Laboratório de Soroepidemiologia e Imunobiologia, Instituto de Medicina Tropical da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.
Laboratório de Soroepidemiologia e Imunobiologia, Instituto de Medicina Tropical da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.
Parasitol Int. 2021 Feb;80:102210. doi: 10.1016/j.parint.2020.102210. Epub 2020 Oct 25.
Quantification of parasites in the context of Chagas disease is required to monitor the treatment with benznidazole, disease-associated cardiomyopathies and graft rejection after heart transplantation. As parasitological exams lack sensitivity, Real Time Polymerase Chain Reaction (rt-PCR) has emerged to evaluate the parasite load in blood samples and cardiac biopsies. However, despite its higher sensitivity, rt-PCR does not provide information on the location and distribution of amastigote nests within infected tissues, the characterization of inflammatory infiltrates or changes to tissue architecture. On the contrary, a sensitive immunohistochemistry technique (IHC) could fill these gaps. In the present study, a quantitative IHC exam was standardized and validated by testing adipose and cardiac tissues of experimentally infected mice containing variable parasite load levels of T. cruzi assessed by a sensitive Sybr Green rt-PCR with kDNA primers. Tissues were divided into four groups according to the parasite load: group A- 100 parasites/50 ng of DNA; group B -10 parasites; group C - around 1 parasite and group D - less than 1 parasite/50 ng/DNA. IHC was able to detect T. cruzi in the four groups, even in group D tissues containing fractions of a single parasite/50 ng of DNA sample according to rt-PCR. In conclusion, a highly sensitivity and reliable quantitative immunohistochemistry technique was developed and is proposed to estimate the percentage of T. cruzi-infected tissue area in chagasic patients presenting with cardiomyopathies, as a complementary test to rt-PCR.
在恰加斯病的背景下，需要对寄生虫进行定量检测，以监测贝那唑嗪治疗、与疾病相关的心肌病和心脏移植后的移植物排斥反应。由于寄生虫学检查缺乏敏感性，实时聚合酶链反应（rt-PCR）已被用于评估血液样本和心脏活检中的寄生虫负荷。然而，尽管 rt-PCR 具有更高的敏感性，但它不能提供关于感染组织内的阿米巴内寄生虫巢的位置和分布、炎症浸润的特征或组织结构变化的信息。相反，一种敏感的免疫组织化学技术（IHC）可以填补这些空白。在本研究中，通过测试含有不同水平的 T. cruzi 寄生虫负荷的实验感染小鼠的脂肪组织和心脏组织，对一种用灵敏的 Sybr Green rt-PCR 检测 kDNA 引物的定量 IHC 检测进行了标准化和验证。组织根据寄生虫负荷分为四组：A 组-100 个寄生虫/50ng 的 DNA；B 组-10 个寄生虫；C 组-约 1 个寄生虫；D 组-少于 1 个寄生虫/50ng/DNA。IHC 能够在四组中检测到 T. cruzi，甚至在 D 组组织中，根据 rt-PCR，含有单个寄生虫/50ng 的 DNA 样本的分数。总之，开发了一种高灵敏度和可靠的定量免疫组织化学技术，并建议将其用于估计患有心肌病的恰加斯病患者中感染组织面积的百分比，作为 rt-PCR 的补充检测。
