Alfonso Eduardo C
Bascom Palmer Eye Institute, Miller School of Medicine, University of Miami, Miami, Florida, USA.
Trans Am Ophthalmol Soc. 2008;106:227-39.
Ocular infections caused by fungal organisms can cause significant ocular morbidity, particularly when diagnosis and treatment are delayed. Rapid and accurate identification of Fusarium species at the subgenus level using current diagnostic standards is timely and insensitive. The purpose of this study is to examine the usefulness of polymerase chain reaction (PCR) analysis of the internal transcribed spacer (ITS) regions (ITS1, 5.8S, and ITS2) in detecting and differentiating Fusarium species from isolates of ocular infections, and to assess the correlation between the genotypic and morphologic classification.
Fifty-eight isolates from 52 patients diagnosed with Fusarium ocular infections were retrieved from storage at the Bascom Palmer Eye Institute's ocular microbiology laboratory. Morphologic classification was determined at both a general and a reference microbiology laboratory. DNA was extracted and purified, and the ITS region was amplified and sequenced. Following DNA sequences, alignment and phylogenetic analysis were done. Susceptibility to antifungal drugs was measured according to the Clinical and Laboratory Standards Institute reference method.
Sequence analysis demonstrated 15 unique sequences among the 58 isolates. The grouping showed that the 58 isolates were distributed among 4 main species complexes. At the species level, morphologic classification correlated with genotypic classification in 25% and 97% of the isolates in a general microbiology and a reference mycology laboratory, respectively.
The sequence variation within the ITS provides a sufficient quantitative basis for the development of a molecular diagnostic approach to the Fusarium pathogens isolated from ocular infections. Morphology based on microscopic and macroscopic observations yields inconsistent results, particularly at nonreference laboratories, emphasizing the need for a more reproducible test with less user-dependent variability. Fusarium solani tends to be more resistant to certain antifungals (azoles).
由真菌病原体引起的眼部感染可导致严重的眼部疾病，尤其是在诊断和治疗延迟时。按照当前诊断标准在亚属水平快速准确鉴定镰刀菌属既不及时也不敏感。本研究的目的是检验聚合酶链反应（PCR）分析内部转录间隔区（ITS）（ITS1、5.8S和ITS2）在检测和区分眼部感染分离株中的镰刀菌属物种方面的实用性，并评估基因型和形态学分类之间的相关性。
从巴斯科姆帕尔默眼科研究所的眼部微生物实验室储存的样本中，检索出52例被诊断为镰刀菌性眼部感染患者的58株分离株。在普通微生物实验室和参考微生物实验室进行形态学分类。提取并纯化DNA，扩增并测序ITS区域。根据DNA序列进行比对和系统发育分析。按照临床和实验室标准协会的参考方法测定对抗真菌药物的敏感性。
序列分析显示58株分离株中有15个独特序列。分组表明58株分离株分布在4个主要物种复合体中。在物种水平上，在普通微生物实验室和参考真菌学实验室中，分别有25%和97%的分离株形态学分类与基因型分类相关。
ITS内的序列变异为开发针对眼部感染分离出的镰刀菌病原体的分子诊断方法提供了充分的定量依据。基于显微镜和肉眼观察的形态学结果不一致，尤其是在非参考实验室，这强调需要一种更具可重复性、用户依赖性变异较小的检测方法。茄病镰刀菌往往对某些抗真菌药（唑类）更具耐药性。
