Noninvasive Diagnosis of Visceral Leishmaniasis:Development and Evaluation of Two Urine-Based Immunoassays for Detection of Leishmania donovani Infection in India

Visceral leishmaniasis (VL), one of the most prevalent parasitic diseasesin the developing world causes serious health concerns. Post kala-azar dermal leishmaniasis (PKDL) is a skin disease which occurs after treatment as a sequel to VL. Parasitological diagnosis involves invasive tissue aspiration which is tedious and painful. Commercially available immunochromatographic rapid diagnostic test such as rK39-RDT is used for field diagnosis of VL, detects antibodiesin serum samples. Urine sample is however, much easier in collection,storage and handling than serum and would be a better alternative where collection of tissue aspirate or blood is impractical. In this study, we have developed and evaluated the performance of two urine-based diagnostic assays, ELISA and dipstick test, and compared the results with serologicalrK39-RDT. Our study shows the capability of urinebased tests in detecting anti-Leishmania antibodies effectively for both VL and PKDL diagnosis. The ability of dipstick test to demonstrate negative results after six months in 90% of the VL cases after treatment could be useful as a test of clinical cure. Urine-based tests can therefore replace the need for invasive practices and ensure better diagnosi

Therapeutic and immunomodulatory activities of short-course treatment of murine visceral leishmaniasis with KALSOME™10, a new liposomal amphotericin B

Visceral leishmaniasis (VL), a potentially fatal disease, is most prevalent in the Indian subcontinent, East Africa and South America. Since the conventional antileishmanial drugs have many limitations we evaluated a new ergosterol rich liposomal amphotericin B formulation, KALSOME™10 for its leishmanicidal efficacy, tolerability and immunomodulatory activity. Normal healthy mice were treated with 3.5 mg/kg single and 7.5 mg/kg single and double doses ofKALSOME™10. Liver and kidney function tests were performed fourteen days after treatment. Next, normal mice were infected with Leishmania donovani amastigotes. Two months post infection they were treated with the above mentioned doses of KALSOME™10 and sacrificed one month after treatment for estimation of parasite burden in the liver and spleen by Limiting Dilution Assay. Leishmanial antigen stimulated splenocyte culture supernatants were collected for cytokine detection through ELISA. Flow cytometric studies were performed on normal animals treated with KALSOME™10, Amphotericin B (AmB) and AmBiosome to compare their immunomodulatory activities. The drug was found to induce no hepato- or nephrotoxicities at the studied doses. Moreover, at all doses, it led to significant reduction in parasite burden in two month infected BALB/c mice, with 7.5 mg/kg double dose resulting in almost complete clearance of parasites from both liver and spleen. Interestingly, the drug at 7.5 mg/kg double dose could almost completely inhibit the secretion of disease promoting cytokines, IL-10 and TGFβ, and significantly elevate the levels of IFNγ and IL-12, cytokines required for control of the disease. Mice treated with KALSOME™10 showed elevated levels of IFNγ and suppressed IL-10 secretion from both CD4+ and CD8+ subsets of T cells, as well as from culture supernatants of splenocytes, compared to that of normal, AmB and AmBisome treated animal Treatment of infected mice with 7.5 mg/kg double dose of KALSOME™10 was safe and effective in clearing the parasites from the sites of infection. The drug maintains the inherent immunomodulatory activities of AmB by effectively suppressing disease promoting cytokines IL-10 and TGFβ, thereby boosting IL-12 and IFNγ levels. This emphasizes KALSOME™10 as a promising drug alternative for lifelong protection from VL