Safety and efficacy of new generation azole antifungals in the management of recalcitrant superficial fungal infections and onychomycosis.

INTRODUCTION: Terbinafine is considered the gold standard for treating skin fungal infections and onychomycosis. However, recent reports suggest that dermatophytes are developing resistance to terbinafine and the other traditional antifungal agents, itraconazole and fluconazole. When there is resistance to terbinafine, itraconazole or fluconazole, or when these agents cannot used, for example, due to potential drug interactions with the patient's current medications, clinicians may need to consider off-label use of new generation azoles, such as voriconazole, posaconazole, fosravuconazole, or oteseconazole. It is essential to emphasize that we do not advocate the use of newer generation azoles unless traditional agents such as terbinafine, itraconazole, or fluconazole have been thoroughly evaluated as first-line therapies. AREAS COVERED: This article reviews the clinical evidence, safety, dosage regimens, pharmacokinetics, and management algorithm of new-generation azole antifungals. EXPERT OPINION: Antifungal stewardship should be the top priority when prescribing new-generation azoles. First-line antifungal therapy is terbinafine and itraconazole. Fluconazole is a consideration but is generally less effective and its use may be off-label in many countries. For difficult-to-treat skin fungal infections and onychomycosis, that have failed terbinafine, itraconazole and fluconazole, we propose consideration of off-label voriconazole or posaconazole.

Multicenter Candida auris outbreak caused by azole-susceptible clade IV in Pernambuco, Brazil.

BACKGROUND: Candida auris is an emerging multidrug-resistant yeast, frequently causing outbreaks in health care facilities. The pathogen persistently colonises human skin and inanimate surfaces such as catheters, aiding to its spread. Moreover, colonisation is a risk factor to develop invasive infection. OBJECTIVES: We investigated 61 C. auris strains isolated from non-sterile human body sites (n = 53) and the hospital environment (n = 8), originating from four different centres in a single Brazilian state. MATERIALS AND METHODS: Antifungal susceptibility testing (AFST) against common antifungals was performed, and resistance-associated genes were evaluated. Genetic relatedness was investigated with short tandem repeat (STR) genotyping and validated with whole-genome sequencing (WGS) single nucleotide polymorphism (SNP) analysis. RESULTS: Antifungal susceptibility testing demonstrated that all isolates were susceptible to azoles, echinocandins and amphotericin B. No mutations were detected in ERG11 and FKS1 genes. With STR typing, isolates were allocated to clade IV and appeared closely related. This was confirmed by WGS SNP analysis of 6 isolates, which demonstrated a maximal difference of only 41 SNPs between these strains. Furthermore, the Brazilian isolates formed a distinct autochthonous branch within clade IV, excluding recent introductions from outside the country. A molecular clock analysis of clade IV isolates from various countries suggests that early in the previous century there was a unique event causing environmental spread of a C. auris ancestor throughout the Latin-American continent, followed by human introduction during the last decades. CONCLUSION: We report the emergence of C. auris patient colonisation in multiple centres by fluconazole-susceptible clade IV close-related strains in Pernambuco State, Brazil.

Azole resistance in Aspergillus fumigatus- comprehensive review.

Aspergillus fumigatus is a ubiquitous filamentous fungus commonly found in the environment. It is also an opportunistic human pathogen known to cause a range of respiratory infections, such as invasive aspergillosis, particularly in immunocompromised individuals. Azole antifungal agents are widely used for the treatment and prophylaxis of Aspergillus infections due to their efficacy and tolerability. However, the emergence of azole resistance in A. fumigatus has become a major concern in recent years due to their association with increased treatment failures and mortality rates. The development of azole resistance in A. fumigatus can occur through both acquired and intrinsic mechanisms. Acquired resistance typically arises from mutations in the target enzyme, lanosterol 14-α-demethylase (Cyp51A), reduces the affinity of azole antifungal agents for the enzyme, rendering them less effective, while intrinsic resistance refers to a natural resistance of certain A. fumigatus isolates to azole antifungals due to inherent genetic characteristics. The current review aims to provide a comprehensive overview of azole antifungal resistance in A. fumigatus, discusses underlying resistance mechanisms, including alterations in the target enzyme, Cyp51A, and the involvement of efflux pumps in drug efflux. Impact of azole fungicide uses in the environment and the spread of resistant strains is also explored.

Development of a Biosafety Level 1 Cellular Assay for Identifying Small-Molecule Antivirals Targeting the Main Protease of SARS-CoV-2: Evaluation of Cellular Activity of GC376, Boceprevir, Carmofur, Ebselen, and Selenoneine.

While research has identified several inhibitors of the main protease (Mpro) of SARS-CoV-2, a significant portion of these compounds exhibit reduced activity in the presence of reducing agents, raising concerns about their effectiveness in vivo. Furthermore, the conventional biosafety level 3 (BSL-3) for cellular assays using viral particles poses a limitation for the widespread evaluation of Mpro inhibitor efficacy in a cell-based assay. Here, we established a BSL-1 compatible cellular assay to evaluate the in vivo potential of Mpro inhibitors. This assay utilizes mammalian cells expressing a tagged Mpro construct containing N-terminal glutathione S-transferase (GST) and C-terminal hemagglutinin (HA) tags and monitors Mpro autodigestion. Using this method, GC376 and boceprevir effectively inhibited Mpro autodigestion, suggesting their potential in vivo activity. Conversely, carmofur and ebselen did not exhibit significant inhibitory effects in this assay. We further investigated the inhibitory potential of selenoneine on Mpro using this approach. Computational analyses of binding energies suggest that noncovalent interactions play a critical role in facilitating the covalent modification of the C145 residue, leading to Mpro inhibition. Our method is straightforward, cost-effective, and readily applicable in standard laboratories, making it accessible to researchers with varying levels of expertise in infectious diseases.

New insides into chimeric and hybrid azines derivatives with antifungal activity.

During the last decades, five or six member rings azaheterocycles compounds appear to be an extremely valuable source of antifungal agents. Their use seems to be a very attractive solution in antifungal therapy and to overcome antifungal resistance in agriculture. The present review highlights the main results obtained in the field of hybrid and chimeric azine (especially pyridine, quinoline, phenanthroline, bypyridine, naphthyridine and their fused derivatives) derivatives presented in scientific literature from the last 10 years, with emphasis on antifungal activity of the mentioned compounds. A special attention was played to hybrid and chimeric azole-azine class, having in view the high antifungal potential of azoles.

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Functional analysis of the Aspergillus fumigatus kinome identifies a druggable DYRK kinase that regulates septal plugging.

More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. Azole antifungals represent first-line therapeutics for most of these infections but resistance is rising, therefore the identification of antifungal targets whose inhibition synergises with the azoles could improve therapeutic outcomes. Here, we generate a library of 111 genetically barcoded null mutants of Aspergillus fumigatus in genes encoding protein kinases, and show that loss of function of kinase YakA results in hypersensitivity to the azoles and reduced pathogenicity. YakA is an orthologue of Candida albicans Yak1, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. We show that YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and to grow in mouse lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit C. albicans Yak1, prevents stress-mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.

Azole Resistance in Veterinary Clinical Aspergillus fumigatus Isolates in the Netherlands.

Aspergillus fumigatus is a saprophytic fungal pathogen that causes opportunistic infections in animals and humans. Azole resistance has been reported globally in human A. fumigatus isolates, but the prevalence of resistance in isolates from animals is largely unknown. A retrospective resistance surveillance study was performed using a collection of clinical A. fumigatus isolates from various animal species collected between 2015 and 2020. Agar-based azole resistance screening of all isolates was followed by in vitro antifungal susceptibility testing and cyp51A gene sequencing of the azole-resistant isolates. Over the 5 year period 16 (11.3%) of 142 A. fumigatus culture-positive animals harbored an azole-resistant isolate. Resistant isolates were found in birds (15%; 2/13), cats (21%; 6/28), dogs (8%; 6/75) and free-ranging harbor porpoise (33%; 2/6). Azole-resistance was cyp51A mediated in all isolates: 81.3% (T-67G/)TR34/L98H, 12.5% TR46/Y121F/T289A. In one azole-resistant A. fumigatus isolate a combination of C(-70)T/F46Y/C(intron7)T/C(intron66)T/M172V/E427K single-nucleotide polymorphisms in the cyp51A gene was found. Of the animals with an azole-resistant isolate and known azole exposure status 71.4% (10/14) were azole naive. Azole resistance in A. fumigatus isolates from animals in the Netherlands is present and predominantly cyp51A TR-mediated, supporting an environmental route of resistance selection. Our data supports the need to include veterinary isolates in resistance surveillance programs. Veterinarians should consider azole resistance as a reason for therapy failure when treating aspergillosis and consider resistance testing of relevant isolates.

Bisphosphonates synergistically enhance the antifungal activity of azoles in dermatophytes and other pathogenic molds.

Superficial infections of the skin, hair, and nails by fungal dermatophytes are the most prevalent of human mycoses, and many infections are refractory to treatment. As current treatment options are limited, recent research has explored drug synergy with azoles for dermatophytoses. Bisphosphonates, which are approved to treat osteoporosis, can synergistically enhance the activity of azoles in diverse yeast pathogens but their activity has not been explored in dermatophytes or other molds. Market bisphosphonates risedronate, alendronate, and zoledronate (ZOL) were evaluated for antifungal efficacy and synergy with three azole antifungals: fluconazole (FLC), itraconazole (ITR), and ketoconazole (KET). ZOL was the most active bisphosphonate tested, displaying moderate activity against nine dermatophyte species (MIC range 64-256 µg/mL), and was synergistic with KET in eight of these species. ZOL was also able to synergistically improve the anti-biofilm activity of KET and combining KET and ZOL prevented the development of antifungal resistance. Rescue assays in Trichophyton rubrum revealed that the inhibitory effects of ZOL alone and in combination with KET were due to the inhibition of squalene synthesis. Fluorescence microscopy using membrane- and ROS-sensitive probes demonstrated that ZOL and KET:ZOL compromised membrane structure and induced oxidative stress. Antifungal activity and synergy between bisphosphonates and azoles were also observed in other clinically relevant molds, including species of Aspergillus and Mucor. These findings indicate that repurposing bisphosphonates as antifungals is a promising strategy for revitalising certain azoles as topical antifungals, and that this combination could be fast-tracked for investigation in clinical trials. IMPORTANCE: Fungal infections of the skin, hair, and nails, generally grouped together as "tineas" are the most prevalent infectious diseases globally. These infections, caused by fungal species known as dermatophytes, are generally superficial, but can in some cases become aggressive. They are also notoriously difficult to resolve, with few effective treatments and rising levels of drug resistance. Here, we report a potential new treatment that combines azole antifungals with bisphosphonates. Bisphosphonates are approved for the treatment of low bone density diseases, and in fungi they inhibit the biosynthesis of the cell membrane, which is also the target of azoles. Combinations were synergistic across the dermatophyte species and prevented the development of resistance. We extended the study to molds that cause invasive disease, finding synergy in some problematic species. We suggest bisphosphonates could be repurposed as synergents for tinea treatment, and that this combination could be fast-tracked for use in clinical therapy.

Study on the Antifungal Activity of Gallic Acid and Its Azole Derivatives against Fusarium graminearum.

The wheat scab caused by Fusarium graminearum (F. graminearum) has seriously affected the yield and quality of wheat in China. In this study, gallic acid (GA), a natural polyphenol, was used to synthesize three azole-modified gallic acid derivatives (AGAs1-3). The antifungal activity of GA and its derivatives against F. graminearum was studied through mycelial growth rate experiments and field efficacy experiments. The results of the mycelial growth rate test showed that the EC50 of AGAs-2 was 0.49 mg/mL, and that of AGAs-3 was 0.42 mg/mL. The biological activity of AGAs-3 on F. graminearum is significantly better than that of GA. The results of field efficacy tests showed that AGAs-2 and AGAs-3 significantly reduced the incidence rate and disease index of wheat scab, and the control effect reached 68.86% and 72.11%, respectively. In addition, preliminary investigation was performed on the possible interaction between AGAs-3 and F. graminearum using density functional theory (DFT). These results indicate that compound AGAs-3, because of its characteristic of imidazolium salts, has potential for use as a green and environmentally friendly plant-derived antifungal agent for plant pathogenic fungi.

Exploring Antiviral Drugs on Monolayer Black Phosphorene: Atomistic Theory and Explainable Machine Learning-Assisted Platform.

Favipiravir (FP) and ebselen (EB) belong to a diverse class of antiviral drugs known for their significant efficacy in treating various viral infections. Utilizing molecular dynamics (MD) simulations, machine learning, and van der Waals density functional theory, we accurately elucidate the binding properties of these antiviral drugs on a phosphorene single-layer. To further investigate these characteristics, this study employs four distinct machine learning models-Random Forest, Gradient Boosting, XGBoost, and CatBoost. The Hamiltonian of antiviral molecules within a monolayer of phosphorene is appropriately trained. The key aspect of utilizing machine learning (ML) in drug design revolves around training models that are efficient and precise in approximating density functional theory (DFT). Furthermore, the study employs SHAP (SHapley Additive exPlanations) to elucidate model predictions, providing insights into the contribution of each feature. To explore the interaction characteristics and thermodynamic properties of the hybrid drug, we employ molecular dynamics and DFT calculations in a vacuum interface. Our findings suggest that this functionalized 2D complex exhibits robust thermostability, indicating its potential as an effective and enabled entity. The observed variations in free energy at different surface charges and temperatures suggest the adsorption potential of FP and EB molecules from the surrounding environment.

Ebselen analogues delay disease onset and its course in fALS by on-target SOD-1 engagement.

Amyotrophic lateral sclerosis (ALS) selectively affects motor neurons. SOD1 is the first causative gene to be identified for ALS and accounts for at least 20% of the familial (fALS) and up to 4% of sporadic (sALS) cases globally with some geographical variability. The destabilisation of the SOD1 dimer is a key driving force in fALS and sALS. Protein aggregation resulting from the destabilised SOD1 is arrested by the clinical drug ebselen and its analogues (MR6-8-2 and MR6-26-2) by redeeming the stability of the SOD1 dimer. The in vitro target engagement of these compounds is demonstrated using the bimolecular fluorescence complementation assay with protein-ligand binding directly visualised by co-crystallography in G93A SOD1. MR6-26-2 offers neuroprotection slowing disease onset of SOD1G93A mice by approximately 15 days. It also protected neuromuscular junction from muscle denervation in SOD1G93A mice clearly indicating functional improvement.

Spectrum of activity and mechanisms of azole-bisphosphonate synergy in pathogenic Candida.

Candidiasis places a significant burden on human health and can range from common superficial vulvovaginal and oral infections to invasive diseases with high mortality. The most common Candida species implicated in human disease is Candida albicans, but other species like Candida glabrata are emerging. The use of azole antifungals for treatment is limited by increasing rates of resistance. This study explores repositioning bisphosphonates, which are traditionally used for osteoporosis, as antifungal synergists that can improve and revitalize the use of azoles. Risedronate, alendronate, and zoledronate (ZOL) were tested against isolates from six different species of Candida, and ZOL produced moderate antifungal activity and strong synergy with azoles like fluconazole (FLC), particularly in C. glabrata. FLC:ZOL combinations had increased fungicidal and antibiofilm activity compared to either drug alone, and the combination prevented the development of antifungal resistance. Mechanistic investigations demonstrated that the synergy was mediated by the depletion of squalene, resulting in the inhibition of ergosterol biosynthesis and a compromised membrane structure. In C. glabrata, synergy compromised the function of membrane-bound multidrug transporters and caused an accumulation of reactive oxygen species, which may account for its acute sensitivity to FLC:ZOL. The efficacy of FLC:ZOL in vivo was confirmed in a Galleria mellonella infection model, where combinations improved the survival of larvae infected with C. albicans and C. glabrata to a greater extent than monotherapy with FLC or ZOL, and at reduced dosages. These findings demonstrate that bisphosphonates and azoles are a promising new combination therapy for the treatment of topical candidiasis. IMPORTANCE: Candida is a common and often very serious opportunistic fungal pathogen. Invasive candidiasis is a prevalent cause of nosocomial infections with a high mortality rate, and mucocutaneous infections significantly impact the quality of life of millions of patients a year. These infections pose substantial clinical challenges, particularly as the currently available antifungal treatment options are limited in efficacy and often toxic. Azoles are a mainstay of antifungal therapy and work by targeting the biosynthesis of ergosterol. However, there are rising rates of acquired azole resistance in various Candida species, and some species are considered intrinsically resistant to most azoles. Our research demonstrates the promising therapeutic potential of synergistically enhancing azoles with non-toxic, FDA-approved bisphosphonates. Repurposing bisphosphonates as antifungal synergists can bypass much of the drug development pipeline and accelerate the translation of azole-bisphosphonate combination therapy.

Azoles activate type I and type II programmed cell death pathways in crop pathogenic fungi.

Triazoles are widely used to control pathogenic fungi. They inhibit the ergosterol biosynthetic pathway, but the precise mechanisms leading to fungicidal activities in many fungal pathogens are poorly understood. Here, we elucidate the mode of action of epoxiconazole and metconazole in the wheat pathogen Zymoseptoria tritici and the rice blast fungus Magnaporthe oryzae. We show that both azoles have fungicidal activity and reduce fluidity, but not integrity, of the plasma membrane. This impairs localisation of Cdc15-like F-BAR proteins, resulting in defective actin ring assembly and incomplete septation. However, mutant studies and pharmacological experiments in vitro and in planta show that azole lethality is due to a combination of reactive oxygen species-induced apoptosis and macroautophagy. Simultaneous inhibition of both programmed cell death pathways abolishes azole-induced cell death. Other classes of ergosterol biosynthesis inhibitors also induce apoptosis and macroautophagy, suggesting that activation of these two cell death pathways is a hallmark of ergosterol synthesis-targeting fungicides. This knowledge will inform future crop protection strategies.

Ebselen improves fungal keratitis through exerting anti-inflammation, anti-oxidative stress, and antifungal effects.

Fungal keratitis is a severely vision-threatening corneal infection, where the prognosis depends on both fungal virulence and host immune defense. Inappropriate host responses can induce substantial inflammatory damage to the cornea. Therefore, in the treatment of fungal keratitis, it is important to concurrently regulate the immune response while efforts are made to eliminate the pathogen. Ebselen is a widely studied organo-selenium compound and has been demonstrated to have antifungal, antibacterial, anti-inflammatory, and oxidative stress-regulatory properties. The effectiveness of ebselen for the treatment of fungal keratitis remains unknown. In this study, ebselen was demonstrated to produce a marked inhibitory effect on Aspergillus fumigatus (A. fumigatus), including spore germination inhibition, mycelial growth reduction, and fungal biofilm disruption. The antifungal activity of ebselen was related to the cell membrane damage caused by thioredoxin (Trx) system inhibition-mediated oxidative stress. On the contrary, ebselen enhanced the antioxidation of Trx system in mammalian cells. Further, ebselen was proven to suppress the expressions of inflammatory mediators (IL-1ß, IL-6, TNF-α, COX-2, iNOS, and CCL2) and reduce the production of oxidative stress-associated indicators (ROS, NO, and MDA) in fungi-stimulated RAW264.7 cells. In addition, ebselen regulated PI3K/Akt/Nrf2 and p38 MAPK signaling pathways, which contributed to the improvement of inflammation and oxidative stress. Finally, we verified the therapeutic effect of ebselen on mouse fungal keratitis. Ebselen improved the prognosis and reduced the fungal burden in mouse corneas. Expressions of inflammatory mediators, as well as the infiltration of macrophages and neutrophils in the cornea were also obviously decreased by ebselen. In summary, ebselen exerted therapeutic effects by reducing fungal load and protecting host tissues in fungal keratitis, making it a promising treatment for fungal infections.

Investigation of Azole Resistance Involving cyp51A and cyp51B Genes in Clinical Aspergillus flavus Isolates.

This study aimed to investigate azole resistance mechanisms in Aspergillus flavus, which involve cyp51A and cyp51B genes. Real-time Reverse Transcriptase qPCR method was applied to determine the overexpression of cyp51A and cyp51B genes for 34 A. flavus isolates. PCR sequencing of these two genes was used to detect the presence of gene mutations. Susceptibility test found sensitivity to voriconazole (VOR) in all strains. 14.7% and 8.8% of isolates were resistant to itraconazole (IT) and posaconazole (POS), respectively, with a cross-resistance in 5.8%. For the double resistant isolates (IT/POS), the expression of cyp51A was up to 17-fold higher. PCR sequencing showed the presence of 2 mutations in cyp51A: a synonymous point mutation (P61P) in eight isolates, which did not affect the structure of CYP51A protein, and another non synonymous mutation (G206L) for only the TN-33 strain (cross IT/POS resistance) causing an amino acid change in the protein sequence. However, we noted in cyp51B the presence of the only non-synonymous mutation (L177G) causing a change in amino acids in the protein sequence for the TN-31 strain, which exhibits IT/POS cross-resistance. A short single intron of 67 bp was identified in the cyp51A gene, whereas three short introns of 54, 53, and 160 bp were identified in the cyp51B gene. According to the models provided by PatchDock software, the presence of non-synonymous mutations did not affect the interaction of CYP51A and CYP51B proteins with antifungals. In our study, the overexpression of the cyp51A and cyp51B genes is the primary mechanism responsible for resistance in A. flavus collection. Nevertheless, other resistance mechanisms can be involved.

Effects of ebselen addition on emotional processing and brain neurochemistry in depressed patients unresponsive to antidepressant medication.

Lithium is an effective augmenting agent for depressed patients with inadequate response to standard antidepressant therapy, but numerous adverse effects limit its use. We previously reported that a lithium-mimetic agent, ebselen, promoted a positive emotional bias-an indicator of potential antidepressant activity in healthy participants. We therefore aimed to investigate the effects of short-term ebselen treatment on emotional processing and brain neurochemistry in depressed patients with inadequate response to standard antidepressants. We conducted a double-blind, placebo-controlled 7-day experimental medicine study in 51 patients with major depressive disorder who were currently taking antidepressants but had an inadequate response to treatment. Participants received either ebselen 600 mg twice daily for seven days or identical matching placebo. An emotional testing battery, magnetic resonance spectroscopy and depression and anxiety rating scales were conducted at baseline and after seven days of treatment. Ebselen did not increase the recognition of positive facial expressions in the depressed patient group. However, ebselen increased the response bias towards fear emotion in the signal detection measurement. In the anterior cingulate cortex, ebselen significantly reduced the concentrations of inositol and Glx (glutamate+glutamine). We found no significant differences in depression and anxiety rating scales between visits. Our study did not find any positive shift in emotional bias in depressed patients with an inadequate response to antidepressant medication. We confirmed the ability of ebselen to lower inositol and Glx in the anterior cingulate cortex. These latter effects are probably mediated through inhibition of inositol monophosphatase and glutaminase respectively.

Adhesive and biofilm-forming Candida glabrata Lebanese hospital isolates harbour mutations in subtelomeric silencers and adhesins.

BACKGROUND: The prevalence of Candida glabrata healthcare-associated infections is on the rise worldwide and in Lebanon, Candida glabrata infections are difficult to treat as a result of their resistance to azole antifungals and their ability to form biofilms. OBJECTIVES: The first objective of this study was to quantify biofilm biomass in the most virulent C. glabrata isolates detected in a Lebanese hospital. In addition, other pathogenicity attributes were evaluated. The second objective was to identify the mechanisms of azole resistance in those isolates. METHODS: A mouse model of disseminated systemic infection was developed to evaluate the degree of virulence of 41 azole-resistant C. glabrata collected from a Lebanese hospital. The most virulent isolates were further evaluated alongside an isolate having attenuated virulence and a reference strain for comparative purposes. A DNA-sequencing approach was adopted to detect single nucleotide polymorphisms (SNPs) leading to amino acid changes in proteins involved in azole resistance and biofilm formation. This genomic approach was supported by several phenotypic assays. RESULTS: All chosen virulent isolates exhibited increased adhesion and biofilm biomass compared to the isolate having attenuated virulence. The amino acid substitutions D679E and I739N detected in the subtelomeric silencer Sir3 are potentially involved- in increased adhesion. In all isolates, amino acid substitutions were detected in the ATP-binding cassette transporters Cdr1 and Pdh1 and their transcriptional regulator Pdr1. CONCLUSIONS: In summary, increased adhesion led to stable biofilm formation since mutated Sir3 could de-repress adhesins, while decreased azole susceptibility could result from mutations in Cdr1, Pdh1 and Pdr1.

Prevalence and genetic diversity of azole-resistant Malassezia pachydermatis isolates from canine otitis and dermatitis: A 2-year study.

Despite previous reports on the emergence of Malassezia pachydermatis strains with decreased susceptibility to azoles, there is limited information on the actual prevalence and genetic diversity of azole-resistant isolates of this yeast species. We assessed the prevalence of azole resistance in M. pachydermatis isolates from cases of dog otitis or skin disease attended in a veterinary teaching hospital during a 2-year period and analyzed the ERG11 (encoding a lanosterol 14-α demethylase, the primary target of azoles) and whole genome sequence diversity of a group of isolates that displayed reduced azole susceptibility. Susceptibility testing of 89 M. pachydermatis isolates from 54 clinical episodes (1-6 isolates/episode) revealed low minimum inhibitory concentrations (MICs) to most azoles and other antifungals, but 11 isolates from six different episodes (i.e., 12.4% of isolates and 11.1% of episodes) had decreased susceptibility to multiple azoles (fluconazole, itraconazole, ketoconazole, posaconazole, ravuconazole, and/or voriconazole). ERG11 sequencing of these 11 azole-resistant isolates identified eight DNA sequence profiles, most of which contained amino acid substitutions also found in some azole-susceptible isolates. Analysis of whole genome sequencing (WGS) results revealed that the azole-resistant isolates from the same episode of otitis, or even different episodes affecting the same animal, were more genetically related to each other than to isolates from other dogs. In conclusion, our results confirmed the remarkable ERG11 sequence variability in M. pachydermatis isolates of animal origin observed in previous studies and demonstrated the value of WGS for disentangling the epidemiology of this yeast species.

We analyzed the prevalence and diversity of azole-resistant Malassezia pachydermatis isolates in a veterinary hospital. A low prevalence of multi-azole resistance (c.10% of isolates and cases) was found. Whole genome and ERG11 sequencing of resistant isolates revealed remarkable genetic diversity.

Treatment of vaginitis caused by non-albicans Candida species.

INTRODUCTION: In the face of increased frequency of non-albicans Candida vulvovaginitis (VVC) reported worldwide, there is a paucity of effective oral and topical antifungal drugs available. Drug selection is further handicapped by an absence of data of clinical efficacy of available antifungal drugs for these infections. AREAS COVERED: In this review, attention is directed at the cause of drug shortage as well as increased frequency of non-albicans Candida (NAC) vulvovaginitis. There is widespread recognition of reduced in vitro azole drug susceptibility in NAC species. Moreover, antifungal susceptibility tests have not been standardized or validated for NAC isolates, hence clinicians rely on an element of empiricism especially given the absence of randomized controlled comparative studies targeting NAC species. Clinical spectrum of NAC species isolates is highly variable with ongoing difficulty in determining a causal role in symptomatic patients. EXPERT OPINION: We have entered the era of demand for Candida species-specific therapy and although consensus treatment guidelines are emerging, new antifungal agents that target these multiple-azole resistant or relatively resistant vaginal NAC species are urgently needed.

Geldanamycin confers fungicidal properties to azole by triggering the activation of succinate dehydrogenase.

AIMS: Azoles have been widely employed for the treatment of invasive fungal diseases; however, their efficacy is diminished as pathogenic fungi tolerate them due to their fungistatic properties. Geldanamycin (GdA) can render azoles fungicidal by inhibiting the ATPase and molecular chaperone activities of heat shock protein 90 (Hsp90). Nonetheless, the clinical applicability of GdA is restricted due to its cytotoxic ansamycin scaffold structure, its induction of cytoprotective heat shock responses, and the conservative nature of Hsp90. Hence, it is imperative to elucidate the mechanism of action of GdA to confer fungicidal properties to azoles and mitigate the toxic adverse effects associated with GdA. MATERIALS AND METHODS: Through various experimental methods, including the construction of gene-deleted Candida albicans mutants, in vitro drug sensitivity experiments, Western blot analysis, reactive oxygen species (ROS) assays, and succinate dehydrogenase activity assays, we identified Hsp90 client proteins associated with the tolerance of C. albicans to azoles. KEY FINDINGS: It was observed that GdA effectively hindered the entry of Hsp90 into mitochondria, resulting in the alleviation of inhibitory effect of Hsp90 on succinate dehydrogenase. Consequently, the activation of succinate dehydrogenase led to an increased production of ROS. within the mitochondria, thereby facilitating the antifungal effects of azoles against C. albicans. SIGNIFICANCE: This research presents a novel approach for conferring fungicidal properties to azoles, which involves specifically disrupting the interaction of between Hsp90 and succinate dehydrogenase rather than employing a non-specific inhibition of ATPase activity of Hsp90.