Antigen-specific assessment of the immunological status of various groups in a leprosy endemic region

Patient and contact samples

Untreated patients (n?=?94) and household contacts (n?=?104) were recruited at the
National Reference Centre for Sanitary Dermatology and Leprosy (CREDESH), Uberlandia,
Minas Gerais, Brazil, a public health care facility in an endemic region where routine
prevention, including Bacillus Calmette–Guérin (BCG) vaccination, household contact
monitoring, early case detection, and treatment are available and under constant supervision.
The Uberlândia municipality had detection rate of 10.81/100.000 inhabitants in 2012
10].

Leprosy patients were diagnosed after thorough dermato-neurological and laboratory
examinations, and classified according Ridley-Jopling five-group system of clinical
manifestations into: tuberculoid (TT), borderline tuberculoid (BT), mid-borderline
(BB), borderline lepromatous (BL) or lepromatous (LL) 11]. For treatment purposes patients were also stratified into paucibacillary (PB), with
up to five skin lesions and a negative bacilloscopy, or MB, with more than five lesions
and/or positive bacilloscopy in accordance with the World Health Organization operational
classification 12].

Household contacts (HHC) who resided with leprosy patients, or had resided with leprosy
patients in the five years prior to diagnosis, were examined for signs or symptoms
that were suggestive of leprosy by physicians with specialized leprosy training. Most
HHC were relatives of their index case (spouse, parent or sibling). HHC were stratified
according to the operational and clinical classifications of their index case.

Samples from the general population

Individuals from the general population (GPop; n?=?2.494) were selected randomly from
seven municipalities in the microregion of Almenara, Minas Gerais, Brazil 13], which had a mean detection rate of 31.32/100.000 inhabitants in 2012 10]. Finger-prick blood spots were collected on Whatman 3 MM paper (Whatman, Maidstone,
UK) and stored at 4 °C until serum was eluted by adding 1 % bovine serum albumin in
1X phosphate buffered saline. Based on estimates of the volume of whole blood in a
2.5 mm filter paper disc, the final dilution of the eluted serum was 1:100 14], 15].

Antibody detection

Antigen-specific IgM and IgG antibodies were measured by indirect enzyme-linked immunosorbent
assays (ELISA) as previously described 3]. Briefly, the four antigens (PGL-1, NDO-HSA, LID-1 and NDO-LID) were used to coat
96-well microtiter plates (LABWARE Manufacture CO, People’s Republic of China); 1 ?g/mL
of LID-1 or 0.2 ?g/mL of NDO-HSA, NDO-LID or PGL-1 was added per well in 100 ?L of
0.1 M sodium carbonate/bicarbonate buffer, pH 9.6, and incubated at 4 °C overnight.
The assay was performed using serum samples at a dilution of 1:300 and whole blood
samples at a dilution of 1:100 14], 15]. After blocking for 1 h at 37 °C, detection antibodies were similarly incubated for
1 h at 37 °C, after which four washes were performed. The wells were then treated
with ortho-phenylenediamine (OPD) substrate, and the absorbance at 492 nm was obtained
using a spectrophotometer plate reader (Molecular Devices, Sunnyvale, CA). To minimize
inter- and intra-test errors, an ELISA index (EI) was calculated as follows: the EI
equals the optical density (OD) of the sample divided by the OD of the cut-off 5]. The cut-off was calculated as an average of three controls negative (individuals
living in low endemic areas leprosy and which showed result of the ML Flow test negative)
plus three times the standard deviation, such that samples with EI values of 1.1 or
above were considered to be positive.

Statistical analysis

Graphs and mean values were generated using GraphPad Prism version 5 (GraphPad Software
Inc., La Jolla, CA, USA), and statistical analysis was performed using Statistical
Package for the Social Sciences (SPSS) version 18 (SPSS Inc., Chicago, IL, USA). Statistical
significance was assessed using nonparametric methods, with the Kruskal-Wallis one-way
(H) analysis of variance used to make comparisons among multiple groups and the Mann–Whitney
U test with Bonferroni correction used to make comparisons between two groups. Spearman’s
coefficient (rho) was used to test the strengths of correlations. Results were considered
statistically significant when p-values ?0.05 were obtained or when p-values ?0.0167 using the Mann–Whitney U test. The concordance among the four antigens was calculated using the Kappa coefficient.
The NDO-HSA antigen was selected as the reference antigen because it was tested in
three groups (patients, HHC and GPop) and because it is one of the most common antigens
used in tests for M. leprae. Kappa values and their interpretation varied as follows: 0, no agreement; 0–0.20,
poor agreement; 0.21-0.40, fair agreement; 0.41-0.60, moderate agreement; 0.61-0.80,
substantial agreement; and 0.81-1.00, almost perfect agreement 16].

Ethics statement

This study conforms to the Declaration of Helsinki and was reviewed and approved by
Research Ethic Committee (CEP) of the Federal University of Uberlandia, Protocol Number
CEP/UFU 138/08, and the Research Ethic Committee (COEP) of the Federal University
of Minas Gerais, Protocol Number ETIC 158/09. All participants signed an informed
consent form and authorized the collection of the samples. The informed consent form
for children under 18 years of age was signed by either a parent or a legal guardian.