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. 2024 Nov;30(11):1447-1452.
doi: 10.1016/j.cmi.2024.07.002. Epub 2024 Jul 14.

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

Jesús Guinea  1 , Eva Alcoceba  2 , Eduardo Padilla  3 , Aída Ramírez  4 , Elena De Carolis  5 , Maurizio Sanguinetti  5 , María Muñoz-Algarra  6 , Teresa Durán-Valle  7 , Inmaculada Quiles-Melero  8 , Paloma Merino  9 , Fernando González-Romo  9 , Aída Sánchez-García  10 , Elia Gómez-García-de-la-Pedrosa  11 , Ana Pérez-Ayala  12 , María Ángeles Mantecón-Vallejo  13 , Javier Pemán  14 , María Soledad Cuétara  15 , Nelly Daniela Zurita  16 , Coral García-Esteban  17 , María Del Carmen Martínez-Jiménez  18 , Miguel Ángel Sánchez Castellano  18 , Elena Reigadas  19 , Patricia Muñoz  19 , Pilar Escribano  20
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Free article

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

Jesús Guinea et al. Clin Microbiol Infect. 2024 Nov.
Free article

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.

Keywords: Candida parapsilosis; Fluconazole-resistant; Genotyping; Y132F.

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