Comparative evaluation of mitochondrial gene markers for molecular diagnosis of Echinococcus granulosus sensu lato in clinical samples.

Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato (s.l.), remains a significant public health concern, particularly in regions reliant on livestock farming. Early and accurate diagnosis is essential for effective disease management, yet traditional diagnostic methods such as imaging and serological assays often lack specificity and sensitivity. Molecular diagnostics, particularly polymerase chain reaction (PCR), offer a promising alternative by enabling the direct detection of E. granulosus s.l. DNA in clinical samples. This study evaluates the diagnostic performance of four primer sets targeting mitochondrial genes-Cytochrome c oxidase subunit 1 (Cox1), Mitochondrial NADH dehydrogenase subunit 1 (NAD1) and Mitochondrial 12s ribosomal RNA (12s rRNA). All primer sets achieved 100% positivity in hydatid cyst fluids and tissues, confirming their utility in detecting E. granulosus s.l. DNA. However, specificity varied, with NAD1 and 12S rRNA primers demonstrating excellent specificity, while Cox1.1 and Cox1.2 exhibited cross-reactivity with Taenia solium and Dirofilaria spp., respectively. Sensitivity analysis revealed NAD1 as the most sensitive marker, detecting as little as 305 fg/µl of parasite DNA. These findings underscore the importance of genetic marker selection in optimizing PCR-based diagnostics. While NAD1 and 12S rRNA primers showed strong potential for clinical application, further refinement of Cox1 primers is necessary to minimize false positives. Our study highlights the power of molecular diagnostics in improving CE detection and emphasizes the need for continuous assay optimization to ensure accurate and reliable parasite identification in endemic settings.