美国皮肤利什曼病：免疫组织化学检测皮肤中利什曼原虫的有效性。
American tegumentary leishmaniasis: effectiveness of an immunohistochemical protocol for the detection of Leishmania in skin.
机构信息
Departamento de Patologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil.
出版信息
PLoS One. 2013 May 21;8(5):e63343. doi: 10.1371/journal.pone.0063343. Print 2013.
BACKGROUND
American tegumentary leishmaniasis (ATL) is endemic in Latin America, where Brazil has over 27 thousand cases per year. The aim of the present study was to develop an immunohistochemical method (IHC) for ATL diagnosis. For this purpose, we used serum from a dog naturally infected with Leishmania (L) infantum (canine hyperimmune serum) as the primary antibody, followed by a detection system with a secondary biotinylated antibody.
METHODOLOGY
Skin samples were obtained from 73 patients in an endemic area of Caratinga, Minas Gerais (MG) State, Brazil all testing positive for ATL with the Montenegro skin test, microscopy, and PCR. Canine hyperimmune serum of a dog naturally infected with Leishmania (L.) infantum was employed as a primary antibody in an immunohistochemical diagnostic method using streptavidin-biotin peroxidase. To assess the specificity of this reaction, IHC assays employing two monoclonal antibodies were carried out. As the polymer-based technology is less time-consuming and labor intensive than the IHC labeled streptavidin-biotin peroxidase method, we compared the two methods for all samples.
RESULTS
The IHC method detected ATL in 67 of the 73 cases (91.8%). Immunolabeled parasites were primarily detected inside macrophages either in the superficial or the deep dermis. Detection was facilitated by the high contrast staining of amastigotes (dark brown) against the light blue background. A lower detection rate (71.2%) was observed with the both of the monoclonal Leishmania antibodies compared to the canine hyperimmune serum. This may have been due to a non-specific background staining observed in all histological samples rendering positive detection more difficult. The higher efficacy of the canine hyperimmune serum in the IHC method was confirmed by the method using streptavidin-biotin peroxidase as well as that with the polymer-based technology (biotin-avidin-free system).
CONCLUSIONS
The data are encouraging with regard to validating IHC as a standard alternative method for ATL diagnosis.
背景
美洲皮肤利什曼病（ATL）在拉丁美洲流行，巴西每年有超过 27000 例病例。本研究旨在建立一种用于 ATL 诊断的免疫组织化学方法（IHC）。为此，我们使用了一条自然感染利什曼原虫（犬超免疫血清）的狗的血清作为一抗，然后使用带有二抗的生物素化抗体检测系统。
方法
从巴西米纳斯吉拉斯州卡塔林加流行地区的 73 名 ATL 患者中获得皮肤样本，所有患者均通过曼氏皮肤试验、显微镜检查和 PCR 检测呈 ATL 阳性。使用一条自然感染利什曼原虫（L.）的犬的超免疫血清作为一抗，在免疫组织化学诊断方法中使用链霉亲和素-生物素过氧化物酶。为了评估该反应的特异性，我们进行了使用两种单克隆抗体的 IHC 检测。由于聚合物技术比 IHC 标记的链霉亲和素-生物素过氧化物酶方法耗时更少且劳动强度更低，因此我们对两种方法进行了所有样本的比较。
结果
IHC 方法检测到 73 例中的 67 例（91.8%）为 ATL。在真皮的浅层或深层，免疫标记的寄生虫主要在巨噬细胞内被检测到。由于在浅蓝色背景下，无鞭毛体（深棕色）的高对比度染色，检测变得更加容易。与犬超免疫血清相比，两种单克隆利什曼抗体的检测率（71.2%）较低。这可能是由于在所有组织学样本中观察到非特异性背景染色，使阳性检测更加困难。用链霉亲和素-生物素过氧化物酶以及基于聚合物的技术（无生物素-亲和素系统）进行的方法证实了犬超免疫血清在 IHC 方法中的更高功效。
结论
IHC 作为 ATL 诊断的标准替代方法具有较高的有效性，这一结果令人鼓舞。
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