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

Abstract

Echinocandins are the recommended treatment for invasive candidiasis due to Candida glabrata. Resistance to echinocandins is known to be caused by nonsynonymous mutations in the hot spot-1 (HS1) regions of the FKS1 and FKS2 genes, which encode a subunit of the ␤-1,3-glucan synthase, the target of echinocandins. Here, we describe the development of a microsphere-based assay using Luminex MagPix technology to identify mutations in the FKS1 HS1 and FKS2 HS1 domains, which confer in vitro echinocandin resistance in C. glabrata isolates. The assay is rapid and can be performed with high throughput. The assay was validated using 102 isolates that had FKS1 HS1 and FKS2 HS1 domains previously characterized by DNA sequencing. The assay was 100% concordant with DNA sequencing results. The assay was then used for high-throughput screening of 1,032 C. glabrata surveillance isolates. Sixteen new isolates with mutations, including a mutation that was new to our collection (del659F), were identified. This assay provides a rapid and cost-effective way to screen C. glabrata isolates for echinocandin resistance.

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

“…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.…”

“…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.…”

“…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.…”

Rapid Detection of FKS -Associated Echinocandin Resistance in Candida glabrata

“…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.…”

“…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.…”