Evaluation of recombinant Leptospira antigen-based enzyme-linked immunosorbent assays for the serodiagnosis of leptospirosis

Abstract

There is an urgent need for development of new serodiagnostic strategies for leptospirosis, an emerging zoonosis with worldwide distribution. We have evaluated the diagnostic utility of five recombinant antigens in enzyme-linked immunosorbent assays (ELISAs) for serodiagnosis of leptospirosis. Sera from 50 healthy residents of a high-incidence region were used to determine cutoff values for 96% specificity. In paired sera from 50 cases of leptospirosis confirmed by the microscopic agglutination test, immunoglobulin G (IgG) but not IgM reacted with the recombinant leptospiral proteins. The recombinant LipL32 IgG ELISA had the highest sensitivities in the acute (56%) and convalescent (94%) phases of leptospirosis. ELISAs based on recombinant OmpL1, LipL41, and Hsp58 had sensitivities of 16, 24, and 18% during the acute phase and 72, 44, and 32% during convalescence, respectively. Compared to sera from healthy individuals, patient sera did not react significantly with recombinant LipL36 (P > 0.05). Recombinant LipL32 IgG ELISA demonstrated 95% specificity among 100 healthy individuals, and specificities ranging from 90 to 97% among 30 dengue patients, 30 hepatitis patients, and 16 patients with diseases initially thought to be leptospirosis. Among 39 Venereal Disease Research Laboratory test-positive individuals and 30 Lyme disease patients, 13 and 23% of sera, respectively, reacted positively with the rLipL32 antigen. These findings indicate that rLipL32 may be an useful antigen for the serodiagnosis of leptospirosis.

FIG. 1

Performance of IgG ELISA at different serum sample dilutions and recombinant protein concentrations. Results are shown for assays with three selected recombinant leptospiral proteins, rHsp58 (left), rLipL32 (center), and rOmpL1 (right). For each point, a mean absorbance value (OD₄₅₀ on the vertical axis) and standard deviation were plotted for serum samples from eight patients with confirmed leptospirosis (solid line) or four healthy control individuals (dashed line). Reactions for wells coated with 5, 25, 50, or 100 ng of recombinant antigen are represented on the horizontal axis. Plates were incubated with sera that were diluted 50- (first row), 100- (second row), or 200- (third row) fold.

FIG. 2

IgG antibody responses in leptospirosis patients and control groups to recombinant leptospiral proteins as determined by ELISA. Results are shown for three selected recombinant antigens, at concentrations of 25 ng of antigen per well (rHsp58 and rOmpL1) or 5 ng of antigen per well (rLipL32). Absorbance (OD₄₅₀) values are shown for reactions with serum samples, diluted 50-fold, from healthy individuals in an endemic region for leptospirosis in Salvador, Brazil (group A, □), or a nonendemic U.S. region (group B, Δ) compared to paired serum samples from 50 patients with confirmed leptospirosis from Salvador, Brazil (group C, acute-phase ● or group D, convalescent-phase ▪). Fifty samples were tested in each group. Median absorbance values for each group of samples are shown as solid lines. The cutoff value, defined as the absorbance value for the 96th percentile of the group of 50 serum samples from healthy control individuals from Salvador, Brazil, are represented as dashed lines.