Seminested PCR for diagnosis of candidemia: Comparison with culture, antigen detection, and biochemical methods for species identification
Seminested PCR for diagnosis of candidemia: Comparison with culture, antigen detection, and biochemical methods for species identification
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DOI：
10.1128/jcm.40.7.2483-2489.2002
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发表时间：
2002-07-01
影响因子：
9.4
通讯作者：
Khan, ZU
中科院分区：
文献类型：
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作者：
Ahmad, S;Khan, Z;Khan, ZU
在临床实验室中快速检测和鉴定念珠菌种类对于血源性念珠菌病患者的治疗极其重要。目前用于检测和鉴定念珠菌的培养和生化方法非常耗时，并且缺乏所需的灵敏度和特异性。在本研究中，我们使用通用和物种特异性引物建立了半巢式 PCR (snPCR)，用于检测血清样本中的念珠菌属。通用外部引物扩增 5.8S 核糖体 DNA (rDNA) 的 3' 末端和 28S rDNA 的 5' 末端，包括内部转录间隔区 2 (ITS2)，从四种常见念珠菌物种（即白色念珠菌、热带念珠菌、光滑念珠菌和近平滑念珠菌）生成 350 至 410 bp 片段。物种特异性引物与每个测试物种的 ITS2 内的独特序列互补，在 snPCR 的再扩增步骤中扩增物种特异性 DNA。在加标血清样本中通过 snPCR 检测念珠菌的灵敏度接近 1 个有机体/ml。对 76 种临床念珠菌分离株进行 snPCR 特异性鉴定念珠菌属的评估显示，与 Vitek 和/或 ID32C 酵母鉴定系统的结果一致率为 99%。对 snPCR 检测培养证实 (n = 12)、疑似 (n = 16) 和表面定植 (n = 10) 患者和健康受试者 (n = 12) 血清中念珠菌属的进一步评估表明，snPCR 结果与健康个体和定植患者血清一致呈阴性。在经培养证实的念珠菌血症患者中，snPCR 结果与血培养结果在阳性和物种鉴定方面完全一致。此外，snPCR 在 5 名患者中检测到由两种念珠菌引起的念珠菌血症，而通过血培养检测到 3 例。在念珠菌血培养阴性的疑似念珠菌血症类别中，9 名患者 (56%) 经 snPCR 检测呈阳性；其中两人患有白色念珠菌和热带念珠菌或光滑念珠菌的双重感染。总之，本研究中开发的 snPCR 对于检测血清样本中的念珠菌属具有特异性，并且比培养法更灵敏。此外，改进对由一种以上念珠菌属物种引起的念珠菌血症病例的检测是另一个优势。
The rapid detection and identification of Candida species in clinical laboratories are extremely important for the management of patients with hematogenous candidiasis. The presently available culture and biochemical methods for detection and species identification of Candida are time-consuming and lack the required sensitivity and specificity. In this study, we have established a seminested PCR (snPCR) using universal and species-specific primers for detection of Candida species in serum specimens. The universal outer primers amplified the 3' end of 5.8S ribosomal DNA (rDNA) and the 5' end of 28S rDNA, including the internally transcribed spacer 2 (ITS2), generating 350- to 410-bp fragments from the four commonly encountered Candida species, viz., C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis. The species-specific primers, complementary to unique sequences within the ITS2 of each test species, amplified species-specific DNA in the reamplification step of the snPCR. The sensitivity of Candida detection by snPCR in spiked serum specimens was close to 1 organism/ml. Evaluation of snPCR for specific identification of Candida species with 76 clinical Candida isolates showed 99% concordant results with the Vitek and/or ID32C yeast identification system. Further evaluation of snPCR for detection of Candida species in sera from culture-proven (n = 12), suspected (n = 16), and superficially colonized (n = 10) patients and healthy subjects (n = 12) showed that snPCR results were consistently negative with sera from healthy individuals and colonized patients. In culture-proven candidemia patients, the snPCR results were in full agreement with blood culture results with respect to both positivity and species identity. In addition, snPCR detected candidemia due to two Candida species in five patients, compared to three by blood culture. In the category of suspected candidemia with negative blood cultures for Candida, nine patients (56%) were positive by snPCR; two of them had dual infection with C. albicans and either C. tropicalis or C. glabrata. In conclusion, the snPCR developed in this study is specific and more sensitive than culture for the detection of Candida species in serum specimens. Moreover, the improved detection of cases of candidemia caused by more than one Candida species is an additional advantage.