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.

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.

Bionomic characterization of Anopheles mosquitoes in the Ethiopian highlands and lowlands.

BACKGROUND: The protective effectiveness of vector control in malaria relies on how the implemented tools overlap with mosquito species-specific compositions and bionomic traits. In Ethiopia, targeted entomological data enabling strategic decision-making are lacking around high-risk migrant worker camps in the lowlands and resident communities in the highlands-resulting in suboptimal malaria control strategies for both populations. This study investigates spatial and temporal mosquito behavior, generating baseline evidence that will improve malaria control for both migrant workers in the lowlands and their home communities in the highlands. METHODS: Hourly Centers for Disease Control and Prevention (CDC) light trap collections were performed indoors and outdoors during the peak (October to December 2022) and minor (March to May 2023) malaria transmission seasons. These seasons coincide with the post-long rain and post-short rain seasons, respectively. Eight resident households were sampled from each of four villages in the highlands and eight households/farm structures on and near farms in four villages in the lowlands. The sampling occurred between 18:00 and 06:00. Spatiotemporal vector behaviors and hourly indoor and outdoor mosquito capture rates, used as a proxy for human biting rates, were calculated for overall catches and for individual species. Adult mosquitoes were identified using morphological keys, and a subset of samples were confirmed to species by sequencing ribosomal DNA internal transcribed spacer region 2 (ITS2) and/or mitochondrial DNA cytochrome c oxidase subunit 1 (Cox1). RESULTS: In the highlands, 4697 Anopheles mosquitoes belonging to 13 morphologically identified species were collected. The predominant species of Anopheles identified in the highlands was An. gambiae sensu lato (s.l.) (n = 1970, 41.9%), followed by An. demeilloni (n = 1133, 24.1%) and An. cinereus (n = 520, 11.0%). In the lowland villages, 3220 mosquitoes belonging to 18 morphological species were collected. Anopheles gambiae s.l. (n = 1190, 36.9%), An. pretoriensis (n = 899, 27.9%), and An. demeilloni (n = 564, 17.5%) were the predominant species. A total of 20 species were identified molecularly, of which three could not be identified to species through comparison with published sequences. In highland villages, the indoor Anopheles mosquito capture rate was much greater than the outdoor rate. This trend reversed in the lowlands, where the rate of outdoor captures was greater than the indoor rate. In both highlands and lowlands, Anopheles mosquitoes showed early biting activities in the evening, which peaked between 18:00 and 21:00, for both indoor and outdoor locations. CONCLUSIONS: The high diversity of Anopheles vectors and their variable behaviors result in a dynamic and resilient transmission system impacting both exposure to infectious bites and intervention effectiveness. This creates gaps in protection allowing malaria transmission to persist. To achieve optimal control, one-size-fits-all strategies must be abandoned, and interventions should be tailored to the diverse spatiotemporal behaviors of different mosquito populations.

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.

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.

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 evaluation of repellency and feeding inhibition of ethno-medicinal plants against major malaria vectors in southern Ethiopia.

BACKGROUND: Plant-based mosquito control methods may use as a supplementary malaria vector control strategy. This study aimed to evaluate the effect of smoking ethno-medicinal plants on indoor density and feeding activity of malaria vectors at early hours of the night and its residual effect after midnight in southern Ethiopia. METHODS: Both field and tent trials were conducted to evaluate the impact of smoking Juniperus procera leaves, Eucalyptus globulus seeds and Olea europaea leaves in Kolla Shara Village from July 2016 to February 2017. For the field trial, five grass-thatched traditional huts (three for ethno-medicinal plants and two as control [only charcoal smoking and non-charcoal smoking]) were used. Indoor host-seeking mosquitoes were collected by CDC light traps. A Latin square design was employed to minimize the bias due to the variation in house location and different sampling nights. For the tent experiment, 25 3-5-day-old starved wild female Anopheles mosquitoes reared from the larvae were released into the tents where a calf was tethered at the mid-point of each tent. RESULTS: A total of 614 Anopheles mosquitoes belonging to 5 species were collected from 5 huts, of which 93.4% was An. arabiensis; O. europaea, E. globulus and J. procera reduced the indoor density of An. arabiensis, with the mean percentage drop of 80%, 73% and 70%, respectively. In the tent trial, smoking of these plants had significant knockdown effects and inhibited feeding on the calves (F = 383.5, DF = 3, P < 0.01). The mean knockdown effect due to O. europaea was relatively high (17.7 ± 0.54; 95% CI 16.8-18.6), while it was only 0.9 ± 0.1 (95% CI 0.29-1.52) in the control tents. All the test plants used in the tent trial caused significantly inhibited feeding activity of An. arabiensis on the host (F = 383.5, DF = 3, P < 0.01). About 94.5%, 89.5% and 86% of mosquitoes were unfed because of the smoking effect of O. europaea, E. globulus and J. procera, respectively, whereas only 19.5% were unfed in the control tent. CONCLUSIONS: Smoking ethno-medicinal plant materials reduced indoor density of malaria vectors and inhibited feeding on calves inside the tents. Thus, plant-based mosquito control methods may play a vital role in reducing mosquito bites in the early hours of the night and thereby reduce residual malaria transmission.

A comparison of PCR and ELISA methods to detect different stages of Plasmodium vivax in Anopheles arabiensis.

BACKGROUND: In characterizing malaria epidemiology, measuring mosquito infectiousness informs the entomological inoculation rate, an important metric of malaria transmission. PCR-based methods have been touted as more sensitive than the current "gold-standard" circumsporozoite (CSP) ELISA. Wider application of PCR-based methods has been limited by lack of specificity for the infectious sporozoite stage. We compared a PCR method for detecting the parasite's mitochondrial (mt) cytochrome oxidase I (COX-I) gene with ELISA for detecting circumsporozoite protein for identification of different life stages of the parasite during development within a mosquito. METHODS: A PCR-based method targeting the Plasmodium mt COX-I gene was compared with the CSP ELISA method to assess infectivity in Anopheles arabiensis colony mosquitoes fed on blood from patients infected with Plasmodium vivax. Mosquitoes were tested at six post-infection time points (days 0.5, 1, 6, 9, 12, 15). The head and thorax and the abdomen for each specimen were tested separately with each method. Agreement between methods at each infection stage was measured using Cohen's kappa measure of test association. RESULTS: Infection status of mosquitoes was assessed in approximately 90 head/thorax and 90 abdomen segments at each time point; in total, 538 head/thorax and 534 abdomen segments were tested. In mosquitoes bisected after 0.5, 1, and 6 days post-infection (dpi), the mt COX-I PCR detected Plasmodium DNA in both the abdomen (88, 78, and 67%, respectively) and head/thorax segments (69, 60, and 44%, respectively), whilst CSP ELISA detected sporozoites in only one abdomen on day 6 post-infection. PCR was also more sensitive than ELISA for detection of Plasmodium in mosquitoes bisected after 9, 12, and 15 dpi in both the head and thorax and abdomen. There was fair agreement between methods for time points 9-15 dpi (κ = 0.312, 95% CI: 0.230-0.394). CONCLUSIONS: The mt COX-I PCR is a highly sensitive, robust method for detecting Plasmodium DNA in mosquitoes, but its limited Plasmodium life-stage specificity cannot be overcome by bisection of the head and thorax from the abdomen prior to PCR. Thus, the mt COX-I PCR is a poor candidate for identifying infectious mosquitoes.

Malaria infection clustered into small residential areas in lowlands of southern Ethiopia.

Malaria is a complex disease and its distribution is not random in endemic areas, and hence areas with low malaria transmission require fine spatial sampling and careful follow-up to identify the hot spots for effective resource utilization to control malaria. The present study is aimed to assess malaria infection in both humans and mosquitoes in a small residential lowland area of southern Ethiopia from July to December 2016. A repeated cross-sectional household survey was conducted in Kolla-Shara Kebele (village) to describe the distribution of malaria and infectious mosquitoes. For the parasitological surveys, a total of 90 households were randomly selected from five sub-villages in equal proportion. About a quarter of the total households included for the surveys were randomly selected for entomological surveys. A P-value of <0.05 was used as a cut-off point for statistical significance. More than a third (35.1%, 46 of 131) febrile cases were microscopically confirmed malaria positive. Above half (58.7%, 27 of 46) of those positive cases were due to P. falciparum and the rest (41.3%, 19 of 46) were due to P. vivax. This study identified two of the five sub-villages as independent clusters with higher risk of malaria infection. Four times higher relative risk (RR) of malaria infection was documented in Abullo sub-village compared to the others (RR = 3.87; P = 0.002). Most of the falciparum malaria cases were aggregated in these sub-villages. About six infectious bites of An. arabiensis per person was recorded during the survey. The infectious bite per person was 17.0 in Abullo and 10.6 in Erze clusters where higher human infections were detected. It is clearly indicated that a smaller portion of the population carry higher malaria cases and infectious bites. Malaria interventions targeting such areas could be effective in the context of malaria elimination strategy in Ethiopia, which consider district as a planning and implementing unit. Future research would preferably be designed to perform long duration of follow-up to identify the appropriate period for interventions and more participants with more heterogeneous villages and districts.

Malaria epidemiology and stratification of incidence in the malaria elimination setting in Harari Region, Eastern Ethiopia.

BACKGROUND: Ethiopia has shown notable progress in reducing the burden of malaria over the past two decades. Because of this progress, the country has shifted efforts from control to elimination of malaria. This study was conducted to analyse the malaria epidemiology and stratification of incidence in the malaria elimination setting in eastern Ethiopia. METHODS: A retrospective study was conducted to analyse the epidemiology of malaria by reviewing the district health office data from 2013 to 2019 in Harari Region. In addition, three years of sub-district level malaria data were used to stratify the malaria transmission intensity. Malaria interventions (Long-lasting insecticidal nets [LLIN] and indoor residual spraying [IRS]) employed were reviewed to analyse the intervention coverage at the Regional level. Descriptive statistics were used to show the malaria transmission in terms of years, season and species of the malaria parasite. Incidence rate per 1000 population and death rate per 1 000 000 population at risk were computed using the total population of each year. RESULTS: In the Harari Region, malaria incidence showed a more pronounced declining trend from 2017 to 2019. Plasmodium falciparum, P. vivax and mixed infections accounted for 69.2%, 30.6% and 0.2% of the cases, respectively. There was an increment in malaria intervention coverage and improved malaria diagnosis. In the year 2019 the coverage of LLIN and IRS in the Region were 93.4% and 85.1% respectively. The annual malaria incidence rate dropped from 42.9 cases per 1000 population in 2013 to 6.7 cases per 1000 population in 2019. Malaria-related deaths decreased from 4.7 deaths per 1 000 000 people annually in 2013 to zero, and there have been no deaths reported since 2015. The malaria risk appears to be heterogeneous and varies between districts. A higher number of malaria cases were recorded in Erer and Jenella districts, which constitute 62% of the cases in the Region. According to the sub-district level malaria stratification, there was shrinkage in the malaria transmission map and about 70% of the sub-districts have achieved elimination targets. CONCLUSIONS: In the Harari Region, malaria morbidity and mortality have been significantly declined. Thus, if this achievement is sustained and scaling-up of the existing malaria prevention and control strategies by focusing on those populations living in the higher malaria transmission districts and sub-districts, planning of malaria elimination from the study area might be feasible.

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.