Development of a Luminex-Based Multiplex Assay for Detection of Mutations Conferring Resistance to Echinocandins in Candida glabrata

Pham, Cau D.; Bolden, Carol B.; Kuykendall, Randall J.; Lockhart, Shawn R.

doi:10.1128/jcm.03378-13

Cited by 40 publications

(32 citation statements)

References 24 publications

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“…This false result would be considered a very major error compared with whole-cell susceptibility testing since our proposed methodology would thus classify a resistant strain as susceptible. However, this molecular mechanism of echinocandin resistance has been described in only few strains worldwide and it is the least common substitution at this residue (8,16,19). Other limitations of this methodology are its inability to detect newly described mutations or other potential non-FKS-linked mechanisms associated with echinocandin resistance and the possibility of changes in epidemiology making the detection of the described mutations useless.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the designed primers would also potentially uncover less-common mutations as Fks1p-F625I (8) and Fks2p-F659Y (10,24), since the primer's 3= ends would not hybridize these mutated sequences. Recently, Pham et al described a high-throughput microsphere-based assay using the Luminex MagPix technology suitable to identify C. glabrata FKS mutants (19). This method would be potentially used as a tool to evaluate a collection of strains in a reference laboratory.…”

Section: Discussionmentioning

confidence: 99%

“…The detection of these FKS mutations has been considered the most accurate way to predict an echinocandin treatment failure (14,18,19). In an effort to improve the detection of echinocandin-resistant C. glabrata isolates, we developed a set of classical PCRs able to detect 10 of the most frequent mutations associated with clinical echinocandin resistance in less than 4 h. The sensitivity and specificity of the method were assessed using a blind collection of C. glabrata clinical isolates comprising echinocandin-resistant and -susceptible strains.…”

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confidence: 99%

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Set of Classical PCRs for Detection of Mutations in Candida glabrata FKS Genes Linked with Echinocandin Resistance

et al. 2014

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Clinical echinocandin resistance among Candida glabrata strains is increasing, especially in the United States. Antifungal susceptibility testing is considered mandatory to guide therapeutic decisions. However, these methodologies are not routinely performed in the hospital setting due to their complexity and the time needed to obtain reliable results. Echinocandin failure in C. glabrata is linked exclusively to Fks1p and Fks2p amino acid substitutions, and detection of such substitutions would serve as a surrogate marker to identify resistant isolates. In this work, we report an inexpensive, simple, and quick classical PCR set able to objectively detect the most common mechanisms of echinocandin resistance in C. glabrata within 4 h. The usefulness of this assay was assessed using a blind collection of 50 C. glabrata strains, including 16 FKS1 and/or FKS2 mutants.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Set of Classical PCRs for Detection of Mutations in Candida glabrata FKS Genes Linked with Echinocandin Resistance

et al. 2014

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“…Thus far, only two studies have reported development of rapid FKS genotyping assays (21,24). Despite good diagnostic performance, both assays suffer from complicated assay design and setup.…”

Section: Discussionmentioning

confidence: 99%

“…While mutations at almost every amino acid position within the FKS hot spot 1 and 2 regions confer some degree of resistance (20), recent studies have shown that the majority of mutations that affect C. glabrata susceptibility to the echinocandins are located within FKS1 and FKS2 HS1 regions. Accordingly, these regions are the primary targets for molecular assays to detect drug resistance (6,21). Given this goal, we have developed a novel diagnostic platform for rapid FKS genotyping of C. glabrata isolates.…”

Section: Discussionmentioning

confidence: 99%

Rapid Detection of FKS -Associated Echinocandin Resistance in Candida glabrata

Zhao

Nagasaki

Kordalewska

et al. 2016

Antimicrob Agents Chemother

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A novel and highly accurate diagnostic assay platform was established for rapid identification of FKS mutations associated with echinocandin resistance in Candida glabrata. The assay platform uses allele-specific molecular beacon and DNA melt analysis following asymmetric PCR. A dual assay for FKS1 and FKS2 was developed to identify within 3 h the most common and clinically relevant resistance-associated mutations, including 8 FKS1 HS1 (wild type [WT], S629P, F625S, D632Y, D632E [T1896G], D632E [T1896A], I634V, and F625F) and 7 FKS2 HS1 (WT, F659del, F659S, F659V, F659L, S663P, and S663F) genotypes. A blinded panel of 188 C. glabrata clinical 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 existing in vitro susceptibility-based assays to identify echinocandin-resistant C. glabrata and holds promise as a surrogate diagnostic method to better direct echinocandin therapy.C andida glabrata is the second leading cause of candidemia in the United States, as well as in northern and eastern areas of Europe (1-3). With rapidly expanded usage of echinocandins, the first-line therapy for invasive candidiasis, an alarming trend of rising echinocandin resistance in C. glabrata has posed a serious clinical challenge (4-6). Detection of echinocandin resistance can be assessed phenotypically, using either microdilution or disc diffusion MIC assays performed in accordance with guidance of the Clinical and Laboratory Standards Institute (CLSI) M27-A3 standard (7) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) Definitive Document Edef 7.2 (8). Current echinocandin clinical breakpoints for C. glabrata were established by the CLSI for all echinocandins and by EUCAST for micafungin and anidulafungin. Breakpoints were specifically developed to identify C. glabrata harboring FKS mutations by considering multiple factors, such as ␤-1,3-D-glucan synthase enzyme kinetics and echinocandin pharmacokinetic and pharmacodynamic data (6, 9). However, despite standardized methodologies, important limitations with the in vitro susceptibility testing cannot be ignored: first, the assay has a time-consuming setup and intrinsically slow turnaround time, requiring 24 to 48 h after isolate recovery; second, interlaboratory variability of caspofungin MICs has limited direct testing of this drug (10); and third, susceptible and resistant populations overlap (11).Clinical echinocandin resistance in C. glabrata is associated with amino acid substitutions caused by mutations in specific hotspot (HS) regions of FKS1 and FKS2, which encode the drug target ␤-1,3-D-glucan synthase. A recent study performed among patients with invasive candidiasis due to C. glabrata has shown that the FKS genotype is a better indicator of echinocandin therapeutic failure than MIC (12).To date, DNA sequencing is the only means to identify mutations within FKS1 and FKS2 genes. Sequence data a...

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Paving the Way to Overcome Antifungal Drug Resistance: Current Practices and Novel Developments for Rapid and Reliable Antifungal Susceptibility Testing

et al. 2021

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treating IFI, rising resistance to these drugs is a cause of major concern. [1,3] For example, the globally emerging multidrug-resistant species Candida auris is now recognized by the Centers for Disease Control and Prevention (CDC) as an urgent threat. [4] Thus, its proper treatment is therefore crucial for both individual therapeutic outcomes and preventing its spread. [5] The increasing and undirected use of antifungals in medicine and agriculture is associated with the rising numbers of acquired resistance in fungi. [3] The readers are referred to the following excellent reviews on fungal pathogens [6,7] and antifungal resistance [3] for further reading. The latter, in combination with the challenges associated with developing novel antifungals, emphasizes the need for rapid disease detection and adequate antifungal therapy protocols. [3,8] Implementing such measures is part of a proper antimicrobial stewardship aimed at reducing the excessive usage of antimicrobial therapies in clinical settings by encouraging physicians to prescribe appropriate antimicrobials only when truly required. [9] Antimicrobial susceptibility testing (AST), specifically antifungal susceptibility testing (AFST), is employed to reveal the susceptibility or resistance of fungal pathogens to clinically relevant antifungals. [10,11] In these tests, pathogenic fungi are exposed to various concentrations of a panel of antifungals to determine the minimum inhibitory concentration (MIC) values, which are typically defined as the lowest drug concentration inhibiting the pathogen's growth. [12] MIC values allow physicians to predict the success of antifungal treatments. [13] As such, rapid AFST is an essential tool to improve antifungal therapy by choosing the correct and most effective antifungal drug in a timely manner. Early therapy initiation is also crucial for enhancing the therapeutic outcome of patients with invasive fungal infections, such as life-threatening bloodstream infections. [14] Clinical AFST is conducted according to standardized methods and protocols, published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) or the Clinical and Laboratory Standards Institute (CLSI). [12,15] Yet, as these gold-standard methods are laborious and typically require at least 24 h (and in many cases, several days) for completion, [12,16] significant research efforts are now being directed toward the development of more expedited phenotypic and molecular AFST techniques. [17,18] The past year has established the link between the COVID-19 pandemic and the global spread of severe fungal infections; thus, underscoring the critical need for rapid and realizable fungal disease diagnostics. While in recent years, health authorities, such as the Centers for Disease Control and Prevention, have reported the alarming emergence and spread of drug-resistant pathogenic fungi and warned against the devastating consequences, progress in the diagnosis and treatment of fungal infections is limited. Early diagnosis and patient-tailored t...

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Development of a Luminex-Based Multiplex Assay for Detection of Mutations Conferring Resistance to Echinocandins in Candida glabrata

Cited by 40 publications

References 24 publications

Set of Classical PCRs for Detection of Mutations in Candida glabrata FKS Genes Linked with Echinocandin Resistance

Set of Classical PCRs for Detection of Mutations in Candida glabrata FKS Genes Linked with Echinocandin Resistance

Rapid Detection of FKS -Associated Echinocandin Resistance in Candida glabrata

Paving the Way to Overcome Antifungal Drug Resistance: Current Practices and Novel Developments for Rapid and Reliable Antifungal Susceptibility Testing

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