Evaluation of the evolution of the in vitro and in vivo experimental infection in hamsters, Mesocricetus auratus, caused by Leishmania (L.) infantum chagasi isolated from patients with visceral leishmaniasis and atypical cutaneous leishmaniasis in the municipality of Amapala, Valle, Honduras

Author(s)

Gabriela Venicia Araujo Flores

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2022-03-09

Examining board members

Marcia Dalastra Laurenti; José Angelo Lauletta Lindoso; Luiz Felipe Domingues Passero; Rodrigo Pedro Pinto Soares

• Advisor: Marcia Dalastra Laurenti

Abstract

In Honduras, infection by L. (L.) infantum chagasi causes, in addition to visceral leishmaniasis (VL), non-ulcerated or atypical cutaneous leishmaniasis (NUCL), in the same geographic area. To better understand the role of the parasite in the pathogenesis of infection caused by L. (L.) infantum chagasi in Central America, the present study aimed to characterize parasites isolated from patients with VL and NUCL in the municipality of Amapala, Valle, Honduras; as well as to evaluate its in vitro interaction with the host cell, and the in vivo evolution of the experimental infection in Mesocricetus auratus. The hsp70 PCR-RFLP, using the restriction enzyme HaeIII, characterized the isolates from peripheral blood of VL patient and skin lesions from patients with NUCL as L. (L.) infantum chagasi. Maxcircle genes showed the existence of two different lineages within the same species, L. (L.) infantum chagasi, one responsible for VL in Brazil and another for NUCL in Honduras, phylogenetically separating viscerotropic and dermotropic strains. The LPGs from all LCNU strains showed to be more pro-inflammatory than those from viscerotropic strains by their ability to induce the production of NO, IL-6 and TNF- in mouse peritoneal macrophages. In in vitro study, a higher macrophage infection index was observed at 32 °C and 34 °C for strains isolated from skin lesions of patients affected by NUCL, with no difference in the time of infection (24 and 48 hours). Regarding viscerotropic strains, a higher infection index was observed at 34 °C and 36 °C, which was even higher 48-hour incubation at 36 °C. In the supernatants of cultures of hamster peritoneal macrophages infected with the different strains studied, low levels of oxygen and nitrogen-derived metabolites, as well as inflammatory cytokines, were detected, which did not show a correlation with the infection index. In the in vivo study, animals subcutaneously infected with the different strains did not develop macroscopic lesions at the inoculation site and histopathological changes in the dermis were very slight. Regarding the histopathological study of the viscera, we observe that the portal mononuclear inflammatory infiltrate and the presence of nodules in the hepatic parenchyma, as well as the proliferation of macrophages in the spleen, increased with the evolution of the infection, in animals inoculated subcutaneously and intraperitoneally, with different strains, dermotropic and viscerotropic. The parasite load in the liver of the animals infected with the different dermotropic strains showed an increase with the time of infection, regardless of the inoculation route used. On the other hand, in the spleen, the parasitic load of dermotropic strains showed an increase in the time of infection in animals inoculated intraperitoneally; while in animals inoculated subcutaneously in addition to the dermotropic strains, the LV-3 strain also showed an increase in the parasite load as a function of time. Humoral immunity, evaluated in the sera of hamsters infected with the different strains of L. (L.) infantum chagasi, showed an increase in total IgG titers as a function of the time of infection, regardless of the inoculation route used. Regarding cellular immunity, we did not observe an expressive increase or decrease of pro and anti-inflammatory cytokines in relation to the healthy control, except for IL-10 that was more evident in all experimental groups. Taken together, our results show genotypic and phenotypic differences between parasites isolated from visceral leishmaniasis and non- ulcerated cutaneous leishmaniasis patients, which partially reflected in in vitro infection, but not in vivo infection where a greater complexity of immune response elements competes for susceptibility of the vertebrate host to infection by L. (L.) infantum chagasi (AU)

• FAPESP's process: 17/24834-9 - Evaluation of the experimental infection in hamster by isolated parasites of atypical cutaneous lesion and human visceral caused by Leishmania (L.) infantum chagasi in the municipality of Amapala, Valle, Honduras
• Grantee: Gabriela Venicia Araujo Flores
• Support Opportunities: Scholarships in Brazil - Doctorate