Effect of pH on the In Vitro Activities of Amphotericin B, Itraconazole, and Flucytosine against Aspergillus Isolates

Abstract

The in vitro susceptibilities of 21 Aspergillus isolates were tested against three antifungal agents in RPMI 1640 and yeast nitrogen base at pH 5.0 and 7.0 by a broth microdilution format of the NCCLS method. The MICs of amphotericin B and itraconazole were higher, while those of flucytosine were lower, at pH 5.0 than at pH 7.0. The poor correlation between in vitro results and clinical outcome could be due to a difference in pH between the in vitro susceptibility test and at the site of infection.In vitro susceptibility testing of filamentous fungi has become increasingly important. A good standard in vitro susceptibility test must give reproducible results, predict the resistance of molds, and correlate with clinical outcome (5).In 1998 the National Committee for Clinical Laboratory Standards (NCCLS) proposed a standard method for the determination of the in vitro antifungal susceptibility of conidium-forming filamentous fungi. This document is now approved (3). Although the standardized NCCLS method has been found to give better inter-and intracenter reproducibility, in vitro antifungal susceptibility testing of filamentous fungi is still faced with several problems such as the correlation of in vitro results with clinical outcome.Clinical outcome may be affected by various factors related to the host, the drugs, the fungus, and their interactions (9). These factors are not taken into account in the in vitro tests, and it may be for this reason that the prediction of antifungal efficacy or failure from in vitro susceptibility tests remains difficult. One of the host-related-factors is the pH at the site of infection. In the human body the pH is carefully regulated at 7.4, but it may be lower at the site of infection due to necrosis (7), the production of organic acids by fungi (6), or lysosome activity of granulocytes and macrophages (1).The aim of this study was to investigate the effect of pH on the in vitro activities of three different antifungal agents against 21 Aspergillus isolates in two different media.Twenty-one clinical Aspergillus isolates were tested: five itraconazole (ITZ)-susceptible and five ITZ-resistant Aspergillus fumigatus isolates, five A. flavus isolates, and six A. terreus isolates. Isolates had been frozen in glycerol broth at Ϫ80°C; they were revived by subculturing twice on Sabouraud glucose agar tubes supplemented with 0.5% chloramphenicol and were incubated for 5 to 7 days at 35°C. All isolates were tested in duplicate on different days.Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used for quality control. Amphotericin B (AMB; BristolMyers Squibb, Woerden, The Netherlands), ITZ (Janssen Pharmaceutica B.V., Tilburg, The Netherlands), and flucytosine (5FC; ICN Pharmaceuticals, Zoetermeer, The Netherlands) were obtained as powders. AMB and ITZ were dissolved in dimethyl sulfoxide, and 5FC was dissolved in distilled water. The final concentrations of the antifungal agents at pH 5.0 ranged from 256 to 0.25 g/ml for AMB, from 16 to 0.016 g/ml for ITZ, and from 1,024 to 0...

In Vitro Activities at pH 5.0 and pH 7.0 and In Vivo Efficacy of Flucytosine against Aspergillus fumigatus

“…Most isolates of Aspergillus species are not susceptible to flucytosine, with MICs typically being Ͼ64 mg/liter (8). However, we previously demonstrated that the activity of flucytosine against Aspergillus species increased when the pH of the medium was lowered from 7.0 to 5.0 (8).…”

Pharmacodynamics and Dose-Response Relationships of Liposomal Amphotericin B against Different Azole-Resistant Aspergillus fumigatus Isolates in a Murine Model of Disseminated Aspergillosis

“…The efficacy of L-AmB was not different between the isolates with an azole resistance mechanism and wild-type controls (P Ͼ 0.05), and no difference in efficacy was found when different azole resistance mechanisms were compared (P Ͼ 0.05). The null hypothesis was not rejected in an F test (P ϭ 0.92, F ϭ 0.2241, DFn [degrees of freedom numerator] ϭ 4, and DFd [degrees of freedom denominator] ϭ 54), indicating that log EC 50 did not significantly differ between the four groups (Table 2). In addition, multiple logistic regression analysis confirmed that there was no significant difference between groups (results not shown).…”

“…Another potentially effective combination is L-AmB plus flucytosine, a combination that has been shown to be effective in central nervous system infections due to Cryptococcus neoformans (48). The efficacy of flucytosine against A. fumigatus infection has long been controversial, but we previously showed efficacy of flucytosine against A. fumigatus infection in our animal model (49) and a good correlation with in vitro activity when the MIC was determined at pH 5 instead of pH 7 (50). Clearly, more research is needed to allow recommendations to be made.…”

Relationship between In Vitro Activities of Amphotericin B and Flucytosine and pH for Clinical Yeast and Mold Isolates

“…We demonstrated that the in vitro antifungal activities of amphotericin B (AMB), itraconazole, and 5FC against A. fumigatus, A. flavus, and A. terreus changed at low pH (14). For both AMB and itraconazole, the in vitro activity decreased when the pH was lowered from 7.0 to 5.0, while for 5FC, the in vitro activity increased when the pH was lowered.…”