Early diagnosis of leptospirosis by immunoglobulin M immunoblot testing

Abstract

There is an urgent need for the development of serodiagnostic approaches with improved sensitivity for patients with acute leptospirosis. Immunoblots were performed on 188 sera collected from 74 patients with laboratory-confirmed early leptospiral infection to detect immunoglobulin M (IgM) antibodies to antigens pooled from 10 leptospiral strains prevalent in Thailand. Sera from patients with other febrile diseases served as controls. IgM reactivity to seven distinct antigens, with apparent molecular masses of 14 to 18, 19 to 23, 24 to 30, 32, 35/36, 37, and 41/42 kDa, was observed. The low-molecular-mass 14- to 18-kDa band was the most frequently detected antigen, being recognized in sera from 82.4% of patients during the first 3 days after the onset of symptoms. We evaluated the accuracy of the IgM immunoblot (IgM-IB) test by using reactivity to the 14- to 18-kDa band and/or at least two bands among the 19- to 23-, 24- to 30-, 32-, 35/36-, 37-, and 41/42-kDa antigens as the diagnostic criterion. The sensitivities of the IgM-IB test and the microscopic agglutination test (MAT) were 88.2% and 2.0%, respectively, with sera from patients 1 to 3 days after the onset of symptoms. In contrast, the IgM-IB test was positive with only 2/48 (4.2%) sera from patients with other febrile illnesses. The high sensitivity and specificity of the IgM-IB test for acute leptospirosis would provide greatly improved diagnostic accuracy for identification of patients who would benefit from early antibiotic intervention. In addition, the antigens identified by the IgM-IB test may serve as components of a rapid, accurate, point-of-care diagnostic test for early leptospirosis.

FIG. 1.

IgM reactivities of sera from patients with leptospirosis. Representative immunoblots are shown in lanes 1 to 9. Symbols (×, ○, and •) indicate the locations of reactive antigen bands in the blots and their sizes on the left side of the figure. Lane 10 shows the locations of molecular size standards.

FIG. 2.

Representative immunoblots for sera from patients without leptospirosis. Sera were obtained from five patients each who were healthy blood donors (A) or had fever due to melioidosis (B), dengue fever (C), hepatitis (D), or malaria (E). The locations of molecular size standards are shown in kilodaltons on the right.

FIG. 3.

Evolution of IgM immunoblot reactivity during leptospirosis. Lanes A, B, and C are immunoblots performed using sera obtained from a single patient 6, 11, and 21 days, respectively, after the onset of fever. The locations of immunoreactive bands are shown on the left. The locations of molecular size standards are shown in kilodaltons on the right.

FIG. 4.

Reactivities of sera from leptospirosis patients with protein antigens. Sera were obtained from patients 2 days (lane 1), 16 days (lane 2), 6 days (lane 4), and 11 days (lane 5) after the onset of leptospirosis. Lane 3 shows the locations of molecular size standards. Lane 6 is an immunoblot of leptospiral antigens probed with antisera to leptospiral proteins GroEL, LipL41, LipL32, and LipL21.