37 The Impact of pH on Antifungal Susceptibility Testing on Vulvovaginal Candidiasis Isolates

Abstract Objectives Vulvovaginal candidiasis (VVC) is a common superficial fungal infection primarily caused by Candida albicans (∼70%) or C. glabrata (∼20%). Antimicrobial susceptibility testing (AST) is not indicated for patients with uncomplicated VVC, however, it is appropriate for compliant patients that fail adequate therapy, including repeat infections or recurrent VVC (rVVC; ≥3 events per year). Unlike traditional AST reference methods performed at a neutral pH, yeasts in vulvovaginal tissue are exposed to antifungals at the normal vaginal pH range of 4-4.5. In this study, we hypothesized that VVC clinical isolates will exhibit elevated Minimum Inhibitory Concentrations (MICs) to antifungals when incubated at a pH = 4 vs pH = 7. Methods The minimum inhibitory concentration (MIC) of VVC (n = 43) and systemic (n = 20) isolates was evaluated at a pH = 4 vs 7 using microbroth dilution antimicrobial susceptibility testing (AST) with growth curves determined utilizing absorbance (OD600nm). Changes in cell wall components were assessed using cell-wall targeting dyes: fluorescently tagged wheat germ agglutinin (chitin), calcofluor white (chitin), and concanavalin A (mannoprotein). The impact of pH on the chemical structure of antifungals was determined utilizing 2D nuclear magnetic resonance (NMR). Results 2D nuclear magnetic resonance (NMR) of fluconazole, flucytosine, isavuconazole, and voriconazole showed no difference in the chemical structure at pH = 4, 5.6, or 7. However, peak shifts were observed in both rezafungin and caspofungin, indicating ionization may impact drug efficacy at a pH = 4. Assessment by microbroth dilution revealed that VVC isolates exhibited significantly increased MICs (>2 doubling dilutions) at a pH of 4 vs 7. At 24h, 23% (5/22) of C. albicans and 52% (11/21) of C. glabrata VVC isolates showed elevated MICs to echinocandins. Similarly, 36% (8/22) of C. albicans and 86% (18/21) of C. glabrata VVC isolates exhibited elevated MICs to azoles (posaconazole, voriconazole, itraconazole, fluconazole, and isavuconazole). Only C. albicans (15%; 2/13) showed increased resistance to flucytosine. pH-dependent MIC trends continued at 48 and 72h. VVC isolates exhibited a delay in initial growth at pH = 4 (13.5 hours) vs pH = 7 (2.9 hours), yet grew significantly more by 72h with an area under the curve at pH = 4 (30.2AU) vs pH = 7 (11.6AU). Co-staining with fluorescently tagged cell wall-targeting dyes identified increased chitin but not mannosylated proteins on the fungal cell surface at a pH = 4 vs 7. Conclusions Given that incubation at a pH = 4 results in a) no shift in the chemical structure of clinically relevant azoles, b) elevated MICs to azoles, and c) a selective fungal growth advantage, MIC data generated by AST performed at a pH = 4 may more accurately identify instances of refractory/recurrent vulvovaginal candidiasis requiring therapeutic optimization. Chart reviews are ongoing to examine the clinical impact of these findings.