Evaluation of an enzyme-linked immunosorbent assay based on crude Leishmania histone proteins for serodiagnosis of human infantile visceral leishmaniasis

Abstract

Human visceral leishmaniasis (VL) is routinely diagnosed by detecting IgG that specifically binds to Leishmania antigens. The enzyme-linked immunosorbent assay (ELISA) remains a widely used method. However, the biggest challenge remains the choice of antigen with the highest specificity and sensitivity. This study is aimed at assessing the diagnostic performances of crude Leishmania histone (CLH) protein-based ELISAs in Mediterranean VL patients. The CLH proteins were biochemically purified from promastigote nuclear extracts. Their reactivities were analyzed by Western blotting (WB) using rabbit polyclonal antibodies against Leishmania recombinant histones and sera from VL patients, respectively. Then, the diagnostic potential of CLH proteins was validated by the CLH-based ELISA using 42 infantile VL patients' sera and 70 control subjects. The CLH-based ELISA performance was compared to that of the soluble Leishmania antigen (SLA)- and the recombinant K39 (rK39)-based ELISAs. Analysis of the WB profile with the use of polyclonal antibodies confirmed the histone origin of low molecular mass proteins (12 to 16 kDa). All VL samples tested presented antibodies reacting against different antigen fractions; however, recognition patterns were different depending on the reactivity of each serum. CLH-based ELISA showed an excellent ability to discriminate between VL cases and healthy controls (97.6% sensitivity and 100% specificity). It had a diagnostic performance similar to that of rK39-based ELISA (97.6% sensitivity and 97.1% specificity, P = 0.5) and a better serodiagnosis accuracy than the SLA-based ELISA (85.7% sensitivity and 90% specificity, P < 0.05). Therefore, crude Leishmania histone extract could be a valuable antigen for clinical use.

Fig 1

Western blotting of CLH proteins. (A) CLH protein extract incubated with rabbit antibodies anti-rH2A, anti-rH2B, anti-rH3, and anti-rH4 protein (lanes 1, 2, 3, and 4, respectively), with sera of VL patients (lanes 5 to 14) and with sera from control subjects (lanes 15 to 17). Molecular mass markers are shown on the left side of the Western blot in kilodaltons. (B) Recognition pattern of a positive serum against 2 batches of Leishmania histone extracts.

Fig 2

Sensitivities and specificities of CLH-, rK39-, and SLA-based ELISAs. (A) ELISA reactivity against CLH proteins. Each dot corresponds to the optical density obtained with an individual serum. Optical densities found in the 2 control groups are shown separately. (B) ROC curves for rK39-, SLA-, and CLH-based ELISAs applied on VL cases versus matched controls.