The reference methodology for evaluating antifungal susceptibility is based on determining the minimum inhibitory concentration (MIC), which is the lowest drug concentration capable of inhibiting fungal growth. However, such MIC data are insufficient to measure antifungal susceptibility if a strain is heteroresistant to the tested drug. In such cases, a minority subpopulation of fungal cells, originating from an initially susceptible lineage, can grow at antifungal drug concentrations above the MIC. In studies on fluconazole heteroresistance in Cryptococcus spp., chromosomal disomy has been shown to result in the overexpression of two genes located on chromosome 1 (Chr1) linked to antifungal resistance: ERG11 and AFR1. This review addresses the underlying mechanisms of antifungal resistance, the evolution of methods for determining antifungal susceptibility, and the clinical implications of Cryptococcus heteroresistance to fluconazole. The analysis of the findings indicated a correlation between heteroresistance and adverse clinical outcomes, although this observation still lacks definite confirmation in the literature. This highlights the need to implement more efficient therapeutic strategies and improve antifungal susceptibility and heteroresistance testing.

Background/Objectives: Cryptococcosis, caused by Cryptococcus neoformans and Cryptococcus gattii species complexes, remains a significant health concern, particularly among immunocompromised patients. The emergence of antifungal resistance and toxicity of conventional treatment underscore the urgent need for novel therapeutic approaches. Combination therapies represent a promising strategy to enhance efficacy and overcome resistance. This study investigated the antifungal activity of five naphthoquinones against nine isolates of Cryptococcus spp. and assessed their synergistic effects with amphotericin B (AmB). Methods: In this study, five selected naphthoquinones were evaluated for their antifungal activity against Cryptococcus spp. isolates using broth microdilution assays to determine minimum inhibitory concentrations (MICs), according to CLSI guidelines. The potential synergistic effect with AmB was assessed using checkerboard assays, with synergy interpreted based on the fractional inhibitory concentration index (FICI). Cytotoxicity was evaluated in MRC-5 human lung fibroblast cells using the MTT assay. Results: Among the compounds tested, 2-methoxynaphthalene-1,4-dione (2-MNQ) demonstrated antifungal activity, with MIC values ranging from 3.12 to 12.5 µg/mL. Checkerboard assays revealed a synergistic interaction between 2-MNQ and AmB, with a fractional inhibitory concentration index (FICI) of 0.27. The combination reduced the MIC of AmB by 4.17-fold. These findings highlight the potential of synthetic naphthoquinones, particularly 2-MNQ, as effective antifungal agents with synergistic properties when combined with AmB. The observed synergy suggests complementary mechanisms, including increased fungal membrane permeability and oxidative stress induction. Conclusions: This study highlights the potential of 2-MNQ and 2,3-DBNQ as antifungal candidates against Cryptococcus spp., with emphasis on the synergistic interaction observed between 2-MNQ and amphotericin B. The findings reinforce the importance of structural modifications in naphthoquinones to enhance antifungal activity and support the need for further preclinical studies investigating combination therapies aimed at improving treatment efficacy in patients with cryptococcosis.

Background: Cryptococcosis remains a severe systemic mycosis, especially among people living with HIV/AIDS, and culture-based molecular data from Amazonas remain limited. Objectives: The aim was to identify which Cryptococcus molecular types circulated among clinical isolates from Amazonas between 2020 and 2024; what were the main clinical characteristics and outcomes of patients infected with Cryptococcus neoformans molecular type VNI and Cryptococcus gattii molecular type VGII; and whether these molecular types differed in selected susceptibility-related and virulence-associated phenotypes. Methods: We analyzed 70 culture-confirmed clinical isolates from patients treated at the Dr. Heitor Vieira Dourado Tropical Medicine Foundation, Manaus, Brazil. Molecular types were assigned by URA5 PCR-RFLP using reference strains. Demographic and clinical variables were retrieved from medical records. A stratified 30-isolate subset comprising 24 VNI and 6 VGII isolates was evaluated for phospholipase activity and melanization. Fluconazole MICs were analyzed for the complete 70-isolate dataset and were determined by broth microdilution according to CLSI M27-A4. Results: VNI predominated (64/70; 91.4%), whereas VGII accounted for 6/70 isolates (8.6%). The series was predominantly composed of men, young adults, and HIV-positive patients. HIV infection was more frequent among VNI cases, whereas VGII included proportionally more HIV-negative patients. In-hospital death occurred in 17/70 patients (24.3%). Fluconazole MICs, phospholipase activity, and melanization were broadly comparable between VNI and VGII, although the small VGII subgroup precluded robust genotype-associated inference. Conclusion: VNI was the dominant molecular type in this severe reference-center cryptococcosis series from Amazonas. Phenotypic findings should be interpreted as exploratory and hypothesis-generating.

Cryptococcosis is a severe systemic mycosis, and relapse may be influenced by fungal subpopulations that survive fluconazole exposure. This retrospective cohort study investigated whether fluconazole minimum inhibitory concentration (MIC) and the heteroresistant (HetR) phenotype of the first available diagnostic cerebrospinal-fluid isolate were associated with recorded relapse within 6 months in 70 patients with neurocryptococcosis from Amazonas, Brazil. One clinical isolate per patient, comprising 64 Cryptococcus neoformans molecular type VNI and 6 Cryptococcus gattii molecular type VGII isolates, was evaluated by broth microdilution and endpoint-based fluconazole heteroresistance level (HFL) testing. Using an operational criterion of HFL/MIC ≥ 8, corresponding to visible growth at least three twofold dilutions above the MIC, 43 of 70 isolates (61.4%) were classified as displaying a HetR phenotype. Six-month follow-up was available for 69 patients. Recorded relapse occurred in 12 of 42 patients infected with HetR isolates (28.6%) and in 4 of 27 patients infected with non-HetR isolates (14.8%). Although relapse was numerically more frequent in the HetR group, the association was not statistically significant (conditional odds ratio, 2.27; exact 95% confidence interval, 0.59–10.96; two-sided Fisher’s exact test, P = .248). The HFL/MIC ratio also did not differ significantly according to recorded relapse status (P = .165). The HetR phenotype of the first available diagnostic isolate, defined operationally in this study as HFL/MIC ≥ 8, was frequent but was not significantly associated with recorded relapse within 6 months in this cohort.