Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue

Abstract

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.

Molecular diagnosis of alveolar echinococcosis in patients based on frozen and formalin-fixed paraffin-embedded tissue samples

“…In cases of Echinococcus-negative samples, multiband patterns appeared on the agarose gels, but the sequenced PCR products were the result of human DNA amplification, certainly due to cross-reaction with the host-DNA during PCR. This phenomenon was previously described by Grimm et al and attributed to a large amount of host cell DNA present in the DNA extract [13]. Moreover, from our study, the use of pure positive controls, isolated from distinct parasite specimens from the host (adult worms and protoscoleces) is confirmed for proper interpretation of results.…”

“…Given the mean time interval between storage and DNA extraction, greater DNA degradation is likely the cause of the absence of amplification. Nucleic acids extracted from paraffin blocks are highly degraded in blocks stored more than 4 years, because DNA stability in conserved FFPE material decreases [ 13 ], with residual fragments of mostly < 300 bp, formation of DNA-protein crosslinks, increasing the sensitivity of DNA to mechanical stress and decrease the accessibility for enzymes. When formalin is oxidized to formic acid, DNA depurination and DNA strand breaks can be observed [ 9 ].…”

Real-time multiplex PCR for human echinococcosis and differential diagnosis

“…We also observed a significant difference between recent and older FFPE samples. The storage time of FFPE samples is critical for PCR results, as previously observed, with a large decrease in PCR performance after four to five years of storage [14,19]. Moreover, the sensitivity of the technique on FFPE samples may be linked to the macroscopic selection of the parasitic zone followed by the extraction of DNA from this zone, as in the estimation of the percentage of tumour cells presenting mutations in pathology examinations [25].…”

“…Further assays using higher DNA volumes were not attempted because of the lack of available left-DNA sample. Moreover, these samples, for which the DNA was extracted >5 years after paraffin inclusion could have become highly degraded during storage, as previously described [14,19], and could hinder echinococcosis diagnosis, especially in cases of diagnostic wandering. An association with other PCR techniques may be necessary in such particular cases with degraded DNA, such as the multiplex PCR assay described by Trachsel et al [37] and the Em-rrn qPCR assay used with success on old samples [19].…”

Advances in the study of molecular identification technology of Echinococcus species

“…PCR has been developed following the demand for molecular detection to diagnose Echinococcus in increasing application areas, such as hydatid disease detection of human and animal, epidemiological investigation, as well as genotyping of Echinococcus, etc. (Armua-Fernandez et al, 2011;Soares et al, 2013;Avcioglu et al, 2017;He et al, 2017;Bittencourt-Oliveira et al, 2018;Gorgani-Firouzjaee et al, 2019;Moradi et al, 2019;Ohiolei et al, 2019;Wan et al, 2020;Grimm et al, 2021).…”