Clinical Microbiology and Infectious Diseases Department, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; CIBER Enfermedades Respiratorias-CIBERES (CB06/06/0058), Madrid, Spain; Faculty of Health Sciences - HM Hospitals, Universidad Camilo José Cela, Madrid, Spain.
Clinical Microbiology Department, Hospital Universitari Son Espases, Mallorca, Spain.
Clin Microbiol Infect. 2024 Nov;30(11):1447-1452. doi: 10.1016/j.cmi.2024.07.002. Epub 2024 Jul 14.
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.
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).
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). Scheme 2 markers did not provide further genotypic discrimination of Y132F fluconazole-resistant genotypes.
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.
我们拟直接对纯培养近平滑念珠菌分离株进行快速、准确的 Y132F ERG11p 取代的分子检测，并评估一种有鉴别能力的基因分型方案来跟踪循环基因型。
从西班牙和意大利的 20 家医院中选择了 223 株近平滑念珠菌（每位患者一株）。分离株为氟康唑耐药（n=94；含有 Y132F ERG11p 取代[n=85]、G458S 取代[n=6]、R398I 取代[n=2]或野生型 ERG11 基因序列）或氟康唑敏感（n=129）。设计并优化了两种针对 A395T 突变的靶向 PCR 格式（常规和实时），从而跳过 DNA 提取，对氟康唑敏感和耐药的纯培养分离株进行检测。比较了两种基因分型方案：方案 1（CP1、CP4a、CP6 和 B 标记物）和方案 2（6A、6B、6C、CP1、CP4a 和 CP6 标记物）。
两种 PCR 格式的筛查均显示出 100%的特异性（氟康唑敏感分离株；n=129/129）和敏感性（Y132F 分离株；n=85/85），但常规 PCR 格式和实时 PCR 格式的结果可分别在 3 小时和 1.5 小时内获得。总体而言，方案 1 显示出比方案 2 更高的遗传多样性，表现在检测到的等位基因数量（n=98；平均值 23，范围 13-38）、观察到和预期的杂合度显著更高，以及身份指数概率（2.5×10）更高。方案 2 标记物不能进一步区分 Y132F 氟康唑耐药基因型。
两种提出的 PCR 格式都可以在 100%的特异性和敏感性的情况下加速对近平滑念珠菌分离株中 Y132F ERG11p 取代的准确检测。此外，我们建议使用 CP1、CP4a、CP6 和 B 微卫星标记物对氟康唑耐药分离株进行基因分型。
