Leptospira DNA detection for the diagnosis of human leptospirosis

Abstract

This study aims to analyse PCR applicability to the diagnosis of human leptospirosis and to compare the sensitivity of two primer pairs in urine and blood samples. PCR with G1/G2 and LP1/LP2 primers was specific and able to detect 10pg of DNA by agarose gel and 1pg by hybridization. Twenty-one serovars, representing 20 serogroups of pathogenic leptospires, were amplified with G1/G2 primers. DNA from two non-pathogenic serovars, Andamana and Patoc, was not amplified. For hybridization, one probe employing DNA from most prevalent leptospires (serovars Icterohaemorrhagiae, Copenhageni, and Autumnalis) was chosen in accordance with the microagglutination titres in patient samples. It was observed that not all serovars hybridized with the PCR products of G1/G2 and LP1/LP2 primer amplification, suggesting heterogeneity in the sequence amplified by these primers. G1/G2-primed amplifications of blood and/or urine samples were shown to be significantly more sensitive (57.6%) than the LP1/LP2 primers (33.3%), P=0.04, when positivity of patients is considered. When each primer pair and only urine samples were considered, PCR positivity was higher for G1/G2 primers than for LP1/LP2 (P=0.007). G1/G2 presented greater sensitivity in urine than in blood, and LP1/LP2 presented greater sensitivity in blood than in urine, although these differences were not statistically significant. The positivity of PCR per patient using both primers in blood and/or urine samples was 63.6%, with 84.4% efficiency. PCR was useful for patients without microagglutination detectable antibodies, for whom it was able to diagnose nine out of 11 patients (81.8%).