Quality Control Guidelines for Amphotericin B, Itraconazole, Posaconazole, and Voriconazole Disk Diffusion Susceptibility Tests with Nonsupplemented Mueller-Hinton Agar (CLSI M51-A Document) for Nondermatophyte Filamentous Fungi

Abstract

Although Clinical and Laboratory Standards Institute (CLSI) disk diffusion assay standard conditions are available for susceptibility testing of filamentous fungi (molds) to antifungal agents, quality control (QC) disk diffusion zone diameter ranges have not been established. This multicenter study documented the reproducibility of tests for one isolate each of five molds (PaecilomycesThe Clinical and Laboratory Standards Institute (CLSI) has developed a reference method for disk diffusion antifungal susceptibility testing of nondermatophyte filamentous fungi (molds; CLSI documents M51-A and M51-S1) (5, 6). These documents describe guidelines for testing the susceptibility of opportunistic molds to triazoles, amphotericin B, and caspofungin. However, the M51 documents do not describe quality control (QC) parameters for any agent-isolate combination (5, 6). For QC purposes, two reference isolates (Candida krusei ATCC 6258 and Paecilomyces variotii ATCC MYA-3630) have been listed (6); data for these two isolates were not collected under M23-A3 guidelines for the selection of QC isolates (3, 9). The purpose of the present multicenter study (eight centers) was to select QC isolates among five molds and one yeast strain and to establish zone diameter limits (in millimeters) for disk diffusion tests of amphotericin B, itraconazole, posaconazole, and voriconazole. MATERIALS AND METHODSStudy design. The design followed the requirements described in the CLSI M23-A3 document for establishing QC ranges (with only one exception, as noted below) (3) and CLSI M51-A guidelines for agar diffusion testing of molds (5, 9); only one disk lot was available for each of the four antifungal agents instead of the recommended two lots from different manufacturers. The inter-and intralaboratory reproducibility of 10 replicate tests performed for each drug and strain combination were determined by the CLSI M51-A disk diffusion method in each laboratory on 10 different days and between three separate batches of nonsupplemented Mueller-Hinton (MH) agars. The same coded set of five molds and the C. krusei ATCC 6258 isolate were sent to each laboratory. Each isolate was tested in each laboratory with amphotericin B, itraconazole, posaconazole and voriconazole following a standard protocol (3, 5) that included the description of the reading conditions to be evaluated at both 24 (Aspergillus fumigatus, A. flavus, P. variotii, and C. krusei) and 48 h (A. terreus and Fusarium verticillioides) (3,5,9).Isolates. Antifungal susceptibility testing. The five molds and the C. krusei isolate were tested by the M51-A disk diffusion method in each center with the three lots of nonsupplemented MH agar (lots 09314, 09315, and 09316B; Hardy Diagnostics, Santa Maria, CA) on 10 different working days (3). A total of 240 zone diameters were determined for each isolate-drug combination. Briefly, mold stock inoculum suspensions were prepared from 7-day cultures grown on potato dextrose agar (Remel, Lenexa, KS) and adjusted spectrophotometrically to optical densit...

Molecular identification and antifungal susceptibility profile of Aspergillus flavus isolates recovered from clinical specimens in Kuwait

“…After applying the discs, the plates were incubated at 35°C for 24 h. Zone diameters were measured at the point where the growth significantly decreased (80 to 100% inhibition) and were recorded to the nearest millimeter. Recently, Espinel-Ingroff et al proposed for the first time quality control and reference zone diameter limits for A. fumigatus (ATCC MYA-3626) for amphotericin B (10-μg disk), itraconazole (10-μg disk), posaconazole (10-μg disk), and voriconazole (10-μg disk) [27]. No quality control ranges were recommended for A. flavus and A. terreus , because some results did not meet M23-A3 document requirements [28].…”

Seasonal Variation of the Antibacterial Activity of some Seaweeds against Multi Drug Resistant Pathogenic bacterial Strains

“…The dried extr acts wer e re-dissolved i n s olvents at a conc entrati on of 50 mg/ml. T hen, t he extr acts wer e t es ted agains t t he p at hogenic bac teria usi ng the d isc diffusion met hod (Espinel-Ingroff et al, 2011). The diameter of i nhibition z ones w as meas ured and c ompared t o determine the best s olvent f or extrac tion of the algal antibact erial material to be us ed in t he f urther experiments.…”

“…The antibact erial ac tivit y of t he seaweed s extrac ts was ass ayed usi ng disk diffusion m et hod (Espinel-Ingroff et al, 2011). T he bact erial suspension (0.5 ml) was spread on s terilized Petri dishes c ontai ning 15 ml of s terilized Muller Hinton agar m edia.…”

Comparison of E Test and Disc Diffusion Methods for Susceptibility Testing of Filamentous Fungi; Experience of a Routine Lab

“…versus triazoles and Amphotericin B (11,12). Nevertheless, ECVs did not predict clinical outcome of therapy yet they may aid in detecting isolates with reduced drug susceptibility (non-wild type strains) harboring resistance mutations (13). The wild type strain is defined here as in other studies as the population of organism/MICs in a species-drug combination with no detectable acquired resistance mechanisms (14,15).…”