The demographic, clinical and microbiological data of patients with candidemia at the "Hopital Universitario La Fe", a tertiary-care hospital in Valencia, Spain, from 1995 to 1997 was analyzed retrospectively. Candida spp. were isolated in blood cultures from 145 patients, 32% of whom were children (25% of these were neonates). The most common species isolated was Candida albicans, followed by Candida parapsilosis, Candida krusei and Candida tropicalis. Risk factors for candidemia included underlying disease, therapy with broad-spectrum antibiotics and the presence of a central venous catheter. The majority of children were treated with amphotericin B, whereas 52% of adults received fluconazole. Overall mortality was 44% (30% in children and 50% in adults), and attributable mortality was 30% (24% in children and 33% in adults). Multivariate analysis indicated that neutropenia, corticosteroid therapy, lack of antifungal treatment, and failure to replace the central venous catheter were factors associated with candidemia-related death. Among the adult population, an APACHE II score greater than 15 predicted candidemia-related death.

No abstract

In a previous study tolerance to amphotericin B (AMB) was found among Candida parapsilosis and C. dubliniensis strains by seeding the whole volumes of wells used for MIC determinations, and minimum fungicidal concentrations (MFC) for non-C. albicans Candida strains were demonstrated to be above the levels safely achievable in serum. As an extension of that study, we performed time-kill assays with 26 blood culture isolates (6 C. albicans, 5 C. parapsilosis, 5 C. krusei, 4 C. glabrata, 3 C. lusitaniae, and 3 C. , and 48 h. MFCs and time-kill patterns were species specific (MFCs, <1 g/ml for all C. dubliniensis and C. albicans isolates except AMB-resistant strain ATCC 200955; MFCs, 2 to >16 g/ml for the other isolates). The times required to reach the fungicidal endpoint (99.9% killing) at four times the MIC were 2 h for C. albicans and C. dubliniensis, 16 h for C. glabrata, 24 h for C. parapsilosis and C. lusitaniae, and >40 h for C. tropicalis and C. krusei. The killing rate increased as the AMB concentration was increased up to 2 g/ml. The highest killing rates were achieved for C. albicans, C. dubliniensis, and C. lusitaniae, while viable C. tropicalis, C. krusei, and C. parapsilosis cells were present after 48 h (MICs, <2 g/ml) when AMB was used at 2 g/ml. Time-kill curves and MFCs can detect viable cells after 48 h when AMB is used at >2 g/ml. The failure of AMB treatment could be due to its poor killing activity against some species at the concentrations reached in patients' serum.Amphotericin B deoxycholate has been considered the "gold standard" for the treatment of invasive fungal infections since its introduction in the 1950s. Although the toxicity of this drug has somewhat limited its use, clinical experience has demonstrated that its less toxic lipid formulations can be used as suitable alternatives to the parental drug (18). Therefore, it is still important to obtain a clearer understanding of the killing patterns of amphotericin B. In general, the pharmacodynamic characteristics of amphotericin B have mainly been evaluated with Candida albicans, and scarce data are available for other Candida spp. In a prior study (4), by using an inoculum larger than that recommended in NCCLS document M27-A2 (16) and subculturing the total volume in the well used for MIC determination, we have shown that the difference between the fungistatic activities and the fungicidal activities could be species dependent among Candida spp. Tolerance to amphotericin B (by using the minimum fungicidal concentration [MFC] determination procedure described above) has been reported among C. parapsilosis and C. dubliniensis strains (4, 27), as have MFCs for non-C. albicans Candida isolates that are above the safe levels achievable in serum (4). Several investigators (17,26,32) have postulated that the MFCs and time-kill curve study results could be more clinically relevant than the MICs of amphotericin B. Because time-kill curves provide a quantitative assessment of fungicidal activity as well as the rapidity of killing over time, t...

The in vitro activities of fluconazole or voriconazole plus terbinafine were evaluated against 20 Candida isolates by the checkerboard, time-kill, and Etest methods. Synergism (C. albicans, C. glabrata, and C. tropicalis) and indifference (C. krusei) were observed. Correlation among methods was good. The Etest is a suitable method to determine drug interactions.The checkerboard and time-kill methods to determine in vitro interactions between drugs are time-consuming and cumbersome for use in clinical laboratories. In order to find a method that facilitates synergistic studies, our aim was dual: (i) to assess the in vitro activities of voriconazole (VRC) and fluconazole (FLC) combined with terbinafine (TRB) against four Candida spp. (resistant or susceptible to FLC and/or TRB) by the checkerboard and time-kill methods and (ii) to compare the results of these methods with those obtained by an Etest-agar dilution technique.Twenty blood isolates (Table 1) were tested. C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were included for quality control.Stock solutions of VRC, FLC (Pfizer, Barcelona, Spain), and TRB (Novartis, Barcelona, Spain) were prepared with the appropriate solvent (dimethyl sulfoxide for VRC and TRB and distilled water for FLC). The final concentrations were 0.002 to 2 g/ml for VRC, 0.06 to 64 g/ml for FLC, and 0.25 to 16 g/ml for TRB. MICs of drugs alone or in combination were determined by the NCCLS M27-A2 method (12) and corresponded to the lowest concentration that showed prominent (Ն50%) growth inhibition and by the Etest method as described below.Drug interactions were assessed by the following three methods described below: broth microdilution checkerboard, timekill, and Etest.(i) Broth microdilution checkerboard. The broth microdilution checkerboard method was performed by using the fractional inhibitory concentration (FIC) index, which is defined as the sum of the MIC of each drug when used in combination divided by the MIC of the drug when used alone. For computation of FIC indices, off-scale MICs were raised to the next highest MIC; synergistic and antagonistic FIC indices were defined as Յ0.5 and Ͼ4, respectively.(ii) Time-kill studies. One isolate of each species was selected, and tests were conducted as previously described (RPMI 1640 medium, 10 5 -CFU/ml inoculum, and 5-ml volume) (8). The drug concentrations tested alone were as follows: VRC, 16 and 1 g/ml; FLC, 32 and 2 g/ml; and TRB, 8 and 2 g/ml. For the combinationsVRC/TRB and FLC/TRB, the drug concentrations were as follows: VRC/TRB, 16/2, 1/2, and 1/8 g/ml; and FLC/TRB, 32/2, 32/8, 2/2, and 2/8 g/ml. At 0, 3, 6, 24, and 48 h, aliquots were removed to determine the number of CFU per milliliter. Synergy was defined as a Ն2-log 10 decrease in CFU per milliliter for a combination compared to the killing with the most active drug alone and an increase of Ն2 log 10 as antagonism. Experiments were conducted in duplicate and on 2 separate days.(iii) Etest studies. RPMI 1640 agar with 2% dextrose and 1, 2, and 8 g of TRB per ml, pre...

The 58-kiloDalton mannoprotein (mp58) on the surface of Candida albicans is highly immunogenic, is expressed by all C. albicans isolates tested, and elicits strong antibody responses during candidiasis. It belongs to a family of immunodominant fungal antigens with representatives also in different species of Aspergillus. The amino acid sequence of the protein portion of mp58 as deduced from the DNA sequence of its encoding gene (FBP1/PRA1) was used to synthesize a complete set of overlapping dodecapeptides (overlap, 7; offset, 5) covalently attached to the surface of derivatized polyethylene pins. The pin-coupled peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by a number of antiserum preparations containing anti-mp58 antibodies. This comprehensive epitope-scanning study revealed the presence of multiple immunoreactive continuous B-cell epitopes within the protein sequence. Regions of increased reactivity included both the amino and carboxy termini of the mature protein (encompassing amino acid residues 16 to 50 and 286 to 299, respectively) and four internal regions spanning amino acids at positions 66 to 92, 121 to 142, 148 to 192, and 211 to 232. Further delineation of epitopic regions and identification of the boundaries of the antigenic sites was performed upon ELISA testing with a second Pepset consisting of completely overlapping 8-mer peptides spanning these reactive regions in the protein moiety of mp58. The highly reactive epitopic region at the C terminus of the protein was further evaluated using both window net and replacement net analyses. A synthetic peptide corresponding to the last 10 amino acid residues at the C terminus of the protein was immunogenic when injected into mice after being coupled to a carrier protein. Moreover, antibodies in the resulting sera specifically recognized the homologus mp58 in ELISAs and immunoblot assays. Delineation of the antibody responses to mp58 could provide the basis for the development of novel immunity-based prophylactic, therapeutic, and diagnostic techniques for the management of candidiasis.Candida albicans is both a commensal and an opportunistic pathogen of humans. Depending on the underlying host defect, this microorganism is able to cause a variety of infections that range from mucosal to life threatening disseminated candidiasis. C. albicans pathogenicity also depends on a complex array of microorganism-related virulence factors (reviewed in reference 15