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

Abstract

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.

Development of Immunochromatography-Based Methods for Detection of Leptospiral Lipopolysaccharide Antigen in Urine

“…The addition of sucrose, which is known as a preservative and resolubilization agent (26), to the conjugate pad made the gold conjugate more stable and flow better. Even though the overall sensitivity and specificity of the ICG-based LFA are still Ͻ90%, they are still higher than those of the IgM dipstick assay (9), which is often used in an initial screening for leptospirosis. In summary, we have developed assays that can detect the presence of Leptospira antigen in the urine of humans and animals and can discriminate it from other bacterial antigens; however, this assay is not able to identify the infecting serovar of Leptospira.…”

“…In this study, we tried to develop a rapid diagnostic assay (i.e., dipstick and ICG-based LFA) for leptospirosis which might fulfill these requirements and be applicable in all countries. Nowadays, the ICG assay for leptospirosis, which is used as one of the diagnostic methods, is mainly used for the detection of antibodies (9,33,34). However, these assays are difficult to use for early diagnosis because of their low sensitivities and the fact that the immune response might not have developed enough to be detected during the early stages of infection.…”

“…Nevertheless, the sensitivity and specificity of these methods were low when performed during the early stage of infection with Leptospira because the appropriate immune response might not yet have been elicited by the time of specimen collection. For example, the dipstick assay (6), which can detect the presence of IgM and is often used in the initial screening of leptospirosis, has low sensitivity when applied to patient serum samples (9). An antigen detection assay might offer an effective solution to this difficulty, because antigen can be detected earlier after infection (10).…”

Leptospirosis in the tropics and in travelers

“…Quantitative PCR techniques, prognostically useful, have shown that higher leptospiral burden (> ~10 4-5 of leptospires/mL of blood) is associated with worse disease [4••,37,38]. Demonstration of the presence of Leptospira by antigen detection to diagnose leptospirosis is currently under development but it remains to be shown whether these antigen detection tests are sufficiently sensitive to diagnose disease caused by different infecting leptospiral strains [39,40].…”

Proteinuria in early detection of human leptospirosis

“…Immunochromatography based methods for detection of Leptospiral lipopolysaccharide antigen in urine have also been developed. 12 The sensitivity and specificity of these methods are often debatable due to prior exposure of the individuals in endemic areas and due to the contaminating nonpathogenic lectospires in the environment.…”