Identification and evaluation of LPS antigen for serodiagnosis of uveitis associated with leptospirosis

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 Leptospira biflexa 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 ìg LPS ml À1 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.

Serological diagnosis of leptospiral uveitis by HbpA IgG ELISA

“…The endemic nature of the disease accounts for the values observed in groups III, IV and V. Sera from all five groups were tested by the conventional 'gold standard' MAT. The predominant infecting serogroups (Icterohaemorrhagiae, Australis and Autumnalis) in MAT-positive leptospiral uveitis (group I) were similar to those reported for systemic leptospirosis patients, in agreement with our earlier reports (Priya et al, 2003(Priya et al, , 2007. Patients with systemic leptospirosis (group II), all of whom were MATpositive, showed high levels of anti-HbpA IgG antibodies, as expected.…”

“…The failure of MAT to identify all clinically proven cases of leptospiral uveitis, as seen in this study and in an earlier study (Priya et al, 2003), both showing a low specificity of 58 %, indicates strongly that MAT cannot be used as the gold standard for diagnosis of leptospiral uveitis, as is currently being done (15) for systemic leptospirosis. There are also reports of the low sensitivity of MAT in diagnosing the latter, even at the optimal stage of systemic leptospiral infection (Limmathurotsakul et al, 2012;Goris et al, 2012).…”

“…The end point was taken as the highest dilution of the serum in which 50 % of the organisms were agglutinated or in which there was a 50 % reduction in the number of organisms compared with the control. Titres ¢1 : 100 were considered as positive (Terpstra et al, 1985;Priya et al, 2003).…”

Early Diagnosis of Leptospirosis by Immunoglobulin M Immunoblot Testing

“…Sera were obtained from patients with acute febrile illness on the day of first visit, day of discharge, and 14 days after the first visit to Loei Provincial Hospital during the period from July through October 2002 (35). A total of 188 serum samples were obtained from 74 patients with leptospirosis confirmed by either culture isolation, MAT, or both.…”

LruA and LruB Antibodies in Sera of Humans with Leptospiral Uveitis

“…Clinical diagnosis of leptospiral uveitis was based on a detailed clinical history, an extensive review of systems, a complete ophthalmic examination, and laboratory and ancillary tests. After clinical diagnosis, MAT was performed following standard procedures (7), using 19 leptospiral serovars obtained from the Royal Tropical Institute, The Netherlands (19). As the sensitivity of the MAT is low, a set of clinical diagnostic predictors, identified using samples from more than 500 seropositive leptospiral uveitis and 4,800 nonleptospiral uveitis cases by multiple logistic regression analysis (21), was used to identify seronegative leptospiral uveitis cases.…”