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Abstract The Democratic Republic of Congo (DRC) suffers from one of the highest malaria burdens worldwide, but information on its Anopheles vector populations is relatively limited. Preventative malaria control in DRC is reliant on pyrethroid-treated nets, raising concerns over the potential impacts of insecticide resistance. We sampled Anopheles gambiae from three geographically distinct populations (Kimpese, Kapolowe and Mikalayi) in southern DRC, collecting from three sub-sites per population and characterising mosquito collections from each for resistance to pyrethroids using WHO tube bioassays. Resistance to each of three different pyrethroids was generally high in An. gambiae with

Abstract Background Indoor residual spraying (IRS) is one of the most effective malaria control tools. However, its application has become limited to specific contexts due to the increased costs of IRS products and implementation programmes. Selective spraying—selective spray targeted to particular areas/surfaces of dwellings—has been proposed to maintain the malaria control and resistance-management benefits of IRS while decreasing the costs of the intervention. Methods A literature search was conducted to find (1) studies that assessed the resting behaviour of Anopheles mosquitoes and (2) studies that evaluated the impact of selective spraying on entomological and malaria outcomes. Additional articles were identified through hand searches of all references cited in articles identified through the initial search. A cost model was developed from PMI VectorLink IRS country programmes, and comparative cost analysis reports to describe the overall cost benefits of selective IRS. Results In some studies, there appeared to be a clear resting preference for certain Anopheles species in terms of the height at which they rested. However, for other species, and particularly the major African malaria vectors, a clear resting pattern was not detected. Furthermore, resting behaviour was not measured in a standardized way. For the selective spray studies that were assessed, there was a wide range of spray configurations, which complicates the comparison of methods. Many of these spray techniques were effective and resulted in reported 25–68% cost savings and reduced use of insecticide. The reported cost savings in the literature do not always consider all of the IRS implementation costs. Using the IRS cost model, these savings ranged from 17 to 29% for programs that targeted Anopheles spp. and 18–41% for programmes that targeted Aedes aegypti. Conclusions Resting behaviour is generally measured in a simplistic way; noting the resting spot of mosquitoes in the morning. This is likely an oversimplification, and there is a need for better monitoring of resting mosquitoes. This may improve the target surface for selective spray techniques, which could reduce the cost of IRS while maintaining its effectiveness. Reporting of cost savings should be calculated considering the entire implementation costs, and a cost model was provided for future calculations.

Abstract 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 (

Abstract From August 2020 to June 2021, we assessed the efficacy of SumiShield 50WG (clothianidin), Fludora Fusion 56.25WP-SB (mixture of clothianidin and deltamethrin) and Actellic 300CS (pirimiphos-methyl) in experimental huts when partially sprayed against wild, free-flying populations of Anopheles gambiae s.l. in Tiassalé, Côte d'Ivoire. A one-month baseline period of mosquito collections was conducted to determine mosquito density and resting behavior in unsprayed huts, after which two treatments of partial indoor residual spraying (IRS) were tested: spraying only the top half of walls + ceilings or only the bottom half of walls + ceilings. These were compared to fully sprayed applications using the three IRS insecticide formulations, during twenty nights per month of collection for nine consecutive months. Mortality was assessed at the time of collection, and after a 24 h holding period (Actellic) or up to 120 h (SumiShield and Fludora Fusion). Unsprayed huts were used as a negative control. The efficacy of each partially sprayed treatment of each insecticide was compared monthly to the fully sprayed huts over the study period with a non-inferiority margin set at 10%. The residual efficacy of each insecticide sprayed was also monitored. A total of 2197 Anopheles gambiae s.l. were collected during the baseline and 17,835 during the 9-month period after spraying. During baseline, 42.6% were collected on the bottom half versus 24.3% collected on the top half of the walls, and 33.1% on the ceilings. Over the nine-month post treatment period, 73.5% were collected on the bottom half of the wall, 11.6% collected on the top half and 14.8% on the ceilings. For Actellic, the mean mortality over the nine-month period was 88.5% [87.7, 89.3] for fully sprayed huts, 88.3% [85.1, 91.4] for bottom half + ceiling sprayed walls and 80.8% [74.5, 87.1] for the top half + ceiling sprayed huts. For Fludora Fusion an overall mean mortality of 85.6% [81.5, 89.7] was recorded for fully sprayed huts, 83.7% [82.9, 84.5] for bottom half + ceiling sprayed huts and 81.3% [79.6, 83.0] for the top half + ceiling sprayed huts. For SumiShield, the overall mean mortality was 86.7% [85.3, 88.1] for fully sprayed huts, 85.6% [85.4, 85.8] for the bottom half + ceiling sprayed huts and 76.9% [76.6, 77.3] for the top half + ceiling sprayed huts. For Fludora Fusion, both iterations of partial IRS were non-inferior to full spraying. However, for SumiShield and Actellic, this was true only for the huts with the bottom half + ceiling, reflecting the resting site preference of the local vectors. The results of this study suggest that partial spraying may be a way to reduce the cost of IRS without substantially compromising IRS efficacy.

In Côte d'Ivoire, there is a high intensity of pyrethroid resistance in malaria vectors which may threaten successful vector control. Molecular studies of the target site mutation in the voltage-gated sodium channel (VGSC) 1014F show that the gene frequencies of these mutations are high, widely spread across Côte d’Ivoire, and even fixed in some areas. To further characterize insecticide resistance in Côte d’Ivoire beyond target site mutations, the metabolic resistance mechanism was explored. Overexpression of the metabolic enzyme cytochrome CYP6M2, CYP6P3, CYP6P4, and CYP6P5 was investigated in Anopheles gambiae (s.s.) and An. coluzzii collected from 30 insecticide resistance monitoring sites across the country in 2021 and 2022. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was carried out using RNA-later preserved 60 surviving mosquitoes from each site after phenotypic susceptibility tests. Additionally, a subsample of about 100 mosquitoes from each site was identified to species by PCR, and the frequency of the VGSC 1014F and 1014S was determined. All four CYPs were found to be overexpressed in Côte d’Ivoire with at least one CYP statistically significantly overexpressed in 27 of the 30 sites investigated compared to the susceptible An. gambiae (s.s.) Kisumu (P

Abstract 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.

Abstract Background The functional survival time of long-lasting insecticidal nets (LLINs), which varies across different field contexts, is critical for the successful prevention of malaria transmission. However, there is limited data on LLIN durability in field settings in Ethiopia. Methods A three-year longitudinal study was conducted to monitor attrition, physical integrity, and bio-efficacy and residual chemical concentration of LLINs in four regions in Ethiopia. World Health Organization (WHO) guidelines were used to determine sample size, measure physical integrity, and calculate attrition rates, and functional survival time. Yearly bio-efficacy testing was done on randomly selected LLINs. An excel tool developed by vector works project was used to calculate the median functional survival time of the LLINs. Predictors of functional survival were identified by fitting binary and multivariate cox proportional hazards model. Results A total of 3,396 LLINs were included in the analysis. A total of 3,396 LLINs were included in the analysis. By the end of 36 months, the proportion of LLINs functionally surviving was 12.9% [95% confidence interval (CI) 10.5, 15.6], the rates of attrition due to physical damage and repurposing were 48.8% [95% confidence interval (CI) 45.0, 52.6] and 13.8% [95% confidence interval (CI) 11.6, 14.6], respectively. The estimated median functional survival time was 19 months (95%CI 17, 21). Factors associated with shorter functional survival time include being in a low malaria transmission setting [Adjusted Hazards Ratio (AHR) (95%CI) 1.77 (1.22, 2.55)], rural locations [AHR (95%CI) 1.83 (1.17, 2.84)], and in a room where cooking occurs [AHR (95%CI) 1.28 (1.05, 1.55)]. Bioassay tests revealed that 95.3% (95%CI 86.4, 98.5) of the LLINs met the WHO criteria of bio-efficacy after 24 months of distribution. Conclusion The LLIN survival time was shorter than the expected three years due to high attrition rates and rapid loss of physical integrity. National malaria programmes may consider, procuring more durable LLINs, educating communities on how to prevent damage of LLINs, and revising the current three-year LLIN distribution schedule to ensure sufficient protection is provided by LLINs against malaria transmission. While this paper contributes to the understanding of determinants impacting functional survival, further research is needed to understand factors for the rapid attrition rates and loss of physical integrity of LLINs in field settings.

Abstract 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.

Abstract Background Entomological surveillance provides critical information on vectors for appropriate malaria vector control and strategic decision-making. The widely documented insecticide resistance of malaria vectors in Côte d’Ivoire requires that any vector control intervention deployment be driven by entomological data to optimize its effectiveness and appropriate resource allocations. To achieve this goal, this study documents the results of monthly vector surveillance and insecticide susceptibility tests conducted in 2019 and a review of all previous entomological monitoring data used to guide vector control decision making. Furthermore, susceptibility to pirimiphos-methyl and clothianidin was assessed in addition to chlorfenapyr and pyrethroids (intensity and piperonyl butoxide (PBO) synergism) tests previously reported. Vector bionomic data were conducted monthly in four sites (Sakassou, Béoumi, Dabakala and Nassian) that were selected based on their reported high malaria incidence. Adult mosquitoes were collected using human landing catches (HLCs), pyrethrum spray catches (PSCs), and human-baited CDC light traps to assess vector density, behaviour, species composition and sporozoite infectivity. Results Pirimiphos-methyl and clothianidin susceptibility was observed in 8 and 10 sites, respectively, while previous data reported chlorfenapyr (200 µg/bottle) susceptibility in 13 of the sites, high pyrethroid resistance intensity and increased mortality with PBO pre-exposure at all 17 tested sites. Anopheles gambiae sensu lato was the predominant malaria vector collected in all four bionomic sites. Vector density was relatively higher in Sakassou throughout the year with mean biting rates of 278.2 bites per person per night (b/p/n) compared to Béoumi, Dabakala and Nassian (mean of 48.5, 81.4 and 26.6 b/p/n, respectively). The mean entomological inoculation rate (EIR) was 4.44 infective bites per person per night (ib/p/n) in Sakassou, 0.34 ib/p/n in Beoumi, 1.17 ib/p/n in Dabakala and 1.02 ib/p/n in Nassian. The highest EIRs were recorded in October in Béoumi (1.71 ib/p/n) and Nassian (3.22 ib/p/n), in July in Dabakala (4.46 ib/p/n) and in May in Sakassou (15.6 ib/p/n). Conclusion Based on all results and data review, the National Malaria Control Programme developed and implemented a stratified insecticide-treated net (ITN) mass distribution in 2021 considering new generation ITNs. These results also supported the selection of clothianidin-based products and an optimal spraying time for the first indoor residual spraying (IRS) campaign in Sakassou and Nassian in 2020.

Abstract Background The Democratic Republic of the Congo (DRC) is the second most malaria-affected country in the world with 21,608,681 cases reported in 2019. The Kongo Central (KC) Province has a malaria annual incidence of 163 cases/per 1000 inhabitants which are close to the national average of 153.4/1000. However, the malaria prevalence varies both between and within health zones in this province. The main objective of this study was to describe the epidemiology and transmission of malaria among children aged 0 to 10 years in the 4 highest endemic health areas in Kisantu Health Zone (HZ) of KC in DRC. Methods A community-based cross-sectional study was conducted from October to November 2017 using multi-stage sampling. A total of 30 villages in 4 health areas in Kisantu HZ were randomly selected. The prevalence of malaria was measured using a thick blood smear (TBS) and known predictors and associated outcomes were assessed. Data are described and association determinants of malaria infection were analysed. Results A total of 1790 children between 0 and 10 years were included in 30 villages in 4 health areas of Kisantu HZ. The overall prevalence in the study area according to the TBS was 14.8% (95% CI: 13.8–16.6; range: 0–53). The mean sporozoite rate in the study area was 4.3% (95% CI: 2.6–6.6). The determination of kdr-west resistance alleles showed the presence of both L1014S and L1014F with 14.6% heterozygous L1014S/L1014F, 84.4% homozygous 1014F, and 1% homozygous 1014S. The risk factors associated with malaria infection were ground or wooden floors aOR: 15.8 (95% CI: 8.6–29.2), a moderate or severe underweight: 1.5 (1.1–2.3) and to be overweight: 1.9 (95% CI: 1.3–2.7). Conclusion Malaria prevalence differed between villages and health areas within the same health zone. The control strategy activities must be oriented by the variety in the prevalence and transmission of malaria in different areas. The policy against malaria regarding long-lasting insecticidal nets should be based on the evidence of metabolic resistance.

Abstract Background The use of synthetic insecticides against mosquitoes may lead to resistance development and potential health hazards in humans and the environment. Consequently, a paradigm needs to shift towards the alternative use of botanical insecticides that could strengthen an insecticide resistance management programme. This study aimed to assess the insecticidal effects aqueous, hexane, and methanol crude leaf extracts of Calpurnia aurea, Momordica foetida, and Zehneria scabra on an insectary colony of Anopheles stephensi larvae and adults. Methods Fresh leaves of C. aurea, M. foetida and Z. scabra were collected and dried, then separately ground to powder. Powdered leaves of test plants were extracted using sonication with aqueous, hexane, and methanol solvents. The extracts were concentrated, and a stock solution was prepared. For comparison, Temephos (Abate®) and control solutions (a mixture of water and emulsifier) were used as the positive and negative controls, respectively. Different test concentrations for the larvae and the adults were prepared and tested according to WHO (2005) and CDC (2010) guidelines to determine lethal concentration (LC) values. Mortality was observed after 24 h exposure. The statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software (Kruskal-Wallis test) and R software (a generalized linear model was used to determine LC50 and LC90 values of the extracts). Results The lowest LC50 values were observed in aqueous extracts of M. foetida followed by Z. scabra extract and C. aurea leaves at 34.61, 35.85, and 38.69 ppm, respectively, against the larvae. Larval mortality was not observed from the hexane extracts and negative control, while the standard larvicide (temephos) achieved 100% mortality. Further, the adulticidal efficacy was greatest for aqueous extract of Z. scabra with LC50 = 176.20 ppm followed by aqueous extract of C. aurea (LC50 = 297.75 ppm). Conclusion The results suggest that the leaf extracts of the three test plants have the potential of being used for the control of vector An. stephensi larvae and adult instead of synthetic mosquitocides. Further studies need to be conducted to identify the active ingredients and their mode of action.

Abstract Background About two out of three Ethiopians are at risk of malaria, a disease caused by the parasites Plasmodium falciparum and Plasmodium vivax. Anopheles stephensi, an invasive vector typically found in South Asia and the Middle East, was recently found to be distributed across eastern and central Ethiopia and is capable of transmitting both P. falciparum and P. vivax. The detection of this vector in the Horn of Africa (HOA) coupled with widespread insecticide resistance requires that new methods of vector control be investigated in order to control the spread of malaria. Wolbachia, a naturally occurring endosymbiotic bacterium of mosquitoes, has been identified as a potential vector control tool that can be explored for the control of malaria transmission. Wolbachia could be used to control the mosquito population through suppression or potentially decrease malaria transmission through population replacement. However, the presence of Wolbachia in wild An. stephensi in eastern Ethiopia is unknown. This study aimed to identify the presence and diversity of Wolbachia in An. stephensi across eastern Ethiopia. Methods DNA was extracted from An. stephensi collected from eastern Ethiopia in 2018 and screened for Wolbachia using a 16S targeted PCR assay, as well as multilocus strain typing (MLST) PCR assays. Haplotype and phylogenetic analysis of the sequenced 16S amplicons were conducted to compare with Wolbachia from countries across Africa and Asia. Results Twenty out of the 184 mosquitoes screened were positive for Wolbachia, with multiple haplotypes detected. In addition, phylogenetic analysis revealed two superclades, representing Wolbachia supergroups A and B (bootstrap values of 81 and 72, respectively) with no significant grouping of geographic location or species. A subclade with a bootstrap value of 89 separates the Ethiopian haplotype 2 from other sequences in that superclade. Conclusions These findings provide the first evidence of natural Wolbachia populations in wild An. stephensi in the HOA. They also identify the need for further research to confirm the endosymbiotic relationship between Wolbachia and An. stephensi and to investigate its utility for malaria control in the HOA. Graphical Abstract

Abstract Background Sub-Saharan Africa has seen substantial reductions in cases and deaths due to malaria over the past two decades. While this reduction is primarily due to an increasing expansion of interventions, urbanisation has played its part as urban areas typically experience substantially less malaria transmission than rural areas. However, this may be partially lost with the invasion and establishment of Anopheles stephensi. A. stephensi, the primary urban malaria vector in Asia, was first detected in Africa in 2012 in Djibouti and was subsequently identified in Ethiopia in 2016, and later in Sudan and Somalia. In Djibouti, malaria cases have increased 30-fold from 2012 to 2019 though the impact in the wider region remains unclear. Methods Here, we have adapted an existing model of mechanistic malaria transmission to estimate the increase in vector density required to explain the trends in malaria cases seen in Djibouti. To account for the observed plasticity in An. stephensi behaviour, and the unknowns of how it will establish in a novel environment, we sample behavioural parameters in order to account for a wide range of uncertainty. This quantification is then applied to Ethiopia, considering temperature-dependent extrinsic incubation periods, pre-existing vector-control interventions and Plasmodium falciparum prevalence in order to assess the potential impact of An. stephensi establishment on P. falciparum transmission. Following this, we estimate the potential impact of scaling up ITN (insecticide-treated nets)/IRS (indoor residual spraying) and implementing piperonyl butoxide (PBO) ITNs and larval source management, as well as their economic costs. Results We estimate that annual P. falciparum malaria cases could increase by 50% (95% CI 14–90) if no additional interventions are implemented. The implementation of sufficient control measures to reduce malaria transmission to pre-stephensi levels will cost hundreds of millions of USD. Conclusions Substantial heterogeneity across the country is predicted and large increases in vector control interventions could be needed to prevent a major public health emergency.

Abstract Background Distribution of long-lasting insecticidal bed nets (LLINs) is one of the main control strategies for malaria. Improving malaria prevention programmes requires understanding usage patterns in households receiving LLINs, but there are limits to what standard cross-sectional surveys of self-reported LLIN use can provide. This study was designed to assess the performance of an accelerometer-based approach for measuring a range of LLIN use behaviours as a proof of concept for more granular LLIN-use monitoring over longer time periods. Methods This study was carried out under controlled conditions from May to July 2018 in Liverpool, UK. A single accelerometer was affixed to the side panel of an LLIN and participants carried out five LLIN use behaviours: (1) unfurling a net; (2) entering an unfurled net; (3) lying still as if sleeping; (4) exiting from under a net; and, (5) folding up a net. The randomForest package in R, a supervised non-linear classification algorithm, was used to train models on 20-s epochs of tagged accelerometer data. Models were compared in a validation dataset using overall accuracy, sensitivity and specificity, receiver operating curves and the area under the curve (AUC). Results The five-category model had overall accuracy of 82.9% in the validation dataset, a sensitivity of 0.681 for entering a net, 0.632 for exiting, 0.733 for net down, and 0.800 for net up. A simplified four-category model, combining entering/exiting a net into one category had accuracy of 94.8%, and increased sensitivity for net down (0.756) and net up (0.829). A further simplified three-category model, identifying sleeping, net up, and a combined net down/enter/exit category had accuracy of 96.2% (483/502), with an AUC of 0.997 for net down and 0.987 for net up. Models for detecting entering/exiting by adults were significantly more accurate than for children (87.8% vs 70.0%; p

Abstract Background The recent detection of the South Asian malaria vector Anopheles stephensi in the Horn of Africa (HOA) raises concerns about the impact of this mosquito on malaria transmission in the region. Analysis of An. stephensi genetic diversity and population structure can provide insight into the history of the mosquito in the HOA to improve predictions of future spread. We investigated the genetic diversity of An. stephensi in eastern Ethiopia, where detection suggests a range expansion into this region, in order to understand the history of this invasive population. Methods We sequenced the cytochrome oxidase subunit I (COI) and cytochrome B gene (CytB) in 187 An. stephensi collected from 10 sites in Ethiopia in 2018. Population genetic, phylogenetic, and minimum spanning network analyses were conducted for Ethiopian sequences. Molecular identification of blood meal sources was also performed using universal vertebrate CytB sequencing. Results Six An. stephensi COI-CytB haplotypes were observed, with the highest number of haplotypes in the northeastern sites (Semera, Bati, and Gewana towns) relative to the southeastern sites (Kebridehar, Godey, and Degehabur) in eastern Ethiopia. We observed population differentiation, with the highest differentiation between the northeastern sites compared to central sites (Erer Gota, Dire Dawa, and Awash Sebat Kilo) and the southeastern sites. Phylogenetic and network analysis revealed that the HOA An. stephensi are more genetically similar to An. stephensi from southern Asia than from the Arabian Peninsula. Finally, molecular blood meal analysis revealed evidence of feeding on cows, goats, dogs, and humans, as well as evidence of multiple (mixed) blood meals. Conclusion We show that An. stephensi is genetically diverse in Ethiopia and with evidence of geographical structure. Variation in the level of diversity supports the hypothesis for a more recent introduction of An. stephensi into southeastern Ethiopia relative to the northeastern region. We also find evidence that supports the hypothesis that HOA An. stephensi populations originate from South Asia rather than the Arabian Peninsula. The evidence of both zoophagic and anthropophagic feeding support the need for additional investigation into the potential for livestock movement to play a role in vector spread in this region. Graphical Abstract

Abstract Background In Ethiopia, despite improvements in coverage and access, utilization of long-lasting insecticidal nets (LLINs) remains a challenge. Different household-level factors have been identified as associated with LLIN use. However, the contribution of LLIN physical integrity to their utilization is not well investigated and documented. This study aimed to assess the association between the physical integrity of LLINs and their use. Methods This study employed a nested case-control design using secondary data from the Ethiopian LLIN durability monitoring study conducted from May 2015 to June 2018. LLINs not used the night before the survey were identified as cases, while those used the previous night were categorized as controls. The physical integrity of LLINs was classified as no holes, good, acceptable, and torn using the proportionate hole index (pHI). A Generalized Estimating Equation (GEE) model was used to assess and quantify the association between LLIN physical integrity and use. The model specifications included binomial probabilistic distribution, logit link, exchangeable correlation matrix structure, and robust standard errors. The factors included in the model were selected first by fitting binary regression, and then by including all factors that showed statistical significance at P-value less than 0.25 and conceptually relevant variables into the multivariate regression model. Results A total of 5277 observations fulfilled the inclusion criteria. Out of these 1767 observations were cases while the remaining 3510 were controls. LLINs that were in torn physical condition had higher odds (AOR [95% CI] = 1.76 [1.41, 2.19]) of not being used compared to LLINs with no holes. Other factors that showed significant association included the age of the LLIN, sleeping place type, washing status of LLINs, perceptions towards net care and repair, LLIN to people ratio, economic status, and study site. Conclusion and recommendation LLINs that have some level of physical damage have a relatively higher likelihood of not being used. Community members need to be educated about proper care and prevention of LLIN damage to delay the development of holes as long as possible and use available LLINs regularly.

Abstract 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. Graphical Abstract

Abstract Background Plasmodium spp. sporozoite rates in mosquitoes are used to better understand malaria transmission intensity, the relative importance of vector species and the impact of interventions. These rates are typically estimated using an enzyme-linked immunosorbent assay (ELISA) utilizing antibodies against the circumsporozoite protein of Plasmodium falciparum, Plasmodium vivax VK210 (P. vivax210) or P. vivax VK247 (P. vivax247), employing assays that were developed over three decades ago. The ELISA method requires a separate assay plate for each analyte tested and can be time consuming as well as requiring sample volumes not always available. The bead-based multiplex platform allows simultaneous measurement of multiple analytes and may improve the lower limit of detection for sporozoites. Methods Recombinant positive controls for P. falciparum, P. vivax210 and P. vivax247 and previously developed circumsporozoite (cs) ELISA antibodies were used to optimize conditions for the circumsporozoite multiplex bead assay (csMBA) and to determine the detection range of the csMBA. After optimizing assay conditions, known amounts of sporozoites were used to determine the lower limit of detection for the csELISA and csMBA and alternate cut-off measures were applied to demonstrate how cut-off criteria can impact lower limits of detection. Sporozoite rates from 1275 mosquitoes collected in Madagascar and 255 mosquitoes collected in Guinea were estimated and compared using the established csELISA and newly optimized csMBA. All mosquitoes were tested (initial test), and those that were positive were retested (retest). When sufficient sample volume remained, an aliquot of homogenate was boiled and retested (boiled retest), to denature any heat-unstable cross-reactive proteins. Results Following optimization of the csMBA, the lower limit of detection was 25 sporozoites per mosquito equivalent for P. falciparum, P. vivax210 and P. vivax247 whereas the lower limits of detection for csELISA were found to be 1400 sporozoites for P. falciparum, 425 for P. vivax210 and 1650 for P. vivax247. Combined sporozoite rates after re-testing of samples that initially tested positive for Madagascar mosquitoes by csELISA and csMBA were 1.4 and 10.3%, respectively, and for Guinea mosquitoes 2% by both assays. Boiling of samples followed by csMBA resulted in a decrease in the Madagascar sporozoite rate to 2.8–4.4% while the Guinea csMBA sporozoite rate remained at 2.0%. Using an alternative csMBA cut-off value of median fluorescence intensity (MFI) of 100 yielded a sporozoite rate after confirmational testing of 3.7% for Madagascar samples and 2.0% for Guinea samples. Whether using csMBA or csELISA, the following steps may help minimize false positives: specimens are appropriately stored and bisected anterior to the thorax-abdomen junction, aliquots of homogenate are boiled and retested following initial testing, and an appropriate cut-off value is determined. Conclusions The csMBA is a cost-comparable and time saving alternative to the csELISA and may help eliminate false negatives due to a lower limit of detection, thus increasing sensitivity over the csELISA. The csMBA expands the potential analyses that can be done with a small volume of sample by allowing multiplex testing where analytes in addition to P. falciparum, P. vivax210 and P. vivax247 can be added following optimization.

Abstract The scale up of indoor residual spraying (IRS) and insecticide treated nets have contributed significantly to global reductions in malaria prevalence over the last two decades. However, widespread pyrethroid resistance has necessitated the use of new and more expensive insecticides for IRS. Partial IRS with pirimiphos-methyl in experimental huts and houses in a village-wide trial was evaluated against Anopheles gambiae s.l. in northern Ghana. Four different scenarios in which either only the top or bottom half of the walls of experimental huts were sprayed, with or without also spraying the ceiling were compared. Mortality of An. gambiae s.l. on partially sprayed walls was compared with the standard procedures in which all walls and ceiling surfaces are sprayed. A small-scale trial was then conducted to assess the effectiveness, feasibility, and cost of spraying only the upper walls and ceiling as compared to full IRS and no spraying in northern Ghana. Human landing catches were conducted to estimate entomological indices and determine the effectiveness of partial IRS. An established transmission dynamics model was parameterized by an analysis of the experimental hut data and used to predict the epidemiological impact and cost effectiveness of partial IRS for malaria control in northern Ghana. In the experimental huts, partial IRS of the top (IRR 0.89, p = 0.13) or bottom (IRR 0.90, p = 0.15) half of walls and the ceiling was not significantly less effective than full IRS in terms of mosquito mortality. In the village trial, the annual entomological inoculation rate was higher for the unsprayed control (217 infective bites/person/year (ib/p/yr)) compared with the fully and partially sprayed sites, with 28 and 38 ib/p/yr, respectively. The transmission model predicts that the efficacy of partial IRS against all-age prevalence of malaria after six months would be broadly equivalent to a full IRS campaign in which 40% reduction is expected relative to no spray campaign. At scale, partial IRS in northern Ghana would have resulted in a 33% cost savings ($496,426) that would enable spraying of 36,000 additional rooms. These findings suggest that partial IRS is an effective, feasible, and cost saving approach to IRS that could be adopted to sustain and expand implementation of this key malaria control intervention.

Abstract Background Sixty percent of the Ethiopia population is at risk of malaria, with the highest prevalence reported in Gambella (6%) and Benishangul-Gumuz (3%) regions. Within these regions are large agricultural developments with high numbers of seasonal migrant workers. The migrant workers are believed to be at increased risk for malaria infection due to their poor living conditions and outdoor activities, but there is little information on their specific behaviours and health risks. This study was conducted to address this gap. Methods Quantitative observations were conducted from September to December 2017 in the Benishangul-Gumuz Region. The nightly routines of mobile migrant workers were observed every month for 4 consecutive months. The study team collected quantitative data including nocturnal behavioural observations of worker living conditions, malaria prevention efforts, and work activities and surveys of worker representatives. Qualitative data was collected from migrant workers, farm managers and local health providers using focus group discussions and semi-structured interviews. Results Migrant workers arrived in the study area during the peak malaria transmission season and the workers in focus groups reported repeated cases of malaria during their stay on the farms. Overall, less than a quarter of the migrant workers were sleeping under a mosquito net by midnight in all 4 observation months. Some work activities also took place outdoors at night. The study additionally found a lack of access to malaria prevention and treatment at the farms and challenges in utilizing local public health facilities. Conclusions There is a need to better address malaria prevention and treatment needs among migrant workers in Ethiopia through outreach from existing healthcare infrastructure and within the farms themselves. This will help prevent malaria transmission both within this population and prevent transmission of malaria back to home communities in lower burden areas in Ethiopia.

Abstract Background Ethiopia has made great strides in malaria control over the last two decades. However, this progress has not been uniform and one concern has been reported high rates of malaria transmission in large agricultural development areas in western Ethiopia. Improved vector control is one way this transmission might be addressed, but little is known about malaria vectors in this part of the country. Methods To better understand the vector species involved in malaria transmission and their behaviour, human landing collections were conducted in Dangur woreda, Benishangul-Gumuz, between July and December 2017. This period encompasses the months with the highest rain and the peak mosquito population. Mosquitoes were identified to species and tested for the presence of Plasmodium sporozoites. Results The predominant species of the Anopheles collected was Anopheles arabiensis (1,733; i.e. 61.3 % of the entire Anopheles), which was also the only species identified with sporozoites (Plasmodium falciparum and Plasmodium vivax). Anopheles arabiensis was collected as early in the evening as 18:00 h-19:00 h, and host-seeking continued until 5:00 h-6:00 h. Nearly equal numbers were collected indoors and outdoors. The calculated entomological inoculation rate for An. arabiensis for the study period was 1.41 infectious bites per month. More An. arabiensis were collected inside and outside worker’s shelters than in fields where workers were working at night. Conclusions Anopheles arabiensis is likely to be the primary vector of malaria in the agricultural development areas studied. High rates of human biting took place inside and outdoor near workers’ residential housing. Improved and targeted vector control in this area might considerably reduce malaria transmission.

Abstract Background Pyrethroid-treated mosquito nets are currently the mainstay of vector control in Côte d’Ivoire. However, resistance to pyrethroids has been reported across the country, limiting options for insecticide resistance management due to the paucity of alternative insecticides. Two types of insecticide-treated nets (ITNs), ITNs with pyrethroids and the synergist piperonyl butoxide (PBO), and Interceptor®G2 nets, a net treated with a combination of chlorfenapyr and alpha-cypermethrin, are believed to help in the control of pyrethroid-resistant mosquitoes. Methods The susceptibility of Anopheles gambiae sensu lato (s.l.) to pyrethroid insecticides with and without pre-exposure to PBO as well as to chlorfenapyr was investigated in fifteen sites across the country. Susceptibility tests were conducted on 2- to 4-day old adult female An. gambiae s.l. reared from larval collections. The resistance status, intensity, and effects of PBO on mortality after exposure to different concentrations of deltamethrin, permethrin and alpha-cypermethrin were determined using WHO susceptibility test kits. In the absence of a WHO-recommended standard protocol for chlorfenapyr, two interim doses (100 and 200 µg/bottle) were used to test the susceptibility of mosquitoes using the CDC bottle assay method. Results Pre-exposure to PBO did not result in full restoration of susceptibility to any of the three pyrethroids for the An. gambiae s.l. populations from any of the sites surveyed. However, PBO pre-exposure did increase mortality for all three pyrethroids, particularly deltamethrin (from 4.4 to 48.9%). Anopheles gambiae s.l. from only one site (Bettie) were susceptible to chlorfenapyr at the dose of 100 µg active ingredient (a.i.)/bottle. At the dose of 200 µg (a.i.)/bottle, susceptibility was only recorded in 10 of the 15 sites. Conclusion Low mosquito mortality was found for pyrethroids alone, and while PBO increased mortality, it did not restore full susceptibility. The vector was not fully susceptible to chlorfenapyr in one third of the sites tested. However, vector susceptibility to chlorfenapyr seems to be considerably higher than for pyrethroids alone or with PBO. These data should be used cautiously when making ITN procurement decisions, noting that bioassays are conducted in controlled conditions and may not fully represent field efficacy where the host-seeking behaviours, which include free-flying activity are known to enhance pro-insecticide chlorfenapyr intoxication to mosquitoes.

Abstract Background Arboviral diseases, including dengue and chikungunya, are major public health concerns in Bangladesh where there have been unprecedented levels of transmission reported in recent years. The primary approach to control these diseases is to control the vector Aedes aegypti using pyrethroid insecticides. Although chemical control has long been practiced, no comprehensive analysis of Ae. aegypti susceptibility to insecticides has been conducted to date. The aim of this study was to determine the insecticide resistance status of Ae. aegypti in Bangladesh and investigate the role of detoxification enzymes and altered target site sensitivity as resistance mechanisms. Methods Eggs of Aedes mosquitoes were collected using ovitraps from five districts across Bangladesh and in eight neighborhoods of the capital city Dhaka, from August to November 2017. CDC bottle bioassays were conducted for permethrin, deltamethrin, malathion, and bendiocarb using 3- to 5-day-old F0–F2 non-blood-fed female mosquitoes. Biochemical assays were conducted to detect metabolic resistance mechanisms, and real-time PCR was performed to determine the frequencies of the knockdown resistance (kdr) mutations Gly1016, Cys1534, and Leu410. Results High levels of resistance to permethrin were detected in all Ae. aegypti populations, with mortality ranging from 0 to 14.8% at the diagnostic dose. Substantial resistance continued to be detected against higher (2×) doses of permethrin (5.1–44.4% mortality). Susceptibility to deltamethrin and malathion varied between populations while complete susceptibility to bendiocarb was observed in all populations. Significantly higher levels of esterase and oxidase activity were detected in most of the test populations as compared to the susceptible reference Rockefeller strain. A significant association was detected between permethrin resistance and the presence of Gly1016 and Cys1534 homozygotes. The frequency of kdr (knockdown resistance) alleles varied across the Dhaka Aedes populations. Leu410 was not detected in any of the tested populations. Conclusions The detection of widespread pyrethroid resistance and multiple resistance mechanisms highlights the urgency for implementing alternate Ae. aegypti control strategies. In addition, implementing routine monitoring of insecticide resistance in Ae. aegypti in Bangladesh will lead to a greater understanding of susceptibility trends over space and time, thereby enabling the development of improved control strategies.

Abstract Background Between 2011 and 2018, an estimated 134.8 million pyrethroid-treated long-lasting insecticidal nets (LLINs) were distributed nationwide in the Democratic Republic of Congo (DRC) for malaria control. Pyrethroid resistance has developed in DRC in recent years, but the intensity of resistance and impact on LLIN efficacy was not known. Therefore, the intensity of resistance of Anopheles gambiae sensu lato (s.l.) to permethrin and deltamethrin was monitored before and after a mass distribution of LLINs in Kinshasa in December 2016, and in 6 other sites across the country in 2017 and 11 sites in 2018. Methods In Kinshasa, CDC bottle bioassays using 1, 2, 5, and 10 times the diagnostic dose of permethrin and deltamethrin were conducted using An. gambiae s.l. collected as larvae and reared to adults. Bioassays were conducted in four sites in Kinshasa province 6 months before a mass distribution of deltamethrin-treated LLINs and then two, six, and 10 months after the distribution. One site in neighbouring Kongo Central province was used as a control (no mass campaign of LLIN distribution during the study). Nationwide intensity assays were conducted in six sites in 2017 using CDC bottle bioassays and in 11 sites in 2018 using WHO intensity assays. A sub-sample of An. gambiae s.l. was tested by PCR to determine species composition and frequency of kdr-1014F and 1014S alleles. Results In June 2016, before LLIN distribution, permethrin resistance intensity was high in Kinshasa; the mean mortality rate was 43% at the 5× concentration and 73% at the 10× concentration. Bioassays at 3 time points after LLIN distribution showed considerable variation by site and time and there was no consistent evidence for an increase in pyrethroid resistance intensity compared to the neighbouring control site. Tests of An. gambiae s.l. in 6 sites across the country in 2017 and 11 sites in 2018 showed all populations were resistant to the diagnostic doses of 3 pyrethroids. In 2018, the intensity of resistance varied by site, but was generally moderate for all three pyrethroids, with survivors at ×5 the diagnostic dose. Anopheles gambiae sensu stricto (s.s.) was the most common species identified across 11 sites in DRC, but in Kinshasa, An. gambiae s.s. (91%) and Anopheles coluzzii (8%) were sympatric. Conclusions Moderate or high intensity pyrethroid resistance was detected nationwide in DRC and is a serious threat to sustained malaria control with pyrethroid LLINs. Next generation nets (PBO nets or bi-treated nets) should be considered for mass distribution.

Abstract Background Several mosquito collection methods are routinely used in vector control programmes. However, they target different behaviours causing bias in estimation of species diversity and abundance. Given the paucity of mosquito trap data in West Africa, we compared the performance of five trap-lure combinations and Human Landing Catches (HLCs) in Guinea. Methods CDC light traps (LT), BG sentinel 2 traps (BG2T), gravid traps (GT) and Stealth traps (ST) were compared in a 5 × 5 Latin Square design in three villages in Guinea between June and July 2018. The ST, a portable trap which performs similarly to a LT but incorporates LEDs and incandescent light, was included since it has not been widely tested. BG2T were used with BG and MB5 lures instead of CO2 to test the efficacy of these attractants. HLCs were performed for 5 nights, but not as part of the Latin Square. A Generalised Linear Mixed Model was applied to compare the effect of the traps, sites and collection times on mosquito abundance. Species identification was confirmed using PCR-based analysis and Sanger sequencing. Results A total of 10,610 mosquitoes were captured across five traps. ST collected significantly more mosquitoes (7096) than the rest of the traps, but resulted in a higher number of damaged specimens. ST and BG2T collected the highest numbers of Anopheles gambiae (s.l.) and Aedes aegypti mosquitoes, respectively. HLCs captured predominantly An. coluzzii (41%) and hybrids of An. gambiae and An. coluzzii (36%) in contrast to the five traps, which captured predominantly An. melas (83%). The rural site (Senguelen) presented the highest abundance of mosquitoes and overall diversity in comparison with Fandie (semi-rural) and Maferinyah Centre I (semi-urban). Our results confirm the presence of four species for the first time in Guinea. Conclusions ST collected the highest number of mosquitoes suggesting this trap may play an important role for mosquito surveillance in Guinea and similar sites in West Africa. We recommend the incorporation of molecular tools in entomological studies since they have helped to identify 25 mosquito species in this area.

Abstract Background The recent detection of the South Asian malaria vector Anopheles stephensi in Ethiopia and other regions in the Horn of Africa has raised concerns about its potential impact on malaria transmission. We report here the findings of a survey for this species in eastern Ethiopia using both morphological and molecular methods for species identification. Methods Adult and larval/pupal collections were conducted at ten sites in eastern Ethiopia and Anopheles specimens were identified using standard morphological keys and genetic analysis. Results In total, 2231 morphologically identified An. stephensi were collected. A molecular approach incorporating both PCR endpoint assay and sequencing of portions of the internal transcribed spacer 2 (ITS2) and cytochrome c oxidase subunit 1 (cox1) loci confirmed the identity of the An. stephensi in most cases (119/124 of the morphologically identified An. stephensi confirmed molecularly). Additionally, we observed Aedes aegypti larvae and pupae at many of the An. stephensi larval habitats. Conclusions Our findings show that An. stephensi is widely distributed in eastern Ethiopia and highlight the need for further surveillance in the southern, western and northern parts of the country and throughout the Horn of Africa.

The number of dengue cases has increased dramatically over the past 20 years and is an important concern, particularly as the trends toward urbanization continue. While the majority of dengue cases are thought to be asymptomatic, it is unknown to what extent these contribute to transmission. A better understanding of their importance would help to guide control efforts. In 2019, a dengue outbreak in La Reunion resulted in more than 18,000 confirmed cases. Between October 2019 and August 2020, 19 clusters were investigated in the south, west, and east of the island, enabling the recruitment of 605 participants from 368 households within a 200 m radius of the home of the index cases (ICs). No active asymptomatic infections confirmed by RT-PCR were detected. Only 15% were possible asymptomatic dengue infections detected by the presence of anti-dengue IgM antibodies. Only 5.3% of the participants had a recent dengue infection confirmed by RT-PCR. Although the resurgence of dengue in La Réunion is very recent (2016), the rate of anti-dengue IgG positivity, a marker of past infections, was already high at 43% in this study. Dengue transmission was focal in time and space, as most cases were detected within a 100-m radius of the ICs, and within a time interval of less than 7 days between infections detected in a same cluster. No particular demographic or socio-cultural characteristics were associated with dengue infections. On the other hand, environmental risk factors such as type of housing or presence of rubbish in the streets were associated with dengue infections.

Vector-borne pathogens cause many human infectious diseases and are responsible for high mortality and morbidity throughout the world. They can also cause livestock epidemics with dramatic social and economic consequences. Due to its high costs, vector-borne disease surveillance is often limited to current threats, and the investigation of emerging pathogens typically occurs after the reports of clinical cases. Here, we use high-throughput sequencing to detect and identify a wide range of parasites and viruses carried by mosquitoes from Cambodia, Guinea, Mali and the USA. We apply this approach to individual Anopheles mosquitoes as well as pools of mosquitoes captured in traps; and compare the outcomes of this assay when applied to DNA or RNA. We identified known human and animal pathogens and mosquito parasites belonging to a wide range of taxa, as well as DNA sequences from previously uncharacterized organisms. Our results also revealed that analysis of the content of an entire trap could be an efficient approach to monitor and identify rare vector-borne pathogens in large surveillance studies. Overall, we describe a high-throughput and easy-to-customize assay to screen for a wide range of pathogens and efficiently complement current vector-borne disease surveillance approaches.

Abstract Background In recent years, the scale-up of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) has greatly reduced malaria transmission. However, malaria remains a global public health concern with the majority of the disease burden in sub-Saharan Africa. Insecticide resistance is a growing problem among Anopheles vector populations, with potential implications for the continued effectiveness of available control interventions. Improved understanding of current resistance levels and underlying mechanisms is essential to design appropriate management strategies and to mitigate future selection for resistance. Methods Anopheles gambiae sensu lato mosquitoes were collected from three villages in Faranah Prefecture, Guinea and their levels of susceptibility to seven insecticides were measured using CDC resistance intensity bioassays. Synergist assays with piperonyl butoxide (PBO) were also undertaken to assess the role of elevated mixed-function oxidases in resistance. Five hundred and sixty-three mosquitoes underwent molecular characterization of vector species, presence of target site mutations (L1014F kdr, N1575Y and G119S Ace-1), Plasmodium falciparum infection, and relative expression of three metabolic genes (CYP6M2, CYP6P3 and GSTD3). Results In Faranah, resistance to permethrin and deltamethrin was observed, as well as possible resistance to bendiocarb. All assayed vector populations were fully susceptible to alpha-cypermethrin, pirimiphos-methyl, clothianidin and chlorfenapyr. Plasmodium falciparum infection was detected in 7.3% (37/508) of mosquitoes tested. The L1014F kdr mutation was found in 100% of a sub-sample of 60 mosquitoes, supporting its fixation in the region. The N1575Y mutation was identified in 20% (113/561) of individuals, with ongoing selection evidenced by significant deviations from Hardy–Weinberg equilibrium. The G119S Ace-1 mutation was detected in 62.1% (18/29) of mosquitoes tested and was highly predictive of bendiocarb bioassay survival. The metabolic resistance genes, CYP6M2, CYP6P3 and GSTD3, were found to be overexpressed in wild resistant and susceptible An. gambiae sensu stricto populations, compared to a susceptible G3 colony. Furthermore, CYP6P3 was significantly overexpressed in bendiocarb survivors, implicating its potential role in carbamate resistance in Faranah. Conclusions Identification of intense resistance to permethrin and deltamethrin in Faranah, is of concern, as the Guinea National Malaria Control Programme (NMCP) relies exclusively on the distribution of pyrethroid-treated LLINs for vector control. Study findings will be used to guide current and future control strategies in the region.

Abstract Insecticide resistance across sub-Saharan Africa may impact the continued effectiveness of malaria vector control. We investigated the association between carbamate and pyrethroid resistance with Anopheles gambiae s.l. parity, Plasmodium falciparum infection, and molecular insecticide resistance mechanisms in Guinea. Pyrethroid resistance was intense, with field populations surviving ten times the insecticidal concentration required to kill susceptible individuals. The L1014F kdr-N1575Y haplotype and I1527T mutation were significantly associated with mosquito survival following permethrin exposure (Prevalence Ratio; PR = 1.92, CI = 1.09–3.37 and PR = 2.80, CI = 1.03–7.64, respectively). Partial restoration of pyrethroid susceptibility following synergist pre-exposure suggests a role for mixed-function oxidases. Carbamate resistance was lower and significantly associated with the G119S Ace-1 mutation. Oocyst rates were 6.8% and 4.2% among resistant and susceptible mosquitoes, respectively; survivors of bendiocarb exposure were significantly more likely to be infected. Pyrethroid resistant mosquitoes had significantly lower parity rates than their susceptible counterparts (PR = 1.15, CI = 1.10–1.21). Our findings emphasize the need for additional studies directly assessing the influence of insecticide resistance on mosquito fitness.

Wolbachia, a widespread bacterium which can influence mosquito-borne pathogen transmission, has recently been detected within Anopheles (An.) species that are malaria vectors in Sub-Saharan Africa. Although studies have reported Wolbachia strains in the An. gambiae complex, apparent low density and prevalence rates require confirmation. In this study, wild Anopheles mosquitoes collected from two regions of Guinea were investigated. In contrast with previous studies, RNA was extracted from adult females (n = 516) to increase the chances for the detection of actively expressed Wolbachia genes, determine Wolbachia prevalence rates and estimate relative strain densities. Molecular confirmation of mosquito species and Wolbachia multilocus sequence typing (MLST) were carried out to analyse phylogenetic relationships of mosquito hosts and newly discovered Wolbachia strains. Strains were detected in An. melas (prevalence rate of 11.6%–16/138) and hybrids between An. melas and An. gambiae sensu stricto (prevalence rate of 40.0%–6/15) from Senguelen in the Maferinyah region. Furthermore, a novel high-density strain, termed wAnsX, was found in an unclassified Anopheles species. The discovery of novel Wolbachia strains (particularly in members, and hybrids, of the An. gambiae complex) provides further candidate strains that could be used for future Wolbachia-based malaria biocontrol strategies.

Durability monitoring of insecticide-treated nets (ITNs) containing a pyrethroid in combination with a second active ingredient (AI) must be adapted so that the insecticidal bioefficacy of each AI can be monitored independently. An effective way to do this is to measure rapid knock down of a pyrethroid-susceptible strain of mosquitoes to assess the bioefficacy of the pyrethroid component and to use a pyrethroid-resistant strain to measure the bioefficacy of the second ingredient. To allow robust comparison of results across tests within and between test facilities, and over time, protocols for bioefficacy testing must include either characterisation of the resistant strain, standardisation of the mosquitoes used for bioassays, or a combination of the two. Through a series of virtual meetings, key stakeholders and practitioners explored different approaches to achieving these goals. Via an iterative process we decided on the preferred approach and produced a protocol consisting of characterising mosquitoes used for bioefficacy testing before and after a round of bioassays, for example at each time point in a durability monitoring study. We present the final protocol and justify our approach to establishing a standard methodology for durability monitoring of ITNs containing pyrethroid and a second AI.

Abstract Background Globally, the Democratic Republic of Congo (DRC) accounted for 9% of malaria cases and 10% of malaria deaths in 2015. As part of control efforts, more than 40 million long-lasting insecticidal nets (LLINs) were distributed between 2008 and 2013, resulting in 70% of households owning one or more LLINs in 2014. To optimize vector control efforts, it is critical to monitor vector behaviour and insecticide resistance trends. Entomological data was collected from eight sentinel sites throughout DRC between 2013 and 2016 in Kingasani, Mikalayi, Lodja, Kabondo, Katana, Kapolowe, Tshikaji and Kalemie. Mosquito species present, relative densities and biting times were monitored using human landing catches (HLC) conducted in eight houses, three times per year. HLC was conducted monthly in Lodja and Kapolowe during 2016 to assess seasonal dynamics. Laboratory data included resistance mechanism frequency and sporozoite rates. Insecticide susceptibility testing was conducted with commonly used insecticides including deltamethrin and permethrin. Synergist bioassays were conducted with PBO to determine the role of oxidases in permethrin resistance. Results In Lodja, monthly Anopheles gambiae s.l. biting rates were consistently high at > 10 bites/person/night indoors and outdoors. In Kapolowe, An. gambiae s.l. dominated during the rainy season, and Anopheles funestus s.l. during the dry season. In all sites, An. gambiae and An. funestus biting occurred mostly late at night. In Kapolowe, significant biting of both species started around 19:00, typically before householders use nets. Sporozoite rates were high, with a mean of 4.3% (95% CI 3.4–5.2) for An. gambiae and 3.3% (95% CI 1.3–5.3) for An. funestus. Anopheles gambiae were resistant to permethrin in six out of seven sites in 2016. In three sites, susceptibility to deltamethrin was observed despite high frequency permethrin resistance, indicating the presence of pyrethroid-specific resistance mechanisms. Pre-exposure to PBO increased absolute permethrin-associated mortality by 24%, indicating that resistance was partly due to metabolic mechanisms. The kdr-1014F mutation in An. gambiae was present at high frequency (> 70%) in three sites (Kabondo, Kingasani and Tshikaji), and lower frequency (

In response to growing concerns over the sustained effectiveness of pyrethroid-only based control tools, new products are being developed and evaluated. Some examples of these are dual-active ingredient (AI) insecticide-treated nets (ITNs) which contain secondary insecticides, or synergist ITNs which contain insecticide synergist, both in combination with a pyrethroid. These net types are often termed ‘next-generation’ insecticide-treated nets. Several of these new types of ITNs are being evaluated in large-scale randomized control trials (RCTs) and pilot deployment schemes at a country level. However, no methods for measuring the biological durability of the AIs or synergists on these products are currently recommended. In this publication, we describe a pipeline used to collate and interrogate several different methods to produce a singular ‘consensus standard operating procedure (SOP)’, for monitoring the biological durability of three new types of ITNs: pyrethroid + piperonyl butoxide (PBO), pyrethroid + pyriproxyfen (PPF), and pyrethroid + chlorfenapyr (CFP). This process, convened under the auspices of the Innovation to Impact programme, sought to align methodologies used for conducting durability monitoring activities of next-generation ITNs.

Abstract Background Indoor residual spraying is one of the key vector control interventions for malaria control in Ethiopia. As malaria transmission is seasonal in most parts of Ethiopia, a single round of spraying can usually provide effective protection against malaria, provided the insecticide remains effective over the entire malaria transmission season. This experiment was designed to evaluate the residual efficacy of bendiocarb, pirimiphos-methyl, and two doses of propoxur on four different wall surfaces (rough mud, smooth mud, dung, and paint). Filter papers affixed to wall surfaces prior to spraying were analyzed to determine the actual concentration applied. Cone bioassays using a susceptible Anopheles arabiensis strain were done monthly to determine the time for which insecticides were effective in killing mosquitoes. Results The mean insecticide dosage of bendiocarb applied to walls was 486 mg/m2 (target 400/mg). This treatment lasted 1 month or less on rough mud, smooth mud, and dung, but 4 months on painted surfaces. Pirimiphos-methyl was applied at 1854 mg/m2 (target 1000 mg/m2), and lasted between 4 and 6 months on all wall surfaces. Propoxur with a target dose of 1000 mg/m2 was applied at 320 mg/m2, and lasted 2 months or less on all surfaces, except painted surfaces (4 months). Propoxur with a target dose of 2000 mg/m2, was applied at 638 mg/m2, and lasted 3 months on rough mud, but considerably longer (5–7 months) on the other substrates. Conclusions It would appear that the higher dose of propoxur and pirimiphos-methyl correspond best to the Ethiopian transmission season, although interactions between insecticide and the substrate should be taken into account as well. However, the insecticide quantification revealed that the dosages actually applied differed considerably from the target dosages, even though care was taken in the mixing of insecticide formulations and spraying of the walls. It is unclear whether this variability is due to initial concentrations of insecticides, poor application, or other factors. Further work is needed to ensure that target doses are correctly applied, both operationally and in insecticide evaluations.

Abstract Background Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) remain the cornerstones of malaria vector control. However, the development of insecticide resistance and its implications for operational failure of preventative strategies are of concern. The aim of this study was to characterize insecticide resistance among Anopheles arabiensis populations in Ethiopia and describe temporal and spatial patterns of resistance between 2012 and 2016. Methods Between 2012 and 2016, resistance status of An. arabiensis was assessed annually during the long rainy seasons in study sites from seven of the nine regions in Ethiopia. Insecticide resistance levels were measured with WHO susceptibility tests and CDC bottle bioassays using insecticides from four chemical classes (organochlorines, pyrethroids, organophosphates and carbamates), with minor variations in insecticides tested and assays conducted between years. In selected sites, CDC synergist assays were performed by pre-exposing mosquitoes to piperonyl butoxide (PBO). In 2015 and 2016, mosquitoes from DDT and deltamethrin bioassays were randomly selected, identified to species-level and screened for knockdown resistance (kdr) by PCR. Results Intense resistance to DDT and pyrethroids was pervasive across Ethiopia, consistent with historic use of DDT for IRS and concomitant increases in insecticide-treated net coverage over the last 15 years. Longitudinal resistance trends to malathion, bendiocarb, propoxur and pirimiphos-methyl corresponded to shifts in the national insecticide policy. By 2016, resistance to the latter two insecticides had emerged, with the potential to jeopardize future long-term effectiveness of vector control activities in these areas. Between 2015 and 2016, the West African (L1014F) kdr allele was detected in 74.1% (n = 686/926) of specimens, with frequencies ranging from 31 to 100% and 33 to 100% in survivors from DDT and deltamethrin bioassays, respectively. Restoration of mosquito susceptibility, following pre-exposure to PBO, along with a lack of association between kdr allele frequency and An. arabiensis mortality rate, both indicate metabolic and target-site mutation mechanisms are contributing to insecticide resistance. Conclusions Data generated by this study will strengthen the National Malaria Control Programme’s insecticide resistance management strategy to safeguard continued efficacy of IRS and other malaria control methods in Ethiopia.

Abstract Background Diseases caused by mosquito-borne pathogens remain an important source of morbidity and mortality in Bangladesh. To better control the vectors that transmit the agents of disease, and hence the diseases they cause, and to appreciate the diversity of the family Culicidae, it is important to have an up-to-date list of the species present in the country. Original records were collected from a literature review to compile a list of the species recorded in Bangladesh. Results Records for 123 species were collected, although some species had only a single record. This is an increase of ten species over the most recent complete list, compiled nearly 30 years ago. Collection records of three additional species are included here: Anopheles pseudowillmori, Armigeres malayi and Mimomyia luzonensis. Conclusions While this work constitutes the most complete list of mosquito species collected in Bangladesh, further work is needed to refine this list and understand the distributions of those species within the country. Improved morphological and molecular methods of identification will allow the refinement of this list in years to come.

Background: Wolbachia, a common insect endosymbiotic bacterium that can influence pathogen transmission and manipulate host reproduction, has historically been considered absent from the Anopheles (An.) genera, but has recently been found in An. gambiae s.l. populations in West Africa. As there are numerous Anopheles species that have the capacity to transmit malaria, we analysed a range of species across five malaria endemic countries to determine Wolbachia prevalence rates, characterise novel Wolbachia strains and determine any correlation between the presence of Plasmodium, Wolbachia and the competing bacterium Asaia. Methods: Anopheles adult mosquitoes were collected from five malaria-endemic countries: Guinea, Democratic Republic of the Congo (DRC), Ghana, Uganda and Madagascar, between 2013 and 2017. Molecular analysis was undertaken using quantitative PCR, Sanger sequencing, Wolbachia multilocus sequence typing (MLST) and high-throughput amplicon sequencing of the bacterial 16S rRNA gene. Results: Novel Wolbachia strains were discovered in five species: An. coluzzii, An. gambiae s.s., An. arabiensis, An. moucheti and An. species A, increasing the number of Anopheles species known to be naturally infected. Variable prevalence rates in different locations were observed and novel strains were phylogenetically diverse, clustering with Wolbachia supergroup B strains. We also provide evidence for resident strain variants within An. species A. Wolbachia is the dominant member of the microbiome in An. moucheti and An. species A but present at lower densities in An. coluzzii. Interestingly, no evidence of Wolbachia/Asaia co-infections was seen and Asaia infection densities were shown to be variable and location dependent. Conclusions: The important discovery of novel Wolbachia strains in Anopheles provides greater insight into the prevalence of resident Wolbachia strains in diverse malaria vectors. Novel Wolbachia strains (particularly high-density strains) are ideal candidate strains for transinfection to create stable infections in other Anopheles mosquito species, which could be used for population replacement or suppression control strategies.

Background: Wolbachia, a common insect endosymbiotic bacterium that can influence pathogen transmission and manipulate host reproduction, has historically been considered absent from the Anopheles (An.) genera, but has recently been found in An. gambiae s.l. populations. As there are numerous Anopheles species that have the capacity to transmit malaria, we analysed a range of species to determine Wolbachia prevalence rates, characterise novel Wolbachia strains and determine any correlation between the presence of Plasmodium, Wolbachia and the competing endosymbiotic bacterium Asaia. Methods: Anopheles adult mosquitoes were collected from five malaria-endemic countries: Guinea, Democratic Republic of the Congo (DRC), Ghana, Uganda and Madagascar, between 2013 and 2017. Molecular analysis of samples was undertaken using quantitative PCR, Sanger sequencing, Wolbachia multilocus sequence typing (MLST) and high-throughput amplicon sequencing of the bacterial 16S rRNA gene. Results: Novel Wolbachia strains were discovered in five species: An. coluzzii, An. gambiae s.s., An. arabiensis, An. moucheti and An. species ‘A’, increasing the number of Anopheles species known to be naturally infected. Variable prevalence rates in different locations were observed and novel strains were phylogenetically diverse, clustering with Wolbachia supergroup B strains. We also provide evidence for resident strain variants within An. species ‘A’. Wolbachia is the dominant member of the microbiome in An. moucheti and An. species ‘A’, but present at lower densities in An. coluzzii. Interestingly, no evidence of Wolbachia/Asaia co-infections was seen and Asaia infection densities were also shown to be variable and location dependent. Conclusions: The important discovery of novel Wolbachia strains in Anopheles provides greater insight into the prevalence of resident Wolbachia strains in diverse malaria vectors. Novel Wolbachia strains (particularly high-density strains) are ideal candidate strains for transinfection to create stable infections in other Anopheles mosquito species, which could be used for population replacement or suppression control strategies.

Summary: Background: The relationship between agriculture, Anopheles mosquitoes, and malaria in Africa is not fully understood, but it is important for malaria control as countries consider expanding agricultural projects to address population growth and food demand. Therefore, we aimed to assess the effect of agriculture on Anopheles biting behaviour and malaria risk in children in rural areas of the Democratic Republic of the Congo (DR Congo). Methods: We did a population-based, cross-sectional, spatial study of rural children (0]=0·89), with the probability of malaria infection increased between 0·2% (95% UI −0·1 to 3·4) and 2·6% (–1·5 to 6·6) given a 15% increase in agricultural cover, depending on other risk factors. The models predicted that large increases in agricultural cover (from 0% to 75%) increase the probability of infection by as much as 13·1% (95% UI −7·3 to 28·9). Increased risk might be due to Anopheles gambiae sensu lato, whose probability of biting indoors increased between 11·3% (95% UI −15·3 to 25·6) and 19·7% (–12·1 to 35·9) with a 15% increase in agriculture. Interpretation: Malaria control programmes must consider the possibility of increased risk due to expanding agriculture. Governments considering initiating large-scale agricultural projects should therefore also consider accompanying additional malaria control measures. Funding: National Institutes of Health, National Science Foundation, Bill & Melinda Gates Foundation, President's Malaria Initiative, and Royster Society of Fellows at the University of North Carolina at Chapel Hill.