Echinococcus tapeworms cause one of the most important parasitoses, echinococcosis, and are widespread worldwide. Humans can become accidental intermediate hosts of larval forms from the Echinococcus granulosus s.l. species complex, the causative agent of cystic echinococcosis, and Echinococcus multilocularis, the causative agent of alveolar echinococcosis. The infection may remain asymptomatic for years, until the larval form, typically developing in the liver or lungs, causes symptoms through tissue compression or metastatic spread. Due to diagnostic challenges, reliable detection is essential for early diagnosis and effective management. At the Laboratory of Parasitology of the Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, molecular diagnosis of echinococcosis is currently performed using conventional PCR and sequencing of PCR products, which is time-consuming, costly, and prone to contamination from previous reactions. Real-time PCR is considered a faster, more specific, sensitive, and cost-effective alternative. In this study, we optimised and validated real-time PCR for detecting Echinococcus spp. DNA in clinical samples and identifying the causative species in human echinococcosis. Optimisation included adjusting primer and probe concentrations; validation involved determining the limit of detection (LOD), repeatability, reproducibility, diagnostic specificity, and sensitivity. The LOD (LOD95) was 0.7028 ng/µL for E. granulosus s.s., 0.00048 ng/µL for E. canadensis, and 0.00013 ng/µL for E. multilocularis. The assay proved to be 100% sensitive and 100% specific, with no cross-reactions between species of Echinococcus and Taenia. Real-time PCR was shown to be a rapid, reliable method comparable to conventional PCR and sequencing. Validation remains a continuous process, requiring regular assessment of test performance and reliability.
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