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

Dudiuk, Catiana; Gamarra, Soledad; Leonardeli, Florencia; Jiménez‐Ortigosa, Cristina; Vitale, Roxana G.; Afeltra, Javier; Perlin, David S.; García‐Effrón, Guillermo

doi:10.1128/jcm.01038-14

Cited by 45 publications

(50 citation statements)

References 30 publications

(36 reference statements)

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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%

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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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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“…Clinical resistance to echinocandins has been associated with hot spot FKS mutations (15,16). Recently, our group published a set of classical PCRs able to detect these mutations (17). The aims of this study were to evaluate the in vitro activities of echinocandins against Argentinian C. glabrata sensu lato strains and to compare the obtained results with those obtained with the described molecular tool.…”

mentioning

confidence: 99%

“…Echinocandin resistance molecular mechanisms were evaluated by using a recently published set of classical PCRs able to identify hot spot mutations at the FKS1 and FKS2 genes. FKS gene sequencing was used to confirm the obtained results (17).…”

mentioning

confidence: 99%

“…The isolates were identified by classical, molecular (internal transcribed spacer [ITS] sequencing and multiplex PCR), and proteomic (matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS]) methods (18)(19)(20). Also, 17 characterized echinocandin-resistant C. glabrata sensu stricto strains harboring different FKS mutations were used as PCR control (17), together with Candida parapsilosis sensu stricto ATCC 22019 and Candida krusei ATCC 6258, which were used as MIC controls (21,22). Anidulafungin (ANF) and caspofungin (CSF) MICs were determined by broth microdilution in accordance with the CLSI M27-A3 and M27-S4 documents (21,22).…”

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Phenotypic and Molecular Evaluation of Echinocandin Susceptibility of Candida glabrata, Candida bracarensis, and Candida nivariensis Strains Isolated during 30 Years in Argentina

Morales-López

Dudiuk

Vivot

et al. 2017

Antimicrob Agents Chemother

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The echinocandin susceptibilities of 122 Candida glabrata complex strains (including 5 Candida nivariensis and 3 Candida bracarensis strains) were evaluated by microdilution and compared with the results from a molecular tool able to detect FKS mutations. No echinocandin resistance was detected. The PCR results coincide with the MIC data in 99.25% of the cases (1 C. glabrata strain was misidentified as resistant) but were 20 h faster. C. nivariensis FKS genes were sequenced and showed differences with C. glabrata FKS genes.

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“…Echinocandin resistance in C. glabrata is associated entirely with fks1p and fks2p amino acid substitutions [25]. Echinocandin failure accredited to mutations in fks1 is linked with raised chitin levels and the lack of a compensatory upsurge in chitin level on echinocandin introduction ( Table 1) [26].…”

Section: Mechanism Of Resistancementioning

confidence: 99%

Echinocandins Versus Dated Antifungals in Combination Against Opportunistic Mycotic Infections

Narwal¹,

Balhara²,

Chaudhary³

et al. 2017

Asian J Pharm Clin Res

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Scientific and clinical reports globally demonstrated that the opportunistic mycotic infections are at major risk to the human fitness. In the past few decades, the development of resistance in microbes to existing antifungals has emphasized on the search of new antimycotic drugs. As a matter of fact, "echinocandins" are new categories of broad-spectrum antifungal enlighten a hope in this direction. Echinocandins are bulky lipopeptides that inhibit the production of β-[1,3]-glucan "a major constituent of fungal cell wall" which ultimately leads to the death of fungal pathogens. In vitro as well as in vivo published reports have demonstrated that the echinocandins exhibit fungicidal activity against most Candida spp., while fungistatic against Aspergillus spp. and exclusively found to be more effective when tested in combination with polyenes/azoles. The present article is expert views on the recent and historical literature available on the antifungal therapies with accessing their impact on the human health. Emphasis is given to the utility of the echinocandins a potental antifungal agent by discussing recent examples of clinical and laboratory studies including the use of improved proteomics approaches to know a bit more about the interaction of human host and fungal pathogens.

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

Cited by 45 publications

References 30 publications

Rapid Detection of FKS -Associated Echinocandin Resistance in Candida glabrata

Rapid Detection of FKS -Associated Echinocandin Resistance in Candida glabrata

Phenotypic and Molecular Evaluation of Echinocandin Susceptibility of Candida glabrata, Candida bracarensis, and Candida nivariensis Strains Isolated during 30 Years in Argentina

Echinocandins Versus Dated Antifungals in Combination Against Opportunistic Mycotic Infections

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