Evaluation of a simple and rapid microscopy technique for detection of Leptospira

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Objectives: Determine the usefulness o f Fontana staining m ethod and dark field microscopy to detect Leptospira species in spiked urine and serum M ethods: Leptospira interrogans serovar icterohaemorrhagiae and canicola were cultured in EMJH (EllinghausenMcCullough-Johnson-Harris) medium to obtain 6*108 organisms/ml (McFarland =2). Organisms were spikedito PBS, alkalinized human urine and serum in triplicates, and serial dilutions were made (6*10%) 6x10'organisms/ml). Smears were prepared using 10 pi o f each dilution. Further centrifuged sediments of urine were used to prepare smears. Slides were stained by modified Fontana method as reported by Gangadhar et al. and examined. Number of Leptospires per field was recorded. M otility of Leptospira were observed using light microscope modified with an in-house dark field stop Results: Characteristic morphology o f Leptospires was observed in PBS, uncentrifuged urine and in urine sediment using m odified Fontana silver staining. Leptospires could not be clearly observed in serum. Leptospires could be detected in Fontana stained smears made from cultures o f 6x103 - 6x106 organisms/ml. At a concentration o f 6x103 organisms/mlat least 1-3 organisms/smear could be detected in PBS and urine (centrifuged and uncentrifuged), while an average of 5 organisms/field could be detetcted in a smear made from cultures o f 6 x l0 5 organisms/ml Inhouse darkfield microscopy was only able to detect motile Leptospires in the urine sediment spiked with 6 x l0 6 organisms/ml and not in uncentrifuged urine or PBS. i Conclusions: Modified Fontana staining was found to be a simple and rapid test for detection o f Leptospira in urine with a detection limit of 6000 organisms/ml