Large scale systemic control short-circuits pathogen transmission by interrupting the sand rat (Psammomys obesus)-to-sand fly (Phlebotomus papatasi) Leishmania major transmission cycle.

Systemic control uses the vertebrate hosts of zoonotic pathogens as "Trojan horses," killing blood-feeding female vectors and short-circuiting host-to-vector pathogen transmission. Previous studies focused only on the effect of systemic control on vector abundance at small spatial scales. None were conducted at a spatial scale relevant for vector control and none on the effect of systemic control on pathogen transmission rates. We tested the application of systemic control, using Fipronil-impregnated rodent baits, in reducing Leishmania major (Kinetoplastida: Trypanosomatidae; Yakimoff & Schokhor, 1914) infection levels within the vector, Phlebotomus papatasi (Diptera: Psychodidae; Scopoli, 1786) population, at the town-scale. We provided Fipronil-impregnated food-baits to all Psammomys obesus (Mammalia:Muridae; Cretzschmar, 1828), the main L. major reservoir, burrows along the southern perimeter of the town of Yeruham, Israel, and compared sand fly abundance and infection levels with a non-treated control area. We found a significant and substantial treatment effect on L. major infection levels in the female sand fly population. Sand fly abundance was not affected. Our results demonstrate, for the first time, the potential of systemic control in reducing pathogen transmission rates at a large, epidemiologically relevant, spatial scale.

Concurrent molecular characterization of sand flies and Leishmania parasites by amplicon-based next-generation sequencing.

BACKGROUND: Phlebotomine sand flies are vectors of Leishmania parasites, which are the causative agents of leishmaniasis. Herein, we developed an amplicon-based next-generation sequencing (Amp-NGS) to characterize sand flies and Leishmania parasites simultaneously targeting partial fragments of 18S rDNA and ITS1 genes, respectively. METHODS: Our assay was optimized using reference sand fly (n = 8) and Leishmania spp. (n = 9) samples and validated using wild-caught sand flies from Palestine. The assay was highly specific, and all DNA references were successfully identified to the species level. RESULTS: Among the wild-caught sand flies (n = 187), Phlebotomus spp. represented 95% of the collected samples (177/187), including Ph. sergenti (147/187, 79%), Ph. papatasi (19/187, 10.2%), Ph. perfiliewi (3/187, 1.6%), Ph. tobbi (2/187, 1.2%) and Ph. syriacus (6/187, 3.2%). Sergentomyia spp. represented only 5% (10/187) of the collected samples and included S. dentata (n = 6), S. fallax (n = 2), S. schwetzi (n = 1) and S. ghesquiere (n = 1). The study observed strong positive correlation between sand fly identification results of the Amp-NGS and morphological identification method (r = 0.84, df = 185, P < 0.001). Some discrepancies between the two methods in the identification of closely related species (i.e. Ph. perfiliewi, Ph. tobbi and Ph. syriacus) were observed. Leishmania DNA was detected and identified as L. tropica in 14 samples (14/187, 7.5%). CONCLUSIONS: Our assay was sensitive to detect (limit of detection was 0.0016 ng/reaction) and identify Leishmania DNA in sand flies, thus representing a new tool for studying sand flies and their associated Leishmania parasites in endemic areas.

A next-generation (DNA) sequencing (NGS)-based method for identifying the sources of sugar meals in mosquito vectors of West Nile virus in Israel.

Mosquitoes of the genus Culex comprise important vectors of pathogenic arboviruses in our region, including West Nile and Rift Valley Fever viruses. To improve our understanding of the epidemiology and transmission dynamics of arboviruses, we need to study the behavior and ecology of their vectors. The feeding patterns of the vector mosquitoes can be very useful in determining how and where to focus control efforts. For example, determining the preferred blood hosts of the females can assist in the implementation of potentially efficacious strategies for focused control of mosquito females. Determining the plants from which both sexes derive their sugar meals can comprise the initial step towards the formulation of efficient lures for trapping mosquitoes. In the past, plant meal identification was based mainly on chemical detection of fructose and microscopical observations of cellulose particles in mosquito guts. More recent studies have utilized DNA barcoding capable of identifying plant food sources. In the current study, we identify multiple plant species from which large numbers of mosquitoes obtained their sugar meals in one experimental procedure. We employed next generation DNA sequencing to sequence the chloroplast specific plant genes atpB and rbcL.

Effects of larval rearing substrates on some life-table parameters of Lutzomyia longipalpis sand flies.

Sand flies are the insects responsible for transmitting Leishmania parasites, the causative agents of leishmaniasis in humans. However, the effects of sand fly breeding sites on their biology and ecology remain poorly understood. Herein, we studied how larval nutrition associated with putative breeding sites of the sand fly Lutzomyia longipalpis affects their oviposition, development, microbiome, and susceptibility to Leishmania by rearing L. longipalpis on substrates collected from an endemic area for leishmaniasis in Brazil. The results showed that female L. longipalpis select the oviposition site based on its potential to promote larval maturation and while composting cashew leaf litter hindered the development, larvae reared on chicken feces developed rapidly. Typical gut microbial profiles were found in larvae reared upon cashew leaf litter. Adult females from larvae reared on substrate collected in chicken coops were infected with Leishmania infantum, indicating that they were highly susceptible to the parasite. In conclusion, the larval breeding sites can exert an important role in the epidemiology of leishmaniasis.

Ecology of Leishmaniasis in an urbanized landscape: Relationship of sand fly densities, and Leishmania tropica infection rates with reservoir host colonies.

Urbanization may influence the transmission of leishmaniasis, which is as a serious public health issue in Palestine. Semi urban environments can provide suitable habitats for the reservoir host species and the vector sand flies to create favorable condition for disease transmission. This study was aimed to evaluating the effect of distance from hyrax (reservoir host) colonies on sand fly (vector) abundance and its relationship to Leishmania infection within a semi urban landscape. In the Aleskan neighborhood the town of Tubas, sand flies were collected in July and September of 2016. Five trapping transects were set running from a rocky area containing hyrax dwelling habitats into an area of inhabited houses. 1051 Phlebotomus sand flies from 9 species were captured. The numbers of Phlebotomus sergenti, the vector species, correlated negatively with the distance from hyrax. Infected, blood engorged female sand flies were captured closer to hyrax colonies than infected, non-fed females. The risk of disease transmission to humans increased with a high density of reservoirs, closer proximity of reservoir hosts, and high densities of sand flies near houses. These results must be taken into account when implementing future interventions to reduce CL in urban environments.

The spatial and temporal distribution, species composition, and host preference of phlebotomine sand flies in the Bethlehem District of Palestine.

In the Palestinian West Bank, leishmaniasis is emerging as a serious public health issue with incidence increasing over time, especially in the western and the northern parts. This study was aimed to evaluating the effect of altitude on sand fly density, temporal and spatial distribution, species composition, and host preference within and between three villages in the Bethlehem District. The three villages occur along an elevation cline, ranging from the disease-free area of Kisan (KIS; 732-782 m ASL), down to the endemic areas of Arab Ar-Rashaiyda (AAR; 522-568 m ASL), and Al'Azazma (AZA; 473-510 m ASL) in the Bethlehem District (southeastern West Bank). Sand flies were trapped monthly from May through October in 2013 in traps located north, south, east, and west of each village. The abundance of sand flies differed among the three villages, with Arab Ar-Rashaiyda (AAR) > Al 'Azazma (AZA) >Kisan (KIS). The sex ratio was even in AAR and AZA, and female biased in KIS. Both male and female Phlebotomus sand fly densities varied with elevation, with greatest densities found at intermediate elevations (AAR). Elevation correlated negatively with the proportion of blood-fed Phlebotomus females. Male Phlebotomus species composition differed among sites, with all the species found in this study present in AZA, while some species were absent from AAR and KIS. The host blood species engorged by P. sergenti were grouped in four categories: human (45.5%), livestock (25%), avian (19.9%), and dog (9.6%). This study provided information about the spatial and temporal distribution, age and species composition, and host preference of sand fly vectors that influence disease transmission. This information will allow us to better target sand flies, to control the disease, and to monitor risk prone areas.

A molecular analysis of sand fly blood meals in a visceral leishmaniasis endemic region of northwestern Ethiopia reveals a complex host-vector system.

BACKGROUND: Visceral leishmaniasis (VL, or "kala-azar") is a major cause of disability and death, especially in East Africa. Its vectors, sand flies (Diptera: Psychodidae: Phlebotominae), are poorly controlled and guarded against in these regions, owing in part to a lack of understanding about their feeding behavior. METHODS: A total of 746 freshly fed female sand flies were collected in five population centers in Kafta Humera (northwestern Ethiopia), where VL is endemic. Flies were collected from habitats that ranged from inside houses to open fields, using light traps and sticky traps. Sources of sand fly blood meals were identified using enzyme-linked immunosorbent assays (ELISA) and DNA amplification with reverse-line blot analysis (PCR-RLB); 632 specimens were screened using ELISA, 408 of which had identifiable blood meals, and 114 were screened using PCR-RLB, 53 of which yielded identifications. Fly species determinations were based on morphology, and those specimens subjected to PCR-RLB were also screened for Leishmania parasites using conventional PCR to amplify the nuclear marker ITS1 (internal transcribed spacer 1) with Leishmania-specific primers. RESULTS: More than three-fourths of all sand flies collected were Phlebotomus orientalis, and the remaining portion was comprised of nine other species. Nearly two-thirds of P. orientalis specimens were collected at village peripheries. The most common blood source for all flies was donkey (33.9% of all identifications), followed by cow (24.2%), human (17.6%), dog (11.8%), and goat or sheep (8.6%); mixtures of blood meals from different sources were found in 28.2% of all flies screened. Unidentified blood meals, presumably from wildlife, not domestic animals, were significantly higher in farm fields. Leishmania parasites were not detected in any of the 114 flies screened, not surprising given an expected infection rate of 1-5 out of 1,000. Meals that included a mixture of human and cow blood were significantly more frequent relative to all cow meals than human blood meals were to non-cow meals, suggesting a zoopotentiative interaction between cows and humans in this system. CONCLUSIONS: Habitat and host preferences of sand fly vectors in Kafta Humera confirmed the finding of previous reports that the main vector in the region, Phlebotomus orientalis, is a highly opportunistic feeder that prefers large animals and is most commonly found at village peripheries. These results were similar to those of a previous study conducted in a nearby region (Tahtay Adiabo), except for the role of cattle on the prevalence of human blood meals. Preliminary examinations of blood meal data from different settings point to the need for additional surveys and field experiments to understand the role of livestock on biting risks.

Development of a next generation DNA sequencing-based multi detection assay for detecting and identifying Leishmania parasites, blood sources, plant meals and intestinal microbiome in phlebotomine sand flies.

Leishmaniasis is a disease caused by Leishmania parasites transmitted by phlebotomine sand flies (Diptera: Psychodidae). Human infections with different Leishmania species cause characteristic clinical manifestations; cutaneous or visceral leishmaniasis. Here we describe the development and application of a Miseq Next GenerationSequencing (NGS)-based Multi Detection Assay (MDA) designed to characterize metagenomics parameters pertinent to the sand fly vectors which may affect their vectorial capacity for Leishmania. For this purpose, we developed a MDA by which, DNA fragments were amplified through polymerase chain reactions (PCR) and then sequenced by MiSeq/NGS. PCR amplification was achieved using some published and some new primers designed specifically for identifying Leishmania spp. (ITS1), sand fly spp. (cytochrome oxidase I), vertebrate blood (Cytochrome b), plant DNA ribulose-1,5-bisphosphate carboxylase large subunit gene (rbcL), and prokaryotic micobiome (16 s rRNA). This MDA/NGS analysis was performed on two species of wild-caught sand flies that transmit different Leishmania spp. in two ecologically distinct, but geographically neighboring locations. The results were analyzed to identify, quantitate and correlate the measured parameters in order to assess their putative importance in the transmission dynamics of leishmaniasis.

Plant-feeding phlebotomine sand flies, vectors of leishmaniasis, prefer Cannabis sativa.

Blood-sucking phlebotomine sand flies (Diptera: Psychodidae) transmit leishmaniasis as well as arboviral diseases and bartonellosis. Sand fly females become infected with Leishmania parasites and transmit them while imbibing vertebrates' blood, required as a source of protein for maturation of eggs. In addition, both females and males consume plant-derived sugar meals as a source of energy. Plant meals may comprise sugary solutions such as nectar or honeydew (secreted by plant-sucking homopteran insects), as well as phloem sap that sand flies obtain by piercing leaves and stems with their needle-like mouthparts. Hence, the structure of plant communities can influence the distribution and epidemiology of leishmaniasis. We designed a next-generation sequencing (NGS)-based assay for determining the source of sand fly plant meals, based upon the chloroplast DNA gene ribulose bisphosphate carboxylase large chain (rbcL). Here, we report on the predilection of several sand fly species, vectors of leishmaniasis in different parts of the world, for feeding on Cannabis sativa We infer this preference based on the substantial percentage of sand flies that had fed on C. sativa plants despite the apparent "absence" of these plants from most of the field sites. We discuss the conceivable implications of the affinity of sand flies for C. sativa on their vectorial capacity for Leishmania and the putative exploitation of their attraction to C. sativa for the control of sand fly-borne diseases.

Genetic diversity of Taenia hydatigena in the northern part of the West Bank, Palestine as determined by mitochondrial DNA sequences.

Cysticercus tenuicollis is the metacestode of canine tapeworm Taenia hydatigena, which has been reported in domestic and wild ruminants and is causing veterinary and economic losses in the meat industry. This study was conducted to determine the sequence variation in the mitochondrial cytochrome c oxidase subunit 1 (coxl) gene in 20 isolates of T. hydatigena metacestodes (cysticercus tenuicollis) collected from northern West Bank in Palestine. Nine haplotypes were detected, with one prevailing (55%). The total haplotype diversity (0.705) and the total nucleotide diversity (0.0045) displayed low genetic diversity among our isolates. Haplotype analysis showed a star-shaped network with a centrally positioned common haplotype. The Tajima's D, and Fu and Li's statistics in cysticercus tenuicollis population of this region showed a negative value, indicating deviations from neutrality and both suggested recent population expansion for the population. The findings of this study would greatly help to implement control and preventive measures for T. hydatigena larvae infection in Palestine.

Some aspects of entomological determinants of Phlebotomus orientalis in highland and lowland visceral leishmaniasis foci in northwestern Ethiopia.

Visceral leishmaniasis (VL) is one of the major public health problems in northwest Ethiopia, mainly in Libo-Kemkem and Metema districts, where Phlebotomus orientalis is the most probable vector of the disease. The aim of this study was to determine the physiological age, host preference and vectorial potential of P. orientalis in the highland and lowland foci of the region. Sand flies were collected using CDC light traps between May 2011 and April 2012 in Libo-Kemkem and October 2012 and September 2013 in Metema from household compounds, farm field and mixed forest. Females belonging to Phlebotomus were dissected for physiological age determination and Leishmania detection and isolation. Leishmania infections in sand flies were investigated using molecular methods. Freshly fed Phlebotomus females were tested to identify blood meal sources using PCR-RLB and ELISA. A total of 1149 (936 from Libo-Kemkem and 213 from Metema) blood unfed female P. orientalis were dissected for age determination. The parity rate was 45.6% and 66.2% in Libo-Kemkem and Metema, respectively. None of 798 female P. orientalis dissected (578 from Libo-Kemkem and 220 from Metema) was infected with Leishmania parasites. A total of 347 P. orientalis specimens collected from Libo-Kemkem were processed using PCR, of which 10 (2.8%) specimens were found with DNA of Leishmania spp. Of a total 491 freshly fed female P. orientalis analyzed for blood meal origins by RLB-PCR and ELISA, 57.6% (67.8% from Libo-Kemkem and 49.8% from Metema) were found to contain bovine blood while 4.9% (3.7% from Libo-Kemkem and 5.7% from Metema) were of human blood. In conclusion, the present study showed parity difference between the two populations of P. orientalis and that both populations have strong zoophilic behavior. Based on the presented evidences, the species is strongly implicated as a vector of kala-azar in both areas. Therefore, vector control should be a component of a strategy to manage visceral leishmaniasis in both study areas.

The substructure of three repetitive DNA regions of Schistosoma haematobium group species as a potential marker for species recognition and interbreeding detection.

BACKGROUND: Schistosoma haematobium is the causative agent of human urogenital schistosomiasis affecting ~112 million people in Africa and the Middle East. The parasite is transmitted by snails of the genus Bulinus, which also transmit other closely related human and animal schistosomes. The accurate discrimination of S. haematobium from species infecting animals will aid effective control and elimination programs. Previously we have shown the utility of different repetitive nuclear DNA sequences (DraI, sh73bp, and sh77bp) for the identification of S. haematobium-group species and inter-repeat sequences for discriminating S. haematobium from S. bovis. RESULTS: In this current study we clarify the structural arrangement and association between the three repetitive sequences (DraI, sh73bp, and sh77bp) in both S. haematobium and S. bovis, with a unique repeat linker being found in S. haematobium (Sh64bp repeat linker) and in S. bovis (Sb30bp repeat linker). Sequence data showed that the 3'-end of the repeat linker was connected to the DraI repetitive sequence array, and at the 5'-end of the repeat linker sh73bp and sh77bp were arranged in an alternating manner. Species-specific oligonucleotides were designed targeting the species-specific repeat linkers and used in a reverse line blot (RLB) hybridization assay enabling differentiation between S. haematobium and S. bovis. The assay was used to discriminate natural infections in wild caught Bulinus globosus. CONCLUSION: This research enabled the characterisation of species-specific DNA regions that enabled the design of species-specific oligonucleotides that can be used to rapidly differentiate between S. haematobium and S. bovis and also have the potential to aid the detection of natural hybridization between these two species.

Minimally invasive microbiopsies: a novel sampling method for identifying asymptomatic, potentially infectious carriers of Leishmania donovani.

Visceral leishmaniasis (VL) is a potentially lethal, sand fly-borne disease caused by protozoan parasites belonging to the Leishmania donovani species complex. There are several adequate methods for diagnosing VL, but the majority of infected individuals remain asymptomatic, comprising potential parasite reservoirs for transmission of the disease. The gold standard for assessing host infectiousness to biting vector insects is xenodiagnosis (i.e. scoring infection rates among insectary-reared insects that had fed on humans suspected of being infected). However, when it comes to sand flies and leishmaniasis, xenodiagnosis is an intricate operation burdened by logistical hurdles and ethical concerns that prevent its effective application for mass screening of widely dispersed communities, particularly in rural regions of underdeveloped countries. Minimally invasive microbiopsy (MB) devices were designed to penetrate the skin to a depth of ∼200µm and absorb blood as well as skin cell lysates, mimicking the mode by which phlebotomine sand flies acquire blood meals, as well as their composition. MBs taken from 137 of 262 volunteers, living in endemic VL foci in Ethiopia, detected Leishmania parasites that could potentially be imbibed by feeding sand flies. Although the volume of MBs was 10-fold smaller than finger-prick blood samples, Leishmania DNA detection rates from MBs were significantly higher, implying that skin, more often than blood, was the source of parasites. Volunteers with histories of VL were almost as likely as healthy volunteers to test positive by MBs (southern Ethiopian focus: 95% CI: 0.35-2.59, P=1.0. northern Ethiopian focus 0.87: 95% CI: 0.22-3.76, P=1), suggesting the importance of asymptomatic patients as reservoirs of L. donovani. Minimally invasive, painless MBs should be considered for reliably and efficiently evaluating both L. donovani infection rates among large numbers of asymptomatic carriers and their infectiousness to blood-feeding sand flies.

Coinfection of Leishmania guyanensis and Human Immunodeficiency Virus-Acquired Immune Deficiency Syndrome: Report of a Case of Disseminated Cutaneous Leishmaniasis in Ecuador.

AbstractReported herein is the first case of Leishmania-human immunodeficiency virus (HIV) coinfection in Ecuador. In Ecuador, HIV infections overlap endemic areas of leishmaniasis. Immunosuppression is a well-established risk factor for developing severe disease. This is a severe case of a 32-year-old man presenting with disseminated pleomorphic ulcers, papules, and cutaneous plaque-like lesions over his whole body. Numerous amastigotes were observed in both skin scrapings and biopsies. The sequence of the cytochrome b gene confirmed the presence of Leishmania guyanensis. The patient was treated but failed to respond to meglumine antimoniate and amphotericin B. Six months later, the patient died due to bacterial septic shock.

Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.

Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR.

Physiological Age Structure and Leishmania spp. Detection in Phlebotomus (Larroussius) orientalis (Parrot, 1936) (Diptera: Psychodidae) at an Endemic Focus of Visceral Leishmaniasis in Northern Ethiopia.

Visceral leishmaniasis (VL) caused by Leishmania donovani is endemic in northern Ethiopia, where P. orientalis is the most important presumed vector. This study was designed to determine the physiological age structure and the occurrence of Leishmania infection in the vector of VL in Tahtay Adiyabo district, northern Ethiopia. Sand flies were collected using CDC light traps from peridomestic and agricultural fields between May 2011 and April 2012 and P. orientalis females were dissected for age determination and detection of Leishmania promastigotes. Sand flies were also analyzed for L. donovani detection using molecular methods. Of 1,282 P. orientalis examined for abdominal stages and age characterization, 66.2%, 28.2%, 4.1%, and 1.6% were unfed, freshly fed, half-gravid, and gravid. Parous rate in unfed females was 34.1% and 35.4% in peridomestic and agricultural fields, respectively. Out of 921 P. orientalis females dissected, one specimen (0.1%) was found naturally infected with promastigotes. Five pools (25 females) of unfed P. orientalis were also found with DNA of Leishmania spp. In particular, a single P. orientalis was positive for L. donovani (0.5%). Based on this and other evidences (abundance, human blood feeding, and xenodiagnostic studies), P. orientalis is the principal vector of VL in this endemic focus.

Estimation of infection prevalence and sensitivity in a stratified two-stage sampling design employing highly specific diagnostic tests when there is no gold standard.

In this work, we describe a two-stage sampling design to estimate the infection prevalence in a population. In the first stage, an imperfect diagnostic test was performed on a random sample of the population. In the second stage, a different imperfect test was performed in a stratified random sample of the first sample. To estimate infection prevalence, we assumed conditional independence between the diagnostic tests and develop method of moments estimators based on expectations of the proportions of people with positive and negative results on both tests that are functions of the tests' sensitivity, specificity, and the infection prevalence. A closed-form solution of the estimating equations was obtained assuming a specificity of 100% for both tests. We applied our method to estimate the infection prevalence of visceral leishmaniasis according to two quantitative polymerase chain reaction tests performed on blood samples taken from 4756 patients in northern Ethiopia. The sensitivities of the tests were also estimated, as well as the standard errors of all estimates, using a parametric bootstrap. We also examined the impact of departures from our assumptions of 100% specificity and conditional independence on the estimated prevalence.

Host-feeding preference of Phlebotomus orientalis (Diptera: Psychodidae) in an endemic focus of visceral leishmaniasis in northern Ethiopia.

BACKGROUND: Blood-feeding behavior studies are important for estimating the efficiency of pathogen transmission and assessing the relative human disease risk. However, in Ethiopia and other parts of East Africa there are large remaining gaps in identifying the feeding habits of Phlebotomus orientalis, the vector of Leishmania donovani. The aim of the study was to determine the blood feeding patterns of P. orientalis in Tahtay Adiyabo district, northern Ethiopia. METHODS: For bloodmeal analysis, sandflies were collected from three different villages of Tahtay Adiyabo district using CDC light traps, sticky traps, and pyrethrum spray catches. Bloodmeal of engorged female sandflies was identified using cytochrome (cyt) b-PCR and reverse-line blotting (RLB) and enzyme linked immunosorbent assay (ELISA) assays. RESULTS: Most (637/641) of the females analyzed were P. orientalis. Successful identification of the host from bloodmeals was achieved in 83.03 and 92.1% using cyt b PCR-RLB and ELISA, respectively. Bloodmeal analysis of P. orientalis females revealed that they have a range of hosts with predominant preference to bovines followed by donkey, human, goat, sheep, dog, and camel. CONCLUSION: Results obtained from bloodmeal analyses demonstrate that the feeding preference of P. orientalis is mainly zoophilic, which could vary depending on the availability of hosts.

Schistosoma haematobium detection in snails by DraI PCR and Sh110/Sm-Sl PCR: further evidence of the interruption of schistosomiasis transmission in Morocco.

BACKGROUND: This is the first study in Morocco to estimate snail infection rates at the last historic transmission sites of schistosomiasis, known to be free from new infection among humans since 2004. Screening of large numbers of snails for infection is one way to confirm that Schistosoma haematobium transmission has stopped and does not resurge. METHODS: A total of 2703 Bulinus truncatus snails were collected from 24 snail habitats in five provinces of Morocco: Errachidia, El Kelaa des Sraghna, Tata, Beni Mellal, and Chtouka Ait Baha. All visible snails were collected with a scoop net or by hand. We used waders and gloves as simple precautions. Snails were morphologically identified according to Moroccan Health Ministry guide of schistosomiasis (1982).All snails were analyzed in pools by molecular tool, using primers from the newly identified repeated DNA sequence, termed DraI, in the S. haematobium group. To distinguish S. bovis and S. haematobium, the snails were analyzed by Sh110/Sm-Sl PCR that was specific of S. haematobium. RESULTS: The results showed that snails from Errachidia, Chtouka Ait Baha, sector of Agoujgal in Tata and sector of Mbarkiya in El kelaa des Sraghna were negative for DraI PCR; but, snails from remaining snail habitats of El Kelaa des Sraghna, Tata and Beni Mellal were positive. This led to suggest the presence of circulating schistosome species (S. haematobium, S. bovis or others) within these positive snail habitats. Subsequently, confirmation with S. haematobium species specific molecular assay, Sh110/Sm-Sl PCR, showed that none of the collected snails were infected by S. haematobium in all historic endemic areas. CONCLUSION: The absence of S. haematobium infection in snails supports the argument of S. haematobium transmission interruption in Morocco.

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.

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.