Droplet Digital PCR Outperforms Standard Tests in Detecting Eye-Dwelling Worm in Dogs and Cats

A parasitic worm that burrows into the tissues around the eyes of dogs and cats has long been a diagnostic headache for veterinarians, but a new study suggests that a more sensitive molecular technique can catch infections that conventional tests miss. Researchers led by Maureen A. Kelly and Guilherme G. Verocai at Texas A&M University, working with collaborators across the United States, validated a droplet digital PCR assay for detecting Onchocerca lupi and then pitted it against the two molecular methods most commonly used in veterinary diagnostic laboratories. The results, published in the journal Parasites & Vectors, show that the digital approach identified substantially more positive cases than either conventional PCR or real-time quantitative PCR, offering what the authors describe as a robust new tool for both clinical diagnosis and large-scale surveillance of this zoonotic parasite.

Onchocerca lupi is a filarial nematode, a threadlike roundworm related to the parasites that cause river blindness in humans. Unlike many of its relatives, however, O. lupi targets the eyes of its hosts. In dogs and cats, infection produces a spectrum of signs ranging from ocular discharge and conjunctivitis to chronic nodular lesions involving the conjunctiva, the sclera, and the retrobulbar space behind the eyeball. The parasite has been documented in southwestern North America as well as in the Old World, and because it can also infect people, it carries genuine public health significance. Perhaps most troubling from a diagnostic standpoint is that many infections remain entirely subclinical, silently persisting in animals that never show obvious signs of disease and therefore never get tested.

The diagnostic toolbox for O. lupi has historically been limited. Confirmation of infection has relied primarily on microscopy and conventional PCR, or cPCR, applied either to adult worms recovered from nodules or to microfilariae, the larval stage, detected in skin snips. Real-time PCR, known as qPCR, has been applied mainly in large epidemiological surveys rather than routine clinical work. Each of these approaches has a critical weakness: amplification of O. lupi DNA can be inhibited by components of the sample matrix, producing false-negative results in animals that are actually infected. In a disease where a missed diagnosis can mean prolonged discomfort for a pet and continued zoonotic risk for its owners, that limitation matters.

Droplet digital PCR, or ddPCR, takes a fundamentally different approach to nucleic acid detection. Instead of relying on amplification curves and threshold comparisons, the technique partitions a sample into thousands of tiny water-in-oil droplets, each of which serves as an individual microreaction vessel. After amplification, each droplet is counted as either positive or negative, and the fraction of positive droplets is converted directly into an absolute number of target DNA copies in the original sample, without the need for a standard curve. This partitioning also dilutes out inhibitors that can derail conventional and real-time PCR reactions, making ddPCR particularly attractive for challenging sample types such as tissue biopsies and archived pathology specimens.

The research team set out with two goals: to validate a novel ddPCR assay capable of detecting O. lupi, and to assess how that assay performed compared with cPCR and qPCR in real clinical cases. The study population consisted of 202 suspected clinical cases submitted from 11 US states, the largest series of suspected O. lupi cases in companion animals assembled in the country to date. Dogs overwhelmingly dominated the caseload, accounting for 97 percent of the samples, or 196 of the 202 animals, while cats made up the remaining 3 percent, or six animals. The samples themselves were diverse: 34 were fragments of adult O. lupi specimens, 103 were subconjunctival nodule biopsies, 23 were interscapular skin snips, three were skin snips from the lower ear, and 39 were formalin-fixed paraffin-embedded tissue samples, the archival material that pathologists routinely generate. Critically, every one of these samples had already been assessed using both cPCR and qPCR, providing a direct head-to-head comparison.

The results were striking. Overall, ddPCR detected O. lupi DNA in 71.3 percent of the clinical cases, identifying 144 of 202 animals as positive. Among the dogs, ddPCR achieved a detection rate of 71.9 percent, or 141 of 196 animals, comfortably outperforming qPCR, which detected 64.8 percent, and conventional PCR, which managed only 54.1 percent. In other words, the digital method found positive results in dozens of animals that the older techniques had called negative. Among the six cats, all three diagnostic tests agreed, confirming infection in half of them. The pattern held across sample types, suggesting that the ddPCR assay’s advantage was not confined to a single kind of specimen but reflected a genuine improvement in analytical sensitivity.

Statistical analysis added nuance to the picture. The researchers used Cohen’s kappa, a measure of agreement between diagnostic methods that corrects for chance agreement, and found moderate agreement between cPCR and qPCR, with a kappa value of 0.74. Agreement between cPCR and ddPCR was fair, at 0.60, while qPCR and ddPCR showed almost perfect agreement, with a kappa of 0.81. A Cochran’s Q test was used to compare the pattern of positive and negative results across the three techniques. These statistics tell a coherent story: ddPCR agrees closely with qPCR but flags additional positives that both older methods miss, consistent with its ability to amplify DNA in samples where inhibitors or very low parasite burdens defeat conventional approaches.

The geographic scope of the study also yielded epidemiological insight. Positive detections were reported in 10 of the 11 states from which samples were submitted, although the authors note that travel history was not always available for the animals, making it difficult to draw firm conclusions about where infections were acquired. The finding nonetheless underscores that suspected O. lupi cases are being seen by veterinary practitioners across a wide swath of the United States, not merely in the southwestern states where the parasite was first documented in North America. For a zoonotic parasite, that broad footprint is a signal that clinicians well beyond the historical endemic zone should keep ocular onchocercosis on their differential lists when dogs and cats present with nodular or inflammatory eye lesions.

One of the assay’s most practical attributes is that it is host-agnostic, meaning it does not depend on the species of the infected animal and can therefore be applied across dogs, cats, and potentially other hosts. Combined with its demonstrated performance across multiple sample types, including the notoriously difficult FFPE archival tissues, this flexibility positions ddPCR as a tool not only for individual clinical diagnosis but also for the kind of large-scale surveillance studies needed to map the parasite’s true distribution. The authors argue that the validated assay supports improved epidemiological understanding and, ultimately, One Health-based prevention strategies that recognize the interconnected health of animals and people.

The study, which was partially funded by the Thomas B. and Jeannette E. Laws McCabe Fund at the University of Pennsylvania and sponsored in part by Elanco Animal Health through the CVBD World Forum Symposium, arrives at a moment when digital PCR is moving from specialized research laboratories into mainstream diagnostic use. For veterinarians confronting a dog with a mysterious conjunctival nodule, the message of this research is concrete: a negative conventional PCR result no longer means the parasite is absent. With a validated ddPCR assay now available, the invisible fraction of O. lupi infections, the subclinical and previously undiagnosed animals that quietly sustain transmission, may finally come into focus, and with it, a clearer picture of how this eye-dwelling worm spreads among the pets and people who share their homes.

Subject of Research: Validation of droplet digital PCR for enhanced molecular diagnosis of zoonotic Onchocerca lupi infection in companion animals

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

Article References: Kelly, M. A., McLean, N. J., Peterson, S., Ferguson, S., Young, T., Foote, B., West, J., Campbell, J., Brown, K., Hakimi, H., Sobotyk, C., Weerarathane, P., & Verocai, G. G. (2026). Enhanced molecular diagnosis of Onchocerca lupi using droplet digital PCR in clinically suspected companion animals. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07605-9

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07605-9

Keywords: Onchocerca lupi, droplet digital PCR, molecular diagnostics, ocular onchocercosis, zoonotic parasites, companion animals, veterinary parasitology, filarial nematode, conventional PCR, real-time PCR, One Health, Parasites & Vectors