Antifungal susceptibility testing

Candida spp. (n = 106) and Aspergillus spp. (n = 67) isolates were tested for susceptibility to CD101, anidulafungin and caspofungin using CLSI and EUCAST BMD methods.^{24–26,28,29} The reference powder of CD101 was obtained from Seachaid Pharmaceuticals (Durham, NC, USA). Stock solutions of all three echinocandins were prepared in DMSO and the final range of concentrations tested was 0.008–16 mg/L.

CLSI BMD testing of Candida spp. was performed and interpreted as outlined in documents M27-A3²⁴ and M27-S4²⁹ by using round-bottomed 96-well plates containing RPMI 1640 medium with 0.2% glucose, inocula of 0.5–2.5 × 10³ cells/mL and incubation at 35°C. MIC values were determined visually after 24 h of incubation. The MIC endpoint criterion was the lowest concentration of drug that caused significant diminution (≥50% inhibition) of growth relative to that of the growth control. EUCAST BMD testing of Candida spp. was performed as outlined in document EDef 7.2²⁶ by using flat-bottomed 96-well plates containing RPMI 1640 broth with 2.0% glucose, inocula of 0.5–2.5 × 10⁵ cells/mL and incubation at 35°C. MIC values were determined spectrophotometrically (at 530 nm) after 24 h of incubation, as the lowest concentration of drug that resulted in ≥50% inhibition of growth relative to that of the growth control.

In vitro testing of Aspergillus susceptibility to CD101, anidulafungin and caspofungin was performed using BMD methods of the CLSI²⁵ and EUCAST.²⁸ Incubation for both methods was at 35°C for 24 h and minimum effective concentrations (MECs) were defined as the lowest concentration of drug in which abnormal, short and branched hyphal clusters were observed in contrast to the long, unbranched, hyphal elements (confluent hyphal growth) seen in the growth control.

MIC and MEC results of CD101 obtained with the CLSI method were compared with those obtained with the EUCAST method in order to determine the EA between MIC values. High off-scale MIC or MEC results were converted into the next highest concentration and low off-scale MIC results were left unchanged. Discrepancies of at least ±2 log₂ dilutions among MIC or MEC results were used to calculate the EA.