Comparative diagnostic evaluation of microscopy and targeted next-generation sequencing of the barcoding gene...

Tick surveillance protocols in public health laboratories have traditionally relied on microscopy for species identification. However, recognition of emerging species with overlapping morphology, loss of expertise, and new technologies have highlighted the need for complementary testing approaches. We performed a comparative diagnostic evaluation assessing accuracy of next-generation sequencing targeting the barcoding portion of cytochrome oxidase subunit I (COI) against traditional microscopy for tick identification. A total of 306 Dermacentor and Ixodes ticks were included in the study. Overall agreement between the methods was >90%. Molecular testing highlighted the challenges with identifying ticks to the species level by morphology alone, especially for rare and/or emerging species. Molecular testing proved useful as a quality assurance tool for microscopy and highlighted the need for targeted continuing education to strengthen morphological analysis expertise. Finally, the COI NGS workflow demonstrated feasibility for integration into routine laboratory sequencing workflows. Our findings support the utility of COI sequencing as a complementary diagnostic approach in reference laboratories where tick identification and surveillance are performed.