Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris, France.
Ophthalmology. 2012 May;119(5):945-50. doi: 10.1016/j.ophtha.2011.10.038. Epub 2012 Feb 18.
The first-line therapy for patients with keratitis is different for bacteria, filamentous fungi, and yeasts. The timely onset of treatments depends on rapid and accurate diagnosis. However, fungal cultures produce high rates of false-negative results. Nucleic acid amplification techniques (polymerase chain reaction [PCR]) improve fungal diagnosis performance, but they require complex postamplification procedures to differentiate filamentous fungi from yeasts or to identify the agent. The objective of this work was to develop a new diagnostic strategy based on real-time PCR high-resolution melting (HRM) analysis that in 1 run (a) detects and semiquantifies yeasts and filamentous fungi, (b) differentiates yeasts from filamentous fungi, and (c) discriminates among relevant species of yeasts.
Experimental study to compare HRM diagnosis performances with microscopic examination of corneal scrapings and fungal culture.
High-resolution melting detection limits and specificity were assessed with (a) isolated strains; (b) agents (other than fungi) producing keratitis; (c) corneal scrapings from fungal keratitis (culture positive and negative); and (d) corneal scrapings from bacterial, viral, or Acanthamoeba keratitis.
The DNA extracted from cornea specimens was mixed with primers diluted in the MeltDoctor HRM Master Mix (Applied Biosystems, Paris, France) in 2 tubes, the first for yeasts, containing the forward primer CandUn (5'CATGCCTGTTTGAGCGTC) and the reverse primer FungUn2 (5'TCCTCCGCTTATTGATATGCT), and the second for filamentous fungi, containing the forward primer FilamUn1 (5'TGCCTGTCCGAGCGTCAT) and FungUn2. Molecular probes were not necessary. The yields of DNA extraction and the PCR inhibitors were monitored by adding internal controls to each sample.
Detection of fungi in corneal samples by HRM.
High-resolution melting consistently detects the equivalent of 0.1 colony-forming units /ml of yeasts and filamentous fungi, differentiates filamentous fungi from yeasts, and discriminates among relevant species of yeasts. High-resolution melting sensitivity and specificity were 100% for culture-positive samples, detecting and characterizing fungi in 7 of 10 culture-negative suspected fungal keratitis.
High-resolution melting is a new, sensitive, specific, and inexpensive test that detects fungi and differentiates filamentous fungi from yeasts directly from clinical specimens in less than 2.30 hours after DNA extraction.
对于细菌、丝状真菌和酵母菌引起的角膜炎，一线治疗方法有所不同。及时开始治疗取决于快速、准确的诊断。然而，真菌培养的假阴性率很高。核酸扩增技术（聚合酶链反应 [PCR]）提高了真菌诊断性能，但需要复杂的扩增后程序来区分丝状真菌和酵母菌，或鉴定病原体。本研究旨在开发一种新的诊断策略，基于实时 PCR 高分辨率熔解（HRM）分析，在一次运行中：（a）检测并半定量酵母菌和丝状真菌，（b）区分酵母菌和丝状真菌，以及（c）区分相关酵母菌种。
比较 HRM 诊断性能与角膜刮片显微镜检查和真菌培养的实验研究。
高分辨率熔解检测限和特异性用（a）分离株；（b）产生角膜炎的其他病原体（真菌除外）；（c）真菌性角膜炎的角膜刮片（培养阳性和阴性）；和（d）细菌性、病毒性或棘阿米巴性角膜炎的角膜刮片进行评估。
从角膜标本中提取的 DNA 与引物混合，稀释于 MeltDoctor HRM Master Mix（Applied Biosystems，巴黎，法国）中，在 2 个管中，第一个管用于酵母菌，包含正向引物 CandUn（5'CATGCCTGTTTGAGCGTC）和反向引物 FungUn2（5'TCCTCCGCTTATTGATATGCT），第二个管用于丝状真菌，包含正向引物 FilamUn1（5'GCGTCCGAGCGTCATGCCTGTCC）和 FungUn2。不需要分子探针。通过向每个样本中添加内对照来监测 DNA 提取的产量和 PCR 抑制剂。
HRM 检测角膜样本中的真菌。
高分辨率熔解一致地检测到相当于 0.1 菌落形成单位/ml 的酵母菌和丝状真菌，将丝状真菌与酵母菌区分开来，并区分相关酵母菌种。对培养阳性样本的高分辨率熔解灵敏度和特异性均为 100%，在 10 例疑似真菌性角膜炎培养阴性的样本中，有 7 例检测和鉴定了真菌。
高分辨率熔解是一种新的、敏感、特异和廉价的检测方法，可直接从临床标本中检测真菌，并在 DNA 提取后不到 2.30 小时内区分丝状真菌和酵母菌。
