Anti-serum validation for use in immunohistochemistry for Trypanosoma cruzi detection

Abstract INTRODUCTION: The detection of Trypanosoma cruzi in tissue samples is important in many situations, such as testing of the reactivation of the infection. The detection of T. cruzi nests in endomyocardial biopsies (EMB) may be useful to evaluate graft rejection. Given their scarcity, such nests are not routinely identified. To increase the diagnosis sensitivity, immunohistochemistry (IHC) may serve as a promising strategy. Here, we validate an antiserum for the detection of T. cruzi infection by IHC. METHODS: We used 1) positive controls (PCs) - 13 EMB, 12 skin biopsies, and 1 heart with T. cruzi nests as sections stained with hematoxylin and eosin (HE); 2) negative controls - a) 10 explant hearts and 10 EMB with no amastigote nests or clinical/laboratory signs of chagasic infection; and b) eight samples with leishmaniasis, toxoplasmosis, or histoplasmosis; and 3) Cases - 31 EMB of chagasic patients with no parasite nests in HE sections but detected positive for T. cruzi DNA by polymerase chain reaction. As a primary antibody, a hyperimmune serum from T. cruzi-infected rabbits was used. RESULTS: IHC results were positive for 21 of 26 PCs (80.8%) and one case of cutaneous leishmaniasis. In 4 of 31 cases, IHC revealed nests (12.9%), which were undetected by conventional histological examination. CONCLUSIONS: This study shows that IHC with the tested antiserum increases the sensitivity of the diagnosis and may be recommended for routine use in EMB analyses of cardiac transplant patients with Chagas disease.引言：在多种临床场景中，检测组织样本中的克氏锥虫（Trypanosoma cruzi）均具有重要意义，例如用于感染复燃的排查。在心内膜心肌活检（endomyocardial biopsies, EMB）标本中检测克氏锥虫寄生灶，可用于评估移植排斥反应。由于此类寄生灶数量稀少，常规组织学检查难以检出。为提升诊断灵敏度，免疫组织化学（immunohistochemistry, IHC）可作为一种颇具前景的检测策略。本研究旨在验证一种可通过IHC检测克氏锥虫感染的抗血清。 方法：1. 阳性对照（positive controls, PCs）：包含13份EMB标本、12份皮肤活检标本，以及1份携带克氏锥虫寄生灶的心脏标本，所有标本均经苏木精-伊红（hematoxylin and eosin, HE）染色；2. 阴性对照：(a) 10份离体心脏标本与10份EMB标本，均未检出无鞭毛体寄生灶，且无恰加斯病感染的临床/实验室证据；(b) 8份分别患有利什曼病、弓形虫病或组织胞浆菌病的标本；3. 病例样本：31份来自恰加斯病患者的EMB标本，此类标本经HE染色未检出寄生虫寄生灶，但通过聚合酶链式反应（polymerase chain reaction）检测证实克氏锥虫DNA呈阳性。本研究使用兔源抗克氏锥虫高免血清作为一抗。 结果：IHC检测结果显示，26份阳性对照标本中有21份呈阳性（阳性率80.8%），另有1例皮肤利什曼病标本呈阳性反应。在31例病例样本中，IHC成功检出4例克氏锥虫寄生灶（占比12.9%），此类寄生灶经常规组织学检查未能发现。 结论：本研究证实，使用本研究验证的抗血清开展IHC检测，可提升克氏锥虫感染的诊断灵敏度，推荐将其用于恰加斯病心脏移植患者EMB标本的常规检测工作中。