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Purpose: We aimed to investigate the clonal relationship and antifungal susceptibility of C. parapsilosis isolated from hospitalized patients and to determine whether it is due to transmission or not and the spread status of resistant isolates., Methods: Between January 2017 and June 2019, totally 277 C parapsilosis isolated from blood, urine and catheter samples of adult or pediatric in-patient (intensive care and service) who applied to Mycology laboratory in our hospital were included in the study. All isolates were identified using conventional methods, API 20 C AUX (Biomerieux, France) semi-automated system and confirmed by MALDI-TOF MS Biotyper Smart (Bruker Daltonik GmbH, Germany). Randomly amplified polymorphic DNA (RAPD) PCR method was used for molecular genotyping of isolates. MIC values for fluconazole, anidulafungin and amphotericin B were determined according to the M27-A3 CLSI broth microdilution reference method guideline., Results: Seven different band patterns (A-G) were detected in 277 isolates by RAPD PCR method. According to the rank order of the isolates, 170 (61.37%) C, 65 (23.47%) A, 18 (6.50%) G, 11 (3.97%) B, six (2.17%) E, two (0.72%) F and one (0.36%) D patterns were determined. When the band patterns of the isolates were evaluated according to the years, it was detected that C pattern continued between 2017 and 2019 and that all isolates continued to spread only as C pattern in 2019. While 211 (76.17%) of the isolates were resistant to fluconazole (≥ 8 µg/ml), two (0.72%) were resistant to amphotericin B (≥ 2 µg/ml) and two (0.72%) were intermediate to anidulafungin., Conclusions: It is noteworthy that the spread of the C pattern in C. parapsilosis strains has increased over the years and is the main pattern isolated from the whole hospital. The detection of high fluconazole resistance in C. parapsilosis isolates in our hospital may also be related to the dominant pattern., Competing Interests: Declarations. Ethical approval: This study was approved by the Noninvasive Research Ethics Committee of Dokuz Eylül University, İzmir, Turkey (Number: 2019/17–21). Human ethics and consent to participate: not applicable (As a retrospective study, we did not apply for a consent to participate in the study from the patients). Clinical trial number: Not applicable. Competing interests: The authors declare no competing interests. Conflict of interest: There are no conflicts of interest for the authors participating in the study., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objectives: Reported amphotericin B resistance rates for Candida auris vary considerably. This may reflect clinically relevant differences in susceptibility, technical issues with testing, or adoption of a clinical breakpoint that bisects the wild-type population. We compared reference methods and two gradient diffusion strips using a shared C. auris strain collection., Methods: Forty C. auris strains from nine U.S. states and ≥3 clades were included. Fourteen MIC data sets were generated using European Committee on Antimicrobial Susceptibility Testing (EUCAST) E.Def 7.4, Clinical and Laboratory Standards Institute (CLSI) M27Ed4, Etest, and MIC gradient test strip (MTS, Liofilchem) MICs. MICs ≤1 mg/L were classified as susceptible., Results: EUCAST and CLSI amphotericin B MIC testing were robust across the included method variables. The modal MIC was 1 mg/L, distributions unimodal and narrow with similar geometric mean (GM)-MICs (0.745-1.072); however, susceptibility classification varied (0-28% resistance). Gradient diffusion strip testing resulted in wider and bimodal distributions for 8/9 data sets. If adopting, per manufacturer's protocol, double inoculation for the Etest method, the modal MIC increased to 2-4 mg/L and resistance rates to 45-63% versus 25-30% with the single inoculation. The EUCAST, CLSI, Etest, and MTS strip MICs correlated to the optical density of drug-free control EUCAST wells, suggesting that some isolates grew better than others and that this was associated with MIC., Discussion: The EUCAST and CLSI MIC results were in close agreement, whereas the strip test showed wider and bimodal distributions with reader to reader and centre to centre variation. Our study adds to the concern for commercial MIC testing of amphotericin B against C. auris and suggests the current breakpoint leads to random susceptibility classification., (Copyright © 2024 European Society of Clinical Microbiology and Infectious Diseases. All rights reserved.)

Candida auris is a growing concern due to its resistance to antifungal drugs, particularly amphotericin B (AMB), detected in 30 to 60% of clinical isolates. However, the mechanisms of AMB resistance remain poorly understood. Here we investigated 441 in vitro- and in vivo-evolved C. auris lineages from 4 AMB-susceptible clinical strains of different clades. Genetic and sterol analyses revealed four major types of sterol alterations as a result of clinically rare variations in sterol biosynthesis genes ERG6, NCP1, ERG11, ERG3, HMG1, ERG10 and ERG12. In addition, aneuploidies in chromosomes 4 and 6 emerged during resistance evolution. Fitness trade-off phenotyping and mathematical modelling identified diverse strain- and mechanism-dependent fitness trade-offs. Variation in CDC25 rescued fitness trade-offs, thereby increasing the infection capacity. This possibly contributed to therapy-induced acquired AMB resistance in the clinic. Our findings highlight sterol-modulating mechanisms and fitness trade-off compensation as risks for AMB treatment failure in clinical settings., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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.

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 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.

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.)

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: 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.

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.)

Candida auris, an emerging and multidrug-resistant fungal pathogen, has led to numerous outbreaks in China. While the resistance mechanisms against azole and amphotericin B have been studied, the development of drug resistance in this pathogen remains poorly understood, particularly in in vivo-generated drug-resistant strains. This study employed pathogen whole-genome sequencing to investigate the epidemiology and drug-resistance mutations of C. auris using 16 strains isolated from two patients. Identification was conducted through Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and antimicrobial susceptibilities were assessed using broth microdilution and Sensititre YeastOne YO10. Whole-genome sequencing revealed that all isolates belonged to the South Asian lineage, displaying genetic heterogeneity. Despite low genetic variability among patient isolates, notable mutations were identified, including Y132F in ERG11 and A585S in TAC1b, likely linked to increased fluconazole resistance. Strains from patient B also carried F214L in TAC1b, resulting in a consistent voriconazole minimum inhibitory concentration of 4 µg/mL across all isolates. Furthermore, a novel frameshift mutation in the SNG1 gene was observed in amphotericin B-resistant isolates compared to susceptible ones. Our findings suggest the potential transmission of C. auris and emphasize the need to explore variations related to antifungal resistance. This involves analyzing genomic mutations and karyotypes, especially in vivo, to compare sensitive and resistant strains. Further monitoring and validation efforts are crucial for a comprehensive understanding of the mechanisms of drug resistance in C. auris., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Fungal implant-associated bone infections are rare but difficult to treat and often associated with a poor outcome for patients. Candida species account for approximately 90% of all fungal infections. In vivo biofilm models play a major role to study biofilm development and potential new treatment options; however, there are only a very few in vivo models to study fungi-associated biofilms. Furthermore, mammalian infection models are replaced more and more due to ethical restrictions with other alternative models in basic research. Recently, we developed an insect infection model with Galleria mellonella larvae to study biofilm-associated infections with bacteria. Here, we further expanded the G. mellonella model to study in vivo fungal infections using Candida albicans and Candida krusei. We established a planktonic and biofilm-implant model to test different antifungal medication with amphotericin B, fluconazole, and voriconazole against the two species and assessed the fungal biofilm-load on the implant surface. Planktonic infection with C. albicans and C. krusei showed the killing of the G. mellonella larvae at 5 × 10 5  colony forming units (CFU). Treatment of larvae with antifungal compounds with amphotericin B and fluconazole showed significant survival improvement against planktonic C. albicans infection, but voriconazole had no effect. Titanium and stainless steel K-wires were preincubated with C. albicans and implanted inside the larvae to induce biofilm infection on the implant surface. The survival analysis revealed significantly reduced survival of the larvae with Candida spp. infection compared to noninfected implants. The treatment with antifungal amphotericin B and fluconazole resulted in a slight and nonsignificant improvement survival of the larvae. The treatment with the antifungal compounds in the biofilm-infection model was not as effective as in the planktonic infection model, which highlights the resistance of fungal biofilms to antifungal compounds like in bacterial biofilms. Scanning electron microscopy (SEM) analysis revealed the formation of a fungal biofilm with hyphae and spores associated with larvae tissue on the implant surface. Thus, our study highlights the use of G. mellonella larvae as alternative in vivo model to study biofilm-associated implant fungal infections and that fungal biofilms exhibit high resistance profiles comparable to bacterial biofilms. The model can be used in the future to test antifungal treatment options for fungal biofilm infections., (© 2023 The Authors. Journal of Orthopaedic Research ® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

In recent decades, Candida albicans have been the main etiological agent of life-threatening invasive infections, characterized by various mechanisms of resistance to commonly used antifungals. One of the strategies to fight Candida infections may be the use of an electromagnetic field. In this study, we examined the influence of the alternating magnetic field of 50 Hz on the cells of C. albicans. We checked the impact of the alternating magnetic field of 50 Hz on the viability, filamentation and sensitivity to fluconazole and amphotericin B of two, differing in hydrophobicity, strains of C. albicans, CAF2-1 and CAF 4-2. Our results indicate that using the alternating magnetic field of 50 Hz reduces the growth of C. albicans. Interestingly, it presents a stronger effect on the hydrophobic strain CAF4-2 than on the hydrophilic CAF2-1. The applied electromagnetic field also affects the permeabilization of the cell membrane. However, it does not inhibit the transformation from yeast to hyphal forms. AMF is more effective in combination with fluconazole rather than amphotericin B. Our findings confirm the hypothesis that the application of the alternating magnetic field of 50 Hz in antifungal therapy may arise as a new option to support the treatment of Candida infections., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Sztafrowski 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.)

Significant variation in minimal inhibitory concentrations (MIC) has been reported for amphotericin B (AMB) and C. auris, depending on the antifungal susceptibility testing (AFST) method. Although the Sensititre YeastOne (SYO) is widely used in routine laboratory testing, data regarding its performance for the AFST of C. auris are scarce. We tested AMB against 65 C. auris clinical isolates with the SYO and the reference methodology by the Clinical and Laboratory Standards Institute (CLSI). The essential agreement (EA, ±1 dilution) between the two methods and the categorical agreement (CA) based on the Centers for Disease Control and Prevention (CDC)'s tentative breakpoint of MIC ≥ 2 mg/L were determined. The SYO wild type upper limit value (WT-UL) was determined using the ECOFFinder. The modal (range) CLSI growth inhibitory MIC was lower than the SYO colorimetric MIC [1(0.25-1) versus 2(1-8) mg/L, respectively]). The CLSI-colorimetric SYO EA was 29% and the CA was 11% (89% major errors; MaE). MaE were reduced when the SYO WT-UL of 8 mg/L was used (0% MaE). Alternatively, the use of SYO growth inhibition endpoints of MIC-1 (75% growth inhibition) or MIC-2 (50% growth inhibition) resulted in 88% CA with 12% MaE and 97% CA with 3% MaE, respectively. In conclusion, SYO overestimated AMB resistance in C. auris isolates when colorimetric MICs, as per SYO instructions and the CDC breakpoint of 2 mg/L, were used. This can be improved either by using the method-specific WT-UL MIC of 8 mg/L for colorimetric MICs or by determining growth inhibition MIC endpoints, regardless of the color. IMPORTANCE Candida auris is an emerging and frequently multidrug-resistant fungal pathogen that accounts for life-threatening invasive infections and nosocomial outbreaks worldwide. Reliable AF is important for the choice of the optimal treatment. Commercial methods are frequently used without prior vigorous assessment. Resistance to AMB was over-reported with the commercial colorimetric method Sensititre YeastOne (SYO). SYO produced MICs that were 1 to 2 twofold dilutions higher than those of the reference CLSI method, resulting in 89% MaE. MaE were reduced using a SYO-specific colorimetric wild type upper limit MIC value of 8 mg/L (0%) or a 50% growth inhibition endpoint (3%)., Competing Interests: The authors declare no conflict of interest.

Objective: Opportunistic fungal infections by Candida species arise among cancer patients due to the weakened immune system following extensive chemotherapy. Prophylaxis with antifungal agents have developed the resistance of Candida spp. to antifungals. Accurate identification of yeasts and susceptibility patterns are main concerns that can directly effect on the treatment of patients., Methods: Over a period of three years, 325 cancer patients suspected to Candida infections were included in the current investigation. The clinical isolates were molecularly identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). All strains, were examined for in vitro susceptibility to the amphotericin B, itraconazole, fluconazole, and anidulafungin according to the CLSI M27 document., Results: Seventy-four cancer patients had Candida infections (22.7%). Candida albicans was the most common species (83.8%). Antifungal susceptibility results indicated that 100% of the Candida isolates were sensitive to amphotericin B; however, 17.6%, 9.4%, and 5.4% of clinical isolates were resistant to anidulafungin, fluconazole, and itraconazole, respectively., Conclusion: The findings of the present work shows a warning increase in resistance to echinocandins. Since all fluconazole resistance isolates were obtained from candidemia, we recommend amphotericin B as the first line therapy for this potentially fatal infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sharifi, Badiee, Abastabar, Morovati, Haghani, Noorbakhsh and Mohammadi.)

Invasive Candida infections threaten human health due to the increasing incidence of resistance to the currently available antifungal agents. Amphotericin B (AMB) is the gold standard therapy to treat these infections. Nevertheless, the use of such substance in the clinic is aggravated by its toxicity. Since AMB binds to membrane sterols, it forms pores on human plasma membranes, mainly in kidney cells, leading to nephrotoxicity. The combination of this drug to a second substance could allow for the use of smaller concentrations of AMB, consequently lowering the probability of adverse effects. This mini-review summarizes information regarding an array of substances that enhance AMB antifungal activity. It may be noticed that several of these compounds target plasma membrane. Interestingly, substances approved for human use also presented combinatory anti-Candida activity with AMB. These data reinforce the potential of associating AMB to another drug as a promising therapeutical alternative to treat Candida infections. Further studies, regarding mechanism of action, pharmacokinetics and toxicity parameters must be conducted to confirm the role of these substances as adjuvant agents in candidiasis therapy.

In recent decades, the incidence of Candida infections has increased in immunocompromised patients. This multicenter study aimed to evaluate in vitro antifungal activities of 8 antifungal agents against the Candida species isolated from 10 university hospitals in Iran. During the period from Dec 2019 to Dec 2021, Candida species were collected from clinical samples of patients. The isolates were identified by PCR restriction fragment length polymorphism and sequencing methods. The antifungal susceptibility tests of each isolate to eight antifungal agents were performed according to the microdilution CLSI M27, M59, and M60 standard methods. A total of 598 Candida strains were isolated from clinical samples. The most commonly isolated Candida species was C. albicans, followed by C. glabrata, C. parapsilosis, Debaryomyces hansenii (Candida famata), C. tropicalis, Pichia kudriavzevii (Candida krusei), C. orthopsilosis, Meyerozyma guilliermondii (Candida guilliermondii), Kluyveromyces marxianus (Candida kefyr), and Clavispora lusitaniae (Candida lusitaniae). MIC 90 values in all Candida species were as follows: 0.25 μg/mL for caspofungin and voriconazole; 0.5 μg/mL for amphotericin B and isavuconazole; 2 μg/mL for itraconazole, luliconazole, and posaconazole; and 16 μg/mL for fluconazole. Although 30/285 C. albicans, 15/31 C. hansenii, 3/12 M. guilliermondii , 67/125 C. glabrata, 5/15 P. kudriavzevii , 6/60 C. parapsilosis, and 5/23 C. tropicalis isolates were multiazole resistant with resistance to 2 to 4 azoles, pan-azole resistance was not observed. According to our data, Candida albicans and C. glabrata were the most frequent species isolated from clinical samples in Iran. Caspofungin and voriconazole, with lower MIC 90 values, are the most effective than other antifungal agents for the treatment of Candida infections in this region. IMPORTANCE Candida species cause severe invasive infections of the heart, brain, eyes, bones, and other parts of the body. Knowledge of regional distributions of causative Candida agents and their antifungal susceptibility patterns can help to monitor resistance to antifungal agents of various species and support local and national surveillance programs. In the present study, C. albicans and C. glabrata were the most frequently isolated species from clinical samples in Iran. Increasing rates of non- albicans Candida isolates from the Iranian population should be looked at as alarming due to various levels of intrinsic MIC values or resistance to various antifungal drugs. Caspofungin and voriconazole are recommended over fluconazole for the treatment of Candida infections in the study region. However, amphotericin B and isavuconazole are also active against the most common Candida species isolated from patients. Pan azole-resistant Candida species were not observed in the present study.

Drug resistance derived from extracellular vesicles (EVs) is an increasingly important research area but has seldom been described regarding fungal pathogens. Here, we characterized EVs derived from a triazole-resistant but amphotericin B-susceptible strain of Candida auris . Nano- to microgram concentrations of C. auris EVs prepared from both broth and solid agar cultures could robustly increase the yeast's survival against both pure and clinical amphotericin B formulations in a dose-dependent manner, resulting in up to 16-fold changes of minimum inhibitory concentration. Meanwhile, this effect was not observed upon addition of these EVs to C. albicans , nor upon addition of C. albicans EVs to C. auris . No change in susceptibilities was observed upon EV treatment for fluconazole, voriconazole, micafungin, and flucytosine. Mass spectrometry indicated the presence of immunogenic-/drug resistance-implicated proteins in C. auris EVs, including alcohol dehydrogenase 1 as well as C. albicans Mp65-like and Xog1-like proteins in high quantities. Based on these observations, we propose a potential species-specific role for EVs in amphotericin B resistance in C. auris . These observations may provide critical insights into treatment of multidrug-resistant C. auris .

Amphotericin B (AmB) is a widely used antifungal agent especially for the therapy of systemic fungal infections. However, the severe side effects of AmB often leads to the premature termination of the treatment. So it is imperative to find the drugs that can both reduce the dosage and enhance the antifungal efficacy of AmB. Here we demonstrated that Nicotinamide (NAM), a cheap and safe vitamin, could enhance the antifungal activities of AmB. We demonstrated the synergistic interaction of NAM and AmB against Candida albicans as well as other Candida spp. and Cryptococcus neoformans . Moreover, NAM could enhance of the activity of AmB against biofilm. This enhancement was also observed in disseminated candidiasis in vivo . Our further study revealed that AmB could induce oxidative damage through the modification of histone acetylation. AmB could inhibit the expression of HST3, an H3K56 deacetylase in C. albicans . The immunoblotting test revealed excessive H3K56ac in AmB-treated fungal cells. Consistantly, the hst3Δ mutant displayed high sensitivity to AmB, while addition of NAM, an H3K56 deacetylation inhibitor, resulted in an even severe inhibition in the growth of this strain. These results indicated that AmB could execute antifungal activity via boosting H3K56ac which was mediated by HST3, and the mechanism for the synergistic interaction of NAM and AmB was based on exacerbating this process, which led to even excessive H3K56ac and oxidative damage. This finding provided theoretical basis for better understanding the antifungal mechanisms of AmB and clinical application of this drug.

Although Amphotericin B (AMB) is considered the most effective anti-mycotic agent for treating Candida infections, its clinical use is limited due to its high toxicity. To address this issue, we developed cholesterol-based dendritic micelles of hyperbranched polyglycerol (HPG), including cholesterol-cored HPG (Chol-HPG) and cholesterol end-capped HPG (HPG@Chol), for AMB delivery. The findings suggested that the presence of cholesterol moieties could control AMB loading and release properties. Dendritic micelles inhibited AMB hemolysis and cytotoxicity in HEK 293 and RAW 264.7 cell lines while increasing antifungal activity against C. albicans biofilm formation. Furthermore, significantly lower levels of renal and liver toxicity biomarkers compared to Fungizone® ensured AMB-incorporated dendritic micelle biosafety, which was confirmed by histopathological evaluations. Overall, the Chol-HPG and HPG@Chol dendritic micelles may be a viable alternative to commercially available AMB formulations as well as an effective delivery system for other poorly soluble antifungal agents., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Candida auris is an emerging multidrug-resistant human fungal pathogen often refractory to treatment by all classes of antifungal drugs. Amphotericin B (AmB) is a fungicidal drug that, despite its toxic side effects, remains a drug of choice for the treatment of drug-resistant fungal infections, including those caused by C. auris. However, the molecular mechanisms underlying AmB resistance are poorly understood. In this study, we present data that suggests membrane lipid alterations and chromatin modifications are critical processes that may contribute to or cause adaptive AmB resistance in clinical C. auris isolates. To determine the plausible cause of increased AmB resistance, we performed RNA-seq of AmB-resistant and sensitive C. auris isolates. Remarkably, AmB-resistant strains show a pronounced enrichment of genes involved in lipid and ergosterol biosynthesis, adhesion, drug transport as well as chromatin remodeling. The transcriptomics data confirm increased adhesion and reduced lipid membrane permeability of AmB-resistant strains compared to the sensitive isolates. The AmB-resistant strains also display hyper-resistance to cell wall perturbing agents, including Congo red, calcofluor white and caffeine. Additionally, we noticed an increased phosphorylation of Mkc1 cell integrity MAP kinase upon AmB treatment. Collectively, these data identify differences in the transcriptional landscapes of AmB-resistant versus AmB-sensitive isolates and provide a framework for the mechanistic understanding of AmB resistance in C. auris.

Aim: Melanin has been linked to pathogenesis in several fungi. They often produce melanin-like pigments in the presence of L-dihydroxyphenylalanine (L-DOPA), but this is poorly studied in Candida glabrata . Methods & materials: C. glabrata was grown in minimal medium with or without L-DOPA supplementation and submitted to a chemical treatment with denaturant and hot acid. Results: C. glabrata turned black when grown in the presence of L-DOPA, whereas cells grown without L-DOPA supplementation remained white. Biophysical properties demonstrated that the pigment was melanin. Melanized C. glabrata cells were effectively protected from azoles and amphotericin B, incubation at 42°C and macrophage killing. Conclusion: In the presence of L-DOPA, C. glabrata produces melanin, increases antifungal resistance and enhances host survival.

Candida albicans is a major cause of human infections, ranging from relatively simple to treat skin and mucosal diseases to systemic life-threatening invasive candidiasis. Fungal infections treatment faces three major challenges: the limited number of therapeutic options, the toxicity of the available drugs, and the rise of antifungal resistance. In this study, we demonstrate the antifungal activity and mechanism of action of peptides ToAP2 and NDBP-5.7 against planktonic cells and biofilms of C. albicans. Both peptides were active against C. albicans cells; however, ToAP2 was more active and produced more pronounced effects on fungal cells. Both peptides affected C. albicans membrane permeability and produced changes in fungal cell morphology, such as deformations in the cell wall and disruption of ultracellular organization. Both peptides showed synergism with amphotericin B, while ToAP2 also presents a synergic effect with fluconazole. Besides, ToAP2 (6.25 µM.) was able to inhibit filamentation after 24 h of treatment and was active against both the early phase and mature biofilms of C. albicans. Finally, ToAP2 was protective in a Galleria mellonella model of infection. Altogether these results point to the therapeutic potential of ToAP2 and other antimicrobial peptides in the development of new therapies for C. albicans infections.

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.)

Since 2016, New York hospitals and health care facilities have faced an unprecedented outbreak of the pathogenic yeast Candida auris We tested over 1,000 C. auris isolates from affected facilities and found high resistance to fluconazole (MIC > 256 mg/liter) and variable resistance to other antifungal drugs. Therefore, we tested if two-drug combinations are effective in vitro against multidrug-resistant C. auris Broth microdilution antifungal combination plates were custom manufactured by TREK Diagnostic System. We used 100% inhibition endpoints for the drug combination as reported earlier for the intra- and interlaboratory agreements against Candida species. The results were derived from 12,960 readings, for 15 C. auris isolates tested against 864 two-drug antifungal combinations for nine antifungal drugs. Flucytosine (5FC) at 1.0 mg/liter potentiated the most combinations. For nine C. auris isolates resistant to amphotericin B (AMB; MIC ≥ 2.0 mg/liter), AMB-5FC (0.25/1.0 mg/liter) yielded 100% inhibition. Six C. auris isolates resistant to three echinocandins (anidulafungin [AFG], MIC ≥ 4.0 mg/liter; caspofungin [CAS], MIC ≥ 2.0 mg/liter; and micafungin [MFG], MIC ≥ 4.0 mg/liter) were 100% inhibited by AFG-5FC and CAS-5FC (0.0078/1 mg/liter) and MFG-5FC (0.12/1 mg/liter). None of the combinations were effective for C. auris 18-1 and 18-13 (fluconazole [FLC] > 256 mg/liter, 5FC > 32 mg/liter) except MFG-5FC (0.1/0.06 mg/liter). Thirteen isolates with a high voriconazole (VRC) MIC (>2 mg/liter) were 100% inhibited by the VRC-5FC (0.015/1 mg/liter). The simplified two-drug combination susceptibility test format would permit laboratories to provide clinicians and public health experts with additional data to manage multidrug-resistant C. auris ., (Copyright © 2020 American Society for Microbiology.)

Candida krusei is one of the most common agents of invasive candidiasis and candidemia worldwide, leading to high morbidity and mortality rates. This species has become a problem due to its intrinsic resistance and reduced susceptibility to azoles and polyenes. Moreover, the number of antifungal drugs available for candidiasis treatment is limited, demonstrating the urgent need for the discovery of novel alternative therapies. In this work, the in vivo and in vitro activities of a new oxadiazole (LMM11) were evaluated against C. krusei. The minimum inhibitory concentration ranged from 32 to 64 μg/mL with a significant reduction in the colony forming unit (CFU) count (~3 log10). LMM11 showed fungicidal effect, similar to amphotericin, reducing the viable cell number (>99.9%) in the time-kill curve. Yeast cells presented morphological alterations and inactive metabolism when treated with LMM11. This compound was also effective in decreasing C. krusei replication inside and outside macrophages. A synergistic effect between fluconazole and LMM11 was observed. In vivo treatment with the new oxadiazole led to a significant reduction in CFU (0.85 log10). Furthermore, histopathological analysis of the treated group exhibited a reduction in the inflammatory area. Taken together, these results indicate that LMM11 is a promising candidate for the development of a new antifungal agent for the treatment of infections caused by resistant Candida species such as C. krusei., Competing Interests: The authors have declared that no competing interests exist.

This study evaluated the activity of echinocandins, azoles and amphotericin B against Candida spp. isolates and other yeasts and characterised azole resistance mechanisms in Candida parapsilosis and Candida tropicalis. Invasive Candida spp. isolates (n = 2936) collected in 60 hospitals worldwide during 2016-2017 underwent antifungal susceptibility testing by broth microdilution. Azole-resistant C. parapsilosis and C. tropicalis were submitted to qPCR for ERG11, CDR1 and MDR1, and the whole genome sequence was analysed. Results of non-susceptibility to echinocandins ranged from 0.0-2.3%, being highest in Candida glabrata. More than 99.0% of the Candida albicans isolates were susceptible to both fluconazole and voriconazole. Fluconazole resistance in C. glabrata was 6.5% overall, being highest in the USA (13.0%). Resistance to voriconazole in Candida krusei was only noted in the USA (5.0%). Azoles inhibited 89.1-91.6% of C. parapsilosis isolates, with most resistant isolates noted in Europe (15.1%), including 36 isolates from Italy (three hospitals), of which 34 harboured Erg11 Y132F mutations and overexpressed MDR1. Azole non-wild-type C. tropicalis (7/227) were found in five countries: 3 isolates from Thailand had the same Erg11 Y132F alteration. Fluconazole non-wild-type isolates were noted among 3/77 (3.9%) Candida dubliniensis, 4/17 (23.5%) Candida guilliermondii, 4/47 (8.5%) Candida lusitaniae and other less common yeast species. Echinocandin use has been recommended over fluconazole for invasive Candida infections. However, azoles are still active against the most common Candida spp. and resistance appears to be restricted to certain geographic regions and associated with Erg11 Y132 alterations in C. parapsilosis and C. tropicalis., (Copyright © 2019 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Combination therapies can be a help to overcome resistance to current antifungals in humans. The combined activity of commercial antifungals and soluble and well-defined low molecular weight chitosan with average degrees of polymerization (DPn) of 17-62 (abbreviated C17 -C62) and fraction of acetylation (FA) of 0.15 against medically relevant yeast strains was studied. The minimal inhibitory concentration (MIC) of C32 varied greatly among strains, ranging from > 5000 μg mL-1 (Candida albicans and C. glabrata) to < 4.9 (C. tropicalis). A synergistic effect was observed between C32 and the different antifungals tested for most of the strains. Testing of several CHOS preparations indicated that the highest synergistic effects are obtained for fractions with a DPn in the 30-50 range. Pre-exposure to C32 enhanced the antifungal effect of fluconazole and amphotericin B. A concentration-dependent post-antifungal effect conserved even 24 h after C32 removal was observed. The combination of C32 and commercial antifungals together or as part of a sequential therapy opens new therapeutic perspectives for treating yeast infections in humans., Competing Interests: The authors have declared that no competing interests exist.

Oral candidiasis as a highly prevalent and recurrent infection in medically compromised individuals is mainly caused by the opportunistic fungal pathogen Candida albicans . This epithelial infection, if not controlled effectively, can progress to life-threatening systemic conditions and complications. The efficacy of current frontline antifungals is limited due to their poor bioavailability and systemic toxicity. As such, an efficient intervention is essential for controlling disease progression and recurrence. Herein, a theranostic nanoplatform (CD-Gu + -AmB) was developed to track the penetration of antifungals and perturb the invasion of C. albicans at oral epithelial tissues, via decorating the homemade red-emissive carbon dots (CD) with positively charged guanidine groups (Gu + ) followed by conjugation with antifungal polyene (amphotericin B, AmB) in a reacting site-controllable manner. The generated CD-Gu + -AmB favorably gathered within the Candida cells and exhibited potent antifungal effects in both planktonic and biofilm forms. It selectively accumulated in the nuclei of human oral keratinocytes and exhibited undetectable toxicity to the host cells. Moreover, we reported for the first time the penetration and exfoliation profiles of CD in a three-dimensional organotypic model of human oral epithelial tissues, demonstrating that the extra- and intracellular accumulation of CD-Gu + -AmB effectively resisted the invasion of C. albicans by forming a "shielding" layer throughout the entire tissue. This study establishes a multifunctional CD-based theranostic nanoplatform functioning as a traceable and topically applied antifungal to arm oral epithelia, thereby shedding light on early intervention of mucosal candidiasis for oral and general health.

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.)

Candida auris is an emerging species of yeast characterized by colonization of skin, persistence in the healthcare environment, and antifungal resistance. C. auris was first described in 2009 from a single isolate but has since been reported in more than 25 countries worldwide. Resistance to fluconazole and amphotericin B is common, and resistance to the echinocandins is emerging in some countries. Antifungal resistance has been shown to be acquired rather than intrinsic and the primary mechanisms of resistance to the echinocandins and azoles have been determined. There are a number of new antifungal agents in phase 2 and phase 3 clinical trials and many have activity against C. auris. This review will discuss what is currently known about antifungal resistance in C. auris, limitations to antifungal susceptibility testing, the mechanisms of resistance, and the new antifungals that are on the horizon., (Published by Elsevier Inc.)

Purpose: To evaluate a new corneal cold storage medium including an antimycotic tablet (Kerasave, AL.CHI.MI.A. S.r.l.)., Methods: Kerasave and tryptone soy broth (control) were inoculated with 10 and 10 colony-forming units (CFU)/mL of 6 Candida isolates (Candida albicans [n = 4], Candida tropicalis [n = 1], and Candida glabrata [n = 1]). Minimum inhibitory concentrations (MICs) were determined using amphotericin B Etest strips. Sterile porcine corneas contaminated with 10 CFU/mL of each isolate were incubated in Kerasave and control at 4°C. Growth rate and Log10 reduction at 4°C at different time intervals were determined for liquid samples and tissue homogenates. Kerasave biocompatibility was assessed according to ISO 10993-5 and ISO 10993-10., Results: No C. albicans or C. tropicalis colonies were recovered from Kerasave inoculated with 10 CFU/mL after incubation for 3 days at 4°C. C. glabrata was inhibited but not killed after 3 days at 4°C. Four of the 6 strains contaminated with 10 CFU/mL demonstrated a significant ≥ 3 Log10 reduction in media and tissue homogenates within 5 days as compared to controls (p < 0.01). Amphotericin B MICs ranged from 0.19 to 0.38 μg/mL for C. albicans (n = 3) and C. tropicalis (n = 1). C. glabrata showed reduced susceptibility (0.5 μg/mL) and 1 C. albicans was resistant to amphotericin B (≥ 1 μg/mL). Kerasave was not cytotoxic, irritating, or sensitizing according to the ISO standards., Conclusions: Kerasave showed high antifungal efficacy against susceptible fungal strains at 4°C in the presence and absence of corneal tissue. Resistant strains to amphotericin B were not eliminated by Kerasave. Kerasave is not cytotoxic, irritating, or sensitizing.

The data on susceptibility to antifungals of new species within Candida glabrata complex are limited. Our study was to enrich a global knowledge of yeast epidemiology and drug resistance. The study was focused on the identification of species within clinical isolates of the C. glabrata complex and on the determination of their resistance to antifungals. Four hundred forty-five clinical C. glabrata sensu lato strains were isolated from different clinical samples at routine mycological exams at the Infant Jesus Teaching Hospital in Warsaw. The identification of the most of tested isolates to species complex level was performed using the ID 32 C system. The identification of C. nivariensis and C. bracarensis species within the C. glabrata complex was performed by DNA sequencing. The MICs of amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole, caspofungin, anidulafungin, and micafungin were determined by E-test. Twenty-four isolates did not have an ITS-1 region, characteristic of C. glabrata sensu stricto and their D1/D2 regions of the 26S rRNA were 99% homologous to C. nivariensis 26S rRNA. No strains of C. bracarensis were recovered. C. nivariensis strains were very susceptible to amphotericin B, anidulafungin, micafungin, and caspofungin. Ninety-two percent of C. nivariensis were resistant to itraconazole. The halves of the strains was resistant to posaconazole. Eighty-three percent of C. nivariensis were susceptible to voriconazole. None of the tested strains were susceptible to fluconazole. In the present study, none of the C. nivariensis strains were simultaneously resistant to azoles and echinocandins. C. nivariensis should be recognized as an emerging pathogen, resistant to azoles., The data on susceptibility to antifungals of new species within Candida glabrata complex are limited. Our study was to enrich a global knowledge of yeast epidemiology and drug resistance. The study was focused on the identification of species within clinical isolates of the C. glabrata complex and on the determination of their resistance to antifungals. Four hundred forty-five clinical C. glabrata sensu lato strains were isolated from different clinical samples at routine mycological exams at the Infant Jesus Teaching Hospital in Warsaw. The identification of the most of tested isolates to species complex level was performed using the ID 32 C system. The identification of C. nivariensis and C. bracarensis species within the C. glabrata complex was performed by DNA sequencing. The MICs of amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole, caspofungin, anidulafungin, and micafungin were determined by E-test. Twenty-four isolates did not have an ITS-1 region, characteristic of C. glabrata sensu stricto and their D1/D2 regions of the 26S rRNA were 99% homologous to C. nivariensis 26S rRNA. No strains of C. bracarensis were recovered. C. nivariensis strains were very susceptible to amphotericin B, anidulafungin, micafungin, and caspofungin. Ninety-two percent of C. nivariensis were resistant to itraconazole. The halves of the strains was resistant to posaconazole. Eighty-three percent of C. nivariensis were susceptible to voriconazole. None of the tested strains were susceptible to fluconazole. In the present study, none of the C. nivariensis strains were simultaneously resistant to azoles and echinocandins. C. nivariensis should be recognized as an emerging pathogen, resistant to azoles.

Due to the antibacterial resistance crisis, developing new antibacterials is of particular interest. In this study, we combined the antifungal drug amphotericin B with 50,520 different small molecule compounds obtained from the Chinese National Compound Library in an attempt to improve its efficacy against Candida albicans persister cells. To systematically study the antifungal effect of each compound, we utilized custom-designed high-throughput microfluidic chips. Our microfluidic chips contained microchannels ranging from 3 µm to 5 µm in width to allow Candida albicans cells to line up one-by-one to facilitate fluorescence-microscope viewing. After screening, we were left with 10 small molecule compounds that improved the antifungal effects of amphotericin B more than 30% against Candida albicans persister cells.

Purpose: Identification of the emerging yeast species Candida nivariensis among presumptively identified Iranian Candida glabrata isolates., Methodology: Clinical C. glabrata species complex isolates from blood (n=71; 33.3 %), urine (n=100; 46.9 %), vaginal swabs (n=20;9.4 %), BAL (n=10; 4.7 %), and sputum (n=12; 5.6 %) from Iran were investigated. Isolates were characterized by CHROMagar, multiplex PCRs, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), amplified fragment length polymorphism (AFLP) fingerprinting, internal transcribed spacer (ITS)/large subunit (LSU) rDNA and FKS1/FKS2 sequencing, and the European Committee on Antimicrobial Susceptibility Testing broth microdilution method. A comprehensive literature review was conducted and all the relevant clinical and microbiological data were collected., Results: Four C. nivariensis isolates were recovered from blood samples of three subjects and were all consistently identified by nine-plex PCR, Bruker MALDI-TOF MS, and LSU and ITS rDNA sequencing. AFLP genotyping clustered the isolates into two groups. Sequencing of the FKS1 and FKS2 hotspots showed no accountable amino acid substitutions. All isolates were susceptible to amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin and micafungin., Conclusion: In total, 4 out of 213 clinical C. glabrata species complex candidemia isolates were C. nivariensis. Improvement of the BioMerieux Vitek MS database is required to accurately identify C. nivariensis and it is advised to alternatively use CHROMagar and/or PCR-based techniques. As other species within the Nakaseomyces clade may cause infection and showed high MIC values for antifungals, inclusion of their spectra into the MALDI-TOF MS database seems relevant. Due to developing resistance to fluconazole and insufficient efficacy of caspofungin, the combination of catheter removal plus treatment with caspofungin, or voriconazole, or micafungin might be effective for patients.

Background: Candida auris is a multidrug-resistant yeast associated with hospital outbreaks worldwide. During 2015-2016, multiple outbreaks were reported in Colombia. We aimed to understand the extent of contamination in healthcare settings and to characterize the molecular epidemiology of C. auris in Colombia., Methods: We sampled patients, patient contacts, healthcare workers, and the environment in 4 hospitals with recent C. auris outbreaks. Using standardized protocols, people were swabbed at different body sites. Patient and procedure rooms were sectioned into 4 zones and surfaces were swabbed. We performed whole-genome sequencing (WGS) and antifungal susceptibility testing (AFST) on all isolates., Results: Seven of the 17 (41%) people swabbed were found to be colonized. Candida auris was isolated from 37 of 322 (11%) environmental samples. These were collected from a variety of items in all 4 zones. WGS and AFST revealed that although isolates were similar throughout the country, isolates from the northern region were genetically distinct and more resistant to amphotericin B (AmB) than the isolates from central Colombia. Four novel nonsynonymous mutations were found to be significantly associated with AmB resistance., Conclusions: Our results show that extensive C. auris contamination can occur and highlight the importance of adherence to appropriate infection control practices and disinfection strategies. Observed genetic diversity supports healthcare transmission and a recent expansion of C. auris within Colombia with divergent AmB susceptibility.

Chitosan, a biopolymer of immense potential, has been well-explored over the past decade in the biomedical field. Despite its numerous biological properties like anti-microbial, anti-inflammatory, anti-diabetic etc., limited studies have been conducted on its ability to protect therapeutic molecules in nano-formulations. Amphotericin B (AMP) is one such therapeutic molecule which requires protection as it possesses inherent limitations of poor water-solubility, susceptibility to oxidation- and/or light-induced degradation etc. Although AMP formulations have been quite successful in treating chronic fungal infections, their high cost, prolonged nature of therapy and instability over longer durations has necessitated the development of alternative carriers. In the present study, a stable nanoparticulate formulation was successfully prepared using low molecular weight chitosan and the anti-fungal agent AMP and this was found to offer protection to the labile AMP. The developed nanocomplexes could prevent degradation of AMP up to six months, in solution form, unlike the native drug which degraded in <24 h. Antifungal studies of the developed nanocomplexes revealed a surface charge-dependent antifungal activity. Furthermore, the nanocomplexes did not affect the viability of mammalian cells and showed excellent intracellular uptake and retention, and hence suggested potential of the nanocomplexes in alleviating systemic fungal infections., (Copyright © 2018. Published by Elsevier B.V.)

Background: Multiple cases of Candida auris infection have been reported with high mortality rates owing to its MDR nature. Rezafungin (previously CD101) is a novel echinocandin with enhanced stability and pharmacokinetics that achieves high plasma drug exposure and allows for once weekly dose administration., Objectives: Evaluate the efficacy of rezafungin in the treatment of disseminated C. auris infection using a mouse model of disseminated candidiasis., Methods: Mice were immunosuppressed 3 days prior to infection and 1 day post-infection. On the day of infection, mice were inoculated with 3 × 107C. auris blastospores via the tail vein. Mice were randomized into four groups (n = 20): rezafungin at 20 mg/kg, amphotericin B at 0.3 mg/kg, micafungin at 5 mg/kg and a vehicle control. Treatments were administered 2 h post-infection. Rezafungin was given additionally on days 3 and 6 for a total of three doses, while the remaining groups were treated every day for a total of seven doses. Five mice from each group were sacrificed on days 1, 4, 7 and 10 of the study. Kidneys were removed from each mouse to determine the number of cfu for each respective day., Results: Rezafungin had significantly lower average log10 cfu/g of tissue compared with amphotericin B- and vehicle-treated mice on all days when kidneys were harvested. Additionally, rezafungin-treated mice had significantly lower average log10 cfu/g of tissue compared with micafungin-treated mice on day 10., Conclusions: Our findings show that rezafungin possesses potent antifungal activity against C. auris in a disseminated model of candidiasis.

Cationic steroid antimicrobials (CSA-ceragenin) are a new class of antimicrobial agent. In vitro activities of CSA-8, CSA-13, CSA-44, CSA-131, and CSA-138 and amphotericin B (AMP-B) were assessed against 50 nonrepeat Candida spp. isolates MICs, MFCs and combination studies were determined. Antibiofilm activities of CSAs, AMP-B, 2 azoles, and 2 echinocandins against Candida albicans were performed. Also, effects of coating the wells of plate with selected CSAs and antifungals were measured. The MIC 50 (μg/mL) values of CSA-8, CSA-13, CSA-44, CSA-131, CSA-138, and AMP-B were 16, 1, 2, 1, 1, and 1, respectively. The MFCs were equal to or 2-fold greater than those of the MICs. Synergistic interactions were mostly seen with CSA-13+ AMP-B combination, whereas the least synergistic interactions were observed with the CSA-131+ AMP-B combination. CSAs inhibited the attachment of Candida biofilms. The studied CSAs and antifungals inhibited C. albicans biofilm formation. In conclusion, CSA-13, CSA-131, and CSA-138 appear to be good candidates (alone or in combination) in the treatment of Candida infections as well as biofilm-related ones., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Systemic candidiasis is a growing health care concern that is becoming even more challenging due to the growing frequency of infections caused by multidrug-resistant (MDR) Candida species. Thus, there is an urgent need for new therapeutic approaches to candidiasis, including strategies bioinspired by insights into natural host defense against fungal pathogens. The antifungal properties of θ-defensins, macrocyclic peptides expressed in tissues of Old World monkeys, were investigated against a panel of drug-sensitive and drug-resistant clinical isolates of Candida albicans and non- albicans Candida species. Rhesus θ-defensin 1 (RTD-1), the prototype θ-defensin, was rapidly and potently fungicidal against drug-sensitive and MDR C. albicans strains. Fungal killing occurred by cell permeabilization that was temporally correlated with ATP release and intracellular accumulation of reactive oxygen species (ROS). Killing by RTD-1 was compared with that by histatin 5 (Hst 5), an extensively characterized anticandidal peptide expressed in human saliva. RTD-1 killed C. albicans much more rapidly and at a >200-fold lower concentration than that of Hst 5. Unlike Hst 5, the anticandidal activity of RTD-1 was independent of mitochondrial ATP production. Moreover, RTD-1 was completely resistant to Candida proteases for 2 h under conditions that rapidly and completely degraded Hst 5. MICs and minimum fungicidal concentrations (MFCs) of 14 natural θ-defensins isoforms against drug-resistant C. albicans isolates identified peptides that are more active than amphotericin B and/or caspofungin against fluconazole-resistant organisms, including MDR Candida auris. These results point to the potential of macrocyclic θ-defensins as structural templates for the design of antifungal therapeutics., (Copyright © 2018 American Society for Microbiology.)

Intercellular Candida glabrata infections are difficult to treat due to poor penetration of drugs into the fungal niche. Delivering amphotericin B (Amp B) into the macrophages where the pathogen inhabits is an effective solution. We are studying the macrophage targeting proficiency of ɩ-carrageenan for the delivery of Amp B using gelatin A nanoparticles (GNPs). The choice of gelatin A was the outcome of in silico inspections where the amino functionalized polymer having the best docking score with Amp B was selected. We prepared a sustained release formulation of amp B loaded carboxymethyl ɩ-carrageenan conjugated gelatin nanoparticles (CMC-Amp B-GNPs) with size 343±12nm and -25±5.3mV zeta potential. The formulations were found to be stable, biocompatible and non-haemolytic. Flow cytometry analysis showed 3 fold higher uptake of CMC-GNPs compared to the GNPs by RAW 264.7 cells. CMC-Amp B-GNPs showed enhanced antifungal activity than bare Amp B and Amp B-GNPs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The current study assesses the potential of functionalised chitosan nanoparticles (CNPs) for proficient macrophage delivery of amphotericin B (AmpB) for the management of Candida glabrata fungemia. Chitosan was functionalised by the method of sulfation by using chlorosulfonic acid and the developed compound was confirmed by FTIR, 1 H NMR and degree of sulfation and CHNS analysis. Amphotericin B encapsulated sulfated chitosan (AmpB-SCNPs), when characterized showed a hydrodynamic diameter of 310 ± 14 nm and zeta potential of 41.5 ± 2 mV. The safety of AmpB-SCNPs was established by the alamar cytotoxicity assay in nanoparticle treated macrophages following 24 h incubation. The AmpB-SCNPs showed a significant increase in the reduction of C. glabrata in comparison with the bare AmpB and AmpB-CNPs (55.2 and 42.7 vs 11.12 cfu/ml) indicating that AmpB-SCNPs could be a promising carrier for specific delivery of AmpB to macrophages for effective treatment of Candida glabrata fungemia., (Copyright © 2018. Published by Elsevier B.V.)

Candida auris is an emerging multidrug-resistant yeast that has been responsible for invasive infections associated with high morbidity and mortality. C. auris strains often demonstrate high fluconazole and amphotericin B MIC values, and some strains are resistant to all three major antifungal classes. We evaluated the susceptibility of 16 C. auris clinical strains, isolated from a wide geographical area, to 10 antifungal agents, including APX001A, a novel agent that inhibits the fungal protein Gwt1 (glycosylphosphatidylinositol-anchored wall transfer protein 1). APX001A demonstrated significantly lower MIC 50 and MIC 90 values (0.004 and 0.031 μg/ml, respectively) than all other agents tested. The efficacy of the prodrug APX001 was evaluated in an immunocompromised murine model of disseminated C. auris infection. Significant efficacy (80 to 100% survival) was observed in all three APX001 treatment groups versus 50% survival for the anidulafungin treatment group. In addition, APX001 showed a significant log reduction in CFU counts in kidney, lung, and brain tissue (1.03 to 1.83) versus the vehicle control. Anidulafungin also showed a significant log reduction in CFU in the kidneys and lungs (1.5 and 1.62, respectively) but did not impact brain CFU. These data support further clinical evaluation of this new antifungal agent., (Copyright © 2018 Hager et al.)

The aim of this study was to identify Candida spp. isolated from candiduria episodes at a tertiary hospital in the Midwest region of Brazil, and to determine their susceptibility profiles to antifungal compounds. From May 2011 to April 2012, Candida spp. isolated from 106 adult patients with candiduria admitted to the University Hospital of the Federal University of Mato Grosso do Sul were evaluated. Both, species identification and susceptibility testing with fluconazole-FLC, voriconazole-VRC, and amphotericin B-AmB were carried out using the Vitek 2. To discriminate species of the C. parapsilosis complex, a RAPD-PCR technique using the RPO2 primer was performed. From the total of 106 isolates, 42 (39.6%) C. albicans and 64 (60.4%) Candida non-albicans (CNA) - 33 C. tropicalis, 18 C. glabrata, 5 C. krusei, 4 C. parapsilosis sensu stricto, 2 C. kefyr, 1 C. lusitaniae, and 1 C. guilliermondii were identified. All isolates were susceptible to AmB and VRC, whereas all C. glabrata isolates presented either resistance (5.6%) or dose-dependent susceptibility (94.4%) to FLC. The study of Candida spp. and their resistance profiles may help in tailoring more efficient therapeutic strategies for candiduria.

Introduction: In recent years, the infection Candida albicans infection worldwide has risen, and the incidence of resistance to traditional antifungal therapies is also increasing. The aim of this study was to evaluate in vitro susceptibility of C. albicans clinical isolates to eight antifungal agents in Ouagadougou., Materials and Methods: A cross-sectional study was conducted from January 2013 to December 2015 at Yalgado Ouédraogo University Teaching Hospital. Two hundred seven strains have been isolated from 347 symptomatic patients received in different clinical services. Samples were cultured on Sabouraud Dextrose Agar supplemented with Cloramphenicol. Isolates were diagnosed as C. albicans using germ tube test, chlamydospore formation on Corn Meal Agar, and Api-Candida test (Biomérieux). Antifungal susceptibility testing was performed by disk diffusion method and isolates classified as susceptible, susceptible dose-dependent and resistant., Results: Three hundred forty-seven (347) patients are included in this study. Two hundred and six (206) out of 347 collected samples (59.36%) were found positive for C. albicans. The strains were mostly isolated from vulvovaginal (49%) and oral infections (40.3%). The highest resistance rates of azoles were obtained with fluconazole (66.5%), itraconazole (52.3%) and ketoconazole (22.9%) when all clinical isolates were included. The resistance rates of fluconazole, itraconazole and ketoconazole remain highest for vulvovaginal and oral isolates. The rate of resistance to the polyene amphotericin B was 32.0% for all clinical isolates and was 56.4% for vulvovaginal strains. Resistance rate to nystatin was 6.3% for all clinical isolates. Cross-resistance analysis with data of all clinical strains revealed that the incidence of resistance to ketoconazole and itraconazole in fluconazole-resistant isolates was significantly higher than recorded for fluconazole-susceptible isolates., Conclusion: In vitro C. albicans antifungal susceptibility test in this study showed relatively high resistance to commonly and widely used azoles (fluconazole, ketoconazole). Most C. albicans clinical isolates were susceptible to nystatin., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Introduction: Incidence and antifungal susceptibility of Candida spp. from two teaching public hospitals are described., Methods: The minimum inhibitory concentrations of fluconazole, voriconazole, itraconazole, and amphotericin B were determined using Clinical Laboratory Standard Institute broth microdilution and genomic differentiation using PCR., Results: Of 221 Candida isolates, 50.2% were obtained from intensive care unit patients; 71.5% were recovered from urine and 9.1% from bloodstream samples. Candida parapsilosis sensu stricto was the most common candidemia agent., Conclusions: We observed variations in Candida species distribution in hospitals in the same geographic region and documented the emergence of non-C. albicans species resistant to azoles.

Yeasts frequently colonize non-sterile sites in the body. The aim of the study was to determine distribution in clinical samples and antifungal susceptibility to five antifungals. From January 2013 through June 2015, 800 isolates were obtained from intensive care unit patients. Candida albicans (58.9%), Candida glabrata (20.4%), Candida krusei (8.6%), and Candida parapsilosis (3.6%) were the leading species. Majority of the C. albicans isolates were susceptible to the fluconazole. Elevated voriconazole minimal inhibitory concentrations (MICs) were observed in isolates exhibiting high fluconazole MICs, most frequently in C. glabrata. Isolates with echinocandins MICs suggesting reduced susceptibility were only sporadic cases with the exception of Trichosporon spp. The amphotericin B MICs were slightly higher for some C. krusei.

The present study was designed to develop a nanoemulsion formulation of Amphotericin B (AmB) for the treatment of skin candidiasis and aspergillosis. Several ingredients were selected on the basis of AmB solubility and compatibility with skin. The formulation that exhibited the best properties was selected from the pseudo-ternary phase diagram. After physicochemical characterization its stability was assessed. Drug release and skin permeation studies were also accomplished. The antifungal efficacy and skin tolerability of developed AmB nanoemulsion was demonstrated. Finally, our results showed that the developed AmB formulation could provide an effective local antifungal effect without theoretical systemic absorption, based on its skin retention capacity, which might avoid related side effect. These results suggested that the nanoemulsion may be an optimal therapeutic alternative for the treatment of skin fungal infections with AmB., (Copyright © 2017 Elsevier Inc. All rights reserved.)