Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis

Background

With widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.

Methodology and Principal Findings

We measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC₅₀±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC₅₀ = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC₅₀ = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC₅₀ = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC₅₀ = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC₅₀ = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC₅₀ = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC₅₀ = 7.05±2.24 µM and 6.18±1.51 µM.

Conclusion

The in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.     

Lipophosphoglycan masks recognition of the Leishmania donovani promastigote surface by human kala-azar serum

Markedly elevated titers of anti-leishmanial antibodies are a hallmark of kala-azar. We investigated the role played by the lipophosphoglycan (LPG) in determining the reactivity of kala-azar serum with the surface of Leishmania donovani promastigotes. In assays performed with liver parasites there was negligible agglutination or fluorescent staining of LPG-bearing promastigotes by kala-azar serum, but strong reactivity in both assays with the use of an L. donovani mutant strain (R2D2) that lacks surface expression of LPG. Immunoprecipitation of lysates of 125I surface-labeled promastigotes indicated that kala-azar serum has reactivity with several surface proteins common to both the wild-type and R2D2 strains, and no reactivity with surface proteins unique to R2D2. Although direct ELISA showed that kala-azar serum recognizes purified promastigote LPG, inhibition ELISA suggested that such recognition is based solely upon reactivity with the normally unexposed core-anchor region of the molecule. We conclude that the poor reactivity of kala-azar serum with the surface of L. donovani promastigotes is caused by its lack of recognition of the exposed phosphodisaccharide repeat units of LPG, which in turn effectively mask the surface molecules that are recognized by kala-azar serum antibodies.     

Characterisation of Bangladeshi Leishmania isolated from kala-azar patients by isoenzyme electrophoresis

To identify the prevalent Leishmania species in Bangladesh, a total of nine patients aged 4-35 years, were studied; six (66.7%) of them were below 20 years of age. All the patients were clinically diagnosed to have visceral leishmaniasis; their haematological profile was in accordance with leishmaniasis and all were improved after treatment with sodium stibogluconate. All the aspirated materials (eight bone marrows and one splenic aspirate) yielded growth of Leishmania parasite in NNN media; Leishman-Donovan bodies were found in seven (77.8%) of them in a Giemsa stained smear. Aldehyde test (AT) was positive in all the nine cases examined, whereas, complement fixation test (CFT) was positive in seven (77.8%) and indirect fluorescent antibody test (IFAT) in eight (88.9%) cases. In this study, five of the nine isolates from kala-azar patients were characterised by isoenzyme analysis comparing with five WHO reference strains viz., Leishmania (Leishmania) donovani (DD8), L.(L.) donovani (HU3), L.(L.) infantum (IPT-1), L.(L.) tropica (K-27) and L.(L.) major (5-ASKH) using cellulose acetate electrophoresis. By analysing 11 soluble isoenzymes it was found that all five WHO reference strains had distinct electrophoretic mobility of the isoenzymes studied. No interspecies difference was observed amongst the five isolates from kala-azar patients examined and their isoenzyme profiles were consistent with WHO reference strain of L.(L.) donovani (DD8) but different from L.(L.) donovani (HU3).     

Characterization of two proteins from Leishmania donovani and their use for vaccination against visceral leishmaniasis

Two proteins from Leishmania donovani, dp72 and gp70-2, have been previously utilized to specifically serodiagnose patients with visceral leishmaniasis. The proteins were shown by ELISA and Western blotting with monoclonal and polyclonal antibodies to be present in both stages of the parasite. Antibodies to gp70-2 recognize in promastigotes multiple discrete bands of similar m.w. which are common to several isolates of L. donovani. The total amount of Ag and number of bands observed per isolate is not constant. Lectin blots with Con A show gp70-2 to be a glycoprotein. Dp72 shows pronounced microheterogeneity between isolates of L. donovani. The Brazilian isolates examined appear to possess a lower m.w. form (64,000 or 68,000) of this molecule. No reactions were observed with dp72 and lectins in Western blots; and neither tunicamycin, N-glycanase, endoglycosidase H nor F affected the migration of [35S]-methionine-labeled protein on SDS-PAGE. A mAb against dp72 also cross-reacted in Western blots with a 60-kDa protein in Leishmania major, Leishmania aethiopica, and Leishmania tropica. No reaction was observed between the purified promastigote surface protease (gp63) and either monoclonal or polyclonal antibodies produced to dp72 or gp70-2. The ability of the pure proteins to provide protection against a challenge by L. donovani amastigotes was examined. BALB/c mice were immunized with gp70-2 and/or dp72 by using Corynebacterium parvum as an adjuvant. Mice immunized with gp70-2 were not protected; however, mice receiving dp72 showed a 81.1% reduction in the liver parasitemia compared with the adjuvant controls.     

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.     

Inference of population structure of Leishmania donovani strains isolated from different Ethiopian visceral leishmaniasis endemic areas

Background

Parasites'' evolution in response to parasite-targeted control strategies, such as vaccines and drugs, is known to be influenced by their population genetic structure. The aim of this study was to describe the population structure of Ethiopian strains of Leishmania donovani derived from different areas endemic for visceral leishmaniasis (VL) as a prerequisite for the design of effective control strategies against the disease.

Methodology/Principal Findings

Sixty-three strains of L. donovani newly isolated from VL cases in the two main Ethiopian foci, in the north Ethiopia (NE) and south Ethiopia (SE) of the country were investigated by using 14 highly polymorphic microsatellite markers. The microsatellite profiles of 60 previously analysed L. donovani strains from Sudan, Kenya and India were included for comparison. Multilocus microsatellite typing placed strains from SE and Kenya (n = 30) in one population and strains from NE and Sudan (n = 65) in another. These two East African populations corresponded to the areas of distribution of two different sand fly vectors. In NE and Sudan Phlebotomus orientalis has been implicated to transmit the parasites and in SE and Kenya P. martini. The genetic differences between parasites from NE and SE are also congruent with some phenotypic differences. Each of these populations was further divided into two subpopulations. Interestingly, in one of the subpopulations of the population NE we observed predominance of strains isolated from HIV-VL co-infected patients and of strains with putative hybrid genotypes. Furthermore, high inbreeding irreconcilable from strict clonal reproduction was found for strains from SE and Kenya indicating a mixed-mating system.

Conclusions/Significance

This study identified a hierarchical population structure of L. donovani in East Africa. The existence of two main, genetically and geographically separated, populations could reflect different parasite-vector associations, different ecologies and varying host backgrounds and should be further investigated.     

Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis

Vaccination against visceral leishmaniasis has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine against visceral leishmaniasis is pressing. In this study, we demonstrate for the first time that a recombinant stage-specific hydrophilic surface protein of Leishmania donovani, recombinant hydrophilic acylated surface protein B1 (HASPB1), is able to confer protection against experimental challenge. Protection induced by rHASPB1 does not require adjuvant and, unlike soluble Leishmania Ag + IL-12, extends to the control of parasite burden in the spleen, an organ in which parasites usually persist and are refractory to a broad range of immunological and chemotherapeutic interventions. Both immunohistochemistry (for IL-12p40) and enzyme-linked immunospot assay (for IL-12p70) indicate that immunization with rHASPB1 results in IL-12 production by dendritic cells, although an analysis of Ab isotype responses to rHASPB1 suggests that this response is not sufficient in magnitude to induce a polarized Th1 response. Although both vaccinated and control-infected mice have equivalent frequencies of rHASPB1-specific CD4(+) T cells producing IFN-gamma, vaccine-induced protection correlates with the presence of rHASPB1-specific, IFN-gamma-producing CD8(+) T cells. Thus, we have identified a novel vaccine candidate Ag for visceral leishmaniasis, which appears to operate via a mechanism similar to that previously associated with DNA vaccination.     

Identification of amastigote-specific antigens of Leishmania donovani using kala-azar patient sera

Antigenic characterization of the soluble fraction of axenic amastigotes of Leishmania donovani ( strain Dd8, causative agent of Indian kala-azar) and their comparison with promastigotes is reported. The axenic amastigotes were assessed for their immunological status employing anti-A2 monoclonal antibody which is extremely specific for L. donovani amastigotes. SDS-PAGE of 35[S] methionine labeled proteins of the two parasite stages exhibited few stage specific and some conserved antigens in both the stages. An increased synthesis of heat shock proteins was observed in axenic amastigotes. Western blot experiments employing sera of kala azar positive patients identified immunodominent antigens of 116,83,26 and 12 kDa in axenic amastigotes which were not present in promastigotes. These amastigote stage specific antigens may have immense potential in immunodiagnosis and prophylaxis of kala-azar.     

Active cutaneous leishmaniasis in Brazil, induced by Leishmania donovani chagasi

L.d. chagasi was isolated from active cutaneous leishmaniasis in both human and canine infections in an endemic area in Rio de Janeiro, Brazil. Both isolates were identified by molecular and immunological characterization of the parasite using three different methods: electrophoretic mobility of isoenzymes; restriction endonuclease fragment analysis of kDNA and serodeme analysis using monoclonal antibodies. This seems to be the first well documented case in the New World of a "viscerotropic" Leishmania inducing a case of cutaneous leishmaniasis. This observation emphasizes that the diagnosis of the etiologic agent of human or canine visceral leishmaniasis based solely upon clinical and epidemiological criteria may lead to erroneous conclusions.     

Leishmania donovani: promastigote--macrophage surface interactions in vitro.

The mechanism by which promastigotes of Leismania donovani enter hamster peritoneal macrophages was studied in vitro by light and electron microscopy. Quantitative light microscope studies showed a time-dependent increase of intracellular parasites, which had no preferable orientation during entry. Scanning and transmission electron microscopy revealed striking host-parasite surface interactions marked by the formation of whorled pseudopodia around the promastigotes in the early stage and engulfment of the parasites akin to normal phagocytosis in the later phase. Early host-parasite interactions were categorized quantitatively by scanning electron microscopy into several types, of which “head-first entry” and “tail-first entry” were approximately equal in frequency of occurrence, confirming light microscope observations. Cytochalasin B at 10 μg/ml prevented the intracellular entry of the parasites and the formation of macrophage-originated pseudopodia normally seen to seize the promastigotes. Killed, but morphologically intact, promastigotes were poorly taken up by macrophages and lacked certain types of interactions normally encountered with macrophage pseudopodia. Motility of promastigotes and their affinity to the surface of macrophages are suggested as elements of importance which parasites contribute to aid the process of their entry. The above results indicate that promastigotes of L. donovani depend on phagocytic activity of macrophages to gain intracellular entrance, but parasite-specific activities and/or properties may also play a role. It is suggested that “facilitated phagocytosis” may be used to describe this unique type of endocytosis associated with leishmania-macrophage interactions.     

Post-translational modification of cellular proteins during Leishmania donovani differentiation

The pathogenic intracellular parasites Leishmania donovani cycle between sand fly gut and the human macrophage phagolysosome, differentiating from extracellular promastigotes to intracellular amastigote forms. Using isobaric tagging for relative and absolute quantifications (iTRAQ/LC-MS/MS) proteomic methodology, we recently described the ordered gene expression changes during this process. While protein abundance changes in Leishmania were documented, little is known about their PTMs. Here we used iTRAQ to detect protein phosphorylation, methylation, acetylation, and glycosylation sites throughout differentiation. We found methylation of arginines, aspartic acids, glutamic acids, asparagines, and histidines. Detected acetylation sites included serines and protein N-terminal acetylations on methionines, serines, alanines, and threonines. Phosphorylations were detected on serines and threonines, but not tyrosines. iTRAQ identified novel fucosylation sites as well as hexosylations. We observed quantity changes in some modifications during differentiation, suggesting a role in L. donovani intracellular development. This study is the first high-throughput analysis of PTM sites dynamics during an intracellular parasitic development.     

Leishmania donovani vs immunity: T-cells sensitized from Leishmania of one donor may modulate their cytokines pattern on re-stimulation with Leishmania from different donor in visceral leishmaniasis

Antimony resistance is frequently encountered during treatment of visceral leishmaniasis (VL) and the differences are well characterized by inadequate IFN-γ dominant type-1 protection mechanisms. The part played by Leishmania parasites derived from antimony treated patients in the outcome of an immune response largely remains to be investigated. In the present study we observed that macrophages of BALB/c mice infected with antimony non-responder (SAG-NR) isolates had a greater amastigote burden than antimony responder (SAG-R) isolates. Later it was observed that antigen from SAG-NR and R L. donovani isolates elicit different cytokine responses in peritoneal blood mononuclear cells (PBMCs) from patients with VL. The production of IFN-γ by T-cells in VL patients increased in response to Leishmania derived from responder patients but this response within same T-cells was lower when sensitized from Leishmania from a non-responder VL patient. On the other hand, IL-4 and IL-10 expression was increased when primed with parasites from non-responder VL source. Such a differential pattern of cytokine expression by the same T-cell population produced to Leishmania from different donors, needs further exploration.     

Leishmania species and zymodemes isolated from endemic areas of cutaneous leishmaniasis in Jordan

Background  

Cutaneous leishmaniasis (CL) is endemic in the Middle Eastern countries. New cases are emerging in areas previously free of the disease. In Jordan, the diagnosis of cases during the 1960s and 1970s was mainly reported in military hospitals in Amman. Endemicity of the disease was ascertained after reporting a total of 524 cases during 1973–1978.