Publications

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    ABSTRACT: We studied breeding sites of Phlebotomus orientalis (Diptera: Psychodidae) the vector of visceral leishmaniasis in northern Ethiopia and Sudan. Although numbers were rather small, 165 sand flies were captured emerging from vertisol cracks. The most productive breeding sites were cracked vertisols, dry river banks and close to trees. No sand flies were caught emerging from sandy clay loam soils in peri-domestic habitats but a few were captured emerging from gaps in a stone wall. Abiotic parameters in vertisols close to trees and in open field from which P. orientalis had emerged, were compared. Soil pH was slightly alkaline and salinity was low. Organic matter contents were similar in both sites. Temperatures and RH remained relatively stable near trees from the end of the rainy season through mid dry season, yet fluctuated markedly at the shallower depth in the open field. During the rainy season, cracks in the soil were sealed resulting in significant lowering of the oxygen concentrations near the tree. Gravimetric water content of soil near trees was lower than open field at shallow depth but similar deeper down. We conclude that ambient conditions suitable for sand fly larvae at shallow depths (45cm) are restricted to areas close to trees. However, deeper in vertisols (90cm) suitable conditions are apparently maintained throughout the dry season even in open fallow fields.
    Acta tropica. 07/2014;
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    ABSTRACT: Protozoan parasites of the genus Leishmania (Kinetoplastida:Trypanosomatidae) cause widespread and devastating human diseases. Visceral leishmaniasis (VL) is endemic in Ethiopia where it has also been responsible for fatal epidemics. It is postulated that genetic exchange in Leishmania has implications for heterosis (hybrid vigour), spread of virulent strains, resistance to chemotherapeutics, and exploitation of different hosts and vectors. Here we analyse 11 natural Ethiopian Leishmania donovani isolates consisting of four putative hybrids, seven parent-like isolates and over 90 derived biological clones. We apply a novel combination of high resolution multilocus microsatellite typing (MLMT, five loci) and multilocus sequence typing (MLST, four loci) that together distinguish parent-like and hybrid L. donovani strains. Results indicate that the four isolates (and their associated biological clones) are genetic hybrids, not the results of mixed infections, each possessing heterozygous markers consistent with inheritance of divergent alleles from genetically distinct Ethiopian L. donovani lineages. The allelic profiles of the putative hybrids may have arisen from a single hybridisation event followed by inbreeding or gene conversion, or alternatively from two or more hybridisation events. Mitochondrial sequencing showed uniparental maxicircle inheritance for all of the hybrids, each possessing a single mitochondrial genotype. Fluorescence activated cell sorting analysis of DNA content demonstrated that all hybrids and their associated clones were diploid. Together the data imply that intra-specific genetic exchange is a recurrent feature of natural L. donovani populations, with substantial implications for the phyloepidemiology of Leishmania.
    International Journal for Parasitology 07/2014; · 3.64 Impact Factor
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    ABSTRACT: Visceral Leishmaniasis (VL) is an important protozoan opportunistic disease in HIV patients in endemic areas. East Africa is second to the Indian subcontinent in the global VL caseload and first in VL-HIV coinfection rate. Because of the alteration in the disease course, the diagnostic challenges, and the poor treatment responses, VL with HIV coinfection has become a very serious challenge in East Africa today. Field experience with the use of liposomal amphotericin B in combination with miltefosine, followed by secondary prophylaxis and antiretroviral drugs, looks promising. However, this needs to be confirmed through clinical trials. Better diagnostic and follow-up methods for relapse and prediction of relapse should also be looked for. Basic research to understand the immunological interaction of the two infections may ultimately help to improve the management of the coinfection.
    PLoS neglected tropical diseases. 06/2014; 8(6):e2869.
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    ABSTRACT: Sand flies belonging to the genus Sergentomyia Franca & Parrot, 1920, are hematophagous insects feeding mostly on reptiles and birds, but some species feed also on mammals including humans. Sergentomyia spp. frequently comprise the vast majority of sand flies trapped along with Phlebotomus spp., the vectors of mammalian leishmaniasis. Within the framework of a project on the ecology and transmission of visceral leishmaniasis in Ethiopia, putative breeding sites of phlebotomine sand flies were studied. Large horizontal sticky traps (LHSTs) covered with sand fly-proof mesh were deployed over cracked vertisol and related habitats for up to 3 nights, and emerging sand flies were collected daily. Emergence traps (ETs) were also adapted to sample other putative breeding sites including tree trunks, termite mounds, rock piles and vertical river banks. Productive breeding sites were identified in the trunks and roots systems of trees, vertisol fields, cracks and burrows in vertisol dry river banks and termite mounds. Emerging flies were also collected form a stone wall and a rock pile situated inside a village. Significantly more Sergentomyia spp. were trapped in vertisols by ETs deployed over root system than in open fields. Similarly, more sand flies emerged from cracks in the vertisol in fallow Sorghum than in fallow sesame fields. Productive breeding sites were characterized by stable micro-climatic conditions. Species composition of emerging sand flies varied with habitat, season and geographical location.
    Acta tropica. 05/2014;
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    ABSTRACT: Antimonials are still being used for visceral leishmaniasis (VL) treatment among HIV co-infected patients in East-Africa due to the shortage of alternative safer drugs like liposomal amphotericin B. Besides tolerability, emergence of resistance to antimonials is a major concern.
    PLoS neglected tropical diseases. 05/2014; 8(5):e2875.
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    ABSTRACT: In East Africa, Phlebotomus orientalis serves as the main vector of Leishmania donovani, the causative agent of visceral leishmaniasis (VL). Phlebotomus orientalis is present at two distant localities in Ethiopia; Addis Zemen where VL is endemic and Melka Werer where transmission of VL does not occur. To find out whether the difference in epidemiology of VL is due to distant compositions of P. orientalis saliva we established colonies from Addis Zemen and Melka Werer, analyzed and compared the transcriptomes, proteomes and enzymatic activity of the salivary glands. Two cDNA libraries were constructed from the female salivary glands of P. orientalis from Addis Zemen and Melka Werer. Clones of each P. orientalis library were randomly selected, sequenced and analyzed. In P. orientalis transcriptomes, we identified members of 13 main protein families. Phylogenetic analysis and multiple sequence alignments were performed to evaluate differences between the P. orientalis colonies and to show the relationship with other sand fly species from the subgenus Larroussius. To further compare both colonies, we investigated the humoral antigenicity and cross-reactivity of the salivary proteins and the activity of salivary apyrase and hyaluronidase. This is the first report of the salivary components of P. orientalis, an important vector sand fly. Our study expanded the knowledge of salivary gland compounds of sand fly species in the subgenus Larroussius. Based on the phylogenetic analysis, we showed that P. orientalis is closely related to Phlebotomus tobbi and Phlebotomus perniciosus, whereas Phlebotomus ariasi is evolutionarily more distinct species. We also demonstrated that there is no significant difference between the transcriptomes, proteomes or enzymatic properties of the salivary components of Addis Zemen (endemic area) and Melka Werer (non-endemic area) P. orientalis colonies. Thus, the different epidemiology of VL in these Ethiopian foci cannot be attributed to the salivary gland composition.
    PLoS Neglected Tropical Diseases 02/2014; 8(2):e2709. · 4.57 Impact Factor
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    ABSTRACT: Anti-leishmanial drug regimens that include a single dose AmBisome® could be suitable for eastern African patients with symptomatic visceral leishmaniasis (VL) but the appropriate single dose is unknown. A multi-centre, open-label, non-inferiority, randomized controlled trial with an adaptive design, was conducted to compare the efficacy and safety of a single dose and multiple doses of AmBisome® for the treatment of VL in eastern Africa. The primary efficacy endpoint was definitive cure (DC) at 6 months. Symptomatic patients with parasitologically-confirmed, non-severe VL, received a single dose of AmBisome® 7.5 mg/kg body weight or multiple doses, 7 times 3 mg/kg on days 1-5, 14, and 21. If interim analyses, evaluated 30 days after the start of treatment following 40 or 80 patients, showed the single dose gave significantly poorer parasite clearance than multiple doses at the 5% significance level, the single dose was increased by 2·5 mg/kg. In a sub-set of patients, parasite clearance was measured by quantitative reverse transcriptase (qRT) PCR. The trial was terminated after the third interim analysis because of low efficacy of both regimens. Based on the intention-to-treat population, DC was 85% (95%CI 73-93%), 40% (95%CI 19-64%), and 58% (95%CI 41-73%) in patients treated with multiple doses (n = 63), and single doses of 7·5 (n = 21) or 10 mg/kg (n = 40), respectively. qRT-PCR suggested superior parasite clearance with multiple doses as early as day 3. Safety data accorded with the drug label. The tested AmBisome® regimens would not be suitable for VL treatment across eastern Africa. An optimal single dose regimen was not identified. www.clinicaltrials.govNCT00832208.
    PLoS Neglected Tropical Diseases 01/2014; 8(1):e2613. · 4.57 Impact Factor
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    ABSTRACT: Visceral leishmaniasis (VL) known as Kala-Azar is a serious systemic disease caused by Leishmania donovani parasites (Trypanosomatidae: Kinetoplastida). The disease is prevalent in the Indian Sub-continent, East Africa and Brazil. In Africa, the worst affected regions are in Sudan, with an estimated 15,000-20,000 cases annually and Ethiopia with 5,000-7,000 cases a year. The main vector of VL in Sudan and Northern Ethiopia is Phlebotomus orientalis, a sand fly frequently found in association with Acacia spp and Balanite spp woodlands. To optimize sampling of sand flies for epidemiological studies in remote areas we tested different means of attraction. Miniature suction traps employing 2AA batteries (3 V) were deployed in the up-draft orientation and baited with chemical light-sticks (Red, Yellow and Green), or bakers' yeast in sugar solution (emitting CO2 and perhaps other attractants). These traps were compared with standard CDC incandescent light traps employing 6 V batteries. Trials were conducted during two consecutive years at different localities around Sheraro, a town in West Tigray, Northern Ethiopia. The sand fly species composition was similar but not identical in the different locations tested with different Sergentomyia spp. predominating. Phlebotomus spp. comprised less than 1% of the catches during the first year trials (November -- December 2011) but increased markedly during the second year trials performed later in the dry season at the height of the sand fly season in February 2012. Although there did not appear to be a species bias towards different light wave-lengths, fermenting yeast in sugar solution attracted relatively more Phlebotomus spp. and Sergentomyia schwetzi. Although the standard 6 V CDC incandescent light traps captured more sand flies, light-weight (~350 g) 3 V suction traps baited with chemical light-sticks were shown to be effective means of monitoring sand flies. Such traps operated without light and baited with yeast in sugar solution caught relatively more Phlebotomus spp.
    Parasites & Vectors 12/2013; 6(1):341. · 3.25 Impact Factor
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    ABSTRACT: BACKGROUND: Leishmaniasis is a clinically and epidemiologically diverse zoonotic disease caused by obligatory, intracellular protozoan parasites of the genus Leishmania. Cutaneous leishmaniasis is the most widely distributed form of the disease characterized by skin lesions. Leishmania aethiopica is considered the predominant etiological agent in Ethiopia. The current study was aimed at developing multilocus microsatellite markers for L. aethiopica isolated from human cutaneous leishmaniasis patients in Ethiopia. RESULTS: L. aethiopica parasites for the study were obtained from Ethiopia and laboratory analysis was conducted at The Ohio State University. DNA was extracted from cultured parasites and an internal transcribed spacer located at the ribosomal region of L. aethiopica genomic DNA was PCR amplified for species identification. Microsatellite markers were identified using multilocus microsatellite typing. We generated an enriched genomic library, and using Primer3 software, designed PCR primers to amplify sequences flanking the detected microsatellites. Subsequent screening of the amplified markers for length variations was performed by gel electrophoresis.Using a variety of molecular methods, 22 different microsatellite markers were identified and tested for typing L. aethiopica strains using a number of clinical isolates. Of the 22 markers tested, 5 were polymorphic and showed distinctive multilocus genotypes, classifying them into four clusters. One marker was found to be specific for L. aethiopica, discriminating it from other species of Leishmania. CONCLUSION: Multilocus microsatellite typing using the markers developed in this study could be useful for epidemiological and population genetic studies of strains of L. aethiopica in order to investigate the structure and dynamics of the corresponding natural foci. It could also help to answer specific clinical questions, such as the occurrence of local and diffuse lesions, strain correlates of parasite persistence after subclinical infection and lesion comparisons from patients suffering from L. aethiopica infections.
    Parasites & Vectors 06/2013; 6(1):160. · 3.25 Impact Factor
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    ABSTRACT: Male Phlebotomus (Synphlebotomus) vansomerenae, specimens were collected together with two other members of the same subgenus between August 2010 and December 2011 in Melka Guba village near Dawa River in Liben district, southeastern Ethiopia. This is the first record of the species in Ethiopia and the first time it has been found outside of Kenya where it was originally described, extending the known distribution of this species in East Africa.
    Journal of Medical Entomology 05/2013; 50(3):659-63. · 1.86 Impact Factor
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    ABSTRACT: BACKGROUND: Phlebotomus orientalis Parrot (Diptera: Psychodidae) is the main vector of visceral leishmaniasis (VL) caused by Leishmania donovani in East Africa. Here we report on life cycle parameters and susceptibility to L. donovani of two P. orientalis colonies originating from different sites in Ethiopia: a non-endemic site in the lowlands - Melka Werer (MW), and an endemic focus of human VL in the highlands - Addis Zemen (AZ). METHODOLOGYPRINCIPAL FINDINGS: Marked differences in life-cycle parameters between the two colonies included distinct requirements for larval food and humidity during pupation. However, analyses using Random Amplified Polymorphic DNA (RAPD) PCR and DNA sequencing of cytB and COI mitochondrial genes did not reveal any genetic differences. F1 hybrids developed successfully with higher fecundity than the parental colonies. Susceptibility of P. orientalis to L. donovani was studied by experimental infections. Even the lowest infective dose tested (2×10(3) per ml) was sufficient for successful establishment of L. donovani infections in about 50% of the P. orientalis females. Using higher infective doses, the infection rates were around 90% for both colonies. Leishmania development in P. orientalis was fast, the presence of metacyclic promastigotes in the thoracic midgut and the colonization of the stomodeal valve by haptomonads were recorded in most P. orientalis females by day five post-blood feeding. CONCLUSIONS: Both MW and AZ colonies of P. orientalis were highly susceptible to Ethiopian L. donovani strains. As the average volume of blood-meals taken by P. orientalis females are about 0.7 µl, the infective dose at the lowest concentration was one or two L. donovani promastigotes per sand fly blood-meal. The development of L. donovani was similar in both P. orientalis colonies; hence, the absence of visceral leishmaniasis in non-endemic area Melka Werer cannot be attributed to different susceptibility of local P. orientalis populations to L. donovani.
    PLoS Neglected Tropical Diseases 04/2013; 7(4):e2187. · 4.57 Impact Factor
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    ABSTRACT: BACKGROUND: Visceral Leishmaniasis (VL) is a disseminated protozoan infection caused by Leishmania donovani parasites which affects almost half a million persons annually. Most of these are from the Indian sub-continent, East Africa and Brazil. Our study was designed to elucidate the role of symptomatic and asymptomatic Leishmania donovani infected persons in the epidemiology of VL in Northern Ethiopia. METHODS: The efficacy of quantitative real-time kinetoplast DNA/PCR (qRT-kDNA PCR) for detecting Leishmania donovani in dried-blood samples was assessed in volunteers living in an endemic focus. RESULTS: Of 4,757 samples, 680 (14.3%) were found positive for Leishmania k-DNA but most of those (69%) had less than 10 parasites/ml of blood. Samples were re-tested using identical protocols and only 59.3% of the samples with 10 parasite/ml or less were qRT-kDNA PCR positive the second time. Furthermore, 10.8% of the PCR negative samples were positive in the second test. Most samples with higher parasitemias remained positive upon re-examination (55/59 =93%). We also compared three different methods for DNA preparation. Phenol-chloroform was more efficient than sodium hydroxide or potassium acetate. DNA sequencing of ITS1 PCR products showed that 20/22 samples were Leishmania donovani while two had ITS1 sequences homologous to Leishmania major. CONCLUSIONS: Although qRT-kDNA PCR is a highly sensitive test, the dependability of low positives remains questionable. It is crucial to correlate between PCR parasitemia and infectivity to sand flies. While optimal sensitivity is achieved by targeting k-DNA, it is important to validate the causative species of VL by DNA sequencing.
    BMC Infectious Diseases 03/2013; 13(1):153. · 3.03 Impact Factor
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    ABSTRACT: The global prevalence of HIV is a major challenge for control of visceral leishmaniasis, a disseminated protozoan infection. In some east African regions, up to 40% of patients with visceral leishmaniasis are co-infected with HIV. Management of visceral leishmaniasis in such patients is complicated by treatment failures and relapses, even while patients are receiving standard antiretroviral therapy. In-vitro studies have consistently documented an inhibitory effect of specific HIV-1 protease inhibitors on leishmania parasites, and the underlying mechanism is partly explained. With the global scaling up of HIV treatment, HIV-1 protease inhibitors are increasingly becoming available for second-line HIV treatment in regions where visceral leishmaniasis and HIV are endemic. However, additional research is needed before HIV-1 protease inhibitors can be taken forward for clinical use against visceral leishmaniasis in HIV-infected patients. Since the effect of protease inhibitors against Leishmania species was generally observed at high drug concentrations, efficacy and dose-response relationships should be studied in animals before these drugs are used in clinical trials. More extensive studies of all available HIV protease inhibitors are needed, including investigation of drug interactions and emergence of drug-resistant parasites. In addition to exploring the full potential of current HIV-1 protease inhibitors against visceral leishmaniasis, leishmania-specific protease inhibitors should be developed.
    The Lancet Infectious Diseases 03/2013; 13(3):251-9. · 19.97 Impact Factor
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    ABSTRACT: The underlying mechanisms resulting in the profound immune suppression characteristic of human visceral leishmaniasis (VL) are not fully understood. Here, we tested the hypothesis that arginase, an enzyme associated with immunosuppression, is higher in patients with VL and contributes to impaired T cell responses. We recruited patients with VL before and after treatment and healthy controls and measured the arginase metabolism in the blood of these individuals. Our results show that arginase activity is significantly higher in the blood of patients with active VL as compared to controls. These high levels of arginase decline considerably once the patients are successfully treated. We identified the phenotype of arginase-expressing cells among PBMCs as neutrophils and show that their frequency was increased in PBMCs of patients before treatment; this coincides with reduced levels of L-arginine in the plasma and decreased expression levels of CD3ζ in T cells.
    PLoS Neglected Tropical Diseases 03/2013; 7(3):e2134. · 4.57 Impact Factor
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    ABSTRACT: Visceral leishmaniasis is a parasitic disease associated with high mortality. The most important foci of visceral leishmaniasis in Ethiopia are in the Northwest and are predominantly associated with high rates of HIV co-infection. Co-infection of visceral leishmaniasis patients with HIV results in higher mortality, treatment failure and relapse. We have previously shown that arginase, an enzyme associated with immunosuppression, was increased in patients with visceral leishmaniasis and in HIV seropositive patients; further our results showed that high arginase activity is a marker of disease severity. Here, we tested the hypothesis that increased arginase activities associated with visceral leishmaniasis and HIV infections synergize in patients co-infected with both pathogens. We recruited a cohort of patients with visceral leishmaniasis and a cohort of patients with visceral leishmaniasis and HIV infection from Gondar, Northwest Ethiopia, and recorded and compared their clinical data. Further, we measured the levels of arginase activity in the blood of these patients and identified the phenotype of arginase-expressing cells. Our results show that CD4(+) T cell counts were significantly lower and the parasite load in the spleen was significantly higher in co-infected patients. Moreover, our results demonstrate that arginase activity was significantly higher in peripheral blood mononuclear cells and plasma of co-infected patients. Finally, we identified the cells-expressing arginase in the PBMCs as low-density granulocytes. Our results suggest that increased arginase might contribute to the poor disease outcome characteristic of patients with visceral leishmaniasis and HIV co-infection.
    PLoS Neglected Tropical Diseases 01/2013; 7(1):e1977. · 4.57 Impact Factor
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    ABSTRACT: Visceral leishmaniasis (VL) caused by Leishmania donovani is a major health problem in Ethiopia. Parasites in disparate regions are transmitted by different vectors, and cluster in distinctive genotypes. Recently isolated strains from VL and HIV-VL co-infected patients in north and south Ethiopia were characterized as part of a longitudinal study on VL transmission. Sixty-three L. donovani strains were examined by polymerase chain reaction (PCR) targeting three regions: internal transcribed spacer 1 (ITS1), cysteine protease B (cpb), and HASPB (k26). ITS1- and cpb - PCR identified these strains as L. donovani. Interestingly, the k26 - PCR amplicon size varied depending on the patient's geographic origin. Most strains from northwestern Ethiopia (36/40) produced a 290 bp product with a minority (4/40) giving a 410 bp amplicon. All of the latter strains were isolated from patients with HIV-VL co-infections, while the former group contained both VL and HIV-VL co-infected patients. Almost all the strains (20/23) from southwestern Ethiopia produced a 450 bp amplicon with smaller products (290 or 360 bp) only observed for three strains. Sudanese strains produced amplicons identical (290 bp) to those found in northwestern Ethiopia; while Kenyan strains gave larger PCR products (500 and 650 bp). High-resolution melt (HRM) analysis distinguished the different PCR products. Sequence analysis showed that the k26 repeat region in L. donovani is comprised of polymorphic 13 and 14 amino acid motifs. The 13 amino acid peptide motifs, prevalent in L. donovani, are rare in L. infantum. The number and order of the repeats in L. donovani varies between geographic regions. HASPB repeat region (k26) shows considerable polymorphism among L. donovani strains from different regions in East Africa. This should be taken into account when designing diagnostic assays and vaccines based on this antigen.
    PLoS Neglected Tropical Diseases 01/2013; 7(1):e2031. · 4.57 Impact Factor
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    ABSTRACT: The underlying mechanisms resulting in the profound immune suppression characteristic of human visceral leishmaniasis (VL) are not fully understood. Here, we tested the hypothesis that arginase, an enzyme associated with immunosuppression, is higher in patients with VL and contributes to impaired T cell responses. We recruited patients with VL before and after treatment and healthy controls and measured the arginase metabolism in the blood of these individuals. Our results show that arginase activity is significantly higher in the blood of patients with active VL as compared to controls. These high levels of arginase decline considerably once the patients are successfully treated. We identified the phenotype of arginase-expressing cells among PBMCs as neutrophils and show that their frequency was increased in PBMCs of patients before treatment; this coincides with reduced levels of L-arginine in the plasma and decreased expression levels of CD3f in T cells.

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