ELISA-based evaluation of antibody response to Leishmania in a region endemic for cutaneous leishmaniasis

Abstract

Leishmaniasis includes several clinical forms. While routine diagnosis of cutaneous leishmaniasis (CL) is by microscopy, an antibody response to CL has been reported in several recent studies. This study evaluated anti-leishmanial immunoglobulin G (IgG) antibody responses as a biomarker of active leishmaniasis and a measure of exposure to Leishmania. Sera from 50 untreated CL patients, 140 patients under treatment and 280 healthy individuals residing in endemic regions collected as part of an epidemiological survey, was analysed with an enzyme-linked immunosorbent assay established in-house using receiver operator characteristic curve at optimized cut-off value. The assay showed high performance as a diagnostic tool in identifying exposure in endemic individuals (sensitivity: 98%, specificity: 90.3%). All patients showed lower antibody levels over time since onset of lesion/s. Antibody levels were higher (p ˂ .01) and persisted for a longer period in untreated patients. In patients under treatment, the level of anti-IgG antibodies was negatively correlated with the total duration the patient had been on treatment. The anti-leishmanial IgG response in Leishmania donovani-induced CL is transient and is unlikely to confer protective immunity. Optimized serological assays may be useful in endemic settings for diagnosis and monitoring the treatment response in CL.

Keywords: ELISA; IgG antibodies; L. donovani; biomarker; cutaneous leishmaniasis; serodiagnosis.

FIGURE 1

Receiver operating characteristic (ROC) curve for indirect ELISA and cut off values for identifying patients from healthy individuals. A: ROC curve of 50 untreated CL patients (Group II) and 267 healthy individuals (Group III and IV), excluded those with a past history of CL (Group V). (AUC=0.997, sensitivity= 98%, specificity=90.3%) B: Levels of anti-leishmanial IgG with cut-off at 0.150. The best cut-off value was calculated at maximum sensitivity and specificity of ELISA absorbance at 405 nm and drawn in a dashed line. Mean level of leishmanial IgG antibodies represented in solid lines.

FIGURE 2

Anti-leishmanial IgG antibodies assessed by indirect ELISA in sera of 140 patients receiving treatment for CL (Group I) and 50 untreated CL patients (Group II), in comparison to persons with no past history of CL and living in a house where no CL cases had been previously reported (Group III), persons with no history of CL but living in a house where CL cases have been diagnosed at the time of sampling or in the past (Group IV), persons with a past history of CL and successfully treated (Group V). Mean absorbance represent the mean level of anti-leishmanial IgG antibodies and shown in solid lines. Each mean value was obtained through triplicate experiments. Cut-off value of indirect ELISA test is shown in a dash line. A two-tailed p value less than 0.05 was considered statistically significant (*p < 0.05).

FIGURE 3

Levels of anti-leishmanial IgG antibodies of seropositive endemic healthy individuals (38/280). Absorbance was measured at 405 nm. Group III: Persons with no past history of CL and living in a house where no CL cases had been previously reported, Group IV: Persons with no history of CL but living in a house where CL cases have been diagnosed at the time of sampling or in the past, Group V: Persons with a past history of CL and successfully treated. No difference was observed between the anti-leishmanial IgG antibody levels of the subgroups (p > 0.05; p value as calculated by Kruskal- Wallis test at 95% confidence interval).

FIGURE 4

Clinical correlations of CL patients with levels of anti-leishmanial antibodies. A: Correlation between the presence of serum anti- leishmanial IgG of untreated CL patients (Pearson’s r= −0.834, p ˂ 0.01) and patients under treatment (Pearson’s r = −0.784, p ˂ 0.01) with the lesion duration/months. B: Correlation of anti-leishmanial IgG antibodies of CL patients under treatment with the treatment duration/months (Pearson’s r= −0.655, p ˂ 0.01). Patients received intra-lesional SSG weekly. C: Correlation between lesion duration and treatment duration of patients under treatment (Pearson’s r= 0.796, p ˂ 0.01). A two-tailed p value less than 0.01 was considered statistically significant.