Inoculum Standardization for Antifungal Susceptibility Testing of Filamentous Fungi Pathogenic for Humans

Abstract

Two methods of inoculum preparation for filamentous fungi were compared: counting with a hematocytometer and spectrophotometric adjustment. One hundred eighty-two filamentous fungi pathogenic for humans were used. Colony counts were done for all inoculum preparations. The agreement between the hematocytometer counts and the colony counts (CFU per milliliter) was 97.2%. The reproducibility between the hematocytometer counts and the colony counts by means of an intraclass correlation coefficient was 0.70. Pearson's correlation index for hematocytometer counts versus colony counts was 0.56, whereas that for optical density versus colony counts was 0.008. Both methods can be used for inoculum size adjustment. However, the use of the spectrophotometric method requires that each species be standardized separately.An increasing prevalence of infections caused by filamentous fungi has been detected in humans in recent years (1). This increasing prevalence has stimulated interest in a reliable and reproducible antifungal susceptibility testing method for molds. Recently, the National Committee for Clinical Laboratory Standards (NCCLS) proposed a reference method (the NCCLS M38-P method) for the susceptibility testing of conidium-forming filamentous fungi (7). The NCCLS method recommends inoculum preparation by a spectrophotometric procedure (2-4, 7). However, it is well known that larger objects such as spores or filaments do not obey the rule that established a proportional relationship between dry weight concentration and optical density (6). In addition, the inoculum size has a great influence on the MICs for filamentous fungi (3-5, 7). Other variables such as the color of the spores can also influence the optical density value.In the work described here, two procedures for inoculum size adjustment were evaluated, and the results were compared with those obtained by colony counting. MATERIALS AND METHODSIsolates. A set of clinical isolates was tested. Each isolate was obtained from a different patient and was sent to the laboratory for identification or antifungal susceptibility testing.The following species were included: 31 Aspergillus fumigatus, 28 Aspergillus flavus, 25 Aspergillus terreus, 16 Aspergillus niger, 18 Scedosporium apiospermum, 27 Scedosporium prolificans, 24 Fusarium solani, and 13 Fusarium oxysporum isolates. The isolates were maintained as a suspension in sterile distilled water at 4°C until testing was performed.Inoculum preparation. The isolates were subcultured from the stock water suspensions on Sabouraud agar and on potato dextrose agar. Isolates of all species except members of the genus Fusarium were incubated at 35°C; members of the genus Fusarium were incubated at 30°C. Inoculum suspensions were prepared from fresh, mature (3-to 5-day-old) cultures grown on Sabouraud agar or potato dextrose agar slants. The colonies were covered with 5 ml of distilled sterile water. Tween 20 (5%) was added to facilitate the preparation of Aspergillus inocula. For Aspergillus spp., the inocula were achieved...

Disseminated Fusariosis Caused by Fusarium verticillioides in an Acute Lymphoblastic Leukemia Patient after Allogeneic Hematopoietic Stem Cell Transplantation

“…This standard is similar to the one published by the CLSI (formerly NCCLS) reference method for broth dilution antifungal susceptibility testing of filamentous fungi, but with the following minor modifications: (i) RPMI 1640 was supplemented with glucose to reach a 2% concentration and (ii) the inoculum sizes were between 1.0 ϫ 10 5 and 5.0 ϫ 10 5 CFU/ml (1,3,16,18). The antifungal susceptibility results for the blood isolate were as follows: amphotericin B, 1 g/ml; itraconazole, Ͼ8 g/ml; voriconazole, 4 g/ml; posaconazole, Ͼ8 g/ml; and terbinafine, 2 g/ml.…”

Optimal Testing Conditions for Determining MICs and Minimum Fungicidal Concentrations of New and Established Antifungal Agents for Uncommon Molds: NCCLS Collaborative Study

“…were below the desired CFU/ml range, these suspensions may require a 10% lower dilution factor than that used for the other species, or the density of these suspensions should be adjusted by using conventional cell counting with a hemacytometer (conidial size is irrelevant). Reliable conidial suspensions can be prepared by both methods (29), but inoculum quantification is highly recommended for confirmation of actual inoculum size (CFU/ml) by either method.…”

Use of Turbidimetric Growth Curves for Early Determination of Antifungal Drug Resistance of Filamentous Fungi

“…Assuming that germination and elongation rates are not affected by population factors and that they are intrinsic characteristics of inocula, independent of inoculum size, the longer lag phases found with smaller inocula are not due to lower germination or elongation rates but are likely due to the fact that the critical turbidity (similar to that of 4,000 70-mlong germinated spores) is reached later by smaller inocula than by larger ones. Therefore, at the end of the lag phases, the germ tubes of spores in small inocula should be longer than the corresponding germ tubes of spores in larger inocula given that the OD is a function of the number of spores or hyphae as well as of their size (12,31).…”