Comparison of Dimethyl Sulfoxide and Water as Solvents for Echinocandin Susceptibility Testing by the EUCAST Methodology

Abstract

Ninety-six strains of Candida, including 29 resistant and 67 susceptible isolates with mutations in the FKS1 and FKS2 genes were tested by the European Committee on Antibiotic Susceptibility Testing EDef 7.1 and 7.2 methodologies to determine the impact on the MIC when water was replaced with dimethyl sulfoxide (DMSO) as the solvent for caspofungin and micafungin. The MICs were significantly lower and the MIC ranges were narrower when DMSO was used as the solvent. The use of DMSO may help to better discriminate between susceptible and resistant populations.

TEXT

The echinocandins, being the preferred systemically active antifungal agents for invasive candidiasis (13), have been licensed for the treatment of candidiasis, and caspofungin (CPF) is also licensed for salvage treatment of aspergillosis (20). Some cases of failures and breakthrough infections associated with elevated MICs of these drugs have been reported (5, 6, 17), which highlight the importance of accurate susceptibility testing methods to detect resistant isolates and then implement effective treatment. EUCAST (European Committee on Antibiotic Susceptibility Testing) and CLSI (Clinical and Laboratory Standards Institute) have standardized procedures to test antifungal susceptibility to echinocandins. Unfortunately, the correlation between these methods and the clinical data is still not satisfactory (3). Several modifications (4), including the revision and lowering of clinical breakpoints by CLSI (16), have been proposed to improve the detection of resistant strains; however, separation of wild-type and resistant isolates remains a problem. Published studies show a high degree of variability of MICs, especially those of CPF (2, 19). Previous studies (3) have pointed out that the use of dimethyl sulfoxide (DMSO) as a solvent (recommended by the manufacturer for anidulafungin [ANF] but not for CPF and micafungin [MCF], despite their hydrophobic nature) could aid in improving the reproducibility of candin susceptibility testing.

In this study, we evaluated the utility of DMSO as a solvent for CPF and MCF compared with that of water by using a collection of 96 Candida sp. isolates.

A total of 96 isolates of six Candida species were included in this study. They were selected for a previous study (3) and classified into two different groups, resistant (29 isolates) and susceptible (67 isolates), according to their FKS genotypes.

EUCAST microdilution testing (1, 18) was performed with all of the strains in duplicate by using DMSO and water to dissolve the drugs. Microtiter plates were read spectrophotometrically at 530 nm after 24 h. The MIC was defined as the lowest drug concentration that inhibited growth to 50% of that of the drug-free control. C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 were used as quality control strains. The antifungal agents used were CPF (Merck & Co., Inc., Rahway, N.J.) and MCF (Astellas Pharma Inc., Tokyo, Japan).

Statistical analyses were performed with the Statistical Package for the Social Sciences (SPSS 18.0; SPSS S.L., Madrid, Spain). Analyses of variance were performed to evaluate the differences between the two solvents with both antifungals. A P value of <0.01 was regarded as statistically significant.

Table 1 shows the MIC range, the geometric mean (GM) MIC, the MIC₅₀ (MIC causing inhibition of 50% of the isolates tested), and the GM MIC ratio (ratio of the GM MICs of resistant and susceptible isolates) of CPF and MCF for each species and solvent. The CPF MIC₅₀s were lower (P = 0.005) and the MICs ranges were narrower (except those for C. tropicalis, which were the same) when using DMSO for all of the species tested, and the MICs were, on average, 1 2-fold dilution lower. The MCF MIC₅₀s were lower with DMSO, except for C. parapsilosis, C. krusei, and C. dubliniensis resistant isolates, for which no differences in the MIC₅₀s were found; thus, the differences were not statistically significant for this drug (P = 0.349). For each species, the differences were statistically significant only for C. parapsilosis, probably because of the lower number of strains of the other species analyzed. When resistant and susceptible strains were compared, the differences were also significant for CPF and susceptible isolates (P = 0.001), while not significant but noticeable for resistant strains (P = 0.037). However, the GM MIC ratios were comparable for most species and both drugs.

Table 1.

MIC^a ranges, GM MICs, and MIC₅₀s of CPF and MCF for the isolates used in this study with water or DMSO as the solvent

Species, susceptibility, and parameter	CPF-water	CPF-DMSO	MCF-water	MCF-DMSO
C. albicans
Rb
nc	10	10	10	10
MIC range	1.0–8.0	0.5–2.0	0.06–2.0	0.03–1.0
GM MIC	2.1	1.2	0.57	0.31
MIC50	2	2	1	0.5
Sd
n	10	10	10	10
MIC range	0.12–0.50	0.12–0.25	0.01–0.03	0.01–0.03
GM MIC	0.25	0.17	0.011	0,015
MIC50	0.25	0.12	0.01	0.01
GM MIC ratio	8.4	7.1	51.8	20.7
C. parapsilosis, S
n	19	19	19	19
MIC range	1.0–8.0	0.5–4.0	0.5–2.0	0.25–2.0
GM MIC	1.8	0.93	0.96	0.83
MIC50	2	1	1	1
C. tropicalis
R
n	4	4	4	4
MIC range	2.0–8.0	0.5–2.0	0.12–1.0	0.12–0.50
GM MIC	4	1.4	0.49	0.35
MIC50	4	2	1	0.5
S
n	15	15	15	15
MIC range	0.25–0.5	0.12–0.25	0.03–0.06	0.01–0.03
GM MIC	0.43	0.2	0.34	0.01
MIC50	0.5	0.25	0.03	0.01
GM MIC ratio	9.3	7	1.4	35
C. glabrata
R
n	11	11	11	11
MIC range	1–16.0	0.5–8.0	0.01–2.0	0.01–2.0
GM MIC	1.1	0.66	0.16	0.13
MIC50	2	1	0.12	0.06
S
n	9	9	9	9
MIC range	0.25–0.50	0.25–0.25	0.01–0.03	0.15–0.15
GM MIC	0.36	0.25	0.02	0.015
MIC50	0.5	0.25	0.03	0.01
GM MIC ratio	3.1	2.64	8	8.7
C. krusei
R
n	3	3	3	3
MIC range	0.50–16.0	0.50–8.0	1.0–4.0	0.50–4.0
GM MIC	4	2.5	2	1.6
MIC50	8	4	2	2
S
n	13	13	13	13
MIC range	0.50–2.0	0.25–0.50	0.12–0.25	0.06–0.12
GM MIC	0.95	0.48	0.13	0.06
MIC50	1	0.5	0.12	0.06
GM MIC ratio	4.2	5.2	15.4	26.7
C. dubliniensis
R
n	1	1	1	1
MIC range	4	2	1	1
GM MIC	NAe	NA	NA	NA
MIC50	NA	NA	NA	NA
S
n	1	1	1	1
MIC range	0.5	0.25	0.03	0.01
GM MIC	NA	NA	NA	NA
MIC50	NA	NA	NA	NA
All isolates
R
n	29	29	29	29
MIC range	0.01–16.0	0.01–8.0	0.01–4.0	0.01–4.0
GM MIC	1.8	1	0.6	0.6
MIC50	2	2	0.5	0.25
S
n	67	67	67	67
MIC range	0.12–8.0	0.12–4.0	0.01–2.0	0.1–2.0
GM MIC	0.69	0.37	0.09	0.06
MIC50	0.5	0.25	0.06	0.01
GM MIC ratio	2.6	2.7	6.7	10

^aMICs are in milligrams per milliliter.

^bR, resistant according to FKS genotype.

^cn, number of isolates tested.

^dS, susceptible according to FKS genotype.

^eNA, not applicable.

Echinocandins have become first-line agents to treat invasive candidiasis. As drug use has increased, more resistant isolates and breakthrough infections have been reported (2, 9, 14, 17). Recent studies reported 2.9% resistance among Candida species and echinocandins (7) and up to 9.3% in C. glabrata (15). Therefore, the importance of antifungal susceptibility testing has increased for the detection of resistant strains. Both EUCAST and CLSI have standardized methods for echinocandins, but until recently, only CLSI had proposed breakpoints. However, several studies found characteristic resistance mechanisms in strains classified as susceptible by these breakpoints (5, 8, 10, 11). Consequently, CLSI revised the breakpoints for these drugs (16), defining species-specific and lower breakpoints. Moreover, commercial kits such as Etest were shown to be more accurate than the EUCAST and CLSI methods in the differentiation of resistant and susceptible isolates (3). Thus, optimization of these methods is warranted in order to provide a better tool that can help direct the best treatment options for patients.

Two studies (4, 12) have demonstrated that addition of bovine serum albumin to the medium increased the power of discrimination between wild-type and mutant isolates. It has also been suggested that the potency of these drugs may be affected by the solvent, given their relatively hydrophobic character. In both EUCAST EDef7.1 and the CLSI standards, water is recommended as the solvent for CPF and MCF while DMSO is preferred for ANF and other hydrophobic antifungals. In this study, we compared the MICs obtained for 96 strains of six Candida species when CPF and MCF were dissolved in DMSO with those obtained when the drugs were dissolved in water. The results showed that with DMSO as the solvent, the MICs were lower than those obtained with water for all of the species and both drugs, except for C. parapsilosis and resistant strains of C. krusei and C. dubliniensis.

From a chemical point of view, echinocandins are more soluble in DMSO, which has a lower dielectric constant than water; this allows them to remain more highly dispersed and accessible to their cellular target (3). This may help account for differences found in MICs between water and this solvent. These differences were more significant for susceptible strains, helping to discriminate between wild-type and mutant isolates. Although GM MIC ratios were comparable between water and DMSO, the variability of the data was reduced with the latter solvent (as the plates are more stable). This will help in establishing a fixed breakpoint, allowing better discrimination between resistant and susceptible strains. In addition, the preservative properties of DMSO help to improve the stability of the stock solutions of these drugs. This solvent replacement will also help in standardizing the method, since all other drugs are dissolved with DMSO.

In summary, the use of DMSO as a solvent for CPF and MCF produces lower MICs and narrower ranges than the use of water alone and increases the ability of the assay to discriminate between susceptible and resistant populations.