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Background Emerging fungal pathogens pose important threats to global public health. The World Health Organization has responded to the rising threat of traditionally neglected fungal infections by developing a Fungal Priority Pathogens List (FPPL). Taking the highest-ranked fungal pathogen in the FPPL, Cryptococcus neoformans , as a paradigm, we review progress made over the past two decades on its global burden, its clinical manifestation and management of cryptococcal infection, and its antifungal resistance. The purpose of this review is to drive research efforts to improve future diagnoses, therapies, and interventions associated with fungal infections. Methods We first reviewed trends in the global burden of HIV-associated cryptococcal infection, mainly based on a series of systematic studies. We next conducted scoping reviews in accordance with the guidelines described in the Preferred Reporting Items for Systematic Reviews and Meta-analyses extension for Scoping Reviews using PubMed and ScienceDirect with the keyword Cryptococcus neoformans  to identify case reports of cryptococcal infections published since 2000. We then reviewed recent updates on the diagnosis and antifungal treatment of cryptococcal infections. Finally, we summarized knowledge regarding the resistance and tolerance of C. neoformans to approved antifungal drugs. Results There has been a general reduction in the estimated global burden of HIV-associated cryptococcal meningitis since 2009, probably due to improvements in highly active antiretroviral therapies. However, cryptococcal meningitis still accounts for 19% of AIDS-related deaths annually. The incidences of CM in Europe and North America and the Latin America region have increased by approximately two-fold since 2009, while other regions showed either reduced or stable numbers of cases. Unfortunately, diagnostic and treatment options for cryptococcal infections are limited, and emerging antifungal resistance exacerbates the public health burden. Conclusion The rising threat of C. neoformans is compounded by accumulating evidence for its ability to infect immunocompetent individuals and the emergence of antifungal-resistant variants. Emphasis should be placed on further understanding the mechanisms of pathogenicity and of antifungal resistance and tolerance. The development of novel management strategies through the identification of new drug targets and the discovery and optimization of new and existing diagnostics and therapeutics are key to reducing the health burden.

Candida albicans is a prevalent human fungal commensal and also a pathogen that causes life-threatening systemic infections. Treatment failures are frequent because few therapeutic antifungal drug classes are available and because drug resistance and tolerance limit drug efficacy. How cells exposed to one stress are later able to better survive other types of stress is not well understood. In eukaryotic organisms, physiological and pathological stresses can disturb endoplasmic reticulum (ER) function, resulting in “ER stress.” Here, we found that exposure to tunicamycin, an inducer of ER stress, resulted in the acquisition of a specific aneuploidy, chromosome 2 trisomy (Chr2x3), in Candida albicans . Importantly, the resulting aneuploidy also conferred cross-tolerance to caspofungin, a first-line echinocandin antifungal, as well as to hydroxyurea, a common chemotherapeutic agent. Exposure to a range of tunicamycin concentrations induced similar ER stress responses. Extra copies of one Chr2 gene, MKK2 , affected both tunicamycin and caspofungin tolerance, while at least 3 genes on chromosome 2 ( ALG7 , RTA2 , and RTA3 ) affected only tunicamycin and not caspofungin responses. Other Chr2 genes ( RNR1 and RNR21 ) affected hydroxyurea tolerance but neither tunicamycin nor caspofungin tolerance. Deletion of components of the protein kinase C (PKC) or calcineurin pathways affected tolerance to both tunicamycin and caspofungin, supporting the idea that the ER stress response and echinocandin tolerance are regulated by overlapping stress response pathways. Thus, antifungal drug tolerance can arise rapidly via ER stress-induced aneuploidy. IMPORTANCE Candida albicans is a prevalent human fungal commensal and also a pathogen that causes life-threatening systemic infections. Treatment failures are frequent because few therapeutic antifungal drug classes are available and because drug resistance and tolerance limit drug efficacy. We found that C. albicans rapidly overcomes the cellular stress induced by the drug tunicamycin by duplicating chromosome 2. Also, chromosome 2 duplication confers tolerance not only to tunicamycin but also to the following two unrelated drugs: caspofungin, an antifungal drug, and hydroxyurea, a chemotherapeutic. Cross tolerance to the three drugs involves different sets of genes, although some genetic pathways affect the tolerance to two of these three drugs. This work highlights a serious concern, namely, that changes in whole chromosome copy number can occur in response to one type of stress, and yet, they may facilitate the emergence of tolerance to multiple drugs, including the few antifungal drug classes available to treat Candida infections.

Antifungal drug tolerance and persistence are being increasingly recognized in fungal pathogens. Accordingly, more and more research is being carried out to characterize and understand these phenomena. However, the terminology and methodology employed in the fungal community lack consensus, particularly for filamentous fungi, as they present further complexities when compared to single-celled microorganisms. Hence, with the aim to ensure consistency in the literature, in this Perspective article, we propose tailored definitions for tolerance and persistence in filamentous fungi and suggest methods to detect and investigate these phenomena in the laboratory.

With the growing population of immunocompromised individuals, opportunistic fungal pathogens pose a global health threat. species, particularly and non-albicans species such as , are the most prevalent pathogenic fungi. Azoles, especially fluconazole, are widely used therapeutic options. This study investigates how adapts to fluconazole, with a focus on understanding the factors regulating fluconazole tolerance and its relationship to resistance. This study compared the factors regulating fluconazole tolerance between and . We analyzed the impact of temperature on fluconazole tolerance, and requirement of calcineurin and Hsp90 for maintenance of fluconazole tolerance. We isolated colonies from edge, inside and outside of inhibition zone in disk diffusion assays. And we exposed strain to high concentrations of fluconazole and investigated the mutants for development of fluconazole resistance and tolerance. We found temperature modulated tolerance in the opposite way in strain YJB-T1891 and strain CG4. Calcineurin and Hsp90 were required for maintenance of fluconazole tolerance in both species. Colonies from inside and outside of inhibition zones did not exhibited mutated phenotype, but colonies isolated from edge of inhibition zone exhibited diverse phenotype changes. Moreover, we discovered that high concentrations (16-128 μg/mL) of fluconazole induce the simultaneous but parallel development of tolerance and resistance in , unlike the sole development of tolerance in . This study highlights that while tolerance to fluconazole is a common response in species, the specific molecular mechanisms and evolutionary pathways that lead to this response vary between species. Our findings emphasize the importance of understanding the regulation of fluconazole tolerance in different species to develop effective therapeutic strategies.

Fungal diseases cause millions of deaths per year worldwide. Antifungal resistance has become a matter of great concern in public health. In recent years rates of non-albicans species have risen dramatically. Candida parapsilosis is now reported to be the second most frequent species causing candidemia in several countries in Europe, Latin America, South Africa and Asia. Rates of acquired azole resistance are reaching a worrisome threshold from multiple reports as in vitro susceptibility testing is now starting also to explore tolerance and heteroresistance to antifungal compounds. With this review, the authors seek to evaluate known antifungal resistance mechanisms and their worldwide distribution in Candida species infections with a specific focus on C. parapsilosis.

ABSTRACT Environmental stress often causes phenotypic changes among pathogenic cryptococci, such as altered antifungal susceptibility, changes in capsule and melanin formation, as well as altered levels of the membrane sterol and antifungal target, ergosterol. We therefore hypothesised that nitrogen limitation, a prevalent environmental stress in the natural habitat of these yeasts, might affect virulence and antifungal susceptibility. We tested the effect of different nitrogen concentrations on capsule, melanin and ergosterol biosynthesis, as well as amphotericin B (AmB) and fluconazole (FLU) susceptibility. This was achieved by culturing cryptococcal strains representing Cryptococcus neoformans and Cryptococcus gattii in media with high (0.53 g/l), control (0.42 g/l) and low (0.21 g/l) NH4Cl concentrations. India ink staining was used to determine capsule thickness microscopically, while melanin and ergosterol content were determined spectrophotometrically. We found that lower nitrogen concentrations enhanced both ergosterol and capsule biosynthesis, while a variable effect was observed on melanisation. Evaluation of drug tolerance using time-kill methodology, as well as tests for FLU heteroresistance, revealed that the low nitrogen cultures had the highest survival percentages in the presence of both AmB and FLU, and showed the highest frequency of FLU heteroresistance, suggesting that nitrogen concentration may indeed influence drug tolerance. Nitrogen concentration affects the virulence factors and antifungal susceptibility of pathogenic cryptococci.

Antimicrobial resistance is a matter of rising concern, especially in fungal diseases. Multiple reports all over the world are highlighting a worrisome increase in azole- and echinocandin-resistance among fungal pathogens, especially in Candida species, as reported in the recently published fungal pathogens priority list made by WHO. Despite continuous efforts and advances in infection control, development of new antifungal molecules, and research on molecular mechanisms of antifungal resistance made by the scientific community, trends in invasive fungal diseases and associated antifungal resistance are on the rise, hindering therapeutic options and clinical cures. In this context, in vitro susceptibility testing aimed at evaluating minimum inhibitory concentrations, is still a milestone in the management of fungal diseases. However, such testing is not the only type at a microbiologist’s disposal. There are other adjunctive in vitro tests aimed at evaluating fungicidal activity of antifungal molecules and also exploring tolerance to antifungals. This plethora of in vitro tests are still left behind and performed only for research purposes, but their role in the context of invasive fungal diseases associated with antifungal resistance might add resourceful information to the clinical management of patients. The aim of this review was therefore to revise and explore all other in vitro tests that could be potentially implemented in current clinical practice in resistant and difficult-to-treat cases.

Abstract Invasive fungal infections are a leading global cause of human mortality. Only three major classes of antifungal drugs are widely used, and resistance to all three classes can arise rapidly. The most widely prescribed antifungal drug, fluconazole, disseminates rapidly and reaches a wide range of concentrations throughout the body. The impact of drug concentration on the spectrum and effect of mutations acquired during adaptation is not known for any fungal pathogen, and how the specific level of a given stress influences the distribution of beneficial mutations has been poorly explored in general. We evolved 144 lineages from three genetically distinct clinical isolates of Candida albicans to four concentrations of fluconazole (0, 1, 8, and 64 μg/ml) and performed comprehensive phenotypic and genomic comparisons of ancestral and evolved populations. Adaptation to different fluconazole concentrations resulted in distinct adaptive trajectories. In general, lineages evolved to drug concentrations close to their MIC50 (the level of drug that reduces growth by 50% in the ancestor) tended to rapidly evolve an increased MIC50 and acquired distinct segmental aneuploidies and copy number variations. By contrast, lineages evolved to drug concentrations above their ancestral MIC50 tended to acquire a different suite of mutational changes and increased in drug tolerance (the ability of a subpopulation of cells to grow slowly above their MIC50). This is the first evidence that different concentrations of drug can select for different genotypic and phenotypic outcomes in vitro and may explain observed in vivo drug response variation.

Lei Chen works in the field of clinical diagnosis of antifungal tolerance and persistence. In this mSphere of Influence, he reflects on how the papers \"Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemia\" by A. Rosenberg, I. V. Ene, M. Bibi, S. Zakin, et al. (Nat Commun 9:2470, 2018, https://doi.org/10.1038/s41467-018-04926-x) and \"Macrophage internalization creates a multidrug-tolerant fungal persister reservoir and facilitates the emergence of drug resistance\" by A. Arastehfar, F. Daneshnia, N. Cabrera, S. Penalva-Lopez, et al. (Nat Commun 14:1183, 2023, https://doi.org/10.1038/s41467-023-36882-6) impacted his work on the development of diagnostic methods for antifungal tolerance and persistence.

Infections caused by Cryptococcus neoformans cause more than 180,000 deaths annually. Estimated 1-year mortality for patients receiving care ranges from 20% in developed countries to 70% in developing countries, suggesting that current treatments are inadequate. Some fungal cells can persist and replicate despite the usage of current antifungal regimens, leading to death or treatment failure. Aging in fungi is associated with enhanced tolerance against antifungals and resistance to killing by host cells. This study shows that age-dependent increase in mitochondrial reactive oxygen species drive changes in the regulation of membrane transporters and ergosterol synthesis, ultimately leading to the heightened tolerance against fluconazole in old C. neoformans cells. Understanding the underlying molecular mechanisms of this age-associated antifungal tolerance will enable more targeted antifungal therapies for cryptococcal infections.