Institute of Pathology, University of Ulm, Ulm, Germany.
Consultant Laboratory for Echinococcosis, Institute for Hygiene and Microbiology, University of Wuerzburg, Wuerzburg, Germany.
PLoS Negl Trop Dis. 2021 Feb 25;15(2):e0009155. doi: 10.1371/journal.pntd.0009155. eCollection 2021 Feb.
Alveolar echinococcosis (AE) is caused by metacestode larva of the tapeworm Echinococcus multilocularis. AE diagnostics currently rely on imaging techniques supported by serology, but unequivocal detection of AE is difficult. Although polymerase chain reaction (PCR)-based methods to detect tapeworm DNA in biopsies have been suggested for several species, no validated protocol adhering to accepted guidelines has so far been presented for AE diagnostics. We herein established a PCR protocol for metacestode biopsies and technically evaluated the method using isolated parasite DNA and cells, biopsies of clinically relevant material, and formalin fixed paraffin-embedded (FFPE) human tissue blocks. We compared the results with an immunochemical (IHC) approach using the monoclonal antibody Em2G11 specific for the antigen Em2 of E. mulitlocularis.
METHODOLOGY/PRINCIPAL FINDINGS: Based on tapeworm 12S rDNA sequences we established and validated a PCR protocol for robust detection of as little as 50 parasite cells per specimen and report 127 cases of positive identification of Echinococcus species in samples from humans and animals. For further validation, we analyzed 45 liver, heart, brain, and soft tissue samples as well as cytological probes of aspirates of FFPE-material from 18 patients with clinically confirmed AE. Of each patient we analyzed (i) fully viable lesions with laminated layer; (ii) tissue with mAbEm2G11-positive small particles of E. multilocularis (spems); (iii) mAbEm2G11-negative tissue adjacent to the main lesion; and (iv) lymph node tissue with mAbEm2G11-positive spems. To identify the areas for the PCR-based approach, we performed IHC-staining with the monoclonal antibody Em2G11. Micro-dissected tissue of these areas was then used for PCR-analysis. 9 of 15 analyzed samples with viable E. multilocularis lesions with laminated layer were positive by PCR. Of this group, all samples preserved for less than 6 years (6/6) were tested positive. 11 of 15 samples of spems and 7 of 9 samples of the control group mAbEm2G11-negative tissue were negative by PCR. We further show that all probes from lymph nodes with spems are PCR negative.
CONCLUSIONS/SIGNIFICANCE: We present a sensitive PCR method for the detection of E. multilocularis in human tissue, particularly in fresh biopsy material and tissue blocks stored for less than 5 years. While the diagnostic sensitivity of material containing only spems was higher using IHC, PCR detection was possible in IHC negative liver tissue and in patients with negative serology. Our results support the view that spems do not contain parasitic DNA or viable cells of the parasite. spems thus most probably do not directly contribute to metastasis formation during AE.
泡型包虫病（AE）是由绦虫细粒棘球绦虫的幼虫引起的。AE 的诊断目前依赖于影像学技术支持的血清学检测，但明确检测 AE 具有一定难度。尽管已经提出了几种基于聚合酶链反应（PCR）的方法来检测活检中的绦虫 DNA，但到目前为止，还没有按照公认的指南制定出针对 AE 诊断的经过验证的方案。本文建立了一种针对包虫蚴活检的 PCR 方案，并使用分离的寄生虫 DNA 和细胞、临床相关材料的活检以及福尔马林固定石蜡包埋（FFPE）的人体组织块对该方法进行了技术评估。我们将结果与使用针对细粒棘球绦虫抗原 Em2 的单克隆抗体 Em2G11 的免疫化学（IHC）方法进行了比较。
方法/主要发现：我们基于绦虫 12S rDNA 序列建立并验证了一种 PCR 方案，可可靠地检测低至每标本 50 个寄生虫细胞，并报告了 127 例从人和动物样本中鉴定出棘球属物种的阳性结果。为了进一步验证，我们分析了 45 例来自 18 例临床确诊 AE 患者的肝、心、脑和软组织样本以及 FFPE 材料的细胞学探针。我们对每位患者的以下样本进行了分析：（i）完全存活的有层状结构的病变；（ii）含有小颗粒 Em2 的 mAbEm2G11 阳性的组织（spems）；（iii）与主要病变相邻的 mAbEm2G11 阴性组织；（iv）含有 mAbEm2G11 阳性 spems 的淋巴结组织。为了确定基于 PCR 的方法的检测区域，我们使用单克隆抗体 Em2G11 进行了 IHC 染色。然后对这些区域的微切割组织进行了 PCR 分析。9/15 例有层状结构的存活的多房棘球蚴病变的样本通过 PCR 呈阳性。在这一组中，所有保存时间少于 6 年（6/6）的样本均呈阳性。15 例 spems 样本中有 11 例和 9 例对照组织 mAbEm2G11 阴性组织的样本通过 PCR 呈阴性。我们进一步表明，所有含有 spems 的淋巴结探针均为 PCR 阴性。
结论/意义：我们提出了一种针对人组织中多房棘球绦虫的敏感 PCR 检测方法，尤其适用于新鲜活检材料和保存时间少于 5 年的组织块。当仅使用 IHC 检测含有 spems 的材料时，其诊断灵敏度更高，但在 IHC 阴性的肝组织和血清学阴性的患者中也可进行 PCR 检测。我们的结果支持 spems 不含有寄生虫 DNA 或寄生虫活细胞的观点。因此，spems 很可能不会直接导致 AE 期间转移的形成。
