Increased Antifungal Drug Resistance in Clinical Isolates of Cryptococcus neoformans in Uganda

Abstract

Cryptococcal antigen screening is recommended among people living with AIDS when entering HIV care with a CD4 count of <100 cells/μl, and preemptive fluconazole monotherapy treatment is recommended for those with subclinical cryptococcal antigenemia. Yet, knowledge is limited of current antimicrobial resistance in Africa. We examined antifungal drug susceptibility in 198 clinical isolates collected from Kampala, Uganda, between 2010 and 2014 using the CLSI broth microdilution assay. In comparison with two previous studies from 1998 to 1999 that reported an MIC50 of 4 μg/ml and an MIC90 of 8 μg/ml prior to widespread human fluconazole and agricultural azole fungicide usage, we report an upward shift in the fluconazole MIC50 to 8 μg/ml and an MIC90 value of 32 μg/ml, with 31% of isolates with a fluconazole MIC of ≥ 16 μg/ml. We observed an amphotericin B MIC50 of 0.5 μg/ml and an MIC90 of 1 μg/ml, of which 99.5% of isolates (197 of 198 isolates) were still susceptible. No correlation between MIC and clinical outcome was observed in the context of amphotericin B and fluconazole combination induction therapy. We also analyzed Cryptococcus susceptibility to sertraline, with an MIC50 of 4 μg/ml, suggesting that sertraline is a promising oral, low-cost, available, novel medication and a possible alternative to fluconazole. Although the CLSI broth microdilution assay is ideal to standardize results, limit human bias, and increase assay capacity, such assays are often inaccessible in low-income countries. Thus, we also developed and validated an assay that could easily be implemented in a resource-limited setting, with similar susceptibility results (P = 0.52).

FIG 1

Effect of growth rate on fluconazole resistance. Relationship between growth rate and fluconazole (A), amphotericin B (B), or sertraline (C) resistance determined by MIC of drug. Growth of fluconazole-resistant isolates (MIC of ≥64 μg/ml) was lower than that of susceptible isolates (≤8 μg/ml) (P = 0.004). Growth did not correlate with increased resistance to amphotericin B or sertraline. Only 1 of the 103 isolates examined exhibited an MIC of ≥1 μg/ml to amphotericin B. OD600, optical density at 600 nm.

FIG 2

Resource-limited assay (RLA) validation. (A) Picture of RLA showing visual determination of fluconazole MIC. MIC determined by RLA compared to the CLSI method for fluconazole (B) and amphotericin B (C). Locations of numbers on the plot indicate the number of isolates which exhibited those MIC values. Dotted line indicates the line of best fit for the data set. There was no significant difference between the CLSI and RLA data (P = 0.521 for fluconazole, P = 0.571 for amphotericin B).

FIG 3

Genotypic makeup of fluconazole MIC groupings based on sequence type. Proportion of each sequence type in the MIC grouping that was susceptible (n = 61) (A), dose-dependent susceptible (n = 33) (B), or resistant (n = 4) (C). Singletons were combined into one group for this analysis.