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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: 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.)

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.)

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.)

The supramolecular-based macrocyclic amphiphiles have fascinating attention and find extensive utilization in the pharmaceutical industry for efficient drug delivery. In this study, we designed and synthesized a new supramolecular amphiphilic macrocycle to serve as an efficient nanocarrier, achieved by treating 4-hydroxybenzaldehyde with 1-bromotetradecane. The derivatized product was subsequently treated with resorcinol to cyclize, resulting in the formation of a calix(4)-resorcinarene-based supramolecular amphiphilic macrocycle. The synthesized macrocycle and intermediate products were characterized using mass spectrometry, IR, and 1 H NMR spectroscopic techniques. The amphotericin-B (Amph-B)-loaded and unloaded amphiphiles were screened for biocompatibility studies, vesicle formation, particle shape, size, surface charge, drug entrapment, in-vitro release profile, and stability through atomic force microscopy (AFM), Zetasizer, HPLC, and FT-IR. Amph-B -loaded macrocycle-based niosomal vesicles were investigated for in-vivo bioavailability in rabbits. The synthesized macrocycle exhibited no cytotoxicity against normal mouse fibroblast cells and was found to be hemocompatible and safe in mice following an acute toxicity study. The drug-loaded macrocycle-based vesicles appeared spherical, nano-sized, and homogeneous in size, with a notable negative surface charge. The vesicles remained stable after 30 days of storage. The results of Amph-B oral bioavailability and pharmacokinetics revealed that the newly tailored niosomal formulation enhanced drug solubility, protected drug degradation at gastric pH, facilitated sustained drug release at the specific target site, and delayed plasma drug clearance. Incorporating such advanced niosomal formulations in the field of drug delivery systems has the potential to revolutionize therapeutic outcomes and improve the quality of patient well-being., Competing Interests: Declaration of Competing Interest The authors claim that there are no known competing financial interests or personal relationships that may have a potential impact on the work described in this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The present work aimed to develop and evaluate AmB-loaded nano-emulsion (AmB-NE) which will augment the solubility of AmB and lead to enhanced anti-leishmanial activity. The composition of AmB-NE was optimized by systematic screening followed by DoE-extreme vertices mixture design. The optimized NE revealed mean droplet size and PDI of 44.19 ± 5.5 nm, 0.265 ± 0.0723, respectively. The NE could efficiently encapsulate AmB with drug content and efficiency 83.509 ± 0.369% and 81.659 ± 0.013%, respectively. The presence of cholesterol and stearyl amine retarded the release (P < 0.0001) of AmB significantly compared to AmB suspension. The AmB-NE and pure AmB suspension demonstrated the IC 50 of 0.06309 μg/mL and 0.3309 μg/mL against L.donovani promastigotes after 48 h incubation. The formulation was robust at all exaggerated stability conditions such as freeze-thaw and centrifugation. These findings indicate that AmB-NE is an attractive approach to treat visceral leishmaniasis with improved activity., 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 © 2023. Published by Elsevier B.V.)

The mechanism of Amphotericin B at the membrane is still subject of debate, with the prevailing hypothesis being the formation of pores. The activity of these pores is influenced by various factors. Recently aggregation in solution and insertion in the membrane had been highlighted as crucial for action of the drug Here we investigated the effect of applied pressure on the activity of Amphotericin B. Our findings demonstrate that applied pressure of 50 mmHg is sufficient to enhance the activity. We interpreted the results as supporting the idea that pressure fractures the membrane and promotes the insertion of the polyene., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Universidad Nacional Autónoma de México. Published by Elsevier B.V. All rights reserved.)

Cutaneous leishmaniasis (CL) is a vector-borne disease characterized by skin lesions that can evolve into high-magnitude ulcerated lesions. Thus, this study aimed to develop an innovative nanoemulsion (NE) with clove oil, Poloxamer® 407, and multiple drugs, such as amphotericin B (AmB) and paromomycin (PM), for use in the topical treatment of CL., Methods: Droplet size, morphology, drug content, stability, in vitro release profile, in vitro cytotoxicity on RAW 264.7 macrophages, and antileishmanial activity using axenic amastigotes of Leishmania amazonensis were assessed for NEs., Results: After optimizing the formulation parameters, such as the concentration of clove oil and drugs, using an experimental design, it was possible to obtain a NE with an average droplet size of 40 nm and a polydispersion index of 0.3, and these parameters were maintained throughout the 365 days. Furthermore, the NE showed stability of AmB and PM content for 180 days under refrigeration (4 °C), presented a pH compatible with the skin, and released modified AmB and PM. NE showed the same toxicity as free AmB and higher toxicity than free PM against RAW 264.7 macrophages. The same activity as free AmB, and higher activity than free PM against amastigotes L. amazonensis., Conclusion: It is possible to develop a NE for the treatment of CL; however, complementary studies regarding the antileishmanial activity of NE should be carried out., 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., (Published by Elsevier B.V.)

Antimicrobial peptides (AMP) represent an alternative in the treatment of fungal infections associated with countless deaths. Here, we report a new AMP, named KWI-19, which was designed based on a peptide encrypted in the sequence of an Inga laurina Kunitz-type inhibitor (ILTI). KWI-19 inhibited the growth of Candida species and acted as a fungicidal agent from 2.5 to 20 μmol L -1 , also showing synergistic activity with amphotericin B. Kinetic assays showed that KWI-19 killed Candida tropicalis cells within 60 min. We also report the membrane-associated mechanisms of action of KWI-19 and its interaction with ergosterol. KWI-19 was also characterized as a potent antibiofilm peptide, with activity against C. tropicalis. Finally, non-toxicity was reported against Galleria mellonella larvae, thus strengthening the interest in all the bioactivities mentioned above. This study extends our knowledge on how AMPs can be engineered from peptides encrypted in larger proteins and their potential as candicidal agents., Competing Interests: Declaration of competing interest The authors declare no competing 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 spread of fungi resistant to conventional drugs has become a threatening problem. In this context, antimicrobial peptides (AMPs) have been considered as one of the main alternatives for controlling fungal infections. Here, we report the antifungal and antibiofilm activity and some clues about peptide RQ18's mechanism of action against Candida and Cryptococcus. This peptide inhibited yeast growth from 2.5 μM and killed all Candida tropicalis cells within 2 h incubation. Moreover, it showed a synergistic effect with antifungal agent the amphotericin b. RQ18 reduced biofilm formation and promoted C. tropicalis mature biofilms eradication. RQ18's mechanism of action involves fungal cell membrane damage, which was confirmed by the results of RQ18 in the presence of free ergosterol in the medium and fluorescence microscopy by Sytox green. No toxic effects were observed in murine macrophage cell lines and Galleria mellonella larvae, suggesting fungal target selectivity. Therefore, peptide RQ18 represents a promising strategy as a dual antifungal and antibiofilm agent that contributes to infection control without damaging mammalian cells., Competing Interests: Declaration of competing interest The authors declare no competing interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Amphotericin B (AmB) is a widely used antifungal agent; however, its clinical application is limited due to severe side effects and nephrotoxicity associated with parenteral administration. In recent years, there has been growing interest in the utilization of food-grade materials as innovative components for nanotechnology-based drug delivery systems. This study introduces gliadin/casein nanoparticles encapsulating AmB (AmB&#95;GliCas NPs), synthesized via antisolvent precipitation. Formulation was refined using a 2 4 factorial design, assessing the influence of gliadin and casein concentrations, as well as organic and aqueous phase volumes, on particle size, polydispersity index (PDI), and zeta potential. The optimal composition with 2 % gliadin, 0.5 % casein, and a 1:5 organic-to-aqueous phase ratio, yielded nanoparticles with a 442 nm size, a 0.307 PDI, a -20 mV zeta potential, and 82 % entrapment efficiency. AmB was confirmed to be amorphous within the nanoparticles by X-ray diffraction. These NPs released AmB sustainably over 96 h, primarily in its monomeric form. Moreover, NPs maintained stability in simulated gastrointestinal fluids with minimal drug release and showed significantly lower hemolytic activity and cytotoxicity on Vero cells than free AmB, suggesting their promise for oral AmB delivery., 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.)

Leishmaniasis is an emerging infectious disease in Thailand, with Leishmania martiniquensis and Leishmania orientalis identified as the primary causative agents among immunocompetent and immunocompromised individuals. Variations in drug susceptibility among different Leishmania species have been reported in different regions. Therefore, drug susceptibility assays are essential to assess the effectiveness of antileishmanial drugs used or potentially used in the affected areas. This study investigated the in vitro drug sensitivity of L. martiniquensis and L. orientalis, along with two reference species causing VL, namely L. donovani and L. infantum, against six antileishmanial drugs. Using a parasite-rescue and transformation assay, the results demonstrated that the IC 50 values of amphotericin B (AmB), miltefosine (MIL), and sodium stibogluconate (Sb(III)) against all Leishmania species tested were within the sensitive range of each drug. On the contrary, the IC 50 values of artemisinin (ART) and dihydroartemisinin (DHA), drugs primarily used for malaria treatment, were outside the sensitive range of the Leishmania species tested except L. infantum. This in vitro study highlights that AmB could effectively exhibit good sensitivity against the intracellular amastigotes of L. martiniquensis and L. orientalis. Also, MIL and Sb(III) could be considered alternative drugs for antileishmanial treatment in Thailand., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objectives: Diutina (Candida) catenulata is an ascomycetous yeast isolated from environmental sources and animals, occasionally infecting humans. The aim of this study is to shed light on the in vitro antifungal susceptibility and genetic diversity of this opportunistic yeast., Methods: Forty-five D. catenulata strains isolated from various sources (including human and environmental sources) and originating from nine countries were included. Species identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and confirmed via internal transcribed spacer ribosomal DNA barcoding. In vitro antifungal susceptibility was determined for seven systemic antifungals via the gradient strip method after 48 hours of incubation at 35°C using Etest® (Biomérieux) or Liofilchem® strips. Isolates exhibiting fluconazole minimal inhibitory concentrations (MICs) of ≥8 μg/mL were investigated for mutations in the ERG11 gene. A novel microsatellite genotyping scheme consisting of four markers was developed to assess genetic diversity., Results: MIC ranges for amphotericin B, caspofungin, micafungin, isavuconazole, and posaconazole were 0.19-1 μg/mL, 0.094-0.5 μg/mL, 0.012-0.064 μg/mL, 0.003-0.047 μg/mL, and 0.006-0.032 μg/mL, respectively. By comparison, a broad range of MICs was noted for fluconazole (0.75 to >256 μg/mL) and voriconazole (0.012-0.38 mg/L), the higher values being observed among clinical strains. The Y132F amino acid substitution, associated with azole resistance in various Candida species (C. albicans, C. tropicalis, C. parapsilosis, and C. orthopsilosis), was the main substitution identified. Although microsatellite typing showed extensive genetic diversity, most strains with high fluconazole MICs clustered together, suggesting human-to-human transmission or a common source of contamination., Discussion: The high rate of acquired fluconazole resistance among clinical isolates of D. catenulata is of concern. In this study, we highlight a link between the genetic diversity of D. catenulata and its antifungal resistance patterns, suggesting possible clonal transmission of resistant isolates., (Copyright © 2022 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Purpose: To investigate the dematiaceous fungal profile of patients with ocular mycoses attending a tertiary eye care hospital in Coimbatore, India METHODS: The identification of dematiaceous fungus based on their morphology, their genotypes, and the measurement of the minimum inhibitory concentrations (MICs) using microdilution method of routinely used antifungal drugs were all compared., Results: A total of 148 dematiaceous fungi were isolated during a study period of 27 months. Isolates were confirmed as Curvularia spp. (n = 98), Exserohilum spp. (n = 32), Alternaria spp. (n = 14), Exophiala spp. (n = 2), Cladosporium sp. (n = 1) and Aureobasidium sp. (n = 1). Out of 50 well grown isolates characterized genotypically based on the amplification and sequencing of the ITS region of the ribosomal RNA gene cluster and subsequent BLAST analysis, Curvularia lunata (n = 24), C. aeria (n = 1), C. spicifera (n = 8), C. hawaiiensis (n = 1), C. maydis (n = 2), C. papendorfii (n = 2), C. geniculata (n = 3), C. tetramera (n = 2) and Exs. rostratum (n = 7) were identified. In vitro antifungal susceptibilities of the most tested dematiaceous isolates showed that voriconazole had a MIC 50 of 0.25 μg ml -1 , while amphotericin B had a MIC 50 of 0.25 μg ml -1 for Curvularia spp. and Alternaria spp., Conclusion: Voriconazole proved to be the most effective drug against the pigmented filamentous fungi, followed by amphotericin B, itraconazole and econazole., Competing Interests: Declaration of interest The authors declare no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Visceral leishmaniasis or Kala-azar is a vector-borne disease caused by an intracellular parasite of the genus leishmania. In India, Amphotericin B (AmB) is a first-line medication for treating leishmaniasis. After a large-scale resistance to pentavalent antimony therapy developed in Bihar state, it was rediscovered as an effective treatment for Leishmania donovani infection. AmB which binds to the ergosterol of protozoan cells causes a change in membrane integrity resulting in ions leakage, and ultimately leading to cell death. The treatment effect of liposomal AmB can be seen more quickly than deoxycholate AmB because, it has some toxic effects, but liposomal AmB is significantly less toxic. Evidence from studies suggested that ABLC (Abelcet) and ABCD (Amphotec) are as effective as l-AmB but Liposomal form (Ambisome) is a more widely accepted treatment option than conventional ones. Nevertheless, the world needs some way more efficient antileishmanial drugs that are less toxic and less expensive for people living with parasitic infections caused by Leishmania. So, academics, researchers, and sponsors need to focus on finding such drugs. This review provides a summary of the chemical, pharmacokinetic, drug-target interactions, stability, dose efficacy, and many other characteristics of the AmB and their various formulations. We have also highlighted the clinically significant aspects of PKDL and VL co-infection with HIV/TB., Competing Interests: Declaration of Competing Interest The authors state that there were no conflicting interests that may be seen as a potential conflict of interest when conducting the review., (Copyright © 2022. Published by Elsevier B.V.)

Objectives: New antifungal drugs, such as olorofim, may overcome the problem of resistance in Aspergillus fumigatus. We here report the activity of olorofim against a set of A. fumigatus sensu lato recently collected in Spain., Methods: A total of 332 A. fumigatus sensu lato clinical isolates collected in a multicentre study conducted in Spain in 2019 and comprising susceptible and resistant isolates to azoles and/or amphotericin B were tested. Isolates distributed among the following species: A. fumigatus sensu stricto (n = 312), Aspergillus lentulus (n = 6), Aspergillus fumigatiaffinis (n = 5), Neosartorya tsurutae (n = 3), Neosartorya udagawae (n = 3), Aspergillus novofumigatus (n = 2), and Aspergillus thermomutatus (n = 1). Azole resistance was found in 44 A. fumigatus sensu stricto isolates that harboured the following cyp51A gene substitutions: TR 34 -L98H (n = 24), G54 (n = 5), TR 46 /Y121F/T289A (n = 1), other mutations (n = 4), and gene wild type (n = 10). Isolates were tested for antifungal susceptibility to olorofim using European Committee on Antimicrobial Susceptibility Testing (EUCAST) E.Def. 9.4 methodology., Results: Olorofim minimum inhibitory concentrations against A. fumigatus sensu stricto isolates ranged from 0.008 to 0.125 mg/L and in vitro activity of the drug was not impacted by the presence of azole/amphotericin B resistance. Azole resistance and amphotericin B resistance was found in 18 and 13 cryptic species isolates, respectively. Olorofim showed high in vitro activity against cryptic species isolates and minimum inhibitory concentrations ranged from 0.004 to 0.016 mg/L, regardless of the presence of resistance to other drugs., Discussion: Olorofim showed in vitro activity against both A. fumigatus sensu stricto and cryptic species clinical isolates and was active against isolates showing resistance to azoles and/or amphotericin B., (Copyright © 2022 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objective: Candida auris has emerged as a health-care-associated and multidrug-resistant fungal pathogen of great clinical concern. As many as 50% of C. auris clinical isolates are reported to be resistant to amphotericin B, but no mechanisms contributing to this resistance have been identified. Here we describe a clinical case in which high-level amphotericin B resistance was acquired in vivo during therapy and undertake molecular and genetic studies to identify and characterize the genetic determinant of resistance., Methods: Whole-genome sequencing was performed on four C. auris isolates obtained from a single patient case. Cas9-mediated genetic manipulations were then used to generate mutant strains harbouring mutations of interest, and these strains were subsequently subjected to amphotericin B susceptibility testing and comprehensive sterol profiling., Results: A novel mutation in the C. auris sterol-methyltransferase gene ERG6 was found to be associated with amphotericin B resistance, and this mutation alone conferred a >32-fold increase in amphotericin B resistance. Comprehensive sterol profiling revealed an abrogation of ergosterol biosynthesis and a corresponding accumulation of cholesta-type sterols in isolates and strains harbouring the clinically derived ERG6 mutation., Conclusions: Together these findings definitively demonstrate mutations in C. auris ERG6 as the first identified mechanism of clinical amphotericin B resistance in C. auris and represent a significant step forward in the understanding of antifungal resistance in this emerging public health threat., (Copyright © 2021 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to characterize the components of the Mycobacterium abscessus massiliense cell envelope and their interactions with amphotericin B (AmB), miltefosine (MIL), and nerolidol (NER). Spin labels analogous to stearic acid and phosphatidylcholine (PC) were distributed on an envelope layer with fluidity comparable to other biological membranes, probably the mycobacterial cell wall, because after treatment with AmB a highly rigid spectral component was evident in the EPR spectra. Methyl stearate analogue spin labels found a much more fluid membrane and did not detect the presence of AmB, except for at very high drug concentrations. Unlike other spin-labeled PCs, the TEMPO-PC spin probe, with the nitroxide moiety attached to the choline of the PC headgroup, also did not detect the presence of AmB. On the other hand, the steroid spin labels were not distributed across the membranes of M. abscessus and, instead, were concentrated in some other location of the cell envelope. Both MIL and NER compounds at 10 μM caused increased fluidity in the cell wall and plasma membrane. Furthermore, NER was shown to have a remarkable ability to extract lipids from the mycobacterial cell wall. The EPR results suggest that the resistance of mycobacteria to the action of AmB must be related to the fact that this drug does not reach the bacterial plasma membrane., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Introduction: Amphotericin B (AmB), used for systemic fungal infections, has a limited clinical application because of its high nephrotoxicity. Natural antioxidant and anti-inflammatory substances have been widely studied for protection against drug-induced nephrotoxicity. α-Bisabolol (BIS) has demonstrated a nephroprotective effect on both in vitro and in vivo models., Aims: The aim of this work was to evaluate the effect of BIS against AmB-induced nephrotoxicity in vitro., Material and Methods: LLC-MK2 cells were pre- and post-treated with non-toxic BIS concentrations and/or AmB IC50 (13.97 μM). Cell viability was assessed by MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] assay. Flow cytometry analyses were used to assess cell death mechanism, production of reactive oxidative stress (ROS) and mitochondrial transmembrane potential. Kidney Injury Molecule-1 (KIM-1) levels were measured via ELISA., Key Findings: The present work showed that BIS pretreatment (125; 62.5 and 31.25 μM) increased cell viability when compared to the group treated only with AmB IC50. AmB treatment induced both necrosis (7-AAD-labeled cells) and late apoptosis (AnxV-labeled). BIS was able to prevent the occurrence of these events. These effects were associated with a decrease of ROS accumulation, improving transmembrane mitochondrial potential and protecting against tubular cell damage, highlighted by the inhibition of KIM-1 release after BIS treatment., Significance: BIS presented a potential effect on model of renal cytotoxicity induced by AmB, bringing perspectives for the research of new nephroprotective agents., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Leishmaniasis is an emerging disease in several countries over the world, especially in tropical regions. In Thailand, Leishmania (Mundinia) martiniquensis is the most frequent cause of visceral leishmaniasis and disseminated cutaneous leishmaniasis among HIV/AIDs patients. Amphotericin B (AmB) is the only drug currently available for the treatment of leishmaniasis in Thailand, but has some limitations like high renal toxicity and the prolonged hospitalization required for the treatment. Moreover, recurrence of the disease has been reported in several cases, indicating that new drugs or treatment strategies should be improved. In this study, Artesunate (ARS) was determined for anti-Leishmania activity against L. martiniquensis in promastigotes and amastigotes. In addition, the combination effects of ARS and AmB against intracellular amastigotes on THP-1 derived macrophages were also investigated for the first time. The result showed that L. martiniquensis was susceptible to ARS in both stages of the parasite. ARS was effective against intracellular amastigotes and safe to macrophage host cells, showing a SI value of 1,065. Furthermore, combination effects of ARS and AmB showed five synergistic combinations with a combination index (CI) value less than 1.0 (0.28-0.92) for intracellular amastigotes ranging from slight synergism to strong synergism. The strong synergistic combination had the highest dose reduction index (DRI), approximately a 9.7-fold reduction in AmB used. None of the treatments in combination had noticeable toxicity to THP-1 derived macrophages in the concentration range examined. The data provided in this study lead to further study in vivo and to develop a novel formulation of drug combinations to improve the outcome of leishmaniasis treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Two platforms based on reduced graphene oxide (rGO) functionalized with Pluronic® P123 (rGO-P123) and polyethyleneimine - PEI (rGO-PEI) polymers and loaded with amphotericin B (AmB) were fabricated and tested against Leishmania amazonensis, which can cause cutaneous and diffuse cutaneous leishmaniasis. The materials rGO-P123 and rGO-PEI were efficiently loaded with AmB - a polyene antibiotic - which resulted in rGO-P123-AmB (0.078 mg per mg of material) and rGO-PEI-AmB (0.086 mg per mg of material). Under near-infrared (NIR) light irradiation, the amount of AmB released from rGO-PEI-AmB at pH 5.0 and 7.4 doubled in comparison to AmB released in the absence of NIR light under identical conditions. It was accompanied by a photothermal effect. Otherwise, rGO-P123-AmB did not show a significant change in AmB released in the presence and absence of NIR light. Cytotoxicity studies in mammalian host macrophages revealed that rGO-PEI and rGO-PEI-AmB were nontoxic to the host cells, whereas rGO-123 and rGO-P123-AmB were very toxic, particularly the latter. Therefore, only rGO-PEI and rGO-PEI-AmB were tested against L. amazonensis promastigotes in the presence and absence of NIR light. In vitro antiproliferative effects revealed that rGO-PEI-AmB showed a more pronounced activity against the parasite than rGO-PEI, which was improved under NIR light irradiation. Scanning-transmission electron microscopy of L. amazonensis promastigotes after incubation with rGO-PEI or rGO-PEI-AmB suggested autophagic and necrotic cell death. Thus, the facile synthesis, high AmB loading capacity and good photothermal effect make the rGO-PEI-AmB platform a promising candidate for the topical treatment of cutaneous leishmaniasis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Acanthamoeba spp. are the causative agents of a sight-threatening infection of the cornea known as Acanthamoeba keratitis (AK). Amphotericin B - deoxycholate (AB) is used in the treatment of infectious keratitis, however, its topical administration has side effects as blepharitis, iritis, and painful instillation. In this context, the preheating of AB can decrease its toxicity by the formation of super aggregates (hAB). hAB associated with a thermoreversible in situ gelling ophthalmic system is a promising option due to the latter biocompatibility, low toxicity, and high residence time on the ocular surface. Our objective was to develop a topical ocular formulation of hAB for the treatment of AK. After heating at 70°C for 20 min, hAB was incorporated into a thermoreversible gelling system. The amebicidal activity of AB and hAB was evaluated against trophozoites and cysts of A. castellanii (ATCC 50492) and a regional clinical isolate (IC01). The results showed that the preheating of AB did not change the pharmacological action of the drug, with the amebicidal effect of AB and hAB under trophozoites and cysts of Acanthamoeba spp. The thermoreversible system remained stable, allowing the increase of drug retention time. For assessment of cytotoxicity, HUVEC (ATCC® CRL-1730) cells were challenged with AB and hAB for 48h. Cell viability was assessed, and hAB did not show cytotoxicity for HUVEC cells. As far as we know this was the first study that showed the preheated AB associated with a thermoreversible in situ gelling ophthalmic system as a promising system for topical ocular topical administration of hAB for AK therapy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objectives: To investigate the in vitro activity of nitroxoline against a molecularly characterized collection of clinical Candida auris isolates., Methods: Thirty-five clinical isolates of C. auris from diverse sources representing all five different C. auris clades were included in the study. Nitroxoline activity was assessed using broth microdilution. Additionally, susceptibility testing by disc diffusion was assessed on RPMI-1640 and Müller-Hinton agar plates. Minimal inhibitory concentrations of the antifungals fluconazole, voriconazole, amphotericin B and anidulafungin were determined., Results: Nitroxoline MICs ranged from 0.125 to 1 mg/L (MIC 50/90 0.25/0.5 mg/L). Compared with amphotericin B (MIC >1 mg/L in 4/35 isolates), anidulafungin (MIC >0.06 mg/L in 26/35 isolates) and fluconazole (MIC >4 mg/L in 31/35 isolates), in vitro activity of nitroxoline was high. Isolates belonging to clade I had marginally lower nitroxoline MICs (range 0.125-0.5 mg/L, mean MIC 0.375 mg/L) compared with clade III (range 0.5-1 mg/L, mean MIC 0.7 mg/L; p = 0.0094). The correlation of MIC and inhibition zones by disc diffusion was good when using RPMI-agar for disc diffusion, with a Pearson's correlation coefficient of -0.74 (95% CI -0.86 to -0.54)., Conclusions: Nitroxoline has excellent in vitro activity against C. auris isolates, with MICs of 0.125-1 mg/L (for comparison, the EUCAST breakpoint for uncomplicated urinary tract infection with Escherichia coli is ≤ 16 mg/L). It is an approved, well-tolerated antimicrobial that achieves high urinary concentrations after oral administration and could be a useful treatment option in C. auris candiduria., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In the past two decades, the increase in microbial resistance to conventional antimicrobials has spurred scientists around the world to search tirelessly for new treatments. Synthetic amino acid-based surfactants constitute a promising alternative to conventional antimicrobial compounds. In this work, two new cationic amino acid-based surfactants were synthesized and their physicochemical, antifungal and antibiofilm properties evaluated. The surfactants were based on phenylalanine-arginine (LPAM) and tryptophan-arginine (LTAM) and prepared from renewable raw materials using a simple chemical procedure. The critical micelle concentrations of the new surfactants were determined by conductivity and fluorescence. Micellization of LPAM and LTAM took place at 1.05 and 0.54 mM, respectively. Both exhibited good antifungal activity against fluconazole-resistant Candida spp. strains, with a low minimum inhibitory concentration (8.2 μg/mL). Their mechanism of action involves alterations in cell membrane permeability and mitochondrial damage, leading to death by apoptosis. Furthermore, when LPAM and LTAM were applied with Amphotericin B, a significant synergistic effect was observed against all the studied Candida strains. These new cationic surfactants are also able to disperse biofilms of Candida spp. at low concentrations. The results indicate that LPAM and LTAM have potential application to combat the advance of fungal resistance as well as microbial biofilms., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cutaneous leishmaniasis (CL) is a significant public health problem caused by different species of Leishmania parasites. Due to low skin permeability, the development of an effective system for delivery of Amphotericin B (AMB), the common effective drug for leishmaniasis treatment, is required to replace the unpleasant and problematic injections. To overcome this problem, a dissolvable microneedle (MN) patch was developed, using biodegradable polymers (a mixture of polyvinylpyrrolidone and carboxymethyl cellulose) for AMB's transdermal delivery. Scanning electron microscopy and fluorescent images showed successful fabrication of the MNs and homogeneous dispersion of the drug into the needles. MNs showed good mechanical properties with the ability to penetrate the rat skin and reach the lower layers. After insertion to the skin, the MNs were rapidly dissolved to release the encapsulated drug, and the resulted micropores in the skin were quickly resealed within 30 min. MN patches showed non-toxicity as exposed to HT-29 cell line. Flow cytometry results showed a potent in vitro leishmanicidal activity of AMB-loaded MN patches against the Leishmania parasites (up to 86% of the parasites' death). Taken together, MN patches might represent a new, efficient and clinically translational approach for transdermal AMB delivery to treat CL., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background & Aims: In the last decade, the role of the microbiota-gut-brain axis in eating behavior and anxiety-depressive disorders has gained increasing attention. Although a gut microbiota dysbiosis has been reported in anorectic patients, its pathophysiological role remains poorly understood. Thus, we aimed to characterize the potential role of gut microbiota by evaluating the effects of its depletion in the Activity-Based Anorexia (ABA) mouse model both in male and female mice., Methods: Male and female C57Bl/6 mice were submitted (ABA group) or not (CT group) to the ABA protocol, which combines access to a running wheel with a progressive limited food access. Gut microbiota was previously depleted or not by a cocktail of antibiotics (ATB) delivered by oral gavages. We monitored body composition, anxiety-like behavior, leptin and adiponectin plasma levels, hypothalamic and hippocampal neuropeptides mRNA levels, as well as dopamine (DRD) and serotonin (5HT1 and 4) receptors mRNA expression., Results: In response to the ABA model, the body weight loss was less pronounced in ATB-treated ABA compared to untreated ABA, while food intake remained unaffected by ATB treatment. ATB-treated ABA exhibited increased fat mass and decreased lean mass compared to untreated ABA both in male and female mice, whereas but plasma adipokine concentrations were affected in a sex-dependent manner. Only male ABA mice showed a reduced anticipatory physical activity in response to ATB treatment. Similarly, anxiety-like behavior was mainly affected in ATB-treated ABA male mice compared to ATB-treated ABA female mice, which was associated with male-specific alterations of hypothalamic CRH mRNA and hippocampal DRD and 5-HT1A mRNA levels., Conclusions: Our study provides evidence that ATB-induced gut microbiota depletion triggers alterations of nutritional and behavioral responses to the activity-based anorexia model in a sex-dependent manner., (Copyright © 2021 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Background: Approximately 10% of people with cystic fibrosis (CF) have mutations that result in little to no CFTR production and thus cannot benefit from CFTR modulators. We previously found that Amphotericin B (AmB), a small molecule that forms anion channels, restored HCO 3 -  secretion and increased host defenses in primary cultures of CF airway epithelia. Further, AmB increased ASL pH in CFTR-null pigs, suggesting an alternative CFTR-independent approach to achieve gain-of-function. However, it remains unclear whether this approach can be effective in people., Methods: To determine whether AmB can impact physiology in people with CF, we first tested whether Fungizone, a clinically approved AmB formulation, could cause electrophysiological effects consistent with anion secretion in primary cultures of CF airway epithelia. We then evaluated the capacity of AmB to change nasal potential difference (NPD), a key clinical biomarker, in people with CF not on CFTR modulators., Results: AmB increased transepithelial Cl - current and hyperpolarized calculated transepithelial voltage in primary cultures of CF airway epithelia from people with two nonsense mutations. In eight people with CF not on CFTR modulators, intranasal Fungizone treatment caused a statistically significant change in NPD. This change was similar in direction and magnitude to the effect of ivacaftor in people with a G551D mutation., Conclusions: Our results provide the first evidence that AmB can impact a clinical biomarker in people with CF. These results encourage additional clinical studies in people with CF to determine whether small molecule anion channels can provide benefit., Competing Interests: Declaration of Competing Interest R.S.C., M.J.W., and M.D.B. are inventors on patent applications PCT/US15/58806, PCT/US18/55435, PCT/US2017/26806, and/or PCT/US20/18/055435, submitted by UIUC, which cover use of AmB to treat CF. cystetic Medicines, a company for which M.J.W. and M.D.B. are founders and consultants, has licensed these patent applications., (Copyright © 2020. Published by Elsevier B.V.)

The use of natural antimicrobial peptides (AMPs) is limited. Modifications of peptides by in silico predictions and computational methods can lead to more accurate designs and reducing their high synthesis costs, instability, and cytotoxicity. In this study, the antifungal properties of CecropinA-Magenin2 (CE-MA) hybrid peptide and its truncated derivatives were evaluated. Eleven C-terminal-truncated derivatives were designed and three of them with 10, 8 and 6 residues namely CMt1, CMt2 and CMt3 were selected through an initial screening based on the prediction of antimicrobial and antifungal activities, toxicity and physicochemical properties. These derivatives and the parental CE-MA peptide were synthesized. Then, based on molecular docking studies, antimicrobial tests and cytotoxicity assays, CMt1 peptide was selected for further studies such as time of killing, combinatorial effects with other drugs and the mechanism of action. The results showed that CE-MA is a weak antifungal peptide but its truncated derivative, CMt1 showed a strong antifungal activity with less toxicity. The results of the ergosterol assay, confocal microscopy and FE-SEM studies indicated that invasion to cell wall and membrane components were the main antifungal mechanisms of CMt1 peptide. Altogether, here we introduce a new truncated peptide with a strong antifungal activity with less toxicity which can be a good candidate for further in vivo and clinical studies to be used as an antifungal drug., Competing Interests: Declaration of competing interest The authors confirm that this article content has no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Brain lesions caused by Cryptococcus neoformans or C. gattii (cryptococcomas) are typically difficult to diagnose correctly and treat effectively, but rapid differential diagnosis and treatment initiation are crucial for good outcomes. In previous studies, cultured cryptococcal isolates and ex vivo lesion material contained high concentrations of the virulence factor and fungal metabolite trehalose. Here, we studied the in vivo metabolic profile of cryptococcomas in the brain using magnetic resonance spectroscopy (MRS) and assessed the relationship between trehalose concentration, fungal burden, and treatment response in order to validate its suitability as marker for early and noninvasive diagnosis and its potential to monitor treatment in vivo. We investigated the metabolites present in early and late stage cryptococcomas using in vivo 1 H MRS in a murine model and evaluated changes in trehalose concentrations induced by disease progression and antifungal treatment. Animal data were compared to 1 H and 13 C MR spectra of Cryptococcus cultures and in vivo data from 2 patients with cryptococcomas in the brain. In vivo MRS allowed the noninvasive detection of high concentrations of trehalose in cryptococcomas and showed a comparable metabolic profile of cryptococcomas in the murine model and human cases. Trehalose concentrations correlated strongly with the fungal burden. Treatment studies in cultures and animal models showed that trehalose concentrations decrease following exposure to effective antifungal therapy. Although further cases need to be studied for clinical validation, this translational study indicates that the noninvasive MRS-based detection of trehalose is a promising marker for diagnosis and therapeutic follow-up of cryptococcomas., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In this report, we discuss the design of a novel collagen/pectin (CP) hybrid composite hydrogel (CPBG) containing in-situ mineralized bioactive glass (BG) particles to simulate an integrative 3D cell environment. Systematic analysis of the CP sol revealed collagen and pectin molecules interacted regardless of both possessing similar net negative charge through the mechanism of surface patch binding interaction. Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) confirmed this associative interaction which resulted in the formation of a hybrid crosslinked network with the BG nanoparticles acting as pseudo crosslink junctions. Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray Analysis (EDAX) and Transmission Electron Microscopy (TEM) results confirmed uniform mineralization of BG particles, and their synergetic interaction with the network. The in-vitro bioactivity tests on CPBG indicated the formation of bone-like hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ) microcrystals on its surface after interaction with simulated body fluid. This hydrogel was loaded with a model antifungal drug amphotericin-B (AmB) and tested against Candida albicans. The AmB release kinetics from the hydrogel followed the Fickian mechanism and showed direct proportionality to gel swelling behavior. Rheological analysis revealed the viscoelastic compatibility of CPBG for the mechanical load bearing applications. Cell viability tests indicated appreciable compatibility of the hydrogel against U2OS and HaCaT cell lines. FDA/PI on the hydrogel portrayed preferential U2OS cell adhesion on hydrophobic hydroxyapatite layer compared to hydrophilic surfaces, thereby promising the regeneration of both soft and hard tissues., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Independent studies from our group and others have provided evidence that sphingolipids (SLs) influence the antimycotic susceptibility of Candida species. We analyzed the molecular SL signatures of drug-resistant clinical isolates of Candida auris, which have emerged as a global threat over the last decade. This included Indian hospital isolates of C. auris, which were either resistant to fluconazole (FLC R ) or amphotericin B (AmB R ) or both drugs. Relative to Candida glabrata and Candida albicans strains, these C. auris isolates were susceptible to SL pathway inhibitors such as myriocin and aureobasidin A, suggesting that SL content may influence azole and AmB susceptibilities. Our analysis of SLs confirmed the presence of 140 SL species within nine major SL classes, namely the sphingoid bases, Cer, αOH-Cer, dhCer, PCer, αOH-PCer, αOH-GlcCer, GlcCer, and IPC. Other than for αOH-GlcCer, most of the SLs were found at higher concentrations in FLC R isolates as compared to the AmB R isolates. SLs were at intermediate levels in FLC R  + AmB R isolates. The observed diversity of molecular species of SL classes based on fatty acyl composition was further reflected in their distinct specific imprint, suggesting their influence in drug resistance. Together, the presented data improves our understanding of the dynamics of SL structures, their synthesis, and link to the drug resistance in C. auris., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

Despite the advancement of new anti-leishmanials, amphotericin B (AmB) prevails as one of the most potent agent in the treatment of visceral leishmaniasis (VL), a neglected tropical disease affecting mostly poverty ridden and underdeveloped regions of the globe. Nonetheless, many patients display intolerance to parenteral AmB, notably at higher dosages. Also, conventional AmB presents an apparently poor absorption. Therefore, to improve AmB bioavailability and overcome multiple barriers for oral delivery of AmB, we fabricated a promising vitamin B 12 -stearic acid (VBS) conjugate coated solid lipid nanoparticles (SLNs) encapsulated with AmB (VBS-AmB-SLNs) by a combination of double emulsion solvent evaporation and thermal sensitive hydrogel techniques. VBS-AmB-SLNs showed a particle size of 306.66 ± 3.35 nm with polydispersity index of 0.335 ± 0.08 while the encapsulation efficiency and drug loading was observed to be 97.99 ± 1.6% and 38.5 ± 5.6% respectively. In vitro drug release showed a biphasic release pattern and chemical stability of AmB was ensured against simulated gastrointestinal fluids. Cellular uptake studies confirmed complete internalization of the formulation. Anti-leishmanial evaluation against intramacrophage amastigotes showed an enhanced efficacy of 94% which was significantly (P < 0.01) higher than conventional AmB without showing any toxic effects on J774A.1 cells. VBS-AmB-SLNs could serve as a potential therapeutic strategy against VL., 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 © 2020 Elsevier B.V. All rights reserved.)

Background: EUCAST has revised the definition of the susceptibility category I from 'Intermediate' to 'Susceptible, Increased exposure'. This implies that I can be used where the drug concentration at the site of infection is high, either because of dose escalation or through other means to ensure efficacy. Consequently, I is no longer used as a buffer zone to prevent technical factors from causing misclassifications and discrepancies in interpretations. Instead, an Area of Technical Uncertainty (ATU) has been introduced for MICs that cannot be categorized without additional information as a warning to the laboratory that decision on how to act has to be made. To implement these changes, the EUCAST-AFST (Subcommittee on Antifungal Susceptibility Testing) reviewed all, and revised some, clinical antifungal breakpoints., Objectives: The aim was to present an overview of the current antifungal breakpoints and supporting evidence behind the changes., Sources: This document is based on the ten recently updated EUCAST rationale documents, clinical breakpoint and breakpoint ECOFF documents., Content: The following breakpoints (in mg/L) have been revised or established for Candida species: micafungin against C. albicans (ATU = 0.03); amphotericin B (S ≤/> R = 1/1), fluconazole (S ≤/> R = 2/4), itraconazole (S ≤/> R = 0.06/0.06), posaconazole (S ≤/> R = 0.06/0.06) and voriconazole (S ≤/> R = 0.06/0.25) against C. dubliniensis; fluconazole against C. glabrata (S ≤/> R = 0.001/16); and anidulafungin (S ≤/> R = 4/4) and micafungin (S ≤/> R = 2/2) against C. parapsilosis. For Aspergillus, new or revised breakpoints include itraconazole (ATU = 2) and isavuconazole against A. flavus (S ≤/> R = 1/2, ATU = 2); amphotericin B (S ≤/> R = 1/1), isavuconazole (S ≤ /> R = 1/2, ATU = 2), itraconazole (S ≤/> R = 1/1, ATU = 2), posaconazole (ATU = 0.25) and voriconazole (S ≤/> R = 1/1, ATU = 2) against A. fumigatus; itraconazole (S ≤/> R = 1/1, ATU = 2) and voriconazole (S ≤/> R = 1/1, ATU = 2) against A. nidulans; amphotericin B against A. niger (S ≤/> R = 1/1); and itraconazole (S ≤/> R = 1/1, ATU = 2) and posaconazole (ATU = 0.25) against A. terreus., Implications: EUCAST-AFST has released ten new documents summarizing existing and new breakpoints and MIC ranges for control strains. A failure to adopt the breakpoint changes may lead to misclassifications and suboptimal or inappropriate therapy of patients with fungal infections., (Copyright © 2020. Published by Elsevier Ltd.)

The present study provides comprehensive proteomics analyses of the response of L. donovani parasite to pharamacological stress in vitro. Identification of differentially expressed proteins with associated molecular functions and metabolic pathways, clearly provides an insight into the potential mechanism of the antileishmanial effects as well as a comparative response of the parasite to Guggul and AmB. Treatment of parasite with AmB results in an enhanced modulatory mechanism to counteract the drug induced stress which may have contributed to relapse. In the case of Guggul treatment, an effective antipromastigote activity was observed, which is being reported for the first time. Thus, a deeper understanding of the molecular pathways in the Leishmania parasite in response to pharmacological stress would help in designing novel and effective strategies in targeting the key molecules essential for parasite survival. It will also help in screening of new lead molecules targeting these vital pathways which could be used as an adjunct therapy along with the limited repertoire of antileishmanial drugs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The most effective medicines available for the treatment of leishmaniasis, a life-threatening disease, exhibit serious toxicological issues. To achieve better therapeutic efficiency while decreasing toxicity associated with amphotericin B (AmB), water-soluble dextrin-AmB (Dex-AmB) formulations were developed. Self-assembled nanocomplexes were formed by dissolving Dex and AmB in alkaline borate buffer, followed by dialysis and either freeze-drying (FD) or nano spray-drying (SD), yielding water dispersible particles with a diameter of 214 nm and 347 nm, respectively. The very simple production process allowed the formation of amorphous inclusion complexes containing 14% of AmB in the form of monomers and water-soluble aggregates. Nanocomplexes were effective against parasites in axenic culture (IC 50 of 0.056 and 0.096 μM for L. amazonensis and 0.030 and 0.044 μM for L. infantum, respectively for Dex-AmB FD and Dex-AmB SD) and in decreasing the intramacrophagic infection with L. infantum (IC 50 of 0.017 and 0.023 μM, respectively for Dex-AmB FD and Dex-AmB SD). Also, the formulations were able to significantly reduce the cytotoxicity of AmB. Overall, this study demonstrates the suitability of dextrin as an AmB carrier and the facile and inexpensive development of a delivery system for the treatment of leishmaniasis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Bloodstream Candida infection is a life-threatening event among ICU admitted patients. This infection is caused by a diverse range of Candida species having varied minimum inhibitory concentrations., Objectives: To identify Candida species causing bloodstream infections with their antifungal susceptibility determination., Methods: Candida species isolated from the blood of ICU admitted patients were identified by phenotypic as well as by molecular methods including PCR-RFLP using MspI restriction enzyme and MALDI TOF MS. The minimum inhibitory concentration of fluconazole, voriconazole, amphotericin B and caspofungin was determined against isolated Candida species by CLSI M27A 3 guidelines., Results: A total of 119 Candida species were isolated. Among them, C. tropicalis(n=29) was the predominant isolate followed by C. parapsilosis(n=18), C. glabrata (n=12), C. krusei (n=11) and C. albicans(n=11). Uncommon Candida species isolated were; Wickerhamomyces anomalus(n=15), Kodaemia ohmeri(n=8), C. lusitaniae (n=5) and C. auris (n=2). A varied antifungal MIC values were observed. Caspofungin had the lowest MIC among the tested antifungals. Increased fluconazole MIC was observed against the isolated Candida species including C. tropicalis. All the isolated C. lusitaniae and C. auris strains have ≥1mcg/ml amphotericin B MIC. In comparison to fluconazole, voriconazole was more effective when tested in vitro., Conclusion: Emergence of uncommon Candida species having varied antifungal MIC warns the physicians to have a prompt, accurate identification with antifungal MIC determination in each case of bloodstream Candida infections., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Leishmaniasis is a widespread tropical infection caused by different species of Leishmania protozoa. Many of the available drugs against the disease are toxic and in certain cases parasite drug resistance is developed. The discovery of drugs for the treatment of leishmaniasis is a pressing concern. In the present work, we describe in vitro studies of the phenolic compound methyl gallate (MG) against Leishmania (Leishmania) amazonensis and its possible mechanisms of action. The in vitro activity of MG was assayed against L. amazonensis (promastigotes, axenic amastigotes, and intramacrophagic amastigotes). Cytotoxicity tests were performed with J774A.1 macrophages and THP-1 cell derived macrophages. To evaluate mechanisms of action, we analyzed cellular TNF-α, IL-12, IFN-γ, IL-10, IL-6, NO, ROS levels, arginase activity, and structural mechanisms (phagocytic and lysosomal activities) involving macrophage activation. Meglumine antimoniate and amphotericin B were used as reference drugs. It was observed that MG effectively inhibited the growth of both promastigote (IC 50 5.71 μM) and amastigote-like forms (EC 50 5.39 μM), with much higher selectivity indexes than the reference drugs, being more benign towards J774A.1 macrophages than meglumine antimoniate and amphotericin B, at 1631- and 70.92-fold respectively, with respect to the promastigote form. Additionally, MG proved to be even more active against intracellular amastigotes of the parasite (EC 50 4.24 μM). Our results showed that antileishmania activity was associated with increased TNF-α, IL-12, NO and ROS levels, as well as decreased IL-6 and decreased arginase activity. In addition, MG induced increased phagocytic capability, and lysosomal volume in macrophages; structural parameters of microbicidal activity. Taken together, our results suggest that MG may be a promising candidate for new drug development against leishmaniasis., 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 © 2020 Elsevier B.V. All rights reserved.)

The emergence of non-Aspergillus mold pathogens has increased notoriously in the last decades with serious health consequences. The options of treatment for these microorganisms often resistant to a wide variety of antifungals is limited. Sertraline is an antidepressant with in vitro and in vivo antifungal properties which has been recently studied as an adjuvant in the treatment of invasive infections. In this study, we evaluated the in vitro interaction of sertraline with voriconazole and amphotericin B against Lomentospora prolificans, Scedosporium spp., Fusarium spp., Paecilomyces spp., Alternaria spp. and Curvularia spp. The minimum inhibitory concentration and minimum fungicidal concentration for sertraline were in the range of 8-32 μg/mL. Sertraline showed antifungal capacity against all fungi tested and synergism in combination with amphotericin B against some strains of Lomentospora prolificans, Scedosporium apiospermum and Alternaria alternata, antagonism with voriconazole against Purpureocillium lilacinum and indifference in both combinations for most of the other strains tested. These results suggest a potential role of sertraline as an adjuvant in the treatment of some of these serious mycoses., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interests., (Copyright © 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

This work was aimed at the production and characterization of a new nanocarrier based on a Sterculia striata polysaccharide (SSP) modified via acylation reaction with propionic anhydride. Nanocapsules of propionated SSP (PSSP) were produced via spontaneous nanoemulsification process and tested as a potential amphotericin B (AMB) nanocarrier. Stable nanoparticles with a very low polydispersity index (0.08-0.29) and high zeta potential (ζ -42.7 to -53.8 mV) were obtained. Particle size was dependent on the degree of substitution and ranged from 205 to 286 nm. A nanocapsule with a degree of substitution (DS) of 2.53 (NC P 2.53) was selected for encapsulation, biocompatibility, and antifungal evaluation against Candida albicans strains. A maximum of 98.3% AMB encapsulation was achieved. Encapsulated AMB was in its monomeric form and showed good biocompatibility and antifungal activity against four C. albicans strains. Data indicate that PSSP has potential as a nanocarrier system for AMB., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cutaneous leishmaniosis (CL) is a parasitic disease in animals and human with no satisfactory treatments and vaccination. Rapamycin is a potent inhibitor of mammalian target of rapamycin (mTOR) with various applications. Here, the effect of rapamycin alone or in combination with two other drugs, namely amphotericin B (AmB) and glucantime, was investigated against Leishmania tropica infection. In vitro viability and electron microscopy evaluation of the parasites showed detrimental changes in their appearance and viability. Treatment with clinically relevant dose of rapamycin (10.2 μg/dose) is able to control the parasite load in BALB/c mice infected with L. tropica. Furthermore, the cytokine profiles showed significant polarization towards Th1 immune response. Surprisingly, combination therapy with either AmB or glucantime was not efficient. Rapamycin is showed an effective alternative therapy against leishmaniosis caused by L. tropica., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Amphotericin B is used for local delivery from polymethylmethacrylate to treat fungal prosthetic joint infections. The optimal amphotericin B formulation and the influence of different poragens in the bone cements are unknown. To investigate the necessary amount of amphotericin B in the bone cement to prevent Candida biofilm several amphotericin B formulations were studied: non-liposomal and liposomal with or without poragen gentamicin. For the non-liposomal formulation, standard bile salt, the sodium deoxycholate, was used and additionally N-methyl-D-glucamine/palmitate was applied. The activity of the released amphotericin B was tested against C. albicans, C. glabrata, C. parapsilosis and C. krusei biofilms with application of the isothermal calorimeter and standard microbiological methods. Compressive strength was measured before and after antifungal elution from the cements. There is less aggregated N-methyl-D-glucamine/palmitate amphotericin B released but its antifungal activity is equivalent with the deoxycholate amphotericin B. The minimum quantity of antifungal preventing the Candida biofilm formation is 12.5 mg in gram of polymer powder for both non-liposomal formulations. The addition of gentamicin reduced the release of sodium deoxycholate amphotericin B. Gentamicin can be added to N-methyl-D-glucamine/palmitate amphotericin B in order to boost the antifungal release. When using liposomal amphotericin B more drug is released. All amphotericin B formulations were active against Candida biofilms. Although compressive strength slightly decreased, the obtained values were above the level of strength recommended for the implant fixation. The finding of this work might be beneficial for the treatment of the prosthetic joint infections caused by Candida spp., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Candida albicans frequently causes variety of superficial and invasive disseminated infections in HIV infected patients. Further, the emergence of non albicans species causing candidiasis predominantly in patients with advanced immune-suppression and drug resistance brings great apprehension. Hence, in this study we evaluate the capability of eugenol (EUG), a natural compound in combination with less toxic concentrations of amphotericin B (AmpB) for enhanced antifungal effects and reduced toxicity. Antifungal activity and time-kill assay were employed according to Clinical Laboratory Standard Institute (CLSI) guidelines with minor modifications on clinical isolates of Candida albicans. To confirm the synergistic interaction of EUG and AmpB, checkerboard experiments were employed. Interestingly, EUG-Amp B combination shows many fold higher anti-candida activity compared to single component treatment. Furthermore, our results depicts reactive oxygen species (ROS) driven killing and mitochondrial hyperpolarisation on treatment. Our data also suggests inhibition of calcium channel by EUG and predicts longer retainment of AmpB. Pronounced cellular damage was observed with combination treatment than to EUG and AmpB alone. Our finding is helpful for the removal of toxic concentrations of antifungal agents., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Leishmaniasis is second most neglected disease after malaria and seems to be a worldwide concern because of increased drug resistance and non-availability of approved vaccine. The underlying molecular mechanism of drug resistance (Amp B) in Leishmania parasites still remains elusive. Herein, the present study investigated differentially expressed secreted proteins of Amphotericin B sensitive (S) and resistant (R) isolate of Leishmania donovani by using label free quantitative LC-MS/MS approach. A total of 406 differentially expressed secreted proteins were found between sensitive (S) and resistant (R) isolate. Among 406 proteins, 32 were significantly up regulated (>2.0 fold) while 22 were down regulated (<0.5 fold) in resistant isolate of L. donovani. Further, differentially expressed proteins were classified into 11 various biological processes. Interestingly, identified up regulated proteins in resistant parasites were dominated in carbohydrate metabolism, stress response, transporters and proteolysis. Western blot and enzymatic activity of identified proteins validate our proteomic findings. Finally, our study demonstrated some new secreted proteins associated with Amp B resistance which provides a basis for further investigations to understand the role of proteins in L. donovani. BIOLOGICAL SIGNIFICANCE: Although great advances have been achieved in the diagnosis and treatment of leishmaniasis, still drug resistance is major hurdle in control of disease. Present study will enhance the deeper understanding of altered metabolic pathways involved in Amp B resistance mechanism and provide possible new proteins which can be potential candidate either for exploring as new drug target or vaccine. Protein-protein interactions highlighted the up-regulated metabolic pathways in resistant parasites which further unravel the adaptive mechanism of parasites., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Leishmaniasis is a poverty-related disease, the chemotherapy of which is based on few drugs. The in vitro macrophage-amastigote model using mouse peritoneal cells, human-monocyte transformed macrophages and immortalized cell lines have been used to test new and safe antileishmanial drugs. Considering the differences for drug sensitivities between these Leishmania infected cells, the efficacy of amphotericin B, pentavalent antimonial, miltefosine and resveratrol was evaluated in a recently developed ex vivo culture of macrophages isolated from mouse lesion induced by L. amazonensis (CD11b + F4/80 + CD68 + CD14 + ) compared with infected peritoneal macrophages (CD11b + F4/80 + CD68 + CD14 + ). The results show that IC50 values of amphotericin B, miltefosine and pentavalent antimonial for parasites in lesional and peritoneal macrophages were similar, although high doses of these compounds did not result in total clearance of parasites in lesional cells (amphotericin B), peritoneal cells (miltefosine) and both cell cultures (pentavalent antimonial). Amastigotes infecting lesional macrophages were more resistant to resveratrol as compared to parasites in peritoneal macrophages. The cytoxicity of miltefosine and resveratrol was higher in infected peritoneal macrophages than in lesional cells. These data suggest that the antileishmanial effect and citotoxicity of some anti leishmanial compounds are dependent of macrophage source and mouse peritoneal macrophages loaded with amastigotes do not represent the lesion cell., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: To provide species distribution and antifungal susceptibility profiles of 358 Trichosporon clinical isolates collected from 24 tertiary-care hospitals., Methods: Species identification was performed by sequencing the IGS1 region of rDNA. Antifungal susceptibility testing for amphotericin B, fluconazole, voriconazole and posaconazole followed the Clinical and Laboratory Standards Institute reference method. Tentative epidemiologic cutoff values (97.5% ECVs) of antifungals for Trichosporon asahii were also calculated., Results: Isolates were cultured mostly from urine (155/358, 43.3%) and blood (82/358, 23%) samples. Trichosporon asahii was the most common species (273/358, 76.3%), followed by T. inkin (35/358, 9.7%). Isolation of non-T. asahii species increased substantially over the last 11 years [11/77 (14.2%) from 1997 to 2007 vs. 74/281, (26.3%) from 2008 to 2018, p0.03]. Antifungal susceptibility testing showed high amphotericin B minimum inhibitory concentrations against Trichosporon isolates, with higher values for T. faecale. The ECV for amphotericin B and T. asahii was set at 4 μg/mL. Among the triazole derivatives, fluconazole was the least active drug. The ECVs for fluconazole and posaconazole against T. asahii were set at 8 and 0.5 μg/mL, respectively. Voriconazole showed the strongest in vitro activity against the Trichosporon isolates; its ECV for T. asahii was set at 0.25 μg/mL after 48 hours' incubation., Conclusions: Trichosporon species diversity has increased over the years in human samples, and antifungal susceptibility profiles were species specific. Trichosporon asahii antifungal ECVs were proposed, which may be helpful to guide antifungal therapy., (Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

An epidemiologic surveillance of non-albicans candidemia for a 6-year period was conducted in Korea. Compared to the published epidemiologic data for the previous 6 years, an increase of C. glabrata (from 21.3% to 28.5%) and a decrease of C. parapsilosis (from 36.5% to 24.7%) were noticed. During the study period, C. tropicalis (36.4%) was most frequently isolated non-albicans Candida, followed by C. glabrata (28.5%), C. parapsilosis (24.7%), and C. krusei (2.6%). Replacement of primary amphotericin B treatment with echinocandins (P < 0.001) eliminated amphotericin B resistance (from 7.8% in 2011 to 0% in 2014)., (Copyright © 2018. Published by Elsevier Ltd.)

New therapeutics against leishmaniasis are desirable, since the current drugs applied against this disease complex presents problems, such as the toxicity, high cost and/or parasite resistance. In the present study, a new fluoroquinoline derivate, namely 7-chloro-N-(4-fluorophenethyl)quinolin-4-amine or GF1061, was evaluated regarding to its in vitro antileishmanial action against Leishmania infantum and L. amazonensis species, as well as by its toxicity in mammalian cells and efficacy in the treatment of infected macrophages. The mechanism of action of this molecule in L. amazonensis and the therapeutic efficacy in infected BALB/c mice were also evaluated. Results showed that GF1061 was effective against both parasite species, showing selectivity index (SI) of 38.7 and 42.7 against L. infantum and L. amazonensis promastigotes, respectively, and of 45.0 and 48.9 against the amastigotes, respectively. Amphotericin B (AmpB), used as control, showed SI values of 6.6 and 8.8 against L. infantum and L. amazonensis promastigotes, respectively, and of 2.2 and 2.7 against the amastigotes, respectively. The molecule was effective in treat infected macrophages, as well as it induced alterations in the mitochondrial membrane potential, increase in the reactive oxygen species production, and in the cell integrity of the parasites. Regarding to the in vivo experiments, BALB/c mice (n = 8 per group) were subcutaneously infected with 10 6 L. amazonensis stationary promastigotes and, 60 days post-infection, they received saline or were treated during 10 days, once a day, with AmpB (1 mg/kg body weight) or GF1061 (5 mg/kg body weight). One day after the treatment, the infected tissue, spleen, liver, and draining lymph node (dLN) of the animals were collected, and the parasite load was evaluated. GF1061-treated mice, as compared to the saline and AmpB groups, showed significant reductions in the parasitism in the infected tissue (66% and 62%, respectively), liver (69% and 44%, respectively), spleen (71% and 38%, respectively), and dLN (72% and 48%, respectively). In conclusion, results suggested that GF1061 may be considered as a possible therapeutic target to be evaluated against leishmaniasis in other mammalian hosts., (Copyright © 2018 Elsevier B.V. All rights reserved.)

P852, a novel cyclic peptide isolated from Bacillus amyloliquefaciens L-H15, showed potent antifungal activity against several major plant fungal pathogens including Fusarium oxysporum. To elucidate the antifungal mechanism, the impact of P852 on the cell morphology and membrane permeabilization of F. oxysporum was studied. By applying electron microscopy and fluorescent techniques, we showed that P852 treatment caused the morphological change of F. oxysporum cells and disrupted its cell structure, including formation of blebs, broken hyphae, deformation of membrane, intracellular organization disruption, pore formation, and cell lysis. Our findings provide insights into the mode of action of P852, which laying a foundation to develop P852 as a novel antifungal agent to control plant fungal pathogens., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Amphotericin B (AmB) is a widely used polyene antifungal agent; however, its poor solubility limits its clinical application. In this study, AmB nanosuspensions were prepared by a high pressure homogenization method (AmB-HPH) and an antisolvent precipitation method (AmB-AP) to improve the drug solubility. To reveal the distinct influences of these two different preparation methods, systematic comparisons of particle size, crystalline state, wettability, in vitro dissolution and in vivo pharmacokinetics on the properties of AmB-HPH and AmB-AP were performed. The results indicated that AmB-AP was in an amorphous state, exhibiting higher saturation solubility and dissolution rate than those of AmB-HPH in the crystalline state. However, the relative bioavailability of AmB-HPH was higher than that of AmB-AP in vivo, which was likely attributed to its better stability. In conclusion, both AmB-HPH and AmB-AP can enhance the solubility and bioavailability of AmB, but the stability of the nanosuspension prepared by the anti-solvent precipitation method should be carefully considered., (Copyright © 2018 Elsevier B.V. All rights reserved.)