Abstract：
OBJECTIVE: 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.
CONCLUSIONS: 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.
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.
CONCLUSIONS: 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.
摘要：
目的：我们提出了直接在纯培养的近平滑梭菌分离株上快速准确地分子检测Y132FERG11p取代的方法。我们还评估了一种鉴别基因分型方案来跟踪循环基因型。
方法：共有来自20家医院的223株近平滑梭菌（各一名患者），位于西班牙和意大利被选中。分离株对氟康唑耐药（n=94；具有Y132FERG11p取代[n=85]，G458S替代[n=6]，R398I替换[n=2]，或野生型ERG11基因序列）或易感氟康唑（n=129）。在对氟康唑敏感和对氟康唑耐药的纯培养分离株上对两种靶向A395T突变PCR格式（常规和实时）进行了工程和优化，从而跳过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)。
结果：使用两种PCR形式进行的筛选均显示出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）值，然而,使用常规和实时PCR格式，结果可在3和1.5小时内获得，分别。总的来说,方案1显示出比方案2更高的遗传多样性，如检测到的等位基因数所示（n=98；平均值23，范围13-38），观察到的和预期的杂合性显著较高，和概率同一性指数(2.5x10-6)。方案2标记没有提供Y132F氟康唑抗性基因型的进一步基因型区分。
结论：两种提出的PCR格式都可以加速对梭菌分离株中取代Y132FERG11p的准确检测，具有100％的特异性和敏感性。此外,我们推荐CP1，CP4a，CP6和B微卫星标记对氟康唑耐药菌株进行基因分型。
方法：共有来自20家医院的223株近平滑梭菌（各一名患者），位于西班牙和意大利被选中。分离株对氟康唑耐药（n=94；具有Y132FERG11p取代[n=85]，G458S替代[n=6]，R398I替换[n=2]，或野生型ERG11基因序列）或易感氟康唑（n=129）。在对氟康唑敏感和对氟康唑耐药的纯培养分离株上对两种靶向A395T突变PCR格式（常规和实时）进行了工程和优化，从而跳过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)。
结果：使用两种PCR形式进行的筛选均显示出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）值，然而,使用常规和实时PCR格式，结果可在3和1.5小时内获得，分别。总的来说,方案1显示出比方案2更高的遗传多样性，如检测到的等位基因数所示（n=98；平均值23，范围13-38），观察到的和预期的杂合性显著较高，和概率同一性指数(2.5x10-6)。方案2标记没有提供Y132F氟康唑抗性基因型的进一步基因型区分。
结论：两种提出的PCR格式都可以加速对梭菌分离株中取代Y132FERG11p的准确检测，具有100％的特异性和敏感性。此外,我们推荐CP1，CP4a，CP6和B微卫星标记对氟康唑耐药菌株进行基因分型。
