Evidence for a role of Anopheles stephensi in the spread of drug- and diagnosis-resistant malaria in Africa.

Anopheles stephensi, an Asian malaria vector, continues to expand across Africa. The vector is now firmly established in urban settings in the Horn of Africa. Its presence in areas where malaria resurged suggested a possible role in causing malaria outbreaks. Here, using a prospective case-control design, we investigated the role of An. stephensi in transmission following a malaria outbreak in Dire Dawa, Ethiopia in April-July 2022. Screening contacts of patients with malaria and febrile controls revealed spatial clustering of Plasmodium falciparum infections around patients with malaria in strong association with the presence of An. stephensi in the household vicinity. Plasmodium sporozoites were detected in these mosquitoes. This outbreak involved clonal propagation of parasites with molecular signatures of artemisinin and diagnostic resistance. To our knowledge, this study provides the strongest evidence so far for a role of An. stephensi in driving an urban malaria outbreak in Africa, highlighting the major public health threat posed by this fast-spreading mosquito.

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 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.

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.

Evaluation of Endod (Phytolacca dodecandra: Phytolaccaceae) as a Larvicide Against Anopheles arabiensis, the Principal Vector of Malaria in Ethiopia.

Malaria control methods rely mostly on adult mosquito control using insecticide-treated nets and indoor residual spraying with insecticides. Plants such as endod (Phytolacca dodecandra) can potentially be used for the control of mosquito larvae as a supplement to adult control methods. Following the discovery of endod, a molluscicide plant, more than 5 decades ago in Ethiopia, subsequent studies have shown that its potency can further be increased by simple procedures such as aging endod berry powder in water. This study was conducted to evaluate the killing effect of fresh and aged endod solution against 4th-stage larvae of Anopheles arabiensis. Laboratory-reared An. arabiensis larvae exposed to different concentrations of endod preparation using distilled or spring water had 50% lethal concentration (LC(50))  =  49.6 ppm and 90% lethal concentration (LC(90))  =  234 ppm for fresh and LC(50)  =  36.4 ppm and LC(90)  =  115.7 ppm for the aged endod solution in distilled water against the laboratory population. Against field-collected larvae of the same species, aged preparations in habitat water resulted in higher LC(50) (472.7 ppm) and LC(90) (691 ppm) values, with only a slight improvement over fresh preparations in habitat water (LC(50)  =  456.2 ppm; LC(90)  =  896.1 ppm). In general, although aged preparations of endod required lower concentrations than fresh to kill at least 90% of the larvae, these concentrations were much higher (12-70×) than that required for schistosome-transmitting snails.

Breeding Sites of Aedes aegypti: Potential Dengue Vectors in Dire Dawa, East Ethiopia.

Background and Objectives. Entomological survey was carried out from May-June to September-October 2014 to investigate the presence of dengue vectors in discarded tires and artificial water containers in houses and peridomestic areas. Methods. A cross-sectional immature stage survey was done indoors and outdoors in 301 houses. Mosquito larval sampling was conducted using pipette or dipper depending on container types. Larvae were identified morphologically and larval indices were also calculated. Results. A total of 750 containers were inspected, and of these 405 were positive for mosquito larvae. A total of 1,873 larvae were collected and morphologically identified as Aedes aegypti (n = 1580: 84.4%) and Culex (n = 293: 15.6%). The larval indices, house index, container index, and breteau index, varied from 33.3 to 86.2, from 23.2 to 73.9, and from 56.5 to 188.9, respectively. Conclusion. Aedes aegypti is breeding in a wide range of artificial containers. To control these mosquitoes, the integration of different methods should be taken into consideration.