5-Flucytosine Longitudinal Antifungal Susceptibility Testing of Cryptococcus neoformans: A Substudy of the EnACT Trial Testing Oral Amphotericin

Abstract

Background: The EnACT trial was a phase 2 randomized clinical trial conducted in Uganda, which evaluated a novel orally delivered lipid nanocrystal (LNC) amphotericin B in combination with flucytosine for the treatment of cryptococcal meningitis. When flucytosine (5FC) is used as monotherapy in cryptococcosis, 5FC can induce resistant Cryptococcus mutants. Oral amphotericin B uses a novel drug delivery mechanism, and we assessed whether resistance to 5FC develops during oral LNC-amphotericin B therapy.

Methods: We enrolled Ugandans with HIV diagnosed with cryptococcal meningitis and who were randomized to receive 5FC and either standard intravenous (IV) amphotericin B or oral LNC-amphotericin B. We used broth microdilution to measure the minimum inhibitory concentration (MIC) of the first and last cryptococcal isolates in each participant. Breakpoints are inferred from 5FC in Candida albicans. We measured cerebral spinal fluid (CSF) 5FC concentrations by liquid chromatography and tandem mass spectrometry.

Results: Cryptococcus 5FC MIC₅₀ was 4 µg/mL, and MIC₉₀ was 8 µg/mL. After 2 weeks of therapy, there was no evidence of 5FC resistance developing, defined as a >4-fold change in susceptibility in any Cryptococcus isolate tested. The median CSF 5FC concentration to MIC ratio (interquartile range) was 3.0 (1.7-5.5) µg/mL. There was no association between 5FC/MIC ratio and early fungicidal activity of the quantitative rate of CSF yeast clearance (R² = 0.004; P = .63).

Conclusions: There is no evidence of baseline resistance to 5FC or incident resistance during combination therapy with oral or IV amphotericin B in Uganda. Oral amphotericin B can safely be used in combination with 5FC.

Keywords: 5-flucytosine resistance; antifungal resistance; antifungal susceptibility testing; cryptococcal meningitis.

Graphical Abstract

This graphical abstract is also available at Tidbit: https://tidbitapp.io/tidbits/5-flucytosine-longitudinal-antifungal-susceptibility-testing-of-cryptococcus-neoformans-a-sub-study-of-the-enact-trial-testing-oral-amphotericin-d56fad6c-7367-43e3-bd40-38b328652247

Figure 1.

Change in flucytosine susceptibility between first and last Cryptococcus isolates. There was minimal change in 5FC MIC between the first and last positive lumbar punctures. A, The percentage of participants who had a 2-fold or 4-fold increase between the first and last positive lumbar punctures. B, Individual lines representing participants, with the 5FC MIC represented by the dot at each time point, with time displayed on the x-axis. Abbreviations: 5FC, flucytosine; MIC, minimum inhibitory concentration.

Figure 2.

Checkerboard of flucytosine susceptibility between the first and last Cryptococcus isolates. In both arms, most participants had a screening and last LP MIC of 4 µg/mL. One participant in the oral amphotericin B arm moved from an MIC of 2 to 8 µg/mL (indicated in the darkest, maroon color shading). Those without a second isolate are recorded as no change. Abbreviations: 5FC, flucytosine; LP, lumbar puncture; MIC, minimum inhibitory concentration.

Figure 3.

Scatterplot depicting the ratio of average 5FC CSF concentration to MIC. The median 5FC to MIC ratio was 3.0, and the mean was 4.7, indicating that the 5FC CSF concentrations were markedly higher than the MIC in the overall population. Points left of the vertical red line indicate 5FC concentrations below the MIC at any point during treatment. There was no relationship between rate of CSF Cryptococcus yeast clearance and the average 5FC CSF concentration to MIC ratio. The blue trendline is based on a linear regression (R² = 0.004). Eleven outliers with EFA >0.6 or 5FC/MIC ratio >10 were removed. Abbreviations: 5FC, flucytosine; CSF, cerebrospinal fluid; EFA, early fungicidal activity; MIC, minimum inhibitory concentration.

Figure 4.

Density plot of average flucytosine (5FC) CSF concentration to Cryptococcus MIC. The average 5FC CSF concentration was rarely less than the MIC for any individual participant. A, Minimal overlap in a density plot of 5FC CSF concentrations and MIC. B, Ratio of 5FC CSF concentrations to 5FC MIC for each participant. A ratio of <1.0 would indicate CSF concentrations of 5FC are below than MIC, which occurred in 13.3% (n = 6) of participants. Abbreviations: 5FC, flucytosine; CSF, cerebrospinal fluid; EFA, early fungicidal activity; MIC, minimum inhibitory concentration.