineoplastic and antifungal drugs. For example, positive cooperativity was seen between the antineoplastic drug 5-fluorouracil and combinations of the antifungal agents amphotericin B and miconazole nitrate. Smaller, and often negative, interactions occurred between the antineoplastic drug cyclophosphamide and antifungal drugs. The levels of drugs required for inhibition in combination drug treatments were critically dependent upon the ratios as well as the absolute concentrations of the drugs tested. Drug combinations were selected which inhibit yeast growth at concentrations far below the individual MIC of the drugs. These combinations may prove of value in clinical treatments of cancer patients infected by Candida.

Interactive effects of antifungal and antineoplastic agents on yeasts commonly prevalent in cancer patients.

The effects of combinations of antifungal and antineoplastic drugs on inhibition of the growth of yeasts which commonly infect cancer patients have been analyzed. It was shown that (i) inhibitory drug combinations could be selected in which all drugs were at levels far below their individual MICs; (ii) interactive effects among antineoplastic and antifungal drugs may be very large; (iii) optimum combinations of drugs for inhibition of yeast growth depended upon both the relative and absolute concentrations of the drugs in the mixture; (iv) drug combinations which were effective at low levels in inhibiting one test yeast were also generally effective against other species, but the levels of susceptibilities and, to a lesser extent, the best ratios of drugs in the test combinations varied with species; and (v) to quantitatively evaluate drug interactions, it is necessary to carefully define and control all experimental conditions, absolute and relative concentrations of drugs used, and the organisms tested.

Effect of antineoplastic agents and X-irradiation on the adherence of Candida spp. to human buccal epithelial cells in vitro.

The role of chemotherapy, X-irradiation and a combination of both on the phenomenon of adherence of yeast to buccal epithelial cells (BEC) was investigated in vitro. Growth of three Candida spp. in the presence of eight of eleven antineoplastic agents led to reduction of adherence of the isolates tested (reduction between 30% and 61% of the control value), and this effect was observed whether exponential or stationary phase Candida cells were used. Exposure of C. albicans to various doses of radiation also led to a reduction in adherence of this yeast to BEC between 31% and 53% of the control value. This reduction was shown to be dose related. Similar results were obtained when BEC were exposed to radiation, and the effects of radiation treatment was accentuated when both yeast and BEC were irradiated simultaneously. Furthermore, treating C. albicans with a combination of chemotherapy and radiation led to the greatest reduction in adherence of yeast to BEC compared to when the yeast was treated with either chemotherapy or radiation alone (reduction between 63% to 74% as compared with control). The possible mechanism/s involved in reduction of adherence of yeast to BEC are discussed.