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Candida auris, an emerging fungal pathogen characterized by multidrug resistance and high-mortality nosocomial infections, poses a serious global health threat. However, the precise and rapid identification and characterization of C. auris remain a challenge. Here, we employed Raman spectroscopy combined with machine learning to identify C. auris isolates and its closely related species as well as to predict antifungal resistance and key virulence factors at the single-cell level. The average accuracy of identification among all Candida species was 93.33%, with an accuracy of 98% for the clinically simulated samples. The drug susceptibility of C. auris to fluconazole and amphotericin B was 99% and 94%, respectively. Furthermore, the phenotypic prediction of C. auris yielded an accuracy of 100% for aggregating cells and 97% for filamentous cells. This proof-of-concept methodology not only precisely identifies C. auris at the clade-specific level but also rapidly predicts the antifungal resistance and biological characteristics, promising a valuable medical diagnostic tool to combat this multidrug-resistant pathogen in the future., Importance: Currently, combating Candida auris infections and transmission is challenging due to the lack of efficient identification and characterization methods for this species. To address these challenges, our study presents a novel approach that utilizes Raman spectroscopy and artificial intelligence to achieve precise identification and characterization of C. auris at the singe-cell level. It can accurately identify a single cell from the four C. auris clades. Additionally, we developed machine learning models designed to detect antifungal resistance in C. auris cells and differentiate between two distinct phenotypes based on the single-cell Raman spectrum. We also constructed prediction models for detecting aggregating and filamentous cells in C. auris , both of which are closely linked to its virulence. These results underscore the merits of Raman spectroscopy in the identification and characterization of C. auris , promising improved outcomes in the battle against C. auris infections and transmission., Competing Interests: The authors declare no conflict of interest.

Amphotericin B (AmpB) is an effective but toxic antifungal drug. Thus, improving its activity/toxicity relationship is of interest. AmpB disrupts fungal membranes by two proposed mechanisms: ergosterol sequestration from the membrane and pore formation. Whether these two mechanisms operate in conjunction and how they could be potentiated remains to be fully understood. Here, we report that gladiolin, a polyketide antibiotic produced by Burkholderia gladioli , is a strong potentiator of AmpB and acts synergistically against Cryptococcus and Candida species, including drug-resistant C. auris . Gladiolin also synergizes with AmpB against drug-resistant fungal biofilms, while exerting no mammalian cytotoxicity. To explain the mechanism of synergy, we show that gladiolin interacts with membranes via a previously unreported binding mode for polyketides. Moreover, gladiolin modulates lipid binding by AmpB and, in combination, causes faster and more pronounced lipid rearrangements relative to AmpB alone which include membrane thinning consistent with ergosterol extraction, areas of thickening, pore formation, and increased membrane destruction. These biophysical data provide evidence of a functional interaction between gladiolin and AmpB at the membrane interface. The data further indicate that the two proposed AmpB mechanisms (ergosterol sequestration and pore formation) act in conjunction to disrupt membranes, and that gladiolin synergizes by enhancing both mechanisms. Collectively, our findings shed light on AmpB's mechanism of action and characterize gladiolin as an AmpB potentiator, showing an antifungal mechanism distinct from its proposed antibiotic activity. We shed light on the synergistic mechanism at the membrane, and provide insights into potentiation strategies to improve AmpB's activity/toxicity relationship., Importance: Amphotericin B (AmpB) is one of the oldest antifungal drugs in clinical use. It is an effective therapeutic, but it comes with toxicity issues due to the similarities between its fungal target (the membrane lipid ergosterol) and its mammalian counterpart (cholesterol). One strategy to improve its activity/toxicity relationship is by combinatorial therapy with potentiators, which would enable a lower therapeutic dose of AmpB. Here, we report on the discovery of the antibiotic gladiolin as a potentiator of AmpB against several priority human fungal pathogens and fungal biofilms, with no increased toxicity against mammalian cells. We show that gladiolin potentiates AmpB by increasing and accelerating membrane damage. Our findings also provide insights into the on-going debate about the mechanism of action of AmpB by indicating that both proposed mechanisms, extraction of ergosterol from membranes and pore formation, are potentiated by gladiolin., Competing Interests: G.L.C. is a shareholder, non-executive director, and consultant of Erebagen Ltd. The other authors declare no competing interests.

The Candida parapsilosis species complex poses a recognized threat to the nosocomial environment. In the scenario of the global rise of resistant strains to antifungals, geraniol, a terpene isolated from different essential oils, has shown promising antimicrobial activity. We evaluated: (1) the effects of geraniol against the C. parapsilosis species complex, in planktonic and biofilm forms; (2) the strains' susceptibility to clinical antifungals and (3) the geraniol interaction with antifungals. Eighteen isolates were subjected to in vitro susceptibility testing by the broth microdilution protocol, using geraniol, amphotericin B, caspofungin, itraconazole and fluconazole to determine the minimum inhibitory concentration (MIC) and subsequently, we measured the fungicidal activity. Geraniol was tested against biofilms by the measurement of the metabolic activity and biomass. Pharmacological interactions were performed by the checkerboard method. Geraniol's MIC range was between 256 and 512 µg/ml. MIC range for clinical antifungals was ≤ 0.031-4 µg/ml. Geraniol also showed antibiofilm activity with average reductions of metabolic activity (38.33%) and biomass (30.69%), at MIC concentration. Furthermore, geraniol showed synergistic/additive effects with antifungals. Briefly, geraniol inhibits both planktonic cells and biofilms of the C. parapsilosis species complex and besides it improves the efficacy of amphotericin B, caspofungin and fluconazole., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Candida albicans, the most common opportunistic pathogenic fungus, is also the main pathogenic organism for oral candidiasis. This condition is particularly prevalent among the elderly, children, and individuals undergoing radiotherapy or suffering from HIV. The lack of new antifungal drugs, and drug resistance coupled with the side effects of current antifungal agents have increased the challenges of clinical antifungal therapies. Polyenes, including amphotericin B and nystatin, are clinical fungicidal drugs, however, their side effects and low solubility have limited their clinical applications. Here, we identified that moxidectin, a novel approved antiparasitic agent, could synergize with both amphotericin B and nystatin to inhibit the growth and biofilm formation of Candida albicans including 60 clinical isolates. The transcriptome and RT-PCR analysis indicated that moxidectin activated the biosynthesis pathway of ergosterol, the direct target of polyenes, further being verified by the loss of the synergistic activities with polyenes against ergosterol pathway mutants, including Δ/Δerg3, Δ/Δerg11 and Δ/Δerg3 Δ/Δerg11. Moxidectin was then confirmed to elevate the ergosterol biosynthesis levels of C. albicans and enhance the binding between cells and polyenes. In a mouse oral candidiasis model, moxidectin combined with low dosages of polyenes to significantly reduce the infection area, colonization of C. albicans and the inflammatory degree of tongue mucosa. Our study originally demonstrated that moxidectin could activate the ergosterol biosynthesis then elevate the ergosterol contents to enhance the antifungal effects of polyenes against C. albicans and its infections. Moxidectin can serve as the candidate potentiator of polyenes for further clinical practice. KEY POINTS: • Moxidectin synergized with polyenes against Candida albicans. • Moxidectin activated the ergosterol biosynthesis of Candida albicans. • Moxidectin combined with polyenes to effectively combat oral candidiasis in mice., Competing Interests: Declarations Ethical approval All procedures were conducted in accordance with the “Guiding Principles in the Care and Use of Animals” (China) and were approved by the Ethics Committee of West China Stomatology Hospital of Sichuan University(WCHSIRB-D-2023–624). Conflicts of interest The authors declare no conflict of interest., (© 2024. The Author(s).)

Exophiala dermatitidis (E. dermatitidis), which causes skin or respiratory disease, is occasionally fatal in immunocompromised patients. Here, we report the unique antifungal potency of terbinafine (TRB), which targets squalene epoxidase, against E. dermatitidis (SQLE ED ) using various in vitro approaches. The versatile antifungal activities, including fungicidal activity, biofilm formation inhibition, biofilm eradication activity, and the combination effect of TRB, posaconazole (PSC), and amphotericin B (AmB) with great antifungal potency against E. dermatitidis were evaluated using crystal violet and cell viability assay. TRB formed an H-bond through Y102 in SQLE ED in the binding model. E. dermatitidis hyphae elongated and attached to a cell scaffold, forming a membrane-like biofilm. TRB and PSC showed more potent antibiofilm activities than AmB, and exhibited post-antifungal effects without incubation against E. dermatitidis conidia, reducing growth at lower concentrations. In contrast, AmB exhibited strong dose- and time-dependent killing and biofilm-eradication activities. The combination of TRB and PSC was more effective than that of TRB and AmB or PSC and AmB. Although the tissue migration of TRB must be considered, these data suggest that TRB and PSC may be useful agents and a potent combination in severely immunocompromised patients with refractory and systemic E. dermatitidis infection., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Fluorescent proteins (FPs) are indispensable tools used for molecular imaging, single-cell dynamics, imaging in infection models, and more. However, next-generation FPs have yet to be characterized in Aspergillus . Here, we characterize 18 FPs in the pathogenic filamentous fungus Aspergillus fumigatus spanning the visible light spectrum. We report on in vivo FP brightness in hyphal and spore morphotypes and show how a fluoropyrimidine-based selection system can be used to iteratively introduce four distinct FPs enabling the simultaneous visualization of the cell membrane, mitochondria, peroxisomes, and vacuoles. Using this strain, we describe and compare the dynamic responses of organelles to stresses induced by voriconazole, amphotericin B, and the novel antifungal drugs olorofim and manogepix. The expansion to the fluorescent genetic toolbox will overcome boundaries in research applications that involve fluorescence imaging in filamentous fungi., Competing Interests: The authors declare no conflict of interest.

The emergence of Candida auris poses a significant health challenge that has led to a new era of multidrug-resistant fungal infections. Invasive infections caused by C. auris are usually associated with remarkable morbidity and mortality. For many years, amphotericin B (AmB) remained the most efficient and the last line of treatment against most hard-to-treat fungal infections. However, strains of C. auris possess extraordinary resistance to most antifungal agents, including AmB. In this study, we screened ~2,600 FDA-approved drugs and clinical compounds to identify the antiemetic drug rolapitant as a promising enhancer to AmB against C. auris . Rolapitant exhibited potent synergistic interactions with AmB against all tested (29/29) C. auris isolates. In a time-kill assay, rolapitant restored the fungicidal activity of AmB within 4 h. Additionally, the synergistic relationship between rolapitant and AmB was observed against other medically crucial Candida , Cryptococcus, and Aspergillus species. A transcriptomic study revealed that exposure to rolapitant affects oxidation reduction processes, ion transporters, and ATP production. Rolapitant triggers an elevation in cytosolic and mitochondrial calcium levels and induces oxidative stress within fungal cells. An ATP luminescence assay confirmed that rolapitant, at sub-inhibitory concentrations, significantly interfered with ATP production in C. auris . Moreover, rolapitant enhanced the in vivo activity of AmB in a mouse model of disseminated C. auris infection, as the combination reduced the fungal burden in murine kidneys by ~1 log (~90%) colony forming units. Our findings warrant further investigation of using rolapitant to overcome AmB resistance in C. auris and other fungal species., Competing Interests: The authors declare no conflict of interest.

The current global scenario presents us with a growing increase in infections caused by fungi, referred to by specialists in the field as a "silent epidemic", aggravated by the limited pharmacological arsenal and increasing resistance to this therapy. For this reason, drug repositioning and therapeutic compound combinations are promising strategies to mitigate this serious problem. In this context, this study investigates the antifungal activity of the non-toxic, low-cost and widely available cationic polyelectrolyte Poly(diallyldimethylammonium chloride) (PDDA), in combination with different antifungal drugs: systemic (amphotericin B, AMB), topical (clioquinol, CLIO) and oral (nitroxoline, NTX). For each combination, different drug:PDDA ratios were tested and, through the broth microdilution technique, the minimum inhibitory concentration (MIC) of these drugs in the different ratios against clinically important Candida species strains was determined. Overall, PDDA combinations with the studied drugs demonstrated a significant increase in drug activity against most strains, reaching MIC reductions of up to 512 fold for the fluconazole resistant Candida krusei (Pichia kudriavzevii). In particular, the AMB-PDDA combination 1:99 was highly effective against AMB-resistant strains, demonstrating the excellent profile of PDDA as an adjuvant/association in novel antifungal formulations with outdated conventional drugs., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Fungal infection poses a major global threat to public health because of its wide prevalence, severe mortality rate, challenges involved in diagnosis and treatment, and the emergence of drug-resistant fungal strains. Millions of people are getting affected by fungal infection, and around 3.8 million people face death per year due to fungal infection, as per the latest report. The polyene antibiotic AmB has an extensive record of use as a therapeutic moiety against systemic fungal infection and leishmaniasis since 1960. AmB has broad-spectrum fungistatic and fungicidal activity. AmB exerts its therapeutic activity at the cellular level by binding to fungal sterol and forming hydrophilic pores, releasing essential cellular components and ions into the extracellular fluid, leading to cell death. Despite using AmB as an antifungal and antileishmanial at a broad scale, its clinical use is limited due to drug-induced nephrotoxicity resulting from binding the aggregated form of the drug to mammalian sterol. To mitigate AmB-induced toxicity and to get better anti-fungal therapeutic outcomes, researchers have developed nanoformulations, self-assembled formulations, prodrugs, cholesterol- and albumin-based AmB formulations, AmB-mAb combination therapy, and AmB cochleates. These formulations have helped to reduce toxicity to a certain extent by controlling the aggregation state of AmB, providing sustained drug release, and altering the physicochemical and pharmacokinetic parameters of AmB. Although the preclinical outcome of AmB formulations is quite satisfactory, its parallel result at the clinical level is insignificant. However, the safety and efficacy of AmB therapy can be improved at the clinical stage by continuous investigation and collaboration among researchers, clinicians, and pharmaceutical companies.

Background: in vitro susceptibility testing for the non-sporulating fungus Madurella mycetomatis is performed with a hyphal suspension as starting inoculum and a viability dye for endpoint reading. Here we compared the performance of four different viability dyes for their use in in vitro susceptibility testing of M. mycetomatis., Methods: To compare the reproducibility and the agreement between the viability dyes 2,3-bis-(2-methoxy-4-nitro-5-sulfphenyl)-2H-tetrazolium-5-carboxanilide salt (XTT), resazurin, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) and luciferin, the in vitro susceptibilities of 14 genetically diverse M. mycetomatis isolates were determined for itraconazole and amphotericin B. The reproducibility, agreement, price and ease of use were compared., Results: Each of the four dyes gave highly reproducible results with >85.7% reproducibility. Percentage agreement ranged between 78.9% and 92.9%. Resazurin was the most economical to use (0.0009 €/minimal inhibitory concentration [MIC]) and could be followed in real time. Luciferin omitted the need to transfer the supernatant to a new 96-well plate, but cost 6.07 €/MIC., Conclusion: All four viability dyes were suitable to determine the in vitro susceptibility of M. mycetomatis against itraconazole and amphotericin B. Based on the high reproducibility, high percentage agreement, price and possibility to monitor in real time, resazurin was the most suited for routine in vitro susceptibility testing in the diagnostic laboratory in mycetoma-endemic countries. Because luminescence could be measured directly without the need to transfer the supernatant to a new 96-well plate, luciferin is suitable for drug-screening campaigns., Lay Summary: To determine the in vitro susceptibility testing in the non-sporulating fungus Madurella mycetomatis, a viability dye is needed for endpoint reading. In this study we tested the viability dyes XTT, resazurin, MTS and luciferin for their use in in vitro susceptibility testing. It appeared that they all could be used but there were differences in time to result and costs associated with them., (© The Author(s) 2024. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Aim. Melanized fungi were rarely studied for their antifungal resistance (AFR) or clinical outcome, despite rising incidence of melanized fungal ocular infections and AFR in general. We report the antifungal resistance patterns, clinical outcome and clinico-microbiological correlation in two commonly isolated melanized fungi from ocular infections, Curvularia lunata and Lasiodiplodia theobromae , at a tertiary eyecare centre in South India. Gap statement . Despite melanized fungi accounting for a significant proportion of ocular fungal infections in the Indian subcontinent, and despite there being a limited selection of effective antifungal agents available for these infections, the existing data and studies on these issues remain sparse. Therefore, this study aimed to investigate the prevalence of antifungal resistance in two of the most common melanized fungal pathogens in ocular infections, Curvularia lunata and Lasiodiplodia theobromae and correlate it with the treatment given and the clinical outcome in patients. Methodology. Electronic medical records provided the clinical data. Standard broth microdilution was performed for antifungal susceptibility testing (AFST) in 30 isolates (17 C . lunata and 13 L . theobromae ) for amphotericin B and natamycin (polyenes): voriconazole, ketoconazole, posaconazole, itraconazole and fluconazole (azoles) and caspofungin (echinocandin). Multidrug resistance (MDR) was defined as resistance to more than or equal to two classes of antifungals. DNA sequencing was performed for the isolates for species confirmation. The multivariate analysis was done for determining poor prognostic factors. Results. AFST showed highest susceptibility of study isolates for voriconazole (83.3% isolates), followed by natamycin (80%), fluconazole (80%), itraconazole (76.7%), ketoconazole (70%), posaconazole (66.7%), caspofungin (66.7%) and lastly amphotericin B (63.3%). All patients empirically received topical natamycin; additional oral ketoconazole/intraocular voriconazole was administered in select few. MDR was strongly associated with poor clinical outcome (multivariate analysis: P = 0.03, odds ratio = 7.8). All patients had microbial keratitis, one progressed to endophthalmitis. Additionally, therapeutic penetrating keratoplasty was required in 40% of cases. Globe salvage was possible in 80% patients, though good visual outcome was seen in only half of them. Both, anatomical and visual outcomes, were poor in 20% of patients. DNA sequencing showed C. lunata as the highest study species. Conclusion. C. lunata and L. theobromae showed varying in vitro antifungal susceptibility and clinical outcome in ocular infections. Voriconazole had significantly higher activity, while amphotericin B had lower activity in vitro for these melanized fungi. MDR isolates showed poorer clinical outcome.

Patients with orofacial clefts are more likely to develop oral fungal diseases due to anatomo-physiological changes and surgical rehabilitation treatment. This case-series study evaluated the genetic diversity and dynamics of oral colonization and spread of C. albicans and C. tropicalis in four patients with orofacial clefts, from the time of hospital admission, perioperative and outpatient follow-up, with specialized physician. Candida biotypes previously identified by CHROMagar Candida and PCR methods were studied by MALDI-TOF MS assays and clustering analyses. Possible correlations with pathogenicity characteristics were observed, including production of hydrolytic exoenzymes and the antifungal sensitivity profiles. Amphotericin B-sensitive and fluconazole-resistant (low frequency) C. tropicalis and C. albicans, including clinically compatible MIC of nystatin, were found in the oral cavity of these patients. Clusters of isolates revealed phenomena of (i) elimination in the operative phase, (ii) maintenance or (iii) acquisition of oral C. tropicalis in the perioperative period and specialized outpatient and medical follow-up. For C. albicans, these phenomena included (i) elimination in the operative phase, (ii) acquisition in the operative phase and propagation from the hospital environment, and (iii) maintenance during hospitalization and operative phase. Amphotericin B and nystatin were shown to be effective in cases of clinical treatment and/or prophylaxis, especially considering the pre-existence of fluconazole-resistant strains. This study confirmed the phenomena of septic maintenance, septic neocolonization and septic elimination involving the opportunistic pathogens. MALDI-TOF MS associated with clustering analysis may assist the monitoring of clinical isolates or groups of epidemiologically important microbial strains in the hospital setting., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Candida auris has emerged as a major multidrug-resistant nosocomial pathogen. The organism exhibits a persistent colonising phenotype, and causes recalcitrant infections often strongly linked to biofilm formation. Alternate strategies are urgently needed to combat this yeast and its biofilm-associated phenotype. This work aimed to evaluate the efficacy of select staphylococcal phenol soluble modulins (PSMs), namely, a truncated version of Staphylococcus aureus PSMα2 shortened by two amino acids at the N-terminal (SaΔ1Δ2PSMα2) and Staphylococcus epidermidis PSMδ against C. auris in vitro and in vivo. The antifungal and antibiofilm activity was tested by broth microdilution and XTT dye reduction assay. Combination effect with antifungal drugs was determined by fractional inhibitory concentration test. The efficacy of combination therapy using SaΔ1Δ2PSMα2 with amphotericin B or caspofungin was evaluated in murine model of C. auris catheter-associated infection. Based on antifungal activity, antibiofilm activity and cytotoxicity data, SaΔ1Δ2PSMα2 exhibited promising activity against C. auris biofilms. Nearly 50 % inhibition in biofilm formation was noted with 0.5-2 μM of the peptide against multiple clinical and C. auris colonizing isolates. It was synergistic with amphotericin B (ΣFIC = 0.281) and caspofungin (ΣFIC = 0.047) in vitro, and improved the activity of voriconazole in voriconazole-resistant C. auris. Combination therapy using amphotericin B or caspofungin (1 μg/ml) with SaΔ1Δ2PSMα2 resulted in 99.5 % reduction in C. auris biofilm in murine model, even when the peptide was used at a concentration that was neither fungicidal nor antibiofilm (0.125 μM; ≈0.26 μg/ml). The study provides insight into the potential utility of SaΔ1Δ2PSMα2-antifungal drug combination against C. auris biofilm-associated infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Previous studies on BSC2 have shown that it enhances yeast cell resistance to AmB via antioxidation and induces multidrug resistance by contributing to biofilm formation. Herein, we found that BSC2 overexpression could reverse the sensitivity of pmp3Δ to AmB and help the tested strains restore the intracellular sodium/potassium balance under exposure to AmB. Meanwhile, overexpression of the chitin gene CHS2 could simulate BSC2 to reverse the sensitivity of pmp3Δ and nha1Δ to high salt or AmB. However, BSC2 overexpression in flo11Δ failed to induce AmB resistance, form biofilms, and affect cell wall biogenesis, while CHS2 overexpression compensated the resistance of flo11Δ to AmB. Additionally, BSC2 levels were positively correlated with maintaining cell membrane integrity under exposure to AmB, CAS, or a combination of both. BSC2 overexpression in nha1Δ exhibited a similar function of CHS2, which can compensate for the sensitivity of the mutant to high salt. Altogether, the results demonstrate for the first time that BSC2 may promote ion equilibrium by strengthening cell walls and inhibiting membrane damage in a FLO path-dependent manner, thus enhancing the resistance of yeast cells to AmB. This study also reveals the possible mechanism of antifungal drugs CAS and AmB combined to inhibit fungi., (Copyright © 2024 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Amphotericin B (AmB) is an antifungal agent administered for the management of serious systemic fungal infections. However, its clinical application is limited because of its water insolubility and side effects. Herein, to apply the minimum dose of AmB that can be used to manage fungal infections, a targeted drug delivery system was designed using lipopeptides and poly(lactide-co-glycolide) (PLGA). Lipopeptides conjugated with PEGylated distearoyl phosphoethanolamine (DSPE) and short peptides via a maleimide-thiol reaction formed nanosized micelles with PLGA and AmB. The antifungal effects of AmB-loaded micelles containing lipopeptides were remarkably enhanced both in vitro and in vivo. Moreover, the intravenous injection of these micelles demonstrated their in vivo targeting capacity of short peptides in a mouse model infected with drug-resistant Candida albicans. Our findings suggest that short antifungal peptides displayed on the surfaces of micelles represent a promising therapeutic candidate for targeting drug-resistant fungal infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background/aim: This experimental study aimed to examine the effectiveness of transdermal antimicrobial photodynamic therapy (APDT) with and without antimicrobial lock therapy (ALT), on catheter biofilms., Methods: S. epidermidis and C. orthopsilosis biofilms were formed within peripheral venous catheters positioned in the marginal ear veins of New Zealand white rabbits. Biofilm formation was confirmed with scanning electron microscopy in two catheters. 24 catheters with staphylococcal biofilms and 24 with fungal biofilms were treated with APDT, ALT or "APDT plus ALT" for five days. Six catheters were separated as controls. APDT was applied with a red colored LED lamp and methylene blue as the photosensitizer. Vancomycin lock solutions were used as ALT for staphylococcal biofilms and amphotericin B for fungal biofilms. The effect of treatment procedures was evaluated by intraluminal biofilm viability testing based on spectrophotometric evaluation, and a quantitative (OD) value was obtained for each catheter., Results: The mean OD values obtained by 600 nm spectrophotometric reading at 24 h (biofilm viability) after "ALT", "APDT" and "ALT plus APDT" procedures were 0.363, 0.151 and 0.128 for S. epidermidis and 0.092, 0.104 and 0.227 for C. orthopsilosis, respectively. All these OD values obtained after treatment procedures were lower than controls for both S. epidermidis (OD: 0,802) and C. orthopsilosis (OD: 0,315), although there were large fluctuations in our results., Conclusions: Our results suggest that transdermal APDT may be an effective method for treating staphylococcal and candida biofilms formed within intravenous catheters in our rabbit ear model. The combined use of APDT and ALT might be beneficial in these staphylococcal biofilms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The global epidemic of drug-resistant Candida auris continues unabated. The initial report on pan-drug resistant (PDR) C. auris strains in a hospitalized patient in New York was unprecedented. PDR C. auris showed both known and unique mutations in the prominent gene targets of azoles, amphotericin B, echinocandins, and flucytosine. However, the factors that allow C. auris to acquire pan-drug resistance are not known. Therefore, we conducted a genomic, transcriptomic, and phenomic analysis to better understand PDR C. auris . Among 1,570 genetic variants in drug-resistant C. auris , 299 were unique to PDR strains. The whole-genome sequencing results suggested perturbations in genes associated with nucleotide biosynthesis, mRNA processing, and nuclear export of mRNA. Whole transcriptome sequencing of PDR C. auris revealed two genes to be significantly differentially expressed-a DNA repair protein and DNA replication-dependent chromatin assembly factor 1. Of 59 novel transcripts, 12 transcripts had no known homology. We observed no fitness defects among multi-drug resistant (MDR) and PDR C. auris strains grown in nutrient-deficient or -enriched media at different temperatures. Phenotypic profiling revealed wider adaptability to nitrogenous nutrients and increased utilization of substrates critical in upper glycolysis and tricarboxylic acid cycle. Structural modeling of a 33-amino acid deletion in the gene for uracil phosphoribosyl transferase suggested an alternate route in C. auris to generate uracil monophosphate that does not accommodate 5-fluorouracil as a substrate. Overall, we find evidence of metabolic adaptations in MDR and PDR C. auris in response to antifungal drug lethality without deleterious fitness costs., Competing Interests: The authors declare no conflict of interest.

We observed an increase in the frequency of Candida auris among invasive candidiasis isolates in the 2022 SENTRY Antifungal Surveillance Program compared to prior years: ≤0.1% before 2018, 0.4%-0.6% from 2018 to 2021, and 1.6% in 2022. C. auris isolates were collected in seven countries, but 28 (35.9%) isolates were recovered in the USA (five states; more common in New York, Texas, and New Jersey) and 26 (33.3%) in Panama. Greece and Turkey had 12 and 9 isolates, respectively. Overall, 82.1% of the isolates were resistant to fluconazole; 17.9% were resistant to amphotericin B; and 1.3% were resistant to caspofungin, anidulafungin, or micafungin (Centers for Disease Control and Prevention tentative resistance breakpoints). Rezafungin inhibited 96.2% of the isolates (Clinical and Laboratory Standards Institute susceptibility breakpoint). Pandrug resistance was not observed, but 17.9% of the isolates were resistant to fluconazole and amphotericin B. South Asian (Clade I) isolates were most common ( n = 40, 51.3%); of these, 97.5% were resistant to fluconazole and 30.0% were resistant to amphotericin B. Thirty (38.5%) isolates belonged to the South American region (Clade IV), and 56.7% of those were resistant to fluconazole and 6.7% to amphotericin B. Seven isolates belonged to the South African Clade III and one to East Asian Clade II. Erg11 (Y132F, K143R, and F126L) and MRR1 (N647T) alterations were detected. One isolate that was resistant to all echinocandins carried an FKS R1354G alteration. Two isolates displayed elevated rezafungin minimum inhibitory concentration (MIC) values but low MIC values against other echinocandins and no FKS alterations. As C. auris is spreading globally, monitoring this species is prudent., Competing Interests: M.C., L.M.D., P.R.R., and C.G.C. were employees at Element Iowa City (JMI Laboratories), where the study was performed. This study was supported by Melinta Therapeutics, which included funding for services related to preparing the manuscript.

Background: Candida auris is an opportunistic pathogen that has become widespread in recent years and shows resistance to multiple drugs. The aim of our study was to determine the antifungal susceptibilities of C. auris isolates, to perform a dendrogram from the mass spectra of strains obtained from matrix-assisted laser desorption ionization-time of flight mass spectrometry in order to evaluate the proteomic similarities of the strains and determine the geographical clade of C. auris strains in this study by the help of Multiplex RT-PCR., Methods: The samples yielded 58 C. auris isolates. MALDI TOF MS (BioMerieux, France) was used for identification of the isolates and Sensititre Yeast One (Thermoscientific) system was used for antifungal susceptibility testing. Dendrograms of strain's spectra were generated by using the RUO/Saramis (BioMerieux, France) database and evaluated through hierarchical clustering analysis. The selected nine strains were examined at the clade level by using Multiplex RT-PCR. Results The susceptibility profile of the strains revealed resistance to Fluconazole in 84% (MICs ≥ 32) and resistance to Amphotericin B in 60% (MIC ≥ 2). All strains were found to be sensitive to Anidulafungin and Micafungin. The dendrogram of the main spectra of C. auris isolates showed a similarity range of 35 - 100%. The nine strains studied were identified as clade 1 (South Asian)., Conclusions: It was determined that C. auris strains were members of geographical clade 1, and the Amphotericin B resistance was found to be higher than expected. This situation poses a threat to critically ill patients.

Leishmaniasis is a parasitic disease spread by the bite of an infected sandfly and caused by protozoan parasites of the genus Leishmania. Currently, there is no vaccine available for leishmaniasis in humans, and the existing chemotherapy methods face various clinical challenges. The majority of drugs are limited to a few toxic compounds, with some parasite strains developing resistance. Therefore, the discovery and development of a new anti-leishmanial compound is crucial. One promising strategy involves the use of nanoparticle delivery systems to accelerate the effectiveness of existing treatments. In this study, Amphotericin B (AmB) was incorporated into functionalized carbon nanotube (f-CNT) and evaluated for its efficacy against Leishmania major in vitro and in a BALB/c mice model. The increase in footpad thickness was measured, and real-time PCR was used to quantify the parasite load post-infection. Levels of nitric oxide and cytokines IL-4 and IFN-γ were also determined. We found that f-CNT-AmB significantly reduced the levels of promastigotes and amastigotes of the Leishmania parasite. The nanoparticle showed strong anti-leishmanial activity with an IC 50 of 0.00494 ± 0.00095 mg/mL for promastigotes and EC 50 of 0.00294 ± 0.00065 mg/mL for amastigotes at 72 h post-infection, without causing harm to mice macrophages. Treatment of infected BALB/c mice with f-CNT-AmB resulted in a significant decrease in cutaneous leishmania (CL) lesion size in the foot pad, as well as reduced Leishmania burden in both lymph nodes and spleen. The levels of nitric oxide and IFN-γ significantly increased in the f-CNT-AmB treated groups. Also, our results showed that the level of IL-4 significantly decreased after f-CNT-AmB treatment in comparison to other groups. In conclusion, our results demonstrate that AmB loaded into f-CNT is significantly more effective than AmB alone in inhibiting parasite propagation and promoting a shift towards a Th1 response., (© 2024 John Wiley & Sons Ltd.)

Candida auris is a multidrug-resistant yeast that has seen a worrying increase during the COVID-19 pandemic. Give7/n this, new therapeutic options, such as controlled-release nanomaterials, may be promising in combating the infection. Therefore, this study aimed to develop amphotericin B (AmB) and micafungin (MICA)-loaded nanoemulsions (NEMA) and evaluated against biofilms of C. auris . Nanoemulsions (NEs) were characterized and determined minimum inhibitory concentration MIC 90 , checkerboard and anti-biofilm. NEMA presented a size of 53.7 and 81.4 nm for DLS and NTA, respectively, with good stability and spherical morphology. MICAmB incorporated efficiency was 88.4 and 99.3%, respectively. The release results show that AmB and MICA obtained a release of 100 and 63.4%, respectively. MICAmB and NEMA showed MIC90 values of 0.015 and 0.031 ug/mL, respectively and synergism. NEMA showed greater metabolic inhibition and morphological changes in mature biofilms. This drugs combination and co-encapsulation proved to be a promising therapy against C. auris biofilms.

Background: Our previous clinical trial demonstrated that antimicrobial photodynamic therapy (aPDT) with methylene blue (MB) and potassium iodide (KI) effectively killed Candida albicans (C. albicans) in adult AIDS patients with oral candidiasis, regardless of biofilm formation or 25S rDNA genotype. This study evaluated changes in antifungal susceptibility and virulence gene expression in C. albicans before and after aPDT, and explored factors related to clinical aPDT efficacy., Methods: Twenty-one adult AIDS patients with C. albicans oral candidiasis were divided into Group a (400 μM MB, N = 11) and Group b (600 μM MB, N = 10). Both groups received two aPDT treatments, where MB was applied for 5 min, followed by 300 mM KI, and illuminated for 30 min (37.29 J/cm²). C. albicans isolates were collected before and after treatment to assess antifungal susceptibility (fluconazole, itraconazole, flucytosine, amphotericin B) and gene expression (CAT1, HWP1). Peripheral blood tests were analyzed for correlations with aPDT efficacy., Results: aPDT reduced minimum inhibitory concentration (MIC) values for amphotericin B, fluconazole, and flucytosine, with significant reductions primarily after the first treatment. MIC reductions differed between groups, with Group a showing greater decreases in flucytosine and fluconazole MICs, and Group b in amphotericin B MICs. No significant changes in CAT1 or HWP1 expression were observed. Clinical efficacy of aPDT negatively correlated with leukocyte and neutrophil levels., Conclusions: aPDT effectively reduces MICs of antifungal drugs against C. albicans isolated from treated patients, particularly after the first treatment. The concentration of MB required to reduce MICs varies among different antifungal drugs. aPDT does not alter CAT1 or HWP1 expression, and its clinical efficacy in eradicating C. albicans is negatively associated with leukocyte and neutrophil levels., Competing Interests: Declaration of competing interest MRH declares the following potential conflicts of interest. Scientific Advisory Boards: Transdermal Cap Inc, Cleveland, OH; Hologenix Inc. Santa Monica, CA; Vielight, Toronto, Canada; JOOVV Inc, Minneapolis-St. Paul MN; Consulting; USHIO Corp, Japan; Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany. The other authors declare there is no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Introduction: Over the past 20 years, significant progress has been made in anti-leishmanial therapy. Three new drugs/formulations are available for the treatment of various forms of leishmaniasis, namely oral miltefosine, paromomycin and liposomal amphotericin B. However, these advances in drug development have added considerable complexity for clinicians including toxicity, emergence of resistance and decreased sensitivity of available drugs. The development of newer drugs with less toxicity and more efficacy is urgently needed., Areas Covered: This review comprehensively examines the latest developments and current status of antileishmanial drugs for the treatment of leishmaniasis across the world. Several new investigational drugs that showed anti-leishmanial activity under in vitro or in vivo conditions and either underwent the phase-I/II clinical trials or are on the verge of entering the trials were reviewed. We also delve into the challenges of drug resistance and discuss the emergence of new and effective antileishmanial compounds., Expert Opinion: The available treatments for leishmaniasis are limited in number, toxic, expensive, and demand extensive healthcare resources. Every available antileishmanial drug is associated with several disadvantages, such as drug resistance and toxicity or high cost. Miltefosine is potentially teratogenic. New antileishmanial drugs/treatment modalities are sorely needed for expanding future treatment options.

Candida albicans invasive candidiasis is considered a global health problem. In such cases, biofilm formation on implanted devices represents a therapeutic challenge and the presence of metabolically inactive persistent cells (PCs) in these communities increases their tolerance to fungicidal drugs. This study investigated the influence of amoxicillin, AMX; cefepime, CEF; gentamicin, GEN; amikacin, AMK; vancomycin, VAN; and ciprofloxacin, CIP; on the production of PCs in biofilms of C. albicans bloodstream isolates. 48 h-mature biofilms ( n  = 6) grown in RPMI-1640 supplemented with antibiotics were treated with 100 μg ml -1 amphotericin B and then evaluated for PCs. Biofilms grown in the presence of antibiotics produced more PCs, up to 10×, when exposed to AMX and CIP; 5 × to CEF; and 6 × to GEN and VAN. The results indicate that antibiotics can modulate PC production in C. albicans biofilms. This scenario may have clinical repercussions in immunocompromised patients under broad-spectrum antibiotic therapy.

Candida sp. infections are a threat to global health, with high morbidity and mortality rates due to drug resistance, especially in immunocompromised people. For this reason, the search for new alternatives is urgent, and in recent years, a combined therapy with natural compounds has been proposed. Considering the biological potential of isoespintanol (ISO) and continuing its study, the objective of this research was to assess the effect of ISO in combination with the antifungals fluconazole (FLZ), amphotericin B (AFB) and caspofungin (CASP) against clinical isolates of C. tropicalis and to evaluate the cytotoxic effect of this compound in the acute phase (days 0 and 14) and chronic phase (days 0, 14, 28, 42, 56, 70 and 84) in female mice ( Mus musculus ) of the Balb/c lineage. The results show that ISO can potentiate the effect of FLZ, AFB and CASP, showing synergism with these antifungals. An evaluation of the mice via direct observation showed no behavioral changes or variations in weight during treatment; furthermore, an analysis of the cytokines IFN-γ and TNF in plasma, peritoneal cavity lavage (PCL) and bronchoalveolar lavage (BAL) indicated that there was no inflammation process. In addition, histopathological studies of the lungs, liver and kidneys showed no signs of toxicity caused by ISO. This was consistent with an analysis of oxaloacetic transaminases (GOT) and pyruvic transaminases (GPT), which remained in the standard range. These findings indicate that ISO does not have a cytotoxic effect at the doses evaluated, placing it as a monoterpene of interest in the search for compounds with pharmacological potential.

Leishmaniasis, attributed to the protozoan parasite Leishmania , manifests in diverse clinical forms, including cutaneous, mucocutaneous, and visceral leishmaniasis; VL constitutes a significant global health menace. Prevalent in tropical and subtropical regions, this affliction disproportionately impacts individuals below the poverty threshold, transmitted through the bite of female sandflies. Existing treatments, such as pentavalent antimony, miltefosine, and Amphotericin B, exhibit limitations. Despite the emergence of liposomal Amphotericin B (AmBisome) as a promising antileishmanial agent, its utility is impeded by adverse effects, elevated production expenses, and cytotoxicity. To address these challenges, our investigation introduces a potential remedy─a citrate-coated gold Amphotericin B nanoparticle formulation. Characterized using dynamic light scattering and transmission electron microscopy, this pioneering formulation exhibited efficacy against L. donovani Ag83 promastigotes as demonstrated by MTT cell viability testing. Evaluating internal reactive oxygen species (ROS) levels and dual staining with acridine orange and ethidium bromide unveiled its consequential impact on cell death. Significantly, our study discloses this novel nanoformulation's unprecedented inhibition of the trypanothione reductase enzyme. The findings posit the citrate-coated gold Amphotericin B nanoformulation as a promising and targeted antileishmanial agent, representing potential advancements in leishmaniasis therapeutics.

Keratitis is a corneal infection caused by various bacteria and fungi. Eye drop treatment of keratitis involves significant challenges due to difficulties in administration, inefficiencies in therapeutic dosage, and frequency of drug applications. All these are troublesome and result in unsuccessful treatment, high cost, time loss, development of drug resistance by microorganisms, and a massive burden on human health and the healthcare system. Most of the antibacterial and antifungal medications are non-water-soluble and/or include toxic drug formulations. Here, the aim was to develop drug-loaded contact lenses with therapeutic dosage formulations and extended drug release capability as an alternative to eye drops, by employing supercritical carbon dioxide (ScCO 2 ) as a drug impregnation solvent to overcome inefficient ophthalmic drug use. ScCO 2 , known as a green solvent, has very low viscosity which provides high mass transfer power and could enhance drug penetration into contact lenses much better with respect to drug loading using other solvents. Here, moxifloxacin (MOX) antibiotic and amphotericin B (AMB) antifungal medicines were separately loaded into commercially available silicone hydrogel contact lenses through 1) drug adsorption from the aqueous solutions and 2) impregnation techniques via ScCO 2 and their efficacies were compared. Drug impregnation parameters, i.e., 8-25 MPa pressure, 310-320 K temperature, 2-16-hour impregnation times, and the presence of ethanol as polar co-solvent were investigated for the optimization of the ScCO 2 drug impregnation process. The highest drug loading and long-term release kinetic from the contact lenses were obtained at 25 MPa and 313 K with 2.5 h impregnation time by using 1 % ethanol (by volume). Furthermore, antibacterial/antifungal activities of the MOX- and AMB-impregnated contact lenses were effective against in vitro Pseudomonas aeruginosa (ATCC 10145) bacteria and Fusarium solani (ATCC 36031) fungus for up to one week. Consequently, the ScCO 2 method can be effectively used to impregnate commercial contact lenses with drugs, and these can then be safely used for the treatment of keratitis. This offers a sustainable delivery system at effective dosage formulations with complete bacterial/fungal inhibition and termination, making it viable for real animal/human applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cladosporium cladosporioides are the pigmented soil fungi containing melanin. The aim of this work was to determine the influence of amphotericin B on free radicals in the natural melanin isolated from pigmented fungi Cladosporium cladosporioides and to compare it with the effect in synthetic DOPA-melanin. Electron paramagnetic resonance (EPR) spectra were measured at X-band (9.3 GHz) with microwave power in the range of 2.2-70 mW. Amplitudes, integral intensities, linewidths of the EPR spectra, and g factors, were analyzed. The concentrations of free radicals in the tested melanin samples were determined. Microwave saturation of EPR lines indicates the presence of pheomelanin in addition to eumelanin in Cladosporium cladosporioides . o-Semiquinone free radicals in concentrations ~10 20 [spin/g] exist in the tested melanin samples and in their complexes with amphotericin B. Changes in concentrations of free radicals in the examined synthetic and natural melanin point out their participation in the formation of amphotericin B binding to melanin. A different influence of amphotericin B on free radical concentration in Cladosporium cladosporioides melanin and in DOPA-melanin may be caused by the occurrence of pheomelanin in addition to eumelanin in Cladosporium cladosporioides . The advanced spectral analysis in the wide range of microwave powers made it possible to compare changes in the free radical systems of different melanin polymers. This study is important for knowledge about the role of free radicals in the interactions of melanin with drugs.

Cryptococcus neoformans is primarily responsible for cases of cryptococcal meningitis in individuals with HIV/AIDS. This study evaluated the susceptibility of C. neoformans obtained from individuals with cryptococcal meningitis associated with HIV/AIDS in Manaus, Amazonas, Brazil, against the action of the antifungals amphotericin B, flucytosine, fluconazole, itraconazole and posaconazole and analyzed it using Multilocus Sequence Typing (MLST) in order to identify the Sequence Types (STs). We analyzed 30 isolates of C. neoformans, from 24 HIV/AIDS patients diagnosed with cryptococcosis from 2017 to 2019 in a reference hospital, in addition to 3 environmental isolates: 1 isolate obtained in the home of a patient and 2 isolates obtained from neighboring homes of patients. 86.6% (n = 26/30) of the clinical isolates were identified as C. neoformans VNI ST93, 6.6% (n = 2/30) as C. neoformans VNI ST5, 3.3% (n = 1/30) as C. neoformans VNI ST32 and 3.3% (n = 1/30) as C. neoformans VNB ST232. The environmental isolates were identified as C. neoformans VNI ST93 (n = 3/3). 96.6% (n = 29/30) isolates were sensitive to amphotericin B, though there was variation in the MIC. 60% (n = 18/30) presented a MIC above the proposed epidemiological cutoff values for one or more antifungals. All environmental isolates were sensitive to the tested antifungals. The MLST showed that there is an important relationship between C. neoformans VNI ST93 and individuals with HIV/AIDS, including in the environmental isolates analyzed. C. neoformans VNB ST232 was observed for the first time in Amazonas. Amphotericin B was proven to be the best drug, but fluconazole and posaconazole also showed relevant action., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Candida spp. can be found in the human microbiome. However, immunocompromised patients are likely to develop invasive Candida infections, with mortality rates higher than 50%. The discovery of C. auris, a species that rapidly acquire antifungal resistance, increased the concern about Candida infections. The limited number of antifungal agents and the high incidence of resistance to them make imperative the development of new antifungal drugs. β-lapachone is a biological active naphthoquinone that displays antifungal activity against C. albicans and C. glabrata. The aim of this study was to evaluate if this substance affects C. auris growth and elucidate its mechanism of action. A fluconazole-resistant C. auris isolate was used in this study. The antifungal activity of β-lapachone was determined through microbroth dilution assays, and its mechanism of action was evaluated using fluorescent probes. Interaction with fluconazole and amphotericin B was assessed by disk diffusion assay and checkerboard. β-lapachone inhibited planktonic C. auris cell growth by 92.7%, biofilm formation by 84.9%, and decrease the metabolism of preformed biofilms by 87.1% at 100 µg/ml. At 100 µg/ml, reductions of 30% and 59% of Calcofluor White and Nile red fluorescences were observed, indicating that β-lapachone affects cell wall chitin and neutral lipids content, respectively. Also, the ratio 590 nm/529 nm of JC-1 decreased 52%, showing that the compound affects mitochondria. No synergism was observed between β-lapachone and fluconazole or amphotericin B. Data show that β-lapachone may be a promising candidate to be used as monotherapy to treat C. auris resistant infections., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Fusarium oxysporum is a cross-kingdom pathogen that infects humans, animals, and plants. The primary concern regarding this genus revolves around its resistance profile to multiple classes of antifungals, particularly azoles. However, the resistance mechanism employed by Fusarium spp. is not fully understood, thus necessitating further studies to enhance our understanding and to guide future research towards identifying new drug targets. Here, we employed an untargeted proteomic approach to assess the differentially expressed proteins in a soil isolate of Fusarium oxysporum URM7401 cultivated in the presence of amphotericin B and fluconazole. In response to antifungals, URM7401 activated diverse interconnected pathways, such as proteins involved in oxidative stress response, proteolysis, and lipid metabolism. Efflux proteins, antioxidative enzymes and M35 metallopeptidase were highly expressed under amphotericin B exposure. Antioxidant proteins acting on toxic lipids, along with proteins involved in lipid metabolism, were expressed during fluconazole exposure. In summary, this work describes the protein profile of a resistant Fusarium oxysporum soil isolate exposed to medical antifungals, paving the way for further targeted research and discovering new drug targets., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Aim: To evaluate the antifungal activity of mangiferin against Candida spp. resistant to fluconazole. Materials & methods: The antifungal activity of mangiferin was assessed using broth microdilution and its interaction with azoles and amphotericin B was evaluated by checkerboard. The activity of mangiferin against Candida spp. biofilms was assessed using the MTT colorimetric assay and its possible mechanism of action was evaluated using flow cytometry. Results: Mangiferin showed activity against Candida albicans, Candida tropicalis and Candida parapsilosis resistant to fluconazole and showed synergism with azoles and amphotericin B. Mangiferin increased the activity of antifungals against Candida biofilms and caused depolarization of the mitochondrial membrane and externalization of phosphatidylserine, suggesting apoptosis. Conclusion: mangiferin combined with antifungals has potential against Candida spp.

The global rate of Amphotericin B (AmB) resistance in Candida auris has surpassed 12%. However, there is limited data on available clinical treatments and microevolutionary analyses concerning reduced AmB sensitivity. In this study, we collected 18 C. auris isolates from five patients between 2019 and 2022. We employed clinical data mining, genomic, and transcriptomic analyses to identify genetic evolutionary features linked to reduced AmB sensitivity in these isolates during clinical treatment. We identified six isolates with a minimum inhibitory concentration (MIC) of AmB below 0.5 µg/mL (AmB 0.5 ) and 12 isolates with an AmB-MIC of 1 µg/mL (AmB 1 ) or ≥ 2 µg/mL (AmB 2 ). All five patients received 24-hour AmB (5 mg/L) bladder irrigation treatment. Evolutionary analyses revealed an ERG3 (c923t) mutation in AmB 1 C. auris . Additionally, AmB 2 C. auris was found to contain a t2831c mutation in the RAD2 gene. In the AmB 1 group, membrane lipid-related gene expression ( ERG1, ERG2, ERG13, and ERG24 ) was upregulated, while in the AmB 2 group, expression of DNA-related genes (e.g. DNA2 and PRI1 ) was up-regulated. In a series of C.auris strains with reduced susceptibility to AmB, five key genes were identified: two upregulated ( IFF9 and PGA6) and three downregulated ( HGT7, HGT13, and PRI32) . In this study, we demonstrate the microevolution of reduced AmB sensitivity in vivo and further elucidate the relationship between reduced AmB sensitivity and low-concentration AmB bladder irrigation. These findings offer new insights into potential antifungal drug targets and clinical markers for the "super fungus", C. auris .

The study investigates the potential of lansoprazole, a proton pump inhibitor, to interfere with fungal respiration and enhance the antifungal activity of amphotericin B against multidrug-resistant Candida auris. The authors administered lansoprazole at concentrations significantly higher than typical therapeutic doses, which demonstrated promising results but also raised concerns about potential toxicity. We suggest incorporating a control group, monitoring toxicity indicators, performing pathological examinations, and conducting cellular assays to improve the study's rigor and reliability. We also highlight the need for further research into the mechanisms of lansoprazole's antifungal activity, its long-term effects on amphotericin B resistance, and potential drug-drug interactions with amphotericin B. Addressing these concerns is crucial for the clinical translation of lansoprazole as an adjuvant to amphotericin B.

Candida auris has emerged as a problematic fungal pathogen associated with high morbidity and mortality. Amphotericin B (AmB) is the most effective antifungal used to treat invasive fungal candidiasis, with resistance rarely observed among clinical isolates. However, C. auris possesses extraordinary resistant profiles against all available antifungal drugs, including AmB. In our pursuit of potential solutions, we screened a panel of 727 FDA-approved drugs. We identified the proton pump inhibitor lansoprazole (LNP) as a potent enhancer of AmB's activity against C. auris. LNP also potentiates the antifungal activity of AmB against other medically important species of Candida and Cryptococcus. Our investigations into the mechanism of action unveiled that LNP metabolite(s) interact with a crucial target in the mitochondrial respiratory chain (complex III, known as cytochrome bc 1 ). This interaction increases oxidative stress within fungal cells. Our results demonstrated the critical role of an active respiratory function in the antifungal activity of LNP. Most importantly, LNP restored the efficacy of AmB in an immunocompromised mouse model, resulting in a 1.7-log (∼98%) CFU reduction in the burden of C. auris in the kidneys. Our findings strongly advocate for a comprehensive evaluation of LNP as a cytochrome bc 1 inhibitor for combating drug-resistant C. auris infections.

Leishmaniases, caused by Leishmania parasites, are widespread and pose significant health risks globally. Visceral leishmaniasis (VL) is particularly prevalent in Brazil, with high morbidity and mortality rates. Traditional treatments, such as pentavalent antimonials, have limitations due to toxicity and resistance. Therefore, exploring new compounds like lectins is crucial. Concanavalin A (ConA) has shown promise in inhibiting Leishmania growth. This study aimed to evaluate its leishmanicidal effect on L. infantum promastigotes and understand its mechanism of action. In vitro tests demonstrated inhibition of promastigote growth when treated with ConA, with IC 50 values ranging from 3 to 5 μM over 24-72 h. This study suggests that ConA interacts with L. infantum glycans. Additionally, ConA caused damage to the membrane integrity of parasites and induced ROS production, contributing to parasite death. Scanning electron microscopy confirmed morphological alterations in treated promastigotes. ConA combined with the amphotericin B (AmB) showed synergistic effects, reducing the required dose of AmB, and potentially mitigating its toxicity. ConA demonstrated no cytotoxic effects on macrophages, instead stimulating their proliferation. These findings reinforce that lectin exhibits promising leishmanicidal activity against L. infantum promastigotes, making ConA a potential candidate for leishmaniasis treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As comparative pharmacokinetic/pharmacodynamic (PK/PD) studies of liposomal amphotericin B (L-AMB) against Candida spp. are lacking, we explored L-AMB pharmacodynamics against different Candida species in an in vitro PK/PD dilution model. Eight Candida glabrata , Candida parapsilosis , and Candida krusei isolates (EUCAST/CLSI AMB MIC 0.125-1 mg/L) were studied in the in vitro PK/PD model simulating L-AMB C max = 0.25-64 mg/L and t 1/2 = 9 h. The model was validated with one susceptible and one resistant Candida albicans isolate. The C max /MIC-log 10 CFU/mL reduction from the initial inoculum was analyzed with the E max model, and Monte Carlo analysis was performed for the standard (3 mg/kg with C max = 21.87 ± 12.47 mg/L) and higher (5 mg/kg with C max = 83 ± 35.2 mg/L) L-AMB dose. A ≥1.5 log 10 CFU/mL reduction was found at L-AMB C max = 8 mg/L against C. albicans , C. parapsilosis , and C. krusei isolates (MIC 0.25-0.5 mg/L) whereas L-AMB C max ≥ 32 mg/L was required for C. glabrata isolates. The in vitro PK/PD relationship followed a sigmoidal pattern ( R 2 ≥ 0.85) with a mean C max /MIC required for stasis of 2.1 for C. albicans (close to the in vivo stasis), 24/17 (EUCAST/CLSI) for C. glabrata , 8 for C. parapsilosis , and 10 for C. krusei . The probability of target attainment was ≥99% for C. albicans wild-type (WT) isolates with 3 mg/kg and for wild-type isolates of the other species with 5 mg/kg. L-AMB was four- to eightfold less active against the included non- C . albicans species than C. albicans . A standard 3-mg/kg dose is pharmacodynamically sufficient for C. albicans whereas our data suggest that 5 mg/kg may be recommendable for the included non- C . albicans species., Competing Interests: The authors declare no conflict of interest.

Introduction. Sporotrichosis is a subcutaneous infection caused by dimorphic Sporothrix species embedded in the clinical clade. Fungi have virulence factors, such as biofilm and melanin production, which contribute to their survival and are related to the increase in the number of cases of therapeutic failure, making it necessary to search for new options. Gap statement. Proton pump inhibitors (PPIs) have already been shown to inhibit the growth and melanogenesis of other fungi. Aim. Therefore, this study aimed to evaluate the effect of the PPIs omeprazole (OMP), rabeprazole (RBP), esomeprazole, pantoprazole and lansoprazole on the susceptibility and melanogenesis of Sporothrix species, and their interactions with itraconazole, terbinafine and amphotericin B. Methodology. The antifungal activity of PPIs was evaluated using the microdilution method, and the combination of PPIs with itraconazole, terbinafine and amphotericin B was assessed using the checkerboard method. The assessment of melanogenesis inhibition was assessed using grey scale. Results. The OMP and RBP showed significant MIC results ranging from 32 to 256 µg ml -1 and 32 to 128 µg ml -1 , respectively. Biofilms were sensitive, with a significant reduction ( P <0.05) in metabolic activity of 52% for OMP and 50% for RBP at a concentration of 512 µg ml -1 and of biomass by 53% for OMP and 51% for RBP at concentrations of 512 µg ml -1 . As for the inhibition of melanogenesis, only OMP showed inhibition, with a 54% reduction. Conclusion. It concludes that the PPIs OMP and RBP have antifungal activity in vitro against planktonic cells and biofilms of Sporothrix species and that, in addition, OMP can inhibit the melanization process in Sporothrix species.

Cryptococcosis is a fungal infection that is becoming increasingly prevalent worldwide, particularly among individuals with compromised immune systems, such as HIV patients. Amphotericin B (AmB) is the first-line treatment mainly combined with flucytosine. The scarcity and the prohibitive cost of this regimen urge the use of fluconazole as an alternative, leading to increased rates of treatment failure and relapses. Therefore, there is a critical need for efficient and cost-effective therapy to enhance the efficacy of AmB. In this study, we evaluated the efficacy of the HIV protease inhibitors (PIs) to synergize the activity of AmB in the treatment of cryptococcosis. Five PIs (ritonavir, atazanavir, saquinavir, lopinavir, and nelfinavir) were found to synergistically potentiate the killing activity of AmB against Cryptococcus strains with ƩFICI ranging between 0.09 and 0.5 against 20 clinical isolates. This synergistic activity was further confirmed in a time-kill assay, where different AmB/PIs combinations exhibited fungicidal activity within 24 hrs. Additionally, PIs in combination with AmB exhibited an extended post-antifungal effect on treated cryptococcal cells for approximately 10 hrs compared to 4 hours with AmB alone. This promising activity against cryptococcal cells did not exhibit increased cytotoxicity towards treated kidney cells, ruling out the risk of drug combination-induced nephrotoxicity. Finally, we evaluated the efficacy of AmB/PIs combinations in the Caenorhabditis elegans model of cryptococcosis, where these combinations significantly reduced the fungal burden of the treated nematodes by approximately 2.44 Log10 CFU (92.4%) compared to the untreated worms and 1.40 Log10 ((39.4%) compared to AmB alone. The cost-effectiveness and accessibility of PIs in resource-limited geographical areas compared to other antifungal agents, such as flucytosine, make them an appealing choice for combination therapy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Alkashef, Seleem. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chronic wounds of significant severity and acute injuries are highly vulnerable to fungal infections, drastically impeding the expected wound healing trajectory. The clinical use of antifungal therapeutic drug is hampered by poor solubility, high toxicity and adverse reactions, thereby necessitating the urgent development of novel antifungal therapy strategy. Herein, this study proposes a new strategy to enhance the bioactivity of small-molecule antifungal drugs based on multifunctional metal nanozyme engineering, using amphotericin B (AmB) as an example. AmB-decorated gold nanoparticles (AmB@AuNPs) are synthesized by a facile one-pot reaction strategy, and the AmB@AuNPs exhibit superior peroxidase (POD)-like enzyme activity, with maximal reaction rates (V max ) 3.4 times higher than that of AuNPs for the catalytic reaction of H 2 O 2 . Importantly, the enzyme-like activity of AuNPs significantly enhanced the antifungal properties of AmB, and the minimum inhibitory concentrations of AmB@AuNPs against Candida albicans (C. albicans) and Saccharomyces cerevisiae (S. cerevisiae) W303 are reduced by 1.6-fold and 50-fold, respectively, as compared with AmB alone. Concurrent in vivo studies conducted on fungal-infected wounds in mice underscored the fundamentally superior antifungal ability and biosafety of AmB@AuNPs. The proposed strategy of engineering antifungal drugs with nanozymes has great potential for enhanced therapy of fungal infections and related diseases., (© 2024 Wiley‐VCH GmbH.)

Background: Vulvovaginal candidiasis is a common fungal infection that affects the female lower genital tract. This study determined the major risk factors associated with vulvovaginal infection (VVI) in the Ashanti region of Ghana and also determined the antifungal resistance patterns of Candida albicans isolates to some antifungals., Methods: Three hundred and fifty (350) high vaginal swab (HVS) samples were collected from women who presented with signs and symptoms of VVI. A structured questionnaire was administered to one hundred and seventy-two (172) of the women. HVS samples were cultured on Sabouraud dextrose agar with 2% chloramphenicol. The polymerase chain reaction was employed to confirm C. albicans. Antifungal susceptibility testing was performed and the susceptibility of C. albicans isolates to fluconazole, clotrimazole, amphotericin B, nystatin, miconazole and 5-flurocytosine were assessed., Results: Vaginal infection was most prevalent amongst females in their reproductive age (21 to 30 years; 63.0%). The study found a significant association between vaginal infections and some risk factors such as sexual practices (p < 0.001), antibiotic misuse (p < 0.05), poor personal hygiene (p < 0.005) and birth control methods (p < 0.049). Out of the 350 HVS samples collected, 112 yielded yeast cells with 65 (58%) identified as C. albicans. The C. albicans isolates were resistant to 5' flucytosine (100%), fluconazole (70%), voriconazole (69.2%), miconazole (58.5%) and nystatin (49.2%). C. albicans isolates were more susceptible to amphotericin B (53.8%) and clotrimazole (45.1%), although an appreciable number of isolates showed resistance (46.1% and 52.3%, respectively)., Conclusion: There should be nationwide education on all associated risk factors of VVI. Also, use of the various antifungal agents in vaginal candidiasis should proceed after antifungal susceptibility testing to ensure efficacious use of these agents., (© 2024. The Author(s).)

Parasites of the genus Leishmania pose a global health threat with limited treatment options. New drugs are urgently needed, and genomic screens have the potential to accelerate target discovery, mode of action, and resistance mechanisms against these new drugs. We describe here our effort in developing a genome-wide CRISPR-Cas9 screen in Leishmania , an organism lacking a functional nonhomologous end joining system that must rely on microhomology-mediated end joining, single-strand annealing, or homologous recombination for repairing Cas9-induced double-stranded DNA breaks. A new vector for cloning and expressing single guide RNAs (sgRNAs) was designed and proven to be effective in a small pilot project while enriching specific sgRNAs during drug selection. We then developed a whole-genome library of 49,754 sgRNAs, targeting all the genes of Leishmania infantum . This library was transfected in L. infantum expressing Cas9, and these cells were selected for resistance to two antileishmanials, miltefosine and amphotericin B. The sgRNAs the most enriched in the miltefosine screen targeted the miltefosine transporter gene, but sgRNAs targeting genes coding for a RING-variant protein and a transmembrane protein were also enriched. The sgRNAs the most enriched by amphotericin B targeted the sterol 24 C methyltransferase genes and a hypothetical gene. Through gene disruption experiments, we proved that loss of function of these genes was associated with resistance. This study describes the feasibility of carrying out whole-genome CRISPR-Cas9 screens in Leishmania provided that a strong selective pressure is applied. Such a screen can be used for accelerating the development of urgently needed antileishmanial drugs.IMPORTANCELeishmaniasis, a global health threat, lacks adequate treatment options and drug resistance exacerbates the challenge. This study introduces a CRISPR-Cas9 screening approach in Leishmania infantum , unraveling mechanisms of drug resistance at a genome-wide scale. Our screen was applied against two main antileishmanial drugs, and guides were enriched upon drug selection. These guides targeted known and new targets, hence validating the use of this screen against Leishmania . This strategy provides a powerful tool to expedite drug discovery as well as potential therapeutic targets against this neglected tropical disease., Competing Interests: The authors declare no conflict of interest.

Cryptococcus neoformans causes cryptococcosis, one of the most prevalent fungal diseases, generally characterized by meningitis. There is a limited and not very effective number of drugs available to combat this disease. In this manuscript, we show the host defense peptide mimetic brilacidin (BRI) as a promising antifungal drug against C. neoformans . BRI can affect the organization of the cell membrane, increasing the fungal cell permeability. We also investigated the effects of BRI against the model system Saccharomyces cerevisiae by analyzing libraries of mutants grown in the presence of BRI. In S. cerevisiae , BRI also affects the cell membrane organization, but in addition the cell wall integrity pathway and calcium metabolism. In vivo experiments show BRI significantly reduces C. neoformans survival inside macrophages and partially clears C. neoformans lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis. We also observed that BRI interacts with caspofungin (CAS) and amphotericin (AmB), potentiating their mechanism of action against C. neoformans . BRI + CAS affects endocytic movement, calcineurin, and mitogen-activated protein kinases. Our results indicate that BRI is a novel antifungal drug against cryptococcosis., Importance: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Cryptococcosis, one of the most prevalent fungal diseases, is generally characterized by meningitis and is mainly caused by two closely related species of basidiomycetous yeasts, Cryptococcus neoformans and Cryptococcus gattii . There are few therapeutic options for treating cryptococcosis, and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a potential antifungal agent against C. neoformans . BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. BRI alone was shown to inhibit the growth of C. neoformans , acting as a fungicidal drug, but surprisingly also potentiated the activity of caspofungin (CAS) against this species. We investigated the mechanism of action of BRI and BRI + CAS against C. neoformans . We propose BRI as a new antifungal agent against cryptococcosis., Competing Interests: Gustavo H. Goldman is a member of the scientific advisory board of Innovation Pharmaceuticals, a company that is conducting clinical trials on brilacidin. Other authors have no conflict of interest.

Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of Leishmania donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme may be a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tulloch et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In recent years, the incidence and drug resistance of Candida parapsilosis have increased. Our study aimed to determine the antifungal sensitivity of C. parapsilosis and the clinical and demographic characteristics of children with candidemia. Two hundred pediatric patients with C. parapsilosis candidemia were included in the study between 1 January 2010 and 1 August 2023. Clinical samples were evaluated on a BACTEC-FX-40 automatic blood culture device (Becton Dickinson, USA). Yeast isolates were identified to the species level via identification cards (YST) using the VITEK 2 Compact (bioMeriéux, France) system. Antifungal susceptibility was performed using antifungal cell cards (AST-YST01). Approval for the study was received from the "University Faculty of Medicine" Hospital Clinical Research Ethics Committee. Non-catheter candidemia was detected in 127 (63.5%) patients, and catheter-related candidemia was detected in 73 (36.5%) patients. It was observed that the patients' history of malignancy, mechanical ventilation, urinary catheter, nasogastric tube, and intensive care unit stay was associated with C. parapsilosis mortality. The mortality rate from candidemia was 9.5%. The most frequently preferred antifungal agents were amphotericin B and fluconazole. The fluconazole drug resistance rate was found to be 6%, and the amphotericin B drug resistance rate was 4%. Because C. parapsilosis candidemia mortality rates can be high depending on risk factors and clinical characteristics, it is important to initiate appropriate and timely antifungal therapy. We think that our study can provide important information about the clinical profiles, distributions, susceptibility profiles, and control of antifungal resistance of C. parapsilosis isolates., Importance: It has been observed that the frequency and antifungal resistance of Candida parapsilosis have increased recently. In our study, we aimed to determine the antifungal sensitivity of C. parapsilosis and the clinical and demographic characteristics of children with candidemia. It was observed that the patients' history of malignancy, mechanical ventilation, urinary catheter, nasogastric tube, and intensive care stay was associated with C. parapsilosis mortality. The mortality rate from candidemia was 9.5%. The most frequently preferred antifungal agents were amphotericin B and fluconazole. The fluconazole drug resistance rate was found to be 6%, and the amphotericin B drug resistance rate was 4%. Because C. parapsilosis candidemia mortality rates can be high depending on risk factors and clinical characteristics, it is important to initiate appropriate and timely antifungal therapy., Competing Interests: The authors declare no conflict of interest.

Aim: Currently, we have limited armamentarium of antifungal agents against Mucorales. There is an urgent need to discover novel antifungal agents that are effective, safe and affordable. Materials & methods: In this study, the anti-Mucorale action of native lactoferrin (LF) and its functional fragments CLF, RR6 and LFcin against three common Mucorale species are reported. The synergistic action of LF with antifungal agents like amphotericin B, isavuconazole and posaconazole was analyzed using checkerboard technique. Results: All the three mucor species showed inhibition when treated with fragments. The checkerboard assay confirmed that native LF showed the best synergistic action against Mucorales in combination with Amphotericin B. Conclusion: These results highlight the potential therapeutic value of native LF against Mucorales.

Background: Candida vulturna is an emerging pathogen belonging to the Metshnikowiaceae family together with Candida auris and Candida haemulonii species complex. Some strains of this species were reported to be resistant to several antifungal agents., Objectives: This study aims to address identification difficulties, evaluate antiungal susceptibilities and explore the molecular mechanisms of azole resistance of Candida vulturna., Methods: We studied five C. vulturna clinical strains isolated in three Colombian cities. Identification was performed by phenotypical, proteomic and molecular methods. Antifungal susceptibility testing was performed following CLSI protocol. Its ERG11 genes were sequenced and a substitution was encountered in azole resistant isolates. To confirm the role of this substitution in the resistance phenotype, Saccharomyces cerevisiae strains with a chimeric ERG11 gene were created., Results: Discrepancies in identification methods are highlighted. Sequencing confirmed the identification as C. vulturna. Antifungal susceptibility varied among strains, with four strains exhibiting reduced susceptibility to azoles and amphotericin B. ERG11 sequencing showed a point mutation (producing a P135S substitution) that was associated with the azole-resistant phenotype., Conclusions: This study contributes to the understanding of C. vulturna's identification challenges, its susceptibility patterns, and sheds light on its molecular mechanisms of azole resistance., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Background: Bacterial aggregation has been shown to occur in synovial fluid which are resistant to high concentrations of antibiotics. Yet the propensity of Candida spp. to form aggregates is unknown., Objective: To assess the ability of numerous Candida spp. to form synovial fluid aggregates and the clinical ramifications of the aggregates., Methods: Nine different Candidal prosthetic joint infection clinical isolates were evaluated for their ability to form aggregates at static and dynamic conditions and their resistance to high concentrations of amphotericin. Furthermore, the ability of tissue plasminogen activator (TPA) to disrupt the aggregates and enhance amphotericin activity was assessed., Results: The results show that all species of Candida spp. evaluated formed aggregates in synovial fluid under dynamic conditions that were resistant to amphotericin. Yet no aggregates formed in tryptic soy broth under any conditions or in synovial fluid under static conditions. As well, when TPA was combined with amphotericin there was a statistically significant decrease (p < .005) in the amount of colony forming units per mL for all Candidal species evaluated. Interestingly, for Candida krusei there was no colony forming units observed after exposure to TPA and amphotericin., Conclusion: Our findings suggest that Candidal species form synovial fluid aggregates that are resistant to high dose amphotericin similar to those that occur with bacteria. However, the varying ability of the different Candida spp. to form hyphae and pseudohyphae compared to yeast cells may have direct impacts on the hardiness of the aggregates and thereby have clinical ramifications with respect to treatment durations., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Purpose: Due to the potential for Aspergillus species to cause lethal infections and the rising rates of antifungal resistance, the significance of antifungal susceptibility tests has increased. We aimed to assess the sensitivities of Aspergillus species to amphotericin B (AMB), voriconazole (VOR), itraconazole (ITZ), and caspofungin (CAS) using disk diffusion (DD) and gradient diffusion (GD) methods and compare them with broth microdilution (BMD) as the reference susceptibility method., Methods: The study involved 62 Aspergillus fumigatus, 28 Aspergillus flavus, and 16 Aspergillus terreus isolates, totaling 106 Aspergillus isolates. BMD and DD methods were performed in accordance with CLSI M38-A2 and CLSI M51-A documents, respectively. The GD method utilized nonsupplemented Mueller Hinton agar (MHA) as the medium., Results: In the BMD method, the lowest minimal inhibitory concentration (MIC) 90 or minimal effective concentration (MEC) 90 values were observed for VOR and CAS (0.5 μg/mL and 0.06 μg/mL, respectively). AMB and ITZ MIC 90 values were both 2 μg/mL. In our comparison of the GD method with the BMD method at ±2 dilution, we observed essential agreement rates of 91.6%, 99.1%, 100%, and 38.6% for AMB, VOR, ITZ, and CAS, respectively. When comparing DD and BMD methods, we found categorical agreement rates of 65.1%, 99.1%, 77.3%, and 100% for AMB, VOR, ITZ, and CAS, respectively. For GD and BMD methods, these rates were 79.2%, 99.1%, 87.8%, and 100%., Conclusions: Given the high essential and categorical agreement rates, we posit that the GD method is a viable alternative to the BMD method for AMB, ITZ and VOR but not for CAS. In addition, the use of nonsupplemented MHA in the GD method proves advantageous due to its cost-effectiveness and widespread availability compared to other growth media., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)