Leishmaniasis: Control and Elimination

Leishmaniasis includes a spectrum of diseases ranging from debilitating cutaneous to fatal visceral infections. This disease is caused by the parasitic protozoa of the genus Leishmania that is transmitted by infected sandflies. Over 1 billion people are at risk of leishmaniasis with an annual incidence of over 2 million cases throughout tropical and subtropical regions in close to 100 countries. Leishmaniasis is the only human parasitic disease where vaccination has been successful through a procedure known as leishmanization that has been widely used for decades in the Middle East. Leishmanization involved intradermal inoculation of live Leishmania major parasites resulting in a skin lesion that following natural healing provided protective immunity to re-infection. Leishmanization is however no longer practiced due to safety and ethical concerns that the lesions at the site of inoculation that can last for months in some people. New genome editing technologies involving CRISPR has now made it possible to engineer safer attenuated strains of Leishmania, which induce protective immunity making way for a second generation leishmanization that can enter into human trials. A major consideration will be how the test the efficacy of a vaccine in the midst of the visceral leishmaniasis elimination program. One solution will be to use the leishmanin skin test (LST) that was also used for decades to determine exposure and immunity to Leishmania. The LST involves injection of antigen from Leishmania in the skin dermis resulting in a delayed type hypersensitivity (DTH) immune reaction associated with a Th1 immune response and protection against visceral leishmaniasis. Reintroduction of novel approaches for leishmanization and the leishmanin skin test can play a major role in eliminating leishmaniasis.

Visceral Leishmaniasis (VL) is the most fatal form of disease leishmaniasis. To date, there are no effective prophylactic measures and therapeutics available against VL. Recently, new immunotherapy-based approaches have been established for the management of VL. Cytokines, which are predominantly produced by helper T cells (Th) and macrophages, have received great attention that could be an effective immunotherapeutic approach for the treatment of human VL. Cytokines play a key role in forming the host immune response and in managing the formation of protective and non-protective immunities during infection. Furthermore, immune response mediated through different cytokines varies from different host or animal models. Various cytokines viz. IFN-γ, IL-2, IL-12, and TNF-α play an important role during protection, while some other cytokines viz. IL-10, IL-6, IL-17, TGF-β, and others are associated with disease progression. Therefore, comprehensive knowledge of cytokine response and their interaction with various immune cells is very crucial to determine appropriate immunotherapies for VL. Here, we have discussed the role of cytokines involved in VL disease progression or host protection in different animal models and humans that will determine the clinical outcome of VL and open the path for the development of rapid and accurate diagnostic tools as well as therapeutic interventions against VL.

The design and development of new pharmaceutical formulations for the existing anti-leishmanial is a new strategic alternate to improve efficacy and safety rather than new drug discovery. Herein hybrid solid lipid nanoparticles (SLN) have been engineered to direct the oral delivery of two anti-leishmanial drugs amphotericin B (AmB) and paromomycin (PM). The combinatorial nanocarriers consist of conventional SLN, antileishmanial drugs (AmB and PM) which have been functionalized with chitosan (Cs) grafted onto the external surface. The Cs-SLN have the mean particle size of 373.9 ± 1.41 nm, polydispersity index (PDI) of 0.342 ± 0.02 and the entrapment efficiency for AmB and PM was found to be 95.20 ± 3.19% and 89.45 ± 6.86 %, respectively. Characterization of SLN was performed by scanning electron microscopy and transmission electron microscopy. Complete internalization of the formulation was observed in Caco-2 cells. Cs-SLN has shown a controlled and slow drug release profile over a period of 72 h and was stable at gastrointestinal fluids, confirmed by simulated gastro-intestinal fluids study. Cs coating enhanced the mucoadhesive property of Cs-SLN. The in-vitro anti-leishmanial activity of Cs-SLN (1 μg/ml) has shown a maximum percentage of inhibition (92.35%) on intra-cellular amastigote growth of L. donovani.

Leishmaniasis is still a serious neglected tropical disease that may cause death in infected individuals. At present, the clinical diagnosis and treatment monitoring still rely on parasitological culture and microscopy that needs experienced technicians. The low sensitivity and inconvenience of microscopic examination could cause misdiagnosis and relapse of leishmaniasis. There is an urgent need for developing a sensitive and easily operated diagnostic method for the diagnosis and disease management of leishmaniasis. Thus, a quantitative real-time PCR (qPCR) based on the conversed regions of kinetoplast minicircle DNA (mkDNA) of Leishmania spp. was developed to detect different species of Leishmania. The designed mkDNA-based qPCR was able to detect as low as one copy of Leishmania mkDNA or DNA from single parasite. It also detected Pan-Leishmania protozoa including Leishmania donovani, Leishmania infantum and Leishmania major without cross-reaction with other pathogen DNAs available in our lab. This method was clinically applied to quantitatively detect skin lesion samples from 20 cutaneous leishmaniasis (CL) and bone marrow and/or PBMC samples from 30 current and cured visceral leishmaniasis (VL) patients, and blood samples from 11 patients with other infections and 5 normal donors as well. Total 20 skin lesion samples from current CL patients and 20 bone marrow and/or PBMC samples from current VL patients were all detected as positive with qPCR without cross-reaction with samples from patients with malaria, brucellosis and dengue or normal donors. Two VL patients with parasite converted to microscopically negative after treatment were detected positive with qPCR. The patients with bigger skin lesion in CL and higher level of immunoglobulin or splenomegaly in VL, had the higher parasite load detected by qPCR. The parasite load was significantly reduced after treatment. In conclusion, the mkDNA-based qPCR assay that we developed in this study can be used not only for diagnosis of both cutaneous and visceral leishmaniasis with high sensitivity and specificity, but also for evaluating the severity and treatment efficacy of this disease, presenting a rapid and accurate tool for clinical surveillance, treatment monitoring and the end point determination of leishmaniasis.