scholarly journals Rapid Detection ofFKS-Associated Echinocandin Resistance in Candida glabrata

2016 ◽  
Vol 60 (11) ◽  
pp. 6573-6577 ◽  
Author(s):  
Yanan Zhao ◽  
Yoji Nagasaki ◽  
Milena Kordalewska ◽  
Ellen G. Press ◽  
Ryan K. Shields ◽  
...  
Keyword(s):  
Candida Glabrata ◽  
Molecular Beacon ◽  
Rapid Identification ◽  
Molecular Diagnostic ◽  
Wild Type ◽  
Asymmetric Pcr ◽  
Content Type ◽  
Allele Specific ◽  
Echinocandin Resistance

ABSTRACTA novel and highly accurate diagnostic assay platform was established for rapid identification ofFKSmutations associated with echinocandin resistance inCandida glabrata. The assay platform uses allele-specific molecular beacon and DNA melt analysis following asymmetric PCR. A dual assay forFKS1andFKS2was developed to identify within 3 h the most common and clinically relevant resistance-associated mutations, including 8FKS1HS1 (wild type [WT], S629P, F625S, D632Y, D632E [T1896G], D632E [T1896A], I634V, and F625F) and 7FKS2HS1 (WT, F659del, F659S, F659V, F659L, S663P, and S663F) genotypes. A blinded panel of 188C. glabrataclinical isolates was tested by both assays. The molecular diagnostic results from the dual assay were 100% concordant with data obtained from DNA sequencing. This platform has the potential to overcome the deficiencies of existingin vitrosusceptibility-based assays to identify echinocandin-resistantC. glabrataand holds promise as a surrogate diagnostic method to better direct echinocandin therapy.

scholarly journals Rapid Detection of ERG11-Associated Azole Resistance and FKS-Associated Echinocandin Resistance in Candida auris

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Xin Hou ◽  
Annie Lee ◽  
Cristina Jiménez-Ortigosa ◽  
Milena Kordalewska ◽  
David S. Perlin ◽  
...  
Keyword(s):  
Rapid Assessment ◽  
Melting Curve ◽  
Rapid Identification ◽  
Molecular Diagnostic ◽  
Melting Curve Analysis ◽  
Azole Resistance ◽  
Candida Auris ◽  
Content Type ◽  
Echinocandin Resistance

ABSTRACT Candida auris is an emerging multidrug-resistant yeast that can cause serious invasive infections. The accurate and rapid assessment of antifungal resistance is important for effective patient management. A novel and highly accurate diagnostic platform was established for the rapid identification of ERG11 mutations conferring azole resistance and FKS1 mutations associated with echinocandin resistance in C. auris. Using allele-specific molecular beacons and DNA melting curve analysis following asymmetric PCR, a duplex ERG11 assay and a simplex FKS1 HS1 assay were developed to identify the most prominent resistance-associated mutations (Y132F and K143R in ERG11; S639F in FKS1 HS1) within 2 h. Assays were validated by testing a panel of 94 C. auris clinical isolates in a blind manner. The molecular diagnostic results from the assays were 100% concordant with DNA sequencing results. This platform has the potential to overcome the deficiencies of existing in vitro susceptibility-based assays to identify azole- and/or echinocandin-resistant C. auris, and thus, it holds promise as a surrogate diagnostic method to direct antifungal therapy more effectively.

scholarly journals Antifungal Activity of SCY-078 and Standard Antifungal Agents against 178 Clinical Isolates of Resistant and Susceptible Candida Species

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Wiley A. Schell ◽  
A. M. Jones ◽  
Katyna Borroto-Esoda ◽  
Barbara D. Alexander
Keyword(s):  
Candida Glabrata ◽  
Antifungal Agents ◽  
Candida Parapsilosis ◽  
Candida Krusei ◽  
Wild Type ◽  
Content Type ◽  
Clinical Resistance ◽  
Excellent Activity ◽  
Candida Lusitaniae

ABSTRACT SCY-078 in vitro activity was determined for 178 isolates of resistant or susceptible Candida albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida lusitaniae, and Candida parapsilosis, including 44 Candida isolates with known genotypic (FKS1 or FKS2 mutations), phenotypic, or clinical resistance to echinocandins. Results were compared to those for anidulafungin, caspofungin, micafungin, fluconazole, and voriconazole. SCY-078 was shown to have excellent activity against both wild-type isolates and echinocandin- and azole-resistant isolates of Candida species.

scholarly journals In Vitro Exposure to Increasing Micafungin Concentrations Easily Promotes Echinocandin Resistance in Candida glabrata Isolates

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
María Ángeles Bordallo-Cardona ◽  
Pilar Escribano ◽  
Elia Gómez G. de la Pedrosa ◽  
Laura Judith Marcos-Zambrano ◽  
Rafael Cantón ◽  
...  
Keyword(s):  
Candida Glabrata ◽  
Content Type ◽  
Inhibition Of Growth ◽  
Direct Exposure ◽  
In Vitro Exposure ◽  
Echinocandin Resistance

ABSTRACT We assessed the in vitro susceptibility of five echinocandin-susceptible Candida glabrata isolates after exposure to micafungin. The direct exposure to plates at different micafungin concentrations resulted in the inhibition of growth at 0.062 μg/ml. The progressive exposure was performed on plates using 0.031 μg/ml of micafungin and sequential propagation on plates containing the next 2-fold concentration; the MICs of micafungin and anidulafungin increased sequentially, and all the isolates became echinocandin resistant, showing fks2 mutations.

scholarly journals De Novo Acquisition of Resistance to SCY-078 in Candida glabrata Involves FKS Mutations That both Overlap and Are Distinct from Those Conferring Echinocandin Resistance

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Cristina Jiménez-Ortigosa ◽  
Winder B. Perez ◽  
David Angulo ◽  
Katyna Borroto-Esoda ◽  
David S. Perlin
Keyword(s):  
Candida Glabrata ◽  
Drug Exposure ◽  
Inhibitory Concentration ◽  
De Novo ◽  
Resistance Mutations ◽  
Content Type ◽  
Kinetic Inhibition ◽  
Echinocandin Resistance ◽  
Orally Active

ABSTRACT SCY-078 is an orally active antifungal whose target is the β-(1,3)-d-glucan synthase (GS). We evaluated the spontaneous emergence of SCY-078-resistant Candida glabrata isolates following drug exposure in vitro. Resistant isolates were analyzed using broth microdilution methodology and FKS sequencing. The kinetic inhibition parameter IC50 (50% inhibitory concentration) was also determined from GS complexes. The spectrum of resistance mutations found suggested a partially overlapping but independent binding site for SCY-078 relative to echinocandins on GS.

scholarly journals Spontaneous Mutational Frequency and FKS Mutation Rates Vary by Echinocandin Agent against Candida glabrata

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Ryan K. Shields ◽  
Ellen G. Kline ◽  
Kelley R. Healey ◽  
Milena Kordalewska ◽  
David S. Perlin ◽  
...  
Keyword(s):  
Candida Glabrata ◽  
Hot Spot ◽  
Mutation Rates ◽  
Wild Type ◽  
Front Line ◽  
Resistance Development ◽  
Content Type ◽  
Mutational Frequency ◽  
Emergence Of Resistance

ABSTRACT Echinocandins are front-line agents for treatment of invasive candidiasis. There are no reported agent-specific differences in Candida mutational frequency of resistance or propensity to develop FKS mutations. The objective of this study was to measure spontaneous and FKS mutation rates among Candida glabrata strains. Twenty bloodstream isolates from patients with or without prior echinocandin exposure were included. Minimum inhibitory concentrations (MICs), minimum fungicidal concentrations (MFCs), and mutation prevention concentrations were higher for caspofungin than for anidulafungin (P < 0.0001) and micafungin (P < 0.0001). Mutational frequencies of resistance at 3× the baseline MIC were highest for caspofungin and lowest for micafungin. A total of 247 isolates were recovered at or above the MFC for caspofungin (n = 159), anidulafungin (n = 74), or micafungin (n = 14). Agent-specific MIC increases were noted for anidulafungin and caspofungin, but not micafungin. Thirty-three percent of isolates harbored hot spot mutations in FKS1 (n = 6) or FKS2 (n = 76). Mutations at the Ser629 (Fks1) or Ser663 (Fks2) loci were more common after selection with anidulafungin or micafungin than with caspofungin (P = 0.003). Four isolates demonstrated >4-fold increases in MICs without FKS hot spot mutations; three of these harbored Fks2 mutations upstream of hot spot 1. The final isolate was FKS1 and FKS2 wild-type, but the 50% inhibitory concentrations of caspofungin and micafungin were increased 2.7- and 8-fold, respectively. In conclusion, micafungin may be superior in vitro to the other agents in limiting the emergence of resistance among C. glabrata. Caspofungin exposure may be most likely to promote resistance development. These data provide a foundation for future investigations of newly developed echinocandin agents.

scholarly journals Assessing Resistance to the Echinocandin Antifungal Drug Caspofungin in Candida albicans by Profiling Mutations in FKS1

2006 ◽  
Vol 50 (6) ◽  
pp. 2058-2063 ◽  
Author(s):  
Sergey V. Balashov ◽  
Steven Park ◽  
David S. Perlin
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Molecular Beacon ◽  
Rapid Identification ◽  
Heterozygous Mutation ◽  
Allele Specific ◽  
Double Amino Acid ◽  
Echinocandin Resistance ◽  
Spontaneous Mutants ◽  
The Relationship

ABSTRACT Resistance of clinical isolates of Candida albicans to the echinocandin drug caspofungin is slowly emerging and is linked to mutations in short conserved regions in the FKS1 gene. The most prominent changes occurred at the serine 645 position in Fks1p with substitutions of proline, tyrosine, and phenylalanine. An allele-specific real-time PCR molecular-beacon assay was developed for rapid identification of drug resistance by targeting FKS1 mutations. Mutations altering serine 645 were reliably identified in both heterozygous and homozygous states. The molecular-beacon assay was used to evaluate two large collections of spontaneous mutants from separate strains of C. albicans with resistance (MICs, >16 μg/ml) to caspofungin with the goal of understanding the relationship between FKS1 mutations and echinocandin resistance. Of 85 resistant isolates recovered, all were identified with mutations in FKS1; 93% showed changes at Ser645, with 62% displaying a characteristic S645P substitution expressed as either a homozygous or a heterozygous mutation in FKS1. Two other prominent amino acid substitutions, S645Y and S645F, were found at frequencies of 22% and 8%, respectively. Three new mutations were also identified: T1922C, G1932T, and C1934G, encoding F641S, L644F, and S645C substitutions, respectively. One strain had the double amino acid substitution L644F and S645C. Allele-specific probes were combined in a multiplex assay for reliable screening of known FKS1 mutations. These data support the importance of FKS1p substitutions in echinocandin resistance and demonstrate the feasibility of applying molecular screening for routine resistance assessment.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals In Vitro Activity of Ibrexafungerp, a Novel Glucan Synthase Inhibitor against Candida glabrata Isolates with FKS Mutations

2019 ◽  
Vol 63 (11) ◽  
Author(s):  
Natalie S. Nunnally ◽  
Kizee A. Etienne ◽  
David Angulo ◽  
Shawn R. Lockhart ◽  
Elizabeth L. Berkow
Keyword(s):  
Candida Glabrata ◽  
Vitro Activity ◽  
Good Activity ◽  
In Vitro Activity ◽  
Broth Microdilution ◽  
Glucan Synthase ◽  
Content Type ◽  
Synthase Inhibitor

ABSTRACT Ibrexafungerp is a first-in-class glucan synthase inhibitor. In vitro activity was determined for 89 Candida glabrata isolates with molecularly identified FKS1 or FKS2 mutations conferring resistance to the echinocandins. All isolates were resistant to at least one echinocandin (i.e., anidulafungin, caspofungin, or micafungin) by broth microdilution. Results for ibrexafungerp were compared with those for each echinocandin. Ibrexafungerp had good activity against all echinocandin-resistant C. glabrata isolates.

scholarly journals Discovery of a Novel Class of Orally Active Antifungal β-1,3-d-Glucan Synthase Inhibitors

2011 ◽  
Vol 55 (11) ◽  
pp. 5099-5106 ◽  
Author(s):  
Scott S. Walker ◽  
Yiming Xu ◽  
Ilias Triantafyllou ◽  
Michelle F. Waldman ◽  
Cara Mendrick ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Glabrata ◽  
Pharmacokinetic Data ◽  
Morphological Response ◽  
Glucan Synthase ◽  
Content Type ◽  
Safety Issues ◽  
Spontaneous Mutants ◽  
Orally Active

ABSTRACTThe echinocandins are a class of semisynthetic natural products that target β-1,3-glucan synthase (GS). Their proven clinical efficacy combined with minimal safety issues has made the echinocandins an important asset in the management of fungal infection in a variety of patient populations. However, the echinocandins are delivered only parenterally. A screen for antifungal bioactivities combined with mechanism-of-action studies identified a class of piperazinyl-pyridazinones that target GS. The compounds exhibitedin vitroactivity comparable, and in some cases superior, to that of the echinocandins. The compounds inhibit GSin vitro, and there was a strong correlation between enzyme inhibition andin vitroantifungal activity. In addition, like the echinocandins, the compounds caused a leakage of cytoplasmic contents from yeast and produced a morphological response in molds characteristic of GS inhibitors. Spontaneous mutants ofSaccharomyces cerevisiaewith reduced susceptibility to the piperazinyl-pyridazinones had substitutions inFKS1. The sites of these substitutions were distinct from those conferring resistance to echinocandins; likewise, echinocandin-resistant isolates remained susceptible to the test compounds. Finally, we present efficacy and pharmacokinetic data on an example of the piperazinyl-pyridazinone compounds that demonstrated efficacy in a murine model ofCandida glabratainfection.

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