Infection of humans by the larval stage of the tapeworms Echinococcus granulosus sensu lato or Echinococcus multilocularis causes the life-threatening zoonoses cystic echinococcosis (CE) and alveolar echinococcosis (AE). Although cystic liver lesions are a hallmark of both diseases, course, prognosis, and patients' management decisively differ between the two. The wide and overlapping spectrum of morphologies and the limited availability of ancillary tools are challenges for pathologists to reliably diagnose and subtype echinococcosis. Here, we systematically and quantitatively recorded the pathologic spectrum in a clinically and molecularly defined echinococcosis cohort (138 specimens from 112 patients). Immunohistochemistry using a novel monoclonal antibody (mAbEmG3) was implemented, including its combined application with the mAbEm2G11. Six morphologic criteria sufficiently discriminated between CE and AE: size of smallest (CE/AE: >2/2 mm) and largest cyst (CE/AE: >25/25 mm), thickness of laminated layer (CE/AE: >0.15/0.15 mm) and pericystic fibrosis (CE/AE: >0.6/0.6 mm), striation of laminated layer (CE/AE: moderate-strong/weak), and number of cysts (CE/AE: 9/>9). Combined immunohistochemistry with mAbEm2G11 (E. multilocularis specific) and mAbEmG3 (reactive in AE and CE) was equally specific as and occasionally more sensitive than polymerase chain reaction. On the basis of these findings, we developed a diagnostic algorithm for the differential diagnosis of echinococcosis. In summary, we have not only identified the means to diagnose echinococcosis with greater certainty, but also defined morphologic criteria, which robustly discriminate between CE and AE. We expect our findings to improve echinococcosis diagnostics, especially of challenging cases, beneficially impacting the management of echinococcosis patients.

BackgroundAlveolar echinococcosis (AE) is caused by the metacestode stage of Echinococcus multilocularis. The inflammatory response to this infection is influenced by the interaction of the parasite with the host. We aimed to analyze human liver lesions infected with Echinococcus multilocularis and the changes of the cellular infiltrates during albendazole (ABZ) treatment.Methodology/Principal findingsWe analyzed liver tissue samples from 8 untreated patients, 5 patients treated with two daily doses of 400 mg ABZ for up to two months and 7 patients treated for more than two months with the same ABZ therapy. A broad panel of monoclonal antibodies was used to characterize the lesion by immunohistochemistry. A change in the cellular infiltrate was observed between the different chemotherapy times. During the initial phases of treatment an increase in CD15+ granulocytes and CD68+ histocytes as well as in small particles of Echinococcus multilocularis (spems) was observed in the tissue surrounding the metacestode. Furthermore, we observed an increase in CD4+ T cells, CD20+ B cells and CD38+ plasma cells during a longer duration of treatment.Conclusions/SignificanceABZ treatment of AE leads to morphological changes characterized by an initial, predominantly acute, inflammatory response which is gradually replaced by a response of the adaptive immune system.

Alveolar echinococcosis (AE) is caused by the intermediate stage of Echinococcus multilocularis. We aimed to correlate computed tomography (CT) data with histology to identify distinct characteristics for different lesion types. We classified 45 samples into five types with the Echinococcus multilocularis Ulm Classification for Computed Tomography (EMUC-CT). The various CT lesions exhibited significantly different histological parameters, which led us to propose a progression model. The initial lesion fit the CT type IV classification, which comprises a single necrotic area with the central located laminated layer, a larger distance between laminated layer and border zone, a small fibrotic peripheral zone, and few small particles of Echinococcus multilocularis (spems). Lesions could progress through CT types I, II, and III, characterized by shorter distances between laminated layer and border zone, more spems inside and surrounding the lesion, and a pronounced fibrotic rim (mostly in type III). Alternatively, lesions could converge to a highly calcified, regressive state (type V). Our results suggest that the CT types mark sequential stages of the infection, which progress over time. These distinct histological patterns advance the understanding of interactions between AE and human host; moreover, they might become prognostically and therapeutically relevant.

Background Alveolar (AE) and cystic echinococcosis (CE) in humans are caused by the metacestode of the tapeworms Echinococcus multilocularis and Echinococcus granulosus sensu lato (s.l.). Immunohistochemistry with the monoclonal antibodies (mAb) Em2G11, specific for AE, and the mAb EmG3, specific for AE and CE, is an important pillar of the histological diagnosis of these two infections. Our aim was to further evaluate mAb EmG3 in a diagnostic setting and to analyze in detail the localization, distribution, and impact of small particles of Echinococcus multilocularis (spems) and small particles of Echinococcus granulosus s.l. (spegs) on lymph nodes. Methodology/principal findings We evaluated the mAb EmG3 in a cohort of formalin-fixed, paraffin embedded (FFPE) specimens of AE (n = 360) and CE (n = 178). These samples originated from 156 AE-patients and 77 CE-patients. mAb EmG3 showed a specific staining of the metacestode stadium of E. multilocularis and E. granulosus s.l. and had a higher sensitivity for spems than mAb Em2G11. Furthermore, we detected spegs in the surrounding host tissue and in almost all tested lymph nodes (39/41) of infected patients. 38/47 lymph nodes of AE showed a positive reaction for spems with mAb EmG3, whereas 29/47 tested positive when stained with mAb Em2G11. Spegs were detected in the germinal centers, co-located with CD23-positive follicular dendritic cells, and were present in the sinuses. Likewise, lymph nodes with spems and spegs in AE and CE were significantly enlarged in size in comparison to the control group. Conclusions/significance mAb EmG3 is specific for AE and CE and is a valuable tool in the histological diagnosis of echinococcosis. Based on the observed staining patterns, we hypothesize that the interaction between parasite and host is not restricted to the main lesion since spegs are detected in lymph nodes. Moreover, in AE the number of spems-affected lymph nodes is higher than previously assumed. The enlargement of lymph nodes with spems and spegs points to an immunological interaction with the small immunogenic particles (spems and spegs) of Echinococcus spp.

Background 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.