Anti-Leishmania activated C-kinase monoclonal antibody immunohistochemical technique for cutaneous leishmaniasis diagnosis.

Abstract

This study aimed to develop and validate an immunohistochemistry (IHC) technique using a monoclonal antibody (mAb) against the Leishmania homolog of activated C kinase (LACK) antigen (anti-LACK IHC) for the diagnosis of cutaneous leishmaniasis (CL). The Leishmania braziliensis LACK sequence (A4HGX7_LEIBR) was analyzed for B-cell epitope prediction, and three antigenic regions were selected to design a multiepitope antigen. The corresponding gene was back-translated, synthesized, and cloned into the pET28a(+) plasmid. Recombinant LACK protein was expressed and used as an immunogen for mAb production by somatic hybridization. The ability of the anti-LACK mAb to recognize native LACK and amastigotes of Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Viannia) guyanensis was confirmed by Western blotting using total parasite extracts and by IHC in skin histological sections from experimentally infected hamsters. Subsequently, the anti-LACK IHC assay was validated using skin lesion samples from 104 patients with suspected CL who attended the outpatient clinic of the Municipal Polyclinic of Teófilo Otoni, Brazil, between 2019 and 2020, using kDNA-PCR as the reference standard. The diagnostic performance of anti-LACK IHC was compared with direct examination (DE) and conventional histopathology (HE). Anti-LACK IHC showed a sensitivity of 59.3% (95% CI: 40.0-83.7), numerically higher than DE (53.7; p = 0.55) and HE (42.6; p = 0.08). The specificity of the anti-LACK IHC was 98.0% (95% CI: 89.5-99.7), equal to the DE (98.0%) and slightly higher to the HE (96.0%), but without statistically significant difference (p = 0.56). The accuracy was of 77.9% (95% CI: 61.9-96.8) with anti-LACK IHC, 75.0% with DE (p = 0.62) and 68.3% with HE (p = 0.12). The combination of anti-LACK IHC with DE or HE numerically increased diagnostic accuracy to 83.7% (p = 0.48) and 79.8% (p = 0.33), respectively. The results demonstrated that anti-LACK IHC was able to detect the main Leishmania species responsible for CL in Brazil and further studies may prove its applicability in the CL diagnosis routine in different Brazilian endemic regions.