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

Abstract

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.

Exposure to live saprophyticLeptospirabefore challenge with a pathogenic serovar prevents severe leptospirosis and promotes kidney homeostasis

“…Our previous studies showed innate immunity engagement during saprophytic L. biflexa infection (14, 15). In addition, L. biflexa extracts can be used to detect Leptospira -specific antibody in up to 67% of serum from patients with clinically confirmed leptospirosis (18) which points to a high degree of immunodominant cross-reactive epitopes between L. biflexa and pathogenic Leptospira . Thus, we hypothesized that these highly cross-reactive immunodominant epitopes may also induce cross-protective immune responses.…”

“…rRNA primers (Forward-CCCGCGTCCGATTAG and Reverse-TCCATTGTGGCCGAACAC) and TAMRA probe (CTCACCAAGGCGACGATCGGTAGC) were used for Leptospira detection using qPCR with a standard curve of 10 5 to 1 Leptospira (16,17). Similarly, qPCR was performed with kidney tissues placed in EMJH to grow live Leptospira after culturing for 5 days and visually quantified under a dark field microscope (20X, Zeiss USA, Hawthorne) on d3 and d5 post culture inoculation.…”

Ocular leptospirosis: lack of awareness among ophthalmologists and challenges in diagnosis

“…Given that IgM/IgD class and IgG3/ IgG1-specific isotypes are produced in the earliest course of infection, immunoglobulin G isotyping may be used in the diagnosis of acute leptospirosis. EIA tests with its speed, ease of use, and accuracy make them good alternatives to the laborious MAT [74].…”

Silent Antibodies Start Talking: Enhanced Lateral Flow Serodiagnosis with Two-Stage Incorporation of Labels into Immune Complexes

“…The advantages of this method are the selection of a specific biomatrix for testing (in contrast with the determination of viruses or bacteria in different organs and tissues), and the similarity of the assay protocols for various infections [ 1 ]. In medical diagnostics, microplate-based enzyme-linked immunosorbent assays (ELISAs), agglutination tests, and other immunoassay formats are successfully used for this purpose [ 2 , 3 , 4 ]. The current trend in the development of diagnostic tools is simplification and acceleration of the testing procedure.…”