Aysheshm Kassahun

Evaluation of PCR procedures for detecting and quantifying Leishmania donovani DNA in large numbers of dried human blood samples from a visceral leishmaniasis focus in Northern Ethiopia

BackgroundVisceral 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.MethodsThe 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.ResultsOf 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.ConclusionsAlthough 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.

Phlebotomus orientalis sand flies from two geographically distant Ethiopian localities: biology, genetic analyses and susceptibility to Leishmania donovani.

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). Methodology/Principal 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 (2×103 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.

Theileria infection in domestic ruminants in northern Ethiopia.

Piroplasmosis caused by different tick-borne hemoprotozoan parasites of the genera Theileria and Babesia is among the most economically important infections of domestic ruminants in sub-Saharan Africa. A survey for piroplasm infection was conducted in three locations in Northern Ethiopia. Of 525 domestic ruminants surveyed, 80% of the cattle, 94% of the sheep and 2% of the goats were positive for different Theileria spp. based on PCR of blood followed by DNA sequencing. Sheep had a significantly higher rate of infection compared with cattle (P<0.0003) and both sheep and cattle had higher rates of infection compared to goats (P<0.0001). Four species of Theileria were detected in cattle: T. velifera, T. mutans, T. orientalis complex and T. annulata with infection rates of 66, 8, 4, and 2%, respectively. This is the first report of T. annulata, the cause of Tropical Theileriosis in Ethiopia. Of the two Theileria spp. detected in small ruminants, T. ovis was highly prevalent (92%) in sheep and rare in goats (1.5%) whereas T. seperata was infrequent in sheep (2%) and rare in goats (0.4%). None of the animals were positive for Babesia spp.; however, Sarcocystis capracanis and S. tenella were detected in one goat and a sheep, respectively. The widespread distribution of Theileria spp. among cattle in northern Ethiopia including the virulent T. annulata and more mildly pathogenic T. mutans and T. orientalis, and the high infection rate in sheep with the usually sub-clinical T. ovis indicate extensive exposure to ticks and transmission of piroplasms with an important economic impact.

Polymorphism in the HASPB Repeat Region of East African Leishmania donovani Strains

Background/Objectives 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. Methodology/Principal Findings 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 patients 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. Conclusions/Significance 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.

Detection of Leishmania donovani and L. tropica in Ethiopian wild rodents

Human visceral (VL, also known as Kala-azar) and cutaneous (CL) leishmaniasis are important infectious diseases affecting countries in East Africa that remain endemic in several regions of Ethiopia. The transmission and epidemiology of the disease is complicated due to the complex life cycle of the parasites and the involvement of various Leishmania spp., sand fly vectors, and reservoir animals besides human hosts. Particularly in East Africa, the role of animals as reservoirs for human VL remains unclear. Isolation of Leishmania donovani parasites from naturally infected rodents has been reported in several endemic countries; however, the status of rodents as reservoirs in Ethiopia remains unclear. Here, we demonstrated natural Leishmania infections in rodents. Animals were trapped in 41 localities of endemic and non-endemic areas in eight geographical regions of Ethiopia and DNA was isolated from spleens of 586 rodents belonging to 21 genera and 38 species. Leishmania infection was evaluated by real-time PCR of kinetoplast (k)DNA and confirmed by sequencing of the PCR products. Subsequently, parasite species identification was confirmed by PCR and DNA sequencing of the 18S ribosomal RNA internal transcribed spacer one (ITS1) gene. Out of fifty (8.2%) rodent specimens positive for Leishmania kDNA-PCR and sequencing, 10 were subsequently identified by sequencing of the ITS1 showing that five belonged to the L. donovani complex and five to L. tropica. Forty nine kDNA-positive rodents were found in the endemic localities of southern and eastern Ethiopia while only one was identified from northwestern Ethiopia. Moreover, all the ten ITS1-positive rodents were captured in areas where human leishmaniasis cases have been reported and potential sand fly vectors occur. Our findings suggest the eco-epidemiological importance of rodents in these foci of leishmaniasis and indicate that rodents are likely to play a role in the transmission of leishmaniasis in Ethiopia, possibly as reservoir hosts.

Disseminated Cutaneous Leishmaniasis Resembling Post-Kala-Azar Dermal Leishmaniasis Caused by Leishmania donovani in Three Patients Co-Infected with Visceral Leishmaniasis and Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome in Ethiopia

We report paired strains of Leishmania parasites, one from the viscera and the other from skin lesions that were isolated from three patients with visceral leishmaniasis and disseminated cutaneous leishmaniasis that were co-infected with human immunodeficiency virus. The causative parasites were characterized by polymerase chain reaction-restriction length polymorphism of the ribosomal DNA internal transcribed spacer 1 and by a panel of multilocus microsatellite markers. We demonstrated that the causative agent was Leishmania donovani in all cases, irrespective of the phenotype of the disease. The paired strains from viscera and skin lesions of the same patients showed genetic identity across the 14 microsatellite markers investigated. These findings demonstrate that the skin lesions in these human immunodeficiency virus-positive patients with visceral leishmaniasis were caused by dissemination of viscerotropic L. donovani parasites as a consequence of severe immunosuppression. However, in all three patients, rapid clearance of the skin lesions was observed after antimonial therapy.

Natural infection of bats with Leishmania in Ethiopia.

The leishmaniases, a group of diseases with a worldwide-distribution, are caused by different species of Leishmania parasites. Both cutaneous and visceral leishmaniasis remain important public health problems in Ethiopia. Epidemiological cycles of these protozoans involve various sand fly (Diptera: Psychodidae) vectors and mammalian hosts, including humans. In recent years, Leishmania infections in bats have been reported in the New World countries endemic to leishmaniasis. The aim of this study was to survey natural Leishmania infection in bats collected from various regions of Ethiopia. Total DNA was isolated from spleens of 163 bats belonging to 23 species and 18 genera. Leishmania infection was detected by real-time (RT) PCR targeting a kinetoplast (k) DNA and internal transcribed spacer one (ITS1) gene of the parasite. Detection was confirmed by sequencing of the PCR products. Leishmania kDNA was detected in eight (4.9%) bats; four of them had been captured in the Aba-Roba and Awash-Methara regions that are endemic for leishmaniasis, while the other four specimens originated from non-endemic localities of Metu, Bedele and Masha. Leishmania isolates from two bats were confirmed by ITS1 PCR to be Leishmania tropica and Leishmania major, isolated from two individual bats, Cardioderma cor and Nycteris hispida, respectively. These results represent the first confirmed observation of natural infection of bats with the Old World Leishmania. Hence, bats should be considered putative hosts of Leishmania spp. affecting humans with a significant role in the transmission.

Susceptibility of clinical isolates of Leishmania aethiopica to miltefosine, paromomycin, amphotericin B and sodium stibogluconate using amastigote-macrophage in vitro model

Cutaneous Leishmaniasis (CL) caused by Leishmania aethiopica is a public health and social problem with a sequel of severe and mutilating skin lesions. It is manifested in three forms: localized CL (LCL), mucosal CL (MCL) and diffuse CL (DCL). Unresponsiveness to sodium stibogluconate (Sb(V)) is common in Ethiopian CL patients. Using the amastigote-macrophage in vitro model the susceptibility of 24 clinical isolates of L. aethiopica derived from untreated patients was investigated. Eight strains of LCL, 9 of MCL, and 7 of DCL patients together with a reference strain (MHOM/ET/82/117/82) were tested against four antileishmanial drugs: amphotericin B, miltefosine, Sb(V) and paromomycin. In the same order of drugs, IC(50) (μg/ml±SD) values for the 24 strains tested were 0.16±0.18, 5.88±4.79, 10.23±8.12, and 13.63±18.74. The susceptibility threshold of isolates originating from the 3 categories of patients to all 4 drugs was not different (p>0.05). Maximal efficacy was superior for miltefosine across all the strains. Further susceptibility test could validate miltefosine as a potential alternative drug in cases of sodium stibogluconate treatment failure in CL patients.