Targeted amplification and MinION nanopore sequencing of key azole and echinocandin resistance determinants of clinically relevant Candida spp. from blood culture bottles

Abstract

The objective of the study was to evaluate the use of targeted multiplex Nanopore MinION amplicon re‐sequencing of key Candida spp. from blood culture bottles to identify azole and echinocandin resistance associated SNPs. Targeted PCR amplification of azole (ERG11 and ERG3) and echinocandin (FKS) resistance‐associated loci was performed on positive blood culture media. Sequencing was performed using MinION nanopore device with R9.4.1 Flow Cells. Twenty‐eight spiked blood cultures (ATCC strains and clinical isolates) and 12 prospectively collected positive blood cultures with candidaemia were included. Isolate species included Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida auris. SNPs that were identified on ERG and FKS genes using Snippy tool and CLC Genomic Workbench were correlated with phenotypic testing by broth microdilution (YeastOne™ Sensititre). Illumina whole‐genome‐sequencing and Sanger‐sequencing were also performed as confirmatory testing of the mutations identified from nanopore sequencing data. There was a perfect agreement of the resistance‐associated mutations detected by MinION‐nanopore‐sequencing compared to phenotypic testing for acquired resistance (16 with azole resistance; 3 with echinocandin resistance), and perfect concordance of the nanopore sequence mutations to Illumina and Sanger data. Mutations with no known association with phenotypic drug resistance and novel mutations were also detected.

Most azole antifungal resistance mutations in the drug target provide cross-resistance and carry no intrinsic fitness cost

“…Whole genome sequence Illumina reads of C. albicans were downloaded from NCBI for four studies: Hirakawa et al 2015 50 (21 genomes), Chew et al 51 2021 (14 genomes), Szarvas et al 2021 53 (30 genomes), and Gong et al 2023 52 (370 genomes). Reads were checked for quality with fastqc v0.12 100 , trimmed with Trimmomatic v0.39 101 , and mapped to the A haplotype of C. albicans SC5314 genome assembly A22-s07-m01-r183 (Candida Genome Database, accessed in August 2023) using bwa-mem2 v2.2.1 102 .…”

“…To evaluate if the deleterious effects we measured in S. cerevisiae can be generalized to natural C. albicans populations, we analyzed 435 genomes from four studies that were publicly available [50][51][52][53] . Although polymorphisms are limited because we study a single gene, we identified variable amino acid substitutions overlapping the genomic dataset and our DMS library.…”

Molecular Diagnostics for Invasive Fungal Diseases: Current and Future Approaches

“…Hence, detection of genes known to confer antifungal resistance or novel resistance mutations is reliant on DNA sequencing. Whilst Sanger sequencing may suffice where a limited number of genes are being analysed, WGS provides a genome-wide view of gene mutations and is also able to detect new combinations of mutations that might otherwise be missed with targeted DNA sequencing [130][131][132]. Even for echinocandin resistance where clinical utility is best understood, no commercial methods are currently available.…”

Molecular Techniques and Target Selection for the Identification of Candida spp. in Oral Samples

“…This approach has provided evidence about mutations, with no known previous association with phenotypic drug resistance of the ERG and FKS genes in Candida spp. [128].…”