Effects of incubation time and buffer concentration on in vitro activities of antifungal agents against Candida albicans

Abstract

Nine selected isolates of Candida albicans were tested for their susceptibilities to amphotericin B and fluconazole by using three methods to assess the effect of incubation time and buffer concentration. By using a microdilution method with 0.0165 M 3-(N-morpholino)propanesulfonic acid (MOPS) and a 24-h incubation time, all of the isolates were found to be susceptible to amphotericin B and fluconazole. After 48 h of incubation, all isolates were still susceptible to amphotericin B. Seven of the nine isolates were resistant to fluconazole, and for the remaining two isolates, MICs increased by fourfold or more but the isolates remained susceptible (MIC, <10 g/ml). The nine isolates, along with three control strains, were further tested against amphotericin B and fluconazole by a standard broth macrodilution method with both 0.165 and 0.0165 M MOPS. The susceptibility results for fluconazole by the broth macrodilution method with the lower MOPS concentration correlated with the results of the 24-h broth microdilution method for determination of susceptibility or resistance in eight of nine tests and with the results of the 48 h broth microdilution method in three of nine tests. The results of the broth macrodilution method with the standard MOPS concentration did not correlate with any of the results obtained by the 24-h broth microdilution but correlated with results of seven of nine tests by the 48-h broth microdilution method. All nine test strains appeared to be susceptible when they were examined by a flow cytometric method. For clinical yeast susceptibility testing in microdilution panels, the 0.0165 M MOPS concentration combined with 24 h of incubation appeared to be the method of choice. The lower MOPS concentration may also be a useful modification to the tentative broth macrodilution method of the National Committee for Clinical Laboratory Standards. Use of the higher buffer concentration or longer incubation time may lead to false in vitro resistance for agents like fluconazole.

Impact of endpoint definition on the outcome of antifungal susceptibility tests with Candida species: 24- versus 48-h incubation and 50 versus 80% reduction in growth

“…For those tests read spectrophotometrically, it is likely that this corresponds better to a 50% rather than an 80% decrease in OD. Many have observed that a 48-h incubation time increases the likelihood that an isolate will demonstrate a trailing effect and a higher MIC [2,3,8]. Furthermore, the data presented here show that this phenomenon appears to be species speci®c (Tables 3, 4, 6).…”

Purification and identification of antibacterial phenolics fromTripodanthus acutifoliusleaves

“…Nevertheless, these differences were not significant (P > 0AE05) when the amount of substance is expressed in lmol ml )1 (concentration units) of each compound, suggesting that the sugar moiety does not contribute to the antibacterial effect. Bacteriostatic, bactericidal or bacteriolytic effects of antibacterial substances on target cells depend on incubation period and bacterial inoculum con-centration (Tornatore et al 1997). The inoculation size in photometric assay was 20-fold greater (because of photometric measurements) than that proposed by the National Committee for Clinical Laboratory Standards (NCCLS 1999), that is the reason why the substances were assayed at concentrations nearly 20-fold MICs (Hartzen et al 1997).…”

In Vitro Antifungal Susceptibility Testing

“…In addition, some Candida isolates do not show trailing effects after 24 hours of incubation but do after 48 hours. [67][68][69] Often, these isolates, called low-high MIC phenotype isolates, are susceptible after 24 hours of incubation (fluconazole MICs ≤ 8 µg/ml) but appear resistant after 48 hours (fluconazole MICs ≥ 64 µg/ml). Of interest, low-high MIC phenotypes isolated from HIV-infected patients with oropharyngeal candidiasis appeared to respond clinically to fluconazole, similar to patients infected with susceptible yeasts.…”

“…71 Some investigators found decreased trailing with a medium of lower pH. 69,72,73 Adjustment of medium pH could be considered to eliminate trailing among Candida sp with azole susceptibility testing.…”