Natarajaseenivasan K, Vijayachari P, Sugunan A P, Sharma S, Sehgal S C
National Leptospirosis Reference Centre, Regional Medical Research Centre (ICMR)Port Blair, India.
Indian J Med Res. 2004 Sep;120(3):151-9.
BACKGROUND & OBJECTIVES: The available serological techniques for the diagnosis of leptospirosis have less sensitivity during the early stage of the disease. Understanding of leptospiral proteins expressed during acute and convalescent phases of leptospirosis, would be help the develop of new serodiagnostic strategies. Therefore, the present study was carried out to identify (i) an antigen that is conserved among the various pathogenic leptospira; (ii) best protein antigen to which immune response can be identified in the acute phase; and (iii) best protein antigen which is present in convalescent sera which can be used for seroepidemiological studies.
Quantitative immunoblot analysis was performed using acute and convalescent phase human sera along with sera from normal healthy individuals and from patients with typhoid, malaria and hepatitis as the controls. All the samples were analyzed for the leptospiral protein recognition by using IgM and IgG immunoblots. Leptospiral cell fractionation was performed using triton X-114 and lysozyme and further the conservation of leptospiral proteins was also performed.
In confirmed cases of leptospirosis, the IgG recognition in acute phase sera was 30.2, 39.5, 27.9, 55.8 and 27.9 per cent for the leptospiral proteins p32, p41/42, p58, p62 and p82 respectively. The IgG has considerably increased to 65.1, 55.8, 46.5, 67.4 and 48.8 per cent against the same proteins during convalescent phase. The IgM recognition was 32.6 , 32.6, 30.2 and 37.2 per cent for acute phase sera and 32.6, 37.2, 44.2 and 41.9 per cent for convalescent phase sera for the leptospiral proteins p14, p25, p32 and p41/42, respectively. Leptospiral proteins like p62 and p82 were recognized among all the control groups with 3.3-15.3 per cent for IgG recognition.
INTERPRETATION & CONCLUSION: Leptospiral protein p32 was found to be highly sensitive and specific and could be useful for the development of newer techniques for diagnosis and seroepidemiological studies. Combination of p32 and p41/42 for IgG and p14, p25, p32, p41/42 for IgM would increase the sensitivity of these techniques further.
现有的用于诊断钩端螺旋体病的血清学技术在疾病早期的敏感性较低。了解钩端螺旋体病急性期和恢复期表达的钩端螺旋体蛋白，将有助于开发新的血清学诊断策略。因此，开展本研究以确定：（i）在各种致病性钩端螺旋体中保守的一种抗原；（ii）在急性期可识别免疫反应的最佳蛋白抗原；（iii）存在于恢复期血清中可用于血清流行病学研究的最佳蛋白抗原。
使用急性期和恢复期人血清以及来自正常健康个体、伤寒患者、疟疾患者和肝炎患者的血清作为对照进行定量免疫印迹分析。使用IgM和IgG免疫印迹分析所有样本对钩端螺旋体蛋白的识别情况。使用曲拉通X - 114和溶菌酶进行钩端螺旋体细胞分级分离，并进一步研究钩端螺旋体蛋白的保守性。
在确诊的钩端螺旋体病病例中，急性期血清对钩端螺旋体蛋白p32、p41/42、p58、p62和p82的IgG识别率分别为30.2%、39.5%、27.9%、55.8%和27.9%。在恢复期，针对相同蛋白的IgG识别率大幅提高至65.1%、55.8%、46.5%、67.4%和48.8%。钩端螺旋体蛋白p14、p25、p32和p41/42在急性期血清中的IgM识别率分别为32.6%、32.6%、30.2%和37.2%，在恢复期血清中的识别率分别为32.6%、37.2%、44.2%和41.9%。在所有对照组中均识别出钩端螺旋体蛋白如p62和p82，IgG识别率为3.3% - 15.3%。
发现钩端螺旋体蛋白p32具有高度敏感性和特异性，可用于开发新的诊断技术和血清流行病学研究。IgG使用p32和p41/42组合，IgM使用p14、p25、p32、p41/42组合将进一步提高这些技术的敏感性。
