Differential immunoglobulin and complement levels in leprosy prior to development of reversal reaction and erythema nodosum leprosum

Background Leprosy is a treatable infectious disease caused by Mycobacterium leprae. However, there is additional morbidity from leprosy-associated pathologic immune reactions, reversal reaction (RR) and erythema nodosum leprosum (ENL), which occur in 1 in 3 people with leprosy, even with effective treatment of M. leprae. There is currently no predictive marker in use to indicate which people with leprosy will develop these debilitating immune reactions. Our peripheral blood mononuclear cell (PBMC) transcriptome analysis revealed that activation of the classical complement pathway is common to both RR and ENL. Additionally, differential expression of immunoglobulin receptors and B cell receptors during RR and ENL support a role for the antibody-mediated immune response during both RR and ENL. In this study, we investigated B-cell immunophenotypes, total and M. leprae-specific antibodies, and complement levels in leprosy patients with and without RR or ENL. The objective was to determine the role of these immune mediators in pathogenesis and assess their potential as biomarkers of risk for immune reactions in people with leprosy. Methodology/findings We followed newly diagnosed multibacillary leprosy cases (n = 96) for two years for development of RR or ENL. They were compared with active RR (n = 35), active ENL (n = 29), and healthy household contacts (n = 14). People with leprosy who subsequently developed ENL had increased IgM, IgG1, and C3d-associated immune complexes with decreased complement 4 (C4) at leprosy diagnosis. People who developed RR also had decreased C4 at leprosy diagnosis. Additionally, elevated anti-M. leprae antibody levels were associated with subsequent RR or ENL. Conclusions Differential co-receptor expression and immunoglobulin levels before and during immune reactions intimate a central role for humoral immunity in RR and ENL. Decreased C4 and elevated anti-M. leprae antibodies in people with new diagnosis of leprosy may be risk factors for subsequent development of leprosy immune reactions

Identifying Leprosy and Those at Risk of Developing Leprosy by Detection of Antibodies against LID-1 and LID-NDO

<div><p>Leprosy is caused by <i>Mycobacterium leprae</i> infection and remains a major public health problem in many areas of the world. Challenges to its timely diagnosis result in delay in treatment, which is usually associated with severe disability. Although phenolic glycolipid (PGL)-I has been reported as auxiliary diagnostic tool, currently there is no serological assay routinely used in leprosy diagnosis. The aim of this study was to evaluate the effectiveness of two related reagents, LID-1 and LID-NDO, for the detection of <i>M</i>. <i>leprae</i> infection. Sera from 98 leprosy patients, 365 household contacts (HHC) and 98 endemic controls from Rio Grande do Norte, Brazil, were evaluated. A subgroup of the HHC living in a hyperendemic area was followed for 7–10 years. Antigen-specific antibody responses were highest in multibacillary (MB) at the lepromatous pole (LL/BL) and lowest in paucibacillary (PB) at the tuberculoid pole (TT/BT). A positive correlation for both anti-LID-1 and anti-LID-NDO antibodies was found with bacterial burden (LID-1, r = 0.84, <i>p</i><0.001; LID-NDO, r = 0.82, <i>p</i><0.001), with higher sensitivity than bacilloscopy. According to <i>Receiver Operating Curve</i>, LID-1 and LID-NDO performed similarly. The sensitivity for MB cases was 89% for LID-1 and 95% for LID-NDO; the specificity was 96% for LID-1 and 88% for LID-NDO. Of the 332 HHC that were followed, 12 (3.6%) were diagnosed with leprosy in a median time of 31 (3–79) months after recruitment. A linear generalized model using LID-1 or LID-NDO as a predictor estimated that 8.3% and 10.4% of the HHC would become a leprosy case, respectively. Together, our findings support a role for the LID-1 and LID-NDO antigens in diagnosing MB leprosy and identifying people at greater risk of developing clinical disease. These assays have the potential to improve the diagnostic capacity at local health centers and aid development of strategies for the eventual control and elimination of leprosy from endemic areas.</p></div

Population from Rio Grande do Norte-Brazil tested for LID-1 and LID-NDO.

<p>Analysis of LID-1 and LID-NDO ELISA as diagnostic tools to identify leprosy patients, to detect asymptomatic infection and to predict leprosy development.</p

Parameters estimated through ROC analysis with regard to three distinct scenarios.

<p>Parameters estimated through ROC analysis with regard to three distinct scenarios.</p

Scatter plot for specific antibody levels and simultaneous tests results.

<p>Each sample is distinguished by an individual marker. The Pearson’s coefficient correlation was r = 0.84 (p<0.001). Dashed lines represent cut-offs to diagnose MB cases according to scenarios 1 (darkest lines) and 2 (lightest lines). See <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004934#pntd.0004934.t002" target="_blank">Table 2</a>. Net sensitivity for scenario 1 = (0+59+4)/66 = 0.951. Net specificity for scenario 1 = 86/98 = 0.878. Net sensitivity for scenario 2 = (3+44+9)/66 = 0.848. Net specificity for scenario 2 = 336/365 = 0.908. (MB: multibacillary; PB: paucibacillary; EC: endemic control; HHC: household contacts).</p

Characterization of the study population.

<p>Characterization of the study population.</p

HHC groups of risk based on antibody levels.

<p>HHC groups of risk based on antibody levels.</p

Linear increase of antibody levels across the leprosy spectrum.

<p>Sera from leprosy patients characterized as TT (tuberculoid), BT (borderline-tuberculoid), BB (borderline-borderline), BL (borderline-lepromatous) or LL (lepromatous) were analyzed for antibodies against LID-1 and LID-NDO. A) The slope of the fitted line (red) suggests an increment of 0.299 on the LID-1 mean, as group moves from TT to LL poles (p<0.001). B) The slope of the fitted line (red) suggests an increment of 0.319 on the LID-NDO mean, as group moves from TT to LL poles (p<0.001).</p

Probability estimates (pi.hat) of asymptomatic infected HHC developing leprosy according to antibody levels.

<p>A total of 332 household contacts living in the hyperendemic area of Mossoró, Rio Grande do Norte, Brazil, were analyzed based on anti-LID-1 (A) and anti-LID-NDO (B) antibody levels. Blue and red triangles represent contacts that developed PB and MB leprosy, respectively. The cut-off values for scenarios 2 and 3 (dashed lines) were used to group individuals by risk (LR: low risk; MR: moderate risk; HR: high risk).</p

Correlation between ELISA results with bacilli index (BI).

<p>Responses against LID-1 (A) and LID-NDO (B) are plotted against the BI (0–6) of leprosy patients. Each sample is distinguished by an individual marker. Strong positive correlations were observed for both anti-LID-1 antibodies (r = 0.84, p<0.001) and anti-LID-NDO (r = 0.82, p<0.001) with the bacterial index. Dashed line represents the cut-off for scenario 1 (0.276 for LID-1 and 0.332 for LID-NDO).</p