A novel approach to reducing hepatotoxicity related to fungal prophylaxis in pediatric lung transplant recipients.

BACKGROUND: Pediatric lung transplant patients are at risk for developing invasive fungal infections post-transplant. No consensus exists on optimal antifungal regimens and voriconazole, a common first-line agent, has been shown to cause hepatotoxicity. We describe a single-center experience utilizing a novel antifungal regimen of intravenous micafungin and nebulized amphotericin B immediately post-transplant with conversion to an azole at the time of hospital discharge and compare it to a historical cohort of patients who received voriconazole monotherapy throughout their immediate post-operative course. METHODS: This is a retrospective review of patients in the age 0-18 who received a lung transplant from June 2016-May 2021. Data points collected included: demographic data, transplant date and discharge date, Aspergillus colonization, type of lung transplant, hospitalization and level of care information, induction and antifungal medication regimen; AST, ALT, GGT, bilirubin, and direct bilirubin at various timepoints; and respiratory and blood culture results. The two patient groups were compared by assessment of changes in LFTs and culture results. RESULTS: Forty-two patients were included in the analysis, with 24 patients receiving micafungin and nebulized amphotericin and 18 patients receiving voriconazole. All patients in both groups experienced a post-operative elevation in at least one transaminase or bilirubin. More patients in the micafungin/amphotericin group had resolution of all abnormal LFTs by 1 month post-transplant (p = .036). Additionally, patients in the micafungin/amphotericin group experienced faster normalization of their LFTs compared with the voriconazole group (p < .001). Ten patients in the micafungin/amphotericin group and five patients in the voriconazole group were found to have fungal growth on culture post-transplant, but this difference was not found to be statistically significant (p = .507). CONCLUSIONS: An antifungal regimen of micafungin and nebulized amphotericin B liposomal may be useful at decreasing the duration of elevated liver enzymes in pediatric patients in the immediate post-lung transplant period when compared with voriconazole monotherapy. Larger prospective studies looking at antifungal regimens in pediatric patients post-lung transplant are warranted.

Synergistic action of synthetic peptides and amphotericin B causes disruption of the plasma membrane and cell wall in Candida albicans.

The objective of this work was to evaluate the combination of synthetic peptides based on the γ-core motif of defensin PvD1 with amphotericin B (AmB) at different concentrations against Candida albicans. We applied the checkerboard assay using different concentrations of the commercial drug AmB and the synthetic peptides γ31-45PvD1++ and γ33-41PvD1++ against C. albicans, aiming to find combinations with synergistic interactions. Between these two interactions involving γ31-45PvD1++ and AmB, an additive effect was observed. One such interaction occurred at concentrations of 0.009 µM of peptide γ31-45PvD1++ and 13.23 µM of AmB and another condition of 0.019 µM of peptide γ31-45PvD1++ and 6.61 µM of AmB. The other two concentrations of the interaction showed a synergistic effect in the combination of synthetic peptide γ31-45PvD1++ and AmB, where the concentrations were 1.40 µM peptide γ31-45PvD1++ and 0.004 µM AmB and 0.70 µM γ31-45PvD1++ peptide and 0.002 µM AmB. We proceeded with analysis of the mechanism of action involving synergistic effects. This examination unveiled a range of impactful outcomes, including the impairment of mitochondrial functionality, compromise of cell wall integrity, DNA degradation, and a consequential decline in cell viability. We also observed that both synergistic combinations were capable of causing damage to the plasma membrane and cell wall, causing leakage of intracellular components. This discovery demonstrates for the first time that the synergistic combinations found between the synthetic peptide γ31-45PvD1++ and AmB have an antifungal effect against C. albicans, acting on the integrity of the plasma membrane and cell wall.

Discovery of Antifungal Norsesquiterpenoids from a Soil-Derived Streptomyces microflavus: Targeting Biofilm Formation and Synergistic Combination with Amphotericin B against Yeast-like Fungi.

Thirty-five norsesquiterpenoids were isolated from the fermentation broth of Streptomyces microflavus from the forest soil of Ailaoshan in China. The structures of new compounds (1-5, 10-26) were elucidated by comprehensive spectroscopic analysis including data from experimental and calculated ECD spectra, as well as Mosher's reagent derivatives method. Norsesquiterpenoids showed different levels of antifungal activity with MIC80 values ranging from 25 to 200 µg/mL against Candida albicans, Candida parapsilosis, and Cryptococcus neoformans. The combining isolated norsesquiterpenoids with amphotericin B resulted in a synergistic interaction against test yeast-like fungi with a fractional inhibitory concentration index < 0.5. Compound 33 significantly inhibited biofilm formation and destroyed the preformed biofilm of fungi. Moreover, 33 downregulated the expression of adhesion-related genes HWP1, ALS1, ALS3, ECE1, EAP1, and BCR1 to inhibit the adhesion of C. albicans. Findings from the current study highlight the potential usage of norsesquiterpenoids from soil-derived Streptomyces for antifungal leads discovery.

Genome-wide analysis of in vivo-evolved Candida auris reveals multidrug-resistance mechanisms.

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.

Low toxicity contributes to Sporothrix globosa invade the skin of patients in low-epidemic areas of China.

OBJECTIVE: This study aims to assess the clinical characteristics of sporotrichosis in low-endemic areas of China, including the prevalence geography, genotypic traits of patients, clinical manifestations, and strain virulence and drug sensitivities. The objective is to improve the currently used clinical management strategies for sporotrichosis. METHODS: Retrospective data were collected from patients diagnosed with sporotrichosis through fungal culture identification. The isolates from purified cultures underwent identification using CAL (Calmodulin) gene sequencing. Virulence of each strain was assessed using a Galleria mellonella (G. mellonella) larvae infection model. In vitro susceptibility testing against commonly used clinical antifungal agents for sporotrichosis was conducted following CLSI criteria. RESULTS: In our low-endemic region for sporotrichosis, the majority of cases (23) were observed in middle-aged and elderly women with a history of trauma, with a higher incidence during winter and spring. All clinical isolates were identified as Sporothrix globosa (S. globosa). The G. mellonella larvae infection model indicated independent and dose-dependent virulence among strains, with varying toxicity levels demonstrated by the degree of melanization of the G. mellonella. Surprisingly, lymphocutaneous types caused by S. globosa exhibited lower in vitro virulence but were more common in affected skin. In addition, all S.globosa strains displayed high resistances to fluconazole, while remaining highly susceptible to terbinafine, itraconazole and amphotericin B. CONCLUSION: Given the predominance of elderly women engaged in agricultural labour in our region, which is a low-epidemic areas, they should be considered as crucial targets for sporotrichosis monitoring. S. globosa appears to be the sole causative agent locally. However, varying degrees of melanization in larvae were observed among these isolates, indicating a divergence in their virulence. Itraconazole, terbinafine and amphotericin B remain viable first-line antifungal options for treating S.globosa infection.

Design of Protease-Responsive Antifungal Liposomal Formulation Decorated with a Lipid-Modified Chitin-Binding Domain.

Candida albicans is a prevalent fungal pathogen that displays antibiotic resistance. The polyene antifungal amphotericin B (AmB) has been the gold standard because of its broad antifungal spectra, and its liposomal formulation, AmBisome, has been used widely and clinically in treating fungal infections. Herein, we explored enhancing the antifungal activity of AmBisome by integrating a small chitin-binding domain (LysM) of chitinase A derived from Pteris ryukyuensis. LysM conjugated with a lipid (LysM-lipid) was initially prepared through microbial transglutaminase (MTG)-mediated peptide tag-specific conjugation of LysM with a lipid-peptide substrate. The AmBisome formulation modified with LysM-lipid conjugates had a size distribution that was comparable to the native liposomes but an increased zeta potential, indicating that LysM-lipid conjugates were anchored to AmBisome. LysM-lipid-modified AmBisome exhibited long-term stability at 4 °C while retaining the capacity to bind chitin. Nevertheless, the antifungal efficacy of LysM-lipid-modified AmBisome against C. albicans was modest. We then redesigned a new LysM-lipid conjugate by introducing a peptide linker containing a thrombin digestion (TD) site at the C-terminus of LysM (LysM-TD linker-lipid), thereby facilitating the liberation of the LysM domain from AmBisome upon the addition of thrombin. This new AmBisome formulation anchored with LysM-TD linker-lipid exhibited superior performance in suppressing C. albicans growth in the presence of thrombin compared with the LysM-lipid formulation. These results provide a platform to design stimuli-responsive AmBisome formulations that respond to external environments and thus advance the treatment of pathogenic fungi infections.

Airway necrosis and granulation tissue formation caused by Rhizopus oryzae leading to severe upper airway obstruction: a case report.

Pulmonary Mucormycosis is a fatal infectious disease with high mortality rate. The occurrence of Mucormycosis is commonly related to the fungal virulence and the host's immunological defenses against pathogens. Mucormycosis infection and granulation tissue formation occurred in the upper airway was rarely reported. This patient was a 60-year-old male with diabetes mellitus, who was admitted to hospital due to progressive cough, sputum and dyspnea. High-resolution computed tomography (HRCT) and bronchoscopy revealed extensive tracheal mucosal necrosis, granulation tissue proliferation, and severe airway stenosis. The mucosal necrotic tissue was induced by the infection of Rhizopus Oryzae, confirmed by metagenomic next-generation sequencing (mNGS) in tissue biopsy. This patient was treated with the placement of a covered stent and local instillation of amphotericin B via bronchoscope. The tracheal mucosal necrosis was markedly alleviated, the symptoms of cough, shortness of breath, as well as exercise tolerance were significantly improved. The placement of airway stent and transbronchial microtube drip of amphotericin B could conduce to rapidly relieve the severe airway obstruction due to Mucormycosis infection.

Characterisation of Modular Polyketide Synthases Designed to Make Pentaene Analogues of Amphotericin B.

Glycosylated polyene macrolides are important antifungal agents that are produced by many actinomycete species. Development of new polyenes may deliver improved antibiotics. Here, Streptomyces nodosus was genetically re-programmed to synthesise pentaene analogues of the heptaene amphotericin B. These pentaenes are of interest as surrogate substrates for enzymes catalysing unusual, late-stage biosynthetic modifications. The previous deletion of amphotericin polyketide synthase modules 5 and 6 generated S. nodosus M57, which produces an inactive pentaene. Here, the chain-terminating thioesterase was fused to module 16 to generate strain M57-16TE, in which cycles 5, 6, 17 and 18 are eliminated from the biosynthetic pathway. Another variant of M57 was obtained by replacing modules 15, 16 and 17 with a single 15-17 hybrid module. This gave strain M57-1517, in which cycles 5, 6, 15 and 16 are deleted. M57-16TE and M57-1517 gave reduced pentaene yields. Only M57-1517 delivered its predicted full-length pentaene macrolactone in low amounts. For both mutants, the major pentaenes were intermediates released from modules 10, 11 and 12. Longer pentaene chains were unstable. The novel pentaenes were not glycosylated and were not active against Candida albicans. However, random mutagenesis and screening may yet deliver new antifungal producers from the M57-16TE and M57-1517 strains.

Antifungal drug resistance in Candida: a special emphasis on amphotericin B.

Invasive fungal infections in humans caused by several Candida species, increased considerably in immunocompromised or critically ill patients, resulting in substantial morbidity and mortality. Candida albicans is the most prevalent species, although the frequency of these organisms varies greatly according to geographic region. Infections with C. albicans and non-albicans Candida species have become more common, especially in the past 20 years, as a result of aging, immunosuppressive medication use, endocrine disorders, malnourishment, extended use of medical equipment, and an increase in immunogenic diseases. Despite C. albicans being the species most frequently associated with human infections, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei also have been identified. Several antifungal drugs with different modes of action are approved for use in clinical settings to treat fungal infections. However, due to the common eukaryotic structure of humans and fungi, only a limited number of antifungal drugs are available for therapeutic use. Furthermore, drug resistance in Candida species has emerged as a result of the growing use of currently available antifungal drugs against fungal infections. Amphotericin B (AmB), a polyene class of antifungal drugs, is mainly used for the treatment of serious systemic fungal infections. AmB interacts with fungal plasma membrane ergosterol, triggering cellular ion leakage via pore formation, or extracting the ergosterol from the plasma membrane inducing cellular death. AmB resistance is primarily caused by changes in the content or structure of ergosterol. This review summarizes the antifungal drug resistance exhibited by Candida species, with a special focus on AmB.

The sphingolipid inhibitor myriocin increases Candida auris susceptibility to amphotericin B.

BACKGROUND: The emergence of the pathogenic yeast Candida auris is of global concern due to its ability to cause hospital outbreaks and develop resistance against all antifungal drug classes. Based on published data for baker's yeast Saccharomyces cerevisiae, sphingolipid biosynthesis, which is essential for maintaining membrane fluidity and formation of lipid rafts, could offer a target for additive treatment. METHODS: We analysed the susceptibility of C. auris to myriocin, which is an inhibitor of the de novo synthesis of sphingolipids in eukaryotic cells in comparison to other Candida species. In addition, we combined sublethal concentrations of myriocin with the antifungal drugs amphotericin B and fluconazole in E-tests. Consequently, the combinatory effects of myriocin and amphotericin B were examined in broth microdilution assays. RESULTS: Myriocin-mediated inhibition of the sphingolipid biosynthesis affected the growth of C. auris. Sublethal myriocin concentrations increased fungal susceptibility to amphotericin B. Isolates which are phenotypically resistant (≥2 mg/L) to amphotericin B became susceptible in presence of myriocin. However, addition of myriocin had only limited effects onto the susceptibility of C. auris against fluconazole. CONCLUSIONS: Our results show that inhibition of de novo sphingolipid biosynthesis increases the susceptibility of C. auris to amphotericin B. This may potentially enhance antifungal treatment options fighting this often resistant yeast pathogen.

[Clinical features and risk factors for invasive fungal sinusitis after allogeneic hematopoietic stem cell transplantation].

Objective: To analyze the clinical characteristics and outcomes of patients with invasive fungal sinusitis (invasive fungal rhinosinusitis, IFR) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and explored the risk factors for IFR after allo-HSCT. Methods: Nineteen patients with IFR after allo-HSCT at Peking University People's Hospital from January 2012 to December 2021 were selected as the study group, and 95 patients without IFR after allo-HSCT during this period were randomly selected as the control group (1:5 ratio) . Results: Nineteen patients, including 10 males and 9 females, had IFR after allo-HSCT. The median age was 36 (10-59) years. The median IFR onset time was 68 (9-880) days after allo-HSCT. There were seven patients with acute myeloid leukemia, five with acute lymphoblastic leukemia, two with myelodysplastic syndrome, two with chronic myeloid leukemia, one with acute mixed-cell leukemia, one with multiple myeloma, and one with T-lymphoblastic lymph node tumor. There were 13 confirmed cases and 6 clinically diagnosed cases. The responsible fungus was Mucor in two cases, Rhizopus in four, Aspergillus in four, and Candida in three. Five patients received combined treatment comprising amphotericin B and posaconazole, one patient received combined treatment comprising voriconazole and posaconazole, nine patients received voriconazole, and four patients received amphotericin B. In addition to antifungal treatment, 10 patients underwent surgery. After antifungal treatment and surgery, 15 patients achieved a response, including 13 patients with a complete response and 2 patients with a partial response. Multivariate analysis revealed that neutropenia before transplantation (P=0.021) , hemorrhagic cystitis after transplantation (P=0.012) , delayed platelet engraftment (P=0.008) , and lower transplant mononuclear cell count (P=0.012) were independent risk factors for IFR after allo-HSCT. The 5-year overall survival rates in the IFR and control groups after transplantation were 29.00%±0.12% and 91.00%±0.03%, respectively (P<0.01) . Conclusion: Although IFR is rare, it is associated with poor outcomes in patients undergoing allo-HSCT. The combination of antifungal treatment and surgery might be effective.

Determination of amphotericin B antifungal susceptibility in Candida strains using an inverted microscope.

INTRODUCTION: Invasive Candida infections have recently shown a significant increase in prevalence and are associated with high mortality rates. Initiating early antifungal treatment in patients with candidemia is vital. The aim of our study was to compare the antifungal susceptibility results of a new method called Flat Plate Method modified from reference "Clinical and Laboratory Standards Institute (CLSI) microdilution method by us with Sensitititre Yeast One colorimetric method and the reference CLSI method. METHODOLOGY: We tested 100 Candida isolates from blood cultures. We followed the CLSI M27-A3 (reference method for broth dilution antifungal susceptibility testing of yeasts; third edition) guidelines for testing in vitro susceptibility to amphotericin B. In the Flat Plate method, 96-well plates were used for evaluation with an inverted microscope. Minimum inhibitory concentration (MIC) values in the SYO method were measured following the manufacturer's instructions. The MIC values obtained by all three methods were considered compatible if they were within ± 2 dilution limits. RESULTS: The SYO method detected C. albicans and C. glabrata with 100% essential agreement, whereas there was 96.29% essential agreement in the case of C. parapsilosis. In the Flat Plate method, the essential agreement with amphotericin B was 91.42%, for C. albicans isolates and 89.47%.for C. glabrata strains. CONCLUSIONS: When determining early antifungal susceptibility using the Flat Plate method, the results are obtained quickly, with high accuracy, and without incurring additional costs. However, there is a need for comprehensive studies comparing different antifungals.

Recurrent disseminated coccidioidal meningitis in two subsequent pregnancies.

OBJECTIVE: Recurrent disseminated coccidioidal meningitis in two subsequent pregnancies is rare and can pose a challenge in ensuring the health of both mother and baby. In this unique case we highlight this rare occurrence and subsequent treatment. CASE REPORT: A 29-year-old G4P1021 with a history of disseminated coccidioidomycosis in a previous pregnancy presented at 8 weeks gestation with nausea, headache, and neck pain. Cerebrospinal fluid analysis was positive for recurrent coccidioidal infection. She was started on Amphotericin and discharged. She re-presented at 30 week's gestation with phonophobia and photophobia, emesis, neck pain and swelling. MRI showed evidence of ventriculomegaly with communicating hydrocephalus. She was treated with therapeutic lumbar punctures throughout her pregnancy and a ventriculoperitoneal shunt following delivery. She had a spontaneous vaginal delivery at 38 weeks and 3 days with no complications. CONCLUSION: This unique case highlights the susceptibility of recurrent disseminated coccidioidomycosis in subsequent pregnancies and treatment thereof.

[Multidisciplinary expert consensus on the clinical rational application of different formulations of amphotericin B (2024 edition)].

Amphotericin B (AmB) is a broad-spectrum and potent polyene antifungal drug for the treatment of invasive fungal diseases (IFDs). Currently, amphotericin B deoxycholate (AmB-D) and three AmB lipid formulations, namely liposomal amphotericin B (L-AmB), amphotericin B colloidal dispersion (ABCD), and amphotericin B lipid complex (ABLC) are available for clinical use. In view of clinical concerns and misperceptions in the selection of different formulations of AmB, the present consensus summarized their pharmaceutical characteristics, antifungal mechanism, pharmacokinetics/phamacodynamics, drug interactions, indications, dosage, local administration, and adverse reactions based on the latest clinical research evidence, guidelines, and clinical experience. This consensus also recommends formulation selection and dosage adjustment for the treatment of target IFDs and in special populations, thereby providing expert consensus for clinical decision-making and standardized application of AmB.

Real-time monitoring of mycelial growth in liquid culture using hyphal dispersion mutant of Aspergillus fumigatus.

Hyphal pellet formation by Aspergillus species in liquid cultures is one of the main obstacles to high-throughput anti-Aspergillus reagent screening. We previously constructed a hyphal dispersion mutant of Aspergillus fumigatus by disrupting the genes encoding the primary cell wall α-1,3-glucan synthase Ags1 and putative galactosaminogalactan synthase Gtb3 (Δags1Δgtb3). Mycelial growth of the mutant in liquid cultures monitored by optical density was reproducible, and the dose-response of hyphal growth to antifungal agents has been quantified by optical density. However, Δags1Δgtb3 still forms hyphal pellets in some rich growth media. Here, we constructed a disruptant lacking all three α-1,3-glucan synthases and galactosaminogalactan synthase (Δags1Δags2Δags3Δgtb3), and confirmed that its hyphae were dispersed in all the media tested. We established an automatic method to monitor hyphal growth of the mutant in a 24-well plate shaken with a real-time plate reader. Dose-dependent growth suppression and unique growth responses to antifungal agents (voriconazole, amphotericin B, and micafungin) were clearly observed. A 96-well plate was also found to be useful for the evaluation of mycelial growth by optical density. Our method is potentially applicable to high-throughput screening for anti-Aspergillus agents.

Lansoprazole interferes with fungal respiration and acts synergistically with amphotericin B against multidrug-resistant Candida auris.

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 bc1). 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 bc1 inhibitor for combating drug-resistant C. auris infections.

Fluconazole-resistant Candida parapsilosis genotypes from hospitals located in five Spanish cities and one in Italy: Description of azole-resistance profiles associated with the Y132F ERG11p substitution.

BACKGROUND: Fluconazole-resistant Candida parapsilosis is a matter of concern. OBJECTIVES: To describe fluconazole-resistant C. parapsilosis genotypes circulating across hospitals in Spain and Rome and to study their azole-resistance profile associated with ERG11p substitutions. PATIENTS/METHODS: We selected fluconazole-resistant C. parapsilosis isolates (n = 528 from 2019 to 2023; MIC ≥8 mg/L according to EUCAST) from patients admitted to 13 hospitals located in five Spanish cities and Rome. Additionally, we tested voriconazole, posaconazole, isavuconazole, amphotericin B, micafungin, anidulafungin and ibrexafungerp susceptibility. RESULTS: Of the 53 genotypes found, 49 harboured the Y132F substitution, five of which were dominating city-specific genotypes involving almost half the isolates. Another genotype involved isolates harbouring the G458S substitution. Finally, we found two genotypes with the wild-type ERG11 gene sequence and one with the R398I substitution. All isolates were fully susceptible/wild-type to amphotericin B, anidulafungin, micafungin and ibrexafungerp. The azole-resistance patterns found were: voriconazole-resistant (74.1%) or voriconazole-intermediate (25.2%), posaconazole-resistant (10%) and isavuconazole non-wild-type (47.5%). Fluconazole-resistant and voriconazole non-wild-type isolates were likely to harbour substitution Y132F if posaconazole was wild type; however, if posaconazole was non-wild type, substitution G458S was indicated if isavuconazole MIC was >0.125 mg/L or substitution Y132F if isavuconazole MIC was ≤0.125 mg/L. CONCLUSIONS: We detected a recent clonal spread of fluconazole-resistant C. parapsilosis across some cities in Spain, mostly driven by dominating city-specific genotypes, which involved a large number of isolates harbouring the Y132F ERG11p substitution. Isolates harbouring substitution Y132F can be suspected because they are non-susceptible to voriconazole and rarely posaconazole-resistant.

Drug Design: Do Not Forget the Supramolecular Factor.

A major challenge currently facing medicinal chemists is designing agents that can selectively destroy drug resistant fungi and bacteria that have begun to emerge. One factor that has been overlooked by virtually all drug discovery/development approaches is the supramolecular factor, in which aggregated forms of a drug candidate exhibit low selectivity in destroying targeted cells while the corresponding monomers exhibit high selectivity. This Perspective discusses how we were led to the supramolecular factor through fundamental studies with simple model systems, how we reasoned that the selectivity of monomers of the antifungal agent amphotericin B should be much greater than the selectivity of the corresponding aggregates, and how we confirmed this hypothesis using derivatives of amphotericin B. In a broader context, these findings provide a strong rationale for considering the supramolecular factor in the design of new drug candidates and the testing of virtually all of them.

Evaluation of the Vitek 2 system for antifungal susceptibility testing of Candida auris using a representative international panel of clinical isolates: overestimation of amphotericin B resistance and underestimation of fluconazole resistance.

Although the Vitek 2 system is broadly used for antifungal susceptibility testing of Candida spp., its performance against Candida auris has been assessed using limited number of isolates recovered from restricted geographic areas. We therefore compared Vitek 2 system with the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method using an international collection of 100 C. auris isolates belonging to different clades. The agreement ±1 twofold dilution between the two methods and the categorical agreement (CA) based on the Centers for Disease Control and Prevention's (CDC's) tentative resistance breakpoints and Vitek 2-specific wild-type upper limit values (WT-ULVs) were determined. The CLSI-Vitek 2 agreement was poor for 5-flucytosine (0%), fluconazole (16%), and amphotericin B (29%), and moderate for voriconazole (61%), micafungin (67%), and caspofungin (81%). Significant interpretation errors were recorded using the CDC breakpoints for amphotericin B (31% CA, 69% major errors; MaEs) and fluconazole (69% CA, 31% very major errors; VmEs), but not for echinocandins (99% CA, 1% MaEs for both micafungin and caspofungin) for which the Vitek 2 allowed correct categorization of echinocandin-resistant FKS1 mutant isolates. Discrepancies were reduced when the Vitek 2 WT-ULV of 16 mg/L for amphotericin B (98% CA, 2% MaEs) and of 4 mg/L for fluconazole (96% CA, 1% MaEs, 3% VmEs) were used. In conclusion, the Vitek 2 system performed well for echinocandin susceptibility testing of C .auris. Resistance to fluconazole was underestimated whereas resistance to amphotericin B was overestimated using the CDC breakpoints of ≥32 and ≥2 mg/L, respectively. Vitek 2 minimun inhibitory concentrations (MICs) >4 mg/L indicated resistance to fluconazole and Vitek 2 MICs ≤16 mg/L indicated non-resistance to amphotericin B.

Searching for Conjugates as New Structures for Antifungal Therapies.

The progressive increase in fungal infections and the decrease in the effectiveness of current therapy explain research on new drugs. The synthesis of compounds with proven antifungal activity, favorable physicochemical and pharmacokinetic properties affecting their pharmaceutical availability and bioavailability, and limiting or eliminating side effects has become the goal of many studies. The publication describes the directions of searching for new compounds with antifungal activity, focusing on conjugates. The described modifications include, among others, azoles or amphotericin B in combination with fatty acids, polysaccharides, proteins, and synthetic polymers. The benefits of these combinations in terms of activity, mechanism of action, and bioavailability were indicated. The possibilities of creating or using nanoparticles, "umbrella" conjugates, siderophores (iron-chelating compounds), and monoclonal antibodies were also presented. Taking into account the role of vaccinations in prevention, the scope of research related to developing a vaccine protecting against fungal infections was also indicated.