Breeding habitats, bionomics and phylogenetic analysis of Aedes aegypti and first detection of Culiseta longiareolata, and Ae. hirsutus in Somali Region, eastern Ethiopia.

INTRODUCTION: Arboviral diseases, such as dengue, chikungunya, yellow fever, and Zika, are caused by viruses that are transmitted to humans through mosquito bites. However, the status of arbovirus vectors in eastern Ethiopia is unknown. The aim of this study was to investigate distribution, breeding habitat, bionomics and phylogenetic relationship of Aedes aegypti mosquito species in Somali Regional State, Eastern Ethiopia. METHODS: Entomological surveys were conducted in four sites including Jigjiga, Degehabur, Kebridehar and Godey in 2018 (October to December) to study the distribution of Ae. aegypti and with a follow-up collection in 2020 (July-December). In addition, an investigation into the seasonality and bionomics of Ae. aegypti was conducted in 2021 (January-April) in Kebridehar town. Adult mosquitoes were collected from indoor and outdoor locations using CDC light traps (LTs), pyrethrum spray collection (PSCs), and aspirators. Larvae and pupae were also collected from a total of 169 water-holding containers using a dipper between October and November 2020 (rainy season) in Kebridehar town. The species identification of wild caught and reared adults was conducted using a taxonomic key. In addition, species identification using mitochondrial and nuclear genes maximum likelihood-based phylogenetic analysis was performed. RESULTS: In the 2018 collection, Ae. aegypti was found in all study sites (Jigjiga, Degahabour, Kebridehar and Godey). In the 2020-2021 collection, a total of 470 (Female = 341, Male = 129) wild caught adult Ae. aegypti mosquitoes were collected, mostly during the rainy season with the highest frequency in November (n = 177) while the lowest abundance was in the dry season (n = 14) for both February and March. The majority of Ae. aegypt were caught using PSC (n = 365) followed by CDC LT (n = 102) and least were collected by aspirator from an animal shelter (n = 3). Aedes aegypti larval density was highest in tires (0.97 larvae per dip) followed by cemented cisterns (0.73 larvae per dip) and the Relative Breeding Index (RBI) was 0.87 and Container Index (CI) was 0.56. Genetic analysis of ITS2 and COI revealed one and 18 haplotypes, respectively and phylogenetic analysis confirmed species identification. The 2022 collection revealed no Ae. aegpti, but two previously uncharacterized species to that region. Phylogenetic analysis of these two species revealed their identities as Ae. hirsutus and Culiseta longiareolata. CONCLUSION: Data from our study indicate that, Ae. aegypti is present both during the wet and dry seasons due to the availability of breeding habitats, including water containers like cemented cisterns, tires, barrels, and plastic containers. This study emphasizes the necessity of establishing a national entomological surveillance program for Aedes in Somali region.

Trend analysis of malaria in urban settings in Ethiopia from 2014 to 2019.

BACKGROUND: Urbanization generally improves health outcomes of residents and is one of the potential factors that might contribute to reducing malaria transmission. However, the expansion of Anopheles stephensi, an urban malaria vector, poses a threat for malaria control and elimination efforts in Africa. In this paper, malaria trends in urban settings in Ethiopia from 2014 to 2019 are reported with a focus on towns and cities where An. stephensi surveys were conducted. METHODS: A retrospective study was conducted to determine malaria trends in urban districts using passive surveillance data collected at health facilities from 2014 to 2019. Data from 25 towns surveyed for An. stephensi were used in malaria trend analysis. Robust linear models were used to identify outliers and impute missing and anomalous data. The seasonal Mann-Kendal test was used to test for monotonic increasing or decreasing trends. RESULTS: A total of 9,468,970 malaria cases were reported between 2014 and 2019 through the Public Health Emergency Management (PHEM) system. Of these, 1.45 million (15.3%) cases were reported from urban settings. The incidence of malaria declined by 62% between 2014 and 2018. In 2019, the incidence increased to 15 per 1000 population from 11 to 1000 in 2018. Both confirmed (microscopy or RDT) Plasmodium falciparum (67%) and Plasmodium vivax (28%) were reported with a higher proportion of P. vivax infections in urban areas. In 2019, An. stephensi was detected in 17 towns where more than 19,804 malaria cases were reported, with most of the cases (56%) being P. falciparum. Trend analysis revealed that malaria cases increased in five towns in Afar and Somali administrative regions, decreased in nine towns, and had no obvious trend in the remaining three towns. CONCLUSION: The contribution of malaria in urban settings is not negligible in Ethiopia. With the rapid expansion of An. stephensi in the country, the receptivity is likely to be higher for malaria. Although the evidence presented in this study does not demonstrate a direct linkage between An. stephensi detection and an increase in urban malaria throughout the country, An. stephensi might contribute to an increase in malaria unless control measures are implemented as soon as possible. Targeted surveillance and effective response are needed to assess the contribution of this vector to malaria transmission and curb potential outbreaks.

An experimental hut study evaluating the impact of pyrethroid-only and PBO nets alone and in combination with pirimiphos-methyl-based IRS in Ethiopia.

BACKGROUND: Pyrethroid resistance observed in populations of malaria vectors is widespread in Ethiopia and could potentially compromise the effectiveness of insecticide-based malaria vector control interventions. In this study, the impact of combining indoor residual spraying (IRS) and insecticide-treated nets (ITNs) on mosquito behaviour and mortality was evaluated using experimental huts. METHODS: A Latin Square Design was employed using six experimental huts to collect entomological data. Human volunteers slept in huts with different types of nets (pyrethroid-only net, PBO net, and untreated net) either with or without IRS (Actellic 300CS). The hut with no IRS and an untreated net served as a negative control. The study was conducted for a total of 54 nights. Both alive and dead mosquitoes were collected from inside nets, in the central rooms and verandah the following morning. Data were analysed using Stata/SE 14.0 software package (College Station, TX, USA). RESULTS: The personal protection rate of huts with PermaNet® 2.0 alone and PermaNet® 3.0 alone was 33.3% and 50%, respectively. The mean killing effect of huts with PermaNet® 2.0 alone and PermaNet® 3.0 alone was 2% and 49%, respectively. Huts with PermaNet® 2.0 alone and PermaNet® 3.0 alone demonstrated significantly higher excito-repellency than the control hut. However, mosquito mortality in the hut with IRS + untreated net, hut with IRS + PermaNet® 2.0 and hut with IRS + PermaNet® 3.0 were not significantly different from each other (p > 0.05). Additionally, pre-exposure of both the susceptible Anopheles arabiensis laboratory strain and wild Anopheles gambiae sensu lato to PBO in the cone bioassay tests of Actellic 300CS sprayed surfaces did not reduce mosquito mortality when compared to mortality without pre-exposure to PBO. CONCLUSION: Mosquito mortality rates from the huts with IRS alone were similar to mosquito mortality rates from the huts with the combination of vector control intervention tools (IRS + ITNs) and mosquito mortality rates from huts with PBO nets alone were significantly higher than huts with pyrethroid-only nets. The findings of this study help inform studies to be conducted under field condition for decision-making for future selection of cost-effective vector control intervention tools.

Wolbachia 16S rRNA haplotypes detected in wild Anopheles stephensi in eastern Ethiopia.

BACKGROUND: About two out of three Ethiopians are at risk of malaria, a disease caused by the parasites Plasmodium falciparum and Plasmodium vivax. Anopheles stephensi, an invasive vector typically found in South Asia and the Middle East, was recently found to be distributed across eastern and central Ethiopia and is capable of transmitting both P. falciparum and P. vivax. The detection of this vector in the Horn of Africa (HOA) coupled with widespread insecticide resistance requires that new methods of vector control be investigated in order to control the spread of malaria. Wolbachia, a naturally occurring endosymbiotic bacterium of mosquitoes, has been identified as a potential vector control tool that can be explored for the control of malaria transmission. Wolbachia could be used to control the mosquito population through suppression or potentially decrease malaria transmission through population replacement. However, the presence of Wolbachia in wild An. stephensi in eastern Ethiopia is unknown. This study aimed to identify the presence and diversity of Wolbachia in An. stephensi across eastern Ethiopia. METHODS: DNA was extracted from An. stephensi collected from eastern Ethiopia in 2018 and screened for Wolbachia using a 16S targeted PCR assay, as well as multilocus strain typing (MLST) PCR assays. Haplotype and phylogenetic analysis of the sequenced 16S amplicons were conducted to compare with Wolbachia from countries across Africa and Asia. RESULTS: Twenty out of the 184 mosquitoes screened were positive for Wolbachia, with multiple haplotypes detected. In addition, phylogenetic analysis revealed two superclades, representing Wolbachia supergroups A and B (bootstrap values of 81 and 72, respectively) with no significant grouping of geographic location or species. A subclade with a bootstrap value of 89 separates the Ethiopian haplotype 2 from other sequences in that superclade. CONCLUSIONS: These findings provide the first evidence of natural Wolbachia populations in wild An. stephensi in the HOA. They also identify the need for further research to confirm the endosymbiotic relationship between Wolbachia and An. stephensi and to investigate its utility for malaria control in the HOA.

Detection and population genetic analysis of kdr L1014F variant in eastern Ethiopian Anopheles stephensi.

Anopheles stephensi is a malaria vector that has been recently introduced into East Africa, where it threatens to increase malaria disease burden. The use of insecticides, especially pyrethroids, is still one of the primary malaria vector control strategies worldwide. The knockdown resistance (kdr) mutation in the IIS6 transmembrane segment of the voltage-gated sodium channel (vgsc) is one of the main molecular mechanisms of pyrethroid resistance in Anopheles. Extensive pyrethroid resistance in An. stephensi has been previously reported in Ethiopia. Thus, it is important to determine whether or not the kdr mutation is present in An. stephensi populations in Ethiopia to inform vector control strategies. In the present study, the kdr locus was analyzed in An. stephensi collected from ten urban sites (Awash Sebat Kilo, Bati, Dire Dawa, Degehabur, Erer Gota, Godey, Gewane, Jigjiga, Semera, and Kebridehar) situated in Somali, Afar, and Amhara regions, and Dire Dawa Administrative City, to evaluate the frequency and evolution of kdr mutations and the association of the mutation with permethrin resistance phenotypes. Permethrin is one of the pyrethroid insecticides used for vector control in eastern Ethiopia. DNA extractions were performed on adult mosquitoes from CDC light trap collections and those raised from larval and pupal collections. PCR and targeted sequencing were used to analyze the IIS6 transmembrane segment of the vgsc gene. Of 159 An. stephensi specimens analyzed from the population survey, nine (5.7%) carried the kdr mutation (L1014F). An. stephensi with kdr mutations were only observed from Bati, Degehabur, Dire Dawa, Gewane, and Semera. We further selected randomly twenty resistant and twenty susceptible An. stephensi mosquitoes from Dire Dawa post-exposure to permethrin and investigated the role of kdr in pyrethroid resistance by comparing the vgsc gene in the two populations. We found no kdr mutations in the permethrin-resistant mosquitoes. Population genetic analysis of the sequences, including neighboring introns, revealed limited evidence of non-neutral evolution (e.g., selection) at this locus. The low kdr mutation frequency detected and the lack of kdr mutation in the permethrin-resistant mosquitoes suggest the existence of other molecular mechanisms of pyrethroid resistance in eastern Ethiopian An. stephensi.

Genetic diversity of Anopheles stephensi in Ethiopia provides insight into patterns of spread.

BACKGROUND: The recent detection of the South Asian malaria vector Anopheles stephensi in the Horn of Africa (HOA) raises concerns about the impact of this mosquito on malaria transmission in the region. Analysis of An. stephensi genetic diversity and population structure can provide insight into the history of the mosquito in the HOA to improve predictions of future spread. We investigated the genetic diversity of An. stephensi in eastern Ethiopia, where detection suggests a range expansion into this region, in order to understand the history of this invasive population. METHODS: We sequenced the cytochrome oxidase subunit I (COI) and cytochrome B gene (CytB) in 187 An. stephensi collected from 10 sites in Ethiopia in 2018. Population genetic, phylogenetic, and minimum spanning network analyses were conducted for Ethiopian sequences. Molecular identification of blood meal sources was also performed using universal vertebrate CytB sequencing. RESULTS: Six An. stephensi COI-CytB haplotypes were observed, with the highest number of haplotypes in the northeastern sites (Semera, Bati, and Gewana towns) relative to the southeastern sites (Kebridehar, Godey, and Degehabur) in eastern Ethiopia. We observed population differentiation, with the highest differentiation between the northeastern sites compared to central sites (Erer Gota, Dire Dawa, and Awash Sebat Kilo) and the southeastern sites. Phylogenetic and network analysis revealed that the HOA An. stephensi are more genetically similar to An. stephensi from southern Asia than from the Arabian Peninsula. Finally, molecular blood meal analysis revealed evidence of feeding on cows, goats, dogs, and humans, as well as evidence of multiple (mixed) blood meals. CONCLUSION: We show that An. stephensi is genetically diverse in Ethiopia and with evidence of geographical structure. Variation in the level of diversity supports the hypothesis for a more recent introduction of An. stephensi into southeastern Ethiopia relative to the northeastern region. We also find evidence that supports the hypothesis that HOA An. stephensi populations originate from South Asia rather than the Arabian Peninsula. The evidence of both zoophagic and anthropophagic feeding support the need for additional investigation into the potential for livestock movement to play a role in vector spread in this region.

An update on the distribution, bionomics, and insecticide susceptibility of Anopheles stephensi in Ethiopia, 2018-2020.

BACKGROUND: Anopheles stephensi, an invasive malaria vector, was first detected in Africa nearly 10 years ago. After the initial finding in Djibouti, it has subsequently been found in Ethiopia, Sudan and Somalia. To better inform policies and vector control decisions, it is important to understand the distribution, bionomics, insecticide susceptibility, and transmission potential of An. stephensi. These aspects were studied as part of routine entomological monitoring in Ethiopia between 2018 and 2020. METHODS: Adult mosquitoes were collected using human landing collections, pyrethrum spray catches, CDC light traps, animal-baited tent traps, resting boxes, and manual aspiration from animal shelters. Larvae were collected using hand-held dippers. The source of blood in blood-fed mosquitoes and the presence of sporozoites was assessed through enzyme-linked immunosorbent assays (ELISA). Insecticide susceptibility was assessed for pyrethroids, organophosphates and carbamates. RESULTS: Adult An. stephensi were collected with aspiration, black resting boxes, and animal-baited traps collecting the highest numbers of mosquitoes. Although sampling efforts were geographically widespread, An. stephensi larvae were collected in urban and rural sites in eastern Ethiopia, but An. stephensi larvae were not found in western Ethiopian sites. Blood-meal analysis revealed a high proportion of blood meals that were taken from goats, and only a small proportion from humans. Plasmodium vivax was detected in wild-collected An. stephensi. High levels of insecticide resistance were detected to pyrethroids, carbamates and organophosphates. Pre-exposure to piperonyl butoxide increased susceptibility to pyrethroids. Larvae were found to be susceptible to temephos. CONCLUSIONS: Understanding the bionomics, insecticide susceptibility and distribution of An. stephensi will improve the quality of a national response in Ethiopia and provide additional information on populations of this invasive species in Africa. Further work is needed to understand the role that An. stephensi will have in Plasmodium transmission and malaria case incidence. While additional data are being collected, national programmes can use the available data to formulate and operationalize national strategies against the threat of An. stephensi.

Anopheles stephensi Mosquitoes as Vectors of Plasmodium vivax and falciparum, Horn of Africa, 2019.

Anopheles stephensi mosquitoes, efficient vectors in parts of Asia and Africa, were found in 75.3% of water sources surveyed and contributed to 80.9% of wild-caught Anopheles mosquitoes in Awash Sebat Kilo, Ethiopia. High susceptibility of these mosquitoes to Plasmodium falciparum and vivax infection presents a challenge for malaria control in the Horn of Africa.

Environmental Factors Associated with Larval Habitats of Anopheline Mosquitoes (Diptera: Culicidae) in Metema District, Northwestern Ethiopia.

BACKGROUND: Malaria is one of the major public health concerns in Ethiopia. Control options available for the management of malaria, include case detection, personal protection and larval source management. Effective control of Anopheles larvae largely depends on understanding of the habitats of the vectors. The aims of this study were to identify the breeding habitats of mosquitoes and characterize the larval habitats in Gende Wuha Town in northwestern Ethiopia. METHODS: Different aquatic habitats were sampled and characterized for anopheline larvae from November 2012 to June 2013. RESULTS: In total, 2784 larvae of Anopheles mosquitoes were collected from various breeding habitats. Microscopic identification of the III and IV instars revealed the presence of seven Anopheles species. Of the Anopheles spp, Anopheles gambiae s.l. (80%) was the most predominant species in the study area. Spearman correlation coefficient results also determined that the density of An. gambiae s.l. increased significantly with habitat temperature (r= 0.346, p< 0.01). Significantly higher An. gambiae s.l. larvae were obtained in non-shaded habitats (z= -3.120, p< 0.05) when compared with shaded habitats. CONCLUSION: The current study demonstrated An. gambiae s.l., the principal malaria vector in the country, is the predominant species in the larval sampling habitats. It was also noted the importance of edge of stream as larva breeding habitats for this species during the dry season of the year. Therefore, attention should be given for this breeding habitat for control of the vector during dry season.

Anopheles larval species composition and characterization of breeding habitats in two localities in the Ghibe River Basin, southwestern Ethiopia.

BACKGROUND: Documentation of the species composition of Anopheles mosquitoes and characterization of larval breeding sites is of major importance for the implementation of larval control as part of malaria vector control interventions in Ethiopia. The aims of this study were to determine the Anopheles larval species composition, larval density, available habitat types and the effects of related environmental and physico-chemical parameters of habitats in the Ghibe River basin of southwestern Ethiopia. METHODS: Anopheles larvae were sampled from November 2014 to October 2016 on a monthly basis and 3rd and 4th instars were identified microscopically to species. The larval habitats were characterized based on habitat perimeter, water depth, intensity of light, water current, water temperature, water pH, water turbidity, distance to the nearest house, vegetation coverage, permanence of the habitat, surface debris coverage, emergent plant coverage, habitat type and substrate type. RESULTS: In total, 9277 larvae of Anopheles mosquitoes and 494 pupae were sampled from borrow pits, hoof prints, rain pools, pools at river edges, pools in drying river beds, rock pools, tire tracks and swamps. Anopheles larval density was highest in pools in drying river beds (35.2 larvae per dip) and lowest in swamps (2.1 larvae per dip) at Darge, but highest in rain pools (11.9 larvae per dip), borrow pits (11.2 larvae per dip) and pools at river edges (7.9 larvae per dip), and lowest in swamps (0.5 larvae per dip) at Ghibe. A total of 3485 late instar Anopheles mosquito larvae were morphologically identified. Anopheles gambiae sensu lato was the primary Anopheles mosquito found in all larval habitats except in swamps. Temperature at the time of sampling and emergent vegetation, were the most important variables for Anopheles mosquito larval density. Anopheles gambiae density was significantly associated with habitats that had smaller perimeters, were sunlit, had low vegetation cover, and a lack of emergent plants. Generally, Anopheles mosquito larval density was not significantly associated with water pH, water temperature, water turbidity, algal content, and larval habitat depth. CONCLUSION: Different species of Anopheles larvae were identified including An. gambiae s.l., the main malaria vector in Ethiopia. Anopheles gambiae s.l. is the most abundant species that bred in most of the larval habitat types identified in the study area. The density of this species was high in sunlit habitat, absence of emergent plants, lack of vegetation near habitat and habitats closer to human habitation. Rainfall plays a great role in determining the availability of breeding habitats. The presence of rain enable to create some of the habitat types, but alter the habitats formed at the edge of the rivers due to over flooding. Controlling the occurrence of mosquito larvae through larval source management during the dry season, targeting the pools in drying river bed and pools formed at the edge of the rivers as the water receded can be very crucial to interrupt the re-emergence of malaria vectors on the onset of rainy season.

Geographical distribution of Anopheles stephensi in eastern Ethiopia.

BACKGROUND: The recent detection of the South Asian malaria vector Anopheles stephensi in Ethiopia and other regions in the Horn of Africa has raised concerns about its potential impact on malaria transmission. We report here the findings of a survey for this species in eastern Ethiopia using both morphological and molecular methods for species identification. METHODS: Adult and larval/pupal collections were conducted at ten sites in eastern Ethiopia and Anopheles specimens were identified using standard morphological keys and genetic analysis. RESULTS: In total, 2231 morphologically identified An. stephensi were collected. A molecular approach incorporating both PCR endpoint assay and sequencing of portions of the internal transcribed spacer 2 (ITS2) and cytochrome c oxidase subunit 1 (cox1) loci confirmed the identity of the An. stephensi in most cases (119/124 of the morphologically identified An. stephensi confirmed molecularly). Additionally, we observed Aedes aegypti larvae and pupae at many of the An. stephensi larval habitats. CONCLUSIONS: Our findings show that An. stephensi is widely distributed in eastern Ethiopia and highlight the need for further surveillance in the southern, western and northern parts of the country and throughout the Horn of Africa.

The effect of ivermectin® on fertility, fecundity and mortality of Anopheles arabiensis fed on treated men in Ethiopia.

BACKGROUND: Insecticide resistance is a growing threat to malaria vector control. Ivermectin, either administered to humans or animals, may represent an alternate strategy to reduce resistant mosquito populations. The aim of this study was to assess the residual or delayed effect of administering a single oral dose of ivermectin to humans on the survival, fecundity and fertility of Anopheles arabiensis in Ethiopia. METHODS: Six male volunteers aged 25-40 years (weight range 64-72 kg) were recruited; four of them received a recommended single oral dose of 12 mg ivermectin and the other two individuals were untreated controls. A fully susceptible insectary colony of An. arabiensis was fed on treated and control participants at 1, 4, 7, 10 and 13 days post ivermectin-administration. Daily mosquito mortality was recorded for 5 days. An. arabiensis fecundity and fertility were measured from day 7 post treatment, by dissection to examine the number of eggs per mosquito, and by observing larval hatching rates, respectively. RESULTS: Ivermectin treatment induced significantly higher An. arabiensis mortality on days 1 and 4, compared to untreated controls (p = 0.02 and p < 0.001, respectively). However, this effect had declined by day 7, with no significant difference in mortality between treated and control groups (p = 0.06). The mean survival time of mosquitoes fed on day 1 was 2.1 days, while those fed on day 4 survived 4.0 days. Mosquitoes fed on the treatment group at day 7 and 10 produced significantly lower numbers of eggs compared to the untreated controls (p < 0.001 and p = 0.04, respectively). An. arabiensis fed on day 7 on treated men also had lower larval hatching rates than mosquitoes fed on days 10 and 13 (p = 0.003 and p = 0.001, respectively). CONCLUSION: A single oral dose of ivermectin given to humans can induce mortality and reduce survivorship of An. arabiensis for 7 days after treatment. Ivermectin also had a delayed effect on fecundity of An. arabiensis that took bloodmeals from treated individuals on day 7 and 10. Additional studies are warranted using wild, insecticide-resistant mosquito populations, to confirm findings and a phase III evaluation among community members in Ethiopia is needed to determine the impact of ivermectin on malaria transmission.

Effect of LongRange™ eprinomectin on Anopheles arabiensis by feeding on calves treated with the drug.

BACKGROUND: Misuse of long-lasting insecticidal nets together with resistance of vectors to most of the insecticides for indoor residual spraying and impregnated nets threaten malaria vector control interventions, requiring search for alternative control methods. Reports have shown that Anopheles mosquitoes die when they feed on endectocidal drugs used to treat humans and animals. A study was designed to investigate the efficacy of LongRange™ (eprinomectin 5%) on laboratory reared Anopheles arabiensis fed on treated calves. METHODS: Anopheles arabiensis from insectary colony was fed on three calves treated with therapeutic dose of LongRange™ eprinomectin (1 ml/50 kg) and on non-treated three other calves as control arm. For the feeding, mosquitoes were placed in paper cups covered with nylon cloth mesh and then allowed to feed on the necks of calves. Subsequently, mosquito survival, fecundity, egg hatchability, larval development and adult emergence were recorded. Data were entered and analysed by using SPSS version 20. The Kaplan-Meier survival analysis and independent sample t-test were used. RESULTS: All mosquitoes that fed on LongRange™ Eprinomectin treated calves died within 7 days following blood ingestion. The drug also slightly affected fecundity and hatchability of An. arabiensis. CONCLUSION: Treating livestock with LongRange™ (eprinomectin 5%) may serve as a supplementary control method for zoophagic An. arabiensis.

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.

Impact of combining indoor residual spraying and long-lasting insecticidal nets on Anopheles arabiensis in Ethiopia: results from a cluster randomized controlled trial.

BACKGROUND: Indoor residual house spraying (IRS) and long-lasting insecticidal nets (LLINs) are the key front-line malaria vector interventions against Anopheles arabiensis, the sole primary malaria vector in Ethiopia. Universal coverage of both interventions has been promoted and there is a growing demand in combinations of interventions for malaria control and elimination. This study compared the impact on entomological outcomes of combining IRS and LLINs with either intervention alone in Adami Tullu district, south-central Ethiopia. The epidemiological outcomes were recently published on a separate paper. METHODS: This factorial, cluster-randomized, controlled trial randomized villages to four study arms: IRS + LLIN, IRS, LLIN, and control. LLINs (PermaNet 2.0) were provided free of charge. IRS with propoxur was applied before the main malaria transmission season in 2014, 2015 and 2016. Adult mosquitoes were collected in randomly selected villages in each arm using CDC light trap catch (LTC) set close to a sleeping person, pyrethrum spray catch (PSC), and artificial pit shelter (PIT), for measuring mosquito host-seeking density (HSD), indoor resting density (IRD), and outdoor resting density (ORD), respectively. Human landing catch (HLC) was performed in a sub-set of villages to monitor An. arabiensis human biting rates (HBR). Mean vector densities and HBR were compared among study arms using incidence rate ratio (IRR) calculated by negative binomial regression. RESULTS: There were no significant differences in mean densities (HSD, IRD, ORD) and HBR of An. arabiensis between the IRS + LLIN arm and the IRS arm (p > 0.05). However, mean HSD, IRD, ORD, and HBR were significantly lower in the IRS + LLIN arm than in the LLIN alone arm (p < 0.05). All An. arabiensis tested for malaria infection were negative for Plasmodium species. For this reason, the entomological inoculation rate could not be determined. CONCLUSIONS: The IRS + LLIN were as effective as IRS alone in reducing densities and HBR of An. arabiensis. However, the effectiveness of the two interventions combined was higher than LLINs alone in reducing densities and HBR of the vector. Added impact of the combination intervention against malaria infectivity rates of An. arabiensis compared to either intervention alone remains unknown and warrants further research. Trial registration PACTR201411000882128. Registered 8 September 2014, https://trialsjournal.biomedcentral.com/articles/10.1186/s13063-016-1154-2.

Species composition, blood meal hosts and Plasmodium infection rates of Anopheles mosquitoes in Ghibe River Basin, southwestern Ethiopia.

BACKGROUND: Vector control interventions using long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) are commonly practiced tools for the control of malaria in Ethiopia. In order to evaluate the effectiveness of these control interventions, and understand the prevailing malaria vectors, their incrimination in disease transmission, and their resting and feeding behavior, we set out to identify the Anopheles species, their blood meal sources, and entomological inoculation rate (EIR) in Ghibe and Darge within the Ghibe River basin, southwestern Ethiopia. METHODS: Adult Anopheles mosquitoes were sampled both indoors and outdoors from January 2015 to October 2016 using Centers for Disease Control and Prevention (CDC) light traps, pyrethrum spray catch (PSC), artificial pit shelters and mouth aspirators. Mosquito species were morphologically identified, and their blood meal sources and malaria sporozoite rates were assessed using enzyme-linked immunosorbent assays. RESULTS: In total, 13 species of Anopheles mosquitoes were identified, among which Anopheles gambiae (s.l.) was the predominant species: 87.9 and 67.7% in Ghibe and Darge, respectively. The mean density of An. gambiae (s.l.) collected per night using CDC light traps was 1.8 and 0.7 outdoors and indoors, respectively, in Ghibe, and 0.125 and 0.07 indoors and outdoors, respectively, in Darge. Anopheles mosquito abundance was higher in houses near the river than in houses far from the river in both study sites. Among Anopheles mosquitoes sampled using CDC light trap catches, 67.6% were unfed and the indoor and outdoor human blood indices of An. gambiae (s.l.) were 58.4 and 15.8%, respectively in Ghibe, while in Darge, they were 57.1 and 50%, respectively. Sporozoite rates were 0.07% for P. vivax and 0.07% for P. falciparum in Ghibe and zero in Darge. In Ghibe, the overall EIRs for P. falciparum and P. vivax were zero and 8.4 infective bites/person/year, respectively, in 2015, while zero and 5.4 infective bites/person/year for P. vivax and P. falciparum, respectively, in 2016. No Plasmodium-positive Anopheles mosquitoes were identified from Darge. CONCLUSIONS: Anopheles gambiae (s.l.), the principal vector of malaria in Ethiopia was the most abundant species both indoors and outdoors, fed both on human and cattle blood and occurred at higher frequencies near rivers. Anopheles gambiae (s.l.) that were circumsporozoite-positive for Plasmodium species were collected from Ghibe, but not Darge.

Long-lasting insecticidal nets and indoor residual spraying may not be sufficient to eliminate malaria in a low malaria incidence area: results from a cluster randomized controlled trial in Ethiopia.

BACKGROUND: Conflicting results exist on the added benefit of combining long-lasting insecticidal nets (LLINs) with indoor residual spraying (IRS) to control malaria infection. The main study objective was to evaluate whether the combined use of LLINs and IRS with propoxur provides additional protection against Plasmodium falciparum and/or Plasmodium vivax among all age groups compared to LLINs or IRS alone. METHODS: This cluster-randomized, controlled trial was conducted in the Rift Valley area of Ethiopia from September 2014 to January 2017 (121 weeks); 44 villages were allocated to each of four study arms: LLIN + IRS, IRS, LLIN, and control. Each week, 6071 households with 34,548 persons were surveyed by active and passive case detection for clinical malaria. Primary endpoints were the incidence of clinical malaria and anaemia prevalence. RESULTS: During the study, 1183 malaria episodes were identified, of which 55.1% were P. falciparum and 25.3% were P. vivax, and 19.6% were mixed infections of P. falciparum and P. vivax. The overall malaria incidence was 16.5 per 1000 person-years of observation time (PYO), and similar in the four arms with 17.2 per 1000 PYO in the LLIN + IRS arm, 16.1 in LLIN, 17.0 in IRS, and 15.6 in the control arm. There was no significant difference in risk of anaemia among the trial arms. CONCLUSIONS: The clinical malaria incidence and anaemia prevalence were similar in the four study groups. In areas with low malaria incidence, using LLINs and IRS in combination or alone may not eliminate malaria. Complementary interventions that reduce residual malaria transmission should be explored in addition to LLINs and IRS to further reduce malaria transmission in such settings. Trial registration PACTR201411000882128 (08 September 2014).

Correction to: Bed nets used to protect against malaria do not last long in a semi-arid area of Ethiopia: a cohort study.

Following publication of the original article [1], the author has flagged errors that affect the scientific content of the article.

Bed nets used to protect against malaria do not last long in a semi-arid area of Ethiopia: a cohort study.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are a key tool for malaria prevention and control. Currently, the recommended serviceable life of an LLIN is 3 years under field conditions. However, field studies show considerable variation in LLIN lifespan, from less than 2 years to more than 4 years. This study aimed to determine the attrition, physical integrity, functional survival, and bio-efficacy of LLINs under field conditions in south-central Ethiopia. METHODS: In October 2014, 7740 LLINs (PermaNet® 2.0) were distributed to 3006 households. Among the distributed LLINs, a cohort study involving 1532 LLINs in 659 households was carried out from October 2014 to November 2016. Data were collected every 6 months by observation, and through interviews with the heads of households. The proportional hole index was used to categorize LLINs as either serviceable or torn. In addition, 120 randomly selected LLINs were tested for bio-efficacy. RESULTS: The overall attrition of LLINs was 96% (n = 993) during the study period. The nets' attrition was mainly due to disposal (64.2%; n = 638). The proportion of LLINs with a hole size 0.5 cm or larger was 79.5% after 24 months. The use of the net on the previous night and having a clean net were associated with a good physical integrity. However, living in a household more than 1 km away from the mosquitoes' breeding site was associated with poor physical integrity. By the 24th month, only 4% of the nets met the criteria for functional survival. The median functional survival time of the nets was 12 months. A longer functional survival was associated with having a clean net, and shorter survival was associated with living in a household more than 1 km away from the mosquitoes' breeding site. The PermaNet® 2.0 met the criteria of effective bio-efficacy up to month 24 after distribution. CONCLUSIONS: The study showed that the median serviceable life of LLINs is only 12 months. However, the bio-efficacy of the LLINs is acceptable for at least 24 months. Therefore, stronger and more efficient LLINs need to be developed for conditions similar to those studied here.