Showing papers on "Visceral leishmaniasis published in 2011"

Whole genome sequencing of multiple Leishmania donovani clinical isolates provides insights into population structure and mechanisms of drug resistance

Abstract: Visceral leishmaniasis is a potentially fatal disease endemic to large parts of Asia and Africa, primarily caused by the protozoan parasite Leishmania donovani. Here, we report a high-quality reference genome sequence for a strain of L. donovani from Nepal, and use this sequence to study variation in a set of 16 related clinical lines, isolated from visceral leishmaniasis patients from the same region, which also differ in their response to in vitro drug susceptibility. We show that whole-genome sequence data reveals genetic structure within these lines not shown by multilocus typing, and suggests that drug resistance has emerged multiple times in this closely related set of lines. Sequence comparisons with other Leishmania species and analysis of single-nucleotide diversity within our sample showed evidence of selection acting in a range of surface- and transport-related genes, including genes associated with drug resistance. Against a background of relative genetic homogeneity, we found extensive variation in chromosome copy number between our lines. Other forms of structural variation were significantly associated with drug resistance, notably including gene dosage and the copy number of an experimentally verified circular episome present in all lines and described here for the first time. This study provides a basis for more powerful molecular profiling of visceral leishmaniasis, providing additional power to track the drug resistance and epidemiology of an important human pathogen.

Diagnosis of visceral leishmaniasis

Abstract: Leishmaniasis is a vector-borne disease with up to 350 million people at risk of infection worldwide. Among its different clinical manifestations, visceral is the most severe form. Since clinical features of visceral leishmaniasis (VL) mimic several other common diseases, accurate diagnosis is crucial as the treatment is associated with significant toxicity. Invasive and risky techniques involving demonstration of the parasites in stained preparations from splenic and bone marrow aspirate is still the gold standard for VL diagnosis. Serological tests using rK39 in ELISA or rapid immunochromatographic format, Direct Agglutination Test (DAT), immunoblotting have issues related to a significant proportion of asymptomatic individuals being positive with these tests and their inability to diagnose relapses as these remain positive for several months to years after cure. PCR is the most common molecular technique successfully used for diagnosis and differentiation of species. Through this review we focus extensively on the comparative utilities of the various diagnostic tools currently available for VL, describing in depth their advantages and disadvantages, addressing the recent advances attained in the field. A simple, rapid, non invasive, accurate and cost effective marker of active VL, which can be used in field conditions, is necessary to improve diagnosis of VL.

Non-ionic surfactant vesicles, niosomes, as a delivery system for the anti-leishmanial drug, sodium stibogluconate.

Abstract: Liver and serum concentrations of antimony in the mouse have been determined after administration of sodium stibogluconate in the free, liposomal and niosomal form. High liver and low serum values were attained by the use of both vesicular formulations. Niosomal sodium stibogluconate was shown to be more active than free drug against experimental murine visceral leishmaniasis, an effect apparently dependent on maintaining high drug levels in the infected reticuloendothelial system.

Visceral Leishmaniasis in the Indian Subcontinent: Modelling Epidemiology and Control

Abstract: Background In the Indian subcontinent, about 200 million people are at risk of developing visceral leishmaniasis (VL) In 2005, the governments of India, Nepal and Bangladesh started the first regional VL elimination program with the aim to reduce the annual incidence to less than 1 per 10,000 by 2015 A mathematical model was developed to support this elimination program with basic quantifications of transmission, disease and intervention parameters This model was used to predict the effects of different intervention strategies

One Health: The global challenge of epidemic and endemic leishmaniasis

Abstract: 'One Health' proposes the unification of medical and veterinary sciences with the establishment of collaborative ventures in clinical care, surveillance and control of cross-species disease, education, and research into disease pathogenesis, diagnosis, therapy and vaccination. The concept encompasses the human population, domestic animals and wildlife, and the impact that environmental changes ('environmental health') such as global warming will have on these populations. Visceral leishmaniasis is a perfect example of a small companion animal disease for which prevention and control might abolish or decrease the suffering of canine and human patients, and which aligns well with the One Health approach. In this review we discuss how surveillance for leishmaniases is undertaken globally through the control of anthroponootic visceral leishmaniasis (AVL) and zoonotic visceral leishmaniasis (ZVL). The ZVL epidemic has been managed to date by the culling of infected dogs, treatment of human cases and control of the sandfly vector by insecticidal treatment of human homes and the canine reservoir. Recently, preventive vaccination of dogs in Brazil has led to reduction in the incidence of the canine and human disease. Vaccination permits greater dog owner compliance with control measures than a culling programme. Another advance in disease control in Africa is provided by a surveillance programme that combines remote satellite sensing, ecological modelling, vector surveillance and geo-spatial mapping of the distribution of vectors and of the animal-to-animal or animal-to-human pathogen transmission. This coordinated programme generates advisory notices and alerts on emerging infectious disease outbreaks that may impede or avoid the spreading of visceral leishmaniasis to new areas of the planet as a consequence of global warming.

IL-10 neutralization promotes parasite clearance in splenic aspirate cells from patients with visceral leishmaniasis.

Abstract: The mechanisms underlying the failure to contain the growth of Leishmania parasites in human visceral leishmaniasis (VL) are not understood. L donovani amastigotes were quantified in cultured splenic aspirate cells to assess the function of IL-10 in lesional tissue ex vivo. In 67 patients with active VL, IL-10 neutralization promoted parasite killing in 73% and complete clearance in 30%, while 18% had more parasites and 9% did not change. The splenic cells secreted increased levels of both tumor necrosis factor α (TNFα) and interferon γ (IFNγ) under IL-10-neutralizing conditions. These findings provide direct support for targeting IL-10 as an approach to therapy in human VL.

Incidence of symptomatic and asymptomatic Leishmania donovani infections in high-endemic foci in India and Nepal: a prospective study

Abstract: Incidence of Leishmania donovani infection and Visceral Leishmaniasis (VL) was assessed in a prospective study in Indian and Nepalese high-endemic villages. DAT-seroconversion was used as marker of incident infection in 3 yearly surveys. The study population was followed up to month 30 to identify incident clinical cases. In a cohort of 9034 DAT-negative individuals with neither active signs nor history of VL at baseline, 42 VL cases and 375 asymptomatic seroconversions were recorded in the first year, giving an infection:disease ratio of 8.9 to 1. In the 18 months' follow-up, 7 extra cases of VL were observed in the seroconverters group (N=375), against 14 VL cases among the individuals who had not seroconverted in the first year (N=8570) (RR=11.5(4.5

Prevalence and Factors Associated with Leishmania infantum Infection of Dogs from an Urban Area of Brazil as Identified by Molecular Methods

Abstract: Background Various factors contribute to the urbanization of the visceral leishmaniasis (VL), including the difficulties of implementing control measures relating to the domestic reservoir. The aim of this study was to determine the prevalence of canine visceral leishmaniasis in an urban endemic area in Brazil and the factors associated with Leishmania infantum infection among seronegative and PCR-positive dogs. Methodology A cross-sectional study was conducted in Belo Horizonte, Minas Gerais, Brazil. Blood samples were collected from 1,443 dogs. Serology was carried out by using two enzyme-linked immunosorbent assays (Biomanguinhos/FIOCRUZ/RJ and “in house”), and molecular methods were developed, including PCR-RFLP. To identify the factors associated with early stages of infection, only seronegative (n = 1,213) animals were evaluated. These animals were divided into two groups: PCR-positive (n = 296) and PCR-negative (n = 917) for L. infantum DNA. A comparison of these two groups of dogs taking into consideration the characteristics of the animals and their owners was performed. A mixed logistic regression model was used to identify factors associated with L. infantum infection. Principal Findings Of the 1,443 dogs examined, 230 (15.9%) were seropositive in at least one ELISA, whereas PCR-RFLP revealed that 356 animals (24.7%) were positive for L. infantum DNA. Results indicated that the associated factors with infection were family income

IL-27 and IL-21 are Associated with T Cell IL-10 Responses in Human Visceral Leishmaniasis

Abstract: IL-10 is believed to underlie many of the immunologic defects in human visceral leishmaniasis (VL). We have identified CD4+CD25−Foxp3− T cells as the major source of IL-10 in the VL spleen. IL-27, a member of the IL-6/IL-12 cytokine family, has been shown to promote development of IL-10–producing T cells, in part by upregulating their production of autocrine IL-21. We investigated whether IL-27 and IL-21 are associated with human VL. IL-27 was elevated in VL plasma, and at pretreatment, spleen cells showed significantly elevated mRNA levels of both IL-27 subunits, IL-27p28 and EBI-3, as well as IL-21, compared with posttreatment biopsies. CD14+ spleen cells were the main source of IL-27 mRNA, whereas CD3+ T cells were the main source of IL-21. IL-27 mRNA could be strongly upregulated in normal donor macrophages with IFN-γ and IL-1β, conditions consistent with those in the VL spleen. Last, a whole-blood assay revealed that most VL patients could produce Ag-specific IFN-γ and IL-10 and that the IL-10 could be augmented with recombinant human IL-21. Thus, proinflammatory cytokines acting on macrophages in the VL spleen have the potential to upregulate IL-27, which in turn can induce IL-21 to expand IL-10–producing T cells as a mechanism of feedback control.

Visceral leishmaniasis: Experimental models for drug discovery

Abstract: Visceral leishmaniasis (VL) or kala-azar is a chronic protozoan infection in humans associated with significant global morbidity and mortality. The causative agent is a haemoflagellate protozoan Leishmania donovani, an obligate intracellular parasite that resides and multiplies within macrophages of the reticulo-endothelial system. Most of the existing anti-leishmanial drugs have serious side effects that limit their clinical application. As an alternate strategy, vaccination is also under experimental and clinical trials. The in vitro evaluation designed to facilitate rapid testing of a large number of drugs has been focussed on the promastigotes milt little attention on the clinically relevant parasite stage, amastigotes. Screening designed to closely reflect the situation in vivo is currently time consuming, laborious, and expensive, since it requires intracellular amastigotes and animal model. The ability to select transgenic Leishmania expressing reporter proteins, such as the green fluorescent proteins (GFP) or the luciferase opened up new possibilities for the development of drug screening models. Many experimental animal models like rodents, dogs and monkeys have been developed, each with specific features, but none accurately reproduces what happens in humans. Available in vitro and in vivo methodologies for antileishmanial drug screening and their respective advantages and disadvantages are reviewed.

Visceral leishmaniasis: elimination with existing interventions.

Abstract: The world's burden of infectious diseases can be substantially reduced by more-effective use of existing interventions. Advances in case detection, diagnosis, and treatment strategies have made it possible to consider the elimination of visceral leishmaniasis in the Indian subcontinent. The priority must now be to effectively implement existing interventions at the community level by actively finding cases in endemic villages and treating them with single-dose liposomal amphotericin B at primary-health-care centres. Once the elimination target of one case per 10,000 population has been reached, combination therapies involving miltefosine and paromomycin can be introduced to ensure long-term availability of several drugs for visceral leishmaniasis and to protect against resistance.

Leishmania antimony resistance: what we know what we can learn from the field

Abstract: Leishmania is the causative agent of various forms of leishmaniasis, a significant cause of morbidity and mortality. The clinical manifestations of the disease range from self-healing cutaneous and mucocutaneous skin ulcers to a fatal visceral form named visceral leishmaniasis or kala-azar. In the absence of any effective vaccine, the only means to treat and control leishmaniasis is affordable medication. The treatment choice is essentially directed by economic considerations; therefore, for a large majority of countries, chemotherapy relies only on the use of cheaper antimonial compounds. The emergence of antimonial therapy failure in India linked to proven parasite resistance has stressed questions about selective factors as well as transmission risk of drug resistance. Unfortunately, in most parts of the world, the frequency of parasite antimony resistance linked to treatment failure is unknown because of a lack of information on Leishmania antimony susceptibility. This information is crucial for addressing the risk of selection and transmission of drug-resistant parasites, particularly in areas where antimony is the only chemotherapeutic alternative. However, the poor knowledge about factors that favor selection of resistant parasites, the multiplicity of the agents that can play a role in the in vivo antileishmanial activity of antimony, and the lack of a standard protocol to diagnose and survey parasite resistance all contribute to insufficient monitoring of antimony resistance. In this review, we discuss on the factors potentially involved in the selection of antimony resistance in the field and discuss on the methods available for its diagnosis.

Structures, Targets and Recent Approaches in Anti-Leishmanial Drug Discovery and Development

Abstract: Recent years have seen a significant improvement in available treatment options for leishmaniasis. Two new drugs, miltefosine and paromomycin, have been registered for the treatment of visceral leishmaniasis (VL) in India since 2002. Combination therapy is now explored in clinical trials as a new treatment approach for VL to reduce the length of treatment and potentially prevent selection of resistant parasites. However there is still a need for new drugs due to safety, resistance, stability and cost issues with existing therapies. The search for topical treatments for cutaneous leishmaniasis (CL) is ongoing. This review gives a brief overview of recent developments and approaches in anti-leishmanial drug discovery and development.

Liposomal amphotericin B treatment of cutaneous leishmaniasis due to Leishmania tropica.

Abstract: Background Cutaneous leishmaniasis (CL) is endemic in Israel, and in the past, has been attributed almost exclusively to Leishmania major. Over the last decade or so, an increase in Leishmania tropica (L. tropica) infections has occurred in several regions of Israel. Topical treatment of Old World CL is usually the rule, however, in some cases systemic treatment is indicated. Liposomal amphotericin B (L-AmB) is efficacious and safe for treating visceral leishmaniasis but its role in treating various forms of CL is yet to be defined. In this study, we summarize the efficacy and safety of L-AmB treatment in a series of Israeli patients with L. tropica infection. Methods Cases of PCR-proven CL caused by L. tropica were treated in an outpatient setting. Treatment schedule consisted of five consecutive days of 3 mg/kg L-AmB, followed by a sixth dose on day 10. Results Thirteen consecutive patients (11 men, two women), received L-AmB. Mean age was 15.3 years; of the 13 patients, 85% had facial lesions. Six had previously failed intralesional sodium stibogluconate treatment and four had failed topical paromomycin treatment. Eleven of 13 patients (84%) achieved complete clinical cure within 2 months. Mean follow-up of 11 months revealed no relapses. Side effects were mild and none terminated treatment prematurely. Limitations A non-randomized study, with a small number of patients. Conclusion Liposomal amphotericin B treatment for L. tropica is effective, well tolerated and cost beneficial in countries where cost of hospital-care is significant.

Diagnosis of Indian visceral leishmaniasis by nucleic acid detection using PCR.

Abstract: Background PCR based diagnosis for Visceral Leishmaniasis (VL), despite numerous published primers, remains far from being applied in the field. The present study was planned to design a Leishmania specific diagnostic assay and to evaluate its sensitivity and specificity on a sample size, which to the best of our knowledge is the largest ever screened in one study. Methods Leishmania specific primers were developed using 18S rRNA gene and their sensitivity was evaluated on 500 parasitologically confirmed patients with VL and 25 Post Kala-azar Dermal Leishmaniasis (PKDL) patients. Specificity was calculated on 250 healthy endemic controls, 250 healthy non endemic controls and 250 non leishmanial diseases like malaria. Results Our PCR assay had a sensitivity of 87.8% (95%CI: 84.1–89.8) using 200 µL of patient's peripheral-blood. Specificity was absolute in non-endemic healthy controls and in subjects with different diseases while in endemic controls it was 84% (95%CI: 78.9–88.0). Its overall specificity was 94.6% (95%CI-92.8–96.1). Conclusions The PCR assay developed is sensitive enough to detect the 18S rRNA gene in an amount equivalent to a single parasite or less in a one million human cell environment. The high sensitivity of this PCR diagnostic test with relatively non-invasive peripheral blood sampling method opens up the possibility of its deployment in field for the routine diagnosis of VL.

KSAC, the First Defined Polyprotein Vaccine Candidate for Visceral Leishmaniasis

Abstract: A subunit vaccine using a defined antigen(s) may be one effective solution for controlling leishmaniasis. Because of genetic diversity in target populations, including both dogs and humans, a multiple-antigen vaccine will likely be essential. However, the cost of a vaccine to be used in developing countries must be considered. We describe herein a multiantigen vaccine candidate comprised of antigens known to be protective in animal models, including dogs, and to be recognized by humans immune to visceral leishmaniasis. The polyprotein (KSAC) formulated with monophosphoryl lipid A, a widely used adjuvant in human vaccines, was found to be immunogenic and capable of inducing protection against Leishmania infantum, responsible for human and canine visceral leishmaniasis, and against L. major, responsible for cutaneous leishmaniasis. The results demonstrate the feasibility of producing a practical, cost-effective leishmaniasis vaccine capable of protecting both humans and dogs against multiple Leishmania species.

Study of parasite kinetics with antileishmanial drugs using real-time quantitative PCR in Indian visceral leishmaniasis

Abstract: Objectives: This study describes parasite kinetics in the blood of visceral leishmaniasis patients treated with liposomal amphotericin B (L-AmB) or a preformed fat emulsion of amphotericin B (ApL) using real-time quantitative PCR (qPCR). Methods: Forty-six patients were treated with a single dose (15 mg/kg of body weight) of either L-AmB (n¼ 13) or ApL (n ¼33). qPCR was used to estimate parasite kinetics by detection of Leishmania donovani DNA using kinetoplast DNA-specific primers in peripheral blood samples using an absolute quantification method. Results: The mean parasite load decreased from baseline (day 0) values of 894.07 and 980.48 to 71.72 and 211.52 parasite genomes/mL at day 7 in L-AmB and ApL groups, respectively, and at day 30 these further declined to 8.30 and 133.98 parasite genomes/mL, respectively. At day 30 post-treatment evaluation, the decline in parasite load was significantly greater (P ¼ 0.024) with L-AmB compared with ApL. Four of 33 patients in the ApL group failed treatment (1 primary failure and 3 relapses) with the presence of parasites, whereas all patients in the L-AmB group were cured at 6 month follow-up. Conclusions: qPCR can be a tool to measure parasite dynamics accurately and provide a marker to measure the efficacy of various drugs. It can be used as a test of cure, allowing us to do away with invasive and risky methods such as splenic or bone marrow aspiration.

Identification of small molecule lead compounds for visceral leishmaniasis using a novel ex vivo splenic explant model system.

Abstract: Background New drugs are needed to treat visceral leishmaniasis (VL) because the current therapies are toxic, expensive, and parasite resistance may weaken drug efficacy. We established a novel ex vivo splenic explant culture system from hamsters infected with luciferase-transfected Leishmania donovani to screen chemical compounds for anti-leishmanial activity. Methodology/Principal Findings This model has advantages over in vitro systems in that it: 1) includes the whole cellular population involved in the host-parasite interaction; 2) is initiated at a stage of infection when the immunosuppressive mechanisms that lead to progressive VL are evident; 3) involves the intracellular form of Leishmania; 4) supports parasite replication that can be easily quantified by detection of parasite-expressed luciferase; 5) is adaptable to a high-throughput screening format; and 6) can be used to identify compounds that have both direct and indirect anti-parasitic activity. The assay showed excellent discrimination between positive (amphotericin B) and negative (vehicle) controls with a Z' Factor >0.8. A duplicate screen of 4 chemical libraries containing 4,035 compounds identified 202 hits (5.0%) with a Z score of <–1.96 (p<0.05). Eighty-four (2.1%) of the hits were classified as lead compounds based on the in vitro therapeutic index (ratio of the compound concentration causing 50% cytotoxicity in the HepG2 cell line to the concentration that caused 50% reduction in the parasite load). Sixty-nine (82%) of the lead compounds were previously unknown to have anti-leishmanial activity. The most frequently identified lead compounds were classified as quinoline-containing compounds (14%), alkaloids (10%), aromatics (11%), terpenes (8%), phenothiazines (7%) and furans (5%). Conclusions/Significance The ex vivo splenic explant model provides a powerful approach to identify new compounds active against L. donovani within the pathophysiologic environment of the infected spleen. Further in vivo evaluation and chemical optimization of these lead compounds may generate new candidates for preclinical studies of treatment for VL.

Evidence that lipopolisaccharide may contribute to the cytokine storm and cellular activation in patients with visceral leishmaniasis

Abstract: Background Visceral leishmaniasis (VL) is characterized by parasite-specific immunosuppression besides an intense pro-inflammatory response. Lipopolisaccharide (LPS) has been implicated in the immune activation of T-cell deficient diseases such as HIV/AIDS and idiopathic lymphocytopenia. The source of LPS is gram-negative bacteria that enter the circulation because of immunological mucosal barrier breakdown. As gut parasitization also occurs in VL, it was hypothesized that LPS may be elevated in leishmaniasis, contributing to cell activation.

Phase IV Trial of Miltefosine in Adults and Children for Treatment of Visceral Leishmaniasis (Kala-Azar) in Bangladesh

Abstract: Miltefosine (target dose of 2.5 mg/kg/day for 28 days) is the recommended treatment for visceral leishmaniasis (kala-azar) in Bangladesh on the basis of data from India. We evaluated miltefosine in a phase IV trial of 977 patients in Bangladesh. At the six-month final follow up, 701 were cured. 24 showed initial treatment failure, and 95 showed treatment failure at 6 months, although 73 of the 95 showed treatment failure solely by the criterion of low hemoglobin values. One hundred twenty-one patients were not assessable. With the conservative assumption that all low hemoglobin values represented treatment failure, the final per protocol cure rate was 85%. Of 13 severe adverse events, 6 led to treatment discontinuation and 7 resulted in deaths, but only 1 death (associated with diarrhea) could be attributed to drug. Nearly all non-serious adverse events were gastrointestinal: vomiting in 25% of patients and diarrhea in 8% of patients. Oral miltefosine is an attractive alternative to intramuscular antimony and intravenous amphotericin B for treatment of kala-azar in Bangladesh.

Persistence of Leishmania donovani Antibodies in Past Visceral Leishmaniasis Cases in India

Abstract: The persistence of anti-Leishmania donovani antibodies in past visceral leishmaniasis (VL) cases was retrospectively assessed by means of the direct agglutination test (DAT) and the rK39 enzyme-linked immunosorbent assay (ELISA). Antibody titers remained high for an extended period of time in past cases of VL. These results highlight the need to carefully elicit the history of patients with VL symptoms.