Improving Molecular Detection of Tick-Borne Pathogens in Citizen-Collected Ticks

This study aimed primarily to evaluate the performance of two Conformité Européenne-In Vitro Diagnostic (CE-IVD) multiplex real-time PCR (rt-PCR) assays for the molecular identification of tick-borne pathogens (TBPs) of human interest on ticks removed from human skin and collected through a citizen science-based approach. As a secondary objective, the aggregated results were used to describe tick species distribution, developmental stages, and seasonal TBP circulation in 2024 in the considered area. The comparison was conducted on 116 tick samples collected in 2024 voluntarily delivered to a hospital in northeastern Italy. Detected TBPs were further confirmed with in-house-validated PCR methods and, where applicable, resolved to the species level. Clinically relevant pathogen species were identified as single infections or coinfections. Overall, 33.6% of tick samples tested positive for at least one TBP, and 6.9% showed coinfections. Kit B exhibited a higher detection rate for <i>Borrelia</i> spp. and <i>Rickettsia</i> spp. targets, partly reflecting its broader diagnostic specificity, while statistically significant differences in cycle threshold values were observed for <i>Anaplasma phagocytophilum</i> detection. The most frequently involved ticks were <i>Ixodes ricinus</i> nymphs, and the most represented area was Verona province. Late spring and early summer were identified as the periods with the highest tick conferment and pathogen diversity. Overall, the results support the use of multiplex real-time PCR commercial kits combined with citizen science-based tick collection as an effective approach for both diagnostic screening and regional surveillance of circulating ticks and TBPs.