{Reference Type}: Journal Article
{Title}: Fluconazole-resistant Candida parapsilosis: fast detection of the Y132F ERG11p substitution, and a proposed microsatellite genotyping scheme.
{Author}: 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;Escribano P;
{Journal}: Clin Microbiol Infect
{Volume}: 0
{Issue}: 0
{Year}: 2024 Jul 14
{Factor}: 13.31
{DOI}: 10.1016/j.cmi.2024.07.002
{Abstract}: <B>OBJECTIVE: </B>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.<BR><B>METHODS: </B>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).<BR><B>RESULTS: </B>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.<BR><B>CONCLUSIONS: </B>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.