A comparison between polymerase chain reaction (PCR) and traditional techniques for the diagnosis of leptospirosis in bovines

Abstract

Leptospirosis is caused by Leptospira, gram negative spirochaetes whose microbiologic identification is difficult due to their low rate of growth and metabolic activity. In Colombia leptospirosis diagnosis is achieved by serological techniques without unified criteria for what positive titers are. In this study we compared polymerase chain reaction (PCR) with microbiological culture and dark field microscopy for the diagnosis of leptospirosis. Microbiological and molecular techniques were performed on 83 samples of urine taken from bovines in the savannahs surrounding Bogotá in Colombia, with presumptive diagnosis of leptospirosis. 117 samples of urine taken from healthy bovines were used as negative controls. 83 samples were MAT positive with titers ≥ 1:50; 81 with titers ≥ 1:100; and 66 with titers ≥ 1:200. 36% of the total samples (73/200) were Leptospira positives by microbiological culture, 32% (63/200) by dark field microscopy and 37% (74/200) by PCR. Amplicons obtained by PCR were 482 base pair long which are Leptospira specific. An amplicon of 262 base pairs typical of pathogenic Leptospira was observed in 71 out of the 74 PCR positive samples. The remaining 3 samples showed a 240 base pair amplicon which is typical of saprophytic Leptospira. PCR as a Leptospira diagnosis technique was 100% sensitive and 99% specific in comparison to microbiological culture. Kappa value of 0.99 indicated an excellent concordance between these techniques. Sensitivity and specificity reported for MAT when compared to microbiological culture was 0.95 and 0.89 with a ≥ 1:50 cut off. PCR was a reliable method for the rapid and precise diagnosis of leptospirosis when compared to traditional techniques in our study. The research presented here will be helpful to improve diagnosis and control of leptospirosis in Colombia and other endemic countries.