Caspofungin Concentrations Research Articles

Effect of Conformational Variability on the Drug Resistance of Candida auris ERG11p and FKS1.

Candida auris infection has been recognized as an urgent threat to antifungal drug resistance, and the Eagle effect of C. auris FKS1 (1,3-β-d-glucan synthase) wild-type isolates has also been noted. The Eagle effect, namely, where higher concentrations of antifungals reduce fungicidal activity relative to lower concentrations, is a confounding factor of apparent antifungal resistance, but the detailed mechanism remains unclear. Here, we present the conformational variability of mutation sites for ERG11p (lanosterol 14α-demethylase) and FKS1 from deep neural network-based prediction along with the reported X-ray crystallographic and cryo-electron microscopy (cryo-EM) structures of antifungals. The sequence variability maps provide valuable insights into the inconsistent correlation between azole resistance and the mysterious Eagle effect with the dispersion of minimal inhibitory concentration (MIC) for echinocandin resistance. The conformational variability prediction supports the hypothesis that mutations K143R of clade I, VF125AL of clade III, and Y132F of clade IV for C. auris ERG11p make the corresponding site variable and that an increased population of invisible variable conformations potentially contributes to triazole resistance. In contrast, the predicted rigid conformation by the S639F mutation of hot spot region 1 (HS1) for FKS1 suggests that caspofungin (CAS) is involved in an uncompetitive inhibition, and a decreased population of the CAS-bound state of FKS1 with Rho1 leads to drug resistance. The predicted variable HS1 region for FKS1 WT isolates and the rigid one for FKS1 S639F mutants support the in vivo drug response and the in vitro MIC dispersion. A plausible mechanism of the Eagle effect is hereby proposed, namely, that a high concentration of CAS with a high membrane affinity reduces the population of the CAS-bound state of FKS1 with Rho1, as well as accompanying events such as aggregation or association depending on the conformational variability of HS1.

Development of Echinocandin Resistance in Candida haemulonii: An Emergent, Widespread, and Opportunistic Fungal Pathogen.

Echinocandins, used for the prevention and treatment of invasive fungal infections, have led to a rise in breakthrough infections caused by resistant Candida species. Among these species, those belonging to the Candida haemulonii complex are rare multidrug-resistant (MDR) yeasts that are frequently misidentified but have emerged as significant healthcare-associated pathogens causing invasive infections. The objectives of this study were to investigate the evolutionary pathways of echinocandin resistance in C. haemulonii by identifying mutations in the FKS1 gene and evaluating the impact of resistance on fitness. After subjecting a MDR clinical isolate of C. haemulonii (named Ch4) to direct selection using increasing caspofungin concentrations, we successfully obtained an isolate (designated Ch4'r) that exhibited a high level of resistance, with MIC values exceeding 16 mg/L for all tested echinocandin drugs (caspofungin, micafungin, and anidulafungin). Sequence analysis revealed a specific mutation in the resistant Ch4'r strain, leading to an arginine-histidine amino acid substitution (R1354H), occurring at the G4061A position of the HS2 region of the FKS1 gene. Compared to the wild-type strain, Ch4'r exhibited significantly reduced growth proliferation, biofilm formation capability, and phagocytosis ratio, indicating a decrease in fitness. Transmission electron microscopy analysis revealed alterations in cell wall components, with a notable increase in cell wall thickness. The resistant strain also exhibited higher amounts (2.5-fold) of chitin, a cell wall-located molecule, compared to the wild-type strain. Furthermore, the resistant strain demonstrated attenuated virulence in the Galleria mellonella larval model. The evolved strain Ch4'r maintained its resistance profile in vivo since the treatment with either caspofungin or micafungin did not improve larval survival or reduce the fungal load. Taken together, our findings suggest that the acquisition of pan-echinocandin resistance occurred rapidly after drug exposure and was associated with a significant fitness cost in C. haemulonii. This is particularly concerning as echinocandins are often the first-line treatment option for MDR Candida species.

Azole non-susceptible C. tropicalis and polyclonal spread of C. albicans in Central Vietnam hospitals.

Candida spp. are responsible for infections ranging from local to systemic, and resistance to antifungal first-line therapy is increasing in non-albicans Candida species. We aimed to determine the etiology of candidiasis and the antifungal resistance of Candida spp. isolated in Hue hospitals, Central-Vietnam. Species identification was performed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry supported by fungal internal-transcribed-spacer amplification and sequencing. Antifungal susceptibility testing was performed by disk diffusion method and minimum inhibitory concentrations of azoles, caspofungin, and amphotericin B against C. tropicalis were determined by broth microdilution. Polymorphism of erg11 gene associated with fluconazole resistance was carried out by polymerase chain reaction and sequencing. Multilocus sequence typing (MLST) was used for typing selected C. albicans isolates. Overall, 196 Candida isolates were detected, mostly C. albicans (48%), followed by C. tropicalis (16%), C. parapsilosis (11%), C. glabrata (9%), C. orthopsilosis (6%) and to a lesser extent another eight species. High rates of resistance to fluconazole and voriconazole (18.8%) were observed in C. tropicalis with five isolates co-resistant to both agents. Y132F and S154F missense mutations in the ERG11 protein were associated with fluconazole-resistance in C. tropicalis (67.7%). Resistance to caspofungin was found in one isolate of C. albicans. MLST identified a polyclonal population of C. albicans with multiple diploid sequence types, and with few lineages showing potential nosocomial spread. Resistance to triazole agents should be considered in C. tropicalis infections in the studied hospitals, and surveillance measures taken to avoid Candida diffusion.

Single-Cell Optical Nanomotion of Candida albicans in Microwells for Rapid Antifungal Susceptibility Testing

Candida albicans is an emerging multidrug-resistant opportunistic pathogen representing an important source of invasive disease in humans and generating high healthcare costs worldwide. The development of a rapid and simple antifungal susceptibility test (AFST) could limit the spread of this disease, increase the efficiency of treatment, and lower the risk of developing resistant strains. We developed a microfluidic chip containing an array of microwells that were designed to trap the cells and perform rapid antifungal susceptibility tests using optical nanomotion detection (ONMD). Yeast cell entrapment in a microwell allows for a very rapid exchange of growth medium with the antifungal, which enables performing single-cell ONMD measurements on the same cell before and after antifungal treatment. The exposure to a low concentration of the antifungal caspofungin or flucanozole induced a significant decrease in the nanomotion signal, demonstrating the high sensitivity of this technique. We used this chip to quantify the real-time response of individual C. albicans cells to the antifungal treatment in as fast as 10 min. This simple and label-free technique could be further developed into a simple-to-use device that allows the performance of fast AFST as part of a routine hospital procedure in developed and also eventually developing world countries.

Analysis of the Localization of Schizosaccharomyces pombe Glucan Synthases in the Presence of the Antifungal Agent Caspofungin.

In recent years, invasive fungal infections have emerged as a common source of infections in immunosuppressed patients. All fungal cells are surrounded by a cell wall that is essential for cell integrity and survival. It prevents cell death and lysis resulting from high internal turgor pressure. Since the cell wall is not present in animal cells, it is an ideal target for selective invasive fungal infection treatments. The antifungal family known as echinocandins, which specifically inhibit the synthesis of the cell wall β(13)glucan, has been established as an alternative treatment for mycoses. To explore the mechanism of action of these antifungals, we analyzed the cell morphology and glucan synthases localization in Schizosaccharomyces pombe cells during the initial times of growth in the presence of the echinocandin drug caspofungin. S. pombe are rod-shaped cells that grow at the poles and divide by a central division septum. The cell wall and septum are formed by different glucans, which are synthesized by four essential glucan synthases: Bgs1, Bgs3, Bgs4, and Ags1. Thus, S. pombe is not only a perfect model for studying the synthesis of the fungal β(1-3)glucan, but also it is ideal for examining the mechanisms of action and resistance of cell wall antifungals. Herein, we examined the cells in a drug susceptibility test in the presence of either lethal or sublethal concentrations of caspofungin, finding that exposure to the drug for long periods at high concentrations (>10 µg/mL) induced cell growth arrest and the formation of rounded, swollen, and dead cells, whereas low concentrations (<10 µg/mL) permitted cell growth with a mild effect on cell morphology. Interestingly, short-term treatments with either high or low concentrations of the drug induced effects contrary to those observed in the susceptibility tests. Thus, low drug concentrations induced a cell death phenotype that was not observed at high drug concentrations, which caused transient fungistatic cell growth arrest. After 3 h, high concentrations of the drug caused the following: (i) a decrease in the GFP-Bgs1 fluorescence level; (ii) altered locations of Bgs3, Bgs4, and Ags1; and (iii) a simultaneous accumulation of cells with calcofluor-stained incomplete septa, which at longer times resulted in septation uncoupling from plasma membrane ingression. The incomplete septa revealed with calcofluor were found to be complete when observed via the membrane-associated GFP-Bgs or Ags1-GFP. Finally, we found that the accumulation of incomplete septa depended on Pmk1, the last kinase of the cell wall integrity pathway.

Antifungal activity and potential mechanism of action of caspofungin in combination with ribavirin against Candida albicans

The number of invasive fungal infections has increased dramatically, resulting in high morbidity and mortality among immunocompromised patients. With increasing use of caspofungin (CAS), resistant strains have emerged frequently and led to limitations in the treatment of patients with severe invasive Candida albicans infections. Combination therapy is an important method to deal with this issue. As such, this study investigated the activity of CAS in combination with ribavirin (RBV) against C. albicans. The results of this in-vitro study showed that the minimum inhibitory concentrations (MICs) of CAS and RBV when they were used as monotherapy were 0.5-1 μg/mL and 2-8 μg/mL, respectively, while the MIC of CAS decreased from 0.5-1 μg/mL to 0.0625-0.25 μg/mL when used in combination with RBV, with a fractional inhibitory concentration index (FICI) ≤0.5. In addition, the RBV+CAS combination group displayed synergistic effects against C. albicans biofilm over 4 h; the sessile MIC (sMIC) of CAS decreased from 0.5-1 µg/mL to 0.0625-0.25µg/mL and the sMIC of RBV decreased from 4-16 µg/mL to 1-2 µg/mL, with FICI <0.5. The survival of C. albicans-infected Galleria mellonella was prolonged, the fungal burden was decreased, and the area of tissue damage was reduced after combination therapy. Further study showed that the mechanisms of action of the synergistic effect were related to the inhibition of biofilm formation, the inhibition of hyphal growth, and the activation of metacaspases, but were not related to the accumulation of reactive oxygen species. It is hoped that these findings will contribute to the understanding of drug resistance in C. albicans, and provide new insights for the application of RBV.

Evaluation of the Sensititre YeastOne and Etest in Comparison with CLSI M38-A2 for Antifungal Susceptibility Testing of Three Azoles, Amphotericin B, Caspofungin, and Anidulafungin, against Aspergillusfumigatus and Other Species, Using New Clinical Breakpoints and Epidemiological Cutoff Values.

Aspergillosis is an invasive fungal disease associated with high mortality. Antifungal susceptibility testing (AFST) is receiving increasing consideration for managing patients, as well as for surveilling emerging drug resistance, despite having time-consuming and technically complex reference methodologies. The Sensititre YeastOne (SYO) and Etest methods are widely utilized for yeasts but have not been extensively evaluated for Aspergillus isolates. We obtained Posaconazole (POS), Voriconazole (VCZ), Itraconazole (ITC), Amphotericin B (AMB), Caspofungin (CAS), and Anidulafungin (AND) minimum inhibitory concentrations (MICs) for both the Etest (n = 330) and SYO (n = 339) methods for 106 sequenced clinical strains. For 84 A. fumigatus, we analyzed the performance of both commercial methods in comparison with the CLSI-AFST, using available cutoff values. An excellent correlation could be demonstrated for Etest-AMB and Etest-VCZ (p < 0.01). SYO-MICs of AMB, VCZ, and POS resulted in excellent essential agreement (>93%), and >80% for AMB, VCZ, and ITC Etest-MICs. High categoric agreement was found for AMB, ITC, and CAS Etest-MICs (>85%) and AMB SYO-MICs (>90%). The considerable number of major/very major errors found using Etest and SYO, possibly related to the proposed cutoffs and associated with the less time-consuming processes, support the need for the improvement of commercial methods for Aspergillus strains.

P059 The overexpression of efflux pump gene cdr1B resulting in voriconazole- and isavuconazole- resistance in Aspergillus fumigatus recovered from a patient with chronic pulmonary aspergillosis in China

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesTriazole resistance in the pathogenic Aspergillus fumigatus has been increasing worldwide, posing a growing therapeutic challenge. To date, triazole resistance in clinical isolates of A. fumigatus causing pulmonary aspergillosis has been mainly attributed to the mutations in the cyp51A gene or its promoter, followed by mutations in cyp51B and hmg1 gene encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. From chronic pulmonary aspergillosis (CPA) patient, we isolated a strain of A. fumigatus (BMU10672) with resistance to voriconazole (VRC) and isavuconazole (ISZ), which was caused by overexpression of efflux pump Cdr1B.MethodsAntifungal susceptibility testing of the isolate of A. fumigatus BMU10672 was performed using the broth microdilution method (CLSI M38-A3), E-test and disk diffusion method. The promoter region and open reading frame of the cyp51A, cyp51B, and hmg1 gene were amplified and sequenced. Then, the expression levels of cyp51A, cyp51B, and efflux pump gene cdr1B with or without being exposed to VRC or ISZ were quantified using real-time PCR, compared with triazole-susceptible A. fumigatus Af293. And the function of efflux pump Cdr1B was tested by efflux pump substrate (Nile red) accumulation assay and efflux pump inhibitor (FK520) assay.ResultsThe minimum inhibitory concentration (MIC) of itraconazole (ITC), VRC, posaconazole (POS), ISZ and amphotericin B (AMB), and the minimal effective concentration (MEC) of caspofungin (CAS) against A. fumigatus BMU10672 was 1 μg/ml, 2 μg/mL, 0.5 μg/mL, 2 μg/mL, 1 μg/mL and 0.125 μg/mL, respectively. The results of E-test and disk diffusion assay were consistent with those of the broth microdilution method (Figs. 1a and b). Together, these results indicate that A. fumigatus BMU10672 is resistant to VRC and ISZ, while being susceptible to ITC, POS, AMB, and CAS. Sequencing of the cyp51A, cyp51B and hmg1 gene of A. fumigatus BMU10672 were all intact. The basal and VRC- or ISZ- induced expression levels of efflux pumps gene cdr1B in A. fumigatus BMU10672 were all higher (> 4-fold) than those in triazole-susceptible A. fumigatus Af293. However, no differences in basal and VRC- or ISZ- induced expression levels of cyp51A gene and cyp51B gene were observed between A. fumigatus BMU10672 and Af293. The efflux pump substrate Nile red accumulation assay showed the A. fumigatus BMU10672 accumulated less Nile red than Af293, confirming that Cdr1B was active at exporting Nile red, while efflux pumps inhibitor FK520 can increase the accumulation of the Nile red in A. fumigatus BMU10672 (Fig. 1c). Inhibition of efflux pumps activity by inhibitor FK520 resulted in a MIC reduction of 4-fold in VRC and ISZ MICs, and 2-fold in ITC and POS, against A. fumigatus BMU10672 (Figs. 1a and b).ConclusionOverexpression of efflux pumps gene cdr1B resulting in VRC- and ISZ- resistance in the clinical isolate of A. fumigatus BMU10672.

Genomic and Molecular Identification of Genes Contributing to the Caspofungin Paradoxical Effect in Aspergillus fumigatus.

ABSTRACTAspergillus fumigatus is a deadly opportunistic fungal pathogen responsible for ~100,000 annual deaths. Azoles are the first line antifungal agent used against A. fumigatus, but azole resistance has rapidly evolved making treatment challenging. Caspofungin is an important second-line therapy against invasive pulmonary aspergillosis, a severe A. fumigatus infection. Caspofungin functions by inhibiting β-1,3-glucan synthesis, a primary and essential component of the fungal cell wall. A phenomenon termed the caspofungin paradoxical effect (CPE) has been observed in several fungal species where at higher concentrations of caspofungin, chitin replaces β-1,3-glucan, morphology returns to normal, and growth rate increases. CPE appears to occur in vivo, and it is therefore clinically important to better understand the genetic contributors to CPE. We applied genomewide association (GWA) analysis and molecular genetics to identify and validate candidate genes involved in CPE. We quantified CPE across 67 clinical isolates and conducted three independent GWA analyses to identify genetic variants associated with CPE. We identified 48 single nucleotide polymorphisms (SNPs) associated with CPE. We used a CRISPR/Cas9 approach to generate gene deletion mutants for seven genes harboring candidate SNPs. Two null mutants, ΔAfu3g13230 and ΔAfu4g07080 (dscP), resulted in reduced basal growth rate and a loss of CPE. We further characterized the dscP phosphatase-null mutant and observed a significant reduction in conidia production and extremely high sensitivity to caspofungin at both low and high concentrations. Collectively, our work reveals the contribution of Afu3g13230 and dscP in CPE and sheds new light on the complex genetic interactions governing this phenotype.IMPORTANCE This is one of the first studies to apply genomewide association (GWA) analysis to identify genes involved in an Aspergillus fumigatus phenotype. A. fumigatus is an opportunistic fungal pathogen that causes hundreds of thousands of infections and ~100,000 deaths each year, and antifungal resistance has rapidly evolved in this species. A phenomenon called the caspofungin paradoxical effect (CPE) occurs in some isolates, where high concentrations of the drug lead to increased growth rate. There is clinical relevance in understanding the genetic basis of this phenotype, since caspofungin concentrations could lead to unintended adverse clinical outcomes in certain cases. Using GWA analysis, we identified several interesting candidate polymorphisms and genes and then generated gene deletion mutants to determine whether these genes were important for CPE. Two of these mutant strains (ΔAfu3g13230 and ΔAfu4g07080/ΔdscP) displayed a loss of the CPE. This study sheds light on the genes involved in clinically important phenotype CPE.

Comparison of simulated candidemia detection during prophylactic antifungal therapy

BackgroundWe investigated the neutralization performance of various automated blood culture systems for antifungal agents with regard to the most commonly isolated Candida species. MethodsIn this study, we evaluated the time to detection (TTD) of simulated candidemia for 6 Candida spp. (C. albicans, C. auris, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis) in 3 automated blood culture systems (BACTEC™ FX, BACT/ALERT® 3D, and BACT/ALERT® VIRTUO®), with or without trough and peak levels of eight antifungal agents (amphotericin B, anidulafungin, caspofungin, fluconazole, itraconazole, micafungin, posaconazole, and voriconazole). ResultsCaspofungin and micafungin significantly prolonged the TTDs for most of the tested strains in the 3 blood culture instruments, especially at peak concentrations. ConclusionPeak concentrations of caspofungin and micafungin influence the performance of blood culture detection systems. Therefore, one should be careful about the possibility of prolonged TTDs for candidemia when using the abovementioned antifungal agents.

Caspofungin Cerebral Penetration and Therapeutic Efficacy in Experimental Cerebral Aspergillosis.

ABSTRACTDespite best available therapy, cerebral aspergillosis is an often-lethal complication of disseminated aspergillosis. There is an urgent need to expand the currently limited therapeutic options. In this study, we assessed cerebral drug exposure and efficacy of caspofungin (CAS) using a lethal infant rat model of cerebral aspergillosis. Eleven-day-old Wistar rats were infected by intracisternal injection of Aspergillus fumigatus conidia. Treatment started after 22 h and was continued for 10 days. Regimens were CAS 1 mg/kg/day intraperitoneally (i.p.), liposomal amphotericin B (L-AmB) 5 mg/kg/day i.p., and both drugs combined at the same dose i.p. Infected controls were given NaCl 0.85% i.p. Primary endpoints assessed were survival, cerebral fungal burden, galactomannan index, and drug concentrations in brain homogenate at 2, 3, 5, and 11 days after infection. Compared to those of controls (4.4 ± 2.7 days), survival times were increased by treatment with CAS alone (10.3 ± 1.7 days; P < 0.0001) and CAS combined with L-AmB (9.3 ± 2.8 days; P < 0.0001). In contrast, survival time of L-AmB-treated animals (4.3 ± 3.1 days) was not different from that of controls. Cerebral fungal burden and galactomannan index declined in all animals over time, without significant differences between controls and treated animals. CAS trough levels in brain tissue were between 0.84 and 1.4 μg/g, concentrations we show to be associated with efficacy. AmB trough levels in brain tissue were higher than the MIC of the A. fumigatus isolate. In summary, CAS concentrations in brain tissue suggest it may be therapeutically relevant and it significantly improved survival in this lethal model of cerebral aspergillosis in nonneutropenic rats. The clinical efficacy of CAS treatment for cerebral aspergillosis merits further study.IMPORTANCE Treatment options for cerebral aspergillosis, an often-lethal disease, are limited. The echinocandins (caspofungin is one of them) are not recommended treatment because their brain tissue penetration is often considered insufficient. In a nursing rat model of cerebral aspergillosis that mimics human disease, we found potentially therapeutically relevant concentrations of caspofungin in brain tissue and prolonged survival of caspofungin-treated animals. The efficacy of caspofungin in the treatment of cerebral aspergillosis documented here, if confirmed in other animal models (especially immunosuppressed murine models) and by using additional Aspergillus isolates across a range of CAS minimal effective concentrations (MECs), would suggest that caspofungin merits further study as a treatment option for patients suffering from aspergillosis disseminated to the brain.

The Caspofungin Paradoxical Effect is a Tolerant "Eagle Effect" in the Filamentous Fungal Pathogen Aspergillus fumigatus.

ABSTRACTCell responses against antifungals other than resistance have rarely been studied in filamentous fungi, while terms such as tolerance and persistence are well-described for bacteria and increasingly examined in yeast-like organisms. Aspergillus fumigatus is a filamentous fungal pathogen that causes a disease named aspergillosis, for which caspofungin (CAS), a fungistatic drug, is used as a second-line therapy. Some A. fumigatus clinical isolates can survive and grow in CAS concentrations above the minimum effective concentration (MEC), a phenomenon known as “caspofungin paradoxical effect” (CPE). Here, we evaluated the CPE in 67 A. fumigatus clinical isolates by calculating recovery rate (RR) values, where isolates with an RR of ≥0.1 were considered CPE+ while isolates with an RR of <0.1 were classified as CPE–. Conidia produced by three CPE+ clinical isolates, CEA17 (RR = 0.42), Af293 (0.59), and CM7555 (0.38), all showed the ability to grow in high levels of CAS, while all conidia produced by the CPE– isolate IFM61407 (RR = 0.00) showed no evidence of paradoxical growth. Given the importance of the calcium/calcineurin/transcription factor-CrzA pathway in CPE regulation, we also demonstrated that all ΔcrzACEA17 (CPE+) conidia exhibited CPE while 100% of ΔcrzAAf293 (CPE–) did not exhibit CPE. Because all spores derived from an individual strain were phenotypically indistinct with respect to CPE, it is likely that CPE is a genetically encoded adaptive trait that should be considered an antifungal-tolerant phenotype. Because the RR parameter showed that the strength of the CPE was not uniform between strains, we propose that the mechanisms which govern this phenomenon are multifactorial.

First electrochemical study of a potent antifungal drug caspofungin: Application to its enhanced voltammetric sensing based on the performance of boron-doped diamond electrode in CTAB-mediated measurements

This present study describes the first electrochemical investigation of caspofungin, which is a semisynthetic echinocandin class of antifungal drug. For this purpose, an electrochemically pretreated (anodically and subsequently cathodically) boron-doped diamond (BDD) electrode was used. The cyclic voltammetric scans showed a single oxidation step that is irreversible and controlled by a dual mechanism of adsorption and diffusion. For the optimization of experimental conditions, the influence of the electrode pretreatment, pH of the supporting electrolyte, concentration of cationic surfactant cetyltrimethylammonium bromide (CTAB), accumulation variables, and instrumental parameters was examined on the current response of caspofungin. The improved sensitivity of the voltammetric measurements was observed due to the synergistic effect of CTAB on the adsorption capacity of BDD electrode in alkaline pH conditions. Employing square-wave adsorptive stripping voltammetry (using open-circuit accumulation for 60 s), the oxidation signal at around +0.45 V allowed to the determination of caspofungin in Britton-Robinson buffer at pH 11.0 containing 1 × 10−4 M CTAB. The process could be used in the concentration range from 2.06 × 10−7 to 1.03 × 10−5 M (from 0.25 to 12.5 μg mL−1 as acetate salt) with a detection limit of 5.27 × 10−8 M (0.064 μg mL−1 as acetate salt). A technique developed here was used successfully to determine caspofungin concentration in commercial parenteral dosage formulations.

Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development.

ABSTRACTCryptococcus neoformans is a major fungal pathogen that often causes life-threatening meningitis in immunocompromised populations. This yeast pathogen is highly resistant to the echinocandin drug caspofungin. Previous studies showed that Cryptococcus lipid translocase (flippase) is required for the caspofungin resistance of that fungus. Mutants with a deleted subunit of lipid flippase, Cdc50, showed increased sensitivity to caspofungin. Here we designed an antifungal peptide targeting the P4-ATPase function. We synthesized stable peptides based on the Cdc50 loop region to identify peptides that can sensitize caspofungin by blocking flippase function and found that myristylated peptides based on the “AS15 sequence” was effective at high concentrations. A modified peptide, “AW9-Ma” showed a MIC of 64 μg/mL against H99 wild type and a fractional inhibitory concentration (FIC) index value of 0.5 when used in combination with caspofungin. Most notably, in the presence of the AW9-Ma peptide, C. neoformans wild type was highly sensitive to caspofungin with a MIC of 4 μg/mL, the same as the cdc50Δ mutant. Further assays with flow cytometry showed inhibition of the lipid flippase enzyme activity and significant accumulation of phosphatidylserine on the cell membrane surface. Using a fluorescently labeled peptide, we confirmed that the peptide co-localized with mCherry-tagged P4-ATPase protein Apt1 in C. neoformans. Structure-activity relationship studies of the AW9 sequence showed that two lysine residues on the peptide are likely responsible for the interaction with the P4-ATPase, hence critical for its antifungal activity.IMPORTANCE The authors have developed a lead compound peptide antifungal drug targeting a protein from the organism Cryptococcus neoformans. Binding of the drug to the target fungal protein causes charged lipid molecules to be retained on the surface. This peptide works in synergy with the existing antifungal drug caspofungin. Echinocandin drugs like caspofungin are one of the few classes of existing antifungals. Due to the high concentrations needed, caspofungin is rarely used to treat C. neoformans infections. The authors believe that their new compound provides a way to lower the concentration of caspofungin needed to treat such infections, thus opening the possibility for greater utility of these antifungal.

Human tissue distribution of caspofungin

Tissue concentrations of caspofungin were determined in nine clinically relevant tissues taken during routine autopsy of 20 patients who had died during caspofungin treatment or within 23 days of cessation. The highest levels were achieved in liver, with concentrations ranging from ≤0.50 to 91.5 µg/g (0.60 µg/g 21 days after the last administration), followed by spleen (<0.25-46.3 µg/g), kidney (<0.25-33.6 µg/g) and lung (<0.25-31.0 µg/g). Intermediate concentrations were found in pancreas, skeletal muscle, thyroid and myocardium. The lowest concentrations were found in brain; caspofungin was only detectable in six of 17 samples. Caspofungin concentrations exceeded the minimum inhibitory concentration values of pathogenic Candida spp. in most of the tissue samples taken from patients who had died during treatment, except in brain samples. These findings warrant clinical outcome studies to establish the optimal treatment for deep-seated candidiasis, and support the current recommendations against echinocandins for treatment of fungal meningoencephalitis.

Therapeutic Drug Monitoring of the Echinocandin Antifungal Agents: Is There a Role in Clinical Practice? A Position Statement of the Anti-Infective Drugs Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology.

Reduced exposure to echinocandins has been reported in specific patient populations, such as critically ill patients; however, fixed dosing strategies are still used. The present review examines the accumulated evidence supporting echinocandin therapeutic drug monitoring (TDM) and summarizes available assays and sampling strategies. A literature search was conducted using PubMed in December 2020, with search terms such as echinocandins, anidulafungin, caspofungin, micafungin, or rezafungin with pharmacology, pharmacokinetics (PKs), pharmacodynamics (PDs), drug-drug interactions, TDM, resistance, drug susceptibility testing, toxicity, adverse drug reactions, bioanalysis, chromatography, and mass spectrometry. Data on PD/PD (PK/PD) outcome markers, drug resistance, PK variability, drug-drug interactions, assays, and TDM sampling strategies were summarized. Echinocandins demonstrate drug exposure-efficacy relationships, and maximum concentration/minimal inhibitory concentration ratio (Cmax/MIC) and area under the concentration-time curve/MIC ratio (AUC/MIC) are proposed PK/PD markers for clinical response. The relationship between drug exposure and toxicity remains poorly clarified. TDM could be valuable in patients at risk of low drug exposure, such as those with critical illness and/or obesity. TDM of echinocandins may also be useful in patients with moderate liver impairment, drug-drug interactions, hypoalbuminemia, and those undergoing extracorporeal membrane oxygenation, as these conditions are associated with altered exposure to caspofungin and/or micafungin. Assays are available to measure anidulafungin, micafungin, and caspofungin concentrations. A limited-sampling strategy for anidulafungin has been reported. Echinocandin TDM should be considered in patients at known risk of suboptimal drug exposure. However, for implementing TDM, clinical validation of PK/PD targets is needed.

Echinocandin Drugs Induce Differential Effects in Cytokinesis Progression and Cell Integrity.

Fission yeast contains three essential β(1,3)-D-glucan synthases (GSs), Bgs1, Bgs3, and Bgs4, with non-overlapping roles in cell integrity and morphogenesis. Only the bgs4+ mutants pbr1-8 and pbr1-6 exhibit resistance to GS inhibitors, even in the presence of the wild-type (WT) sequences of bgs1+ and bgs3+. Thus, Bgs1 and Bgs3 functions seem to be unaffected by those GS inhibitors. To learn more about echinocandins’ mechanism of action and resistance, cytokinesis progression and cell death were examined by time-lapse fluorescence microscopy in WT and pbr1-8 cells at the start of treatment with sublethal and lethal concentrations of anidulafungin, caspofungin, and micafungin. In WT, sublethal concentrations of the three drugs caused abundant cell death that was either suppressed (anidulafungin and micafungin) or greatly reduced (caspofungin) in pbr1-8 cells. Interestingly, the lethal concentrations induced differential phenotypes depending on the echinocandin used. Anidulafungin and caspofungin were mostly fungistatic, heavily impairing cytokinesis progression in both WT and pbr1-8. As with sublethal concentrations, lethal concentrations of micafungin were primarily fungicidal in WT cells, causing cell lysis without impairing cytokinesis. The lytic phenotype was suppressed again in pbr1-8 cells. Our results suggest that micafungin always exerts its fungicidal effect by solely inhibiting Bgs4. In contrast, lethal concentrations of anidulafungin and caspofungin cause an early cytokinesis arrest, probably by the combined inhibition of several GSs.

Human plasma protein levels alter the in vitro antifungal activity of caspofungin: An explanation to the effect in critically ill?

Recent studies have shown low caspofungin concentrations in critically ill patients. In some patients, the therapeutic target, area under the total plasma concentration curve in relation to the minimal inhibition concentration (AUCtot /MIC), seems not to be achieved and therapeutic drug monitoring (TDM) has been proposed. Caspofungin is highly protein-bound and the effect of reduced plasma protein levels on pharmacodynamics has not been investigated. Fungal killing activity of caspofungin in vitro was investigated under varying levels of human plasma protein. Time-kill studies were performed with clinically relevant caspofungin concentrations of 1-9mg/L on four blood isolates of C.glabrata, three susceptible and one strain with reduced susceptibility, in human plasma and plasma diluted to 50% and 25% using Ringer's acetate. Enhanced fungal killing of the three susceptible strains was observed in plasma with lower protein content (p<.001). AUCtot /MIC required for a 1 log10 CFU/ml kill at 24h in 50% and 25% plasma was reduced with 36+12 and 80+9%, respectively. The maximum effect was seen at total caspofungin concentrations of 4-9×MIC. For the strain with reduced susceptibility, growth was significantly decreased at lower protein levels. Reduced human plasma protein levels increase the antifungal activity of caspofungin in vitro, most likely by increasing the free concentration. Low plasma protein levels in critically ill patients with candidemia might explain a better response to caspofungin than expected from generally accepted target attainment and should be taken into consideration when assessing TDM based on total plasma concentrations.

Heterogeneity in the transcriptional response of the human pathogen Aspergillus fumigatus to the antifungal agent caspofungin.

Aspergillus fumigatus is the main causative agent of invasive pulmonary aspergillosis (IPA), a severe disease that affects immunosuppressed patients worldwide. The fungistatic drug caspofungin (CSP) is the second line of therapy against IPA but has increasingly been used against clinical strains that are resistant to azoles, the first line antifungal therapy. In high concentrations, CSP induces a tolerance phenotype with partial reestablishment of fungal growth called CSP paradoxical effect (CPE), resulting from a change in the composition of the cell wall. An increasing number of studies has shown that different isolates of A. fumigatus exhibit phenotypic heterogeneity, including heterogeneity in their CPE response. To gain insights into the underlying molecular mechanisms of CPE response heterogeneity, we analyzed the transcriptomes of two A. fumigatus reference strains, Af293 and CEA17, exposed to low and high CSP concentrations. We found that there is a core transcriptional response that involves genes related to cell wall remodeling processes, mitochondrial function, transmembrane transport, and amino acid and ergosterol metabolism, and a variable response related to secondary metabolite (SM) biosynthesis and iron homeostasis. Specifically, we show here that the overexpression of a SM pathway that works as an iron chelator extinguishes the CPE in both backgrounds, whereas iron depletion is detrimental for the CPE in Af293 but not in CEA17. We next investigated the function of the transcription factor CrzA, whose deletion was previously shown to result in heterogeneity in the CPE response of the Af293 and CEA17 strains. We found that CrzA constitutively binds to and modulates the expression of several genes related to processes involved in CSP tolerance and that crzA deletion differentially impacts the SM production and growth of Af293 and CEA17. As opposed to the ΔcrzACEA17 mutant, the ΔcrzAAf293 mutant fails to activate cell wall remodeling genes upon CSP exposure, which most likely severely affects its macrostructure and extinguishes its CPE. This study describes how heterogeneity in the response to an antifungal agent between A. fumigatus strains stems from heterogeneity in the function of a transcription factor and its downstream target genes.

Pharmacokinetics/Pharmacodynamics of Caspofungin in Plasma and Peritoneal Fluid of Liver Transplant Recipients.

The weaker diffusion of echinocandins in the peritoneal fluid (PF) could promote Candida-resistant isolates. The aim of this study was to analyze the pharmacokinetics (PK)/pharmacodynamics (PD) of caspofungin in plasma and PF samples from liver transplant recipients. Liver transplant patients received caspofungin as postoperative prophylaxis. Caspofungin concentrations were quantified in plasma and PF samples on days 1, 3, and 8. Data were analyzed using nonlinear mixed-effect modeling and Monte Carlo simulations. Area under the curve (AUC) values for plasma and PF were simulated under three dosing regimens. Probabilities of target attainment (PTAs) were calculated using area under the unbound plasma concentration-time curve from 0 to 24 h at steady state (fAUC0-24)/MIC ratios, with MICs ranging from 0.008 to 8 mg/L. All of the patients included were monitored weekly for Candida colonization and for Candida infections. Twenty patients were included. The median daily dose of caspofungin was 0.81 mg/kg. Plasma (n = 395) and PF (n = 50) concentrations at steady state were available. A two-compartment model with first-order absorption and elimination was described. Our two-compartment model with first-order absorption and elimination produced an effective PK/PD relationship in plasma, achieving a PTA of ≥90% with MICs ranging from 0.008 to 0.12 mg/L for Candida albicans and Candida glabrata. In PF, PTAs at D8 were optimal only for a MIC of 0.008 mg/L in patients weighing 60 kg under the three dosing regimens. Among the 16 patients colonized, all MIC values were below the maximal concentration (Cmax) in plasma but not in PF. PF concentrations of caspofungin were low. Simulations showed that the PTAs for Candida spp. in PF were not optimal, which might suggest a potential risk of resistance.