Showing papers on "Visceral leishmaniasis published in 2017"

Drug resistance and treatment failure in leishmaniasis: A 21st century challenge

Abstract: Reevaluation of treatment guidelines for Old and New World leishmaniasis is urgently needed on a global basis because treatment failure is an increasing problem. Drug resistance is a fundamental determinant of treatment failure, although other factors also contribute to this phenomenon, including the global HIV/AIDS epidemic with its accompanying impact on the immune system. Pentavalent antimonials have been used successfully worldwide for the treatment of leishmaniasis since the first half of the 20th century, but the last 10 to 20 years have witnessed an increase in clinical resistance, e.g., in North Bihar in India. In this review, we discuss the meaning of “resistance” related to leishmaniasis and discuss its molecular epidemiology, particularly for Leishmania donovani that causes visceral leishmaniasis. We also discuss how resistance can affect drug combination therapies. Molecular mechanisms known to contribute to resistance to antimonials, amphotericin B, and miltefosine are also outlined.

Chemotherapy of leishmaniasis: present challenges

Abstract: Cutaneous and visceral leishmaniasis are amongst the most devastating infectious diseases of our time, affecting millions of people worldwide. The treatment of these serious diseases rely on a few chemotherapeutic agents, most of which are of parenteral use and induce severe side-effects. Furthermore, rates of treatment failure are high and have been linked to drug resistance in some areas. Here, we reviewed data on current chemotherapy practice in leishmaniasis. Drug resistance and mechanisms of resistance are described as well as the prospects for applying drug combinations for leishmaniasis chemotherapy. It is clear that efforts for discovering new drugs applicable to leishmaniasis chemotherapy are essential. The main aspects on the various steps of drug discovery in the field are discussed.

Leishmaniasis: A Review on Parasite, Vector and Reservoir Host

Abstract: Leishmaniasis is a vector-borne disease affecting millions of people worldwide. The disease is caused by different species of Leishmania, and it is manifested by three major clinical forms namely cutaneous, muco-cutaneous and visceral leishmaniasis. This review is aimed to address briefly the parasite, the vector and the reservoir aspects in transmission of leishmaniasis in different regions of the world. The complexity of transmission of the disease lays on the complex life cycle of the parasite which involves sand fly vectors and mammalian reservoirs. The transmission can either be zoonotic and/or anthroponotic through the bite of an infected female sand fly. There is uneven global distribution of the disease often because of the various distribution patterns of the parasite, the vector and the reservoir host species. Various sand fly species in the genus Phlebotomus and Lutzomyia are responsible for transmission of leishmaniasis in the old and new worlds. Animal reservoirs are important for maintaining infections in various areas, and hence are important for zoonotic and rural/sylvatic transmission of the disease. A high prevalence of infection has been reported from small to large domestic and wild mammals. Control and elimination of leishmaniasis require detection of human and animal cases, identification of reservoir hosts, and implementation of effective vector control strategies in areas.

A third generation vaccine for human visceral leishmaniasis and post kala azar dermal leishmaniasis: First-in-human trial of ChAd63-KH

Abstract: Background Visceral leishmaniasis (VL or kala azar) is the most serious form of human leishmaniasis, responsible for over 20,000 deaths annually, and post kala azar dermal leishmaniasis (PKDL) is a stigmatizing skin condition that often occurs in patients after successful treatment for VL. Lack of effective or appropriately targeted cell mediated immunity, including CD8+ T cell responses, underlies the progression of VL and progression to PKDL, and can limit the therapeutic efficacy of anti-leishmanial drugs. Hence, in addition to the need for prophylactic vaccines against leishmaniasis, the development of therapeutic vaccines for use alone or in combined immuno-chemotherapy has been identified as an unmet clinical need. Here, we report the first clinical trial of a third-generation leishmaniasis vaccine, developed intentionally to induce Leishmania-specific CD8+ T cells. Methods We conducted a first-in-human dose escalation Phase I trial in 20 healthy volunteers to assess the safety, tolerability and immunogenicity of a prime-only adenoviral vaccine for human VL and PKDL. ChAd63-KH is a replication defective simian adenovirus expressing a novel synthetic gene (KH) encoding two Leishmania proteins KMP-11 and HASPB. Uniquely, the latter was engineered to reflect repeat domain polymorphisms and arrangements identified from clinical isolates. We monitored innate immune responses by whole blood RNA-Seq and antigen specific CD8+ T cell responses by IFNγ ELISPOT and intracellular flow cytometry. Findings ChAd63-KH was safe at intramuscular doses of 1x1010 and 7.5x1010 vp. Whole blood transcriptomic profiling indicated that ChAd63-KH induced innate immune responses characterized by an interferon signature and the presence of activated dendritic cells. Broad and quantitatively robust CD8+ T cell responses were induced by vaccination in 100% (20/20) of vaccinated subjects. Conclusion The results of this study support the further development of ChAd63-KH as a novel third generation vaccine for VL and PKDL. Trial registration This clinical trial (LEISH1) was registered at EudraCT (2012-005596-14) and ISRCTN (07766359).

Laboratory confirmed miltefosine resistant cases of visceral leishmaniasis from India.

Abstract: Miltefosine unresponsive and relapse cases of visceral leishmaniasis (VL) are increasingly being reported. However, there has been no laboratory confirmed reports of miltefosine resistance in VL. Here, we report two laboratory confirmed cases of VL from India. Two patients with VL were referred to us with suspected VL. The first patient was a native of the VL endemic state of Bihar, but residing in Delhi, a VL non-endemic area. He was treated with broad-spectrum antibiotics and antipyretics but was unresponsive to treatment. The second patient was from Jharkhand state in eastern India (adjoining Bihar), another endemic state for VL. He was refractory to anti-leishmanial treatment, which included administration of miltefosine. Following investigation, both patients were serologically positive for VL, and blood buffy coat from both patients grew Leishmania donovani. The isolates derived from both cases were characterized for their drug susceptibility, genetically characterised, and SNPs typed for LdMT and LdROS gene expression. Both patients were successfully treated with amphotericin B. The in vitro drug susceptibility assays carried out on both isolates showed good IC50 values to amphotericin B (0.1 ± 0.0004 μg/ml and 0.07 ± 0.0019 μg/ml). One isolate was refractory to SbIII with an IC50 of > 200 μM while the second isolate was sensitive to SbIII with an IC50 of 36.70 ± 3.2 μM. However, in both the isolates, IC50 against miltefosine was more than 10-fold higher (> 100 μM) than the standard strain DD8 (6.8 ± 0.1181 μM). Furthermore, genetic analyses demonstrated single nucleotide polymorphisms (SNPs) (354Tyr↔Phe and 1078Phe↔Tyr) in the LdMT gene of the parasites. Here, we document two laboratory confirmed cases of miltefosine resistant VL from India. Our finding highlights the urgent need to establish control measures to prevent the spread of these strains. We also propose that LdMT gene mutation analysis could be used as a molecular marker of miltefosine resistance in L. donovani.

The Equivocal Role of Th17 Cells and Neutrophils on Immunopathogenesis of Leishmaniasis.

Abstract: Advances in the understanding of leishmaniasis progression indicate that cellular interactions more complex than the Th1/Th2 paradigmdefine the course of infection. Th17 cells are a crucial modulator of adaptive immunity against Leishmania parasites acting mainly on neutrophil recruitment and playing a dual role at the site of infection. This review describes the roles of both these cell typesin linking innate defenseresponses to the establishment of specific immunity. We focus on the Th17–neutrophil interaction as a crucial component of anti-Leishmania immunity, and the clinical evolution of cutaneous or visceral leishmaniasis.To date, information obtained through experimental models and patient evaluations suggests that the influence of the presence of interleukin (IL)-17 (the main cytokine produced by Th17 cells) and neutrophilsduring Leishmania infections is strictly dependent on the tissue (skin or liver/spleen) and parasite species. Also, the time at which neutrophils are recruited, and the persistence of IL-17 in the infection microenvironment, may also be significant. A clearer understanding of these interactionswill enable better measurement ofthe influence of IL-17 and its regulators,and contribute to the identification of disease/resistance biomarkers.

Chemotherapeutics of visceral leishmaniasis: present and future developments.

Abstract: Treatment of Visceral Leishmaniasis (VL), a neglected tropical disease, is very challenging with few treatment options. Long duration of treatment and drug toxicity further limit the target of achieving VL elimination. Chemotherapy remains the treatment of choice. Single dose of liposomal amphotericin B (LAmB) and multidrug therapy (LAmB + miltefosine, LAmB + paromomycin (PM), or miltefosine + PM) are recommended treatment regimen for treatment of VL in Indian sub-continent. Combination therapy of pentavalent antimonials (Sbv) and PM in East Africa and LAmB in the Mediterranean region/South America remains the treatment of choice. Various drugs having anti-leishmania properties are in preclinical phase and need further development. An effective treatment and secondary prophylaxis of HIV-VL co-infection should be developed to decrease treatment failure and drug resistance.

Phlebotomine sand fly-borne pathogens in the Mediterranean Basin: Human leishmaniasis and phlebovirus infections.

Abstract: Pathogens transmitted to humans by phlebotomine sand flies are neglected, as they cause infectious diseases that are not on the priority list of national and international public health systems. However, the infections caused by protozoa of the Leishmania genus and viruses belonging to the Phlebovirus genus (family Phenuiviridae)-the most significant group of viruses transmitted by sand flies-have a relevant role for human pathology. These infections are emerging in the Mediterranean region and will likely spread in forthcoming decades, posing a complex threat to human health. Four species and 2 hybrid strains of Leishmania are pathogenic for humans in the Mediterranean Basin, with an estimated annual incidence of 239,500-393,600 cases of cutaneous leishmaniasis and 1,200-2,000 cases of visceral leishmaniasis. Among the phleboviruses, Toscana virus can cause neuroinvasive infections, while other phleboviruses are responsible for a typical "3-day fever"; the actual incidence of Phlebovirus infections in the Mediterranean area is unknown, although at least 250 million people are exposed. Here, we reviewed the current literature on epidemiology of sand fly-borne infections in the Mediterranean Basin, with a focus on humans. Our analysis indicates the need for increased public health activities directed to determine the disease burden of these infections as well as to improve their surveillance. Among the emerging challenges concerning sand fly-borne pathogens, the relationships between sand fly-borne protozoa and viruses should be considered in future studies, including epidemiological links between Leishmania and phleboviruses as well as the conditional capacity for these pathogens to be involved in interactions that may evolve towards increased virulence.

Antileishmanial Activity of Pyrazolopyridine Derivatives and Their Potential as an Adjunct Therapy with Miltefosine.

Abstract: A series of pyrazolo(dihydro)pyridines was synthesized and evaluated for antileishmanial efficacy against experimental visceral leishmaniasis (VL). Among all compounds, 6d and 6j exhibited better activity than miltefosine against intracellular amastigotes. Compound 6j (50 mg/kg/day) was further studied against Leishmania donovani/BALB/c mice via the intraperitoneal route for 5 days and displayed >91 and >93% clearance of splenic and liver parasitic burden, respectively. Combination treatment of 6j with a subcurative dose of miltefosine (5 mg/kg) in BALB/c mice almost completely ameliorated the disease (>97% inhibition) by augmenting nitric oxide generation and shifting the immune response toward Th1. Furthermore, investigating the effect of 6j on Leishmania promastigotes revealed that it induced molecular events, such as a loss in mitochondrial membrane potential, externalization of phosphatidylserine, and DNA fragmentation, that ultimately resulted in the programmed cell death of the parasite. These resu...

Comparative genomics of canine-isolated Leishmania (Leishmania) amazonensis from an endemic focus of visceral leishmaniasis in Governador Valadares, southeastern Brazil

Abstract: Leishmaniasis is a highly diverse group of diseases caused by kinetoplastid of the genus Leishmania. These parasites are taxonomically diverse, with human pathogenic species separated into two subgenera according to their development site inside the alimentary tract of the sand fly insect vector. The disease encompasses a variable spectrum of clinical manifestations with tegumentary or visceral symptoms. Among the causative species in Brazil, Leishmania (Leishmania) amazonensis is an important etiological agent of human cutaneous leishmaniasis that accounts for more than 8% of all cases in endemic regions. L. (L.) amazonensis is generally found in the north and northeast regions of Brazil. Here, we report the first isolation of L. (L.) amazonensis from dogs with clinical manifestations of visceral leishmaniasis in Governador Valadares, an endemic focus in the southeastern Brazilian State of Minas Gerais where L. (L.) infantum is also endemic. These isolates were characterized in terms of SNPs, chromosome and gene copy number variations, confirming that they are closely related to a previously sequenced isolate obtained in 1973 from the typical Northern range of this species. The results presented in this article will increase our knowledge of L. (L.) amazonensis-specific adaptations to infection, parasite survival and the transmission of this Amazonian species in a new endemic area of Brazil.

Increased miltefosine tolerance in clinical isolates of Leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress.

Abstract: Background Miltefosine (MIL) is an oral antileishmanial drug used for treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Recent reports indicate a significant decline in its efficacy with a high rate of relapse in VL as well as post kala-azar dermal leishmaniasis (PKDL). We investigated the parasitic factors apparently involved in miltefosine unresponsiveness in clinical isolates of Leishmania donovani. Methodology L. donovani isolated from patients of VL and PKDL at pretreatment stage (LdPreTx, n = 9), patients that relapsed after MIL treatment (LdRelapse, n = 7) and parasites made experimentally resistant to MIL (LdM30) were included in this study. MIL uptake was estimated using liquid chromatography coupled mass spectrometry. Reactive oxygen species and intracellular thiol content were measured fluorometrically. Q-PCR was used to assess the differential expression of genes associated with MIL resistance. Results LdRelapse parasites exhibited higher IC50 both at promastigote level (7.92 ± 1.30 μM) and at intracellular amastigote level (11.35 ± 6.48 μM) when compared with LdPreTx parasites (3.27 ± 1.52 μM) and (3.85 ± 3.11 μM), respectively. The percent infectivity (72 hrs post infection) of LdRelapse parasites was significantly higher (80.71 ± 5.67%, P<0.001) in comparison to LdPreTx (60.44 ± 2.80%). MIL accumulation was significantly lower in LdRelapse parasites (1.7 fold, P<0.001) and in LdM30 parasites (2.4 fold, P<0.001) when compared with LdPreTx parasites. MIL induced ROS levels were significantly lower (p<0.05) in macrophages infected with LdRelapse while intracellular thiol content were significantly higher in LdRelapse compared to LdPreTx, indicating a better tolerance for oxidative stress in LdRelapse isolates. Genes associated with oxidative stress, metabolic processes and transporters showed modulated expression in LdRelapse and LdM30 parasites in comparison with LdPreTx parasites. Conclusion The present study highlights the parasitic factors and pathways responsible for miltefosine unresponsiveness in VL and PKDL.

Nanotized Curcumin and Miltefosine, a Potential Combination for Treatment of Experimental Visceral Leishmaniasis.

Abstract: Leishmaniasis chemotherapy remains very challenging due to high cost of the drug and its associated toxicity and drug resistance, which develops over a period of time. Combination therapies (CT) are now in use to treat many diseases, such as cancer and malaria, since it is more effective and affordable than monotherapy. CT are believed to represent a new explorable strategy for leishmaniasis, a neglected tropical disease caused by the obligate intracellular parasite Leishmania In the present study, we investigated the effect of a combination of a traditional Indian medicine (ayurveda), a natural product curcumin and miltefosine, the only oral drug for visceral leishmaniasis (VL) using a Leishmania donovani-hamster model. We developed an oral nanoparticle-based formulation of curcumin. Nanoformulation of curcumin alone exhibited significant leishmanicidal activity both in vitro and in vivo In combination with miltefosine, it exhibited a synergistic effect on both promastigotes and amastigotes under in vitro conditions. The combination of these two agents also demonstrated increased in vivo leishmanicidal activity accompanied by increased production of toxic reactive oxygen/nitrogen metabolites and enhanced phagocytic activity. The combination also exhibited increased lymphocyte proliferation. The present study thus establishes the possible use of nanocurcumin as an adjunct to antileishmanial chemotherapy.

Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment.

Abstract: Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19-100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16-94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55-89.16%) for VL samples, 100% (95%CI, 69.15-100%) for RVL samples, and 52.5% (95% CI, 36.13-68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98-100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L. donovani parasites in VL, PKDL and RVL patients, as well as being capable of assessing response to treatment. As such, this real time PCR assay represents an important contribution in efforts to eliminate VL.

Transcriptional Profiling in Experimental Visceral Leishmaniasis Reveals a Broad Splenic Inflammatory Environment that Conditions Macrophages toward a Disease-Promoting Phenotype.

Abstract: Visceral Leishmaniasis (VL), caused by the intracellular protozoan Leishmania donovani, is characterized by relentlessly increasing visceral parasite replication, cachexia, massive splenomegaly, pancytopenia and ultimately death. Progressive disease is considered to be due to impaired effector T cell function and/or failure of macrophages to be activated to kill the intracellular parasite. In previous studies, we used the Syrian hamster (Mesocricetus auratus) as a model because it mimics the progressive nature of active human VL. We demonstrated previously that mixed expression of macrophage-activating (IFN-γ) and regulatory (IL-4, IL-10, IL-21) cytokines, parasite-induced expression of macrophage arginase 1 (Arg1), and decreased production of nitric oxide are key immunopathologic factors. Here we examined global changes in gene expression to define the splenic environment and phenotype of splenic macrophages during progressive VL. We used RNA sequencing coupled with de novo transcriptome assembly, because the Syrian hamster does not have a fully sequenced and annotated reference genome. Differentially expressed transcripts identified a highly inflammatory spleen environment with abundant expression of type I and type II interferon response genes. However, high IFN-γ expression was ineffective in directing exclusive M1 macrophage polarization, suppressing M2-associated gene expression, and restraining parasite replication and disease. While many IFN-inducible transcripts were upregulated in the infected spleen, fewer were induced in splenic macrophages in VL. Paradoxically, IFN-γ enhanced parasite growth and induced the counter-regulatory molecules Arg1, Ido1 and Irg1 in splenic macrophages. This was mediated, at least in part, through IFN-γ-induced activation of STAT3 and expression of IL-10, which suggests that splenic macrophages in VL are conditioned to respond to macrophage activation signals with a counter-regulatory response that is ineffective and even disease-promoting. Accordingly, inhibition of STAT3 activation led to a reduced parasite load in infected macrophages. Thus, the STAT3 pathway offers a rational target for adjunctive host-directed therapy to interrupt the pathogenesis of VL.

Ly6Chi inflammatory monocytes promote susceptibility to Leishmania donovani infection.

Abstract: Ly6Chi inflammatory monocytes (iMO) are critical for host defense against toxoplasmosis and malaria but their role in leishmaniasis is unclear. In this study, we report a detrimental role of Ly6Chi iMOs in visceral leishmaniasis (VL) caused by Leishmania donovani. We demonstrate that Ly6Chi iMOs are continuously recruited into the spleen and liver during L. donovani infection and they are preferential targets for the parasite. Using microarray-based gene expression profiling, we show that Ly6Chi iMOs isolated from the infected liver and spleen have distinct phenotypic and activation profiles. Furthermore, we demonstrate that blocking the recruitment of Ly6Chi iMOs into the liver and spleen during L. donovani infection using a CCR2 antagonist reduces the frequency of the pathogenic IFN-γ/IL10 dual producer CD4+ T cells in the spleen and leads to a significant reduction in parasite loads in the liver and spleen. Using STAT1-/- mice we show that STAT1 is critical for mediating the recruitment of Ly6Chi iMOs into organs during L. donovani infection, and adaptive transfer of wild type Ly6Chi iMOs into STAT1-/- recipients renders them susceptible to disease. Our findings reveal an unexpected pathogenic role for Ly6Chi iMOs in promoting parasite survival in VL and open the possibility of targeting this population for host-directed therapy during VL.

Leishmaniasis in Thailand: A Review of Causative Agents and Situations

Abstract: Before 1999, leishmaniasis was considered an imported disease in Thailand. Since then, autochthonous leishmaniasis was reported in both immmunocompetent and immmunocompromised patients especially in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS). A new species was identified and named as Leishmania siamensis consisting of two lineages, that is, lineages TR and PG. Analysis of isoenzymes has clarified the more commonly detected L. siamensis lineage PG as Leishmania martiniquensis (MON-229), a species originally reported from the Martinique Island, whereas the L. siamensis lineage TR has been identified as the true novel species, L. siamensis (MON-324). Both cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) have been found among Thai patients. Disseminated CL and VL could be presented in some reported patients who had HIV/AIDS coinfection. So far, only sporadic cases have been reported; thus, the true prevalence of leishmaniasis should be determined in Thailand among the high-risk populations such as people with HIV/AIDS. A recent survey among animals identified L. martiniquensis DNA in black rats (Rattus rattus) suggesting a potential animal reservoir. In addition, L. martiniquensis DNA was identified in Sergentomyia gemmea and Sergentomyia barraudi, the predominant sandfly species in the affected areas. However, further studies are needed to prove that these sandflies could serve as the vector of leishmaniasis in Thailand.

Identification of Potential MHC Class-II-Restricted Epitopes Derived from Leishmania donovani Antigens by Reverse Vaccinology and Evaluation of Their CD4+ T-Cell Responsiveness against Visceral Leishmaniasis.

Abstract: Visceral leishmaniasis (VL) is one of the most neglected tropical diseases for which no vaccine exists. In spite of extensive efforts, no successful vaccine is available against this dreadful infectious disease. To support vaccine development, an immunoinformatics approach was applied to screen potential MHC class-II-restricted epitopes that can activate the immune cells. Initially, 37 epitopes derived from six stage-dependent, overexpressed antigens were predicted, which were presented by at least 26 diverse MHC class-II allele. Based on a population coverage analysis and human leukocyte antigen cross-presentation ability, six of the 37 epitopes were selected for further analysis. Stimulation with synthetic peptide alone or as a cocktail triggered intracellular IFN-γ production. Moreover, specific IgG antibodies were detected in the serum of active VL cases against P1, P4, P5, and P6 in order to evaluate the peptide effect on the humoral immune response. Additionally, most of the peptides, except P2, were found to be non-inducers of CD4+ IL-10 against both active VL as well as treated VL subjects. This finding suggests there is no role of these peptides in the pathogenesis of Leishmania. Peptide immunogenicity was validated in BALB/c mice immunized with a cocktail of synthetic peptide emulsified in complete Freund's adjuvant/incomplete Freund's adjuvant. The immunized splenocytes induced strong spleen cell proliferation upon parasite re-stimulation. Furthermore, increased IFN-γ, interleukin-12, IL-17, and IL-22 production augmented with elevated nitric oxide (NO) synthesis is thought to play a crucial role in macrophage activation. In this investigation, we identified six MHC class-II-restricted epitope hotspots of Leishmania antigens that induce CD4+ Th1 and Th17 responses, which could be used to potentiate a human universal T-epitope vaccine against VL.

Infection-adapted emergency hematopoiesis promotes visceral leishmaniasis.

Abstract: Cells of the immune system are derived from hematopoietic stem cells (HSCs) residing in the bone marrow. HSCs become activated in response to stress, such as acute infections, which adapt the bone marrow output to the needs of the immune response. However, the impact of infection-adapted HSC activation and differentiation on the persistence of chronic infections is poorly understood. We have examined here the bone marrow outcome of chronic visceral leishmaniasis and show that the parasite Leishmania donovani induces HSC expansion and skews their differentiation towards non-classical myeloid progenitors with a regulatory phenotype. Our results further suggest that emergency hematopoiesis contributes to the pathogenesis of visceral leishmaniasis, as decreased HSC expansion results in a lower parasite burden. Conversely, monocytes derived in the presence of soluble factors from the infected bone marrow environment are more permissive to infection by Leishmania. Our results demonstrate that L. donovani is able to subvert host bone marrow emergency responses to facilitate parasite persistence, and put forward hematopoiesis as a novel therapeutic target in chronic infections.

Skin parasite landscape determines host infectiousness in visceral leishmaniasis

Abstract: Increasing evidence suggests that the infectiousness of patients for the sand fly vector of visceral leishmaniasis is linked to parasites found in the skin. Using a murine model that supports extensive skin infection with Leishmania donovani, spatial analyses at macro-(quantitative PCR) and micro-(confocal microscopy) scales indicate that parasite distribution is markedly skewed. Mathematical models accounting for this heterogeneity demonstrate that while a patchy distribution reduces the expected number of sand flies acquiring parasites, it increases the infection load for sand flies feeding on a patch, increasing their potential for onward transmission. Models representing patchiness at both macro- and micro-scales provide the best fit with experimental sand fly feeding data, pointing to the importance of the skin parasite landscape as a predictor of host infectiousness. Our analysis highlights the skin as a critical site to consider when assessing treatment efficacy, transmission competence and the impact of visceral leishmaniasis elimination campaigns.Parasitemia has been considered the main determinant of visceral leishmaniasis transmission. By combining imaging, qPCR and experimental xenodiagnoses with mathematical models, Doehl et al. argue that the patchy landscape of parasites in the skin is necessary to explain infectiousness.

Field trial of efficacy of the Leish-tec® vaccine against canine leishmaniasis caused by Leishmania infantum in an endemic area with high transmission rates.

Abstract: Background: Because domestic dogs are reservoir hosts for visceral leishmaniasis (VL) in Brazil, one of the approaches used to reduce human disease incidence is to cull infected dogs. However, the results of controlled intervention trials based on serological screening of dogs and killing of seropositive animals are equivocal. A prophylactic vaccine to protect dogs from being infectious to the sand fly vector could be an effective strategy to provide sustained control. Here, we investigated whether a currently licensed commercial subunit rA2 protein-saponin vaccine (Leish-tec®) had an additional effect to dog culling on reducing the canine infectious populations. Methodology/principal findings: This prospective study was conducted in an L. infantum highly endemic area of southeast Brazil. At the onset of the intervention, all of the eligible dogs received through subcutaneous route a three-dose vaccine course at 21-day intervals and a booster on month 12. For the purpose of comparison, newly recruited healthy dogs were included as the exposed control group. To ascertain vaccine-induced protection, dogs were screened on clinical and serological criteria every 6 months for a 2-year follow-up period. Antibody-based tests and histopathological examination of post-mortem tissue specimens from euthanized animals were used as a marker of infection. The standardized vaccine regime, apart from being safe, was immunogenic as immunized animals responded with a pronounced production of anti-A2-specific IgG antibodies. It should be noted the mean seroconversion time for infection obtained among immunized exposed dogs (~ 18 months), which was twice as high as that for unvaccinated ones (~ 9 months). After two transmission cycles completed, the cumulative incidence of infection did differ significantly (P = 0.016) between the vaccinated (27%) and unvaccinated (42%) dogs. However, the expected efficacy for the vaccine in inducing clinical protection was not evident since 43% of vaccine recipients developed disease over time. Our estimates also indicated that immunoprophylaxis by Leish-tec® vaccine in addition to dog culling might not have an impact on bringing down the incidence of canine infection with L. infantum in areas of high transmission rates. Conclusions/significance: Leish-tec® as a prophylactic vaccine showed promise but needs to be further optimized to be effective in dogs under field conditions, and thereby positively impacts human incidence.

Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection

Abstract: Leishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application. We have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL). The assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse. The study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid molecular diagnosis of VL and PKDL with potential for application in assessment of cure.

Clinical and immunopathological findings during long term follow-up in Leishmania infantum experimentally infected dogs

Abstract: Canine Visceral Leishmaniasis (CVL) is caused by Leishmania infantum, which in the New World is transmitted by Lutzomyia longipalpis. While prospective clinical and immunological assessments of dogs experimentally challenged with L. infantum have been previously reported over a relatively short follow-up period, the long-term characterization of infected animals has not been performed to date. We evaluated dogs in a subclinical state for six years following experimental infection with L. infantum and Lu. longipalpis saliva, via an intradermal route, to characterize clinical, parasitological and immunological parameters arising from L. infantum experimental infection. We also assess these parameters in a group of naturally infected animals. The immune profiles of the experimentally and naturally infected animals exhibited increases of IFN-γ, IL-6 and IL-18, and decreases in TNF, IL-2, IL-8 and CXCL1, compared to controls. Our results indicate that over a six-year follow-up post-challenge, subclinically infected dogs presented low CVL clinical scores despite the persistence of Leishmania parasites in the lymph nodes, spleen and skin. Similarities observed among immune profiles in the context of experimental and natural infection seem to suggest that an enduring activation of the host immune response may lead to the control of parasite growth, thereby limiting disease severity.

Safety and Effectiveness of Sodium Stibogluconate and Paromomycin Combination for the Treatment of Visceral Leishmaniasis in Eastern Africa: Results from a Pharmacovigilance Programme

Abstract: Introduction In 2010, WHO recommended a new first-line treatment for visceral leishmaniasis (VL) in Eastern Africa. The new treatment, a combination of intravenous (IV) or intramuscular (IM) sodium stibogluconate (SSG) and IM paromomycin (PM) was an improvement over SSG monotherapy, the previous first-line VL treatment in the region. To monitor the new treatment’s safety and effectiveness in routine clinical practice a pharmacovigilance (PV) programme was developed.

Vaccination with poly(D,L-lactide-co-glycolide) nanoparticles loaded with soluble Leishmania antigens and modified with a TNFα-mimicking peptide or monophosphoryl lipid A confers protection against experimental visceral leishmaniasis.

Abstract: Visceral leishmaniasis (VL) persists as a major public health problem, and since the existing chemotherapy is far from satisfactory, development of an effective vaccine emerges as the most appropriate strategy for confronting VL. The development of an effective vaccine relies on the selection of the appropriate antigen and also the right adjuvant and/or delivery vehicle. In the present study, the protective efficacy of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs), which were surface-modified with a TNFα-mimicking eight-amino-acid peptide (p8) and further functionalized by encapsulating soluble Leishmania infantum antigens (sLiAg) and monophosphoryl lipid A (MPLA), a TLR4 ligand, was evaluated against challenge with L. infantum parasites in BALB/c mice. Vaccination with these multifunctionalized PLGA nanoformulations conferred significant protection against parasite infection in vaccinated mice. In particular, vaccination with PLGA-sLiAg-MPLA or p8-PLGA-sLiAg NPs resulted in almost complete elimination of the parasite in the spleen for up to 4 months post-challenge. Parasite burden reduction was accompanied by antigen-specific humoral and cellular immune responses. Specifically, injection with PLGA-sLiAg-MPLA raised exclusively anti-sLiAg IgG1 antibodies post-vaccination, while in p8-PLGA-sLiAg-vaccinated mice, no antibody production was detected. However, 4 months post-challenge, in mice vaccinated with all the multifunctionalized NPs, antibody class switching towards IgG2a subtype was observed. The study of cellular immune responses revealed the increased proliferation capacity of spleen cells against sLiAg, consisting of IFNγ-producing CD4+ and CD8+ T cells. Importantly, the activation of CD8+ T cells was exclusively attributed to vaccination with PLGA NPs surface-modified with the p8 peptide. Moreover, characterization of cytokine production in vaccinated-infected mice revealed that protection was accompanied by significant increase of IFNγ and lower levels of IL-4 and IL-10 in protected mice when compared to control infected group. Conclusively, the above nanoformulations hold promise for future vaccination strategies against VL.

A Vaccine Therapy for Canine Visceral Leishmaniasis Promoted Significant Improvement of Clinical and Immune Status with Reduction in Parasite Burden.

Abstract: Herein, we evaluated the treatment strategy employing a therapeutic heterologous vaccine composed of antigens of Leishmania braziliensis associated with MPL adjuvant (LBMPL vaccine) for visceral leishmaniasis (VL) in symptomatic dogs naturally infected by L. infantum. Sixteen dogs received immunotherapy with MPL adjuvant (n=6) or with a vaccine composed of antigens of L. braziliensis associated with MPL (LBMPL vaccine therapy, n=10). Dogs were submitted to an immunotherapeutic scheme consisting of 3 series composed of 10 subcutaneous doses with 10-day interval between each series. The animals were evaluated before (T0) and 90 days after treatment (T90) for their biochemical/hematological, immunological, clinical and parasitological variables. Our major results showed that the vaccine therapy with LBMPL was able to restore and normalize main biochemical (urea, AST, ALP and billirrubin) and hematological (erythrocytes, hemoglobin, hematocrit and platelets) parameters. In addition, in an ex vivo analysis using flow cytometry, dogs treated with LBMPL vaccine showed increased CD3+ T-lymphocytes and their subpopulations (TCD4+ and TCD8+), reduction of CD21+ B-lymphocytes, increased NK-cells (CD5-CD16+) and CD14+ monocytes. Under in vitro conditions, the animals developed a strong antigen-specific lymphoproliferation mainly by TCD4+ and TCD8+ cells; increasing in both TCD4+IFN-+ and TCD8+IFN-+ as well as reduction of TCD4+IL-4+ and TCD8+IL-4+ lymphocytes with an increased production of TNF- and reduced levels of IL-10. Concerning the clinical signs of CVL, the animals showed an important reduction in the number and intensity of the disease signs; increase body weight as well as reduction of splenomegaly. In addition, the LBMPL immunotherapy also promoted a reduction in parasite burden assessed by Real Time PCR. In the bone marrow, we observed seven times less parasites in LBMPL animals compared with MPL group. The skin tissue showed a reduction in parasite burden in LBMPL dogs 127.5 times higher than MPL. As expected, with skin parasite reduction promoted by immunotherapy, we observed a blocking transmission to sand flies in LBMPL dogs with only three positive dogs after xenodiagnosis. The results obtained in this study highlighted the strong potential for the use of this heterologous vaccine therapy as an important strategy for VL treatment.

Splenic CD4+ T Cells in Progressive Visceral Leishmaniasis Show a Mixed Effector-Regulatory Phenotype and Impair Macrophage Effector Function through Inhibitory Receptor Expression

Abstract: Visceral leishmaniasis (VL), caused by infection with the intracellular protozoan Leishmania donovani, is a chronic progressive disease with a relentlessly increasing parasite burden in the spleen, liver and bone marrow. The disease is characterized by fever, splenomegaly, cachexia, and pancytopenia, and progresses to death if not treated. Control of Leishmania infection is mediated by Th1 (IFNγ-producing) CD4+ T cells, which activate macrophages to produce nitric oxide and kill intracellular parasites. However, despite expansion of CD4+ T cells and increased IFNγ expression in the spleen, humans with active VL do not control the infection. We used an experimental model of chronic progressive VL in hamsters, which mimics clinical and pathological features seen in humans, to better understand the mechanisms that lead to progressive disease. Transcriptional profiling of the spleen during chronic infection revealed expression of markers of both T cell activation and inhibition. CD4+ T cells isolated from the spleen during chronic progressive VL showed mixed expression of Th1 and Th2 cytokines and chemokines, and were marginally effective in controlling infection in an ex vivo T cell-macrophage co-culture system. Splenic CD4+ T cells and macrophages from hamsters with VL showed increased expression of inhibitory receptors and their ligands, respectively. Blockade of the inhibitory receptor PD-L2 led to a significant decrease in parasite burden, revealing a pathogenic role for the PD-1 pathway in chronic VL. PD-L2 blockade was associated with a dramatic reduction in expression of host arginase 1, but no change in IFNγ and inducible nitric oxide synthase. Thus, the expression of counter-regulatory molecules on splenic CD4+ T cells and macrophages promotes a more permissive macrophage phenotype and attenuates intracellular parasite control in chronic progressive VL. Host-directed adjunctive therapy targeting the PD-1 regulatory pathway may be efficacious for VL.