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.

Fig 1. Specificity analysis of candidate serodiagnostic biomarkers.

Human IgG specific to purified L. interrogans recombinant proteins compared to L. interrogans whole cell extract (WCE) using a serum panel from healthy individuals (n = 12 samples, diluted 1:50) from a non-endemic area for leptospirosis (FL, US). Statistics by Mann-Whitney test (unpaired, exact): comparison between each recombinant protein and WCE, * p<0.005, ** p<0.0001, ns, not significant.

Fig 2. Diagnostic performance of each L. interrogans recombinant protein.

Human IgG specific to rLigA, rLigB and rLipL32 in four MAT+ leptospirosis sera panels, AO (LDA/HBO) n = 49/50, AO (HLA) n = 49/50, PT (AZ) n = 43, PT (LIS) n = 100 and one control panel, Healthy n = 33/34. Statistics by Ordinary One-Way ANOVA between each leptospirosis panel and healthy control and by ROC Area Under the Curve (AUC).

Fig 3. Diagnostic performance of combined recombinant proteins.

Human IgG specific for rLigA or LigB or LipL32 in four MAT+ leptospirosis sera panels, AO (LDA/HBO) n = 50, AO (HLA) n = 50, PT (AZ) n = 43, PT (LIS) n = 100 and one control panel, Healthy n = 33. A. Scatter plot of all sera panels and B. ROC curve of each sera panel. Statistics by Ordianry One-Way ANOVA and ROC AUC.

Fig 4. Diagnostic performance of each Leptospira serovar extract.

Human IgG specific to L. interrogans Copenhageni FioCruz L1-130 (LiC), L. kirschneri Mozdok 61H (LkM), L. borgpetersenii Arborea (LbA) and L. biflexa Patoc 1 (LbP) in four MAT+ leptospirosis sera panels, AO (LDA/HBO) n = 43, AO (HLA) n = 43, PT (AZ) n = 42–43, PT (LIS) n = 40–43 and one control panel, Healthy n = 43. Statistics by Ordinary One-Way ANOVA between each leptospirosis panel and healthy control and by ROC Area Under the Curve (AUC).

Fig 5. Diagnostic performance of combined extracts of Leptospira serovars.

Human IgG specific to L. interrogans Copenhgeni FioCruz L1-130 (LiC), L. borgpetersenii Arborea (LbA), L. kirschneri Mozdoc 61H (LkM) and L. biflexa Patoc 1 (LbP) in four MAT+ leptospirosis sera panels, AO (LDA/HBO) n = 36, AO (HLA) n = 42, PT (AZ) n = 43, PT (LIS) n = 43 and one control panel, Healthy n = 43. A. Scatter plot of all sera panels and B. ROC curve of each sera panel. Statistics by Ordinary One-Way ANOVA and ROC AUC.

Fig 6. Analysis of immunoglobulin class M, D and A, and IgG isotypes in MAT+ LiC IgG+ sera panels (AUC>0.995).

Scatter plots of L. interrogans specific IgM, IgD, IgA and IgG3, IgG1, IgG2, IgG4 in a serum panel from Portugal (PT-AZ, n = 43) and a panel from Angola (AO-HLA, n = 29). Statistics by Welch’s t test.