Milena Kordalewska

Rapid and Accurate Molecular Identification of the Emerging Multidrug-Resistant Pathogen Candida auris

ABSTRACT Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality. C. auris is commonly misidentified as several different yeast species by commercially available phenotypic identification platforms. Thus, there is an urgent need for a reliable diagnostic method. In this paper, we present fast, robust, easy-to-perform and interpret PCR and real-time PCR assays to identify C. auris and related species: Candida duobushaemulonii, Candida haemulonii, and Candida lusitaniae. Targeting rDNA region nucleotide sequences, primers specific for C. auris only or C. auris and related species were designed. A panel of 140 clinical fungal isolates was used in both PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The identification results from the assays were 100% concordant with DNA sequencing results. These molecular assays overcome the deficiencies of existing phenotypic tests to identify C. auris and related species.

A multicentre study of antifungal susceptibility patterns among 350 Candida auris isolates (2009–17) in India: role of the ERG11 and FKS1 genes in azole and echinocandin resistance

Background Candida auris has emerged globally as an MDR nosocomial pathogen in ICU patients. Objectives We studied the antifungal susceptibility of C. auris isolates (n = 350) from 10 hospitals in India collected over a period of 8 years. To investigate azole resistance, ERG11 gene sequencing and expression profiling was conducted. In addition, echinocandin resistance linked to mutations in the C. auris FKS1 gene was analysed. Methods CLSI antifungal susceptibility testing of six azoles, amphotericin B, three echinocandins, terbinafine, 5-flucytosine and nystatin was conducted. Screening for amino acid substitutions in ERG11 and FKS1 was performed. Results Overall, 90% of C. auris were fluconazole resistant (MICs 32 to ≥64 mg/L) and 2% and 8% were resistant to echinocandins (≥8 mg/L) and amphotericin B (≥2 mg/L), respectively. ERG11 sequences of C. auris exhibited amino acid substitutions Y132 and K143 in 77% (n = 34/44) of strains that were fluconazole resistant whereas WT genotypes, i.e. without substitutions at these positions, were observed in isolates with low fluconazole MICs (1-2 mg/L) suggesting that these substitutions confer a phenotype of resistance to fluconazole similar to that described for Candida albicans. No significant expression of ERG11 was observed, although expression was inducible in vitro with fluconazole exposure. Echinocandin resistance was linked to a novel mutation S639F in FKS1 hot spot region I. Conclusions Overall, 25% and 13% of isolates were MDR and multi-azole resistant, respectively. The most common resistance combination was azoles and 5-flucytosine in 14% followed by azoles and amphotericin B in 7% and azoles and echinocandins in 2% of isolates.

Rapid Detection of FKS-Associated Echinocandin Resistance in Candida glabrata

ABSTRACT 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.

The gastrointestinal tract is a major source of echinocandin drug resistance in a murine model of Candida glabrata colonization and systemic dissemination

ABSTRACT Candida species are a part of the human microbiome and can cause systemic infection upon immune suppression. Candida glabrata infections are increasing and have greater rates of antifungal resistance than other species. Here, we present a C. glabrata gastrointestinal (GI) colonization model to explore whether colonized yeast exposed to caspofungin, an echinocandin antifungal, develop characteristic resistance mutations and, upon immunosuppression, breakthrough causing systemic infection. Daily therapeutic dosing (5 mg/kg of body weight) of caspofungin resulted in no reduction in fecal burdens, organ breakthrough rates similar to control groups, and resistance rates (0 to 10%) similar to those reported clinically. Treatment with 20 mg/kg caspofungin initially reduced burdens, but a rebound following 5 to 9 days of treatment was accompanied by high levels of resistance (FKS1/FKS2 mutants). Although breakthrough rates decreased in this group, the same FKS mutants were recovered from organs. In an attempt to negate drug tolerance that is critical for resistance development, we cotreated mice with daily caspofungin and the chitin synthase inhibitor nikkomycin Z. The largest reduction (3 log) in GI burdens was obtained within 3 to 5 days of 20 mg/kg caspofungin plus nikkomycin treatment. Yet, echinocandin resistance, characterized by a novel Fks1-L630R substitution, was identified following 5 to 7 days of treatment. Therapeutic caspofungin plus nikkomycin treatment left GI burdens unchanged but significantly reduced organ breakthrough rates (20%; P < 0.05). Single-dose pharmacokinetics demonstrated low levels of drug penetration into the GI lumen posttreatment with caspofungin. Overall, we show that C. glabrata echinocandin resistance can arise within the GI tract and that resistant mutants can readily disseminate upon immunosuppression.