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

IF 10.9 1区医学 Q1 INFECTIOUS DISEASES

Clinical Microbiology and Infection Pub Date : 2024-07-14 DOI:10.1016/j.cmi.2024.07.002

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引用次数: 0

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.

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耐氟康唑的副丝状念珠菌：Y132F ERG11p置换的快速检测和拟议的微卫星基因分型方案。

目的：我们建议直接在纯培养的副猪嗜血杆菌分离株上对 Y132F ERG11p 取代进行快速准确的分子检测。我们还评估了一种用于追踪循环基因型的鉴别性基因分型方案：方法：我们从西班牙和意大利的 20 家医院共筛选出 223 株副丝状菌分离株（每株一个病人）。分离株为氟康唑耐药株（n=94；携带 Y132F ERG11p 替代[n=85]、G458S 替代[n=6]、R398I 替代[n=2]或野生型 ERG11 基因序列）或氟康唑敏感株（n=129）。在对氟康唑敏感和对氟康唑耐药的纯培养分离物上设计并优化了两种靶向-A395T 突变 PCR 格式（常规和实时），从而避免了 DNA 提取。比较了两种基因分型方案：方案 1（CP1、CP4a、CP6 和 B 标记）和方案 2（6A、6B、6C、CP1、CP4a 和 CP6 标记）：使用这两种 PCR 方法进行的筛选显示出 100%的特异性（氟康唑敏感分离物；n=129/129）和灵敏度（Y132F 分离物；n=85/85），然而，传统 PCR 方法和实时 PCR 方法分别在 3 小时和 1.5 小时内得到结果。总体而言，方案 1 比方案 2 显示出更高的遗传多样性，具体表现在检测到的等位基因数量（n=98；平均 23，范围 13-38）、观察到的和预期的杂合度明显更高，以及同一性概率指数（2.5x10-6）。方案 2 标记不能进一步区分 Y132F 氟康唑抗性基因型：结论：所提出的两种 PCR 模式都能加快准确检测副丝虫分离株中的 Y132F ERG11p 替换，特异性和灵敏度均为 100%。此外，我们还推荐使用 CP1、CP4a、CP6 和 B 微卫星标记对氟康唑耐药分离株进行基因分型。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Clinical Microbiology and Infection 医学-传染病学

CiteScore

25.30

自引率

2.10%

发文量

441

审稿时长

2-4 weeks

期刊介绍： Clinical Microbiology and Infection (CMI) is a monthly journal published by the European Society of Clinical Microbiology and Infectious Diseases. It focuses on peer-reviewed papers covering basic and applied research in microbiology, infectious diseases, virology, parasitology, immunology, and epidemiology as they relate to therapy and diagnostics.