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

Jenny Knapp,Solange Bresson-Hadni,Celia Turco,Sophie Felix,Carine Richou,Bruno Heyd,Alexandre Doussot,Sandra Courquet,Florent Demonmerot,Franck Monnien,Séverine Valmary-Degano,Lucie Bourgeois,Séverine Lallemand,Laurence Millon

doi:10.1051/parasite/2022004

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.

Highlights

Alveolar echinococcosis (AE) is a serious and rare zoonosis, endemic to the northern hemisphere, with increasing incidence
AE is caused by E. multilocularis, while cystic echinococcosis (CE) is caused by three species of the E. granulosus complex (E. granulosus sensu stricto, E. canadensis and E. ortleppi), and neotropical echinococcosis is caused by E. vogeli and E. oligarthra
For all positive controls, obtained from pure materials (E. multilocularis, E. granulosus and T. solium), a band on the agarose gel was visualized at the expected size and the expected species for each specific PCR with a DNA concentration at 1 ng/lL, except for the NAD1 JB11.5 PCR (C) and the COX1 PCR (H), with a DNA concentration increased at 5 ng/lL, because of weak bands observed

Summary

Introduction

Alveolar echinococcosis (AE) is a serious and rare zoonosis, endemic to the northern hemisphere, with increasing incidence. The taxonomy of the genus Echinococcus has been established and includes 8 to 10 species, according to the latest publications, of which six have been described to be pathogenic for humans: E. multilocularis, E. granulosus sensu stricto, E. canadensis, E. ortleppi, E. vogeli, and E. oligarthra [32, 38, 40]. The clinical diagnosis is confirmed by serology in 95% of cases with an enzyme-linked immune sorbent assay (ELISA) based on E. multilocularis antigens (Em2 and Em18) and immunoblotting tests (western blot) [41]. According to the recommendations of the WHO-Informal Working Group on Echinococcosis (WHOIWGE) [7], probable cases are defined by clinical and epidemiological history, imaging findings, and serology positive for AE; only histopathology and molecular biology exams make a confirmed AE diagnosis (Fig. 1)

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