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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.

SUBMITTER: Knapp J

PROVIDER: S-EPMC8812296 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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

Knapp Jenny J Lallemand Séverine S Monnien Franck F Felix Sophie S Valmary-Degano Séverine S Courquet Sandra S Demonmerot Florent F Heyd Bruno B Turco Celia C Doussot Alexandre A Bourgeois Lucie L Bresson-Hadni Solange S Richou Carine C Millon Laurence L

Parasite (Paris, France) 20220202

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 lo ...[more]

PMID: 35113014

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Project description:BackgroundWe have developed a gene expression assay (Whole-Genome DASL), capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE) specimens.Methodology/principal findingsWe demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF) and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3-1.5 and 1.5-2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is approximately 3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2) approximately 0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2) approximately 0.86 and R(2) approximately 0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2)>0.98) with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2) approximately 0.92), with approximately 71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2) approximately 0.80, while still maintaining a correlation of R(2) approximately 0.75 with standard FFPE inputs (200 ng).Conclusions/significanceTaken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material) or when minimally invasive procedures are performed (e.g. biopsied specimens).

Dataset Information

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

Publications

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

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