Seasonal dynamics of Anopheles stephensi and its implications for mosquito detection and emergent malaria control in the Horn of Africa.

Invasion of the malaria vector Anopheles stephensi across the Horn of Africa threatens control efforts across the continent, particularly in urban settings where the vector is able to proliferate. Malaria transmission is primarily determined by the abundance of dominant vectors, which often varies seasonally with rainfall. However, it remains unclear how An. stephensi abundance changes throughout the year, despite this being a crucial input to surveillance and control activities. We collate longitudinal catch data from across its endemic range to better understand the vector's seasonal dynamics and explore the implications of this seasonality for malaria surveillance and control across the Horn of Africa. Our analyses reveal pronounced variation in seasonal dynamics, the timing and nature of which are poorly predicted by rainfall patterns. Instead, they are associated with temperature and patterns of land use; frequently differing between rural and urban settings. Our results show that timing entomological surveys to coincide with rainy periods is unlikely to improve the likelihood of detecting An. stephensi. Integrating these results into a malaria transmission model, we show that timing indoor residual spraying campaigns to coincide with peak rainfall offers little improvement in reducing disease burden compared to starting in a random month. Our results suggest that unlike other malaria vectors in Africa, rainfall may be a poor guide to predicting the timing of peaks in An. stephensi-driven malaria transmission. This highlights the urgent need for longitudinal entomological monitoring of the vector in its new environments given recent invasion and potential spread across the continent.

Molecular Confirmation of Anopheles stephensi Mosquitoes in the Al Hudaydah Governorate, Yemen, 2021 and 2022.

We detected malaria vector Anopheles stephensi mosquitoes in the Al Hudaydah governorate in Yemen by using DNA sequencing. We report 2 cytochrome c oxidase subunit I haplotypes, 1 previously found in Ethiopia, Somalia, Djibouti, and Yemen. These findings provide insight into invasive An. stephensi mosquitoes in Yemen and their connection to East Africa.

Detection of Invasive Anopheles stephensi Mosquitoes through Molecular Surveillance, Ghana.

The invasive Anopheles stephensi mosquito has rapidly expanded in range in Africa over the past decade. Consistent with World Health Organization guidelines, routine entomologic surveillance of malaria vectors in Accra, Ghana, now includes morphologic and molecular surveillance of An. stephensi mosquitoes. We report detection of An. stephensi mosquitoes in Ghana.

Loop-Mediated Isothermal Amplification Assay to Detect Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Spread of the Anopheles stephensi mosquito, an invasive malaria vector, threatens to put an additional 126 million persons per year in Africa at risk for malaria. To accelerate the early detection and rapid response to this mosquito species, confirming its presence and geographic extent is critical. However, existing molecular species assays require specialized laboratory equipment, interpretation, and sequencing confirmation. We developed and optimized a colorimetric rapid loop-mediated isothermal amplification assay for molecular An. stephensi species identification. The assay requires only a heat source and reagents and can be used with or without DNA extraction, resulting in positive color change in 30-35 minutes. We validated the assay against existing PCR techniques and found 100% specificity and analytical sensitivity down to 0.0003 nanograms of genomic DNA. The assay can successfully amplify single mosquito legs. Initial testing on samples from Marsabit, Kenya, illustrate its potential as an early vector detection and malaria mitigation tool.

Leech, potato, and tomato carboxypeptidase inhibitors against Anopheles stephensi carboxypeptidase B1 and B2.

Carboxypeptidase B (CPB) in Anopheles spp. breaks down blood and releases free amino acids, which promote Plasmodium sexual development in the mosquito midgut. Our goal was to computationally assess the inhibitory effectiveness of carboxypeptidase inhibitors obtained from tomato, potato (CPiSt), and leech against the Anopheles stephensi CPBAs1 and CPBAs2 enzymes. The tertiary structures of CPB inhibitors were predicted and their interaction mode with CPBAs1 and CPBAs2 were examined using molecular docking. Next, this data was compared with four licensed medications that are known to reduce the Anopheles' CPB activity. Molecular dynamics simulations were used to evaluate the stability of complexes containing CPiSt and its mutant form. Both CPiSt and its mutant form showed promise as possible candidates for further evaluations in the paratransgenesis technique for malaria control, based on the similar bindings of CPiSt and CPiSt-Mut to the active sites of CPBAs1 and CPBAs2, as well as their binding affinity in comparison to the drugs.

Long-lasting residual efficacy of Actellic®300CS and Icon®10CS on different surfaces against Anopheles stephensi, an invasive malaria vector.

BACKGROUND: Anopheles stephensi, a malaria-transmitting mosquito species, has developed resistance to various insecticides such as DDT, Dieldrin, Malathion, and synthetic pyrethroids. To combat this issue, the World Health Organization (WHO) suggests using Actellic®300CS and Icon®10CS for Indoor Residual Spraying to tackle pyrethroid-resistant mosquitoes. The aim of this research project was to evaluate the susceptibility of An. stephensi to certain insecticides at the diagnostic concentration + intensity 5x diagnostic concentration (5XDC) assays in Iran and to study the lasting effectiveness of Actellic®300CS and Icon®10CS against this particular malaria vector. METHODS: This study assessed the susceptibility of An. stephensi populations in southern Iran to various insecticides, including deltamethrin 0.05%, DDT 4%, malathion 5%, bendiocarb 0.1%, a synergist assay with PBO 4% combined with deltamethrin 0.05%, and an intensity assay using 5x the diagnostic concentration of deltamethrin (0.25%) and bendiocarb 0.5%. Laboratory cone bioassay tests were conducted to determine the residual effectiveness of Actellic®300 and Icon®10CS insecticides on different surfaces commonly found in households, such as cement, mud, plaster, and wood. The tests were carried out following the WHO test kits and standard testing protocols. RESULTS: The An. stephensi populations in Bandar Abbas were found to be susceptible to malathion 5% and deltamethrin 0.25% (5XDC), but exhibited resistance to DDT, standard concentration of deltamethrin, and both standard and intensity concentrations of bendiocarb. In laboratory cone bioassay tests, An. stephensi mortality rates when exposed to Actellic®300CS and Icon®10CS on different surfaces remained consistently more than 80%. Actellic®300CS achieved more than 80% mortality on all substrates for the entire 300-day post-spraying period. Conversely, Icon®10CS maintained mortality rates more than 80% on plaster and wood surfaces for 165 days and on mud and cement surfaces for 270 days post-spraying. Both Actellic®300CS and Icon®10CS demonstrated 100% mortality within 72 h of each test on all surfaces throughout the entire 300-day post-spraying period. CONCLUSION: The study shows the varying levels of resistance of An. stephensi Bandar Abbas population to different insecticides and demonstrates the consistent performance of Actellic®300CS in controlling these mosquitoes on various surfaces. The findings suggest that long-lasting CS formulations may be more effective for malaria vector control compared to the current options. Further research is needed to validate these findings in field settings and assess the impact of these insecticides on malaria transmission.

Preliminary monitoring of knockdown resistance (kdr) mutation in Anopheles stephensi: insights from a malarious area in Southeastern Iran.

BACKGROUND: Anopheles stephensi is recognized as the main malaria vector in Iran. In recent years, resistance to several insecticide classes, including organochlorine, pyrethroids, and carbamate compounds, has been reported for this medically important malaria vector. The main objective of the present study was to evaluate the insecticide susceptibility status of An. stephensi collected from the southern part of Iran, and to clarify the mechanism of resistance, using bioassay tests and molecular methods comparing the sequence of susceptible and resistant mosquitoes. METHODS: Mosquito larvae were collected from various larval habitats across six different districts (Gabrik, Sardasht, Tidar, Dehbarez, Kishi and Bandar Abbas) in Hormozgan Provine, located in the southern part of Iran. From each district standing water areas with the highest densities of Anopheles larvae were selected for sampling, and adult mosquitoes were reared from them. Finally, the collected mosquito species were identified using valid keys. Insecticide susceptibility of An. stephensi was tested using permethrin 0.75%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, and DDT 4%, following the World Health Organization (WHO) test procedures for insecticide resistance monitoring. Additionally, knockdown resistance (kdr) mutation in the voltage-gated sodium channel (vgsc) gene was sequenced and analysed among resistant populations to detect possible molecular mechanisms of observed resistance phenotypes. RESULTS: The susceptibility status of An. stephensi revealed that resistance to DDT and permethrin was found in all districts. Furthermore, resistance to all tested insecticides in An. stephensi was detected in Gabrik, Sardasht, Tidar, and Dehbarez. Analysis of knockdown resistance (kdr) mutations at the vgsc did not show evidence for the presence of this mutation in An. stephensi. CONCLUSION: Based on the results of the current study, it appears that in An. stephensi from Hormozgan Province (Iran), other resistance mechanisms such as biochemical resistance due to detoxification enzymes may be involved due to the absence of the kdr mutation or non-target site resistance. Further investigation is warranted in the future to identify the exact resistance mechanisms in this main malaria vector across the country.

Artificial nighttime lighting impacts Plasmodium falciparum mature stage V gametocytes infectivity in Anopheles stephensi.

BACKGROUND: Malaria is one of the most important vector-borne diseases of humans with an estimated 241 million cases worldwide in 2020. As an urban and periurban mosquito species, Anopheles stephensi is exposed to artificial human stimuli like light that can alter many aspects of mosquito behaviour, physiology and metabolism. Therefore, fluctuations in the light environment may influence the host, parasite and/or mosquito biology and hence modulate risk for disease transmission. In this study, the effect of artifitial light at night on mosquito infectivity by Plasmodium falciparum during the first hours of blood digestion was tested. METHODS: A total of three independent standard membrane feeding assays were performed to artificially fed septic and aseptic mosquitoes with P. falciparum infected blood. After blood feeding, females were transferred to incubators with different photoperiod cycles, so digestion occurred under day artificial light or dark. At 7 and 16 days post blood feeding, mosquitoes were dissected for midguts and salivary glands, respectively. Percentage of mosquitoes fed, percentage of prevalence and P. falciparum oocyst intensity between septic and aseptic mosquitoes in the two different photoperiod regimes, were compared using a Kruskal-Wallis test followed by a Dunn´s multiple comparison test . RESULTS: The exposition of mosquitoes to light after they took an infected blood meal has a negative effect on the successful progression of P. falciparum in the mosquito midgut. Antibiotic treatment significantly incremented the number of oocysts per midgut. Photophase significantly reduced the median oocyst intensity in both septic and aseptic mosquitoes. The percentage of oocyst reduction, understood as the percentage of reduction in the mean oocyst intensity of the parasite in the mosquito midgut between photophase and scotophase, was 51% in the case of aseptic mosquitoes and 80% for septic mosquitoes, both in the photophase condition. CONCLUSION: Although there are still many gaps in the understanding of parasite-mosquito interactions, these results support the idea that light can, not only, influence mosquito biting behaviour but also parasite success in the mosquito midgut. Hence, light can be considered an interesting additional mosquito-control strategy to reduce mosquito-borne diseases.

Possible potential spread of Anopheles stephensi, the Asian malaria vector.

BACKGROUND: Anopheles stephensi is native to Southeast Asia and the Arabian Peninsula and has emerged as an effective and invasive malaria vector. Since invasion was reported in Djibouti in 2012, the global invasion range of An. stephensi has been expanding, and its high adaptability to the environment and the ongoing development of drug resistance have created new challenges for malaria control. Climate change is an important factor affecting the distribution and transfer of species, and understanding the distribution of An. stephensi is an important part of malaria control measures, including vector control. METHODS: In this study, we collected existing distribution data for An. stephensi, and based on the SSP1-2.6 future climate data, we used the Biomod2 package in R Studio through the use of multiple different model methods such as maximum entropy models (MAXENT) and random forest (RF) in this study to map the predicted global An. stephensi climatically suitable areas. RESULTS: According to the predictions of this study, some areas where there are no current records of An. stephensi, showed significant areas of climatically suitable for An. stephensi. In addition, the global climatically suitability areas for An. stephensi are expanding with global climate change, with some areas changing from unsuitable to suitable, suggesting a greater risk of invasion of An. stephensi in these areas, with the attendant possibility of a resurgence of malaria, as has been the case in Djibouti. CONCLUSIONS: This study provides evidence for the possible invasion and expansion of An. stephensi and serves as a reference for the optimization of targeted monitoring and control strategies for this malaria vector in potential invasion risk areas.

An Investigation into the Larvicidal Activity of Biologically Synthesized Silver and Copper Oxide Nanoparticles Against Mosquito Larvae.

This study is primarily focused on the synthesis of silver and copper oxide nanoparticles utilizing the extract of Ipomoea staphylina plant and their larvicidal activity against specific larvae. Notably, Anopheles stephensi and Aedes aegypti are significant disease vectors responsible for transmitting diseases such as malaria, dengue fever, Zika virus, and chikungunya (Anopheles stephensi), and dengue and Zika (Aedes aegypti). These mosquitoes have a substantial impact on urban areas, influencing disease transmission dynamics. In an effort to control these larvae, we have pursued the synthesis of a herbal-based nanomedicine derived from I. staphylina, a valuable herb in traditional medicine. Our successful synthesis of silver and CuO nanoparticles followed environmentally sustainable green chemistry methodologies. The I. staphylina plant extract played a dual role as a reducing agent and dopant, aligning with principles of sustainability. We employed X-ray diffraction (XRD) analysis to validate the nanoparticle structure and size, while field-emission scanning electron microscopy (FE-SEM) revealed well-defined nanostructures. Elemental composition was determined through energy-dispersive X-ray (EDX) analysis, and UV-visible spectroscopy provided insights into the bandgap energy (3.15 eV for silver, 1.2 eV for CuO nanoparticles). These nanoparticles exhibited robust larvicidal activity, with CuO nanoparticles surpassing silver nanoparticles in terms of LC50 and LC90 values. Moreover, the developmental toxicity of CuO and Ag NPs was evaluated in zebrafish embryos as part of non-target eco-toxicological studies conducted in a standard laboratory environment. These findings underscore the potential utility of these nanoparticles as highly effective and environmentally friendly natural pesticides, offering cost-effectiveness and ecological benefits.

Detection of Anopheles stephensi Mosquitoes by Molecular Surveillance, Kenya.

The Anopheles stephensi mosquito is an invasive malaria vector recently reported in Djibouti, Ethiopia, Sudan, Somalia, Nigeria, and Ghana. The World Health Organization has called on countries in Africa to increase surveillance efforts to detect and report this vector and institute appropriate and effective control mechanisms. In Kenya, the Division of National Malaria Program conducted entomological surveillance in counties at risk for An. stephensi mosquito invasion. In addition, the Kenya Medical Research Institute conducted molecular surveillance of all sampled Anopheles mosquitoes from other studies to identify An. stephensi mosquitoes. We report the detection and confirmation of An. stephensi mosquitoes in Marsabit and Turkana Counties by using endpoint PCR and morphological and sequence identification. We demonstrate the urgent need for intensified entomological surveillance in all areas at risk for An. stephensi mosquito invasion, to clarify its occurrence and distribution and develop tailored approaches to prevent further spread.

Molecular Tools for Early Detection of Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Reports of the expansion of the Asia malaria vector Anopheles stephensi mosquito into new geographic areas are increasing, which poses a threat to the elimination of urban malaria. Efficient surveillance of this vector in affected areas and early detection in new geographic areas is key to containing and controlling this species. To overcome the practical difficulties associated with the morphological identification of immature stages and adults of An. stephensi mosquitoes, we developed a species-specific PCR and a real-time PCR targeting a unique segment of the second internal transcribed spacer lacking homology to any other organism. Both PCRs can be used to identify An. stephensi mosquitoes individually or in pooled samples of mixed species, including when present in extremely low proportions (1:500). This study also reports a method for selective amplification and sequencing of partial ribosomal DNA from An. stephensi mosquitoes for their confirmation in pooled samples of mixed species.

Effects of Seasonal Conditions on Abundance of Malaria Vector Anopheles stephensi Mosquitoes, Djibouti, 2018-2021.

We describe the influence of seasonal meteorologic variations and rainfall events on Anopheles stephensi mosquito populations during a 40-month surveillance study at a US military base in Djibouti. Focusing surveillance and risk mitigation for An. stephensi mosquitoes when climatic conditions are optimal presents an opportunity for malaria prevention and control in eastern Africa.

Laboratory-based efficacy evaluation of Bacillus thuringiensis var. israelensis and temephos larvicides against larvae of Anopheles stephensi in ethiopia.

BACKGROUND: Malaria, transmitted by the bite of infective female Anopheles mosquitoes, remains a global public health problem. The presence of an invasive Anopheles stephensi, capable of transmitting Plasmodium vivax and Plasmodium falciparum parasites was first reported in Ethiopia in 2016. The ecology of An. stephensi is different from that of Anopheles arabiensis, the primary Ethiopian malaria vector, and this suggests that alternative control strategies may be necessary. Larviciding may be an effective alternative strategy, but there is limited information on the susceptibility of Ethiopian An. stephensi to common larvicides. This study aimed to evaluate the efficacy of temephos and Bacillus thuringiensis var. israelensis (Bti) larvicides against larvae of invasive An. stephensi. METHODS: The diagnostic doses of two larvicides, temephos (0.25 ml/l) and Bti (0.05 mg/l) were tested in the laboratory against the immature stages (late third to early fourth stages larvae) of An. stephensi collected from the field and reared in a bio-secure insectary. Larvae were collected from two sites (Haro Adi and Awash Subuh Kilo). For each site, three hundred larvae were tested against each insecticide (as well as an untreated control), in batches of 25. The data from all replicates were pooled and descriptive statistics prepared. RESULTS: The mortality of larvae exposed to temephos was 100% for both sites. Mortality to Bti was 99.7% at Awash and 100% at Haro Adi site. CONCLUSIONS: Larvae of An. stephensi are susceptible to temephos and Bti larvicides suggesting that larviciding with these insecticides through vector control programmes may be effective against An. stephensi in these localities.

Mapping current and future thermal limits to suitability for malaria transmission by the invasive mosquito Anopheles stephensi.

BACKGROUND: Anopheles stephensi is a malaria-transmitting mosquito that has recently expanded from its primary range in Asia and the Middle East, to locations in Africa. This species is a competent vector of both Plasmodium falciparum and Plasmodium vivax malaria. Perhaps most alarming, the characteristics of An. stephensi, such as container breeding and anthropophily, make it particularly adept at exploiting built environments in areas with no prior history of malaria risk. METHODS: In this paper, global maps of thermal transmission suitability and people at risk (PAR) for malaria transmission by An. stephensi were created, under current and future climate. Temperature-dependent transmission suitability thresholds derived from recently published species-specific thermal curves were used to threshold gridded, monthly mean temperatures under current and future climatic conditions. These temperature driven transmission models were coupled with gridded population data for 2020 and 2050, under climate-matched scenarios for future outcomes, to compare with baseline predictions for 2020 populations. RESULTS: Using the Global Burden of Disease regions approach revealed that heterogenous regional increases and decreases in risk did not mask the overall pattern of massive increases of PAR for malaria transmission suitability with An. stephensi presence. General patterns of poleward expansion for thermal suitability were seen for both P. falciparum and P. vivax transmission potential. CONCLUSIONS: Understanding the potential suitability for An. stephensi transmission in a changing climate provides a key tool for planning, given an ongoing invasion and expansion of the vector. Anticipating the potential impact of onward expansion to transmission suitable areas, and the size of population at risk under future climate scenarios, and where they occur, can serve as a large-scale call for attention, planning, and monitoring.

First report of Anopheles stephensi from southern Ethiopia.

BACKGROUND: Anopheles stephensi is an emerging exotic invasive urban malaria vector in East Africa. The World Health Organization recently announced an initiative to take concerted actions to limit this vector's expansion by strengthening surveillance and control in invaded and potentially receptive territories in Africa. This study sought to determine the invasion of An. stephensi in southern Ethiopia. METHODS: A targeted entomological survey, both larvae and adult, was conducted in Hawassa City, southern Ethiopia between November 2022 and February 2023. Anopheles larvae were reared to adults for species identification. CDC light traps and BG Pro traps were used indoors and outdoors overnight at selected houses to collect adult mosquitoes in the study area. Prokopack aspirator was employed to sample indoor resting mosquitoes in the morning. Adults of An. stephensi was identified using morphological keys and then confirmed by PCR. RESULTS: Larvae of An. stephensi were found in 28 (16.6%) of the 169 potential mosquito breeding sites surveyed. Out of 548 adult female Anopheles mosquitoes reared from larvae, 234 (42.7%) were identified as An. stephensi morphologically. A total of 449 female anophelines were caught, of which 53 (12.0%) were An. stephensi. Other anopheline species collected in the study area included Anopheles gambiae sensu lato (s.l.), Anopheles pharoensis, Anopheles coustani, and Anopheles demeilloni. CONCLUSION: This study confirmed the presence of An. stephensi in southern Ethiopia. The presence of both larval and adult stages of this mosquito attests that this species established sympatric colonization with native vector species such as An. gambiae (s.l.) in southern Ethiopia. The findings warrant further investigation on the ecology, behaviour, population genetics, and role of An. stephensi in malaria transmission in Ethiopia.

Laboratory evaluation of the efficacy of deltamethrin-laced attractive toxic sugar bait formulation on Anopheles stephensi.

BACKGROUND: Attractive toxic sugar bait (ATSB) is a promising "attract and kill"-based approach for mosquito control. It is a combination of flower nectar/fruit juice to attract the mosquitoes, sugar solution to stimulate feeding, and a toxin to kill them. Selecting an effective attractant and optimizing concentration of toxicant is significant in the formulation of ATSB. METHODS: Current study formulated an ATSB using fruit juice, sugar and deltamethrin, a synthetic pyrethroid. It was evaluated against two laboratory strains of Anopheles stephensi. Initial studies evaluated comparative attractiveness of nine different fruit juices to An. stephensi adults. Nine ASBs were prepared by adding fermented juices of plum, guava, sweet lemon, orange, mango, pineapple, muskmelon, papaya, and watermelon with 10% sucrose solution (w/v) in 1:1 ratio. Cage bioassays were conducted to assess relative attraction potential of ASBs based on the number of mosquito landings on each and the most effective ASB was identified. Ten ATSBs were prepared by adding the identified ASB with different deltamethrin concentrations (0.015625-8.0 mg/10 mL) in 1:9 ratio. Each ATSB was assessed for the toxic potential against both the strains of An. stephensi. The data was statistically analysed using PASW (SPSS) software 19.0 program. RESULTS: The cage bioassays with nine ASBs revealed higher efficacy (p < 0.05) of Guava juice-ASB > Plum juice-ASB > Mango juice-ASB in comparison to rest of the six ASB's. The bioassay with these three ASB's ascertained the highest attractancy potential of guava juice-ASB against both the strains of An. stephensi. The ATSB formulations resulted in 5.1-97.9% mortality in Sonepat (NIMR strain) with calculated LC30, LC50, and LC90 values of 0.17 mg deltamethrin/10 mL, 0.61 mg deltamethrin/10 mL, and 13.84 mg deltamethrin/10 mL ATSB, respectively. Whereas, 6.12-86.12% mortality was recorded in the GVD-Delhi (AND strain) with calculated LC30, LC50, and LC90 values of 0.25 mg deltamethrin/10 mL, 0.73 mg deltamethrin/10 mL and 10.22 mg deltamethrin/10 mL ATSB, respectively. CONCLUSION: The ATSB formulated with guava juice-ASB and deltamethrin (0.0015625-0.8%) in 9:1 ratio showed promising results against two laboratory strains of An. stephensi. Field assessment of these formulations is being conducted to estimate their feasibility for use in mosquito control.

Public health impact of the spread of Anopheles stephensi in the WHO Eastern Mediterranean Region countries in Horn of Africa and Yemen: need for integrated vector surveillance and control.

BACKGROUND: Anopheles stephensi is an efficient vector of both Plasmodium falciparum and Plasmodium vivax in South Asia and the Middle East. The spread of An. stephensi to countries within the Horn of Africa threatens progress in malaria control in this region as well as the rest of sub-Saharan Africa. METHODS: The available malaria data and the timeline for the detection of An. stephensi was reviewed to analyse the role of An. stephensi in malaria transmission in Horn of Africa of the Eastern Mediterranean Region (EMR) in Djibouti, Somalia, Sudan and Yemen. RESULTS: Malaria incidence in Horn of Africa of EMR and Yemen, increased from 41.6 in 2015 to 61.5 cases per 1000 in 2020. The four countries from this region, Djibouti, Somalia, Sudan and Yemen had reported the detection of An. stephensi as of 2021. In Djibouti City, following its detection in 2012, the estimated incidence increased from 2.5 cases per 1000 in 2013 to 97.6 cases per 1000 in 2020. However, its contribution to malaria transmission in other major cities and in other countries, is unclear because of other factors, quality of the urban malaria data, human mobility, uncertainty about the actual arrival time of An. stephensi and poor entomological surveillance. CONCLUSIONS: While An. stephensi may explain a resurgence of malaria in Djibouti, further investigations are needed to understand its interpretation trends in urban malaria across the greater region. More investment for multisectoral approach and integrated surveillance and control should target all vectors particularly malaria and dengue vectors to guide interventions in urban areas.

Confirmation of the presence of Anopheles stephensi among internally displaced people's camps and host communities in Aden city, Yemen.

BACKGROUND: Declines in global malaria cases and deaths since the millennium are currently challenged by multiple factors including funding limitations, limits of, and resistance to vector control tools, and also recent spread of the invasive vector species, Anopheles stephensi-especially into novel urban settings where malaria rates are typically low. Coupled with general increases in urbanization and escalations in the number of conflicts creating rapid and unplanned population displacement into temporary shelter camps within host urban areas, particularly in the Middle East and sub-Saharan Africa, increased urban malaria is a major threat to control and elimination. METHODS: Entomological monitoring surveys (targeting Aedes aegypti) of water containers across urban areas hosting internally displaced people (IDP) communities in Aden city, Yemen, were performed by The MENTOR Initiative, a non-governmental organisation. As part of these surveys in 2021 23 larvae collected and raised to adults were morphologically identified as An. stephensi. Twelve of the samples were sent to Liverpool School of Tropical Medicine for independent morphological assessment and genetic analysis by sequencing the ribosomal ITS2 region and the mitochondrial COI gene. RESULTS: All twelve samples were confirmed morphologically and by sequence comparison of the single ITS2 and COI haplotype detected to the NCBI BLAST database as An. stephensi. Phylogenetic analysis with comparable COI sequences indicated close relationship to haplotypes found in Djibouti and Ethiopia. CONCLUSION: The study results confirm the presence of An. stephensi in Yemen. Confirmation of the species in multiple urban communities hosting thousands of IDPs living in temporary shelters with widescale dependency on open water containers is of particular concern due to the vulnerability of the population and abundance of favourable breeding sites for the vector. Proactive monitoring and targeted integrated vector management are required to limit impacts in this area of typically low malaria transmission, and to prevent further the spread of An. stephensi within the region.

Resistance of Anopheles stephensi to selected insecticides used for indoor residual spraying and long-lasting insecticidal nets in Ethiopia.

BACKGROUND: Malaria, transmitted by the bite of infective female Anopheles mosquitoes, remains a global public health problem. The presence of invasive Anopheles stephensi, capable of transmitting Plasmodium vivax and Plasmodium falciparum, was first reported in Ethiopia in 2016. The ecology of this mosquito species differs from that of Anopheles arabiensis, the primary malaria vector in Ethiopia. This study aimed to evaluate the efficacy of selected insecticides, which are used in indoor residual spraying (IRS) and selected long-lasting insecticidal nets (LLINs) for malaria vector control against adult An. stephensi. METHODS: Anopheles stephensi mosquitoes were collected as larvae and pupae from Awash Subah Kilo Town and Haro Adi village, Ethiopia. Adult female An. stephensi, reared from larvae and pupae collected from the field, aged 3-5 days were exposed to impregnated papers of IRS insecticides (propoxur 0.1%, bendiocarb 0.1%, pirimiphos-methyl 0.25%), and insecticides used in LLINs (alpha-cypermethrin 0.05%, deltamethrin 0.05% and permethrin 0.75%), using diagnostic doses and WHO test tubes in a bio-secure insectary at Aklilu Lemma Institute of Pathobiology, Addis Ababa University. For each test and control tube, batches of 25 female An. stephensi were used to test each insecticide used in IRS. Additionally, cone bioassay tests were conducted to expose An. stephensi from the reared population to four brands of LLINs, MAGNet™ (alpha-cypermethrin), PermaNet® 2.0 (deltamethrin), DuraNet© (alpha-cypermethrin) and SafeNet® (alpha-cypermethrin). A batch of ten sugar-fed female mosquitoes aged 2-5 days was exposed to samples taken from five positions/sides of a net. The data from all replicates were pooled and descriptive statistics were used to describe features of the data. RESULTS: All An. stephensi collected from Awash Subah Kilo Town and Haro Adi village (around Metehara) were resistant to all tested insecticides used in both IRS and LLINs. Of the tested LLINs, only MAGNet™ (alpha-cypermethrin active ingredient) caused 100% knockdown and mortality to An. stephensi at 60 min and 24 h post exposure, while all other net brands caused mortality below the WHO cut-off points (< 90%). All these nets, except SafeNet®, were collected during LLIN distribution for community members through the National Malaria Programme, in December 2020. CONCLUSIONS: Anopheles stephensi is resistant to all tested insecticides used in IRS and in the tested LLIN brands did not cause mosquito mortality as expected, except MAGNet. This suggests that control of this invasive vector using existing adult malaria vector control methods will likely be inadequate and that alternative strategies may be necessary.