A key intrinsic resistance mechanism is the ineffective binding of antifungal drugs to targets. For example, variations in the structure of the enzyme sterol 14α-demethylases (SDMs) in certain fungi result in decreased binding affinity for azole antifungals, rendering these drugs ineffective (20). SDM is a key enzyme in fungal ergosterol biosynthesis. SDM catalyses the conversion of lanosterol or eburicol to a 14α-demethylated product (20). The binding of azole antifungals to SDM depletes ergosterol and decreases the integrity of the lipid bilayer of the fungal membrane (20). Certain amino acid (AA) substitutions in the ligand-binding pocket of SDMs decrease the binding of azole antifungals. For example, two natural AA substitutions, F129 and V291, in Mucormycota SDMs cause intrinsic resistance to short-tail azoles such as fluconazole and voriconazole (21). The T301I substitution in the SDM of A. fumigatus causes intrinsic resistance to fluconazole (22).

Efflux pump activity is a key mechanism of intrinsic resistance. Drugs need to reach a certain level in their cellular targets. Intrinsically resistant fungi exhibit high efflux pump activity, which actively expels antifungal agents from the cell, decreasing their intracellular concentrations and diminishing their efficacy (23). A common efflux system in fungi involves the ATP-binding cassette (ABC) superfamily and major facilitator superfamily (MFS) (23). Fungi express multiple ABCs that are key to their physiology, particularly for the expulsion of toxic metabolites. For example, N. glabratus has 18 ABC-encoding genomes and A. fumigatus has 50 genomes (24,25). In C. auris, four efflux pump encoding genes, Subsequent to Nucleotide Q21 (SNQ21), Subsequent to Nucleotide Q22 (SNQ22), Multidrug Resistance 1 (MDR1) and Candida Drug Resistance 1 (CDR1), contribute to fluconazole resistance (26).

Fungal cell wall and membrane composition serve key roles in intrinsic resistance. For example, variations in sterol content affect the binding and efficacy of polyene antifungals such as amphotericin B. Environmental molds such as those from Mucoromycota and Lomentospora spp. have unique cell wall structures that limit the penetration and action of azole antifungals, thereby contributing to their intrinsic resistance (21). Mucoromycota phyla such as Rhizopus and Mucor species have alternative sterol synthesis pathways (21). Duplication of the gene encoding FK506-sensitivity gene (FKS) synthetase, which synthesizes the cell wall component (1,3)-d-glucan, is responsible for the intrinsic resistance of Mucor circinelloides to micafungin (27). Fusarium species also exhibit resistance to several antifungal agents, including azoles, because of their unique cell wall structures, which impede drug penetration, in addition to their robust efflux pump systems (28).

The intrinsic resistance of fungal species highlights the need to develop novel antifungal agents that bypass the resistance mechanisms inherent to these fungi. Additionally, the accurate and timely identification of intrinsically resistant fungal species is key for effective treatment, emphasizing the need for advanced diagnostic tools to identify resistance profiles. Some therapeutic strategies can be employed using alternative drugs or combining them with efflux pump inhibitors or other antifungals that target cell walls.

Point mutations are a key genetic mechanism through which fungi acquire resistance to antifungal agents. Point mutations occur at a rate of 1×10⁻⁶−1×10⁻⁸/cell/generation and involve changes in a single nucleotide base pair within the DNA sequence (41,42). Such alterations lead to modifications in the structure or function of the target protein, thereby diminishing the efficacy of antifungal drugs. Point mutations have clinical implications as they often result in resistance to commonly used antifungal agents, complicating the management of fungal infection.

Point mutations can result in changes in the genetic code, including missense, nonsense and silent mutations. Missense mutations, which result in the substitution of one AA with another in the protein product, alter the structure of the target enzyme or protein, thereby affecting its interaction with antifungal drugs (43,44). Nonsense mutations introduce a premature stop codon into the DNA sequence, producing a truncated and often non-functional protein (43,44). Although silent mutations do not change the AA sequence of a protein, they affect protein folding or stability, potentially influencing drug susceptibility (43,44).

Point mutations confer resistance to antifungal agents in clinically relevant fungal species. One example is the ERG11 gene in C. albicans (Fig. 1A). The ERG11 gene encodes 14α-SDM, an enzyme targeted by azole antifungals such as fluconazole (45). Point mutations in ERG11 lead to AA substitutions that reduce its binding affinity for azole drugs, thereby conferring drug resistance (45). For example, the Y132H mutation in ERG11, which changes a tyrosine to a histidine at position 132, alters the structure of the enzyme and decreases drug binding, resulting in fluconazole resistance (45). Other point mutations in the ERG11 gene of C. albicans (including K143Q, Y205E, A255V, E260V, N435V, G472R and D502E) decrease the ability of fluconazole and voriconazole to inhibit the drug target (45).

Similarly, mutations in the FKS genes, which encode subunits of β-1,3-D-glucan synthase, lead to decreased susceptibility to echinocandin antifungals. In N. glabratus, the S629P mutation in FKS2, in which serine is replaced by proline at position 629, affects the interaction of the enzyme with the drug, thus conferring resistance to echinocandins (46). In A. fumigatus, mutations, such as S678P, in FKS1 confer resistance by altering the drug-binding site of the enzyme (47). In C. albicans, mutations in the gene encoding FKS synthetase, the target of echinocandins, decrease susceptibility to these antifungal agents (48). Another example is the point mutation in the CYP51A gene of A. fumigatus. Point mutations in CYP51A lead to azole resistance, particularly to itraconazole (49). The G54E mutation, which changes glycine to glutamic acid at position 54, markedly decreases the efficacy of itraconazole and other azoles by altering enzyme structure (49).

Among resistance mechanisms, point mutations that target genes, particularly ERG11 and FKS1/FKS2, are the most clinically relevant. These mutations directly impair drug-target binding, resulting in resistance (46,47). Their genomic stability ensures resistance persists in the absence of antifungal pressure. Clinically, such mutations are among the most frequently observed in treatment-refractory infection and are often associated with elevated MICs that render standard antifungal regimens ineffective (45–48,50,51). Consequently, infection caused by fungi harbouring these mutations is harder to treat and requires alternative or combination therapies, highlighting the serious clinical impact.

Gene duplication and transposon insertion are key genetic mechanisms that contribute to antifungal resistance. These alterations lead to the increased expression of resistance genes or disruption of regulatory pathways that control drug susceptibility, thereby enhancing the ability of fungi to survive in the presence of antifungal agents. These mechanisms occur at a relatively high frequency of 1×10⁻³−1×10⁻⁴/cell/generation, making them notable drivers of resistance in fungal populations (41,42).

Gene duplication involves the creation of one or more copies of a gene within the genome, leading to the upregulation of genes that confer resistance to antifungal agents. Increased gene expression can enhance the production of proteins that degrade the antifungal drug, modify the drug target or expel the drug from the cell, thereby decreasing its efficacy. A notable example of gene duplication is the MDR1 gene in C. albicans and C. dubliniensis (52,53). The MDR1 gene encodes the MFS transporter involved in drug efflux. Duplication of the MDR1 gene leads to the upregulation of the efflux pump, increasing the expulsion of azole antifungals such as fluconazole from the cell, thereby conferring resistance (52,53). Clinical isolates of C. albicans with duplicated MDR1 genes exhibit high levels of resistance to fluconazole, the most commonly used antifungal (52,53). Similarly, duplication of the ERG11 gene in N. glabratus (Fig. 1B), which encodes 14α-SDM, results in overproduction of the enzyme and is associated with resistance to multiple azole antifungals, making infections difficult to treat (54). An increased amount of the target enzyme can sequester the drug, decreasing its inhibitory effect and leading to drug resistance.

Transposon insertions involve the movement of transposable elements or ‘jumping genes’, within the genome. These elements insert themselves into various genomic locations, disrupting genes or regulatory regions and leading to altered gene expression. Transposon insertions can enhance the expression of resistance genes or disrupt the genes involved in susceptibility to antifungal agents.

The clinical and public health importance of transposon insertions in antifungal resistance has been observed in C. neoformans, which causes 15% of HIV-associated mortalities worldwide (55). The standard treatment for C. neoformans combines rapamycin and FK506 (Fig. 1C) (56). Sequencing of drug target genes revealed C. neoformans LINE-1-like element (CNL1) transposon insertions in an isolate of C. neoformans are resistant to rapamycin and FK506 (56). Cnl1 transposon insertion can cause resistance to 5-flucytosine, which is the standard therapy for cryptococcal meningitis and encephalitis (56).

Aneuploidies, defined as the gain or loss of entire chromosomes or large chromosomal segments, are key contributors to antifungal resistance due to their ability to cause widespread changes in gene expression (57,58). Unlike point mutations or gene duplication, these large-scale genomic alterations can upregulate resistance genes or disrupt cell pathways, thereby enhancing fungal survival under drug pressure. Fungi exhibit high genomic plasticity, making aneuploidy a relatively frequent adaptive mechanism. It arises from errors in chromosomal segregation during cell division and typically results in increased expression of drug targets, efflux pumps or other resistance-associated genes (59). This enables rapid and reversible resistance during antifungal treatment.

One of the most studied examples of aneuploidy in antifungal resistance involves C. albicans (60–66). In C. albicans, fluconazole resistance is frequently associated with the formation of an isochromosome consisting of the left arm of chromosome 5, which contains the ERG11 gene (target of fluconazole) and transcriptional activator of efflux pump CDR gene 1 (TAC1) (60–62). This chromosomal alteration increases resistance by enhancing drug target abundance and efflux activity. Similarly, in C. neoformans, duplication of chromosome 1 is associated with fluconazole resistance through upregulation of ERG11 and azole fungal resistance 1 (AFR1), particularly under antifungal stress conditions (Fig. 1D) (67,68).

Aneuploidy in fungal pathogens has notable clinical implications, as it enables rapid gene expression changes that confer drug resistance and complicate treatment. This can lead to treatment failure, prolonged infection and the need for combination or novel antifungal therapy (69). Rapid acquisition of aneuploidy under antifungal pressure allows fungi to adapt quickly, increasing resistance risk during therapy. Therefore, advanced diagnostic techniques, such as karyotyping, comparative genomic hybridization and next-generation sequencing, are key for detecting aneuploidy, guiding treatment and monitoring resistance emergence.

Aneuploidy also helps fungi to mitigate decreased growth and survival due to the acquisition of antifungal resistance. Resistant strains frequently exhibit slower proliferation rates compared with wild-type strains under drug-free conditions. This has been observed, for example, in C. albicans with ERG11 mutations, where the resistant strains demonstrate decreased proliferation rates and virulence in the absence of azole or echinocandin exposure (70). However, in certain cases, compensatory mutations or genomic adaptations through aneuploidy can partially or fully restore fitness, allowing resistant strains to proliferate at rates comparable with wild-type strains even without antifungal agents (63,65). For example, C. albicans strains exhibit distinct transcriptional profiles that lead to altered proliferation and oxidative balance relative to euploid strains (65). Acquisition of aneuploidy in C. albicans can lead to increased fitness during the evolution of antifungal drug resistance. Specifically, the formation of an isochromosome 5L, which amplifies the ERG11 and TAC1 genes, confers fluconazole resistance while also enhancing proliferation rates in the absence of the drug (63).

LOH is a key genetic mechanism underlying antifungal resistance. LOH refers to mitotic recombination in which one allele at a specific locus is lost, converting heterozygous into homozygous loci (71). Compared with other resistance mechanisms, LOH may occur at higher frequencies under antifungal pressure, particularly in diploid fungi, facilitating rapid adaptation by unmasking recessive resistance-conferring mutations (72). The baseline rates of LOH is 1×10⁻⁶−1×10⁻⁷/cell division in C. albicans, but these rates can increase ≥285-fold, under antifungal stress such as fluconazole exposure, reaching frequencies of 1×10⁻³−1×10⁻⁴ (72). These elevated rates are markedly higher compared with those of point mutations (~1×10⁻⁹) and suggest that LOH is a key driver of rapid adaptation and antifungal resistance in diploid fungal pathogens (72). This genetic alteration can unmask the recessive mutations that confer resistance and enhance the ability of an organism to survive in the presence of antifungal agents. LOH can occur through numerous mechanisms, including mitotic recombination, gene conversion and chromosomal deletion (71). This process is particularly relevant in diploid organisms where heterozygous loci are common.

In diploid fungi, LOH can lead to the unmasking of resistance-conferring mutations that were hidden by a dominant wild-type allele. When a diploid organism loses one copy of a gene, the remaining allele is expressed (73). An organism exhibits a resistant phenotype if the retained allele carries a resistance mutation (73). This allows fungi to adapt quickly to antifungal pressures through advantageous mutations.

An example of the effect of LOH on antifungal resistance is the MDR1 gene in C. albicans (Fig. 1E). The MDR1 gene encodes an MFS transporter involved in drug efflux (74). LOH can lead to the loss of the wild-type allele and retention of a mutant MDR1 allele, resulting in overexpression of the efflux pump and enhanced resistance to azole antifungals (74). Similarly, the LOH in the Δ5,6-desaturase (ERG3P) gene in C. albicans, involved in the ergosterol biosynthesis pathway, causes resistance to amphotericin B (75). Mutations in ERG3 alter the cell membrane composition and LOH can unmask these mutations, leading to decreased susceptibility to amphotericin B and posing treatment challenges (75). Another example is the LOH-mating-type-like (MTL) locus, which causes resistance to azole antifungal agents (76). Single nucleotide polymorphism (SNP) analysis in azole-resistant C. albicans isolates revealed that replacing an MTL locus with a duplicate of the other locus results in two homozygous copies of the MTL locus (76).

LOH of fungal pathogens has clinical implications. LOH can lead to rapid and substantial changes in gene expression and unmasking of resistance mutations that complicate treatment strategies. Advanced diagnostic tools such as comparative genomic hybridization and next-generation sequencing are key for identifying LOH events in clinical isolates, guiding appropriate antifungal therapy and monitoring the emergence of resistance. SNP and Southern blot analyses can also be used to detect common LOH.

Hypermutator lineages are a notable genetic mechanism contributing to antifungal resistance. Elevated mutation rates characterize these lineages due to defects in DNA repair mechanisms, particularly those involved in mismatch repair (MMR) (77). The increased mutation frequency enhances the genetic diversity within a fungal population, providing a larger pool of mutations from which resistance-conferring changes can arise (77). This mechanism is particularly concerning in clinical settings, as it can lead to the rapid development of resistance to multiple antifungal agents, complicating treatment.

One of the well-documented examples of the hypermutator lineage involves N. glabratus (Fig. 1F) (78–81). Mutations in MutS Homolog 2 (MSH2) gene, a key component of the MMR system, lead to hypermutator phenotypes (78,79). The MSH2 gene is responsible for recognizing and initiating the repair of mismatched nucleotides during DNA replication (78,79). Clinical isolates of N. glabratus with MSH2 mutations exhibit elevated mutation rates that rapidly lead to resistance to multiple antifungal agents, including azoles, echinocandins and polyenes (78,79). These hypermutator strains are challenging to treat because of their ability to quickly adapt to antifungal pressures.

In C. neoformans, hypermutator lineages are associated with mutations in the MSH2 and MutL Homolog 1 genes, which are involved in the MMR pathway. These mutations result in defective repair systems and increased genetic variability (56,82). Hypermutator strains of C. neoformans develop resistance to antifungal agents, such as fluconazole and amphotericin B, more rapidly than non-hypermutator strains (56,82). This rapid adaptation poses challenges to the management of cryptococcal infection, particularly in immunocompromised patients.

Although less commonly reported than C. neoformans and N. glabratus, hypermutator phenotypes have also been observed in A. fumigatus (77,79,83). Mutations in the DNA repair gene MSH lead to increased mutation rates and the rapid emergence of resistance (83). A study of environmental and clinical isolates of A. fumigatus revealed that hypermutator strains quickly develop resistance to azole antifungals, which are commonly used to treat aspergillosis (83). Additionally, genetic instability caused by MSH mutations increases virulence (83). The presence of these strains in clinical settings underscores the need for monitoring and development of alternative therapeutic strategies.

The emergence of hypermutator lineages of fungal pathogens has key clinical implications. These strains rapidly develop resistance to multiple antifungal agents, making infection more difficult to treat and increasing the risk of treatment failure (56,78,79,82,83). The elevated mutation rates in hypermutator lineages lead to a high degree of genetic variability, which can result in the selection of resistance-conferring mutations under antifungal pressure (56,78,79,82,83).

Phenotypical heterogeneity in fungi arises through numerous mechanisms. A notable example is biofilm formation. Fungal cells in biofilms exist in a sessile, multicellular state and produce an extracellular matrix that protects them from antifungal agents (85,86). This matrix serves as a physical barrier that reduces the effective concentration of the drug reaching fungal cells within the biofilm (85,86). Biofilms are problematic in clinical settings because they are associated with chronic infection and are difficult to eradicate (85,86). Species such as C. albicans can form biofilms on medical devices such as catheters and prosthetic valves, leading to persistent infection (85,86).

Another mechanism underlying phenotypical heterogeneity involves persister cells. Within a fungal population, a small fraction of cells enter a dormant state, becoming persister cells (10,84). These cells are highly tolerant of antifungal treatment because their reduced metabolic activity makes them less susceptible to drugs targeting active cell processes (10,84). Persister cells do not carry genetic mutations that confer resistance and their tolerance arises from a reversible phenotype (10,84). This makes them challenging as they can survive treatment and later repopulate once the antifungal pressure is removed. Persisters have been extensively studied in Candida spp. and Cryptococcus spp (84,87,88,91). Studies have reported the occurrence of persistent C. neoformans in clinical isolates from patients with cryptococcal meningitis and pneumonia treated with amphotericin B, a standard treatment for cryptococcosis (87,88).

Fungi activate stress response pathways that alter their phenotype and increase tolerance to antifungal agents. Adaptive responses to stress in human hosts are key for pathogen survival and virulence. For example, the heat shock response lead to the upregulation of chaperone proteins that help the cell cope with the damage caused by antifungal drugs (89). Another example is A. fumigatus, when they are exposed to reactive oxygen species (ROS) produced by immune cells such as macrophages and neutrophils. Analysis of the transcriptomes of A. fumigatus revealed that ROS exposure induces the expression of catalases and superoxide dismutases for survival in an oxidative environment (92). In C. albicans, the activation of the calcineurin pathway in response to azole treatment increases tolerance to the drug, allowing cells to survive otherwise lethal conditions (90).

Histones are proteins around which DNA is wrapped, forming chromatin. Post-translational histone modification, such as acetylation, methylation, phosphorylation and ubiquitination, influences chromatin structure and gene expression (17,93). Histone acetylation, typically associated with gene activation, is catalysed by histone acetyltransferases and reversed by HDACs (17,93). HDAC inhibitors alter the antifungal resistance by altering the expression of resistance genes.

Histone modifications are among the most studied epigenetic mechanisms in fungi and are linked to antifungal drug resistance, biofilm formation and virulence regulation (94–99). There are seven genes of HDAC in C. neoformans, two of which serve key roles in resistance and virulence. Histone Deacetylase 1 (HDA1) and HDAC genes are required for the survival of C. neoformans in a cultured macrophage model and a mouse model of infection (94,95). Fluconazole works synergistically with HDAC inhibitors in an in vitro infection model of C. neoformans (96). This highlights the fact that HDAC genes modulate the survival and susceptibility of C. neoformans to the antifungal agent.

In C. albicans, three classes of HDAC have been identified: class I, which include proteins similar to yeast Reduced Potassium Dependency 3 (RPD3); class II which consist of proteins related to HDA1; and class III which are NAD⁺-dependent. SET domain protein 3 (SET3) and Histone Deacetylase of the Rpd3S Complex 2 (HOS2) are HDAC genes that are clinically important in C. albicans, particularly for promoting biofilm formation (97). The deletion of SET3 and HOS2 decreases biofilm formation by C. albicans (97). SET3C complex interacts with master regulator genes of biofilms, such as Negative Regulator Gene 1 (NRG1), Biofilm Regulator Gene 1 (BRG1), TEA/ATTS domain-containing transcription factor 1 (TEC1), Non-Disjunction and Telomere Defect 80 (NDT80) and Regulator of Biofilm 1 (ROB1) (98). HDA1 and RPD3 increase during the early phase of azole resistance in C. albicans and decrease during the later phase (99). HDAC inhibitors increase the sensitivity of C. albicans to azole antifungals by altering the expression of stress response and drug resistance genes (99).

Although less characterized in fungi compared with higher eukaryotes, DNA methylation serves a key role in transcriptional repression of antifungal target genes and may contribute to stable yet reversible drug resistance. DNA methylation involves the addition of a methyl group to cytosine residues in DNA, typically leading to gene suppression. Although DNA methylation has been well studied in higher eukaryotes, its role in fungi is less clear (100–103). DNA methylation affects the expression of the genes involved in antifungal resistance (93). Changes in methylation patterns can lead to the silencing or activation of genes that help the fungus survive in the presence of antifungal drugs (93).

In C. neoformans, DNA methylation patterns are associated with the resistance to antifungal agents. The DNA methylation in C. neoformans is mediated by the maintenance DNA methyltransferase Dnmt5, which catalyses the deposition of 5-methylcytosine primarily at centromeric and repetitive regions (104). Although this organism lacks a canonical de novo methyltransferase, Dnmt5 maintains heritable methylation patterns across generations (104). Disruption of Dnmt5 causes global hypomethylation, chromosomal instability and altered expression of stress-responsive genes (104). These epigenetic changes may indirectly modulate the expression of genes associated with antifungal resistance, including those encoding efflux pumps and cell wall remodelling proteins. While the spectrum of fungal DNA methyltransferases remains to be characterized, emerging evidence suggests that epigenetic silencing via methylation may contribute to reversible antifungal tolerance (93,104,105).

Chromatin remodelling allows dynamic control of gene expression in response to antifungal stress, enabling fungi to adjust resistance pathways without permanent genetic mutations. Chromatin remodelling complexes alter the structure of chromatin, making certain regions of the genome more or less accessible to transcription factors and other regulatory proteins. These complexes facilitate the expression of resistance genes by repositioning nucleosomes and exposing regulatory regions of the DNA (17,93). In C. albicans, the chromatin remodelling Switch/sucrose non-fermenting complex 5 (SNF5) subunit is key for tolerance to azoles and amphotericin B under hypoxic stress (106). A study revealed that hypoxia potentiates the antifungal effects of azoles and amphotericin B in C. albicans (106). Remodelling complexes reposition nucleosomes to expose or hide regulatory regions, thereby influencing the expression of resistance genes.

Identification of antifungal resistance driven by genetic mechanisms presents diagnostic challenges. The diversity of genetic alterations requires comprehensive and precise diagnostic tools. Traditional methods such as culture-based assays and susceptibility testing may not detect specific genetic changes that confer resistance. Advanced molecular techniques, such as PCR and next-generation and whole-genome sequencing, are essential for detecting specific genetic mutations and alterations (107,108). Techniques such as chromatin immunoprecipitation and bisulfite sequencing are necessary to identify epigenetic changes such as histone modification and DNA methylation patterns that contribute to resistance (109,110). However, these advanced diagnostic tools are expensive and not readily available in all clinical settings, particularly in resource-limited regions. Ensuring accessibility and affordability of these technologies is key for their widespread implementation. Additionally, the time required to perform and analyze advanced diagnostic tests can delay the initiation of appropriate antifungal therapy, potentially worsening patient outcomes. Therefore, rapid diagnostic methods are required to identify resistance mechanisms.

Development of novel antifungal drugs is challenging due to several interrelated scientific, clinical and economic barriers. Fungi are eukaryotic organisms, sharing key processes with human cells. This limits the availability of pathogen-specific targets and raises the risk of host toxicity (111). Clinically, antifungal drug development must address diverse fungal species, several of which form biofilms, reside in immune-privileged or poorly perfused sites (such as the central nervous system and eyes) and demonstrate intrinsic or acquired resistance to existing therapies (112). Economically, antifungal infections represent a smaller market share than bacterial or viral disease, resulting in lower commercial incentive for pharmaceutical investment (113). In addition, the regulatory pathways for antifungal agents are complex and complicated by the need for robust efficacy data in severely ill and immunocompromised populations, where trial enrolment and design are difficult.

Genetic and epigenetic resistance mechanisms complicate treatment of fungal infection. Fungi with hypermutator phenotypes or multiple resistance mechanisms may exhibit resistance to several classes of antifungal drug, thereby limiting the treatment options. This necessitates the use of combination therapy or novel antifungal agents to manage infections effectively. Moreover, epigenetic changes and phenotypical heterogeneity can lead to dynamic and reversible resistance, thereby complicating treatment strategies (17,93). Fungal cells may revert to a resistant state following an initial response to therapy, leading to the recurrence of infection. Higher doses of antifungal agents or prolonged treatment required to overcome resistance can increase the risk of toxicity and adverse side effects (114,115). Balancing efficacy and safety is a key consideration in antifungal therapy.

Numerous strategies can be used to address the clinical challenges posed by genetic and epigenetic resistance mechanisms. Combinations of antifungal agents with different mechanisms of action can help overcome resistance by simultaneously targeting multiple pathways (116–118). For example, combining azoles with echinocandins or vorinostats may be more effective against biofilm-associated infections than azoles alone (119,120). Continued research and development of antifungal drugs with novel targets are essential to expand the arsenal against resistant fungal strains. Agents that bypass existing resistance mechanisms or target epigenetic modifications hold promise as effective treatments. Developing drugs that modulate epigenetic changes, such as histone deacetylase or DNA methylation inhibitors, can sensitize resistant fungal cells to existing antifungal agents. These approaches offer novel avenues for overcoming resistance.

Numerous strategies are under investigation to address antifungal resistance. Combination therapy (such as azoles with echinocandins or HDAC inhibitors) can enhance efficacy and delay resistance emergence (121). Antifungal classes such as orotomides (for example, olorofim) and glycosylphosphatidylinositol (GPI)-anchor pathway inhibitors (such as fosmanogepix) have demonstrated activity against resistant strains in preclinical and clinical trials (122–124). Adjunctive immunotherapy, including IFN-γ and monoclonal antibodies, offers potential benefit in immunocompromised hosts (125). Antifungal stewardship programs also serve a key role in optimizing antifungal use and minimizing resistance pressure (126).

The identification of novel antifungal drug candidates is achieved through numerous complementary approaches. High-throughput screening of large chemical libraries has led to the discovery of olorofim, a dihydroorotate dehydrogenase inhibitor developed by screening agents targeting fungal pyrimidine biosynthesis, and is in phase III trials against invasive A. fumigatus infections (124,127). Drug repurposing has resulted in identifying sertraline (an antidepressant) with activity against C. neoformans, and tamoxifen, which targets calmodulin in Cryptococcus spp (128,129). In silico modelling and structure-based design have facilitated the development of manogepix (APX001A), which targets GPI-anchored wall transfer protein 1 (GWT1) and has demonstrated broad-spectrum antifungal activity (130). Natural product screening identified ibrexafungerp (SCY-078), a semi-synthetic derivative of enfumafungin, a triterpenoid glycoside (131). Ibrexafungerp inhibits β-1,3-glucan synthase and has been approved for vulvovaginal candidiasis by the U.S. Food and Drug Administration (FDA) in 2021 (131,132). Furthermore, structure-guided drug design based on fungal enzyme crystal structures has supported the optimization of such agents for potency and selectivity (131).

Improving the delivery of antifungal agents to the infection site can increase drug efficacy and decrease resistance. Strategies such as liposomal formulations, nanoparticles and localized delivery systems have been explored to enhance drug delivery (133,134). Developing rapid and accurate diagnostic tools is key for the timely identification of resistance mechanisms and guiding appropriate therapy. Point-of-care diagnostics and streamlined genomic assays help clinicians quickly adapt treatment strategies. By understanding and targeting the underlying mechanisms of resistance, clinicians may improve patient outcomes and effectively manage resistant fungal infection.

To overcome the limitations of conventional antifungal therapy, novel drug delivery systems have been developed to enhance targeted delivery and therapeutic efficacy. Liposomal formulations, such as liposomal amphotericin B, encapsulate antifungal agents within lipid bilayers, enhancing drug solubility, decreasing toxicity and enabling targeted delivery (133,135). A randomized controlled trial in 2022, conducted within resource-limited settings in Africa (Botswana, Malawi, South Africa, Uganda, and Zimbabwe) demonstrated that a single high-dose (10 mg/kg) liposomal amphotericin B regimen, combined with flucytosine and fluconazole, was non-inferior to the conventional 7-day amphotericin B regimen in treating HIV-associated cryptococcal meningitis (136). The simplified regimen markedly decreased adverse events, including nephrotoxicity and anaemia, while maintaining comparable survival outcomes (136). This highlights the clinical efficacy and improved safety profile of liposomal formulations in resource-limited settings, where the use of simplified regimen may decrease the treatment cost.

Nanoparticles can be engineered to respond to stimuli such as pH, temperature or specific enzymes, facilitating drug release at the site of infection, especially where biofilm is involved (134,137). A pre-clinical study demonstrated that ~100 nm diameter liposomal nanoparticles loaded with anidulafungin enhance the solubility and antifungal targeting (138). The formulations were tested in vitro against C. albicans, showing equivalent MICs to free drug for planktonic cells and superior efficacy in disrupting biofilms, decreasing fungal burden by ≥99% (138). In vivo evaluation using a Galleria mellonella infection model demonstrated notably improved survival rates (33–67%) in larvae treated with liposomal formulations compared with 25% in the free drug group, without causing haemolysis, indicating both efficacy and safety (138). A randomized clinical trial evaluated a topical gel containing fluconazole-loaded solid lipid nanoparticles (SLNs) for the treatment of pityriasis versicolor (139). The SLN-based gel demonstrated considerably increased clinical and mycological cure rates compared with conventional fluconazole gel, with enhanced drug retention and patient compliance (139). These findings support the clinical utility of nanoparticle-based topical delivery systems in improving antifungal treatment outcomes.

Localized delivery systems, including hydrogels and coated catheters, enable sustained and concentrated antifungal release at the infection site, particularly for biofilm- or device-related infections (140). In a comparative in vitro biofilm colonization model, central venous catheters (CVCs) and peripherally inserted central catheters impregnated with a novel chlorhexidine-minocycline-rifampin (CHX-M/R) combination were evaluated against resistant bacteria (methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, Pseudomonas aeruginosa) and fungi (C. albicans, N. glabratus) (140). The CHX-M/R-coated catheters completely inhibited biofilm colonization by all tested pathogens at all time points and revealed considerably superior efficacy compared with first-generation M/R catheters, CHX-silver sulfadiazine-treated CVC and uncoated controls (140). The aforementioned studies support the potential of CHX-M/R-coated catheters for sustained, broad-spectrum antimicrobial and antifungal protection, particularly in preventing biofilm-associated device infection.

Overall, the clinical implications of antifungal resistance driven by genetic and epigenetic mechanisms present notable diagnostic and therapeutic challenges. Addressing these challenges requires a multifaceted approach, including the development of advanced diagnostics, novel antifungal agents, combination therapy and strategies targeting epigenetic modifications.