The Use of Immunofluorescence in the Diagnosis of Human Leptospirosis by A Genus-Specific Antigen

Torten, M.; Shenberg, E; Hoeden, J. Van Der

doi:10.1093/infdis/116.5.537

Cited by 34 publications

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“…Several other alternatives of antibody detection assays have subsequently been developed for early diagnosis of leptospirosis. These include the hemolytic test (8), the slide agglutination test (14), the indirect hemagglutination assay (19,28), the indirect immunofluorescence test (32), the microcapsule agglutination test (2), the indirect enzyme-linked immunosorbent assay (ELISA) for immunoglobulin M (IgM) antibodies (1,22), the dot-ELISA for IgM (23,26,36), the LEPTO Stick (15), and the lateral flow assay (27). While these methods are much simpler than the MAT and many of them are currently available commercially, they still need a lag period after the infection before the antibodies become detectable, and once incited, the antibodies stay for a long time, even after the pathogenic organisms have been eliminated.…”

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confidence: 99%

Diagnosis of Human Leptospirosis by Monoclonal Antibody-Based Antigen Detection in Urine

Saengjaruk¹,

Chaicumpa²,

Watt

et al. 2002

J Clin Microbiol

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Hybridomas secreting specific monoclonal antibodies (MAb) to all members of the genus Leptospira (clone LF9) and those that are specific only to the pathogenic species (clones LD5 and LE1) were produced. MAb LF9, which was immunoglobulin G1 (IgG1), reacted to a 38-kDa component of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell lysates of all Leptospira spp., while MAb LD5 and MAb LE1, which were IgG1 and IgG2a, respectively, reacted to the 35-to 36-kDa components of all serogroups of the pathogenic species of Leptospira. The MAb LD5 was used in a dot blot-enzyme-linked immunosorbent assay (dot-ELISA) for detecting Leptospira antigen in urine samples serially collected from two groups of patients diagnosed with leptospirosis, i.e., 36 clinically diagnosed patients and 25 Leptospira culture confirmed patients. Their serum samples were tested serologically by IgM Dipstick assay, indirect immunofluorescence assay (IFA), and/or microscopic agglutination test (MAT). Urine samples of 26 patients diagnosed with other illnesses and 120 healthy individuals served as controls. For the first group of patients, who had been ill for an average of 3.4 days before hospitalization, the IgM Dipstick test, IFA, and MAT were positive for 69.4, 70.0, and 85.7% of patients, while the Leptospira antigenuria tested by the MAb-based dot-ELISA was positive for 75.0, 88.9, 97.2, 97.2, and 100% of patients on days 1, 2, 3, 7, and 14 of hospitalization, respectively. All but 1 of 11 patients whose serum samples collected on the first day of hospitalization were IgM seronegative, were positive by urine antigen test on day 1. This is strong evidence that detection of antigen in urine can provide diagnostic information that could be useful in directing early therapeutic intervention. The MAT was positive in 10 of 12 patients (83.3%) of the 25 culture-positive Leptospira patients who had been ill for an average of 5.04 days before hospitalization, and the Leptospira antigen was found in 64.0, 84.0, 96.0, 100, 100, 100, and 100% of the patients' urine samples collected on days 1, 2, 3, 4, 5, 6, and 7 of hospitalization, respectively. Leptospira antigenuria was found in 3 of the 26 patients diagnosed with other illnesses and 1 of the 120 healthy controls. The reasons for this positivity are discussed. The detection of antigen in urine by the monoclonal antibody-based dot-ELISA has high potential for rapid, sensitive, and specific diagnosis of leptospirosis at a low cost.

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confidence: 99%

Diagnosis of Human Leptospirosis by Monoclonal Antibody-Based Antigen Detection in Urine

Saengjaruk¹,

Chaicumpa²,

Watt

et al. 2002

J Clin Microbiol

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“…Complement fixation test (Wolff, 1954), Enzyme linked immunosorbent assay (Vanasco et al, 2001), the macroscopic agglutination test (Galton et al, 1958), the microcapsule agglutination test (Cui et al, 1991), the indirect hemagglutination assay (Levett and Whittington, 1998) the dipstick assay (Smits et al, 1999) are some of the methods and other methods (Torten et al, 1966). The Polymerase chain reaction based diagnostic tests for leptospirosis are speed, sensitive and an indication of current infection status (Zuerner and Bolin, 1997;Zuerner et al, 1995) each assay has its own advantages, drawbacks and limitations Levett, 2001).…”

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confidence: 99%

Characterization, Comparison Study of Outer Membrane Protein OMPL1 of Pathogenic Leptospira Species for Disease Diagnosis

Muthiah¹,

Kumar²,

Ramadass³

2015

Research J. of Microbiology

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Leptospirosis is an acute bacterial infection which affects humans and a wide range of animals this bacterial disease is caused by pathogenic members of the genus Leptospira. It is now known that this is only one of more than 200 pathogenic serovars distributed between 7 species and 23 serogroups worldwide. Commercial leptospiral vaccines rely on bacterins and inactivated whole cells. A major component of immunity resulting from whole cell vaccines is a humoral immune response resulted against the serovar specific carbohydrate antigen of leptospiral lipopolysaccharide. Cross protection against many of the 250 serovars of pathogenic Leptospira species is lacking though the immunity generated is readily demonstrable. Hence there is an urgent need for development of an improved leptospiral vaccine. Variations in the carbohydrate side chains of LPS are responsible for the antigenic diversity observed among Leptospiral serovars. The present study was undertaken to develop a recombinant fusion protein from Leptospira as antigen by the following processes. Identification and purification of gene encoding the leptospiral outer membrane OMPL1 by PCR. Cloning and expression of the OMPL1 gene in a suitable vector system. Purification and characterization of the expressed recombinant leptospiral outer membrane protein OMPL1. Standardization of recombinant antigen based diagnostic study.

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“…Among them are the complement fixation test (33), several enzyme-linked immunosorbent assay formats (1,29), the macroscopic slide agglutination test (14), the microcapsule agglutination test (9), the indirect hemagglutination assay (20), the dipstick assay (27), and other methods (3,15,22,30). Each assay has its own advantages, drawbacks, and limitations (4,18).…”

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New Approach for Serological Testing for Leptospirosis by Using Detection of Leptospira Agglutination by Flow Cytometry Light Scatter Analysis

Yitzhaki¹,

Barnea²,

Keysary³

et al. 2004

J Clin Microbiol

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Leptospirosis is considered an important reemerging infectious disease worldwide. The standard and most widespread method for the diagnosis of leptospirosis is the microscopic agglutination test (MAT). This test is laborious and time-consuming, and the interpretation of the results is subjective. In the present work we describe an application of flow cytometry (FCM) as a tool for the serological diagnosis of leptospirosis. The analysis is based on the sensitivity of FCM to the size and shape of the bacteria analyzed by measurement of light scatter parameters: forward scatter (FSC) and side scatter (SSC). The addition of positive serum to an infecting leptospiral serovar results in a shift of the light scatter parameter to a different location with higher FSC and SSC values, indicating the formation of leptospiral aggregates. By using immunofluorescent staining, we have shown that the large particles formed are the agglutinated leptospires. Quantification of the agglutination process has been achieved by calculating an agglutination factor (A f ), based on changes in the light scatter parameters measured by FCM. A f enables us to determine the specificity of the serological reaction of the patient serum with each leptospiral serovar. In this work, 27 serum samples from 18 leptospirosis patients were tested by both the MAT and the FCM techniques, in which each serum sample was tested against 13 serovars. Twenty-six human serum samples derived from patients with a variety of other defined illnesses were used as negative controls and enabled us to define the A f threshold value as <9.3 for negative patients, while any value higher than that would be a positive result for leptospirosis. Compared to MAT, the FCM technique was found to be more specific and sensitive, especially in identifying the serogroup in the acute phase of the disease. The whole process was found to be rapid and took less than 1.5 h. Moreover, FCM analysis is objective and can be automated for the handling of large numbers of samples.

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The Use of Immunofluorescence in the Diagnosis of Human Leptospirosis by A Genus-Specific Antigen

Cited by 34 publications

References 0 publications

Diagnosis of Human Leptospirosis by Monoclonal Antibody-Based Antigen Detection in Urine

Diagnosis of Human Leptospirosis by Monoclonal Antibody-Based Antigen Detection in Urine

Characterization, Comparison Study of Outer Membrane Protein OMPL1 of Pathogenic Leptospira Species for Disease Diagnosis

New Approach for Serological Testing for Leptospirosis by Using Detection of Leptospira Agglutination by Flow Cytometry Light Scatter Analysis

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