A comparison of diagnostic methods for canine Ehrlichiosis: Microscopy and RNases hybridization-assisted amplification technology compared with the quantitative polymerase chain reaction.

Background and aim: Canine vector-borne pathogens, particularly blood parasites, pose significant health threats to domestic dogs, ranging from subclinical infections to severe systemic diseases. In Thailand, microscopic examination remains the standard diagnostic method, despite its limitations. This study aimed to (i) determine the prevalence of major canine vector-borne pathogens in Bangkok, Thailand during the 2024 rainy season and (ii) evaluate the diagnostic performance of microscopy and a novel RNase hybridization-assisted amplification (RHAM) test kit in detecting canine Ehrlichiosis, compared to the quantitative polymerase chain reaction (qPCR) gold standard.

Materials and methods: A total of 134 whole blood samples were collected from clinically suspected dogs. Microscopy was performed on Giemsa-stained blood smears, and the RHAM test kit was employed for nucleic acid detection. qPCR served as the reference method. Sensitivity, specificity, accuracy, and precision of each diagnostic method were calculated relative to qPCR results.

Results: Microscopic examination revealed the following infection prevalences: Ehrlichia spp. (26.12%), Babesia spp. (4.48%), Hepatozoon canis (6.72%), Anaplasma spp. (0.75%), Dirofilaria immitis (3.73%), and Brugia spp. (3.73%). Compared with qPCR, microscopy demonstrated a sensitivity of 51.47%, specificity of 87.88%, accuracy of 69.40%, and precision of 81.39% for Ehrlichiosis detection. In contrast, the RHAM test kit achieved markedly higher diagnostic metrics: Sensitivity (91.18%), specificity (98.48%), accuracy (94.78%), and precision (98.41%). Notably, the RHAM kit provided rapid, user-friendly detection, approximating qPCR diagnostic performance, although its sensitivity slightly declined in samples with very low pathogen titers.

Conclusion: This study highlights the continued high prevalence of Ehrlichiosis among dogs in Bangkok during the rainy season. Although microscopy remains practical, its diagnostic limitations are significant. The RHAM test kit demonstrated excellent sensitivity and specificity, offering a rapid and accurate alternative for Ehrlichiosis detection, particularly suitable for resource-limited settings lacking qPCR capabilities. Adoption of the RHAM assay could improve early diagnosis and management of canine Ehrlichiosis at grassroots veterinary facilities.