Fig 1.
Flow chart of study enrollment and outcomes.
HHC: household contacts; PB: paucibacillary leprosy; MB: multibacillary leprosy; RR+: active reversal reaction; ENL+: active erythema nodosum leprosum; Reaction -/-: patients that did not develop reactions during the follow-up; RR-/+: patient that developed RR during the follow-up; ENL-/+: patient that developed ENL during the follow-up. Isolated neuritis without cutaneous inflammation was not included as a “reaction” outcome.
Table 1.
Characterization of the study population.
Fig 2.
CD32 expression in B cells and plasmablasts.
The frequency of CD19+ B cells as a subset of lymphocytes in the subtypes of leprosy are shown in 2A with CD19+CD27+CD20- plasmablasts as a subset of B cells in 2B. Frequency of CD32B (FcγRIIb) in B cells (2C) and plasmablasts (2D) were decreased in MB leprosy compared to HHC. Comparison was with ANOVA followed by Tukey’s multiple comparisons test for 4A and 4D. Kruskal-Wallis followed by Dunn’s multiple comparisons test was used for 4B and 4C. * p ≤ 0.05, ** p< 0.01. The horizontal bar is the mean and the vertical bars the standard error of the mean (SEM).
Fig 3.
Plasmablast populations and CD21 expression in leprosy immune reactions.
Plasmablast populations were proportionally greater in people with ENL (3A) and fewer of these plasmablasts were CD21+ compared to non-reaction groups (3B). Comparison was made between MB patients that do not develop immune reactions in 2-years of follow-up (Reaction-/-) with those with MB who developed RR (RR-/+) or ENL (ENL -/+) (ANOVA or Kruskal-Wallis test). Those patients who developed reactions during the cohort were also compared with patients with active immune reactions (RR+ and ENL+) (Student’s t test or Mann-Whitney test). * p ≤ 0.05. The horizontal bar is the mean and the vertical bars the standard error of the mean (SEM).
Fig 4.
CD21 in the skin lesions of leprosy patients.
The figure shows representative photos (4A) and quantitative measurement of fluorescence (4B). Each photo is a compilation of 15 slices of 1.25 μM confocal micrographs, as previously described [15]. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI). Individuals with active RR (n = 3) and active ENL (n = 3) were compared with individuals with the same clinical forms of leprosy but without reactions: BT controls (n = 7) and BL/LL controls (n = 3). Differences in fluorescence between groups were assessed with ANOVA followed by Tukey multiple-comparison test. *** p< 0.001. The horizontal bars represent means and the vertical bars the standard error of the mean (SEM).
Fig 5.
Immunoglobulins and complement components in leprosy and leprosy reactions.
IgM levels were higher in ENL -/+ at the time of leprosy diagnosis, but the mean IgM level was lower during acute ENL (5A) with a similar pattern for IgG1 levels (5B). C3d-CIC were increased in people at leprosy diagnosis who went on to develop ENL (5C). C4 was lower in RR -/+ and ENL -/+, but higher during acute RR and acute ENL (5D). Comparison was made between MB patients that do not develop immune reactions in a 2-years follow-up (Reaction-/-) with those that developed RR (RR -/+) or ENL (ENL -/+) during the follow-up (ANOVA or Kruskal-Wallis test). Those patients that developed reactions during the follow-up were also compared with patients with active presentation of immune reactions (RR+ and ENL+) (Student’s t test or Mann-Whitney test). *, p ≤ 0.05, **, p < 0.01, ****, p < 0.0001. The horizontal bars represent mean value and the vertical bars the standard error of the mean (SEM).
Fig 6.
Anti-M. leprae antibodies in the peripheral blood of MB patients prior to and and during leprosy reactions.
The OD of LID-NDO ELISA was positively correlated with M. leprae bacterial index by dermal smears (Pearson’s correlation, p<0.0001) (6A). The bacterial index at diagnosis was higher in those patients who developed ENL during the follow-up (ENL-/+) when compared to the bacterial index of people who did not develop reactions (Reaction -/-) or those who developed RR (RR-/+) (Kruskal-Wallis test followed by Dunn multiple comparison test, p<0.001) (6B), as was the LID-NDO OD (ANOVA followed by Dunn multiple comparison test, p≤0.05) (6C). In Fig 6C, the horizontal bars represent mean value and the vertical bars the standard error of the mean (SEM). * p ≤ 0.05, *** p< 0.001, **** p< 0.0001.