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 3)-β-d-葡聚糖敏感的凝血因子 g 和马蹄 creb 的前凝血酶通过将鲎阿米巴细胞裂解物应用到葡聚糖硫酸琼脂糖 CL-6B 柱上进行分级，然后与显色底物 (BOC- Leu Gly-Arg-pNA) 制备用于 (1 3)-β-D-葡聚糖测定的试剂。该试剂对商业 (1 3)-β-D-葡聚糖表现出线性剂量依赖性响应，并且足够灵敏，可以检测 pg/ml 物质，变异系数小于 4%。该测定实际上与多种病原真菌多糖发生反应，包括白色念珠菌、犬小孢子菌、红色毛癣菌和烟曲霉。通过事先用高氯酸处理血浆，几乎回收了血液中掺入的所有葡聚糖。然后，我们测量了 183 名因可能败血症而接受血培养的免疫功能低下患者的血浆葡聚糖水平。所有这些患者均患有白血病和恶性淋巴瘤等血液系统恶性肿瘤作为基础疾病。其中，13 例的葡聚糖浓度高于正常值：4 例真菌培养呈阳性（3 例念珠菌病和 1 例隐球菌病），4 例经尸检证实深部真菌病（1例念珠菌病、1例曲霉菌病、2例不明），另外5例临床诊断为深部真菌病真菌病，因为对抗真菌剂有明确的有利反应。不存在经培养证实的深部真菌病，且血浆 (1 3)-β-d-葡聚糖浓度不高。这些结果表明，本方法是诊断机会性系统性真菌病的高度敏感的筛查。此外，随着患者使用抗真菌药物治疗临床情况有所改善，升高的阿鲁聚糖水平也随之降低。这表明通过该方法检测到的葡聚糖来自真菌，并且该测试对于检查药物的效果以及跟踪临床过程很有用。