Echinocandin Drugs Research Articles

Emergence of echinocandin-resistant Candida spp. in a hospital setting: a consequence of 10 years of increasing use of antifungal therapy?

Since their introduction in the 2000s, echinocandin drugs have become widely used for the treatment and prophylaxis of invasive fungal infections and, notably, invasive candidiasis. Although cases of breakthrough candidiasis in patients receiving echinocandins have been reported, clinical failure during echinocandin treatment due to the acquisition of resistance by a normally susceptible Candida spp. isolate is considered rare. To date, no publications have been published correlating the use of echinocandins and the emergence of echinocandin resistance among Candida species. So, our goal is to report an initial analysis of echinocandin use in relation to the emergence of resistant Candida isolates. We report here a single-centre experience of the emergence of eight resistant isolates belonging to normally susceptible Candida species in six patients receiving echinocandins. We describe the context and analyse the use of echinocandins over the previous decade. For seven of these isolates, we identified FKS gene mutations involved in decreased susceptibility. Seven isolates were obtained in 2011, on the heels of a ten-fold increase in caspofungin use over the preceding decade. In contrast, in 2012, the use of echinocandins decreased in our institution by 19.5 % and, in that year, only one Candida-resistant isolate was detected, despite the stable global epidemiology of invasive candidaemia. This work underlines the necessity of improving the prescription of antifungal drugs. Improvement in the monitoring of strain susceptibility should also be considered in order to better detect the emergence of resistant or non-susceptible yeast strains.

Breakthrough Candidemia Due to Multidrug-Resistant Candida glabrata during Prophylaxis with a Low Dose of Micafungin

We identified a case of breakthrough candidemia in a 25-year-old patient receiving micafungin prophylaxis (50 mg/day). Five Candida glabrata isolates were obtained from blood cultures and were classified as multidrug-resistant isolates, since all of them exhibited high MICs for echinocandin and azole drugs. A mutation (S663F) in hot spot 1 of the FKS2 gene was found in all five isolates. This mutation yielded a 1,3-β-D-glucan synthase enzyme with highly reduced sensitivities to echinocandin drugs.

Multi-drug resistant oral Candida species isolated from HIV-positive patients in South Africa and Cameroon

Candida species are a common cause of infection in immune-compromised HIV-positive individuals, who are usually treated with the antifungal drug, fluconazole, in public hospitals in Africa. However, information about the prevalence of drug resistance to fluconazole and other antifungal agents on Candida species is very limited. This study examined 128 Candida isolates from South Africa and 126 Cameroonian Candida isolates for determination of species prevalence and antifungal drug susceptibility. The isolates were characterized by growth on chromogenic and selective media and by their susceptibility to 9 antifungal drugs tested using the TREK™ YeastOne9 drug panel (Thermo Scientific, USA). Eighty-three percent (82.8%) of South African isolates were Candida albicans (106 isolates), 9.4% were Candida glabrata (12 isolates), and 7.8% were Candida dubliniensis (10 isolates). Of the Cameroonian isolates, 73.02% were C. albicans (92 isolates); 19.05% C. glabrata (24 isolates); 3.2% Candida tropicalis (4 isolates); 2.4% Candida krusei (3 isolates); 1.59% either Candida kefyr, Candida parapsilopsis, or Candida lusitaneae (2 isolates); and 0.79% C. dubliniensis (1 isolate). Widespread C. albicans resistance to azoles was detected phenotypically in both populations. Differences in drug resistance were seen within C. glabrata found in both populations. Echinocandin drugs were more effective on isolates obtained from the Cameroon than in South Africa. A multiple-drug resistant C. dubliniensis strain isolated from the South African samples was inhibited only by 5-flucytosine in vitro on the YO9 panel. Drug resistance among oral Candida species is common among African HIV patients in these 2 countries. Regional surveillance of Candida species drug susceptibility should be undertaken to ensure effective treatment for HIV-positive patients.

Candida glabrata

Chronic recurrent vulvovaginal candidiasis caused by Candida glabrata is still rare in comparison to C. albicans infection, but therapy remains more difficult. Standard agents as fluconazole or itraconazole often fail, as well as the newer systemic triazoles like voriconazole or posaconazole. Micafungin is a new echinocandin drug with a wide antifungal spectrum including rare Candida species. No clinical trials with micafungin in chronic recurrent vulvovaginal candidiasis have been undertaken. We present the initial results employing a new therapy regimen consisting of micafungin in combination with topical ciclopirox olamine. All 14 patients with chronic recurrent vulvovaginal candidiasis caused by C. glabrata were treated successfully.

Lethal small bowel necrosis due to aspergillosis during acute promyelocytic leukemia induction

Lethal small bowel necrosis due to aspergillosis during acute promyelocytic leukemia induction

Determination of micafungin and anidulafungin in human plasma: UV- or mass spectrometric quantification?

Micafungin and anidulafungin are two newer antifungal drugs from the echinocandine class. They are used as monotherapy or in combination with azole-antifungal drugs. The optimized clinical treatment course for the echinocandin drugs with regard to the different infection types and patient subgroups (renal or hepatic impairment, overweight) is still under debate. Therefore, an easy and rugged assay for these two drugs is highly desirable. We here present a method for the quantification of micafungin or anidulafungin in human plasma, applying protein precipitation as sample preparation, reversed phase separation of the analytes and UV-detection and simultaneous tandem mass spectrometry. Anidulafungin served as I.S. for micafungin quantification and vice versa. The method was validated in the calibration ranges from 0.1 μg/ml to 20 μg/ml for both substances. Intra-day precision and accuracies recorded with the UV-detector were 1.80% and 2.65% for micafungin and 4.30% and 10.44% for anidulafungin at the 0.1 μg/ml level. The respective data at the 1 μg/ml level were 2.25% and −0.83% for micafungin and 4.35% and −1.85% for anidulafungin and at the 20 μg/ml level 0.97% and −2.98% for micafungin and 1.04% and 4.74% for anidulafungin, respectively. With the mass spectrometer, because of the unique properties of the analyte molecules, no acceptable validation results could be achieved. Therefore, the mass spectrometric chromatograms served only as identity confirmation of the observed UV-peaks.

Echinocandin-Resistant Candida: Molecular Methods and Phenotypes

We now have a decade of experience with echinocandin drugs. Large-scale epidemiologic antifungal surveillance studies have demonstrated that caspofungin, micafungin, and anidulafungin retain high potency on clinical isolates of Candida, and resistance remains relatively low. Yet reports of breakthrough infections involving strains with a high minimum inhibitory concentration (MIC) are mounting. Mechanism-specific resistance involving amino acid substitutions in the Fks subunit(s) of the drug target glucan synthase results in reduced enzyme sensitivity to drug and high MICs. The mechanism affects all three drugs and is encountered in all Candida species, as well as in Aspergillus. An initial susceptibility testing breakpoint failed to adequately distinguish wild-type susceptible isolates from fks mutant resistant strains. Considering data from epidemiologic, microbiologic, pharmacokinetic/pharmacodynamic, biochemical, and genetic studies that better capture resistant isolates with fks genotypes has resulted in a proposed new breakpoint which provides a more reliable measure of probable therapeutic success.

Current Perspectives on Echinocandin Class Drugs

It has been nearly a decade since caspofungin was approved for clinical use as the first echinocandin class antifungal agent, followed by micafungin and anidulafungin. The echinocandin drugs target the fungal cell wall by inhibiting the synthesis of β-1,3-D-glucan, a critical cell wall component of many pathogenic fungi. They are fungicidal for Candida spp. and fungistatic for moulds, such as Aspergillus fumigatus, where they induce abnormal morphology and growth properties. The echinocandins have a limited antifungal spectrum but are highly active against most Candida spp., including azole-resistant strains and biofilms. As they target glucan synthase, an enzyme absent in mammalian cells, the echinocandins have a favorable safety profile. They show potent MIC and epidemiological cutoff values against susceptible Candida and Aspergillus isolates, and the frequency of resistance is low. When clinical breakthrough occurs, it is associated with high MIC values and mutations in Fks subunits of glucan synthase, which can reduce the sensitivity of the enzyme to the drug by several thousand-fold. Such strains were not adequately captured by an early clinical breakpoint for susceptibility prompting a revised lower value, which addresses the FKS resistance mechanism and new pharmacokinetic/pharmacodynamic studies. Elevated MIC values unlinked to therapeutic failure can occur and result from adaptive cell behavior, which is FKS-independent and involves the molecular chaperone Hsp90 and the calcineurin pathway. Mutations in FKS1 and/or FKS2 alter the kinetic properties of glucan synthase, which reduces the relative fitness of mutant strains causing them to be less pathogenic. The echinocandin drugs also modify the cell wall architecture exposing buried glucans, which in turn induce a variety of important host immune responses. Finally, the future for glucan synthase inhibitors looks bright with the development of new orally active compounds.

Improved Detection of Candida sp. fks Hot Spot Mutants by Using the Method of the CLSI M27-A3 Document with the Addition of Bovine Serum Albumin

Echinocandins are highly bound to serum proteins, altering their antifungal properties. The addition of 50% human serum to the MIC assay improves the identification of echinocandin-resistant Candida spp. harboring fks hot spot mutations. However, this modification cannot readily be applied to the method of the CLSI M27-A3 document due to safety and standardization difficulties. The aim of this study was to evaluate commercial bovine serum albumin (BSA) as a safe and standardized alternative to human serum. A collection of 28 echinocandin-susceptible strains, 10 Candida parapsilosis sensu lato strains (with naturally reduced echinocandin susceptibility), and 40 FKS hot spot mutants was used in this work. When RPMI 1640 was used for susceptibility testing, wild-type strains and fks mutants showed MIC range overlaps (-2, -1, and -3 2-fold-dilution steps separated these populations for anidulafungin, caspofungin, and micafungin, respectively). On the other hand, the addition of BSA to RPMI 1640 differentially increased echinocandin MIC values for these groups of strains, allowing better separation between populations, with no MIC range overlaps for any of the echinocandin drugs tested. Moreover, the use of RPMI-BSA reduced the number of fks hot spot mutant isolates for which MIC values were less than or equal to the upper limit for the wild type (very major errors) from 9, 2, and 7 with RPMI alone to 3, 0, and 3 for anidulafungin, caspofungin, and micafungin, respectively. When RPMI-BSA was used to study the susceptibility of C. parapsilosis sensu lato species to echinocandins, the strains behaved as anidulafungin- and micafungin-resistant isolates (MIC, ≥8 μg/ml). These data support the need for a revision of the CLSI protocol for in vitro testing of echinocandin susceptibility in order to identify all or most of the fks hot spot mutants. Also, caspofungin could be used as a surrogate marker of reduced susceptibility to echinocandins.

In vitrointeractions among echinocandins againstAspergillus fumigatus: lack of concordance among methods

Drug combinations are increasingly being explored as a means of improving outcomes in cases of invasive aspergillosis. However, the optimal methods for assessing in vitro drug combinations are unclear. We investigated whether echinocandin drug combinations have potentially useful interactions against Aspergillus fumigatus. We explored the in vitro interactions of three echinocandins (caspofungin [CSP], anidulafingin [ANF], and micafungin [MCF]) by three methods, i.e., (i) checkerboard assay, (ii) disk diffusion assay, and (iii) E-test/agar dilution. The checkerboard experiments revealed different interactions between echinocandin pairs depending on the drug combination tested. Specifically, the combination of CSF and either ANF or MCF yielded a fractional inhibitory concentration (FIC) index range of 0.15-2.0. Whereas the combination of MCF and ANF yielded an FIC index range of 0.19-0.31, consistent with synergistic interaction. In contrast, all echinocandin pairs appeared indifferent by the disk diffusion method. Moreover, the combination of MCF and ANF appeared antagonistic when the two drugs were tested using the E-test/agar dilution method with both the FKS1 wild-type and echinocandin-resistant fks1 mutant strains. Our results highlight methodological problems inherent in in vitro antifungal combination testing. We did not find compelling evidence of inter-echinocandin synergy that could serve as a basis for further in vivo experimentation.

Profiling the Aspergillus fumigatus Proteome in Response to Caspofungin

The proteomic response of Aspergillus fumigatus to caspofungin was evaluated by gel-free isobaric tagging for relative and absolute quantitation (iTRAQ) as a means to determine potential biomarkers of drug action. A cell fractionation approach yielding 4 subcellular compartment fractions was used to enhance the resolution of proteins for proteomic analysis. Using iTRAQ, a total of 471 unique proteins were identified in soluble and cell wall/plasma membrane fractions at 24 and 48 h of growth in rich media in a wild-type drug-susceptible strain. A total of 122 proteins showed at least a 2-fold change in relative abundance following exposure to caspofungin (CSF) at just below the minimum effective concentration (0.12 μg/ml). The largest changes were seen in the mitochondrial hypoxia response domain protein (AFUA_1G12250), the level of which decreased >16-fold in the secreted fraction, and ChiA1, the level of which decreased 12.1-fold in the cell wall/plasma membrane fraction. The level of the major allergen and cytotoxin AspF1 was also shown to decrease by 12.1-fold upon the addition of drug. A subsequent iTRAQ analysis of an echinocandin-resistant strain (fks1-S678P) was used to validate proteins specific to drug action. A total of 103 proteins in the 2 fractions tested by iTRAQ were differentially expressed in the wild-type susceptible strain but not significantly changed in the resistant strain. Of these potential biomarkers, 11 had levels that changed at least 12-fold. Microarray analysis of the susceptible strain was performed to evaluate the correlation between proteomics and genomics, with a total of 117 genes found to be changing at least 2-fold. Of these, a total of 22 proteins with significant changes identified by iTRAQ also showed significant gene expression level changes by microarray. Overall, these data have the potential to identify biomarkers that assess the relative efficacy of echinocandin drug therapy.

Efficacy and safety of micafungin for treating febrile neutropenia in hematological malignancies

Less toxic antifungal drugs are required for empirical antifungal therapy. Micafungin is an echinocandin drug that is effective against both Candida and Aspergillus, and preliminary clinical studies have shown good antifungal activity. We prospectively examined the effect and safety of micafungin against febrile neutropenia with suspected fungal infection in 53 patients (median age, 56 years) who had undergone chemotherapy. The administered dose of micafungin was 150 mg/day, and its effect was evaluated as fever resolution as well as the results of chest imaging and serum fungal tests. Micafungin levels were measured on day 4 after the first administration using high-performance liquid chromatography. We also measured trough levels of micafungin. Underlying diseases comprised acute lymphoblastic leukemia (n = 4), acute myeloid leukemia (n = 20), multiple myeloma (n = 3), and non-Hodgkin's lymphoma (n = 26). The overall efficacy of micafungin was 70%. Breakthrough fungal infections were documented in two (3.8%) patients, both of whom died of invasive mycosis. None of the patients were switched to other antifungal drugs due to events unrelated to adverse effects. Plasma levels of micafungin and the degree of hepatic or renal dysfunction did not correlate. Micafungin is safe and effective for the empirical antifungal therapy of febrile neutropenia in patients with hematological malignancies.

Prévalence de Candida parapsilosis, C. orthopsilosis et de C. metapsilosis au sein des candidémies au CHU de Nantes et profil de sensibilité aux échinocandines par la méthode E-test ® : étude rétrospective de cinq ans (2004–2009)

Aim of the study To determine the prevalence of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis among candidemia at Nantes University Hospital and to evaluate the in vitro susceptibility of the isolates against three echinocandin drugs (caspofungin, micafungin and anidulafungin). Material and methods Retrospective study (march 2004 to july 2009) of 178 cases of candidemia corresponding to 183 Candida spp. strains identified by means of routine phenotypical methods. Re-identification of C. parapsilosis sensu lato isolates was performed by ITS rDNA sequencing analysis. Minimal inhibitory concentrations (MIC) were determined by E-test ®. All echinocandin non-susceptible isolates (MIC > 2 μg/mL) were analyzed for the presence/absence of FKS1 mutations associated with resistance. Results During this period, C. parapsilosis sensu lato was responsible for 27 candidemia, ranging at the second most common Candida species after C. albicans ( n = 99, 54.1%). Neither isolates belong to C. orthopsilosis nor C. metapsilosis. According to the literature, all the isolates displayed high MICs against the three echinocandin drugs. All the isolates displayed both susceptibility (MIC ≤ 2 μg/mL) and a good agreement between MICs read at 24 h and 48 h for caspofungin and micafungin (MIC 50 = 0.75 μg/mL, MIC 90 = 1.5 μg/mL). Surprisingly, whereas most of the strains were susceptible to anidulafungin at 24 h (MIC 50 = 1 μg/mL, MIC 90 = 1.5 μg/mL), 14 (52 %) displayed non-susceptibility, despite the lack of mutation associated with resistance on FKS1, when reading was performed at 48 h (MIC 50 = 3 μg/mL, MIC 90 = 12 μg/mL). Conclusion Prevalence of C. orthopsilosis and C. metapsilosis in patients with candidemia is low at Nantes University Hospital. The difficulty encountered with MIC reading by E-test ® are discussed.

FKS Mutations and Elevated Echinocandin MIC Values among Candida glabrata Isolates from U.S. Population-Based Surveillance

Candida glabrata is the second leading cause of candidemia in the United States. Its high-level resistance to triazole antifungal drugs has led to the increased use of the echinocandin class of antifungal agents for primary therapy of these infections. We monitored C. glabrata bloodstream isolates from a population-based surveillance study for elevated echinocandin MIC values (MICs of ≥0.25 μg/ml). From the 490 C. glabrata isolates that were screened, we identified 16 isolates with an elevated MIC value (2.9% of isolates from Atlanta and 2.0% of isolates from Baltimore) for one or more of the echinocandin drugs caspofungin, anidulafungin, and micafungin. All of the isolates with elevated MIC values had a mutation in the previously identified hot spot 1 of either the glucan synthase FKS1 (n = 2) or FKS2 (n = 14) gene. No mutations were detected in hot spot 2 of either FKS1 or FKS2. The predominant mutation was mutation of FKS2-encoded serine 663 to proline (S663P), found in 10 of the isolates with elevated echinocandin MICs. Two of the mutations, R631G for FKS1 and R665G for FKS2, have not been reported previously for C. glabrata. Multilocus sequence typing indicated that the predominance of the S663P mutation was not due to the clonal spread of a single sequence type. With a rising number of echinocandin therapy failures reported, it is important to continue to monitor rates of elevated echinocandin MIC values and the associated mutations.

Effect of Candida glabrata FKS1 and FKS2 Mutations on Echinocandin Sensitivity and Kinetics of 1,3-β- d -Glucan Synthase: Implication for the Existing Susceptibility Breakpoint

Thirteen Candida glabrata strains harboring a range of mutations in hot spot regions of FKS1 and FKS2 were studied. The mutations were linked to an echinocandin reduced susceptibility phenotype. Sequence alignments showed that 11 out of the 13 mutants harbored a mutation in FKS1 or FKS2 not previously implicated in echinocandin reduced susceptibility in C. glabrata. A detailed kinetic characterization demonstrated that amino acid substitutions in Fks1p and Fks2p reduced drug sensitivity in mutant 1,3-beta-D-glucan synthase by 2 to 3 log orders relative to that in wild-type enzyme. These mutations were also found to reduce the catalytic efficiency of the enzyme (Vmax) and to influence the relative expression of FKS genes. In view of the association of FKS mutations and reduced susceptibility of 1,3-beta-D-glucan synthase, an evaluation of the new CLSI echinocandin susceptibility breakpoint was conducted. Only 3 of 13 resistant fks mutants (23%) were considered anidulafungin or micafungin nonsusceptible (MIC > 2 microg/ml) by this criterion. In contrast, most fks mutants (92%) exceeded a MIC of >2 microg/ml with caspofungin. However, when MIC determinations were performed in the presence of 50% serum, all C. glabrata fks mutants showed MICs of > or = 2 microg/ml for the three echinocandin drugs. As has been observed with Candida albicans, the kinetic inhibition parameter 50% inhibitory concentration may be a better predictor of FKS-mediated resistance. Finally, the close association between FKS1/FKS2 hot spot mutations provides a basis for understanding echinocandin resistance in C. glabrata.

Correlating Echinocandin MIC and Kinetic Inhibition of fks1 Mutant Glucan Synthases for Candida albicans : Implications for Interpretive Breakpoints

A detailed kinetic characterization of echinocandin inhibition was performed for mutant 1,3-beta-d-glucan synthase enzymes from clinical isolates of Candida albicans with nine different FKS1 mutations resulting in high MICs. Among 14 mutant Fks1p enzymes studied, the kinetic parameters 50% inhibitory concentration and K(i) increased 50-fold to several thousandfold relative to those for the wild type. Enzymes with mutations at Ser645 (S645P, S645Y, and S645F) within hot spot 1 showed the most prominent decrease in sensitivity, while those with mutations at the N- and C-terminal ends of hot spot 1 generally retained greater sensitivity to all three drugs. Kinetic inhibitions by caspofungin, micafungin, and anidulafungin were comparable among the fks1 mutant enzymes, although absolute values did vary with specific mutations. Amino acid substitutions in Fks1p did not alter K(m) values, although some mutations decreased the V(max). Given the association of FKS1 mutations with clinical resistance, an evaluation of the kinetic parameters for the inhibition of mutant 1,3-beta-D-glucan synthase as a function of the MIC enabled an independent evaluation of the recently adopted susceptibility breakpoint for echinocandin drugs. Overall, a breakpoint MIC of >or=2 microg/ml for caspofungin captured nearly 100% of fks1 C. albicans strains when a kinetic inhibition rise threshold of <or=50-fold for the K(i) was used as a measure of susceptibility. A similar MIC breakpoint for micafungin and anidulafungin was less inclusive, and a projected MIC of >or=0.5 microg/ml was required for >95% coverage of clinical isolates. However, when MIC determinations were performed in the presence of 50% serum, all fks1 mutants showed MIC values of >or=2 microg/ml for the three echinocandin drugs. The 1,3-beta-D-glucan synthase kinetic inhibition data support the proposed susceptibility breakpoint for caspofungin in C. albicans, but a lower susceptibility breakpoint (<or=0.5 microg/ml) may be more appropriate for anidulafungin and micafungin. Overall, the data indicate that MIC testing with caspofungin may serve as a surrogate marker for resistance among the class of echinocandin drugs.

CYP56 (Dit2p) in Candida albicans : Characterization and Investigation of Its Role in Growth and Antifungal Drug Susceptibility

The complete DNA sequence of Candida albicans DIT2, encoding cytochrome P450 family 56 (CYP56), was obtained, and heterologous expression was achieved in Escherichia coli, where CYP56 was targeted to the membrane fraction. In reconstituted assays with the purified enzyme, CYP56 was shown to catalyze the conversion of N-formyl tyrosine into N,N'-bisformyl dityrosine, a reaction that was dependent on cytochrome P450 reductase, NADPH, and oxygen, yielding a turnover of 21.6 min(-1) and a k(s) of 26 microM. The Hill number was calculated as 1.6, indicating that two molecules of the substrate could bind to the protein. Azole antifungals could bind to the heme of CYP56 as a sixth ligand with high affinity. Both chromosomal alleles of CYP56 were disrupted using the SAT1 flipper technique, and CYP56 was found to be nonessential for cell viability under the culture conditions investigated. Susceptibility to azole drugs that bind to cytochromes P450 was tested, and the mutant showed unaltered susceptibility. However, the mutant showed increased susceptibility to the echinocandin drug caspofungin, suggesting an alteration in 1,3-glucan synthase and/or cell wall structure mediated by the presence of dityrosine. Phenotypically, the wild-type and mutant strains were morphologically similar when cultured in rich yeast extract-peptone-dextrose medium. However in minimal medium, the cyp56Delta mutant strain exhibited hyphal growth, in contrast to the wild-type strain, which grew solely in the yeast form. Furthermore, CYP56 was essential for chlamydospore formation.

Use of a serum-based antifungal susceptibility assay to predict thein vivoefficacy of novel echinocandin compounds

In vitro susceptibility assays of antifungal activity do not always accurately predict in vivo efficacy. As well as having a clear clinical importance, the ability to predict efficacy is also essential for effective screening of novel drug compounds. Initial screening of novel compounds must often be based on in vitro data. The present report describes the use of serum-MIC, an in vitro test of antifungal susceptibility, to accurately predict in vivo efficacy of echinocandin drugs in a mouse model of disseminated candidiasis. The basis of the serum-MIC method was to measure the inhibitory activity of a test compound against Candida albicans hyphal growth in the presence of pooled mouse serum. For 13 previously uncharacterized echinocandin compounds, as well as for the known echinocandin drugs, micafungin and caspofungin, serum-MIC determinations were shown to give better correlation to efficacy in the animal model than conventional, CLSI standard, in vitro antifungal susceptibility tests. The most accurate prediction of efficacy was obtained when the serum-MIC was adjusted in relation to the serum concentration at 30 min post-treatment. Furthermore, when the efficacy of micafungin was determined by measuring C. albicans kidney burden in the mouse model of infection, the adjusted serum-MIC consistently reflected the effective serum concentrations. Our data indicate that determination of serum-MIC values will facilitate prediction of the in vivo potency of new antifungal compounds such as novel echinocandins.

A Naturally Occurring Proline-to-Alanine Amino Acid Change in Fks1p in Candida parapsilosis , Candida orthopsilosis , and Candida metapsilosis Accounts for Reduced Echinocandin Susceptibility

Candida parapsilosis has emerged as a common cause of invasive fungal infection, especially in Latin America and in the neonatal setting. C. parapsilosis is part of a closely related group of organisms that includes the species Candida orthopsilosis and Candida metapsilosis. All three species show elevated MICs for the new echinocandin class drugs caspofungin, micafungin, and anidulafungin relative to other Candida species. Despite potential impacts on therapy, the mechanism behind this reduced echinocandin susceptibility has not been determined. In this report, we investigated the role of a naturally occurring Pro-to-Ala substitution at amino acid position 660 (P660A), immediately distal to the highly conserved hot spot 1 region of Fks1p, in the reduced-echinocandin-susceptibility phenotype. Kinetic inhibition studies demonstrated that glucan synthase from the C. parapsilosis group was 1 to 2 logs less sensitive to echinocandin drugs than the reference enzyme from C. albicans. Furthermore, clinical isolates of C. albicans and C. glabrata which harbor mutations at this equivalent position also showed comparable 2-log decreases in target enzyme sensitivity, which correlated with increased MICs. These mutations also resulted in 2.4- to 18.8-fold-reduced V(max) values relative to those for the wild-type enzyme, consistent with kinetic parameters obtained for C. parapsilosis group enzymes. Finally, the importance of the P660A substitution for intrinsic resistance was confirmed by engineering an equivalent P647A mutation into Fks1p of Saccharomyces cerevisiae. The mutant glucan synthase displayed characteristic 2-log decreases in sensitivity to the echinocandin drugs. Overall, these data firmly indicate that a naturally occurring P660A substitution in Fks1p from the C. parapsilosis group accounts for the reduced susceptibility phenotype.

Stage-specific innate immune recognition ofAspergillus fumigatusand modulation by echinocandin drugs

The pulmonary innate immune system clears inhaled Aspergillus fumigatus conidia (spores) from terminal airways. Failure to control conidial germination in immune compromised hosts can result in hyphal tissue invasion and fatal disease. Insight into the molecular recognition of A. fumigatus by host leukocytes indicates that the innate immune system exploits obligate changes in fungal cell wall composition that occur at the first stage of germination, conidial swelling. Germinating spores activate at least two host signal transduction pathways. Surface exposure of fungal beta-(1,3) glucan, a polysaccharide constituent of the fungal cell wall, triggers dectin-1 signaling by host phagocytes. Spore germination leads to the induction of Toll-like receptor (TLR) signaling as well. This stage-specific recognition mechanism focuses host antifungal responses on cells with the potential for tissue invasion and may serve to limit potentially deleterious effects of inflammation in space and time. Fungal beta-(1,3) glucan not only activates host innate immune responses but also represents the target of echinocandin drugs. The activity of echinocandin drugs has largely been understood on the basis of pharmacologic growth inhibition of yeast and moulds, resulting in lysis of yeast cells and stunting of dysmorphic hyphae. The recognition that fungal beta-1,3 glucan activates dectin-1 signaling suggests that echinocandin drugs may exert immune modulatory effects by altering innate immune responses to drug-treated fungal cells, a view supported by recent data from studies on C. albicans, A. fumigatus, and non-Aspergillus moulds.