Avelar, D. M. d. | bioRxiv Science

bioRxiv2025

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

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.

immunology↗

bioRxiv2025

Diversity of trypanosomatids in small mammals from a forest fragment at the wildlife-urban interface in southeastern Brazil: implications for public health surveillance

This study investigated the diversity of trypanosomatids infecting small mammals in a fragmented forest landscape in southeastern Brazil, aiming to clarify their role in transmission cycles and assess potential public health risks. Eighteen individuals from three species - Didelphis albiventris (n = 8), Marmosops incanus (n = 4), and Nectomys squamipes (n = 6) - were captured in the Mata da Tapera Municipal Natural Park (MT), located within the Espinhaco Range Biosphere Reserve, Minas Gerais State. Xenodiagnosis using Lutzomyia longipalpis and Rhodnius neglectus, followed by 18S rDNA nested PCR, enabled the detection of Leishmania infantum, L. braziliensis, and Trypanosoma cruzi DTU TcI in insects fed on naturally infected hosts. Tissue and organ samples were also analyzed by in vitro culture and molecular diagnostics. Leishmania infantum was identified in D. albiventris (n = 2), L. braziliensis in M. incanus (n = 1), T. cruzi TcI in D. albiventris (n = 2), and Trypanosoma lainsoni in both D. albiventris and N. squamipes (n = 1 each), the latter representing the first record of this parasite in that rodent species. This integrated approach provides a valuable model for zoonotic surveillance at the wildlife-urban interface, enhancing diagnostic sensitivity and species identification. These findings confirm the presence of zoonotic trypanosomatids in a highly anthropized landscape and highlight transmission risks to humans and domestic animals, particularly in an area with active ecotourism, underscoring the need for targeted surveillance within a One Health framework. Author summaryIn many parts of Brazil, forested areas are getting smaller and more fragmented due to urban growth. These changes increase the contact between wild animals, domestic animals, and humans, creating new opportunities for diseases to spread. In this study, we examined small wild mammals living in a forest fragment close to human settlements in southeastern Brazil to find out if they carried parasites that can also infect humans. We used a combination of techniques, including allowing laboratory-raised insects to feed on these animals, to detect the presence of different parasites. We found that these mammals were naturally infected with species of Leishmania and Trypanosoma, including those that cause leishmaniasis and Chagas disease in humans. We also detected Trypanosoma lainsoni, a lesser-known parasite, for the first time in one rodent species. Our findings suggest that small mammals living in areas where forests meet cities may play an important role in maintaining and spreading parasites that affect public health. This study shows the importance of monitoring wildlife in changing environments and supports the idea that human, animal, and environmental health are deeply connected.

molecular biology↗