Comparative and collaborative evaluation of standardization of antifungal susceptibility testing for filamentous fungi

Abstract

The purpose of the study was to evaluate the interlaboratory agreement of broth dilution susceptibility methods for five species of conidium-forming (size range, 2 to 7 m) filamentous fungi. The methods used included both macro-and microdilution methods that were adaptations of the proposed reference method of the National Committee for Clinical Laboratory Standards for yeasts (m27-P). The MICs of amphotericin B, fluconazole, itraconazole, miconazole, and ketoconazole were determined in six centers by both macro-and microdilution tests for 25 isolates of Aspergillus flavus, Aspergillus fumigatus, Pseudallescheria boydii, Rhizopus arrhizus, and Sporothrix schenckii. All isolates produced clearly detectable growth within 1 to 4 days at 35؇C in the RPMI 1640 medium. Colony counts of 0.4 ؋ 10 6 to 3.3 ؋ 10 6 CFU/ml (mean, 1.4 ؋ 10 6 CFU/ml) were demonstrated in 90% of the 148 inoculum preparations. Overall, good intralaboratory agreement was demonstrated with amphotericin B, fluconazole, and ketoconazole MICs (90 to 97%). The agreement was lower with itraconazole MICs (59 to 79% median). Interlaboratory reproducibility demonstrated similar results: 90 to 100% agreement with amphotericin B, fluconazole, miconazole, and ketoconazole MICs and 59 to 91% with itraconazole MICs. Among the species tested, the MICs for S. schenckii showed the highest variability. The results of the study imply that it may be possible to develop a reference method for antifungal susceptibility testing of filamentous fungi.Despite the lower volume of serious infections caused by filamentous fungi compared with that of serious infections caused by yeasts, the performance of antifungal susceptibility testing for these opportunistic pathogens is important in the clinical laboratory (8). Progress has been made in developing guidelines for the antifungal susceptibility testing of yeasts to deal with the standardization of different testing parameters such as inoculum preparation, medium composition and pH, length of incubation, and endpoint criteria (3,5,6,(9)(10)(11)(12). The standardization of these antifungal susceptibility testing steps has led to increased interlaboratory reproducibility which has opened the possibility of developing standards by adopting and tailoring these steps for antifungal susceptibility tests for the filamentous fungi. The first priority of the National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Susceptibility Tests was to develop a standard for the preparation of inoculum suspensions. Among four procedures evaluated, a spectrophotometric method was recommended as the procedure that gives the least variable results for the preparation of inoculum suspensions of yeast cells (11). This recommendation has been substantiated by ensuing collaborative studies of the subcommittee and other studies (2,3,5,6,12).The spectrophotometric method has been evaluated further in a single laboratory for the preparation of conidial suspensions of selected medically important filamentous fungi (1). In t...

Eficiência terapêutica das formulações lipídicas de anfotericina B

“…Em relação aos fungos filamentosos, a AB possui amplo espectro de ação, incluindo Aspergillus fumigatus (MIC 1,0-2,0 mg/mL), Aspergillus flavus (MIC 1,0 mg/mL) (Espinel-Ingroff et al, 1997;Arikan et al, 1999), Fusarium oxysporum (MIC 2,0 mg/mL), Fusarium solani (MIC 1,0-2,0 mg/mL), Rhizopus arrhizuz (MIC 0,2-0,5 mg/mL) (Espinel-Ingroff et al, 1997) e Paracoccidioides brasiliensis (1,0-0,25 mg/mL) (Hahn, Hamdan, 2000).…”

Comparison of the E-test with the NCCLS M38-P Method for Antifungal Susceptibility Testing of Common and Emerging Pathogenic Filamentous Fungi

“…On the basis of data from several studies (12,13), this document recommends the use of (i) standard RPMI 1640 broth; (ii) nongerminated conidial inoculum suspensions of approximately 10 4 CFU/m1; and (iii) incubation at 35°C for 24 h (Rhizopus spp. ), 48 h (Aspergillus spp., Fusarium spp., and other opportunistic molds), and 72 h (S. apiospermum).…”

Biocatalysts in Control of Phytopatogenic Fungi and Methods for Antifungal Effect Detection

“…The methodology is useful for testing common filamentous fungi or yeasts, including the dermatophytes. The fungi encompass Aspergillus spp., Fusarium spp., Rhizopus spp., P. boydii, S. prolificans, S. chenckii (Espinel-Ingroff et al, 1995), Trichophyton, Microsporum, Epidermophyton spp. (Ghannoum et al, 2004), Candida spp.…”

“…1). These assays were performed using enzymes at 20 µg/ml (Cano et al, 2008) and syringomycin E at 2 µg/ml (Espinel-Ingroff et al, 1995) as final concentrations. Results confirm the hypothesis that vanadate addition leads to keep ATP of dead cells due to ATPases inhibition.…”