Acquired resistance or tolerance? - in search of mechanisms underlying changes in the resistance profile of Candida albicans and Candida parapsilosis as a result of exposure to methotrexate.

The increasing prevalence of fungal strains showing acquired resistance and multidrug resistance is an increasing therapeutic problem, especially in patients with a severely weakened immune system and undergoing chemotherapy. What is also extremely disturbing is the similarity of the resistance mechanisms of fungal cells and other eukaryotic cells, including human cells, which may contribute to the development of cross-resistance in fungi in response to substances used in e.g. anticancer treatment. An example of such a drug is methotrexate, which is pumped out of eukaryotic cells by ABC transmembrane transporters - in fungi, used to remove azoles from fungal cells. For this reason, the aim of the study was to analyze the expression levels of genes: ERG11, MDR1 and CDR1, potentially responsible for the occurrence of cross-resistance in Candida albicans and Candida parapsilosis as a result of fungal exposure to methotrexate (MTX). In vitro exposure of C. albicans and C. parapsilosis strains to methotrexate showed a high increase in resistance to fluconazole and a partial increase in resistance to voriconazole. Analysis of the expression of resistance genes showed varied responses of the tested strains depending on the species. In the case of C. albicans, an increase in the expression of the MDR1 gene was observed, and a decrease in ERG11 and CDR1. However, for C. parapsilosis there was an increase in the expression of the CDR1 gene and a decrease in ERG11 and MDR1. We noted the relationship between the level of resistance to voriconazole and the level of ERG11 gene expression in C. albicans. This indicates that this type of relationship is different for each species. Our research confirms that the mechanisms by which fungi acquire resistance and develop cross-resistance are highly complex and most likely involve several pathways simultaneously. The emergence of multidrug resistance may be related to the possibility of developing tolerance to antimycotics by fungi.

Four-Arm δ-Ornithine-Based Polypeptoids Resensitize Voriconazole against Azole-Resistant C. albicans.

Worldwide Candida albicans infections cause a huge burden in healthcare and the efficacy of traditional antifungals is diminished because of the rapid development of antifungal resistance. It is necessary to develop new antifungals or new strategies to make multidrug-resistant (MDR) C. albicans to resensitize to existing antifungal drugs. In this work, a series of 4-arm polypeptoids (FAPs) were synthesized through grafting linear ε-l-lysine or δ-ornithine-based oligopeptides to a trimeric lysine core. The most potent 4R-O7 exhibited excellent activities toward three sensitive and two MDR C. albicans strains with MIC values as low as 24-48 µg/mL (vs 375 µg/mL for ε-polylysine, ε-PL). The mechanism studies revealed that 4R-O7 penetrated the cell membrane and generated ROS to kill cells. 4R-O7 exhibited a synergistic effect (FICI < 0.5) with voriconazole (VOR) and also assisted VOR to restore its efficacy to MDR C. albicans. In addition, the combined use of 4R-O7 and VOR significantly improved the elimination efficacy of mature C. albicans biofilms and enhanced the potency in a mouse subcutaneous C. albicans infection model.

Impression Cytologic Evaluation of the Conjunctiva in Patients Treated with Topical 1% Voriconazole

Objectives: The aim of the present study was to evaluate any conjunctival metaplastic changes by impression cytology in patients who underwent topical 1% voriconazole treatment for severe fungal keratitis. Materials and Methods: The study was conducted at Ege University Faculty of Medicine, Departments of Ophthalmology and Medical Pathology. Patients who were treated with 1% topical voriconazole for fungal keratitis for at least 3 months were included. The used topical voriconazole treatment was initiated as one drop every hour and was tapered according to clinical improvement in all patients. Treatment was continued 4 times a day for at least 3 months. Impression cytology samples were collected at least 3 months after cessation of topical voriconazole from the affected eyes and from the fellow eyes as a control group. Collected specimens were transferred to the pathology department for evaluation and grading (Nelson's grading system). Results: The mean age of the patients was 57.68±17.32 years (range, 22-87 years). The impression cytology grade of the inferior bulbar conjunctiva was 1.73±0.77 (range, 0-3) in the study group and 1.19±0.98 (range, 0-3) in the control group (p=0.03). The impression cytology grade of the temporal bulbar conjunctiva was 1.69±0.73 (range, 0-3) in the study group and 1.15±0.88 (range, 0-3) in the control group (p=0.02). The impression cytology grades of the nasal and superior bulbar conjunctiva did not differ statistically (p values 0.13 and 0.17, respectively). Conclusion: Topical voriconazole is an effective broad-spectrum antifungal drug, but it induces conjunctival squamous metaplasia. Clinicians should be aware of this possible side effect of topical voriconazole and should carefully evaluate the conjunctiva of treated patients at each visit to detect possible metaplastic changes.

Analysis of the relationship between drug susceptibility of Cryptococcus neoformans isolates and mortality in HIV-negative cryptococcal meningitis.

OBJECTIVES: The relationship between antifungal susceptibility and mortality of cryptococcal meningitis (CM) in HIV-negative patients is poorly understood. METHODS: We conducted a retrospective analysis of 1-year follow-up of 200 HIV-negative CM patients with an initial cerebrospinal fluid (CSF) culture for Cryptococcus neoformans. According to the cut-off values of minimum inhibitory concentration (MIC), two groups of five antifungal agents were classified: amphotericin B (AmB), ≤0.5 µg/mL, >0.5 µg/mL; 5-flucytosine (5-FC), ≤4 µg/mL, >4 µg/mL; fluconazole (FLU), ≤4 µg/mL, >4 µg/mL; itraconazole (ITR), ≤0.125 µg/mL, >0.125 µg/mL; and voriconazole (VOR), <0.25 µg/mL, ≥0.25 µg/mL. Comparisons were performed to analyse clinical features, laboratory, modified Rankin Scale (mRS) scores, and CSF findings under different prognosis outcomes in 1-year. RESULTS: All of Cryptococcus neoformans isolates were sensitive to AmB and VOR, most of them were sensitive to 5-FC and FLU (95.5% and 90.5%, respectively) while only 55.0% of them were susceptible to ITR. Minimum inhibitory concentrations of ITR and VOR were significantly related to baseline mRS scores. All-cause mortality was not significantly related to MICs in Cryptococcus neoformans strains. The combination of actual antifungal agents and two groups of the MICs values for antifungal agents had no significant effects on all-cause mortality. CONCLUSION: Most Cryptococcus neoformans isolates were sensitive to AmB, VOR, 5-FC, and FLU. Because of the small number of deaths, we are not able to comment on whether MIC is associated with mortality of CM in HIV-negative patients.

Alarming Increase of Azole-Resistant Candida Causing Blood Stream Infections in Oncology Patients in Egypt.

Candidemia is a life-threatening invasive fungal infection in immunocompromised patients. The widespread use of azoles and the shift toward non-albicans Candida (NAC) species remarkably increase azole resistance in developing countries. We aimed to study candidemia trends and associated risk factors in oncology patients since they vary geographically, and rapid and appropriate treatment improves outcomes. Vitek 2 was used to identify the Candida species, and the E-test determined their susceptibility to azoles. Candida was the cause of 3.1% (n = 53/1701) of bloodstream infections (BSIs) during a 1-year study. Candida tropicalis was the most predominant species among the 30 candidemia episodes studied (36.7%), followed by C. albicans (33.3%). However, C. krusei, C. guilliermondii, C. pelliculosa, C. parapsilosis, C. famata, and C. inconspicua accounted for 30.0% of the isolates. An increased risk of NAC BSI was significantly associated with chemotherapy and leucopenia (P = 0.036 and 0.016, respectively). However, the multivariable analysis revealed that leucopenia was the only independent risk factor (P = 0.048). Fluconazole and voriconazole resistance were 58.3% and 16.7%, with NAC species showing higher resistance rates than C. albicans. Both fluconazole and voriconazole minimum inhibitory concentration (MIC) median values were higher in NAC than in C. albicans, but only voriconazole was significantly higher (0.220 versus 0.048 µg/ml, P = 0.047). In conclusion, the increased prevalence of NAC BSIs and incredibly high fluconazole resistance rates in cancer patients emphasize the necessity of antifungal stewardship to preserve voriconazole effectiveness, continued surveillance of candidemia, and future studies into azole resistance molecular mechanisms.

Evaluation of the inhibitory effect of azoles on pharmacokinetics of lenvatinib in rats both in vivo and in vitro by UPLC-MS/MS.

BACKGROUND: Lenvatinib is a multitargeted tyrosine kinase inhibitor used in the treatment of a variety of solid tumors. This study aims to investigate the potential pharmacokinetic interactions between lenvatinib and various azoles (ketoconazole, voriconazole, isavuconazole and posaconazole) when orally administered to rats. METHODS: A total of 30 Sprague-Dawley rats were randomly allocated into five groups and administered 20 mg/kg of ketoconazole, voriconazole, isavuconazole and 30 mg/kg of posaconazole and 0.5% CMC-Na, through gavage for a duration of 7 days prior to the commencement of the experiment. On the final day, the rats were given 10 mg/kg of lenvatinib. The blood concentration of lenvatinib was determined using UPLC-MS-MS. In vitro lenvatinib were incubated with azoles and rat liver microsomes (RLMs) or human liver microsomes (HLMs). Molecular docking was lastly used to examine the binding strength of the enzymes and ligands with Autodock Vina. RESULTS: AUC and Cmax of lenvatinib significantly increased with each of the azoles (p < 0.05), whereas CLz/F decreased 0.83-flod, 0.41-fold (p < 0.05) and 0.72-fold (p < 0.01) in voriconazole, isavuconazole and ketoconazole in rats. The IC50 of lenvatinib with the azoles were 0.237, 1.300, 0.355 and 2.403 µM in RLMs and 0.160, 1.933, 3.622 and 1.831 µM in HLMs. Molecular docking analysis suggested that azoles exhibited a strong binding ability towards the target enzymes. CONCLUSION: It is imperative to acknowledge the potential drug-drug interactions mediated by CYP3A4 between azoles and lenvatinib, as these interactions hold significant implications for their clinical utilization.

Antifungal susceptibility patterns for Aspergillus, Scedosporium, and Exophiala isolates recovered from cystic fibrosis patients against amphotericin B, and three triazoles and their impact after long-term therapies.

In cystic fibrosis (CF) patients, fungal colonization of the respiratory tract is frequently found. Aspergillus fumigatus, Scedosporium genus, and Exophiala dermatitidis are the most commonly isolated moulds from the respiratory tract secretions of CF patients. The aim of this 5-year surveillance study was to identify trends in species distribution and susceptibility patterns of 212 mould strains identified as Aspergillus spp., Scedosporium spp., and Exophiala spp., isolated from sputum of 63 CF patients who received long-term therapy with itraconazole (ITR) and/or voriconazole (VRC). The Aspergillus isolates were identified as members of the sections Fumigati (n = 130), Flavi (n = 22), Terrei (n = 20), Nigri (n = 8), Nidulantes (n = 1), and Usti (n = 1). Among the 16 species of the genus Scedosporium, 9 were S. apiospermum, 3 S. aurantiacum, and 4 S. boydii. Among the 14 Exophiala species, all were molecularly identified as E. dermatitidis. Overall, 94% (15/16) of Scedosporium spp., 50% (7/14) of E. dermatitidis, and 7.7% (14/182) of Aspergillus spp. strains showed high MIC values (≥8 µg/ml) for at least one antifungal. Particularly, 8.9% (19/212) of isolates showed high MIC values for amphotericin B, 11.7% (25/212) for ITR, 4.2% (9/212) for VRC, and 3.3% (7/212) for posaconazole. In some cases, such as some A. fumigatus and E. dermatitidis isolates recovered from the same patient, susceptibility to antifungal azoles decreased over time. We show that the use of azoles for a long time in CF patients causes the selection/isolation of mould strains with higher MIC values.

The use of azoles for a long time in cystic fibrosis patients causes the selection/isolation of Aspergillus, Scedosporium, and Exophiala species with higher MIC values.

Effects of the CYP3A inhibitors, voriconazole, itraconazole, and fluconazole on the pharmacokinetics of osimertinib in rats.

Background: Osimertinib, as third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), is the first-line treatment approved to treat advanced T790M mutation-positive tumors. Triazole antifungals are therapeutic drugs for cancer patients to reduce the risk of opportunistic fungal infections. Our objective was to investigate whether three triazole antifungals (voriconazole, itraconazole, and fluconazole) could change the pharmacokinetics of osimertinib in rats. Methods: The adult male Sprague-Dawley rats were randomly divided into four groups (n = 6): control (0.3% CMC-Na), and voriconazole (20 mg/kg), itraconazole (20 mg/kg), or fluconazole (20 mg/kg) combined with osimertinib (10 mg/kg) group. Tail vein blood samples were collected into heparin tubes at various time points within 0-48 h after osimertinib administration. Osimrtinib's plasma concentration was detected using HPLC-MS/MS system equipped with a Waters XBridge C18 column, with the mobile phase consisting of acetonitrile and 0.2% formic acid water at a flow rate of 0.5 mL/min. Results: Co-administration with voriconazole or fluconazole increased the Cmax of osimertinib by 58.04% and 53.45%, respectively; the AUC0-t increased by 62.56% and 100.98%, respectively. However, when co-administered with itraconazole, the Cmax and AUC0-t of osimertinib only increased by 13.91% and 34.80%, respectively. Conclusions: Our results revealed that the pharmacokinetics of osimertinib were significantly changed by voriconazole and fluconazole in rats, whereas it was slightly affected by itraconazole. This work will contribute to a more comprehensive understanding of the pharmacokinetic properties of osimertinib when co-administered with triazole antifungals.

Aneuploidy Formation in the Filamentous Fungus Aspergillus flavus in Response to Azole Stress.

Aspergillus flavus is a mycotoxigenic fungus that contaminates many important agricultural crops with aflatoxin B1, the most toxic and carcinogenic natural compound. This fungus is also the second leading cause of human invasive aspergillosis, after Aspergillus fumigatus, a disease that is particularly prevalent in immunocompromised individuals. Azole drugs are considered the most effective compounds in controlling Aspergillus infections both in clinical and agricultural settings. Emergence of azole resistance in Aspergillus spp. is typically associated with point mutations in cyp51 orthologs that encode lanosterol 14α-demethylase, a component of the ergosterol biosynthesis pathway that is also the target of azoles. We hypothesized that alternative molecular mechanisms are also responsible for acquisition of azole resistance in filamentous fungi. We found that an aflatoxin-producing A. flavus strain adapted to voriconazole exposure at levels above the MIC through whole or segmental aneuploidy of specific chromosomes. We confirm a complete duplication of chromosome 8 in two sequentially isolated clones and a segmental duplication of chromosome 3 in another clone, emphasizing the potential diversity of aneuploidy-mediated resistance mechanisms. The plasticity of aneuploidy-mediated resistance was evidenced by the ability of voriconazole-resistant clones to revert to their original level of azole susceptibility following repeated transfers on drug-free media. This study provides new insights into mechanisms of azole resistance in a filamentous fungus. IMPORTANCE Fungal pathogens cause human disease and threaten global food security by contaminating crops with toxins (mycotoxins). Aspergillus flavus is an opportunistic mycotoxigenic fungus that causes invasive and noninvasive aspergillosis, diseases with high rates of mortality in immunocompromised individuals. Additionally, this fungus contaminates most major crops with the notorious carcinogen, aflatoxin. Voriconazole is the drug of choice to treat infections caused by Aspergillus spp. Although azole resistance mechanisms have been well characterized in clinical isolates of Aspergillus fumigatus, the molecular basis of azole resistance in A. flavus remains unclear. Whole-genome sequencing of eight voriconazole-resistant isolates revealed that, among other factors, A. flavus adapts to high concentrations of voriconazole by duplication of specific chromosomes (i.e., aneuploidy). Our discovery of aneuploidy-mediated resistance in a filamentous fungus represents a paradigm shift, as this type of resistance was previously thought to occur only in yeasts. This observation provides the first experimental evidence of aneuploidy-mediated azole resistance in the filamentous fungus A. flavus.

Microwave-Assisted Synthesis of Aminophosphonic Derivatives and Their Antifungal Evaluation against Lomentospora prolificans.

Lomentospora prolificans is a pathogenic and multidrug-resistant fungus that can infect both immunocompetent and immunocompromised patients, with mortality rates up to 87%. The World Health Organization (WHO) included this fungal species in its first list of 19 priority fungal pathogens, which focused on fungal pathogens that can cause invasive acute and subacute systemic fungal infections. Therefore, there is a growing interest in finding new therapeutic alternatives. In this work, the synthesis of twelve α-aminophosphonates by the microwave-assisted Kabachnik-Fields reaction and twelve α-aminophosphonic acids by a monohydrolysis reaction is reported. All compounds were evaluated by the agar diffusion method as a preliminary screening in comparison with voriconazole, showing inhibition halos for compounds 7, 11, 13, 22 and 27. The five active compounds in the preliminary tests were evaluated against five strains of L. prolificans following protocol M38-A2 from CLSI. The results showed that these compounds exhibit antifungal activity in the concentration range of 900->900 µg/mL. Cytotoxicity against healthy COS-7 cells was also evaluated by the MTT assay, and it was shown that compound 22 was the least cytotoxic, with a viability of 67.91%, comparable to the viability exhibited by voriconazole (68.55%). Docking studies showed that the possible mechanism of action of the active compounds could be through the inhibition of the enzyme lanosterol-14-alpha-demethylase in an allosteric hydrophobic cavity.

In Vitro and In Vivo Evaluation of Synergistic Effects of Everolimus in Combination with Antifungal Agents on Exophiala dermatitidis.

To investigate the combined function of the novel oral mTOR inhibitor, everolimus, with antifungal agents and their potential mechanisms against Exophiala dermatitidis, the CLSI microliquid-based dilution method M38-A2, chequerboard technique, and disk diffusion testing were performed. The efficacy of everolimus was evaluated in combination with itraconazole, voriconazole, posaconazole, and amphotericin B against 16 clinically isolated strains of E. dermatitidis. The synergistic effect was determined by measuring the MIC and fractional inhibitory concentration index. Dihydrorhodamine 123 was used for the quantification of ROS levels. The differences in the expression of antifungal susceptibility-associated genes were analyzed following different types of treatment. Galleria mellonella was used as the in vivo model. While everolimus alone showed minimal antifungal effects, combinations with itraconazole, voriconazole, posaconazole, or amphotericin B resulted in synergy in 13/16 (81.25%), 2/16 (12.5%), 14/16 (87.75%), and 5/16 (31.25%) of isolates, respectively. The disk diffusion assay revealed that the combination of everolimus and antifungal drugs showed no significant increase in the inhibition zones compared with the single agent, but no antagonistic effects were observed. Combination of everolimus and antifungal agents resulted in increased ROS activity (everolimus + posaconazole versus posaconazole [P < 0.05], everolimus + amphotericin B versus amphotericin B [P < 0.002]). Simultaneously, compared to mono-treatment, the combination of everolimus + itraconazole suppressed the expression of MDR2 (P < 0.05) and the combination of everolimus + amphotericin B suppressed the expression of MDR3 (P < 0.05) and CDR1B (P < 0.02). In vivo, combinations of everolimus and antifungal agents improved survival rates, particularly the combination of everolimus + amphotericin B (P < 0.05). In summary, the in vivo and in vitro experiments performed in our study suggest that the combination of everolimus with azoles or amphotericin B can have synergistic effects against E. dermatitidis, potentially due to the induction of ROS activity and inhibition of efflux pumps, providing a promising new approach for the treatment of E. dermatitidis infections. IMPORTANCE Cancer patients with E. dermatitidis infection have high mortality if untreated. Clinically, the conventional treatment of E. dermatitidis is poor due to the long-term use of antifungal drugs. In this study, we have for the first time investigated the interaction and action mechanism of everolimus combined with itraconazole, voriconazole, posaconazole, and amphotericin B on E. dermatitidis in vitro and in vivo, which provided new ideas and direction for further exploring the mechanism of drug combination and clinical treatment of E. dermatitidis.

Voriconazole Eye Drops: Enhanced Solubility and Stability through Ternary Voriconazole/Sulfobutyl Ether ß-Cyclodextrin/Polyvinyl Alcohol Complexes.

Voriconazole (VCZ) is a broad-spectrum antifungal agent used to treat ocular fungal keratitis. However, VCZ has low aqueous solubility and chemical instability in aqueous solutions. This study aimed to develop VCZ eye drop formulations using cyclodextrin (CD) and water-soluble polymers, forming CD complex aggregates to improve the aqueous solubility and chemical stability of VCZ. The VCZ solubility was greatly enhanced using sulfobutyl ether ß-cyclodextrin (SBEßCD). The addition of polyvinyl alcohol (PVA) showed a synergistic effect on VCZ/SBEßCD solubilization and a stabilization effect on the VCZ/SBEßCD complex. The formation of binary VCZ/SBEßCD and ternary VCZ/SBEßCD/PVA complexes was confirmed by spectroscopic techniques and in silico studies. The 0.5% w/v VCZ eye drop formulations were developed consisting of 6% w/v SBEßCD and different types and concentrations of PVA. The VCZ/SBEßCD systems containing high-molecular-weight PVA prepared under freeze-thaw conditions (PVA-H hydrogel) provided high mucoadhesion, sustained release, good ex vivo permeability through the porcine cornea and no sign of irritation. Additionally, PVA-H hydrogel was effective against the filamentous fungi tested. The stability study revealed that our VCZ eye drops provide a shelf-life of more than 2.5 years at room temperature, while a shelf-life of only 3.5 months was observed for the extemporaneous Vfend® eye drops.

Antiamoebic Susceptibility in Acanthamoeba Keratitis: Comparison of Isolates From South India and Northern California.

PURPOSE: Outcomes of Acanthamoeba keratitis are often worse in India than in the United States. The goal of this study was to determine whether antiamoebic susceptibility patterns were different when comparing Acanthamoeba isolates from India with those of the United States. METHODS: Acanthamoeba isolates were obtained from corneal scrapings of 43 patients with infectious keratitis seen at the Francis I. Proctor Foundation (N = 23) and Aravind Eye Hospital (N = 20) from 2008 through 2012 and plated on growth media. A previously described minimum cysticidal concentration (MCC) assay was performed by a single laboratory technician to assess susceptibility to 5 antiamoebic agents for all isolates. Testing was conducted in triplicate, with the median MCC chosen for analyses. RESULTS: The MCC (µg/mL) of polyhexamethylene biguanide was 6.25 [IQR 5.47-12.5] for Aravind isolates and 6.25 [IQR 6.25-9.375] for Proctor isolates ( P = 0.75), corresponding values were 6.25 [IQR 3.125-6.25] and 3.125 [IQR 3.125-9.375] for chlorhexidine ( P = 0.81), 2500 [IQR 2500-5000] and 5000 [IQR 1250-20,000] for voriconazole ( P = 0.25), 15.6 [IQR 15.6-39.0625] and 15.6 [IQR 15.6-31.25] for hexamidine ( P = 0.92), and 15.6 [IQR 7.81-15.6] and 15.6 [IQR 7.81-31.25] for propamidine ( P = 0.42). CONCLUSIONS: This study found no statistically significant differences in antiamoebic susceptibility of Indian versus US samples from Acanthamoeba keratitis clinical isolates. These findings suggest that differences in antiamoebic susceptibility are likely not responsible for differential outcomes in Acanthamoeba keratitis between the 2 locations.

A fatal case of pneumonia caused by Thermothelomycesthermophila.

Thermothelomyces thermophila (formerly Myceliophthora thermophila) is usually found in soil and specifically compost as an environmental dematiaceous fungus. Here, we report the first case of invasive pulmonary infection caused by T. thermophila in a pediatric patient with acute lymphoblastic leukemia. T. thermophila was serially cultured from bronchoalveolar lavage (BAL) fluid and sputum samples obtained from this patient with respiratory symptoms. The patient received antifungal treatment with liposomal amphotericin B (160 mg daily) and itraconazole (200 mg daily) combination therapy, but she died. By the antifungal susceptibility testing, low minimum inhibitory concentrations (MIC) were observed for itraconazole (MIC 0.06 µg/mL), voriconazole (MIC 0.12 µg/mL), and posaconazole (MIC 0.03 µg/mL) but high MIC was observed with amphotericin B (MIC 4.0 µg/mL). Since T. thermophila is usually found in the environment, it can be considered as a contaminant and may cause difficulties in diagnosis. Therefore, it is necessary to confirm the potential of pathogen through repeated culture and to conduct an antifungal susceptibility testing to find a suitable antifungal agent.

In Vitro Interactions of Antifungal Agents and Everolimus Against Aspergillus Species.

Multiple cellular activities, including protein and lipid synthesis, ribosome biogenesis, and metabolic processes, are regulated by the target of rapamycin (TOR) pathway. Recent research suggests that the TOR might play an important role in various physiological functions of pathogenic fungi, such as nutrient sensing, stress response, and cell cycle progression. Given their robust immunosuppressant and antitumor activities, TOR inhibitors are widely used in clinical settings. In the present study, a microdilution checkerboard-based approach was employed to assess the interactions between the oral mammalian target of rapamycin (mTOR) inhibitor everolimus (EVL) and antifungal agents in the treatment of Aspergillus species derived from 35 clinical isolates in vitro. The results revealed that EVL exhibited promising inhibitory synergy with itraconazole (ITC), posaconazole (POS), and amphotericin B (AMB) for 85.7%, 74.2%, and 71.4%, respectively. In contrast, EVL exhibited minimal synergistic inhibitory activity (14.3%) when applied in combination with voriconazole (VRC). Antagonistic interactions were not observed. In vivo experiments conducted in Galleria mellonella revealed that EVL in combination with antifungal agents improved the larva survival rates in the ITC, VRC, POS, and AMB groups by 18.3%, 13.3%, 26.7%, and 13.3%, respectively. These data suggest that the combination treatment with antifungal agents and antifungal agents holds promise as a means of alleviating clinical aspergillosis.

Synergy Between Pseudomonas aeruginosa Filtrates And Voriconazole Against Aspergillus fumigatus Biofilm Is Less for Mucoid Isolates From Persons With Cystic Fibrosis.

Persons with cystic fibrosis (CF) frequently suffer from Pseudomonas aeruginosa and Aspergillus fumigatus co-infections. There is evidence that co-infections with these interacting pathogens cause airway inflammation and aggravate deterioration of lung function. We recently showed that P. aeruginosa laboratory isolates synergistically interact with the anti-fungal azole voriconazole (VCZ), inhibiting biofilm metabolism of several A. fumigatus laboratory strains. Interaction was usually mediated via pyoverdine, but also via pyocyanin or pyochelin. Here we used planktonic filtrates of 7 mucoid and 9 non-mucoid P. aeruginosa isolates from CF patients, as well as 8 isolates without CF origin, and found that all of these isolates interacted with VCZ synergistically at their IC50 as well as higher dilutions. CF mucoid isolates showed the weakest interactive effects. Four non-mucoid P. aeruginosa CF isolates produced no or very low levels of pyoverdine and did not reach an IC50 against forming A. fumigatus biofilm; interaction with VCZ still was synergistic. A VCZ-resistant A. fumigatus strain showed the same level of susceptibility for P. aeruginosa anti-fungal activity as a VCZ-susceptible reference strain. Filtrates of most Pseudomonas isolates were able to increase anti-fungal activity of VCZ on a susceptible A. fumigatus strain. This was also possible for the VCZ-resistant strain. In summary these data show that clinical P. aeruginosa isolates, at varying degrees, synergistically interact with VCZ, and that pyoverdine is not the only molecule responsible. These data also strengthen the idea that during co-infections of A. fumigatus and P. aeruginosa lower concentrations of VCZ might be sufficient to control fungal growth.

Aspergillus tubingensis Endocarditis: A Case Report and Review of the Literature.

Aspergillus endocarditis is a rare infection that may affect immunocompetent patients following heart valve replacement or heart surgery. We report the case of a 39 year old woman with a history of intravenous drug use who developed endocarditis with direct examination of the resected valve and vegetation showing the presence of mycelia. Cultures were positive for an Aspergillus of section Nigri, which was subsequently identified as Aspergillus tubingensis by sequencing. The clinical course was favorable following surgery and prolonged antifungal therapy (8 months in total). Antifungal susceptibility testing showed good in vitro activity of amphotericin B, voriconazole and echinocandins against planktonic cells of this A. tubingensis isolate. However, only amphotericin B displayed significant activity against biofilms. In vitro combinations of voriconazole or amphotericin B with echinocandins did not meet the criteria of synergism. Our review of the literature identified 17 other cases of endocarditis attributed to Aspergillus of section Nigri with an overall mortality rate of 57% (100% in the absence of surgery). Endocarditis caused by Aspergillus niger and related cryptic species are rare events, for which surgical management appears to be crucial for outcome. While amphotericin B was the only antifungal drug displaying significant anti-biofilm activity, the type and duration of antifungal therapy remain to be determined.

Differences in kinetics of tacrolimus concentration after letermovir discontinuation by type of concomitant azole antifungal.

Letermovir is commonly used for CMV prophylaxis after allogeneic hematopoietic cell transplantation (HCT). Pharmacokinetic studies have shown an increase in tacrolimus exposure among healthy volunteers who took letermovir. However, studies in HCT recipients are needed because these patients are typically using concomitant antifungals with different degrees of CYP3A4 inhibition that may further interact with tacrolimus pharmacokinetics. In this study, we retrospectively evaluated the kinetics of tacrolimus concentration after letermovir discontinuation by type of concomitant azole antifungal in 57 HCT recipients. The median fold change in tacrolimus concentration-to-dose (C/D) ratio after discontinuing letermovir was 0.64 (range 0.43-0.99) with fluconazole and 1.10 (range 0.59-1.73) with voriconazole (p < 0.001). The tacrolimus C/D ratio decreased ≥ 30% after discontinuing letermovir (p < 0.001) in 66% of patients on fluconazole and 9% on voriconazole. Among patients whose tacrolimus C/D ratio decreased ≥ 30%, three (9%) patients in the fluconazole group and one (4%) in the voriconazole group experienced worsening of GVHD. Careful monitoring of tacrolimus concentration is important after letermovir discontinuation to avoid worsening of GVHD due to decreased tacrolimus concentration.

The effects of antifungal therapy on the recurrence of aspergillus infection after pulmonary aspergilloma resection: a study protocol for a single-center, prospective, non-blind, randomized, 24-month, parallel group study.

BACKGROUND: In recent years, the incidence of pulmonary aspergilloma has increased. The harm of aspergilloma is life-threatening massive hemoptysis, and the conventional treatment is surgical treatment. However, whether the antifungal treatment after surgery is required and the course of treatment before and after surgery are still unclear. METHODS: In this study, patients with pulmonary aspergilloma confirmed pathologically after surgery will be selected as subjects to conduct a single-center, randomized, parallel grouping, prospective, 2-year clinical study. Through regular visits, the recurrence of aspergillus infection, quality of life, lung function indicators, safety of antifungal therapy and other indicators were recorded to evaluate the recurrence risk of aspergillus infection and safety of antifungal agents. Cox proportional risk regression model was used to analyze the influencing factors of antifungal therapy on aspergillus infection recurrence after aspergillus bulbectomy. Cox multiple regression model was used for optimal model fitting, and regression coefficient (ß), relative risk (RR) and 95% confidence interval of RR were calculated. DISCUSSION: The study will explore whether antifungal therapy could improve the quality of life, reduce the recurrence of aspergillus infection, and ultimately improve the prognosis of patients with aspergilloma. The study results will provide high-quality evidence-based medical evidence for the formulation, revision and optimization of international and domestic clinical guidelines and expert consensus on chronic aspergillus lung disease, effectively improve the clinical treatment effect of aspergilloma, and form the latest concept of diagnosis and treatment of aspergilloma. TRIAL REGISTRATION: The trial was registered on the Chinese Clinical Trial Registry website ( https://www.chictr.org.cn/showprojen.aspx?proj=33231 ). Registration number: ChiCTR1800019990.

Formulation, optimization, and nephrotoxicity evaluation of an antifungal in situ nasal gel loaded with voriconazole‒clove oil transferosomal nanoparticles.

Fungal infections of the paranasal cavity are among the most widely spread illnesses nowadays. The aim of the current study was to estimate the effectiveness of an in situ gel loaded with voriconazole‒clove oil nano-transferosomes (VRC-CO-NT) in enhancing the activity of voriconazole against Aspergillus flavus, which causes rhinosinusitis. The nephrotoxic side effects of voriconazole may be reduced through the incorporation of the clove oil, which has antioxidant activity that protects tissue. The Box‒Behnken design was applied to formulate the VRC-CO-NT. The particle size, entrapment efficiency, antifungal inhibition zone, and serum creatinine concentration were considered dependent variables, and the soybean lecithin, VRC, and CO concentrations were considered independent ones. The final optimized formulation was loaded into a deacetylated gellan gum base and evaluated for its gelation, rheological properties, drug release profile, permeation capabilities, and in vivo nephrotoxicity. The optimum formulation was determined to be composed of 50 mg/mL lecithin, 18 mg/mL VRC, and 75 mg/mL CO, with a minimum particle size of 102.96 nm, an entrapment efficiency of 71.70%, an inhibition zone of 21.76 mm, and a serum creatinine level of 0.119 mmol/L. The optimized loaded in situ gel released 82.5% VRC after 12 hours and resulted in a 5.4-fold increase in drug permeation. The in vivo results obtained using rabbits resulted in a nonsignificant differentiation among the renal function parameters compared with the negative control group. In conclusion, nasal in situ gel loaded with VRC-CO-NT is considered an efficient novel carrier with enhanced antifungal properties with no signs of nephrotoxicity.