Clinical evaluation of a validated droplet digital PCR assay for the detection of Ehrlichia canis in ocular and blood specimens from seroreactive dogs.

Abstract

Limited data are available on the diagnostic value of ocular specimens in Ehrlichia canis-infected dogs. This study evaluated the diagnostic sensitivity of conjunctival swabs compared with whole blood in dogs clinically suspected of canine monocytic ehrlichiosis (CME). Forty-one seropositive dogs (as determined by the IDEXX SNAP 4Dx Plus test) exhibiting clinical signs consistent with CME were enrolled. DNA was extracted from all specimens and tested using three PCR methods: conventional PCR (cPCR), TaqMan real-time PCR (qPCR), and a previously validated droplet digital PCR (ddPCR) targeting the disulfide bond formation protein (dsb) gene. In blood samples, cPCR, qPCR, and ddPCR detected E. canis DNA in 22/41 (53.7%), 25/41 (61.0%), and 33/41 (80.5%) cases, respectively. In conjunctival swabs, detection rates were 0/41, 11/41 (26.8%), and 27/41 (65.9%), respectively. All PCR methods showed higher sensitivity in blood than conjunctival samples, with ddPCR yielding the highest overall detection. Bacterial load in blood was significantly higher than in conjunctival swabs (median 2.68 vs. 0.76 copies/&#xb5;L, P&#x202f;<&#x202f;0.01). ddPCR detection in blood showed moderate agreement with qPCR (&#x3ba; = 0.55, 95% CI; 0.28-0.82, P&#x202f;<&#x202f;0.01) and cPCR (&#x3ba; = 0.44, 95% CI; 0.2-0.68, P&#x202f;<&#x202f;0.01). In conjunctival swabs, ddPCR showed fair agreement with qPCR (&#x3ba; = 0.32, 95% CI; 0.15 - 0.53, P&#x202f;<&#x202f;0.01). Higher blood E. canis bacterial loads were significantly associated with lower hematocrit (HCT) (mean 29%, P&#x202f;=&#x202f;0.03) and lower intraocular pressure (IOP; median 9&#x202f;mmHg, P&#x202f;=&#x202f;0.04). In conclusion, ddPCR proved to be a highly sensitive and precise diagnostic tool for detecting E. canis. Although conjunctival swabs are less invasive, their diagnostic yield requires highly sensitive detection tools. Notably, the level of E. canis DNAemia was associated with the severity of anemia and reduced IOP, highlighting the potential of ddPCR for assessing disease severity in CME.