Utility of a modified silver staining technique for detection of Leptospira

Background: Leptospira are spiral thin and highly motile pathogenic bacteria that are best visualized by dark ground microscopy. Although these bacteria are not stained by the Gram’s stain, the Fontana stain, which is a silver impregnation staining method, can be used successfully for light microscopy. It is important to investigate the usefulness of Fontana stain method for direct demonstration of Leptospira in human body fluids. Objectives: To determine the usefulness and sensitivity of a modified Fontana silver staining method for microscopic detection of Leptospira in clinical specimens. Methodology: 6×10 8 organisms/ml of Leptospira interrogans serovar Icterohaemorrhagiae and Canicola were spiked into PBS (Phosphate Buffered Saline), alkalinized urine and serum in triplicate and serial dilutions were made (6×10 6 to 6×10 1 organisms/ml). Smears were prepared using 10 µl of each dilution. In addition, centrifuged sediment of urine were also used to prepare smears. Slides were stained by modified Fontana method as reported by Gangadhar et al.(1998) and examined. Numbers of leptospires per field (×100) were recorded. Results: Leptospira spiked in PBS and urine appeared as thin slender bacteria with characteristic hooked ends after Fontana staining under the light microscope. Serum could not be used for the detection of Leptospira by this method. Leptospires could be detected by staining the spiked PBS and urine at 6x10 3 – 6x10 6 organisms/ml. Conclusion: Leptospires could be detected by Fontana staining in spiked PBS, urine (uncentrifuged and sediment). Serum was not suitable for detection of leptospires by Fontana staining. The detection limit of leptospires in PBS and urine by Fontana stain was found to be 6000 organisms/ml.


INTRODUCTION
Leptospirosis is a potentially fatal zoonotic infection with a worldwide distribution, caused by Leptospira species. 1,2 Leptospirosis is endemic in Sri Lanka with outbreaks of disease generally associated with the paddy cultivation seasons and monsoon periods. Many mammalian species can harbor leptospires in their kidneys and act as reservoirs for transmission of infection. Rodents, cattle, buffaloes, horses, sheep, goats, pigs and dogs are the most common reservoirs. [3][4][5] The disease is transmitted mainly by direct or indirect contact with water sources or soil contaminated with urine of infected animals. 6 Major life threatening complications of leptospirosis reported in recent outbreaks in Sri Lanka which include renal failure 7 , haemorrhage into most organs and tissues and multi organ failure. 8 Early diagnosis of leptospirosis is critical as early administration of antimicrobials improve prognosis. Laboratory diagnosis of leptospirosis involves culture, Polymerase Chain Reaction (PCR), Microscopic Agglutination Test (MAT) and detection of leptospires in clinical samples by histological, histochemical or immuno staining techniques. 9 However, the availability of these diagnostic opportunities are limited to specialized laboratories and are expensive and time consuming. Rapid immunochromatographic assays to detect Leptospira specific IgM antibodies are useful in presumptive diagnosis of leptospirosis 10 but are not available in the public sector in Sri Lanka. Due to the unavailability of early diagnostic assays currently in Sri Lanka, the diagnosis of leptospirosis is mainly based on clinical judgment.
Direct demonstration of Leptospira in clinical specimens by microscopy can provide an early and rapid diagnostic opportunity. However observation of live motile leptospires require the use of a dark ground microscope and can be unreliable due to presence of leptospira like artifacts in the specimen. 11 Leptospires are spiral and thin bacteria which cannot be stained by the Gram stain. However, leptospires in body fluids and tissues can be stained and visualized using silver impregnation methods. The modified Fontana technique can be used for staining of leptospira and is a relatively inexpensive method where stained organisms can be observed using a conventional light microscope which is available in a basic laboratory setting. In this study, we had evaluated the usefulness of a modified Fontana silver staining technique by Gangadhar and Rajsekhar(1998) 12,13 for detection of leptospires in urine and serum.

Objective
To determine the usefulness of a modified Fontana silver staining method for observation of morphology and enumeration of Leptospira.

Materials and Methods:
Bacterial Strains and specimens Cultures of Leptospira interrogans serovar Icterohaemorrhagiae and Canicola were obtained from the National Leptospira Reference Laboratory, Medical Research Institute (MRI), Colombo. Stock cultures were maintained at room temperature in Ellinghausen-McCullough-Johnson-Harris(EMJH) medium.
Preparation of Leptospira spiked urine and serum specimens Confluent bacterial cultures of Leptospira interrogans serovar Icterohaemorrhagiae and Leptospira interrogans serovar Canicola cultured in EMJH medium were used for the experiments. Optical Density (OD) of cultures were measured using spectrophotometer at 420 nm and OD was adjusted to McFarland 2 which is equivalent to 6 × 10 8 organisms/ ml. Serial tenfold dilutions of Leptospira cultures were prepared in PBS, serum and urine in triplicates.
Fresh urine samples were alkalinized using 4 N NaOH until pH was 6-7 and added in triplicate to two sets of tubes for spiking. A serum specimen which tested negative for Leptospira IgM and IgG was used for spiking. Tenfold serial dilutions of leptospires were prepared in PBS, urine and serum. Spiked urine specimen was used as follows: one set of spiked urine was used for preparation of smears while the other set was centrifuged at 3000 rpm for 5 minutes. The centrifuged sediments were used for preparation of smears. Smears were prepared using 10 µl of urine suspension or sediment on pre-labeled clean glass slides and air dried. Experiments were repeated three times to ensure reproducibility.
Modified Fontana silver staining Technique Smears were stained using modified Fontana silver staining technique as described by Gangadhar and Rajsekhar (1998). 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. Subsequently, the slides were dipped in absolute alcohol for 3 minutes. The under surface of the slides were cleaned using filter paper and the smears air dried. The slides were dipped in the mordant containing 1 g phenol (Sigma) and 5 g tannic acid (BDH) made upto 100ml with distilled water, in a hot water bath pre-heated to 75 º C and kept for 1 minute. The slides were rinsed in distilled water and dipped in ammoniated silver solution, which was pre-heated to 75 º C in a hot water bath and kept for 1 minute. The ammoniated silver solution was prepared by dissolving 0.6g of silver nitrate(Sigma) in 100ml of distilled water. Sixty ml aliquot of this solution was mixed with few drops of 10% ammonia solution and shaken to obtain a brown precipitate. Further ammonia solution was added till the precipitate dissolved. Finally small quantities of silver nitrate solution were added from the 40 ml aliquot to the above ammoniated solution till a stable precipitate reappeared.
After rinsing the slides in distilled water,the slides were air dried and examined under oil immersion objective of a bright field microscope.

Results
Fontana staining method of leptospires resulted in dark brown stained bacteria in a white or pale yellow background. The Fontana stain revealed thin and spiral organisms usually containing one or two hooked ends when observed under 100x. (Figure 1).The morphology of leptospires were clearly visible in smears prepared from spiked PBS and spiked urine specimens. However it was not possible to identify Leptospires spiked to serum using the modified Fontana silver stain method due to the high background staining which was attributed to the high protein content of the serum. Due to the nonspecific staining it was not possible to differentiate the leptospires from the background in serum.
Leptospires could be detected in Fontana stained smears made from spiked cultures containing 6x103-6x10 6 organisms/ml. At a concentration of 6x10 3 organisms/ml, at least 1-3 organisms/smear could be detected in PBS and urine (centrifuged and uncentrifuged), while an average of 5 organisms/field could be detected in a smear made from spiked cultures of 6 x 10 5 organisms /ml ( Table 1). The results indicate that at least 6000 organisms/ml should be present in the urine for detection by Fontana staining when examining the whole smear. It is recommended to observe the whole smear in order to improve the sensitivity of the method.

Discussion
Rapid, simple to perform and inexpensive diagnostic tests which can be carried out in a resource poor setting will be of great value to support the clinical diagnosis in developing countries, enabling initiation of antimicrobial therapy in the early disease stages. The Microscopic Agglutination Test is considered as the gold standard for diagnosis of leptospirosis. 9 This test is currently available only at the national leptospirosis reference center (MRI, Colombo). MAT requires culture and maintenance of live leptospira strains and is a technically demanding procedure. PCR which can detect Leptospira specific DNA and isolation of leptospira by culture are also technically demanding and expensive. Furthermore, culture may take upto one month to become positive, 6 thereby limiting its usefulness in clinical practice.

A B C
Direct demonstration of leptospira in clinical specimens can be useful in early diagnosis. Light microscopy for detection of Leptospira has been reported by Babudieri (1965) using Giemsa stain and silver stain. 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. However the stain will also interact with protein fibrils which can be misinterpreted as leptospires during microscopy. In addition, in protein rich specimens (serum) or in the presence of cells (urine sediment), the strong colour of silver staineddeposits will mask the fine slender stained leptospires. Therefore observation of leptospires in such smears will be limited to areas containing thin smears with low background staining.
In this study, depending on the concentration of organisms spiked to the test sample, it was necessary to observe the whole smear in samples containing 6000 organisms/ml and at least an average of 30 fields when the specimen contained 6 x 10 4 organisms/ml for identification. The stain enabled observation of the typical hooked ends of Leptospira interrogans which supported the microscopic identification of the organism. Using the modified Fontana staining of the two serotypes, Canicola and Icterohaemorrhagiae, visualization of the latter was clear compared to Canicola which may be due to the difference in the staining properties and size variation.
Quantitative PCR has demonstrated between 10 2 to 10 6 leptospira/ml in blood 8 and between 10 1 to 10 6 leptospira/ml in urine 15 . The bacteria are known to colonize the proximal renal tubules of kidneys and are excreted intermittently in the urine from the second week onwards up to a month or even upto one year. 16 In our study, the ability to detect above 6000 organisms/ml in urine using the modified Fontana staining suggests its possible diagnostic utility although having a low sensitivity. A study by Rodriguez et al (2013) is the only recent study reporting the usefulness of the modified Fontana method developed by Gangadhar and Rajsekhar (1998). In their study, serial dilutions of Leptospira were spiked into urine and results of modified Fontana staining was compared with culture and dark field microscopy. 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. 6 Dark ground microscopy may be used for detection of live motile leptospires in body fluids. However the presence of lysed red blood cells (RBC), fibrils and other bacteria may give false positive results by this method, thereby limiting its usefulness in diagnosis. Further, dark ground microscopy is unavailable in most laboratories due to the high cost, expense and limited utility. The diagnostic utility of dark ground microscopy is therefore low. The results of the present study suggest that the modified Fontana stain can be useful in resource poor settings as a rapid and low cost preliminary test for diagnosis of leptospirosis.
However the silver impregnation staining method cannot be used for quantitative enumeration as the smear may vary in thickness giving high background in different areas limiting the observation of leptospires. Serum or blood specimens cannot be used for modified Fontana staining as it is high in protein content. Serum specimens gave a high background by modified Fontana stain and therefore investigators were unable to observe the leptospires in smears of spiked serum in comparison with smears made from leptospira spiked urine specimens. On the other hand urine specimens also contain some proteins and there is a possibility of false positive results. The intermittent secretion of leptospires in urine is another limiting factor which may give rise to false negative results. However it should be considered that in the absence of the availability of rapid diagnostic assays, modified Fontana stain may offer supportive evidence for diagnosis in patients with high leptospiraemia.
In conclusion, the modified Fontana staining method is a simple and rapid test for detection of leptospira in urine. This method enabled microscopic observation of 6000 organisms/ml in PBS, and urine (uncentrifuged specimen and centrifuged sediment).