Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Fluconazole (FLZ) has emerged as a highly successful agent in the management of systemic infections of Candida. Cure rates for symptomatic candidiasis following single 150-mg FLZ dose therapy exceed 90%. In vitro, however, FLZ is fungistatic only in a narrow pH range and is not effective at vaginal pH, 4.2. This study evaluated the effect of FLZ on Candida albicans under in vitro conditions resembling the vaginal microenvironment, using vagina-simulative medium (VS). We found that FLZ was fungicidal for C. albicans in VS, but not in other media at the same pH, 4.2. In VS, FLZ was fungicidal at concentrations of >8 g/ml and reduced viability by greater than 99.9%. Analysis of the components of VS indicated that 17 mM acetic acid, a concentration achieved in the vagina, was responsible for the synergistic, fungicidal effect. This effect was not seen at neutral pH. Other substrates were not effective substitutes for acetic acid; however, short-chained carboxylic acids, glyoxylate and malonate, were effective. Most strains of C. albicans that were resistant to FLZ under standard conditions were killed by FLZ plus acetate. Other species of Candida were also killed, except C. krusei and C. glabrata. This study shows that FLZ has fungicidal activity for Candida species under in vitro conditions that mimic the vaginal microenvironment. This raises the possibility that FLZ may also have fungicidal effects during treatment of vaginal candidiasis. Elucidating the mechanism by which FLZ and acetate interact may disclose vulnerable pathways that could be exploited in drug development.

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“…Clumping altered colony counts by at most twofold. Vital dye staining with methylene blue followed a standard protocol (16).…”

Section: Methodsmentioning

confidence: 99%

Fungicidal Activity of Fluconazole against Candida albicans in a Synthetic Vagina-Simulative Medium

Moosa¹,

Sobel²,

Elhalis

et al. 2004

Antimicrob Agents Chemother

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“…The mixture was let stand for 15 minutes at room temperature with occasional mixing and observed by epifluorescence microscopy (Zeiss, NAG2) with excitation lamp of 490 nm and sweeping filter of 520 nm. The percentage of fluorescent viable yeasts was determined by counting at least 100 yeasts in ten different fields 5,13 .…”

Section: Viability Studiesmentioning

confidence: 99%

Excretion-secretion products and proteases from live Sporothrix schenckii yeast phase: immunological detection and cleavage of human IgG

Rosa

Gezuele

Calegari

et al. 2009

Rev. Inst. Med. trop. S. Paulo

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suMMARYAntigenic preparations from Sporothrix schenckii usually involve materials from mixed cultures of yeast and mycelia presenting cross-reactions with other deep mycoses. We have standardized pure yeast phase with high viability of the cells suitable to obtain specific excretion-secretion products without somatic contaminations. These excretion-secretion products were highly immunogenic and did not produce noticeable cross-reactions in either double immunodiffusion or Western blot. The antigenic preparation consists mainly of proteins with molecular weights between 40 and 70 kDa, some of them with proteolytic activity in mild acidic conditions. We also observed cathepsin-like activity at two days of culture and chymotrypsin-like activity at four days of culture consistent with the change in concentration of different secreted proteins. The proteases were able to cleave different subclasses of human IgG suggesting a sequential production of antigens and molecules that could interact and interfere with the immune response of the host.

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“…In 1964, Pedroso found that semianaerobic conditions in synthetic media were an important factor in the isolation of P. brasiliensis (228 (126,249). Data obtained with a synthetic medium (257) supplemented with culture filtrates (spent medium) and horse serum markedly improved the plating efficiency (66), and this medium has been used in several experimental designs (37,63).…”

Section: Microbiologymentioning

confidence: 99%

Paracoccidioidomycosis: an update

1993

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This review summarizes knowledge on various aspects of paracoccidioidomycosis. Mycelial propagules, chlamydospores, and arthroconidia exhibit thermal dimorphism; arthroconidia are infectious in animals and, by electron microscopy, appear well provided for survival. The mycelial-to-yeast-phase transformation requires a strict control of glucan synthesis probably mediated by membrane enzymes. Hormonal influences on the transformation of the fungus (mycelium or conidium to yeast phase) have been demonstrated. Estrogen-binding proteins have been detected in the fungal cytosol, and during the transformation novel proteins are produced as a result of estradiol incorporation. Clinical forms have been better defined on the basis of better experimental models. Emphasis has been placed on the lungs as the portal of entry and on the existence of silent pulmonary infections. A specific Paracoccidioides brasiliensis antigen, the 43-kDa glycoprotein (Gp43), has been identified, characterized, and cloned. This has led to improved reproducibility and specificity of serologic tests. The depression of cell-mediated immune responses has been associated with severe disease in humans and in the experimental host. T-cell subsets in patients' tissues were characterized by means of monoclonal antibodies, and a reduced CD4/CD8 ratio was demonstrated. This has been related to alterations in lymphokine and tumor necrosis factor production, production of antigen-antibody complexes, etc. Amphotericin B has provided effective therapy. Azole derivatives have also improved prognosis and facilitated therapy. Itraconazole is presently the drug of choice, yet incapacitating sequelae (mainly pulmonary fibrosis) still constitute major problems.

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Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Cited by 54 publications

References 20 publications

Fungicidal Activity of Fluconazole against Candida albicans in a Synthetic Vagina-Simulative Medium

Fungicidal Activity of Fluconazole against Candida albicans in a Synthetic Vagina-Simulative Medium

Excretion-secretion products and proteases from live Sporothrix schenckii yeast phase: immunological detection and cleavage of human IgG

Paracoccidioidomycosis: an update

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