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Background Mosquito-feeding assays that assess transmission of Plasmodium from man-to-mosquito typically use laboratory mosquito colonies. The microbiome and genetic background of local mosquitoes may be different and influence Plasmodium transmission efficiency. In order to interpret transmission studies to the local epidemiology, it is therefore crucial to understand the relationship between infectivity in laboratory-adapted and local mosquitoes. Methods We assessed infectivity of Plasmodium vivax-infected patients from Adama, Ethiopia, using laboratory-adapted (colony) and wild-caught (wild) mosquitoes raised from larval collections in paired feeding experiments. Feeding assays used 4–6 day-old female Anopheles arabiensis mosquitoes after starvation for 12 h (colony) and 18 h (wild). Oocyst development was assessed microscopically 7 days post-feeding. Wild mosquitoes were identified morphologically and confirmed by genotyping. Asexual parasites and gametocytes were quantified in donor blood by microscopy. Results In 36 paired experiments (25 P. vivax infections and 11 co-infections with P. falciparum), feeding efficiency was higher in colony (median: 62.5%; interquartile range, IQR: 47.0–79.0%) compared to wild mosquitoes (median: 27.8%; IQR: 17.0–38.0%; Z = 5.02; P < 0.001). Plasmodium vivax from infectious individuals (51.6%, 16/31) infected a median of 55.0% (IQR: 6.7–85.7%; range: 5.5–96.7%; n = 14) of the colony and 52.7% (IQR: 20.0–80.0%; range: 3.2–95.0%; n = 14) of the wild mosquitoes. A strong association (ρ(16) = 0.819; P < 0.001) was observed between the proportion of infected wild and colony mosquitoes. A positive association was detected between microscopically detected gametocytes and the proportion of infected colony (ρ(31) = 0.452; P = 0.011) and wild (ρ(31) = 0.386; P = 0.032) mosquitoes. Conclusions Infectivity assessments with colony and wild mosquitoes yielded similar infection results. This finding supports the use of colony mosquitoes for assessments of the infectious reservoir for malaria in this setting whilst acknowledging the importance of mosquito factors influencing sporogonic development of Plasmodium parasites.

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.

Plasmodium falciparum and P. vivax co-exist at different endemicity levels across Ethiopia. For over two decades Artemether-Lumefantrine (AL) is the first line treatment for uncomplicated P. falciparum, while chloroquine (CQ) is still used to treat P. vivax. It is currently unclear whether a shift from CQ to AL for P. falciparum treatment has implications for AL efficacy and results in a reversal of mutations in genes associated to CQ resistance, given the high co-endemicity of the two species and the continued availability of CQ for the treatment of P. vivax. This study thus assessed the prevalence of Pfcrt-K76T and Pfmdr1-N86Y point mutations in P. falciparum. 18S RNA gene based nested PCR confirmed P. falciparum samples (N = 183) collected through community and health facility targeted cross-sectional surveys from settings with varying P. vivax and P. falciparum endemicity were used. The proportion of Plasmodium infections that were P. vivax was 62.2% in Adama, 41.4% in Babile, 30.0% in Benishangul-Gumuz to 6.9% in Gambella. The Pfcrt-76T mutant haplotype was observed more from samples with higher endemicity of P. vivax as being 98.4% (61/62), 100% (31/31), 65.2% (15/23) and 41.5% (22/53) in samples from Adama, Babile, Benishangul-Gumuz and Gambella, respectively. However, a relatively higher proportion of Pfmdr1-N86 allele (77.3–100%) were maintained in all sites. The observed high level of the mutant Pfcrt-76T allele in P. vivax co-endemic sites might require that utilization of CQ needs to be re-evaluated in settings co-endemic for the two species. A country-wide assessment is recommended to clarify the implication of the observed level of variation in drug resistance markers on the efficacy of AL-based treatment against uncomplicated P. falciparum malaria., Graphical abstract Image 1

Additional file 3: Table S3. GEE model for association of malaria infection prevalence using all methods combined (nPCR and/or microscopy/RDT) among community survey samples with sample characteristics such as gender, age category, level of endemicity from 2016 to 2020, Ethiopia.

Additional file 2: Table S2. Concordance of RDT and Microscopy detected samples compared to nPCR among the study participants, 2016–2020.

Additional file 1: Table S1. School based prevalence of asymptomatic malaria in selected sites from different transmission settings using nPCR and microscopy/RDT from 2016 to 2018, Ethiopia.

Background As countries move to malaria elimination, detecting and targeting asymptomatic malaria infections might be needed. Here, the epidemiology and detectability of asymptomatic Plasmodium falciparum and Plasmodium vivax infections were investigated in different transmission settings in Ethiopia. Method: A total of 1093 dried blood spot (DBS) samples were collected from afebrile and apparently healthy individuals across ten study sites in Ethiopia from 2016 to 2020. Of these, 862 were from community and 231 from school based cross-sectional surveys. Malaria infection status was determined by microscopy or rapid diagnostics tests (RDT) and 18S rRNA-based nested PCR (nPCR). The annual parasite index (API) was used to classify endemicity as low (API > 0 and Results In community surveys, the overall prevalence of asymptomatic Plasmodium infections by microscopy/RDT, nPCR and all methods combined was 12.2% (105/860), 21.6% (183/846) and 24.1% (208/862), respectively. The proportion of nPCR positive infections that was detectable by microscopy/RDT was 48.7% (73/150) for P. falciparum and 4.6% (2/44) for P. vivax. Compared to low transmission settings, the likelihood of detecting infections by microscopy/RDT was increased in moderate (Adjusted odds ratio [AOR]: 3.4; 95% confidence interval [95% CI] 1.6–7.2, P = 0.002) and high endemic settings (AOR = 5.1; 95% CI 2.6–9.9, P Conclusions Conventional diagnostics missed nearly half of the asymptomatic Plasmodium reservoir detected by nPCR. The detectability of infections was particularly low in older age groups and low transmission settings. These findings highlight the need for sensitive diagnostic tools to detect the entire parasite reservoir and potential infection transmitters.

Background: As countries move to malaria elimination, detecting and targeting asymptomatic malaria infections might be needed. Here, we investigated their spatial distribution and detectability in P. falciparum and P. vivax co-endemic settings in Ethiopia.Method: A total of 1093 dried blood spot (DBS) samples were collected from afebrile and apparently healthy individuals across ten study sites in Ethiopia from 2016 to 2020. Of these, 862 were from community and 231 from school based cross-sectional surveys. Malaria infection status was determined by microscopy, rapid diagnostics tests (RDT) and 18S rRNA nested PCR (nPCR). The annual parasite index (API) was used to classify endemicity as low (API>0 andResults: In community surveys, the overall prevalence of asymptomatic malaria infections by microscopy/RDT, nPCR and all methods combined was 12.2% (105/860), 21.6% (183/846) and 24.1% (208/862), respectively. The proportion of nPCR positive infections that was detectable by microscopy/RDT was 48.7(73/150) for P. falciparum and 4.6(2/44) for P. vivax. Compared to low transmission settings, the likelihood of detecting infections by microscopy/RDT was increased in moderate (AOR: 3.4; 95%CI:1.6-7.2, P=0.002) and high endemic settings (AOR=5.1; 95%CI=2.6-9.9, PConclusion: Conventional diagnostics missed nearly half of the asymptomatic malaria reservoir detected by nPCR. The detectability of infections was particularly low in older age groups and low endemic settings. These findings highlight the need for sensitive diagnostic tools to detect the entire parasite reservoir and potential infection transmitters.

Anopheles stephensi, an efficient Asian malaria vector, recently spread into the Horn of Africa and may increase malaria receptivity in African urban areas. We assessed occurrence, genetic complexity, blood meal source and infection status of An. stephensi in Awash Sebat Kilo town, Ethiopia. We used membrane feeding assays to assess competence of local An. stephensi to P. vivax and P. falciparum isolates from clinical patients. 75.3% of the examined waterbodies were infested with An. stephensi developmental stages that were genetically closely related to isolates from Djibouti and Pakistan. Both P. vivax and P. falciparum were detected in wild-caught adult An. stephensi. Local An. stephensi was more receptive to P. vivax compared to a colony of An. arabiensis. We conclude that An. stephensi is an established vector in this part of Ethiopia, highly permissive for local P. vivax and P. falciparum isolates and presents an important new challenge for malaria control.Summary of the articleAn. stephensi, a metropolitan malaria vector that recently expanded to the Horn of African, was highly susceptible to local P. falciparum and P. vivax isolates from Ethiopia and may increase malariogenic potential of rapidly expanding urban settings in Africa.

House is the major site for malaria infection where most human-vector contact takes place. Hence, improving housing might reduce the risk of malaria infection by limiting house entry of vectors. This study aimed to explore the impact of screening doors and windows with wire meshes on density and entomological inoculation rate (EIR) of malaria vector, and malaria incidence, and assess the acceptability, durability, and cost of the intervention. The susceptibility status of malaria vector was also assessed. A two-arm randomized trial was done in Arba Minch Town, southwest Ethiopia. 92 houses were randomly included in the trial. The baseline entomological and malaria prevalence data were collected. The mosquito sampling was done twice per household per month by Centers for Diseases Control and Prevention (CDC) light traps for six months. The baseline prevalence of malaria was assessed by testing 396 (83% of the 447 study participants) household members in all the eligible houses. The 92 houses were then randomized into control and intervention groups using mosquito and malaria prevalence baseline data to make the two groups comparable except the intervention. Then, we put wire-mesh on doors and windows of 46 houses. Post-screening mosquito collection was done in each household twice per month for three months. Each household member was visited twice per month for six months to assess malaria episodes. The frequency of damage to different structure of screening was measured twice. In-depth interview was conducted with 24 purposely selected household heads from intervention group. Speciation of Anopheles mosquito was done by morphological key, and the circum-sporozoite proteins (CSPs) analysis was done using enzyme-linked immunosorbent assay. A generalized estimating equation with a negative binomial distribution was used to assess the impact of the intervention on the indoor density of vectors. Clinical malaria case data were analyzed using Poisson regression with generalized linear model. Screening doors and windows reduced the indoor density of An. arabiensis by 48% (mean ratio of intervention to control = 0.85/1.65; 0.52) (P = .001). Plasmodium falciparum CSP rate was 1.6% (3/190) in the intervention houses, while it was 2.7% (10/372) in the control houses. The protective efficacy of screening intervention from CSP positive An. arabiensis was 41% (mean ratio of intervention to control = 1.6/2.7; 0.59), but was not statistically significant (P = .6). The EIR of An. arabiensis was 1.91 in the intervention group, whereas it was 6.45 in the control group. 477 participants were followed for clinical malaria (50.1% from intervention and 49.9% from the control group). Of 49 RDT positive cases, 45 were confirmed to be positive with microscopy. 80% (n = 36) cases were due to P. falciparum and the rest 20% (n = 9) were due to P. vivax . The incidence of P. falciparum in the intervention group was lower (IRR: 0.39, 95% CI: 0.2–0.80; P = .01) than in the control group. Using incidence of P. falciparum infection, the protective efficacy of intervention was 61% (95% CI: 18–83; P = .007). 97.9% of screened windows and 63.8% of screened doors were intact after eleven months of installation. Malaria mosquito was resistance (mortality rate of 75%) to the insecticide used for bed nets treatment. Almost all participants of intervention arm were willing to continue using screened doors and windows. Screening doors and windows reduced the indoor exposure to malaria vectors. The intervention is effective, durable and well-accepted. Hence, the existing interventions can be supplemented with house screening intervention for further reduction and ultimately elimination of malaria by reducing insecticide pressure on malaria vectors. However, further research could be considered in broad setting on different housing improvement and in the way how to scale-up for wider community.

Background Following successful malaria control during the last decade, Ethiopia instituted a stepwise malaria elimination strategy in selected low-transmission areas. Methods Cross-sectional surveys were conducted in Babile district, Oromia, Ethiopia from July to November 2017 to evaluate malaria infection status using microscopy and nested polymerase chain reaction (nPCR) and serological markers of exposure targeting Plasmodium falciparum and Plasmodium vivax apical membrane antigen-1 (AMA-1). Results Parasite prevalence was 1.2% (14/1135) and 5.1% (58/1143) for P. falciparum and 0.4% (5/1135) and 3.6% (41/1143) for P. vivax by microscopy and nPCR, respectively. Antibody prevalence was associated with current infection by nPCR for both P. falciparum (p

There is a reduction in the burden of malaria globally due to extensive application of bed nets, indoor residual spraying, effective drugs and diagnostic tools. To sustain the gain and accelerate the current reduction, there is a need for locally adapted effective, sustainable and community accepted supplementary intervention. Growing evidence suggests that improving housing reduce indoor density of vectors and incidence of malaria. This study assessed the effect of house screening intervention on indoor malaria transmission, and its durability, and community acceptance. A randomized control trial with two arms (houses with screened doors and windows and without these interventions) was conducted in Arba Minch Town, southwestern Ethiopia. Both epidemiological and entomological data were collected to evaluate the impact of screening doors and windows on incidence of malaria and indoor density of malaria vectors. The frequency of damage to different structure of screening was measured in two round examinations with pre-prepared checklist. In-depth interviews with semi-structured questioner in Amharic language were conducted with purposely selected household heads from intervention group. A total of 477 participants, 50.1% (n = 239) inhabitants of screened houses and 49.9% (n= 238) inhabitants of unscreened houses, were followed for six months. Of 45 microscopically confirmed clinical malaria cases, 80 % (n = 36) were Plasmodium falciparum whereas the rest 20 % (n = 9) were Plasmodium vivax. The incidence of P. falciparum malaria was low [Incidence Rate Ratio (IRR) 0.39, 95% Confidence Interval (CI): 0.2 - 0.80, p = 0.01] among inhabitants of houses with screened doors and windows compared to those living in unscreened houses. The Protective Efficacy (PE) of screening doors and windows from clinical P. falciparum malaria was 61 % (95% CI: 18 - 83, p = 0.007). The mean indoor density of anopheline was 0.79 (95% Walad CI: 0.49, 1.25, p = 0.02) per CDC light trap per night, while it was 0 .35 (95% Walad CI: 0.22, 0.57) in unscreened houses. The screened houses had 56% fewer anopheline and 54 % fewer culicine compared to control houses. Among screened houses 97.9 % of screened windows and 63.8 % of screened doors were intact at eleven months of installation. Almost all participants of in-depth interviews were willing to continue using the screening of doors and windows. Screening of doors and windows are effective, durable and well-accepted means to control indoor malaria transmission. Hence, currently available malaria control interventions can beXII supplemented with screening doors and windows with wire-mesh for further reducing and ultimate eliminating of malaria.