A review on new natural and synthetic anti-leishmanial chemotherapeutic agents and current perspective of treatment approaches.

Leishmaniases are considered among the most neglected yet dangerous parasitic diseases worldwide. According to the recent WHO report (Weekly Epidemiological Record, Sep, 2021), 200 countries and territories reported leishmanises cases in 2020; of which 89 (45%) for CL, and 79 (40%) for VL were endemic. Indian subcontinent (India, Bangladesh and Nepal), one of the three eco-epidemiological hotspots of VL, currently reported 18% of the total cases of VL worldwide. Eastern Mediterranean region and the Region of the Americas together reported >90% of the new CL cases, of which >80% were from Afghanistan, Algeria, Brazil, Colombia, Iraq, Pakistan and the Syrian Arab Republic. While considering the current therapeutic options, conventional anti-leishmanial drugs have long been proved to be toxic and/or expensive and have resulted in extensive drug resistance in India. Recent searches for novel anti-leishmanial drugs have led to find out the prime cellular targets and metabolic pathways to bridge the gap between the known facts and unexplored data. Cutting edge knowledge based drug designing has simplified the search for novel molecules with leishmanicidal efficacy by identifying ligand-receptor interactions and has accelerated the cost effective primary discovery of molecules through computational validation against Leishmaniases. This review focuses on the limitations of conventional drugs, and discusses the chemotherapeutic potential of many novel natural and synthetic anti-leishmanial agents reported since the last decade. It is also interpreted that some of the reported molecules might be tested singly or as a part of combinatorial therapy on pre-clinical and clinical level.

A Composite Recombinant Salivary Proteins Biomarker for Phlebotomus argentipes Provides a Surveillance Tool Postelimination of Visceral Leishmaniasis in India.

Incidence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) has declined by more than 95% since initiation of the elimination program in 2005. As the ISC transitions to the postelimination surveillance phase, an accurate measurement of human-vector contact is needed to assure long-term success. To develop this tool, we identified PagSP02 and PagSP06 from saliva of Phlebotomus argentipes, the vector of Leishmania donovani in the ISC, as immunodominant proteins in humans. We also established the absence of cross-reactivity with Phlebotomus papatasi saliva, the only other human-biting sand fly in the ISC. Importantly, by combining recombinant rPagSP02 and rPagSP06 we achieved greater antibody recognition and specificity than single salivary proteins. The receiver operating characteristics curve for rPagSP02 + rPagSP06 predicts exposure to Ph. argentipes bites with 90% specificity and 87% sensitivity compared to negative control sera (P >.0001). Overall, rPagSP02 + rPagSP06 provides an effective surveillance tool for monitoring vector control efforts after VL elimination.

Development of Immunological Assays Based on Leishmania donovani Antigen for Diagnosis of Canine Visceral Leishmaniasis and Their Multicenter Evaluation in Brazil and Italy.

Canine visceral leishmaniasis (CVL) due to Leishmania infantum infection is a zoonotic disease prevalent in the areas of South America and the Mediterranean. Infected dogs as reservoirs can contribute to disease transmission and can be a scourge to public health. Therefore, early diagnosis of infected dogs may play a pivotal role in circumscribing disease progression. Invasive tissue aspiration and insufficient serological methods impair a single assay for prompt CVL diagnosis. In the present study, we aimed to evaluate the potential of Leishmania donovani isolated membrane protein, LAg, for the diagnosis of CVL through immunological assays. Initially, enzyme-linked immunosorbent assay was done with Brazilian dog sera to evaluate the performance of LAg in diagnosing CVL and found sensitivity and specificity of 92.50% and 95%, respectively. The study further confirmed the diagnostic efficacy of LAg in a dipstick format. The dipstick test of canine sera from three centers in Brazil and one center in Italy collectively showed sensitivity values in the range of 53.33% to 100% in recognizing symptomatic dogs and specificity values between 75% and 100% to rule out healthy dogs. Moreover, a rapid immunochromatographic test was developed and optimized using LAg. This test was able to identify 94.73% of CVL of Brazilian origin with specificity of 97.29%. The current results highlight the reactive potential of the L. donovani antigen, LAg, for L. infantum CVL diagnosis and support our previous findings, which suggest the utility of LAg for the diagnosis of both L. donovani and L. infantum human VL in a variety of endemic regions. LAg as a diagnostic candidate may be employed to identify comprehensive CVL cases in epidemiological areas.

Chlorogenic acid acts upon Leishmania donovani arresting cell cycle and modulating cytokines and nitric oxide in vitro.

AIMS: Visceral leishmaniasis (VL), caused by Leishmania donovani in India, is fatal if untreated, having serious concern of limited chemotherapeutic options. In this study, we evaluated antileishmanial efficacy of purified chlorogenic acid (CGA) against promastigotes and intracellular amastigotes infected into RAW264.7 macrophages. METHODS AND RESULTS: Chlorogenic acid was effective both on promastigotes (IC50  = 78.394 µmol/L, i.e. 27.75 µg/mL) and intracellular amastigotes (ED50  = 26.752 µmol/L, i.e. 9.47 µg/mL). In promastigotes, significant retardation in mitotic growth was caused both by cell-death and reduction of metabolic activity, evidenced by propidium-iodide uptake and MTT assay, respectively. Flow cytometric analysis revealed that retardation of mitotic growth was due to cell-cycle arrest at G1/S checkpoint. Complete clearance of amastigotes from infected RAW264.7 cells, assessed by microscopic counting, was achieved with 60 µmol/L (21.24 µg/mL) CGA for 24 hours, with negligible toxicity to host macrophages. This parasite clearing efficacy was comparable to 1.0 µg/mL (1.082 µmol/L) Amphotericin B, and 20 µmol/L Miltefosine, two standard antileishmanial drugs. Cytokine-ELISA revealed that elevated IL-10 production by infected macrophages was reduced after parasite clearance. Consequently, IL-12, TNF and NO (assayed by Griess test) production by macrophages were significantly increased after successful resolution of infection. CONCLUSION: Chlorogenic acid might emerge as a potential antileishmanial drug.

A multicentric evaluation of dipstick test for serodiagnosis of visceral leishmaniasis in India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain.

Visceral leishmaniasis (VL) is one of the leading infectious diseases affecting developing countries. Colloidal gold-based diagnostic tests are rapid tools to detect blood/serum antibodies for VL diagnosis. Lack of uniformity in the performance of these tests in different endemic regions is a hurdle in early disease diagnosis. This study is designed to validate a serum-based dipstick test in eight centres of six countries, India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain with archived and fresh sera from 1003 subjects. The dipstick detects antibodies against Leishmania donovani membrane antigens (LAg). The overall sensitivity and specificity of the test with 95% confidence intervals were found to be 97.10% and 93.44%, respectively. The test showed good sensitivity and specificity in the Indian subcontinent (>95%). In Brazil, Ethiopia, and Spain the sensitivity and specificity of the dipstick test (83.78-100% and 79.06-100%) were better as compared to the earlier reports of the performance of rK39 rapid test in these regions. Interestingly, less cross-reactivity was found with the cutaneous form of the disease in Spain, Brazil, and Sri Lanka demonstrating 91.58% specificity. This dipstick test can therefore be a useful tool for diagnosing VL from other symptomatically similar diseases and against cutaneous form of leishmaniasis.

Discovery of novel quinolinone adenosine A2B antagonists.

A novel series of quinolinone-based adenosine A(2B) receptor antagonists was identified via high throughput screening of an encoded combinatorial compound collection. Synthesis and assay of a series of analogs highlighted essential structural features of the initial hit. Optimization resulted in an A(2B) antagonist (2i) which exhibited potent activity in a cAMP accumulation assay (5.1 nM) and an IL-8 release assay (0.4 nM).

IL-10- and TGF-beta-mediated susceptibility in kala-azar and post-kala-azar dermal leishmaniasis: the significance of amphotericin B in the control of Leishmania donovani infection in India.

Visceral leishmaniasis (VL) or kala-azar is known to be associated with a mixed Th1-Th2 response, and effective host defense requires the induction of IFN-gamma and IL-12. We address the role of the differential decline of IL-10 and TGF-beta in response to sodium antimony gluconate (SAG) and amphotericin B (AmB), the therapeutic success of SAG and AmB in Indian VL, and the significance of IL-10 and TGF-beta in the development and progression of post-kazla-azar dermal leishmaniasis (PKDL). In the active disease, PBMC from VL patients showed suppressed Ag-specific lymphoproliferation, IFN-gamma and IL-12 production, and elevation of IL-10 and TGF-beta. Cure corresponded with an elevation in IFN-gamma and IL-12 production and down-regulation of IL-10 and TGF-beta. Both CD4(+) and CD8(+) T cells were involved in IFN-gamma and IL-10 production. Interestingly, the retention and maintenance of residual IL-10 and TGF-beta in some SAG-treated individuals and the elevation of IL-10 and TGF-beta in PKDL, a sequel to kala-azar, probably reflects the role of these cytokines in reactivation of the disease in the form of PKDL. Contrastingly, AmB treatment of VL resulted in negligible TGF-beta levels and absolute elimination of IL-10, reflecting the better therapeutic activity of AmB and its probable role in the recent decline in PKDL occurrences in India. Moreover, elucidation of immune responses in Indian PKDL patients revealed a spectral pattern of disease progression where disease severity could be correlated inversely with lymphoproliferation and directly with TGF-beta, IL-10, and Ab production. In addition, the enhancement of CD4(+)CD25(+) T cells in active VL, their decline at cure, and reactivation in PKDL suggest their probable immunosuppressive role in these disease forms.

Immune responses in kala-azar.

Human infection with Leishmania results in diverse clinical and immunopathological situations. The capacity of the parasites to cause this wide range of disease manifestations depends upon their ability to evade the immune defense mechanisms by performing a well-tuned orchestra of hostparasite interactions inside the macrophages. While updated knowledge focus on the key role of cell-mediated immunity (CMI) in protection, the survival strategies of the parasites leads to the suppression of CMI which can further be aggravated by the co-infections with HIV, tuberculosis etc. The present review describes the immune mechanisms in human leishmaniasis with a special attention to visceral leishmaniasis or kala-azar, one of the most important epidemiological health problems in Indian subcontinent. Modulations of the both humoral and cell-mediated immune responses during asymptomatic infections, active disease and after successful chemotherapy are discussed. The components responsible for the regulation of the critical balance of Th1/Th2 type of responses are re-evaluated. Co-infection of HIV and visceral leishmaniasis and their interdependence has been addressed. Although the specific role of an elevated humoral response in kala-azar is yet to be established, attempts for its application in diagnosis, precisely for the development of field diagnostic techniques, are presented. Also discussed are attempts to utilize the immunogenic potentials of different leishmanial antigens in the development of anti-leishmanial vaccines.

Leishmania promastigote membrane antigen-based enzyme-linked immunosorbent assay and immunoblotting for differential diagnosis of Indian post-kala-azar dermal leishmaniasis.

Diagnosis of post-kala-azar dermal leishmaniasis (PKDL), caused by Leishmania donovani, is difficult, as the dermal lesions are of several types and resemble those caused by other skin diseases, especially leprosy. Since the disease generally appears very late after the clinical cure of kala-azar in India, it is also difficult to correlate PKDL with a previous exposure to L. donovani. Very few attempts have been made so far to diagnose PKDL serologically, and the diagnostic methods vary in their sensitivities and specificities. Diagnosis of PKDL through sophisticated PCR methods, although highly sensitive, has limited practical use. We have developed a serodiagnostic method using an enzyme-linked immunosorbent assay to detect specific immunoglobulin (Ig) isotypes and IgG subclass antibodies in the sera of Indian PKDL patients. Our assay, which uses L. donovani promastigote membrane antigens, was 100% sensitive for the detection of IgG and 96.7% specific for the detection of IgG and IgG1. Optical density values for individual patients, however, demonstrated wide variations. Western blot analysis based on IgG reactivity could differentiate patients with PKDL from control subjects, which included patients with leprosy, patients from areas where kala-azar is endemic, and healthy subjects, by the detection of polypeptides of 67, 72, and 120 kDa. The recognition patterns of the majority of serum samples from patients with PKDL were also distinct from those of the serum samples from patients with visceral leishmaniasis (VL), at least for a 31-kDa polypeptide. To further differentiate patients with PKDL from those with active and cured VL, we analyzed the specific titers of the Ig isotypes and IgG subclasses. High levels of IgG, IgG1, IgG2, and IgG3 antibodies significantly differentiated patients with PKDL from patients cured of VL. The absence of antileishmanial IgE and IgG4 in patients with PKDL differentiated these patients from those with active VL. These results imply intrinsic differences in the antibodies generated in the sera from patients with PKDL and VL.

Caspase 3 regulates phosphatidylserine externalization and phagocytosis of oxidatively stressed erythrocytes.

The appearance of phosphatidylserine (PS) on the outer surface of red cells is an important signal for their uptake by macrophages. We report for the first time that procaspase 3 present in the anucleated mature human erythrocyte is activated under oxidative stress induced by t-butylhydroperoxide leading to impairment of the aminophospholipid translocase, PS externalization and increased erythrophagocytosis. This is the first report linking caspase 3 activation to inhibition of flippase activity and uptake of red cells by macrophages.