Detection and Identification of Microsporidia in Stool

人们对确定粪便标本对检测由微孢子虫引起的胃肠道疾病的诊断价值很感兴趣，并评估了各种染色方式在粪便标本中显示微孢子虫的能力。不幸的是，感染人类的各种微孢子虫只有通过在组织标本的电子显微镜上可视化生物特有的亚细胞形态特征才能准确地识别。由于目前正在开发的治疗方案的疗效可能在某种程度上是物种特有的，而且由于有必要进行侵入性程序来获得适当的标本以进行电子显微镜(EM)，因此需要一种比EM更广泛的鉴定方法。我们发表了一份报告，描述了我们的聚合酶链式反应(PCR)扩增微孢子虫rRNA基因，然后限制性内切酶消化的产物，作为一种方法鉴定新鲜粪便中的微孢子虫。我们正在继续修改检测方法，以便在福尔马林固定的粪便标本中检测到微孢子虫。使用聚合酶链式反应扩增的DNA和物种特异性DNA探针的Southern杂交格式已经被开发用于微孢子虫的物种形成。该项目的最后阶段是确定我们化验的灵敏度，以及标本在福尔马林中保存的时间长度，并仍然可以检测到微孢子虫。我们已经证明，微孢子虫在福尔马林或聚乙烯醇中长时间储存会降低通过聚合酶链式反应扩增的机会。我们建议在福尔马林固定后清洗粪便标本，并将清洗后的标本保存在磷酸盐缓冲盐水中。我们开发了一种从粪便中提取微孢子虫DNA的更短、更便宜、更简单的方法。

There is considerable interest in determining the diagnostic value of stool specimens for detecting gastrointestinal disease caused by Microsporidia organisms, and a variety of staining modalities have been evaluated for their ability to visualize microsporidia in stool preparations. Unfortunately, the various species of microsporidia that infect humans can be identified accurately only by visualizing characteristic subcellular morphologic features of organisms in electron micrographs of tissue specimens. Because the efficacy of treatment regimens currently in development may be somewhat species-specific, and because invasive procedures are necessary to obtain appropriate specimens for electron microscopy (EM), a more widely available approach to identification than EM is required. We have published a report which describes our polymerase chain reaction (PCR) assay for amplification of microsporidial rRNA genes followed by restriction endonuclease digestion of PCR products as a method for identifying microsporidia in fresh stool. We are continuing to modify the assay to allow detection of microsporidia in formalin-fixed stool specimens. A Southern blot format using PCR-amplified DNA and species-specific DNA probes has been developed for use in microsporidial speciation. The final stage of the project has been to determine the sensitivity of our assay and the length of time the specimens can be stored in formalin and still allow detection of the microsporidia. We have demonstrated that prolonged storage of microsporidia in formalin or polyvinyl alcohol reduces the chances of amplification by PCR. We suggest washing stool specimens after fixation in formalin and storage of the washed specimen in phosphate-buffered saline. We have developed a shorter, cheaper, and simpler method for extraction of microsporidial DNA from stool.