Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue.
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 detec...
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ftdoajarticles:oai:doaj.org/article:9a56b7b6983246c1b0681b299ef46ed2 2023-05-15T15:14:06+02:00 Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue. Johannes Grimm Julian Krickl Annika Beck Juliane Nell Monika Bergmann Dennis Tappe Beate Grüner Thomas Fe Barth Klaus Brehm 2021-02-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0009155 https://doaj.org/article/9a56b7b6983246c1b0681b299ef46ed2 EN eng Public Library of Science (PLoS) https://doi.org/10.1371/journal.pntd.0009155 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0009155 https://doaj.org/article/9a56b7b6983246c1b0681b299ef46ed2 PLoS Neglected Tropical Diseases, Vol 15, Iss 2, p e0009155 (2021) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2021 ftdoajarticles https://doi.org/10.1371/journal.pntd.0009155 2022-12-31T11:50:15Z 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 ... Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 15 2 e0009155 |
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English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Johannes Grimm Julian Krickl Annika Beck Juliane Nell Monika Bergmann Dennis Tappe Beate Grüner Thomas Fe Barth Klaus Brehm Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
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 ... |
format |
Article in Journal/Newspaper |
author |
Johannes Grimm Julian Krickl Annika Beck Juliane Nell Monika Bergmann Dennis Tappe Beate Grüner Thomas Fe Barth Klaus Brehm |
author_facet |
Johannes Grimm Julian Krickl Annika Beck Juliane Nell Monika Bergmann Dennis Tappe Beate Grüner Thomas Fe Barth Klaus Brehm |
author_sort |
Johannes Grimm |
title |
Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue. |
title_short |
Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue. |
title_full |
Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue. |
title_fullStr |
Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue. |
title_full_unstemmed |
Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue. |
title_sort |
establishing and evaluation of a polymerase chain reaction for the detection of echinococcus multilocularis in human tissue. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2021 |
url |
https://doi.org/10.1371/journal.pntd.0009155 https://doaj.org/article/9a56b7b6983246c1b0681b299ef46ed2 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 15, Iss 2, p e0009155 (2021) |
op_relation |
https://doi.org/10.1371/journal.pntd.0009155 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0009155 https://doaj.org/article/9a56b7b6983246c1b0681b299ef46ed2 |
op_doi |
https://doi.org/10.1371/journal.pntd.0009155 |
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PLOS Neglected Tropical Diseases |
container_volume |
15 |
container_issue |
2 |
container_start_page |
e0009155 |
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1766344593709727744 |