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Establishment of a New Laboratory Test for Fungemia Using Horseshoe-crab Coagulation Factor G

鲎凝固因子 G 真菌血症实验室检测新方法的建立

基本信息

批准号：
61571116
负责人：
OBAYASHI Taminori
金额：
$ 1.41万
依托单位：
Jichi Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1988
项目状态：
已结题

项目摘要

(1 3)-beta-d-glucan-sensitive coagulation factor g and proclotting enzyme of the horseshoe creb were fractionated by applying Tachypleus amebocyte lysate to a dextran-sulfate Sepharose CL-6B colum, and then combined with a chromogenic substrate (BOC-Leu Gly-Arg-pNA) to prepare a reagent for (1 3)-beta-D-glucan assay. This reagent showed a linear dose-dependant response to commercial (1 3)-beta-D-glucan and was sensitive enouhgh to betect a pg/ml matter of the substance with a coefficient of variation less than 4%. The assay actually reacted with various polysaccharides of pathogenic fungi including Candida albicans, Microsporum canis,Trichophyton rubrum, and Aspergillus fumigatus. Almost all glucan spiked in the blood was recovered by treating plasma with perchloric acid before hand. Then we measured a plasma glucan level in 183 immunocompromised patients wto underwent blood culture for possible sepsis. All of these patients had hematological malignancies like leukemia and malignant lymphomas as an underlying disease.Of these, 13 had a higher concentration of glucan than normal: 4 were culture-positive for fungi (3 candidiasis and 1 cryptococosis), 4 were autopsy-verified deep mycoses (1 candidiasis, 1 aspergillosis, and 2 unidentifitied), and the other 5 were clinically diagnosed as deep mycoses because of a definite favourable response to antifungal agents. There were no culture-proven deep mycoses without high plasma (1 3)-beta-d-glucan concentration. These results showed that the present method is a highly sensitive screening for the diagnosis of opportunistic systemic mycosis. In addition, the elevated alucan levels decreased as the patients improved clinically with institution of antifungal medication. This suggests that the glucan detected by this method is derived from fungi, and that the test is useful for checking the effect of the drug as well as for following clinical course.

(1)用葡聚糖琼脂糖凝胶CL-6B柱分离马蹄形CREB的凝血因子g和凝血酶，然后与显色底物(BOC-Leu Gly-Arg-PNA)结合，制备(1，3)-β-D-葡聚糖检测试剂。该试剂对市售的(1，3)-β-D-葡聚糖呈线性剂量依赖性反应，对pg/ml的物质具有较高的灵敏度，变异系数小于4%。该检测实际上与包括白色念珠菌、犬小孢子菌、红色毛霉菌和烟曲霉在内的各种病原真菌的多糖发生反应。几乎所有在血液中添加的葡聚糖都是通过事先用高氯酸处理血浆来恢复的。然后，我们检测了183名免疫功能低下的患者的血浆葡聚糖水平，WTO接受了血培养，以确定可能发生的脓毒症。所有这些患者都以白血病和恶性淋巴瘤等血液系统恶性肿瘤为基础疾病。其中13例葡聚糖浓度高于正常：4例真菌培养阳性(3例念珠菌病和1例隐球菌病)，4例尸检证实的深部真菌病(1例念珠菌病，1例曲霉病，2例不明原因)，其余5例临床诊断为深部真菌病，因为对抗真菌药物有明确的反应。在没有高血浆(1，3)-β-d-葡聚糖浓度的情况下，没有培养证实的深部真菌病。这些结果表明，本方法是诊断机会性全身真菌病的一种高度敏感的筛查方法。此外，随着患者临床症状的改善和抗真菌药物的使用，升高的白蛋白水平降低。这表明，用这种方法检测到的葡聚糖来自真菌，这一测试对于检查药物的效果以及后续的临床病程是有用的。