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 β-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.

FIG 1

Schematic diagram of the FKS1 HS1 region and the corresponding probes designed to detect it. The amino acids comprising the hot spot region are shown boxed directly above the corresponding codon. The nucleotides encoding the mutations detected in this assay are shown in bold type.

FIG 2

Schematic diagram of the FKS2 HS1 region and the corresponding probes designed to detect it. The amino acids comprising the hot spot region are shown boxed directly above the corresponding codon. The nucleotides encoding the mutations detected in this assay are shown in bold type.