Sensitive and specific immunohistochemical diagnosis of human alveolar echinococcosis with the monoclonal antibody Em2G11

Abstract

Background: Alveolar echinococcosis (AE) is caused by the metacestode stage of Echinococcus multilocularis. Differential diagnosis with cystic echinococcosis (CE) caused by E. granulosus and AE is challenging. We aimed at improving diagnosis of AE on paraffin sections of infected human tissue by immunohistochemical testing of a specific antibody.

Methodology/principal findings: We have analysed 96 paraffin archived specimens, including 6 cutting needle biopsies and 3 fine needle aspirates, from patients with suspected AE or CE with the monoclonal antibody (mAb) Em2G11 specific for the Em2 antigen of E. multilocularis metacestodes. In human tissue, staining with mAb Em2G11 is highly specific for E. multilocularis metacestodes while no staining is detected in CE lesions. In addition, the antibody detects small particles of E. multilocularis (spems) of less than 1 µm outside the main lesion in necrotic tissue, liver sinusoids and lymphatic tissue most probably caused by shedding of parasitic material. The conventional histological diagnosis based on haematoxylin and eosin and PAS stainings were in accordance with the immunohistological diagnosis using mAb Em2G11 in 90 of 96 samples. In 6 samples conventional subtype diagnosis of echinococcosis had to be adjusted when revised by immunohistology with mAb Em2G11.

Conclusions/significance: Immunohistochemistry with the mAb Em2G11 is a new, highly specific and sensitive diagnostic tool for AE. The staining of small particles of E. multilocularis (spems) outside the main lesion including immunocompetent tissue, such as lymph nodes, suggests a systemic effect on the host.

Figure 1. Immunohistochemical staining modalities of the monoclonal antibody (mAb) Em2G11 for metacestodes of Echinococcus multilocularis.

Figure 1A: In metacestodes grown in a Mongolian jird, the antibody strongly marks the laminated layer (single arrow left below). The germinal layer and calcareous corpuscles are strongly stained (two arrows and single arrow right) as well as the precipitated cyst fluid. The area oft the rostellum is superimposed with a positive reacting layer (dashed line) while the inner part of the protoscolex did not react with the monoclonal antibody; bar =  50 µm. 1B, C: In human liver, the Em2 antigen is strongly positive in the slender laminated layer of E. multilocularis. The staining reveals a tubular and infiltrative growth pattern (arrows). In contrast, the laminated layer of E. granulosus is much broader (arrows), no staining is detected by mAb Em2G11; bar =  1000 µm.

Figure 2. Immunohistochemical staining modalities of the monoclonal antibody (mAb) Em2G11 for metacestodes of Echinococcus multilocularis.

Figure 2A: E. multilocularis lesion in human liver tissue. The antigen is detected in the laminated layer (two arrows, right) and in the necrotic area around the lesion (dashed lined area, right). The antibody detects small particles of E. multilocularis (spems) up two 1.5 mm away from the main lesion in a small liver vessel (small area marked with a dashed line on the left). Insert left highlights this lesion at a higher magnification showing a specific staining of spems. Insert right shows specific staining in lymphoid tissue of a regional lymph node on the surface of cells (arrows; bar =  750 µm; bar insert =  40 µm). B: In contrast, no staining is observed in caseous necrosis of tuberculosis (arrows low) and in bronchial epithelial tissue (arrows high; bar =  50 µm). C: Serial section of an aspirate from the liver. C shows a PAS staining of a strongly positive laminated layer. C′: Staining of the section with mAb Em2G11 reveals a strong positivity of the laminated layer and of the necrotic tissue with spems (dashed lined area; bar =  500 µm). D: PAS staining of brain tissue showing the laminated layer of an E. multilocularis metacestode. D′: The laminated layer is strongly positive for mAb Em2G11 even after 60 years of formalin fixation (bar =  50 µm).