Decreasing fluconazole susceptibility of clinical South African Cryptococcus neoformans isolates over a decade

Abstract

Fluconazole is used in combination with amphotericin B for induction treatment of cryptococcal meningitis and as monotherapy for consolidation and maintenance treatment. More than 90% of isolates from first episodes of cryptococcal disease had a fluconazole minimum inhibitory concentration (MIC) ≤4 μg/ml in a Gauteng population-based surveillance study of Cryptococcus neoformans in 2007–2008. We assessed whether fluconazole resistance had emerged in clinical cryptococcal isolates over a decade. We prospectively collected C. neoformans isolates from 1 January through 31 March 2017 from persons with a first episode of culture-confirmed cryptococcal disease at 37 South African hospitals. Isolates were phenotypically confirmed to C. neoformans species-complex level. We determined fluconazole MICs (range: 0.125 μg/ml to 64 μg/ml) of 229 C. neoformans isolates using custom-made broth microdilution panels prepared, inoculated and read according to Clinical and Laboratory Standards Institute M27-A3 and M60 recommendations. These MIC values were compared to MICs of 249 isolates from earlier surveillance (2007–2008). Clinical data were collected from patients during both surveillance periods. There were more males (61% vs 39%) and more participants on combination induction antifungal treatment (92% vs 32%) in 2017 compared to 2007–2008. The fluconazole MIC₅₀, MIC₉₀ and geometric mean MIC was 4 μg/ml, 8 μg/ml and 4.11 μg/ml in 2017 (n = 229) compared to 1 μg/ml, 2 μg/ml and 2.08 μg/ml in 2007–2008 (n = 249) respectively. Voriconazole, itraconazole and posaconazole Etests were performed on 16 of 229 (7%) C. neoformans isolates with a fluconazole MIC value of ≥16 μg/ml; only one had MIC values of >32 μg/ml for these three antifungal agents. Fluconazole MIC₅₀ and MIC₉₀ values were two-fold higher in 2017 compared to 2007–2008. Although there are no breakpoints, higher fluconazole doses may be required to maintain efficacy of standard treatment regimens for cryptococcal meningitis. Cryptococcus neoformans, a pathogenic fungal species-complex with an environmental niche, is the most common cause of meningitis among HIV-seropositive adults in sub-Saharan Africa. Fluconazole is recommended in combination with amphotericin B for induction treatment of cryptococcal meningitis and as monotherapy for consolidation and maintenance treatment. Fluconazole is also commonly prescribed to HIV-seropositive individuals for other indications; fluconazole exposure may result in secondary resistance if patients have concurrent active cryptococcal disease. Azole fungicides used in agriculture may potentially drive primary cryptococcal resistance when the fungus is exposed to these fungicides in the environment. We aimed to determine fluconazole MICs in 2017 and compare these values to those obtained in a 2007–2008 South African survey to assess whether fluconazole resistance had emerged in C. neoformans over a decade. We found that the proportion of isolates with an MIC of ≥16 μg/ml increased from 0% in 2007–2008 to 7% in 2017. MIC₅₀ and MIC₉₀ values were also two-fold higher in 2017 compared to 2007–2008. These study findings provided evidence for higher fluconazole dose recommendations (in combination with amphotericin B for the induction phase and as monotherapy for consolidation and maintenance phases) in the 2019 Southern African guideline for HIV-associated cryptococcosis.