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 that employs an anti- Leishmania-activated C kinase (LACK) antigen (anti-LACK) monoclonal antibody (mAb) 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 Leishmania-LACK was expressed and used as an immunogen for mAb production via somatic hybridization. The ability of the anti-LACK mAb to recognize native LACK and Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Viannia) guyanensis amastigotes was confirmed in total extracts and skin histological sections from experimentally infected hamsters using Western blotting and IHC techniques, respectively. The anti-LACK IHC was then validated on skin lesion samples from 104 suspected CL patients attending the outpatient clinic of the Municipal Polyclinic of Teofilo Otoni between 2019 and 2020, using kDNA-PCR as reference test. The diagnostic performance of anti-LACK IHC was compared to direct (DE) and histopathological (HE) examinations. The anti-LACK mAb successfully recognized native LACK in soluble antigens and detected amastigotes of the three Leishmania species in histological sections from experimentally infected hamsters. Moreover, anti-LACK IHC presented sensitivity of 59.3% (95% CI: 40 - 83.7), specificity of 98% (95% CI: 89.5- 99.7), and accuracy of 77.9% (95% CI: 61.9 - 96.8), higher than that presented by DE and HE techniques, although differences were not statistically significant. Agreement analysis yielded a Kappa index of 0.56 (95% CI: 0.42-0.71) considering CL cases compared with kDNA-PCR results. The combination of DE or HE with anti-LACK IHC increased diagnostic accuracy to 83.7% (p: 0.48) and 79.8% (p: 0.33), respectively. In this initial study, the anti-LACK IHC detected the main Leishmania species causing CL in Brazil, yet further improvement promises enhanced diagnostic performance of this technique.