Detection of Echinococcus granulosus sensu lato microRNAs in cystic echinococcosis patients: An exploratory study using quantitative PCR and digital PCR

BACKGROUND: Cystic echinococcosis (CE) is a zoonosis caused by the larval stage of Echinococcus granulosus sensu lato (s.l.). The adult parasite typically resides in the intestine of canids, while the larval stage, a fluid-filled cyst, primarily resides in the liver and lungs of ungulates and humans. The diagnosis of abdominal CE in humans is mainly based on ultrasound (US) complemented by serology, but both techniques present limitations. Therefore, new diagnostic methods are needed. MicroRNAs (miRNAs) are potential suitable biomarkers for parasitic diseases, as well as for the diagnosis and cyst staging of CE. The objective of this study was to evaluate the presence of three E. granulosus s.l. miRNAs (egr-let-7-5p, egr-miR-10a-5p, and egr-miR-71-5p) in the serum of CE patients using novel TaqMan-based quantitative PCR (qPCR) and digital PCR (dPCR) assays. METHODOLOGY/PRINCIPAL FINDINGS: Serum samples from 25 patients with CE, 10 patients with non-CE hepatic lesions, and 10 patients with other parasitic infections were collected. In addition, four cyst fluids were also obtained. Total small RNAs were extracted, reverse-transcribed into cDNA, and amplified. Echinococcus granulosus s.l. miRNAs were detected by qPCR and dPCR in cyst fluid samples, and by dPCR in serum samples. In detail, egr-let-7-5p, egr-miR-10a-5p, and egr-miR-71-5p were amplified in 52%, 48%, and 36% of CE samples, respectively (range: 0.20- 2.86 copies/µL). No egr-miRNAs were detected in patients with non-CE hepatic lesions, whereas egr-miR-71-5p was amplified in only one Schistosoma spp. patient. In association with US for CE diagnosis, dPCR assay showed the highest performance when a single E. granulosus s.l. miRNA was amplified, achieving a sensitivity of 84% (95% CI 63.9-95.5) and specificity of 95% (95% CI 75.0-99.9). Increasing the number of positive miRNAs required for a positive result reduced sensitivity substantially (40% with two miRNAs, 95% CI 21.1-61.3; 12% with three miRNAs, 95% CI 2.5-31.2). CONCLUSIONS: In conclusion, although this study's explorative nature and the limited sample size, the detection of E. granulosus s.l. miRNAs in the serum of CE patients proved feasible, potentially supporting the medical decision-making process in association with US for CE diagnosis.