Fluconazole-resistant Candida parapsilosis: fast detection of the Y132F ERG11p substitution, and a proposed microsatellite genotyping scheme

Guinea, Jesús; Alcoceba, Eva; Padilla, Eduardo; Ramírez, Aída; De Carolis, Elena; Sanguinetti, Maurizio; Muñoz-Algarra, María; Durán-Valle, Teresa; Quiles-Melero, Inmaculada; Merino, Paloma; González-Romo, Fernando; Sánchez-García, Aída; Gómez-García-de-la-Pedrosa, Elia; Pérez-Ayala, Ana; Mantecón-Vallejo, María Ángeles; Pemán, Javier; Cuétara, María Soledad; Zurita, Nelly Daniela; García-Esteban, Coral; Martínez-Jiménez, María del Carmen; Sánchez Castellano, Miguel Ángel; Reigadas, Elena; Muñoz, Patricia; Escribano, Pilar

doi:10.1016/j.cmi.2024.07.002

Clinical Microbiology and Infection

2024

DOI: 10.1016/j.cmi.2024.07.002

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Fluconazole-resistant Candida parapsilosis: fast detection of the Y132F ERG11p substitution, and a proposed microsatellite genotyping scheme

Jesús Guinea,

Eva Alcoceba,

Eduardo Padilla

et al.

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High‐Resolution Melting Assay to Detect the Mutations That Cause the Y132F and G458S Substitutions at the ERG11 Gene Involved in Azole Resistance in Candida parapsilosis

Trevijano‐Contador,

López‐Peralta,

López‐López

et al. 2024

Mycoses

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BackgroundCandida parapsilosis is a pathogenic yeast that has reduced susceptibility to echinocandins and ranks as the second or third leading cause of candidaemia, depending on the geographical region. This yeast often causes nosocomial infections, which are frequently detected as outbreaks. In recent years, resistance to azoles in C. parapsilosis has increased globally, primarily due to the accumulation of mutations in the ERG11 gene.ObjectivesIn this study, we have developed an assay based on real‐time PCR and high‐resolution melting (HRM) curve analysis to detect two of the most prevalent mutations at ERG11 that confer resistance to fluconazole (Y132F and G458S).MethodsWe designed allele‐specific oligonucleotides that selectively bind to either the wild type or mutated sequences and optimised the conditions to ensure amplification of the specific allele, followed by detection via high‐resolution melting (HRM) analysis.ResultsThe designed oligonucleotides to detect the Erg11Y132F and Erg11G458S mutations produced specific amplification of either WT or mutated alleles. We conducted a duplex real‐time PCR combining oligonucleotides for the wild‐type sequences in one mix, and oligonucleotides for the mutated alleles in another. Following this, we performed an analysis of the HRM curve to identify the amplified allele in each case. This technique was blindly evaluated on a set of 114 C. parapsilosis isolates, all of which were unequivocally identified using our approach.ConclusionThis technique offers a new method for the early detection of azole resistance mechanism in C. parapsilosis.

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High‐Resolution Melting Assay to Detect the Mutations That Cause the Y132F and G458S Substitutions at the ERG11 Gene Involved in Azole Resistance in Candida parapsilosis

Trevijano‐Contador,

López‐Peralta,

López‐López

et al. 2024

Mycoses

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Follow the Path: Unveiling an Azole Resistant Candida parapsilosis Outbreak by FTIR Spectroscopy and STR Analysis

De Carolis,

Magrì,

Camarlinghi

et al. 2024

JoF

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Accurate identification and rapid genotyping of Candida parapsilosis, a significant opportunistic pathogen in healthcare settings, is crucial for managing outbreaks, timely intervention, and effective infection control measures. This study includes 24 clinical samples and 2 positive environmental surveillance swabs collected during a fluconazole-resistant Candida parapsilosis outbreak at the Tuscany Rehabilitation Clinic (Clinica di Riabilitazione Toscana, CRT), located in the province of Arezzo, Italy. Fourier-transform infrared (FTIR) spectroscopy, genetic sequencing of the ERG11 gene, and short tandem repeat (STR) analysis was applied to track the fluconazole-resistant C. parapsilosis outbreak at the CRT facility. FTIR analysis clustered the isolates into two major groups, correlating with resistance-associated ERG11 mutations (Y132F and R398I), azole resistance levels, and year of isolation. The combined use of FTIR spectroscopy and STR typing provided a comprehensive approach to identify and track fluconazole-resistant C. parapsilosis isolates, which identified specific clusters of genetically similar isolates. By comparison with feasible molecular techniques, we conclude that FTIR spectroscopy applied in real time can inform targeted infection control strategies and aid in the effective management of nosocomial infections.

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Fluconazole-resistant Candida parapsilosis: fast detection of the Y132F ERG11p substitution, and a proposed microsatellite genotyping scheme

Cited by 2 publications

References 22 publications

High‐Resolution Melting Assay to Detect the Mutations That Cause the Y132F and G458S Substitutions at the ERG11 Gene Involved in Azole Resistance in Candida parapsilosis

High‐Resolution Melting Assay to Detect the Mutations That Cause the Y132F and G458S Substitutions at the ERG11 Gene Involved in Azole Resistance in Candida parapsilosis

Follow the Path: Unveiling an Azole Resistant Candida parapsilosis Outbreak by FTIR Spectroscopy and STR Analysis

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