1887

Abstract

Leptospirosis is a widespread zoonotic disease that affects all mammals in different parts of the world. Though there are many commercial kits available for the diagnosis of systemic leptospirosis, the nature of the antigen has not been described. Therefore, identification of a specific antigen is important. Since ocular involvement in leptospirosis has been reported, there is a need to identify and characterize the leptospiral antigen for diagnosis of uveitis associated with past leptospiral infection (leptospiral uveitis) and for confirming the clinical diagnosis. Seven-day-old culture of serovar Patoc was used for preparing the antigen. The present study included serum samples from 81 patients with clinical criteria for leptospiral uveitis, 15 cataract controls and 15 non-leptospiral uveitis controls. Serum samples were assayed by ELISA using our antigenic preparation and by a microscopic agglutination test (MAT) using 19 serovars. The antigen prepared had 280 μg LPS ml and no detectable amount of protein. Silver-staining of SDS-PAGE for protein and LPS, dot blot and Western blot analysis and proteinase K and periodate treatment showed that LPS (13–21 kDa and 28 kDa) in our preparation was the relevant antigen for serodiagnosis. IgG antibodies showed reactivity in both leptospiral uveitis patients and controls. However, on the basis of IgM response to LPS, 48 % of the leptospiral uveitis patients were significantly positive compared with controls; 58 % of leptospiral uveitis patients and none of the controls were positive for MAT. When MAT and IgM ELISA results were considered together, 77 % were significantly positive. LPS is identified as a candidate antigen for serodiagnosis of leptospiral uveitis and has sensitivity and specificity of 48 and 90 %, respectively, in ELISA for IgM antibodies. Confirmation of clinical diagnosis with a specific laboratory test would help to initiate the most appropriate treatment for leptospiral uveitis.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.05120-0
2003-08-01
2024-04-25
Loading full text...

Full text loading...

/deliver/fulltext/jmm/52/8/JM520810.html?itemId=/content/journal/jmm/10.1099/jmm.0.05120-0&mimeType=html&fmt=ahah

References

  1. Adler B, Murphy A. M, Locarnini S. A, Faine S. 1980; Detection of specific anti-leptospiral immunoglobulins M and G in human serum by solid-phase enzyme-linked immunosorbent assay. J Clin Microbiol 11:452–457
    [Google Scholar]
  2. Arimitsu Y, Kobayashi S, Akama K, Matuhasi T. 1982; Development of a simple serological method for diagnosing leptospirosis: a microcapsule agglutination test. J Clin Microbiol 15:835–841
    [Google Scholar]
  3. Blackmore D. K, Schollum L. M, Moriarty K. M. 1984; The magnitude and duration of titres of leptospiral agglutinins in human sera. N Z Med J 97:83–86
    [Google Scholar]
  4. Bloom H, Beier H, Gross H. S. 1987; Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93–99 [CrossRef]
    [Google Scholar]
  5. Chapman A. J, Adler B, Faine S. 1988; Antigens recognised by the human immune response to infection with Leptospira interrogans serovar hardjo . J Med Microbiol 25:269–278 [CrossRef]
    [Google Scholar]
  6. Chu K. M, Rathinam R, Namperumalsamy P, Dean D. 1998; Identification of Leptospira species in the pathogenesis of uveitis and determination of clinical ocular characteristics in south India. J Infect Dis 177:1314–1321 [CrossRef]
    [Google Scholar]
  7. Cumberland P, Everard C. O. R, Levett P. N. 1999; Assessment of the efficacy of an IgM-ELISA and microscopic agglutination test (MAT) in the diagnosis of acute leptospirosis. Am J Trop Med Hyg 61:731–734
    [Google Scholar]
  8. Dana M. R. 2002; Leptospirosis. In Diagnosis and Treatment of Uveitis . pp 273–277 Edited by Foster C. S., Vitale A. T. Philadelphia: W. B. Saunders;
  9. Dubois M, Gilles K. A, Hamilton J. K, Rebers P. A, Smith F. 1956; Colorimetric method for the determination of sugar and related substances. Anal Chem 28:350–356 [CrossRef]
    [Google Scholar]
  10. Eapen C. K, Sugathan S, Kuriakose M, Abdoel T, Smits H. L. 2002; Evaluation of the clinical utility of a rapid blood test for human leptospirosis. Diagn Microbiol Infect Dis 42:221–225 [CrossRef]
    [Google Scholar]
  11. Engstrom S. M, Shoop E, Johnson R. C. 1995; Immunoblot interpretation criteria for serodiagnosis of early Lyme disease. J Clin Microbiol 33:419–427
    [Google Scholar]
  12. Faine S, Adler B, Bolin C, Perolat P. 1999 Leptospira and Leptospirosis , 2nd edn. Melbourne: MediSci;
    [Google Scholar]
  13. Farr R. W. 1995; Leptospirosis. Clin Infect Dis 21:1–8 [CrossRef]
    [Google Scholar]
  14. Flannery B, Costa D, Carvalho F. P. 8 other authors 2001; Evaluation of recombinant Leptospira antigen-based enzyme-linked immunosorbent assays for the serodiagnosis of leptospirosis. J Clin Microbiol 39:3303–3310 [CrossRef]
    [Google Scholar]
  15. Gallagher S. R, Smith J. A. 1994; One-dimensional gel electrophoresis of proteins. In Current Protocols in Immunology pp. 8.4.1–8.4.19 Edited by Coligan J. E., Kruisbeek A. M., Margulies D. H., Shevach E. M., Strober W. Chichester: Wiley;
    [Google Scholar]
  16. Gerhardt W, Keller H. 1986; Evaluation of test data from clinical studies.I. Terminology, graphic interpretation, diagnostic strategies, and selection of sample groups. Scand J Clin Lab Investig Suppl 181:5–45
    [Google Scholar]
  17. Goossens H. A. T, van der Bogaard A. E, Nohlmans M. K. E. 1999; Evaluation of fifteen commercially available serological tests for diagnosis of Lyme borreliosis. Eur J Clin Microbiol Infect Dis 18:551–560 [CrossRef]
    [Google Scholar]
  18. Guerreiro H, Croda J, Flannery B, Mazel M, Matsunaga J, Galvao Reis M, Levett P. N, Ko A. I, Haake D. A. 2001; Leptospiral proteins recognized during the humoral immune response to leptospirosis in humans. Infect Immun 69:4958–4968 [CrossRef]
    [Google Scholar]
  19. Gussenhoven G. C, van der Hoorn M. A, Goris M. G, Terpstra W. J, Hartskeerl R. A, Mol B. W, van Ingen C. W, Smits H. L. 1997; LEPTO dipstick, a dipstick assay for detection of Leptospira -specific immunoglobulin M antibodies in human sera. J Clin Microbiol 35:92–97
    [Google Scholar]
  20. Hauser U, Wilske B. 1997; Enzyme-linked immunosorbent assays with recombinant internal flagellin fragments derived from different species of Borrelia burgdorferi sensu lato for the serodiagnosis of Lyme neuroborreliosis. Med Microbiol Immunol 186:145–151 [CrossRef]
    [Google Scholar]
  21. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
    [Google Scholar]
  22. Levett P. N, Whittington C. U. 1998; Evaluation of the indirect hemagglutination assay for diagnosis of acute leptospirosis. J Clin Microbiol 36:11–14
    [Google Scholar]
  23. Levett P. N, Branch S. L, Whittington C. U, Edwards C. N, Paxton H. 2001; Two methods for rapid serological diagnosis of acute leptospirosis. Clin Diagn Lab Immunol 8:349–351
    [Google Scholar]
  24. Lowry O. H, Rosebrough N. J, Farr A. L, Randall R. J. 1951; Protein measurement with Folin phenol reagent. J Biol Chem 193:265–275
    [Google Scholar]
  25. Magnarelli L. A, Ijdo J. W, Padula S. J, Flavell R. A, Fikrig E. 2000; Serologic diagnosis of Lyme borreliosis by using enzyme-linked immunosorbent assays with recombinant antigens. J Clin Microbiol 38:1735–1739
    [Google Scholar]
  26. Martins M. G, Matos K. T. F, da Silva M. V, de Abreu M. T. 1998; Ocular manifestations in the acute phase of leptospirosis. Ocul Immunol Inflamm 6:75–79 [CrossRef]
    [Google Scholar]
  27. Matsuo K, Isogai E, Araki Y. 2000a; Occurrence of [←3)-β-d-Man p -(1←4)-β-d-Man p -(1←] n units in the antigenic polysaccharides from Leptospira biflexa serovar patoc strain Patoc I. Carbohydr Res 328:517–524 [CrossRef]
    [Google Scholar]
  28. Matsuo K, Isogai E, Araki Y. 2000b; Utilization of exocellular mannan from Rhodotorula glutinis as an immunoreactive antigen in diagnosis of leptospirosis. J Clin Microbiol 38:3750–3754
    [Google Scholar]
  29. Rathinam S. R, Rathnam S, Selvaraj S, Dean D, Nozik R. A, Namperumalsamy P. 1997; Uveitis associated with an epidemic outbreak of leptospirosis. Am J Ophthalmol 124:71–79 [CrossRef]
    [Google Scholar]
  30. Ribeiro M. A, Sakata E. E, Silva M. V, Camargo E. D, Vaz A. J, De Brito T. 1992; Antigens involved in the human antibody response to natural infections with Leptospira interrogans serovar copenhageni . J Trop Med Hyg 95:239–245
    [Google Scholar]
  31. Schmidt B. L, Edjlalipour M, Luger A. 2000; Comparative evaluation of nine different enzyme-linked immunosorbent assays for determination of antibodies against Treponema pallidum in patients with primary syphilis. J Clin Microbiol 38:1279–1282
    [Google Scholar]
  32. Sehgal S. C, Vijayachari P, Sharma S, Sugunan A. P. 1999; LEPTO dipstick: a rapid and simple method for serodiagnosis of acute leptospirosis. Trans R Soc Trop Med Hyg 93:161–164 [CrossRef]
    [Google Scholar]
  33. Silva M. V, Camargo E. D, Batista L, Vaz A. J, Brandao A. P, Nakamura P. M, Negrao J. M. 1995; Behaviour of specific IgM, IgG and IgA class antibodies in human leptospirosis during the acute phase of the disease and during convalescence. J Trop Med Hyg 98:268–272
    [Google Scholar]
  34. Smits H. L, Ananyina Y. V, Chereshsky A. 17 other authors 1999; International multicenter evaluation of the clinical utility of a dipstick assay for detection of Leptospira -specific immunoglobulin M antibodies in human serum specimens. J Clin Microbiol 37:2904–2909
    [Google Scholar]
  35. Smits H. L, van der Hoorn M. A. W. G, Goris M. G. A, Gussenhoven G. C, Yersin C, Sasaki D. M, Terpstra W. J, Hartskeerl R. A. 2000; Simple latex agglutination assay for rapid serodiagnosis of human leptospirosis. J Clin Microbiol 38:1272–1275
    [Google Scholar]
  36. Smits H. L, Eapen C. K, Sugathan S. 8 other authors 2001; Lateral-flow assay for rapid serodiagnosis of human leptospirosis. Clin Diagn Lab Immunol 8:166–169
    [Google Scholar]
  37. Terpstra W. J, Ligthart G. S, Schoone G. J. 1985; ELISA for the detection of specific IgM and IgG in human leptospirosis. J Gen Microbiol 131:377–385
    [Google Scholar]
  38. Towbin H, Staehelin T, Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354 [CrossRef]
    [Google Scholar]
  39. Tsai C. M, Frasch C. E. 1982; A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 119:115–119 [CrossRef]
    [Google Scholar]
  40. Vinh T, Shi M.-H, Adler B, Faine S. 1989; Characterization and taxonomic significance of lipopolysaccharides of Leptospira interrogans serovar hardjo . J Gen Microbiol 135:2663–2673
    [Google Scholar]
  41. Wanyangu S. W, Palmer M. F, Zochowski W. J, Waitkins S. A. 1987; Comparison of the DIFCO and Patoc I slide antigens in the screening of leptospirosis. Comp Immunol Microbiol Infect Dis 10:155–161 [CrossRef]
    [Google Scholar]
  42. Winslow W. E, Merry D. J, Pirc M. L, Devine P. L. 1997; Evaluation of a commercial enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody in diagnosis of human leptospiral infection. J Clin Microbiol 35:1938–1942
    [Google Scholar]
  43. Xu Y, Murray B. E, Weinstock G. M. 1998; A cluster of genes involved in polysaccharide biosynthesis from Enterococcus faecalis OG1RF. Infect Immun 66:4313–4323
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.05120-0
Loading
/content/journal/jmm/10.1099/jmm.0.05120-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error