Uezato H, Hagiwara K, Hosokawa A, Maruno M, Nonaka S, Oshiro M, Nakashima Y, Furuya M, Hashiguchi Y
Department of Dermatology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan.
J Dermatol. 1998 Oct;25(10):623-31. doi: 10.1111/j.1346-8138.1998.tb02470.x.
In this study, detection rates of Leishmania parasites from human skin were compared among three different types of specimens, formalin-fixed, ethanol-fixed, and frozen, by polymerase chain reaction (PCR) and Southern blotting. For this purpose, we used biopsy specimens collected from 19 leishmaniasis patients and performed PCR and Southern hybridization with the probe specific for Leishmania (Viannia) braziliensis complex. Among these 19, 16 specimens were from cutaneous leishmaniasis (CL), one, diffuse cutaneous leishmaniasis (DCL) and 2, mucocutaneous leishmaniasis (MCL) and were formalin-fixed and paraffin-embedded. The causative agents for one case of CL and one case of DCL were already identified as L. (Leishmania) complex. Six specimens of CL were preserved in 100% ethanol. Two specimens of MCL were frozen tissues. PCR using the formalin-fixed and paraffin-embedded specimens revealed positive bands at 70 bp in 9 (47.4%) out of 19 specimens of CL, MCL and DCL. Southern blotting detected the signals in 12 (63.2%) out of the 19. PCR using the 100% ethanol-fixed specimens revealed positive bands in 4 (66.7%) out of 6, and Southern blotting also detected the signals in 4 (66.7%) out of the 6. PCR and Southern blotting using 2 frozen specimens of MCL were always positive (100%). Although we failed to detect significant differences by Chi-square test between the results from the formalin-fixed, paraffin-embedded specimens and those from 100% ethanol-fixed ones, we concluded that ethanol-fixed specimens, convenient for transportation and storage, would be more useful for diagnosis of leishmaniasis by PCR in a developing country.
在本研究中，通过聚合酶链反应（PCR）和Southern印迹法，比较了三种不同类型标本（福尔马林固定、乙醇固定和冷冻）中人体皮肤利什曼原虫寄生虫的检出率。为此，我们使用了从19例利什曼病患者采集的活检标本，并使用针对巴西利什曼原虫（Viannia）复合体的特异性探针进行PCR和Southern杂交。在这19例中，16例标本来自皮肤利什曼病（CL），1例来自弥漫性皮肤利什曼病（DCL），2例来自黏膜皮肤利什曼病（MCL），均为福尔马林固定并石蜡包埋。1例CL和1例DCL的病原体已被鉴定为利什曼原虫（Leishmania）复合体。6例CL标本保存在100%乙醇中。2例MCL标本为冷冻组织。使用福尔马林固定并石蜡包埋的标本进行PCR检测时，19例CL、MCL和DCL标本中有9例（47.4%）在70 bp处显示阳性条带。Southern印迹法在19例中有12例（63.2%）检测到信号。使用100%乙醇固定的标本进行PCR检测时，6例中有4例（66.7%）显示阳性条带，Southern印迹法在6例中也有4例（66.7%）检测到信号。使用2例MCL冷冻标本进行PCR和Southern印迹法检测始终呈阳性（100%）。尽管我们通过卡方检验未能检测到福尔马林固定、石蜡包埋标本与100%乙醇固定标本结果之间的显著差异，但我们得出结论，对于发展中国家通过PCR诊断利什曼病而言，乙醇固定的标本便于运输和储存，将更有用。
