Trailing end-point phenotype of Candida spp. in antifungal susceptibility testing to fluconazole is eliminated by altering incubation temperature

According to the Clinical and Laboratory Standards Institute (CLSI) (formerly the National Committee for Clinical Laboratory Standards) standardized method for antifungal susceptibility testing M27-A2, the MIC for fluconazole is defined as the lowest concentration of drug causing an 80 % inhibition of growth relative to a drug-free control (CLSI, 2002). Proposed breakpoints suggest that susceptible isolates have an MIC ¡8 mg ml, susceptible-dose dependent isolates have an MIC of 16–32 mg ml and resistant isolates have an MIC ¢64 mg ml (CLSI, 2002). Standard testing reports an MIC at 48 h, and this result is usually within 1–2 doubling dilutions of the 24 h value. Most isolates of Candida albicans are susceptible to fluconazole with a 48 h MIC ¡1 mg ml (Pfaller & Diekema, 2004). However, when tested by the CLSI method, certain isolates have been found to be susceptible at 24 h, usually with an MIC ¡1 mg ml, but highly resistant at 48 h with an MIC ¢64 mg ml, which may be referred to as a trailing end point or tolerance (Pfaller & Diekema, 2004). In one study, pH was found to affect trailing in certain isolates (Marr et al., 1999). Mechanisms have been proposed to explain trailing, including activation of calcineurin and altered regulation of genes mediating resistance (Sanglard et al., 2003; Lee et al., 2004). Various studies have suggested that these isolates are actually clinically susceptible (Revankar et al., 1998; Rex et al., 1998). Other Candida species may also demonstrate trailing end points. We evaluated whether altering the incubation temperature in the CLSI method may affect the in vitro susceptibility testing of such isolates to fluconazole. The clinical isolates used were known to exhibit trailing end points by previous testing.