Enhanced molecular diagnosis of Onchocerca lupi using droplet digital PCR in clinically suspected companion animals

Abstract Background Onchocerca lupi , a zoonotic filarial nematode documented in southwestern North America and the Old World, is associated with ocular onchocerciosis in dogs and cats. Clinical signs range from ocular discharge and conjunctivitis to chronic nodular lesions involving the conjunctiva, sclera, and retrobulbar space. Many infections remain subclinical and undiagnosed. Current diagnostic approaches rely on microscopy and conventional PCR (cPCR) to confirm adult worms in nodules or microfilariae in skin snips. Real-time PCR (qPCR) has been applied primarily in large-scale epidemiological studies. However, amplification of O. lupi DNA may be inhibited, leading to false-negative results. Highly sensitive techniques such as droplet digital PCR (ddPCR) can enable the detection of parasite DNA in various biological samples through absolute quantification. The objectives of this study were to: I) validate a novel ddPCR assay that detects O. lupi , and II) assess the performance of the ddPCR assay compared to cPCR and qPCR in suspected clinical cases. Methods Following assay validation and optimization, 202 suspected clinical cases from 11 US states were assessed. Of these clinical cases, 97% (n = 196/202) were dogs, and 3% (n = 6/202) were cats. Samples included adult specimen O. lupi fragments (n = 34/202), subconjunctival nodule biopsies (n = 103/202), interscapular skin snips (n = 23/202), lower ear skin snips (n = 3/202), and formalin-fixed paraffin-embedded (FFPE) tissue samples (n = 39/202), all previously assessed using cPCR and qPCR. Statistical analysis was performed to assess agreement using Cohen’s kappa (κ), and Cochran’s Q test was used to compare the pattern of positive/negative results of the three diagnostic techniques. Results Overall, the ddPCR detected 71.3% (n = 144/202) of clinical cases as positive for O. lupi , including 71.9% of dog samples (n = 141/196), outperforming qPCR (64.8%; n = 127/196) and cPCR (54.1%; n = 106/196). Additionally, we confirmed O. lupi infection in 50% (n = 3/6) of the cat samples across all three diagnostic tests. Statistical analysis showed moderate agreement between cPCR + qPCR (κ = 0.74), fair agreement between cPCR + ddPCR (κ = 0.60), and almost perfect agreement between qPCR + ddPCR (κ = 0.81). This study represents the largest series of suspected O. lupi clinical cases in companion animals in the US to date, with positive detections reported in 10 of 11 sampled states, although travel history was not always available. Conclusion The validated, host-agnostic ddPCR assay demonstrated superior sensitivity across multiple sample types and provides a robust tool for clinical diagnosis and large-scale surveillance studies, supporting improved epidemiological understanding and One Health-based prevention strategies.