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

Abstract

Confirmed diagnosis of alveolar echinococcosis (AE) is based on pathological criteria and molecular evidence. This parasite-borne disease, caused by the cestode Echinococcus multilocularis, sparingly involves humans as a dead-end host. In humans, the parasite mainly colonizes the liver but can colonize any organ and cause atypical forms, often difficult to characterize clinically. Moreover, molecular methods may be suitable to make the diagnosis of AE in cases of atypical forms, extra-hepatic localizations, or immunosuppressed patients. The aim of this study was to determine the most relevant published PCR techniques, for diagnosis of AE in patients and adopt the best strategy for molecular diagnosis depending on the nature of the tested sample. In this study, we evaluated nine end-point PCR assays and one real-time PCR assay (qPCR), targeting mitochondrial genes, using a total of 89 frozen or formalin-fixed paraffin-embedded (FFPE) samples from either 48 AE or 9 cystic echinococcosis patients. Targeted fragment-genes ranged from 84 to 529 bp. Six PCR assays were able to amplify the DNA of 100% of the frozen AE-samples and for one PCR, 69.8% of the FFPE AE-samples. The 16S rrnL PCR (84 bp) was positive in PCR for 77% of the AE samples and in qPCR for 86.5%. The sensitivity of the PCR assays was higher for fresh samples and FFPE samples stored for less than 5 years. The qPCR assay further increased sensitivity for the tested samples, confirming the need for the development of an Echinococcus spp. qPCR to improve the molecular diagnosis of echinococcoses.

Real-time multiplex PCR for human echinococcosis and differential diagnosis

“…The panel was composed of 46 AE (15 frozen tissues, 31 FFPE specimens) and 35 CE lesions (19 frozen tissues, 16 FFPE specimens), collected from 1997 to 2021, with the geographical origin of the patients when available (Table 1). The Echinococcus species of the sample material have been confirmed by either species-specific PCRs, qPCR or via sequencing in previous studies [19] (Table 1). The species of the AE lesions were identified as E. multilocularis and those of the CE samples as E. granulosus s.s. (n = 29), E. canadensis (n = 4), and E. ortleppi (n = 2).…”

“…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].…”

“…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].…”

“…# qPCR positive once from the duplicate. y Sample not described in Knapp et al[19] but tested with the same PCR techniques. $…”

Disseminated alveolar echinococcosis in a patient diagnosed by metagenomic next-generation sequencing: A case report

“…Molecular diagnosis of AE by PCR or qPCR based on native biopsy material or formalin-fixed paraffin-embedded tissue samples is feasible and sensitive, especially in patients with atypical manifestations, extrahepatic localizations and immunosuppression ( 24 , 25 ). As a rapidly developing molecular diagnostic technique, mNGS enables rapid detection and comprehensive identification of pathogens directly from clinical samples without prior presumption, with a higher sensitivity and accuracy.…”