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Background The primary malaria vector-control interventions, indoor residual spraying and long-lasting insecticidal nets, are effective against indoor biting and resting mosquito species. Consequently, outdoor biting and resting malaria vectors might elude the primary interventions and sustain malaria transmission. Varied vector biting and resting behaviour calls for robust entomological surveillance. This study investigated the bionomics of malaria vectors in rural south-east Zambia, focusing on species composition, their resting and host-seeking behaviour and sporozoite infection rates. Methods The study was conducted in Nyimba District, Zambia. Randomly selected households served as sentinel houses for monthly collection of mosquitoes indoors using CDC-light traps (CDC-LTs) and pyrethrum spray catches (PSC), and outdoors using only CDC-LTs for 12 months. Mosquitoes were identified using morphological taxonomic keys. Specimens belonging to the Anopheles gambiae complex and Anopheles funestus group were further identified using molecular techniques. Plasmodium falciparum sporozoite infection was determined using sandwich enzyme-linked immunosorbent assays. Results From 304 indoor and 257 outdoor light trap-nights and 420 resting collection, 1409 female Anopheles species mosquitoes were collected and identified morphologically; An. funestus (n = 613; 43.5%), An. gambiae sensu lato ( s.l. )(n = 293; 20.8%), Anopheles pretoriensis (n = 282; 20.0%), Anopheles maculipalpis (n = 130; 9.2%), Anopheles rufipes (n = 55; 3.9%), Anopheles coustani s.l. (n = 33; 2.3%), and Anopheles squamosus (n = 3, 0.2%). Anopheles funestus sensu stricto ( s.s .) (n = 144; 91.1%) and Anopheles arabiensis (n = 77; 77.0%) were the dominant species within the An. funestus group and An. gambiae complex, respectively. Overall, outdoor CDC-LTs captured more Anopheles mosquitoes (mean = 2.25, 95% CI 1.22–3,28) than indoor CDC-LTs (mean = 2.13, 95% CI 1.54–2.73). Fewer resting mosquitoes were collected with PSC (mean = 0.44, 95% CI 0.24–0.63). Sporozoite infectivity rates for An. funestus, An. arabiensis and An. rufipes were 2.5%, 0.57% and 9.1%, respectively. Indoor entomological inoculation rates (EIRs) for An. funestus s.s , An. arabiensis and An. rufipes were estimated at 4.44, 1.15 and 1.20 infectious bites/person/year respectively. Outdoor EIRs for An. funestus s.s. and An. rufipes at 7.19 and 4.31 infectious bites/person/year, respectively. Conclusion The findings of this study suggest that An. rufipes may play an important role in malaria transmission alongside An. funestus s.s. and An. arabiensis in the study location. Graphical Abstract

Background Community Health Workers (CHWs) play a crucial role in malaria control efforts, yet their contributions to large-scale field trials remain understudied. This research examined the management of CHWs recruited to support a phase III trial of Attractive Targeted Sugar Baits (ATSBs) in Western Zambia. The study aimed to understand the extent to which CHW involvement in the trial aligned with best practices and challenges faced by CHWs implementing health promotion activities. Methods A literature review, review of Zambia Ministry of Health Guidelines, and review of WHO guidelines was conducted to identify best practices and common challenges for CHWs contributing to the delivery of malaria interventions. Subsequently ATSB trial documents were reviewed to compare CHW involvement and CHW challenges experienced during the ATSB trial from 2021 to 2023. A comparative analysis was utilized to assess the involvement of CHWs in the ATSB trial against literature review findings, specific to CHW recruitment, training, supervision, incentivization, and community support. Results Five best practices for CHW delivery of interventions were identified: participatory and gender equitable recruitment and selection; comprehensive training; incentivization for enhanced motivation and performance; high quality supportive supervision; and fostering community support. Five common challenges for involving CHWs in programme delivery were identified: inadequate and poor-quality supervision of CHWs; low compensation and motivation; logistical constraints; workload and multiplicity of roles, and community challenges. The analysis found that ATSB trial practices largely aligned with literature best practices and established guidelines, particularly in training and incentive structures. However, challenges were identified in achieving gender balance (32% female CHWs in year 2), community involvement in CHW selection, and coordination between CHWs and research team members. CHW involvement was a key factor towards successfully implementing the trial protocol. Conclusions Experiences with CHWs involvement in the ATSB trial in Zambia suggest that some facets of field research readily facilitate adherence to best practices for CHW recruitment and management. Additionally, field research that is adequately funded and characterized by rigorous implementation may avoid some of the common challenges faced by CHWs implementing health promotion activities. Nonetheless, some CHW cadre challenges appear universal to programmatic and research contexts, including an imbalance in gender representation favouring male participation in CHW opportunities. More documentation of research experiences may be needed to understand CHW involvement and experiences in field research outside of randomized controlled trials.

Background Attractive targeted sugar bait (ATSB) stations containing bait (to attract) and ingestion toxicant (to kill) sugar-foraging mosquitoes are hypothesized to reduce malaria transmission by shortening the lifespan of Anopheles vectors. Methods A two-arm cluster-randomized controlled trial (cRCT) was conducted in Western Province Zambia. Seventy clusters of 250–350 households were assigned (1:1) by restricted randomization to an intervention arm (ATSB) or control arm (no ATSB) in the context of standard of care vector control (insecticide-treated nets and/or indoor residual spraying). Two ATSB stations (Westham Sarabi, 0.11% dinotefuran w/w) were maintained on exterior walls of eligible household structures for a 7-month deployment period (December-June) during the high malaria transmission season. The primary outcome was clinical malaria incidence among two consecutive seasonal cohorts of children aged 1–14 years, followed-up monthly from January-June in 2022 and 2023. Secondary outcome was Plasmodium falciparum prevalence among individuals aged over six months. Analysis compared clinical malaria incidence and prevalence between arms among the intention-to-treat population. Results ATSB coverage, assessed by cross-sectional survey, was 98.3% in March–April 2022 and 89.5% in March–April 2023. 4494 children contributed any follow-up time to the cohort, with 2313 incident malaria cases in the intervention arm (1.28 per child per six-month transmission season), and 2449 in the control arm (1.38 per child-season). The incidence rate ratio between the two arms was 0.91 (95% CI 0.72–1.15, p  = 0.42). 2536 individuals participated in cross-sectional surveys, with prevalence of P. falciparum 50.7% in the intervention arm and 53.5% in the control arm. The odds ratio between the two arms was 0.89 (95% CI 0.66–1.18, p  = 0.42). Secondary covariable-adjusted and subgroup analyses did not substantially alter the findings. No serious adverse events associated with the intervention were reported. Conclusions Two ATSB stations deployed per eligible structure for two consecutive transmission seasons did not result in a statistically significant reduction in clinical malaria incidence among children aged 1–14 years or in P. falciparum prevalence in rural western Zambia. Further studies are needed to assess the efficacy of ATSB stations in different settings and with different deployment strategies. Trial registration The trial is registered with Clinicaltrials.gov (NCT04800055).

Background House screening remains conspicuously absent in national malaria programs despite its recognition by the World Health Organization as a supplementary malaria vector-control intervention. This may be attributed, in part, to the knowledge gap in screen durability or longevity in local climatic conditions and community acceptance under specific cultural practices and socio-economic contexts. The objectives of this study were to assess the durability of window and door wire mesh screens a year after full house screening and to assess the acceptability of the house screening intervention to the participants involved. Methods This study was conducted in Nyimba district, Zambia and used both quantitative and qualitative methods of data collection and analysis. Both direct observation and questionnaires were employed to assess the durability of the screens and the main reasons for damage. Findings on damage were summarized as percentages. Focus group discussions were used to assess people’s knowledge, perceptions, and acceptability of the closing eaves and house screening intervention. Deductive coding and inductive coding were used to analyse the qualitative data. Results A total of 321 out of 400 (80.3%) household owners of screened houses were interviewed. Many window screens (90.3%) were intact. In sharp contrast, most door screens were torn ( n  = 150; 46.7%) or entirely removed ( n  = 55; 17.1%). Most doors ( n  = 114; 76%) had their wire mesh damaged or removed on the bottom half. Goats (25.4%), rust (17.6%) and children (17.1%) were cited most as the cause of damage to door screens. The focus group discussion elicited positive experiences from the participants following the closing of eaves and screening of their windows and doors, ranging from sleeping peacefully due to reduced mosquito biting and/or nuisance and having fewer insects in the house. Participants linked house screening to reduced malaria in their households and community. Conclusion This study demonstrated that in rural south-east Zambia, closing eaves and screening windows and doors was widely accepted. Participants perceived that house screening reduced human-vector contact, reduced the malaria burden and nuisance biting from other potentially disease carrying insects. However, screened doors are prone to damage, mainly by children, domestic animals, rust, and termites.

Background Attractive Targeted Sugar Baits (ATSBs) are a proposed new vector control tool for malaria that contain sugar and an ingestion toxicant, and are designed to attract and kill sugar-feeding mosquitoes. During a two-arm cluster randomized Phase III trial conducted in Zambia to test the efficacy of ATSB stations on malaria incidence, ATSB stations deployed on eligible household structures within intervention clusters were routinely monitored to ensure their good physical condition and high coverage. This study investigates trends in prevalence and rate of damage to ATSB stations during year 2 of the two-year trial. Methods The analysis was conducted using monitoring data collected in year 2, which included types of damage observed, location, and date of removal and/or replacement of ATSB stations. The study evaluated temporal trends in the prevalence of overall damage and different damage types among 68,299 ATSB stations deployed. A profile of all ATSB stations installed on each structure was constructed, and spatial analyses conducted on overall damage and different damage types observed on 18,890 structures. Mixed effects regression analyses were conducted to investigate drivers of damage to ATSB stations on these structures. Results Prevalence of overall damage and different damage types was temporally and spatially heterogeneous. Among damaged ATSB stations observed during monitoring, tears and mold had the highest prevalences on average, with tears maintaining above 50.0% prevalence through most of the monitoring period, while mold prevalence increased steadily during the first few months, peaking in February. Overall, 45.6% of structures had at least one damaged ATSB station, however this varied spatially across the trial site. Both structure characteristics and environmental factors significantly impacted the odds and rate of damage to ATSB stations on structures, including: ATSB stations’ level of protection from rainfall and sunshine; roof and wall material of the structure; night-time temperature; rainfall; enhanced vegetation index, and land cover. Conclusion Damage to ATSB stations in this setting was common and was temporally and spatially heterogeneous. This has implications on operational feasibility, sustainability, and cost of future deployment. Further research is required to understand the mechanisms of damage, and to minimize prevalence and rate of damage to ATSB stations.

Background Vector control is the most important malaria prevention strategy in Zambia. Attractive Targeted Sugar Baits (ATSB) are a potential new tool for vector control in this setting, which, if efficacious, would be intended to supplement insecticide-treated bed nets (ITNs) and indoor residual spraying (IRS). ATSBs target and kill sugar feeding mosquitoes, potentially limiting the spread of malaria. No information on the cost or cost-effectiveness of deployment of ATSB stations is currently available. Methods A cluster randomized control trial (cRCT) was carried out in Western Province, Zambia to assess the efficacy of Sarabi v.1.2 ATSB stations in a highly malarious setting. Costs associated with the procurement, distribution, maintenance, and disposal of the ATSB stations were collected over a two-year period. These costs were assessed alongside the main trial efficacy outcomes to determine cost-effectiveness and potential budget impact on the deployment of ATSB stations in this setting. Total costs, incremental costs, incremental cost-effectiveness ratios (ICER) and budget impact were estimated using trial data. One-way, scenario and probabilistic sensitivity analysis were performed to further determine the impact of assumptions and uncertainty on cost-effectiveness estimates, and the potential cost implications of alternative deployment scenarios. Sub-group analysis was performed to determine the impact of deployment in settings with the most favorable effect scenarios. Results The total cost of the intervention in the context of the cRCT was USD 1,261,515. ATSB cost accounted for 46% of the total cost followed by personnel (25%), supplies and transport (13% each), equipment (2%) and storage (1%). Over the two year (14-month intervention) this resulted in an estimated ICER of USD 79 per malaria incident case averted or USD 919 per disability-adjusted life year (DALY) averted and USD 10.08 per person-year protected. In a subset of high-density ATSB clusters ICER was USD 42 per incident case averted and USD 493 per DALY averted and USD 4.35 per person-year protected. Probabilistic sensitivity analysis indicated that deployment in areas with higher structure density may be more cost-effective, especially if potential cost-savings are considered. However, effect estimates in this subgroup were highly uncertain and not statistically significant. While the scenario appeared more cost-effective than the base case on the cost-effectiveness acceptability curve (CEAC), the probability of cost-effectiveness reached only around 70%, falling short of the commonly used 80% threshold and remaining relatively weak. Conclusions ATSB Sarabi v.1.2 as deployed in western Zambia were not likely to be cost -effective. ATSB would need to demonstrate higher or more certain efficacy along with affordable alternative distribution strategies prior to any deployment at scale. Trial registration The trial is registered on clinicaltrials.gov under registration number: NCT04800055.

The zoophilic and exophilic traits of outdoor-biting Anopheles have led to this group largely being overlooked for their role in malaria transmission, despite several species now recognized as locally important in regions of sub-Saharan Africa. Given the current limitations with identification of these understudied species, it is crucial to accurately correlate morphological features to molecular data. Here, we produced high quality reference sequence data for representative understudied anopheline species to better understand the phylogenetic relationships between under- and well-studied vectors of malaria. For mitochondrial genome assembly, shallow shotgun sequencing was implemented on single mosquito specimens and phylogenetic analyses were performed on the concatenated protein coding genes of the mitogenomes using a Bayesian approach. This study generated 10 complete mitogenomes focusing on less studied taxa with an average length 15,380 bp and A-T content of 77.4% consistent with other anophelines containing 37 genes. Bayesian inference analysis yielded four main clades with molecular dating indicating that well-studied malaria vectors diverged from outdoor-biting species more than 63 million years ago. These findings support the taxonomic grouping of mosquitoes belonging to the Anopheles genus based on morphological characteristics and can provide molecular diagnostics for species identification enabling more precise and adept interventions for malaria control.

Background Understanding the composition, host feeding patterns, and infection status of all potential vectors is essential to understand changing disease transmission dynamics and inform future vector control strategies . This study assessed anopheline species composition, abundance, distribution, and host feeding preferences in Southern Province, Zambia to identify potential contributors to residual malaria transmission beyond primary vectors. Methods Adult mosquitoes were collected along a 100 km transect between the districts of Pemba, Monze and Gwembe in Southern Province, Zambia, an area of low malaria transmission. Mosquitoes were sampled from 12 randomly selected households at 5 km intervals along the transect. Centers for Disease Control and Prevention light traps were used to collect adult mosquitoes indoors and outdoors. Mosquitoes were identified using morphological keys and molecular approaches. A multiplexed PCR assay was utilized to identify mammalian hosts and Plasmodium falciparum (Pf) sporozoite infection was determined using an enzyme linked immunosorbent assay. Results A total of 5697 female anopheline mosquitoes were collected from both indoor and outdoor traps, revealing a diverse range of species. Nine species of Anopheles mosquitoes were identified, dominated by Anopheles rufipes (n = 1527, 26.8%), and An. pretoriensis (n = 1427, 25.1%). Host blood meal analysis of 401 visibly blood-fed mosquitoes revealed that Anopheles species in the study area fed primarily on goats (n = 176; 62.5%), humans (n = 55; 19.4%) and cows (n = 29; 10.2%). The human blood index across all captured anophelines was estimated at 0.22. Human blood meals were detected in secondary malaria vectors, with notably high HBI values of An. pretoriensis and An. rufipes mosquitoes captured outdoors near goat pens. Out of the 5697 female anophelines tested for sporozoite infectivity, one An. rufipes specimen was positive for P. falciparum. Conclusion This study demonstrates that malaria transmission risk in the surveyed areas may extend beyond well-recognized primary vectors. Species that are not well studied, often exophagic and assumed to be zoophagic, were found to have fed on humans and the study showed some preliminary evidence of P. falciparum infectivity. These findings indicate that peri-domestic human activity may sustain residual transmission despite high Insecticidal-Treated Nets and indoor residual spraying coverage.

Background The primary vector control interventions in Zambia are long-lasting insecticidal nets and indoor residual spraying. Challenges with these interventions include insecticide resistance and the outdoor biting and resting behaviours of many Anopheles mosquitoes. Therefore, new vector control tools targeting additional mosquito behaviours are needed to interrupt transmission. Attractive targeted sugar bait (ATSB) stations, which exploit the sugar feeding behaviours of mosquitoes, may help in this role. This study evaluated the residual laboratory bioefficacy of Westham prototype ATSB® Sarabi v.1.2.1 Bait Station (Westham Ltd., Hod-Hasharon, Israel) in killing malaria vectors in Western Province, Zambia, during the first year of a large cluster randomized phase-III trial (Clinical Trials.gov Identifier: NCT04800055). Methods This was a repeat cross-sectional study conducted within three districts, Nkeyema, Kaoma, and Luampa, in Western Province, Zambia. The study was conducted in 12 intervention clusters among the 70 trial clusters (35 interventions, 35 controls) between December 2021 and June 2022. Twelve undamaged bait stations installed on the outer walls of households were collected monthly (one per cluster per month) for bioassays utilizing adult female and male Anopheles gambiae sensu stricto (Kisumu strain) mosquitoes from a laboratory colony. Results A total of 84 field-deployed ATSB stations were collected, and 71 ultimately met the study inclusion criteria for remaining in good condition. Field-deployed stations that remained in good condition (intact, non-depleted of bait, and free of dirt as well as mold) retained high levels of bioefficacy (mean induced mortality of 95.3% in males, 71.3% in females, 83.9% combined total) over seven months in the field but did induce lower mortality rates than non-deployed ATSB stations (mean induced mortality of 96.4% in males, 87.0% in females, 91.4% combined total). There was relatively little variation in corrected mortality rates between monthly rounds for those ATSB stations that had been deployed to the field. Conclusion While field-deployed ATSB stations induced lower mortality rates than non-deployed ATSB stations, these stations nonetheless retained relatively high and stable levels of bioefficacy across the 7-month malaria transmission season. While overall mean mosquito mortality rates exceeded 80%, mean mortality rates for females were 24 percentage points lower than among males and these differences merit attention and further evaluation in future studies. The duration of deployment was not associated with lower bioefficacy. Westham prototype ATSB stations can still retain bioefficacy even after deployment in the field for 7 months, provided they do not meet predetermined criteria for replacement.

Background Anopheles squamosus is a widespread mosquito species in sub-Saharan Africa. It is a potential vector for human malaria parasites and has been found naturally infected with Plasmodium falciparum and Plasmodium vivax . Morphological identification is challenging even with pristine specimens and current molecular methods such as the use of the internal transcribed spacer 2 (ITS2) polymerase chain reaction (PCR) cannot distinguish An. squamosus from morphologically similar Anopheles species . Described in the following methods is the development and validation of a new PCR assay that will reliably identify An. squamosus . Methods Multiple alignments of previously published ITS2 contig sequences in NCBI from An. squamosus and An. species 11 and 15, were used to identify candidate ITS2 regions for primer design. Six sets of primers were evaluated overall for specificity of species identification. The one set with An. squamosus species-specific amplification was tested using 78 specimens morphologically identified from Zambia and South Africa. Results A new assay consisting of a forward (ITS2-ASQ-R10, 5’-CCC TCG AAG GGT GCT GTG-3’) and reverse (ITS2-ASQ-R10 5’-AAT CCA CGG TGT GAT GGC-3’) primer reliably (> 94.9%) amplified an ITS2 fragment of 301 bp length for An. squamosus . The An. squamosus- specific primer set can be multiplexed with existing ITS2 assays frequently used for anopheline species identification. Conclusions The development of this robust PCR assay for An. squamosus is vital to accurate identification of this species in malaria vector surveillance efforts. Improved understanding of the anopheline community composition will lead to better targeted methods of vector eradication and malaria prevention. To further the validation of this ITS2 PCR assay, more species of Anopheles should be compared in addition to An. squamosus collected in different regions. To refine and optimize the PCR process with these primers, touchdown PCR can be used to increase specificity. Applying genomic tools to correctly identify An. squamosus will allow for a better understanding of their role in malaria transmission and may lead to genomic insights into what influences their behaviour, thus leading to new innovations in malaria elimination.