Spatiotemporal distribution and bionomics of Anopheles stephensi in different eco-epidemiological settings in Ethiopia.

BACKGROUND: Malaria is a major public health concern in Ethiopia, and its incidence could worsen with the spread of the invasive mosquito species Anopheles stephensi in the country. This study aimed to provide updates on the distribution of An. stephensi and likely household exposure in Ethiopia. METHODS: Entomological surveillance was performed in 26 urban settings in Ethiopia from 2021 to 2023. A kilometer-by-kilometer quadrant was established per town, and approximately 20 structures per quadrant were surveyed every 3 months. Additional extensive sampling was conducted in 50 randomly selected structures in four urban centers in 2022 and 2023 to assess households' exposure to An. stephensi. Prokopack aspirators and CDC light traps were used to collect adult mosquitoes, and standard dippers were used to collect immature stages. The collected mosquitoes were identified to species level by morphological keys and molecular methods. PCR assays were used to assess Plasmodium infection and mosquito blood meal source. RESULTS: Catches of adult An. stephensi were generally low (mean: 0.15 per trap), with eight positive sites among the 26 surveyed. This mosquito species was reported for the first time in Assosa, western Ethiopia. Anopheles stephensi was the predominant species in four of the eight positive sites, accounting for 75-100% relative abundance of the adult Anopheles catches. Household-level exposure, defined as the percentage of households with a peridomestic presence of An. stephensi, ranged from 18% in Metehara to 30% in Danan. Anopheles arabiensis was the predominant species in 20 of the 26 sites, accounting for 42.9-100% of the Anopheles catches. Bovine blood index, ovine blood index and human blood index values were 69.2%, 32.3% and 24.6%, respectively, for An. stephensi, and 65.4%, 46.7% and 35.8%, respectively, for An. arabiensis. None of the 197 An. stephensi mosquitoes assayed tested positive for Plasmodium sporozoite, while of the 1434 An. arabiensis mosquitoes assayed, 62 were positive for Plasmodium (10 for P. falciparum and 52 for P. vivax). CONCLUSIONS: This study shows that the geographical range of An. stephensi has expanded to western Ethiopia. Strongly zoophagic behavior coupled with low adult catches might explain the absence of Plasmodium infection. The level of household exposure to An. stephensi in this study varied across positive sites. Further research is needed to better understand the bionomics and contribution of An. stephensi to malaria transmission.

Dynamics of asymptomatic Plasmodium falciparum and Plasmodium vivax infections and their infectiousness to mosquitoes in a low transmission setting of Ethiopia: a longitudinal observational study.

OBJECTIVE: A 15-month longitudinal study was conducted to determine the duration and infectivity of asymptomatic qPCR-detected Plasmodium falciparum and Plasmodium vivax infections in Ethiopia. METHOD: Total parasite and gametocyte kinetics were determined by molecular methods; infectivity to Anopheles arabiensis mosquitoes by repeated membrane feeding assays. Infectivity results were contrasted with passively recruited symptomatic malaria cases. RESULTS: For P. falciparum and P. vivax infections detected at enrolment, median durations of infection were 37 days (95% confidence interval [CI], 15-93) and 60 days (95% CI, 18-213), respectively. P. falciparum and P. vivax parasite densities declined over the course of infections. From 47 feeding assays on 22 asymptomatic P. falciparum infections, 6.4% (3/47) were infectious and these infected 1.8% (29/1579) of mosquitoes. No transmission was observed in feeding assays on asymptomatic P. vivax mono-infections (0/56); one mixed-species infection was highly infectious. Among the symptomatic cases, 4.3% (2/47) of P. falciparum and 73.3% (53/86) of P. vivax patients were infectious to mosquitoes. CONCLUSION: The majority of asymptomatic infections were of short duration and low parasite density. Only a minority of asymptomatic individuals were infectious to mosquitoes. This contrasts with earlier findings and is plausibly due to the low parasite densities in this population.

Naturally acquired antibodies to gametocyte antigens are associated with reduced transmission of Plasmodium vivax gametocytes to Anopheles arabiensis mosquitoes.

Naturally acquired antibodies may reduce the transmission of Plasmodium gametocytes to mosquitoes. Here, we investigated associations between antibody prevalence and P. vivax infectivity to mosquitoes. A total of 368 microscopy confirmed P. vivax symptomatic patients were passively recruited from health centers in Ethiopia and supplemented with 56 observations from asymptomatic P. vivax parasite carriers. Direct membrane feeding assays (DMFA) were performed to assess mosquito infectivity; for selected feeds these experiments were also performed after replacing autologous plasma with malaria naïve control serum (n=61). The prevalence of antibodies against 6 sexual stage antigens (Pvs47, Pvs48/45, Pvs230, PvsHAP2, Pvs25 and PvCelTOS) and an array of asexual antigens was determined by ELISA and multiplexed bead-based assays. Gametocyte (ρ< 0.42; p = 0.0001) and parasite (ρ = 0.21; p = 0.0001) densities were positively associated with mosquito infection rates. Antibodies against Pvs47, Pvs230 and Pvs25 were associated with 23 and 34% reductions in mosquito infection rates (p<0.0001), respectively. Individuals who showed evidence of transmission blockade in serum-replacement DMFAs (n=8) were significantly more likely to have PvsHAP2 or Pvs47 antibodies. Further studies may demonstrate causality for the observed associations, improve our understanding of the natural transmission of P. vivax and support vaccine development.

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.

The epidemiology and detectability of asymptomatic plasmodium vivax and plasmodium falciparum infections in low, moderate and high transmission settings in 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 < 5), moderate (API ≥ 5 and < 100) and high transmission (API ≥ 100) and detectability of infections was assessed in these settings. 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 < 0.001). After adjustment for site and correlation between observations from the same survey, the likelihood of detecting asymptomatic infections by microscopy/RDT (AOR per year increase = 0.95, 95% CI 0.9-1.0, P = 0.013) declined with age. 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.

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.

Comparison of infectivity of Plasmodium vivax to wild-caught and laboratory-adapted (colonized) Anopheles arabiensis mosquitoes in Ethiopia.

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.

Prevalence of Plasmodium falciparum Pfcrt and Pfmdr1 alleles in settings with different levels of Plasmodium vivax co-endemicity in Ethiopia.

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.

Serological evidence for a decline in malaria transmission following major scale-up of control efforts in a setting selected for Plasmodium vivax and Plasmodium falciparum malaria elimination in Babile district, Oromia, Ethiopia.

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<0.001) and P. vivax (p=0.014) and showed an age-dependent increase (p<0.001, for both species). Seroconversion curves indicated a decline in malaria exposure 15 y prior to sampling for P. falciparum and 11.5 y prior to sampling for P. vivax, broadly following malaria incidence data from district health offices, with higher antibody titres in adults than children for both species. CONCLUSIONS: Malaria transmission declined substantially in the region with continuing heterogeneous but measurable local transmission, arguing in favour of continued and tailored control efforts to accelerate the progress towards elimination efforts.

Exploring the impact of house screening intervention on entomological indices and incidence of malaria in Arba Minch town, southwest Ethiopia: A randomized control trial.

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.