Pathobiology and Medical Diagnosis Laboratory, Mehregan Hospital, Kerman, Iran.
Department of Medical Parasitology and Mycology, Kerman University of Medical Sciences, Kerman, Iran.
J Clin Lab Anal. 2020 Oct;34(10):e23444. doi: 10.1002/jcla.23444. Epub 2020 Jul 12.
Candida species are considered as the cause of one of the most important opportunistic fungal diseases. Accurate identification of Candida species is important because of antifungal susceptibility patterns are different among these species, so proper identification helps in the selection of antifungal drugs for the prevention and treatment. Phenotypic methods for identification of Candida species, which are widely used in clinical microbiology laboratories, have some limitations. Real-time PCR followed by the high-resolution melting analysis (HRMA) is a novel approach for the rapid recognition of pathogenic fungi. Molecular phylogeny is essential for obtaining a better understanding of the evolution of the genus Candida and the identification of the relative degree of the Candida species. The purpose of this study was molecular identification of Candida isolates by Real-time PCR-high-resolution melting analysis and investigation of the genetic diversity of Candida species.
Two hundred and thirty-two Candida isolates including 111 Candida isolates obtained from 96 HIV/AIDS patients and 121 Candida isolates obtained from 98 non-HIV persons were identified by real-time PCR and high-resolution melting curve analysis. To evaluate genetic diversity and relationships among Candida species, PCR products of nine clinical Candida isolates, as a representative of each kind of species, were randomly selected for DNA sequence analysis.
In HIV/AIDS patients, six species of Candida spp. were identified as follows: C albicans (n = 64; 57.7%), C glabrata (n = 31; 27.92%), C parapsilosis (n = 9; 8.1%), C tropicalis (n = 4; 3.6%), C krusei (n = 2; 1.8%), and C kefyr (n = 1; 0.90%). In non-HIV persons, we identified eight species of Candida including C albicans (n = 46; 38.33%) followed by C glabrata and C krusei (each one, n = 18; 15%), C tropicalis (n = 13; 10.83%), C lusitaniae (n = 12; 5.17%), C parapsilosis (n = 10; 4.31%), and C kefyr and C guillermondii (each one, n = 2; 1.66%). Also, the phylogenetic analysis showed the presence of two main clades and six separate subclades. Accordingly, about 88.9% of the isolates were located in clade I and 11.10% of the studied isolates were in clade II.
Real-time PCR followed by high-resolution melting analysis (HRMA) is known as a reliable, fast, and simple approach for detection and accurate identification of Candida species, especially in clinical samples.
假丝酵母菌属被认为是最重要的机会性真菌感染病的原因之一。由于不同种属的抗真菌药敏模式不同，因此准确鉴定假丝酵母菌属非常重要，这有助于选择预防和治疗用的抗真菌药物。目前，临床微生物学实验室广泛使用的表型方法在鉴定假丝酵母菌属方面存在一些局限性。实时 PCR 结合高分辨率熔解曲线分析（HRMA）是一种快速识别致病真菌的新方法。分子系统发育对于更好地了解假丝酵母菌属的进化以及鉴定假丝酵母菌属的亲缘关系程度至关重要。本研究的目的是通过实时 PCR-HRMA 对假丝酵母菌属进行分子鉴定，并研究假丝酵母菌属的遗传多样性。
我们对 232 株假丝酵母菌属进行鉴定，其中 111 株来自 96 例 HIV/AIDS 患者，121 株来自 98 例非 HIV 患者。通过实时 PCR 和高分辨率熔解曲线分析对其进行鉴定。为了评估假丝酵母菌属种间的遗传多样性和关系，我们随机选择了 9 株临床分离的假丝酵母菌属的 PCR 产物进行 DNA 序列分析。
在 HIV/AIDS 患者中，鉴定出 6 种假丝酵母菌属，分别为：白假丝酵母菌（n=64；57.7%）、光滑假丝酵母菌（n=31；27.92%）、近平滑假丝酵母菌（n=9；8.1%）、热带假丝酵母菌（n=4；3.6%）、克柔假丝酵母菌（n=2；1.8%）和季也蒙假丝酵母菌（n=1；0.90%）。在非 HIV 患者中，我们鉴定出 8 种假丝酵母菌属，包括白假丝酵母菌（n=46；38.33%），其次是光滑假丝酵母菌和克柔假丝酵母菌（各 18 株；15%）、热带假丝酵母菌（n=13；10.83%）、卢特氏假丝酵母菌（n=12；5.17%）、近平滑假丝酵母菌（n=10；4.31%）、季也蒙假丝酵母菌和凯氏假丝酵母菌（各 2 株；1.66%）。此外，系统发育分析显示存在两个主要分支和六个独立的分支。因此，约 88.9%的分离株位于分支 I，11.10%的研究分离株位于分支 II。
实时 PCR 结合高分辨率熔解曲线分析（HRMA）是一种可靠、快速、简单的检测和准确鉴定假丝酵母菌属的方法，特别是在临床样本中。
