Details

Autor(en) / Beteiligte

• Liao, Robert Steven

Titel

Vitality and mortality of Candida albicans

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2002

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Invasive infection with Candida albicans is a major source of morbidity and mortality. Amphotericin B and fluconazole are the antifungal agents most commonly used to treat serious C. albicans infections. Therapy with amphotericin is often prolonged and refractory, and the correlation between MICs and clinical outcome is uncertain. Endpoint determination for fluconazole susceptibility testing is also problematic and a major source of error. We hypothesized that by studying C. albicans viability we could improve our understanding of why amphotericin B is less effective than would be expected of a fungicidal antifungal agent, and also correlate these insights to improving antifungal susceptibility testing. A number of assays were developed that allowed for a unique overall view of C. albicans viability by separately assessing parameters directly associated with vitality and mortality. The loss of C. albicans viability was shown to be a gradual process that could be delineated into different stages between alive and dead. The loss of replication and the ability to form colony forming units was shown to be an early event in the death of C. albicans. In vitro incubation with amphotericin B, at concentrations usually achieved in the serum with normal dosing, showed that C. albicans exists in a viable nonreplicating state that was capable of resuscitation and growth after additional incubation for 15 hr at 22°C. This capacity to recover and grow in vitro indicated that amphotericin B should not be considered fungicidal at 0.5 μg/mL. Sequential combination treatment with amphotericin in vitro followed by miconazole was shown to be effective in achieving fungicidal inhibition and eliminating this rescue mechanism. Several vitality assays were also integrated into the current methods used to assess antifungal susceptibility testing in order to improve endpoint determinations. Measurement of C. albicans intracellular adenosine 5′-triphosphate allowed for rapid susceptibility testing with amphotericin B within 30 minutes. The fluorescent dye, 5,6-carboxyfluorescein diacetate, was also incorporated into the microdilution method and was shown to provide objective and quantifiable endpoints for susceptibility testing a wide range of antifungal agents at 24 h and 48 h that were reproducible and easier to interpret than the existing methods.