Teshome Gebre-Michael

Hiv viral load and response to antileishmanial chemotherapy in co-infected patients

OBJECTIVE To investigate whether clearance of Leishmania parasites from tissue aspirate smears in patients with HIV and visceral leishmaniasis (VL) co-infection treated with pentavalent antimonials is influenced by initial HIV viral load and to assess the effect of active VL on HIV viral load and replication in vivo. METHODS Leishmania parasites were identified in Giemsa-stained smears prepared from tissue aspirates. Parasite index was determined by quantifying Leishmania donovani bodies in smears. HIV-1 RNA was quantitated by using the nucleic acid sequence-based amplification technique with a limit of detection of 500 copies/ml. All patients were treated with pentavalent antimonials at 20 mg pentavalent antimony (Sb(V))/kg daily for a total of 28 days. None of the patients received specific anti-retroviral therapy. RESULTS Seventeen patients (73.9%) showed good initial response to anti-leishmanial treatment and the remaining six (26.1%) had very poor response. Among the good responders, 11 (64.7%) had no demonstrable Leishmania donovani bodies in post-therapy tissue aspirate smear preparations, and in the remaining six (35.3%) their parasite loads were reduced to very low levels. Patients with poor response had persistently high parasite index despite completion of anti-leishmanial chemotherapy. Poor responders had pre-treatment median HIV viral load that was >160-fold higher than responders to anti-leishmanial chemotherapy; [410000 copies/ml (quartile range, 33000-530000) and 2500 copies/ml (quartile range 500-297500), respectively]. Furthermore, compared with pre-treatment viral concentrations, patients with good response showed marked reduction in post-treatment viral load. In contrast, post-treatment HIV viral concentrations were markedly increased among patients with poor response to anti-leishmanial therapy. CONCLUSIONS The results suggest that pre-treatment HIV viral load influences response to anti-leishmanial chemotherapy and active VL is associated with increased viral replication in vivo, supporting the notion that dual infection plays an important role in the pathogenesis and disease progression of either infection.

Insecticide resistance in Anopheles arabiensis (Diptera: Culicidae) from villages in central, northern and south west Ethiopia and detection of kdr mutation

BackgroundAnopheles arabiensis is the major vector of malaria in Ethiopia. Malaria vector control in Ethiopia is based on selective indoor residual spraying using DDT, distribution of long lasting insecticide treated nets and environmental management of larval breeding habitats. DDT and pyrethroid insecticides are neurotoxins and have a similar mode of action on the sodium ion channel of insects. It was therefore necessary to verify the insecticide susceptibility status of An. arabiensis, to better understand the status of cross-resistance between DDT and the pyrethroids in this species as well as to detect a resistant gene.MethodsStandard WHO insecticide susceptibility tests were conducted on adults reared from larval and pupal collections from breeding sites at three villages namely: Sodere in the Rift Valley, Gorgora in the north and Ghibe River Valley in the south west of Ethiopia. The occurrence of cross-resistance between pyrethroids and DDT was determined using a DDT selected laboratory colony originally collected from Gorgora. Phenotypically characterized mosquitoes were tested for the presence of knockdown resistance (kdr) alleles using the standard polymerase chain reaction assay.ResultsAll An. gambiae s.l. specimens assayed by PCR were identified as An. arabiensis. The knockdown and mortality results showed An. arabiensis resistance to DDT in all villages, resistance to deltamethrin and permethrin in the Ghibe River Valley and permethrin resistance in Gorgora. Bioassay susceptibility tests also indicated the presence of cross-resistance between DDT and permethrin, but not between DDT and deltamethrin. The knockdown resistance (kdr) mutation of leucine to phenylalanine in the sodium ion channel gene was detected in populations from Gorgora and the Ghibe River Valley.ConclusionSince An. arabiensis shows high levels of resistance to DDT in all villages tested and varying pyrethroid resistance in Gorgora and the Ghibe River valley, precautionary measures should be taken in future vector control operations. Moreover, the status of resistance in other locations in Ethiopia and the spread of resistant gene (s) should be investigated.

The isolation of Leishmania tropica and L. aethiopica from Phlebotomus (Paraphlebotomus) species (Diptera: Psychodidae) in the Awash Valley, northeastern Ethiopia.

In a survey of Leishmania infections in phlebotomine sandflies in a highly suspected focus of leishmaniasis in the Awash Valley (northeastern Ethiopia) between January 1994 and August 1997, a total of 3307 females of 11 Phlebotomus species (P. orientalis, P. fantalensis, P. saevus, P. sergenti, P. gemetchi, P. alexandri, P. bergeroti, P. duboscqi, P. arabicus, P. martini, and P. rodhaini) were dissected. Promastigotes were detected in 17 females of three species (11 P. saevus, 4 P. sergenti and 2 P. arabicus). Of these, only two P. saevus (one from Upper Awash and one from Middle Awash) and three P. sergenti (from Upper Awash) positives were successfully isolated in culture and were typed by isoenzyme analysis. Four isolates (two each from P. saevus and P. sergenti) were identified as new zymodemes (Z) of L. tropica and one isolate from P. sergenti was typed as a new zymodeme of L. aethiopica. This is the first finding of natural infections of P. saevus and P. arabicus and the first evidence for the former to be a vector of L. tropica. This is also the first time P. sergenti has been implicated in L. tropica transmission in Ethiopia; the isolation of L. aethiopica from a Paraphlebotomus species (P. sergenti) is also a new record. The possible presence of human cutaneous leishmaniasis (L. tropica and L. aethiopica), and wild reservoir host(s) of the parasites, especially rock hyrax (Procavia capensis) in the Upper and Middle Awash Valley remain to be determined.

Further studies on the phlebotomine sandflies of the kala-azar endemic lowlands of Humera-Metema (north-west Ethiopia) with observations on their natural blood meal sources

BackgroundVisceral leishmaniasis (VL) has been known to exist in northwest Ethiopia (Humera-Metema lowlands) since the early 1970s associated with large scale agricultural development activities, often resulting in outbreaks. The latest outbreak of the disease that has started around 1995 in both regions, has led to the present preliminary entomological surveys (1996-2005) the results of which are reported here. Sandflies were collected using CDC light traps and Phlebotomus females were dissected for Leishmania detection and isolation; freshly fed Phlebotomus females collected were subsequently tested for their blood meal sources using ELISA. All Phlebotomus collections were identified to species.ResultsDuring the surveys (1996-2005), a total of 1963 sandflies of six Phlebotomus species (P. orientalis, P. papatasi, P. bergeroti, P. duboscqi, P. rodhaini and P. alexandri) were recorded from the study areas: the predominant species was P. orientalis in both localities. None of the total 618 P. orientalis females dissected (506 from Metema and 112 from Humera), nor the total 114 females of four other species dissected (P. papatasi, P. duboscqi, P. bergeroti and P. rodhaini) was infected with Leishmania promastigotes. ELISA-based blood meal analysis of 273 fresh fed P. orientalis females collected from Metema revealed a remarkably high bovine blood feeds (92%) with only 2.2% of human blood feeds.ConclusionBased on abundance and other circumstantial evidences (its proven role in Sudan), P. orientalis is the most likely vector of VL in northwest Ethiopia, pending further clarifications. The zoophagic feeding behaviour of P. orientalis detected in the present study could have epidemiological significance, but more investigations are required in this and other behavioural characteristics towards appropriate management of the vector.

Blood meal origins and insecticide susceptibility of Anopheles arabiensis from Chano in South-West Ethiopia

BackgroundAnopheles arabiensis, the main malaria vector in Ethiopia, shows both anthropophilic and zoophilic behaviours. Insecticide resistance is increasing, and alternative methods of vector control are needed. The objectives of this study were to determine the blood meal origins and the susceptibility to insecticides of An. arabiensis from Chano village near Arba Minch in South-West Ethiopia.MethodsBlood meal sources of anopheline mosquitoes collected using Centers for Disease Control and Prevention (CDC) light traps and pyrethrum spray catches (PSC) from human dwellings, and hand-held mouth aspirators from outdoor pit shelters were analysed using a direct enzyme-linked-immunosorbent assay (ELISA). The susceptibility of An. arabiensis to pyrethroid insecticides (alphacypermethrin, lambdacyhalothrin, deltamethrin, and cyfluthrin) and DDT was assessed using females reared from larval and pupal collections from natural breeding sites.ResultsThe blood meal origins of 2967 freshly fed Anopheles mosquitoes were determined. An. arabiensis was the predominant species (75%), and it fed mainly on cattle. The densities of both freshly fed An. arabiensis and those fed on human blood followed similar seasonal patterns. The overall human blood index (HBI) of An. arabiensis, including mixed blood meals, was 44% and the bovine blood index (BBI) was 69%. The HBI of An. arabiensis from CDC light trap collections was 75% and this was higher than those for PSC (38%) and outdoor pit shelter collections (13%), while the BBI was 65% for PSC, 68% for outdoor pit shelters and 72% for CDC light traps. More freshly fed and human blood-fed An. arabiensis were sampled from houses close to the shore of Lake Abaya (the major breeding site).A high proportion of An. arabiensis was resistant to the pyrethroid insecticides, with a mortality rate of 56% for lambdacyhalothrin, 50% for cyfluthrin and alphacypermethrin, 47% for deltamethrin, and 10% for DDT.ConclusionAnopheles arabiensis is the predominant species of anopheline mosquito in this region, and cattle are the main source of its blood meals. The greater tendency of this species to feed on cattle justifies the application of insecticides on cattle to control it. However, An. arabiensis has already developed resistance to the available pyrethroid insecticides, and alternative insecticides are needed for malaria vector control.

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.

Comparative Analysis of Salivary Gland Transcriptomes of Phlebotomus orientalis Sand Flies from Endemic and Non-endemic Foci of Visceral Leishmaniasis

Background 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. Methodology/Principal Findings 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. Conclusions 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.

Laboratory and field evaluation of neem (Azadirachta indica A. Juss) and Chinaberry (Melia azedarach L.) oils as repellents against Phlebotomus orientalis and P. bergeroti (Diptera: Psychodidae) in Ethiopia

The study evaluated the efficacy of neem (Azadirachta indica A. Juss.) and Chinaberry (Melia azedarach L.) seed oils as repellents against laboratory and field populations of some sandflies in Ethiopia. In the laboratory, concentrations of 2% and 5% neem oil in coconut oil tested against Phlebotomus orientalis (vector of visceral leishmaniasis) provided 96.28% (95% CI=95.60-96.97) protection up to a mean time of 7h and 20 min and 98.26% (95% CI=93.46-104. 07) protection up to 9h, respectively. Similarly, M. azedarach oil at 2% concentration produced 95.13% (95% CI=90.74-99.52) protection for the same duration (7h and 20 min), while the 5% oil gave 96.20 (95% CI=86.98-105.41) protection for 8h and 20 min against the same species with no significant difference in percentage protection between the two oils at 2% and 5% concentrations. In the field tests with only neem oil (A. indica) against field populations of P. orientalis and P. bergeroti, similar high level of repellencies were recorded with about the same duration of protection. Application of both neem and Chinaberry oils can be safe and low-cost means of personal protection against sandfly bites in endemic areas of Ethiopia, if the community is advised and encouraged to grow the plants abundantly.

Characterization of breeding sites of Phlebotomus orientalis – The vector of visceral leishmaniasis in northwestern Ethiopia

We studied breeding sites of Phlebotomus orientalis (Diptera: Psychodidae) the vector of visceral leishmaniasis in northern Ethiopia. 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.

Phlebotomus (Phlebotomus) duboscqi (Diptera: Phlebotominae), naturally infected with Leishmania major in southern Ethiopia

In a survey of Leishmania infections in phlebotomine sandflies in visceral leishmaniasis focus at Aba Roba, Segen Valley, southern Ethiopia, Phlebotomus duboscqi was found naturally infected with L. major (zymodeme MON-74). This is the first isolation of L. major from a vector sandfly in Ethiopia, from an area where human and/or rodent infections with L. major are, so far, unknown.