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Thirteen Candida glabrata strains harboring a range of mutations in hot spot regions of FKS1 and FKS2 were studied. The mutations were linked to an echinocandin reduced susceptibility phenotype. Sequence alignments showed that 11 out of the 13 mutants harbored a mutation in FKS1 or FKS2 not previously implicated in echinocandin reduced susceptibility in C. glabrata . A detailed kinetic characterization demonstrated that amino acid substitutions in Fks1p and Fks2p reduced drug sensitivity in mutant 1,3-β- d -glucan synthase by 2 to 3 log orders relative to that in wild-type enzyme. These mutations were also found to reduce the catalytic efficiency of the enzyme ( V max ) and to influence the relative expression of FKS genes. In view of the association of FKS mutations and reduced susceptibility of 1,3-β- d -glucan synthase, an evaluation of the new CLSI echinocandin susceptibility breakpoint was conducted. Only 3 of 13 resistant fks mutants (23%) were considered anidulafungin or micafungin nonsusceptible (MIC > 2 μg/ml) by this criterion. In contrast, most fks mutants (92%) exceeded a MIC of >2 μg/ml with caspofungin. However, when MIC determinations were performed in the presence of 50% serum, all C. glabrata fks mutants showed MICs of ≥2 μg/ml for the three echinocandin drugs. As has been observed with Candida albicans , the kinetic inhibition parameter 50% inhibitory concentration may be a better predictor of FKS -mediated resistance. Finally, the close association between FKS1/FKS2 hot spot mutations provides a basis for understanding echinocandin resistance in C. glabrata .

Our purpose was to develop and characterize an enzyme-linked immunosorbent assay (ELISA) which could measure the concentration of amphotericin B in serum. Amphotericin B was assayed by competition ELISA. Multiwell ELISA plates coated with amphotericin B (1.0 micrograms/ml) conjugated to bovine serum albumin were used to test replicates of serum samples spiked with amphotericin B. Purified rabbit polyclonal antibody against amphotericin B (1.4 micrograms/ml) was added subsequent to the instillation of samples spiked with unknown amounts of amphotericin B. Experiments were performed to test the sensitivity, specificity, precision, and accuracy of the assay. The ability to measure lipid-associated amphotericin B was also evaluated in preliminary studies. Analysis of reference samples containing amphotericin B yielded a traditional sigmoidal curve. The limits of detection were 0.15 to 156 micrograms/ml. The sensitivity of the assay was affected by light and temperature exposure. Assay specificity was altered only by the presence of nystatin, a polyene antifungal agent similar to amphotericin B. Intrarun (coefficient of variation = 3.0%) and interrun (coefficient of variation = 12.8%) coefficients of variation were calculated and were comparable to those in similar assays. The assay's correlation coefficient (r = 0.907) demonstrated a statistically significant correlation between the optical density of the sample and the concentration of drug in the sample. The amphotericin B ELISA's ease, precision, and overall accuracy suggest that this assay could be used for assessments of serum amphotericin B concentrations. Multiple research questions concerning the role of serum amphotericin B concentrations in toxicity and efficacy have gone unanswered because of the labor-intensive nature of the assays which have been available to date. The ability to easily and rapidly measure 40 duplicate samples containing amphotericin B should also prove to be a distinct advantage for clinical research or reference laboratories in addressing these questions.

Thirteen Candida glabrata strains harboring a range of mutations in hot spot regions of FKS1 and FKS2 were studied. The mutations were linked to an echinocandin reduced susceptibility phenotype. Sequence alignments showed that 11 out of the 13 mutants harbored a mutation in FKS1 or FKS2 not previously implicated in echinocandin reduced susceptibility in C. glabrata. A detailed kinetic characterization demonstrated that amino acid substitutions in Fks1p and Fks2p reduced drug sensitivity in mutant 1,3-beta-D-glucan synthase by 2 to 3 log orders relative to that in wild-type enzyme. These mutations were also found to reduce the catalytic efficiency of the enzyme (Vmax) and to influence the relative expression of FKS genes. In view of the association of FKS mutations and reduced susceptibility of 1,3-beta-D-glucan synthase, an evaluation of the new CLSI echinocandin susceptibility breakpoint was conducted. Only 3 of 13 resistant fks mutants (23%) were considered anidulafungin or micafungin nonsusceptible (MIC2 microg/ml) by this criterion. In contrast, most fks mutants (92%) exceeded a MIC of2 microg/ml with caspofungin. However, when MIC determinations were performed in the presence of 50% serum, all C. glabrata fks mutants showed MICs ofor = 2 microg/ml for the three echinocandin drugs. As has been observed with Candida albicans, the kinetic inhibition parameter 50% inhibitory concentration may be a better predictor of FKS-mediated resistance. Finally, the close association between FKS1/FKS2 hot spot mutations provides a basis for understanding echinocandin resistance in C. glabrata.

We describe a case of recurring Candida glabrata infection in a 68-year-old African-American female on caspofungin therapy. The initial isolate was susceptible, but isolates recovered during following relapses were not. All isolates were clonal, and high-MIC strains contained a mutation in the highly conserved hot spot 1 region of Fks1p.

Fluconazole concentrations in the serum and prostate of human volunteers undergoing transurethral resection for benign prostatic hypertrophy were measured. There was a high correlation (r = 0.783) between serum (mean = 6.6 micrograms/ml) and tissue (mean = 1.9 micrograms/g) fluconazole concentrations, and these data were used to construct a model for local tissue concentrations.