Amphotericin B (AmB) is a polyene antifungal that, despite its severe dose-limiting toxicity, is the most effective drug to treating invasive fungal infections. The drug's toxicity arises from its poor selectivity for ergosterol over cholesterol and its tendency to aggregate. Understanding the relationship between toxicity and aggregation is challenging due to the drug's complex mechanism of action, yet imperative to improve the safety profiles of existing and new polyene-based antifungal drugs. In this review, we critically examine and summarise studies that investigate the aggregation of AmB and its relationship to toxicity. As the aggregation behaviour of AmB is highly sensitive to environmental conditions, we provide an in-depth analysis of sample preparation, characterisation of aggregates and control experiments. While data from haemolysis, animal, and cell-based studies consistently show that oligomeric AmB is more toxic than larger aggregates, findings on the toxicity of monomeric AmB inconsistent. Our collated data underscores the need for careful consideration of sample preparation and proper use of vehicle controls when studying the complex relationship between the supramolecular organisation of AmB and its biological effects.

Nakaseomyces glabratus (formerly Candida glabrata) is a leading cause of invasive candidiasis and rapidly develops antifungal drug resistance during treatment. An increasing number of clinical isolates show reduced susceptibility to echinocandins and azoles, leaving amphotericin B (AMB) as a last therapeutic option. Resistance of N. glabratus to this drug is rare, and its underlying mechanisms are still not fully understood. Here, we describe two independent multidrug-resistant bloodstream isolates displaying resistance to AMB and anidulafungin (ANF), as well as a reduced susceptibility to azoles. We performed whole-genome sequencing and sterol profiling on nine clinical N. glabratus isolates, which were resistant to ANF and displayed resistance or low susceptibility to fluconazole (FLU) and AMB. We identified loss-of-function mutations in the genes ERG3 and ERG4, which could be linked to ergosterol depletion and AMB resistance. The transcriptional response of the reference strain CBS138 and an AMB[R] + ANF[R] isolate was analyzed by RNA-seq, revealing that ergosterol depletion also contributed to upregulation of ERG and ABC transporter genes, which might explain the low FLU susceptibility. Surprisingly, the AMB[R] isolates displayed severe fitness defects, and one of them was fully virulent in a Galleria mellonella infection model. Our results indicate that ergosterol depletion in N. glabratus leads to AMB resistance without affecting fitness or virulence.

The emergence of multidrug-resistant fungal pathogens from urinary tract infections (UTIs) poses a growing challenge in clinical settings. Here, we report a case of a complicated UTI caused by Nakaseomyces glabratus (Candida glabrata) that progressed to urosepsis, leading to the emergence of an isolate carrying simultaneous loss-of-function mutations in ERG3 and ERG11, and abrogated ergosterol biosynthesis. Together with a missense mutation in FUR1-likely responsible for 5-fluorocytosine resistance-this constellation confers resistance to all viable UTI antifungals: azoles, amphotericin B, and flucytosine. Engineered ERG3Δ + ERG11Δ strains recapitulated this multidrug resistance and revealed profound fitness costs that come with it, challenging the assumption that high-cost mutations are unlikely to persist during infection. Among fitness trade-offs, we detected collateral sensitivity to nitroxoline, a commonly used urinary tract antibiotic with potent antifungal activity and a unique mechanism of action. This study provides the first clinical evidence of an elusive mechanism of hyper-multidrug resistance in N. glabratus and highlights nitroxoline as a promising repurposing agent for treating multidrug-resistant fungal infections of the urinary tract.

Scedosporium apiospermum is an emerging cause of invasive mold infection in immunocompromised hosts, often with central nervous system involvement and limited susceptibility to amphotericin B. We describe a 36-year-old lung transplant recipient who presented with fever, meningismus, and multiple enhancing brain lesions nine months post-transplant. Cerebrospinal fluid studies, including metagenomic next-generation sequencing (mNGS), were negative. Cardiac imaging revealed a pedunculated right ventricular septal mass, and plasma cell-free DNA (cfDNA) testing (Karius) identified S. apiospermum. Subsequent brain biopsy and thrombectomy confirmed the diagnosis by histopathology and culture. Following surgical removal of the cardiac mass and treatment with voriconazole, the patient improved with near resolution of brain lesions. This case highlights disseminated S. apiospermum endocarditis diagnosed by plasma cfDNA despite negative CSF mNGS, underscoring that site-specific mNGS may be falsely negative in compartmentalized infections. Plasma cfDNA testing can complement conventional and tissue-based diagnostics for early detection of disseminated mold infections in transplant recipients.

BACKGROUND TALAROMYCES MARNEFFEI: (T. marneffei, formerly Penicillium marneffei) is a rare fatal fungus endemic in Southeast Asia and southern China. T. marneffei infections mainly occur in HIV-infected adults, and commonly involves the skin, lung, and reticuloendothelial system. T. marneffei infections of isolated central nervous system (CNS) in immunocompetent pediatric patients in nonendemic areas have rarely been reported. CASE PRESENTATION: We report a rare case of T. marneffei-induced disseminated encephalomyelitis in an immunocompetent girl from a nonendemic area of Eastern China. The main clinical manifestations were abdominal pain with distension and abnormal gait. Contrast-enhanced magnetic resonance imaging (MRI) revealed both brain and spinal cord lesions. The infection status of T. marneffei was quickly determined via the metagenomic next-generation sequencing (mNGS) of spinal cord biopsy tissue. T. marneffei induced disseminated encephalomyelitis was diagnosed. Following successful antifungal treatment with amphotericin B liposomes and voriconazole, the child recovered gradually. To date, only 3 cases of T. marneffei infection of the central nervous system in non-HIV-infected pediatric patients have been reported in the literature. Among them, one child had inborn errors of immunity, and the other two children were from endemic areas. Moreover, the clinical manifestations of those 3 reported cases were disseminated with common infection sites in the lungs. our patient represents a unique case of an immunocompetent child from a nonendemic area with isolated CNS infection. CONCLUSIONS: We report this rare case and aim to promote pediatric clinicians' recognition of T. marneffei isolated CNS infection in immunocompetent pediatric patients from nonendemic regions. Furthermore, the early use of mNGS is recommended when non-HIV-infected pediatric patients present with unexplained clinical manifestations and poor response to conventional treatments. Timely diagnosis and appropriate antifungal therapy can improve patient prognosis.

Antifungal drug resistance is a growing global health concern, yet treatment options remain limited to just four classes: polyenes, azoles, allylamines, and echinocandins. Cryptococcus species are responsible for life-threatening meningoencephalitis and pulmonary infections, which cause over 140,000 deaths annually. The high mortality of Cryptococcus is driven by limited drug access, toxicity, persistence, and emerging resistance. To better understand antifungal resistance and identify novel therapeutics, we profiled a genetically diverse panel of Cryptococcus isolates spanning all major lineages (VNI, VNIII, VNIV, VGI-VGVI). Phenotypic assays were used to assess susceptibility to fluconazole (FLZ), 5-fluorocytosine (5-FC), and amphotericin B (AmpB), as well as the epigenetic inhibitors CPTH2 (a histone acetyltransferase inhibitor) and SAHA (a histone deacetylase inhibitor). We found lineage- and strain-specific variation in antifungal susceptibility, with disc-diffusion and microdilution assays yielding consistent patterns. Pharmacodynamic modelling revealed potent fungicidal activity of CPTH2 across all tested lineages. DNA content and chitin levels varied significantly by lineage and treatment: FLZ and CPTH2 induced DNA content increases in susceptible strains, suggesting a stress response, while 5-FC-induced chitin remodelling was most pronounced in resistant strains. Multivariate analysis identified chitin induction as a principal driver of phenotypic variance under antifungal stress. Our findings reveal lineage-specific antifungal responses in Cryptococcus, highlight the antifungal potential of epigenetic inhibitors such as CPTH2, and underscore the utility of phenotypic profiling to inform drug development and resistance surveillance.

Mucormycosis has a poor prognosis in immunocompromised individuals. Rhino-orbital cerebral mucormycosis spreads directly from the nasal cavity to the paranasal sinuses and then to the adjacent organs, resulting in a wide range of symptoms and differential diagnoses. For patients with risk factors, it is important to consider this disease and proceed with early testing and diagnosis. Treatment involves three key elements: surgical debridement, administration of antifungal drugs, and control of the underlying disease. Amphotericin B liposomal preparations are the first choice of antifungal drugs. Antifungal drug administration is a long-term treatment.

CNS candidiasis typically occurs in patients with candidemia, especially immunocompromised hosts or patients with neurosurgical CNS devices. The gold standard for diagnosis is identification of Candida growth in CSF culture. For initial treatment, liposomal amphotericin B with or without flucytosine is recommended. If the isolate is susceptible to initial treatment, fluconazole is added. When possible, CNS devices should be removed. Autosomal recessive CARD9 deficiency is a primary immunodeficiency disorder associated with chronic mucocutaneous or invasive candidiasis, especially drug-resistant or recurrent candidal meningitis. In patients presenting with primary candidal meningitis in the absence of known immunodeficiency, CARD9 deficiency should be considered. A history of chronic mucocutaneous candidiasis provides an important diagnostic clue.

Fungemia caused by rare fungal species has been increasingly recognized in both immunocompromised and immunocompetent children. Our aim is to characterize the clinical and microbiological features of rare fungal bloodstream infections in hospitalized pediatric patients. A retrospective, descriptive study was conducted including all cases of fungemia due to rare yeasts in patients admitted to two tertiary pediatric hospitals between 2021 and 2025. Fungal species identification, anatomical sites of infection, antifungal therapeutic regimens, and clinical outcomes were systematically analyzed. In total, 13 cases of rare fungal bloodstream infections were identified among 13 patients from 2,555 blood cultures obtained during the study period, representing an incidence of 0.5%. The predominant species isolated were Saccharomyces cerevisiae (n=5), Wickerhamomyces anomalus (n=3), Candid orthopsilosis (n=2), Yarrowia lipolytica (n=1), Meyerozyma guilliermondii (n=1), and Trichosporon asahii (n=1). The median age at infection was 31 months (range: 0-178 months), with 92.3% (12/13) of patients presenting underlying medical conditions. The median interval from hospital admission to fungemia onset was 19 days (range: 0-73 days). Amphotericin B deoxycholate was the most frequently employed initial antifungal agent (n=5), followed by liposomal amphotericin B (n=4). Modification of antifungal therapy was required in five cases (38.5%). Microbiological clearance was achieved in all patients on subsequent blood cultures. The 30-day all-cause mortality rate was 15.4% (2/13). We concluded that rare fungal bloodstream infections occurred predominantly in patients with underlying comorbidities and prolonged hospital stays; nevertheless, the majority showed favorable clinical responses with appropriate antifungal therapy and achieved fungal eradication from the bloodstream.

Cryptococcus laurentii is an emerging opportunistic yeast pathogen associated with unpredictable antifungal susceptibility and biofilm-mediated resistance. This study investigated the therapeutic potential and synergistic interactions of standard antifungals (fluconazole, amphotericin B) and off-target drugs against clinical isolates of C. laurentii. Three clinical isolates recovered from blood (n = 2) and cerebrospinal fluid (n = 1) were confirmed by phenotypic characteristics and PCR amplification of the 18 S rRNA gene (~ 480 bp). Biofilm formation was screened using Congo red agar and quantified via crystal violet assay. Susceptibility testing employed CLSI-guided disc/well diffusion and broth microdilution methods for antifungals and 14 off-target drugs. Minimum biofilm inhibitory concentrations (MBICs) were determined, and synergistic effects were evaluated using checkerboard assays (fractional inhibitory concentration index, FICI) in planktonic cells and biofilms, supplemented by coverslip biofilm reduction assay. Isolates showed moderate biofilm production (mean OD595 = 0.32 ± 0.008), fluconazole susceptibility (mean MIC = 0.77 µg/mL), and amphotericin B resistance (mean MIC = 4.48 µg/mL). Diclofenac sodium exhibited the strongest off-target activity (mean MIC = 1.32 µg/mL). Checkerboard assays revealed synergy between fluconazole + amphotericin B (FICI = 0.5) and fluconazole + diclofenac sodium (FICI = 0.32) in planktonic cells, with additive effects against biofilms (FICI = 0.9). The fluconazole-diclofenac combination significantly disrupted biofilm matrix. Diclofenac sodium enhances fluconazole efficacy and disrupts biofilms, supporting drug repurposing as an adjuvant strategy for managing biofilm-associated C. laurentii infections.

Amphotericin B (AmB), the drug of choice for fungal keratitis, displays poor aqueous solubility, physiological instability, and pH-dependent aggregation causing ocular irritation. This study aimed to develop novel reconstitutable bioresponsive Nanocubosomal in situ gelling system (NCG) of AmB to address the above-mentioned limitations. Initially, 13 batches of Nanocubosomal Dispersion (NCD) were formulated using Quality by Design-driven central composite design with glyceryl monooleate as lipid, poloxamer 407 as surfactant, and dimethyl sulfoxide as solvent, employing high-shear homogenization and ultrasonication. The optimised NCD exhibited nanoscale size (158.9 ± 2.6 nm), low polydispersity (0.162 ± 0.007), high entrapment efficiency (91.2 ± 1.1%), and colloidal stability (zeta potential -32.8 ± 1.3 mV). Due to low pH-solubility (0.2 ± 0.03 µg/mL) at pH 7.4 and moderate lipophilicity (log P: 0.46 ± 0.01), AmB would get preferentially localised in lipid domain. The optimised batch of NCD was finally lyophilised with Carbopol 934 (0.3%w/v) and mannitol (5%w/v) before gamma sterilisation to obtain NCG. NCG demonstrated bioresponsive behaviour, maintaining low pre-instillation viscosity (12 ± 0.9 mPa.s) while instantly transforming into resilient pseudoplastic gel (22,230 ± 310 mPa.s) at ocular pH that is likely to prolong ocular retention. The in-vitro drug release from NCG at 24 h was found to be significantly lower compared to marketed lyophilised Amfocare® (p < 0.0001), indicating sustained release behaviour. NCG displayed significantly enhanced (p < 0.01) antifungal activity against Candida albicans compared to Amfocare®. Draize testing in rabbits ruled out possibility of ocular irritation, demonstrating improved tolerability of NCG. Overall, NCG, by its ability to maintain AmB in monomeric form, represents a promising, stable, effective, irritation-free in situ gelling lipid-based platform for treatment of fungal keratitis.