Comparison of polymerase chain reaction with conventional methods for the diagnosis of leptospirosis

Abstract

A study was undertaken to standardize the peR technique for the diagnosis of leptospirosis and to compare the efficacy of peR with DFM and culture in the diagnosis of leptospirosis. Two sets of primers, namely, primers A and B derived from Leptospira interrogans serovar canicola rrs (16S) gene and G I and G2 derived from genomic library of L. interrogans serovar icterohaemorrhagiae, were evaluated to amplify 12 reference strains of leptospires representing the serovars viz., australis, rachmati.; canicola, grippotyphosa, hardjo, hebdomadis, icterohaemorrhagiae, pomona, poi, pyrogenes, tarassovi and patoc. The primers A and B specifically amplified all the serovars tested, while the primers Gland G2 failed to amplify the serovar patoc. The primers A and B which amplified a 331 base pair fragment of leptospiral DNA were used for the routine detection of leptospires in clinical samples. Restriction enzyme digestion of the primer A and B amplified product with the enzymes Dde I and MnI I and direct sequencing established the identity of the amplified product. A total of 192 samples were collected from different sources like human, dogs and bovines with suspected history of leptospirosis and from rodents. All were tested by peR and the positivity ranged from 33.3 to 54.2 per cent. Of the samples collected 125 samples were tested by all the three techniques viz., peR, DFM and culture and the results were compared. The peR technique was found to be more sensitive, specific and rapid, over conventional methods as it detected 41.6 per cent, compared to 25.6 per cent by DFM and 2.4 per cent by culture, of the samples tested.