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•Leishmania infantum is the etiologic agent of zoonotic visceral leishmaniasis, which can be fatal.•Visceral leishmaniasis treatment may be toxic, and resistance has been reported.•There are currently no vaccines available for humans.•Transgenic L. infantum could be an excellent vaccine candidate.•Prime-boosted mice have shown maximized protection. The etiologic agents of visceral leishmaniasis are Leishmania infantum and Leishmania donovani. Despite the variety of drugs available to treat leishmaniasis, most lead to serious adverse effects, and resistance to these drugs has been reported. Currently, no leishmaniasis vaccine is available for humans. That is why the group developed transgenic L. infantum promastigote lines, which express toxic proteins after differentiation into amastigotes. That is why group developed the pFL-AMA plasmid and transfected it into L. Infantum promastigotes. This plasmid was expressed only in the amastigote form of the parasite. Sequences encoding toxic proteins (active bovine trypsin and egg avidin) were inserted in this plasmid, and the transfected parasites died after the differentiation process. In this study, two immunization protocols were performed in BALB/c mice: prime and prime-boost immunization prior to challenge with the wild-type L. infantum (WT). The parasite burdens in the spleen, liver, and bone marrow were evaluated to verify immunological protection. Histopathological analysis of the spleen and liver and the humoral immune response were also performed. The data showed that the parasite burden was reduced in prime-boosted mice in the spleen, liver, and bone marrow, indicating that mice immunized with two doses of the transfected parasites were satisfactorily protected. High levels of IgG, IgG1, and IgG2a antibodies were observed, as well as the presence of anti-inflammatory cytokine Interleukine-10 and pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF-α) and Interferon-γ (IFN – γ) suggesting a Th1/Th2 mix response, in addition to the presence of multinucleated giant cells in the spleen and lymphocyte infiltration in the liver. Therefore, L. infantum transfected with a toxic plasmid is an excellent vaccine candidate against visceral leishmaniasis and the application of a boost before the challenge promotes greater protection against WT L. infantum infection.

Background Escalation in the use of biologic agents including tumor necrosis factor (TNF)-α inhibitors to treat immune-mediated inflammatory diseases (IMID) is linked to higher susceptibility of severe infections caused by intracellular pathogens, including Leishmania . Methods This multicentric prospective study assessed the presence of Leishmania spp. infection among patients with IMID under treatment with biologic agents in two Italian clinical centers. We utilized a combination of diagnostic tests: real-time PCR for the detection of parasitic kinetoplast DNA in peripheral blood, Western blot for the identification of serum IgG antibodies, and a Whole blood assay to assess cytokine and chemokine responses following stimulation with parasitic antigen. Results A total of 126 patients residing in Italy were enrolled. Patients testing positive in at least one assay were classified as Leishmania -positive. Of the 125 asymptomatic individuals, 25 (20%) tested positive for Leishmania infection, revealing a significant rate of subclinical infection. The most frequent marker of infection was positive serology (15/126, 12%) followed by a detectable cell-mediated immune response (9/125, 7%). Parasitic DNA was detected in 3 patients (2%). Conclusions This study showed a high prevalence of asymptomatic Leishmania infection in Italian patients with IMID under treatment with biologic agents, with a north-to-south gradient. Given the risk of disease reactivation, these patients may benefit from close monitoring. Further research is warranted to clarify the clinical implications of these findings.