1. Fluconazole-resistant Candida parapsilosis: fast detection of the Y132F ERG11p substitution, and a proposed microsatellite genotyping scheme.
- Author
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Guinea J, Alcoceba E, Padilla E, Ramírez A, De Carolis E, Sanguinetti M, Muñoz-Algarra M, Durán-Valle T, Quiles-Melero I, Merino P, González-Romo F, Sánchez-García A, Gómez-García-de-la-Pedrosa E, Pérez-Ayala A, Mantecón-Vallejo MÁ, Pemán J, Cuétara MS, Zurita ND, García-Esteban C, Martínez-Jiménez MDC, Sánchez Castellano MÁ, Reigadas E, Muñoz P, and Escribano P
- Subjects
- Humans, Microbial Sensitivity Tests, Candidiasis microbiology, Spain, Italy, Genotyping Techniques methods, Sensitivity and Specificity, Amino Acid Substitution, Fungal Proteins genetics, Polymerase Chain Reaction methods, Drug Resistance, Fungal genetics, Candida parapsilosis genetics, Candida parapsilosis drug effects, Candida parapsilosis classification, Candida parapsilosis isolation & purification, Fluconazole pharmacology, Antifungal Agents pharmacology, Microsatellite Repeats, Genotype
- Abstract
Objectives: We propose fast and accurate molecular detection of the Y132F ERG11p substitution directly on pure-cultured Candida parapsilosis isolates. We also assessed a discriminative genotyping scheme to track circulating genotypes., Methods: A total of 223 C. parapsilosis isolates (one patient each) from 20 hospitals, located in Spain and Italy were selected. Isolates were fluconazole-resistant (n = 94; harbouring the Y132F ERG11p substitution [n = 85], the G458S substitution [n = 6], the R398I substitution [n = 2], or the wild-type ERG11 gene sequence) or fluconazole-susceptible (n = 129). Two targeted-A395T-mutation PCR formats (conventional and real-time) were engineered and optimized on fluconazole-susceptible and fluconazole-resistant pure-cultured isolates, thus skipping DNA extraction. Two genotyping schemes were compared: Scheme 1 (CP1, CP4a, CP6, and B markers), and Scheme 2 (6A, 6B, 6C, CP1, CP4a, and CP6 markers)., Results: The screening performed using both PCR formats showed 100% specificity (fluconazole-susceptible isolates; n = 129/129) and sensitivity (Y132F isolates; n = 85/85) values; however, results were available in 3 and 1.5 hours with the conventional and real-time PCR formats, respectively. Overall, Scheme 1 showed higher genetic diversity than Scheme 2, as shown by the number of alleles detected (n = 98; mean 23, range 13-38), the significantly higher observed and expected heterozygosity, and the probability of identity index (2.5 × 10
-6 ). Scheme 2 markers did not provide further genotypic discrimination of Y132F fluconazole-resistant genotypes., Conclusion: Both proposed PCR formats allow us to speed up the accurate detection of substitution Y132F ERG11p in C. parapsilosis isolates with 100% specificity and sensitivity. In addition, we recommend CP1, CP4a, CP6, and B microsatellite markers for genotyping fluconazole-resistant isolates., (Copyright © 2024 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)- Published
- 2024
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