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

Abstract

Leptospirosis is a vaccine-preventable bacterial zoonotic disease caused by pathogenic Leptospira species. The efficacy of Leptospira canine vaccines is assessed by challenging vaccinated and control dogs with virulent serovars of Leptospira, followed by detection of Leptospira 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 lipL32 gene and those obtained with the reference culture method. The specificity of the qPCR assay was confirmed by negative results for nonpathogenic Leptospira 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 (P < 0.001) and urine samples (P = 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 Leptospira spp. in urine and blood samples during vaccine efficacy studies, without loss of sensitivity.

Keywords: clinical study; lipL32 gene; pathogenic Leptospira spp.; real-time PCR.

FIG 1

Schematic representation of the onset of immunity and duration of immunity clinical study design. Time point zero (T0) was 2 weeks after the second injection of L3 or 3 weeks after Veriscan vaccination for the onset-of-immunity (OOI) studies and about 1 year for the duration-of-immunity (DOI) studies. Red arrows represent time points for blood samples, and yellow arrows represent time points for urine samples. Lc, Leptospira interrogans serovar Canicola; Lg, Leptospira interrogans serovar Grippotyphosa; Li, Leptospira interrogans serovar Icterohaemorrhagiae.

FIG 2

Results from six onset of immunity studies in which dogs were challenged with L. Canicola (Lc), L. Grippotyphosa (Lg), or L. Icterohaemorrhagiae (Li), as indicated. Number of positive dogs per study group with leptospirae in blood and urine samples detected using culture method (white bars) and quantitative PCR (qPCR) assay (black bars).

FIG 3

Example of results with culture method (white bars) and qPCR assay (black bars) in blood and urine samples from one of the duration-of-immunity studies (study 7), in which dogs were challenged with L. Grippotyphosa (Lg). The numbers of positive dogs at each time point in the vaccinated and control groups are shown.