The Prevalence of Persister Cells and Heteroresistance Subpopulations in <i>Candida albicans</i> Isolates from HIV-Positive Patients with Oral Candidiasis: Emerging Challenges in Treatment

Abstract

Background: Oral candidiasis remains a major challenge in managing HIV-infected patients, particularly in tropical regions such as Ahvaz. In addition to classical resistance, nonclassical mechanisms, including persister cells and heteroresistance, may contribute to antifungal treatment failure. Objectives: This study aimed to determine the prevalence of persister cells and heteroresistance in Candida albicans isolates recovered from HIV-positive patients with oral candidiasis in Ahvaz, and to evaluate their potential associations with clinical and immunological parameters. Methods: In this cross-sectional study, 101 clinical isolates of C. albicans were recovered from oral samples of HIV-positive patients with oral candidiasis who were referred to the Behavioral Diseases Counseling Center in Ahvaz, southwestern Iran. After obtaining written informed consent, comprehensive medical histories, including prior antifungal use, were collected. Oral samples were cultured on CHROMagar™ Candida, and species identification was performed using PCR-RFLP with the MspI enzyme. Antifungal susceptibility testing was performed according to the clinical and Laboratory Standards Institute (CLSI) M27-A3 and M27-S4 microdilution guidelines. Persister cells were identified by exposure to high-dose amphotericin B followed by regrowth analysis. Heteroresistant cells were assessed using population analysis profiling and area under the curve (AUC) calculations, with high heteroresistance defined as AUC ≥ 25.76 (75th percentile). Host immunological parameters (CD4 counts and viral loads) and antifungal susceptibility profiles (minimum inhibitory concentrations (MICs) of amphotericin B and fluconazole) were documented to evaluate potential clinical correlates. Results: Among 101 patients, persister-cell phenotypes were distributed as follows: high persister, 1% (n = 1); low persister, 95% (n = 96); and non-persister, 4% (n = 4). Amphotericin B MIC values were higher in the low-persister group than in the non-persister group; however, the difference was not statistically significant (P = 0.131). Heteroresistance profiling identified high heteroresistance (AUC ≥ 25.76) in 21.78% (n = 22), low heteroresistance in 64.35% (n = 65), and no heteroresistance in 13.86% (n = 14). CD4 counts differed significantly among the heteroresistance groups (P = 0.014), and Bonferroni-adjusted pairwise comparisons showed a significant difference between the non-heteroresistance and low-heteroresistance groups. Fluconazole MIC and viral load did not differ significantly among the heteroresistance groups. Conclusions: Persister cells and heteroresistant subpopulations were prevalent among C. albicans isolates from HIV-positive patients with oral candidiasis. The heteroresistance phenotype was significantly associated with the CD4 count; however, this cross-sectional association should be interpreted with caution. These findings support rigorous detection and monitoring of these resistant phenotypes, as well as continued viral-load surveillance, to improve clinical management.