Enzyme immunoassays (EIA) for serodiagnosis of human leptospirosis: Specific IgG3/IgG1 isotyping may further inform diagnosis of acute disease

The laborious microscopic agglutination test (MAT) is the gold standard serologic test for laboratory diagnosis of leptospirosis. We developed EIA based serologic assays using recombinant proteins (rLigA, rLigB, rLipL32) and whole-cell extracts from eight Leptospira serovars as antigen and assessed the diagnostic performance of the new assay within each class, against MAT positive (MAT+) human sera panels from Portugal/PT (n = 143) and Angola/AO (n = 100). We found that a combination of recombinant proteins rLigA, rLigB and rLipL32 correctly identified antigen-specific IgG from patients with clinical and laboratory confirmed leptospirosis (MAT+) with 92% sensitivity and ~ 97% specificity (AUC 0.974) in serum from the provinces of Luanda (LDA) and Huambo (HBO) in Angola. A combination of whole cell extracts of L. interrogans sv Copenhageni (LiC), L. kirschneri Mozdok (LkM), L. borgpetersenii Arborea (LbA) and L. biflexa Patoc (LbP) accurately identified patients with clinical and laboratory confirmed leptospirosis (MAT+) with 100% sensitivity and ~ 98% specificity for all provinces of Angola and Portugal (AUC: 0.997 for AO/LDA/HBO, 1.000 for AO/HLA, 0.999 for PT/AZ and 1.000 for PT/LIS). Interestingly, we found that MAT+ IgG+ serum from Angola had a significantly higher presence of IgD and that IgG3/IgG1 isotypes were significantly increased in the MAT+ IgG+ serum from Portugal. Given that IgM/IgD class and IgG3/IgG1 specific isotypes are produced in the earliest course of infection, immunoglobulin G isotyping may be used to inform diagnosis of acute leptospirosis. The speed, ease of use and accuracy of EIA tests make them excellent alternatives to the laborious and expensive MAT for screening acute infection in areas where circulating serovars of pathogenic Leptospira are well defined.Author summary: Leptospirosis is a potential fatal zoonotic disease with worldwide distribution caused by a bacterium found in contaminated sources of water and soil. Diagnosis of leptospirosis is tentatively based on evaluation of fever and myalgia in patients presenting at the hospital in areas of endemicity, and it is rarely confirmed in most parts of the world due to lack of affordable diagnostic tests. We developed a highly sensitive and specific serologic test for leptospirosis using ubiquitous enzyme immunoassay technology. In areas where circulating serovars of pathogenic Leptospira are well defined, versatile enzyme immunoassays can be adapted and operated at a fraction of the cost of the cumbersome gold standard Microscopic Agglutination Test and should be explored as accurate serodiagnostic tools for leptospirosis. Addition of specific immunoglobulin G isotyping (IgG3/IgG1) can further inform diagnosis of acute leptospirosis.