Detection of leptospires from infected urine and tissue samples in vitro by modified Fontana silver stain

Abstract

Introduction: The microbiological diagnosis of leptospirosis comprises bacteriological and serological methods. The former ones allow the direct detection of leptospires and are considered presumptive with the exception of culture. Therefore, they constitute invaluable tools for rapid diagnosis, mainly in samples from deceased subjects. Objective: To evaluate a modified Fontana silver staining method in experimentally infected samples. Material and methods: Human and animal (hamster) urine samples were experimentally infected with different strains of Leptospira interrogans sensu lato. Liquid culture medium, leptospira cultures, experimentally infected and non-infected human urine samples, clarified and non-clarified imprints, and clarified and non-clarified suspension smears from tissues of experimentally infected and non-infected hamsters were applied for the ass essment of silver staining. The analytical sensitivity of the assay was compared with dark field microscopy and culture. Other bacterial and fungi species were also used. Results: The modified Fontana silver staining allowed the accurate observation of the well-defined leptospire helical structure. On leptospire cultures from infected human samples, we could observe until (1-10) × 10 3 leptospires/ml, higher sensitivity in comparison with direct dark field microscopy and lower in comparison with culture. The best results in tissues were obtained on clarified imprints and non-clarified suspension smears. Morphological and stainable structures compatible with leptospires were not observed in the samples without them. Conclusion: This procedure allowed differentiating the characteristic morphology of leptospires. As its application suggests, it consists of a simple and easily conducted procedure with stable reagents.

Detection of Leptospira in cattle and dogs in Dakahlia Governorate

“…Here, Leptospires were detected in urine smears stained by Fontana Method as long, thin, brownish black spirochaetes against yellow background. This is in agreement with previous studies by (Rodríguez et al, 2013;Gunasekara et al, 2017).…”

“…Leptospira is thin and spiral bacteria, which cannot be stained by the Gram stain. Silver Impregnation Method "Fontana Method" is used for staining smears by deposition of silver on Leptospira, which leads to increase its thickness, and then Leptospira can be demonstrated by light microscopy as brownish black organisms against yellow background (Gunasekara et al, 2017;Lather et al, 2009;Rodríguez et al, 2013). Fontana Method is a relatively inexpensive method, the stained bacteria can be observed by using the ordinary light microscope, which is available in a basic laboratory setting (Gunasekara et al, 2017).…”

Utility of a modified silver staining technique for detection of Leptospira

“…14 Silver stain impregnation staining methods can be applied to body fluids for detection of leptospires. In this technique, chemically reducing surface properties of leptospires reduce silver ion to its metallic state, 12 where the deposition appear as black or brown in colour. This enables visualization of the slender spiral bacteria using a conventional light microscope.…”

“…Similar to our study they were able to detect presence of leptospires by staining in smears containing >10 3 organisms/ml and found 100% sensitivity at and above the concentration of (1-10) × 10 5 organisms/ml in culture, natural urine (uncentrifuged and sediment) and alkalinized urine (uncentrifuged and sediment) . 12 Live leptospires are generally visualized by dark ground microscopy, while phase contrast microscopy is also a possible means of visualization.…”

“…12,13 Briefly, smears were fixed by dipping in a fixing agent for two minutes containing 1 ml of Glacial acetic acid(Merck) and 2 ml of 40% formaldehyde (Sigma) solution made up to 100 ml in distilled water. The slides were removed using a pair of forceps and blotted onto a tissue paper.…”