A multiplex PCR-based sequencing method for the diagnosis and differentiation of echinococcosis using plasma samples, a proof-of-concept study

Echinococcosis poses a significant public health threat, and its diagnosis relies on imaging. Non-invasive plasma detection of Echinococcus cell-free DNA (cfDNA) offers a promising diagnostic approach. We developed a low-cost multiplex PCR panel with 230 primer pairs targeting Echinococcus mitochondrial genomes and nuclear DNA repeat regions. We validated the panel using gradient dilutions of two pathogen species' DNA and 81 plasma samples (53 patients and 28 controls), differentiating species through sequencing analysis. Our method detected Echinococcus genomic DNA at an input as low as 1 fg per reaction, and tests of clinical samples showed a sensitivity of 68.42% and specificity of 92.86%. Based on our detection, 80% of the clinically confirmed cases were correctly identified as positive echinococcosis cases, and cfDNA levels correlated significantly with lesion size. This study highlights the potential of targeted cfDNA sequencing for non-invasive echinococcosis diagnosis and species differentiation and offers a promising tool for doubtful cases.