Quantitative Real-Time PCR Assays for the Detection of Pathogenic Species in Urine and Blood Samples in Canine Vaccine Clinical Studies: a Rapid Alternative to Classical Culture Methods.

Leptospirosis is a vaccine-preventable bacterial zoonotic disease caused by pathogenic Leptospira species. The efficacy of canine vaccines is assessed by challenging vaccinated and control dogs with virulent serovars of , followed by detection of in blood and urine. We assessed the consistency between results obtained for urine and blood samples from clinical studies with species-specific real-time quantitative PCR (qPCR) targeting the gene and those obtained with the reference culture method. The specificity of the qPCR assay was confirmed by negative results for nonpathogenic and for several canine viruses, bacteria, and parasites. The results from the two methods were compared using McNemar's test, kappa coefficient (κ), and percentage of agreement analyses. The results for numbers of positive and negative dogs were similar, with no false-negative results with the qPCR assay. For both blood and urine, there was strong agreement between the culture method and qPCR results (κ = 0.68 [95% confidence interval (CI), 0.62 to 0.74] and κ = 0.65 [95% CI, 0.59 to 0.71], respectively). However, there was a statistically significant difference between blood samples ( < 0.001) and urine samples ( = 0.028). The negative percentage agreements were 97% and 84% and the positive percentage agreements were 68% and 83% for blood and urine samples, respectively. Although the cell culture method is the recommended gold standard, our results show that qPCR assay is a valid alternative method for the rapid and specific detection of pathogenic spp. in urine and blood samples during vaccine efficacy studies, without loss of sensitivity.