Real-time multiplex PCR for human echinococcosis and differential diagnosis

Abstract

Molecular identification of rare human infectious pathogens appears to be one of the most relevant current methods for rapid diagnosis and management of patients. PCR techniques, in particular real-time quantitative PCR, are best suited for the detection of DNA from the pathogens, even at low concentrations. Echinococcosis infections are due to helminths of the Echinococcus genus, with closely related species involved in parasitic lesions affecting animals and, accidentally, humans. We developed a multiplex qPCR (MLX qPCR) assay allowing for the detection of four Echinococcus species involved in Europe in alveolar echinococcosis (AE) and cystic echinococcosis (CE) (Echinococcus multilocularis, E. granulosus sensu stricto, E. ortleppi, and E. canadensis), based on short mitochondrial targets. A collection of 81 fresh and formalin-fixed paraffin-embedded tissues (FFPE) of AE and CE lesions was assembled. The qPCR assays were performed in triplex for Echinococcus spp. detection, associated with a qPCR inhibitor control. A duplex qPCR was also designed to enable diagnosis of two other dead-end helminthiases (cysticercosis (Taenia solium), and toxocariasis (Toxocara cati and T. canis)). The sensitivity of the qPCR was assessed and ranged from 1 to 5 × 10−4 ng/μL (seven PCR assays positive), corresponding to 37–42 cycles for quantifiable DNA. The specificity was 100% for all the targets. This multiplex qPCR, adapted to low amounts of DNA can be implemented in the laboratory for the rapid molecular diagnosis of Echinococcosis species.

Who Let the Dogs Out? Unmasking the Neglected: A Semi-Systematic Review on the Enduring Impact of Toxocariasis, a Prevalent Zoonotic Infection

“…Molecular techniques have high analytical specificity and shorter turnaround times than other diagnostic methods. Polymerase chain reaction (PCR)-based assays using a variety of genetic markers (e.g., ITS-1 and ITS-2 regions of rDNA) have now been developed and have enabled the identification and phylogenetic analysis of T. canis, T. cati, and other ascarids [234]. Vitellogenin (Vg), recognized as a reservoir of both amino acids and lipids within the eggs of the parasites, is acknowledged to hold a pivotal role in the embryonic development of a diverse spectrum of organisms.…”

First cases of alveolar echinococcosis in dogs in Poland

“…In fluid sediment from cyst cavities or liver lesions, protoscolex of Echinococcus can be found [25]. The morphology of tapeworm protoscolexes are characteristic of the Echinococcus genus; therefore, the final recognition of the species is based on the PCR reaction [5,9,26].…”

High species diversity of Echinococcus spp. in wild mammals of Namibia

“…To achieve realistic prevalence estimates, detection methods need improvement in terms of sensitivity. Application of qPCR methods as recently developed are likely to provide a way forward ( Maksimov et al, 2020 ; Knapp et al, 2023 ), although the diversity of Echinococcus spp., as found in this study, will be a challenge for routine application with large numbers of samples.…”