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Abstract Background Current front line malaria vector control methods such as indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs), rely upon the preference of many primary vectors to feed and/or rest inside human habitations where they can be targeted with domestically-applied insecticidal products. We studied the human biting behaviour of the malaria vector Anopheles funestus Giles and the potential malaria vector Anopheles quadriannulatus Theobald in Luangwa valley, south-east Zambia. Methods Mosquitoes were collected by human landing catch in blocks of houses with either combined use of deltamethrin-based IRS and LLINs or LLINs alone. Human behaviour data were collected to estimate how much exposure to mosquito bites indoors and outdoors occurred at various times of the night for LLIN users and non-users. Results Anopheles funestus and An. quadriannulatus did not show preference to bite either indoors or outdoors: the proportions [95% confidence interval] caught indoors were 0.586 [0.303, 0.821] and 0.624 [0.324, 0.852], respectively. However, the overwhelming majority of both species were caught at times when most people are indoors. The proportion of mosquitoes caught at a time when most people are indoors were 0.981 [0.881, 0.997] and 0.897 [0.731, 0.965], respectively, so the proportion of human exposure to both species occuring indoors was high for individuals lacking LLINs (An. funestus: 0.983 and An. quadriannulatus: 0.970, respectively). While LLIN users were better protected, more than half of their exposure was nevertheless estimated to occur indoors (An. funestus: 0.570 and An. quadriannulatus: 0.584). Conclusions The proportion of human exposure to both An. funestus and An. quadriannulatus occuring indoors was high in the area and hence both species might be responsive to further peri-domestic measures if these mosquitoes are susceptible to insecticidal products.

Abstract Background The Zambian Malaria Control Programme with the Roll Back Malaria (RBM) partners have developed the current National Malaria Strategic Plan (NMSP 2006–2011) which focuses on prevention based on the Integrated Vector Management (IVM) strategy. The introduction and implementation of an IVM strategy was planned in accordance with the World Health Organization (WHO) steps towards IVM implementation namely Introduction Phase, Consolidation Phase and Expansion Phase. Achievements IVM has created commitment for Legal and Regulatory policy review, monitoring, Research and a strong stewardship by the chemical suppliers. It has also leveraged additional resources, improved inter-sectoral collaboration, capacity building and enhanced community participation which facilitated a steady scaling up in coverage and utilisation of key preventive interventions. Thus, markedly reducing malaria incidence and case fatalities in the country. Conclusion Zambia has successfully introduced, consolidated and expanded IVM activities. Resulting in increased coverage and utilization of interventions and markedly reducing malaria-related morbidity and mortality while ensuring a better protection of the environment.

Background: Malaria elimination requires closely co-ordinated action between neighbouring countries. In Southern Africa several countries have reduced malaria to low levels, but the goal of elimination has eluded them thus far. The Southern Africa Development Community (SADC) Malaria Elimination Eight (E8) initiative was established in 2009 between Angola, Botswana, Eswatini, Mozambique, Namibia, South Africa, Zambia, and Zimbabwe to coordinate malaria interventions aiming to eliminate malaria by 2030. Cross-border coordination is important in malaria elimination settings as it strengthens surveillance, joint planning and implementation, knowledge exchange and optimal use of resources. This paper describes how this collaboration is realized in practice, its achievements and challenges, and its significance for malaria elimination prospects., Methods: The ministers of health of the E8 countries oversee an intergovernmental technical committee supported by specialist working groups consisting of technical personnel from member countries and partner institutions. These technical working groups are responsible for malaria elimination initiatives in key focus areas such as surveillance, vector control, diagnosis, case management, behaviour change and applied research. The technical working groups have initiated and guided several collaborative projects which lay essential groundwork for malaria elimination., Results: The E8 collaboration has yielded achievements in the following key areas. (1) Establishment and evaluation of malaria border health posts to improve malaria services in border areas and reduce malaria among resident and, mobile and migrant populations. (2) The development of a regional malaria microscopy slide bank providing materials for diagnostic training and proficiency testing. (3) A facility for regional external competency assessment and training of malaria microscopy trainers in collaboration with the World Health Organization. (4) Entomology fellowships that improved capacity in entomological surveillance; an indoor residual spraying (IRS) training of trainers' scheme to enhance the quality of this core intervention in the region. (5) Capacity development for regional malaria parasite genomic surveillance. (6) A mechanism for early detection of malaria outbreak through near real time reporting and a quarterly bulletins of malaria incidence in border districts., Conclusions: The E8 technical working groups system embodies inter-country collaboration of malaria control and elimination activities. It facilitates sustained interaction between countries through a regional approach. The groundwork for elimination has been laid, but the challenge will be to maintain funding for collaboration at this level whilst reducing reliance on international donors and to build capacities necessary to prepare for malaria elimination., (© 2024. The Author(s).)

Background: A malaria slide bank (MSB) is a useful asset for any malaria microscopy testing laboratory to have access to. However, it is not feasible for every country to have its own MSB. If countries are able to pool their resources, a regional MSB is a viable solution. This paper describes the methodology, costing and lessons learnt of establishing and maintaining an MSB over a 3-year period, for a Southern Africa Development Community region., Methods: A national reference laboratory in South Africa was granted funding for setting up the MSB; it possessed experienced staff and suitable resources. Two additional full-time personnel were employed to carry out the activities of this project. Strict protocols for donor/patient blood sample screening, smear preparation, mass staining, quality control and slide validation were followed. Slides from the MSB were used for training and proficiency testing purposes. The initial and recurrent yearly costs to set up and maintain the MSB were calculated., Results: Over 35 months, 154 batches (26,623 slides) were prepared; the majority were Plasmodium falciparum. Ninety-two percent (141/154) of batches passed internal quality control, and 89% (93/104) passed external validation. From these slides, two training slide sets and six proficiency testing slide sets were sent out. The initial year's cost to establish an MSB was calculated at approximately $165,000, and the recurrent year-on-year cost was $130,000., Conclusions: The key components for maintaining a high-quality MSB are consistent funding, competent staff and adherence to standardized protocols. Travel to malaria-endemic areas for access to non-falciparum malaria species, and dilution of P. falciparum blood to desired parasite densities, are extremely useful to ensure variety. The MSB created here supported multiple laboratories in eight countries, and has the potential to expand., (© 2021. The Author(s).)

Advances in genomics have led to an appreciation that introgression is common, but its evolutionary consequences are poorly understood. In recent species radiations the sharing of genetic variation across porous species boundaries can facilitate adaptation to new environments and generate novel phenotypes, which may contribute to further diversification. Most Anopheles mosquito species that are of major importance as human malaria vectors have evolved within recent and rapid radiations of largely nonvector species. Here, we focus on one of the most medically important yet understudied anopheline radiations, the Afrotropical Anopheles funestus complex (AFC), to investigate the role of introgression in its diversification and the possible link between introgression and vector potential. The AFC comprises at least seven morphologically similar species, yet only An. funestus sensu stricto is a highly efficient malaria vector with a pan-African distribution. Based on de novo genome assemblies and additional whole-genome resequencing, we use phylogenomic and population genomic analyses to establish species relationships. We show that extensive interspecific gene flow involving multiple species pairs has shaped the evolutionary history of the AFC since its diversification. The most recent introgression event involved a massive and asymmetrical movement of genes from a distantly related AFC lineage into An. funestus , an event that predated and plausibly facilitated its subsequent dramatic geographic range expansion across most of tropical Africa. We propose that introgression may be a common mechanism facilitating adaptation to new environments and enhancing vectorial capacity in Anopheles mosquitoes., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Whereas data on insecticide resistance and its underlying mechanisms exist for parts of Zambia, data remain limited in the southern part of the country. This study investigated the status of insecticide resistance, metabolic mechanisms, and parasite infection in Anopheles funestus along Lake Kariba in southern Zambia. Indoor-resting mosquitoes were collected from 20 randomly selected houses within clusters where a mass drug administration trial was conducted and raised to F1 progeny. Non-blood-fed 2- to 5-day-old female An. funestus were exposed to WHO insecticide-impregnated papers with 0.05% deltamethrin, 0.1% bendiocarb, 0.25% pirimiphos-methyl, or 4% dichloro-diphenyl-trichloroethane (DDT). In separate assays, An. funestus were pre-exposed to piperonyl butoxide (PBO) to determine the presence of monooxygenases. Wild-caught An. funestus that had laid eggs for susceptibility assays were screened for circumsporozoite protein of Plasmodium falciparum by ELISA, and sibling species were identified by polymerase chain reaction. Anopheles funestus showed resistance to deltamethrin and bendiocarb but remained susceptible to pirimiphos-methyl and DDT. The pre-exposure of An. funestus to PBO restored full susceptibility to deltamethrin but not to bendiocarb. The overall sporozoite infection rate in An. funestus populations was 5.8%. Detection of pyrethroid and carbamate resistance in An. funestus calls for increased insecticide resistance monitoring to guide planning and selection of effective insecticide resistance management strategies. To prevent the development of resistance and reduce the underlying vectorial capacity of mosquitoes in areas targeted for malaria elimination, an effective integrated vector management strategy is needed.

Background: Despite rapid upscale of insecticide-treated nets (ITNs) and indoor residual spraying (IRS), malaria remains a major source of morbidity and mortality in Zambia. Uptake and utilization of these and novel interventions are often affected by knowledge, attitudes and practices (KAP) amongst persons living in malaria-endemic areas. The aims of this study were to assess malaria KAP of primary caregivers and explore trends in relation to ITN use, IRS acceptance and mosquito density in two endemic communities in Luangwa and Nyimba districts, Zambia., Methods: A cohort of 75 primary caregivers were assessed using a cross-sectional, forced-choice malaria KAP survey on ITN use, IRS acceptance and initial perception of a novel spatial repellent (SR) product under investigation. Entomological sampling was performed in participant homes using CDC Miniature Light Traps to relate indoor mosquito density with participant responses., Results: Ninety-nine percent of participants cited bites of infected mosquitoes as the route of malaria transmission although other routes were also reported including drinking dirty water (64%) and eating contaminated food (63%). All caregivers agreed that malaria was a life-threatening disease with the majority of caregivers having received malaria information from health centers (86%) and community health workers (51%). Cumulatively, self-reported mosquito net use was 67%. Respondents reportedly liked the SR prototype product but improvements on color, shape and size were suggested. Overall, 398 mosquitoes were captured from light-trap collections, including 49 anophelines and 349 culicines. Insecticide treated nets use was higher in households from which at least one mosquito was captured., Conclusions: The current study identified misconceptions in malaria transmission among primary caregivers indicating remaining knowledge gaps in educational campaigns. Participant responses also indicated a misalignment between a low perception of IRS efficacy and high stated acceptance of IRS, which should be further examined to better understand uptake and sustainability of other vector control strategies. While ITNs were found to be used in study households, misperceptions between presence of mosquitoes and bite protection practices did exist. This study highlights the importance of knowledge attitudes and practice surveys, with integration of entomological sampling, to better guide malaria vector control product development, strategy acceptance and compliance within endemic communities.

Background: Across most of sub-Saharan Africa, malaria is transmitted by mosquitoes from the Anopheles gambiae complex, comprising seven morphologically indistinguishable but behaviourally-diverse sibling species with ecologically-distinct environmental niches. Anopheles gambiae and An. arabiensis are the mostly widely distributed major malaria vectors within the complex, while An. quadriannulatus is sparsely distributed., Methods: Indoor residual spraying (IRS) with the organophosphate pirimiphos-methyl (PM) was conducted four times between 2011 and 2017 in the Luangwa Valley, south-east Zambia. Anopheles mosquitoes were repeatedly collected indoors by several experiments with various objectives conducted in this study area from 2010 onwards. Indoor mosquito collection methods included human landing catches, Centres for Disease Control and Prevention miniature light traps and back pack aspirators. Anopheles gambiae complex mosquitoes were morphologically identified to species level using taxonomic keys, and to molecular level by polymerase chain reaction. These multi-study data were collated so that time trends in the species composition of this complex could be assessed., Results: The proportion of indoor An. gambiae complex accounted for by An. quadriannulatus declined from 95.1% to 69.7% following two application PM-IRS rounds with an emulsifiable concentrate formulation from 2011 to 2013, while insecticidal net utilisation remained consistently high throughout that period. This trend continued after two further rounds of PM-IRS with a longer-lasting capsule suspension formulation in 2015 and 2016/2017, following which An. quadriannulatus accounted for only 4.5% of the complex. During the same time interval there was a correspondingly steady rise in the proportional contribution of An. arabiensis to the complex, from 3.9 to 95.1%, while the contribution of nominate An. gambiae remained stable at ≤ 0.9%., Conclusion: It seems likely that An. arabiensis is not only more behaviourally resilient against IRS than An. gambiae, but also than An. quadriannulatus populations exhibiting indoor-feeding, human-feeding and nocturnal behaviours that are unusual for this species. Routine, programmatic entomological monitoring of dynamic vector population guilds will be critical to guide effective selection and deployment of vector control interventions, including supplementary measures to tackle persisting vectors of residual malaria transmission like An. arabiensis.

Background: The effectiveness of long-lasting insecticidal-treated nets (LLINs) and indoor residual spraying (IRS) for malaria control is threatened by resistance to commonly used pyrethroid insecticides. Rotations, mosaics, combinations, or mixtures of insecticides from different complementary classes are recommended by the World Health Organization (WHO) for mitigating against resistance, but many of the alternatives to pyrethroids are prohibitively expensive to apply in large national IRS campaigns. Recent evaluations of window screens and eave baffles (WSEBs) treated with pirimiphos-methyl (PM), to selectively target insecticides inside houses, demonstrated malaria vector mortality rates equivalent or superior to IRS. However, the durability of efficacy when co-applied with polyacrylate-binding agents (BA) remains to be established. This study evaluated whether WSEBs, co-treated with PM and BA have comparable wash resistance to LLINs and might therefore remain insecticidal for years rather than months., Methods: WHO-recommended wire ball assays of insecticidal efficacy were applied to polyester netting treated with or without BA plus 1 or 2 g/sq m PM. They were then tested for insecticidal efficacy using fully susceptible insectary-reared Anopheles gambiae mosquitoes, following 0, 5, 10, 15, then 20 washes as per WHO-recommended protocols for accelerated ageing of LLINs. This was followed by a small-scale field trial in experimental huts to measure malaria vector mortality achieved by polyester netting WSEBs treated with BA and 2 g/sq m PM after 0, 10 and then 20 standardized washes, alongside recently applied IRS using PM., Results: Co-treatment with BA and either dosage of PM remained insecticidal over 20 washes in the laboratory. In experimental huts, WSEBs treated with PM plus BA consistently killed similar proportions of Anopheles arabiensis mosquitoes to PM-IRS (both consistently ≥ 94%), even after 20 washes., Conclusion: Co-treating WSEBs with both PM and BA results in wash-resistant insecticidal activity comparable with LLINs. Insecticide treatments for WSEBs may potentially last for years rather than months, therefore, reducing insecticide consumption by an order of magnitude relative to IRS. However, durability of WSEBs will still have to be assessed in real houses under representative field conditions of exposure to wear and tear, sunlight and rain.

Background: Long-lasting, insecticidal nets (LLINs) and indoor residual spraying (IRS) are the most widely accepted and applied malaria vector control methods. However, evidence that incremental impact is achieved when they are combined remains limited and inconsistent., Methods: Fourteen population clusters of approximately 1000 residents each in Zambia's Luangwa and Nyimba districts, which had high pre-existing usage rates (81.7 %) of pyrethroid-impregnated LLINs were quasi-randomly assigned to receive IRS with either of two pyrethroids, namely deltamethrin [Wetable granules (WG)] and lambdacyhalothrin [capsule suspension (CS)], with an emulsifiable concentrate (EC) or CS formulation of the organophosphate pirimiphos methyl (PM), or with no supplementary vector control measure. Diagnostic positivity of patients tested for malaria by community health workers in these clusters was surveyed longitudinally over pre- and post-treatment periods spanning 29 months, over which the treatments were allocated and re-allocated in advance of three sequential rainy seasons., Results: Supplementation of LLINs with PM CS offered the greatest initial level of protection against malaria in the first 3 months of application (incremental protective efficacy (IPE) [95 % confidence interval (CI)] = 0.63 [CI 0.57, 0.69], P < 0.001), followed by lambdacyhalothrin (IPE [95 % CI] = 0.31 [0.10, 0.47], P = 0.006) and PM EC (IPE, 0.23 [CI 0.15, 0.31], P < 0.001) and then by deltamethrin (IPE [95 % CI] = 0.19 [-0.01, 0.35], P = 0.064). Neither pyrethroid formulation provided protection beyond 3 months after spraying, but the protection provided by both PM formulations persisted undiminished for longer periods: 6 months for CS and 12 months for EC. The CS formulation of PM provided greater protection than the combined pyrethroid IRS formulations throughout its effective life IPE [95 % CI] = 0.79 [0.75, 0.83] over 6 months. The EC formulation of PM provided incremental protection for the first 3 months (IPE [95 % CI] = 0.23 [0.15, 0.31]) that was approximately equivalent to the two pyrethroid formulations (lambdacyhalothrin, IPE [95 % CI] = 0.31 [0.10, 0.47] and deltamethrin, IPE [95 % CI] = 0.19 [-0.01, 0.35]) but the additional protection provided by the former, apparently lasted an entire year., Conclusion: Where universal coverage targets for LLIN utilization has been achieved, supplementing LLINs with IRS using pyrethroids may reduce malaria transmission below levels achieved by LLIN use alone, even in settings where pyrethroid resistance occurs in the vector population. However, far greater reduction of transmission can be achieved under such conditions by supplementing LLINs with IRS using non-pyrethroid insecticide classes, such as organophosphates, so this is a viable approach to mitigating and managing pyrethroid resistance.

Malaria is a life-threatening disease that caused more than 400,000 deaths in sub-Saharan Africa in 2015. Mass prevention of the disease is best achieved by vector control which heavily relies on the use of insecticides. Monitoring mosquito vector populations is an integral component of control programs and a prerequisite for effective interventions. Several individual methods are used for this task; however, there are obstacles to their uptake, as well as challenges in organizing, interpreting and communicating vector population data. The Horizon 2020 project "DMC-MALVEC" consortium will develop a fully integrated and automated multiplex vector-diagnostic platform (LabDisk) for characterizing mosquito populations in terms of species composition, Plasmodium infections and biochemical insecticide resistance markers. The LabDisk will be interfaced with a Disease Data Management System (DDMS), a custom made data management software which will collate and manage data from routine entomological monitoring activities providing information in a timely fashion based on user needs and in a standardized way. The ResistanceSim, a serious game, a modern ICT platform that uses interactive ways of communicating guidelines and exemplifying good practices of optimal use of interventions in the health sector will also be a key element. The use of the tool will teach operational end users the value of quality data (relevant, timely and accurate) to make informed decisions. The integrated system (LabDisk, DDMS & ResistanceSim) will be evaluated in four malaria endemic countries, representative of the vector control challenges in sub-Saharan Africa, (Cameroon, Ivory Coast, Ethiopia and Zambia), highly representative of malaria settings with different levels of endemicity and vector control challenges, to support informed decision-making in vector control and disease management.

The understanding of malaria vector species in association with their bionomic traits is vital for targeting malaria interventions and measuring effectiveness. Many entomological studies rely on morphological identification of mosquitoes, limiting recognition to visually distinct species/species groups. Anopheles species assignments based on ribosomal DNA ITS2 and mitochondrial DNA COI were compared to morphological identifications from Luangwa and Nyimba districts in Zambia. The comparison of morphological and molecular identifications determined that interpretations of species compositions, insecticide resistance assays, host preference studies, trap efficacy, and Plasmodium infections were incorrect when using morphological identification alone. Morphological identifications recognized eight Anopheles species while 18 distinct sequence groups or species were identified from molecular analyses. Of these 18, seven could not be identified through comparison to published sequences. Twelve of 18 molecularly identified species (including unidentifiable species and species not thought to be vectors) were found by PCR to carry Plasmodium sporozoites - compared to four of eight morphological species. Up to 15% of morphologically identified Anopheles funestus mosquitoes in insecticide resistance tests were found to be other species molecularly. The comprehension of primary and secondary malaria vectors and bionomic characteristics that impact malaria transmission and intervention effectiveness are fundamental in achieving malaria elimination.

Background: Measurement of densities of host-seeking malaria vectors is important for estimating levels of disease transmission, for appropriately allocating interventions, and for quantifying their impact. The gold standard for estimating mosquito-human contact rates is the human landing catch (HLC), where human volunteers catch mosquitoes that land on their exposed body parts. This approach necessitates exposure to potentially infectious mosquitoes, and is very labour intensive. There are several safer and less labour-intensive methods, with Centers for Disease Control light traps (LT) placed indoors near occupied bed nets being the most widely used., Methods: This paper presents analyses of 13 studies with paired mosquito collections of LT and HLC to evaluate these methods for their consistency in sampling indoor-feeding mosquitoes belonging to the two major taxa of malaria vectors across Africa, the Anopheles gambiae sensu lato complex and the Anopheles funestus s.l. group. Both overall and study-specific sampling efficiencies of LT compared with HLC were computed, and regression methods that allow for the substantial variations in mosquito counts made by either method were used to test whether the sampling efficacy varies with mosquito density., Results: Generally, LT were able to collect similar numbers of mosquitoes to the HLC indoors, although the relative sampling efficacy, measured by the ratio of LT:HLC varied considerably between studies. The overall best estimate for An. gambiae s.l. was 1.06 (95% credible interval: 0.68-1.64) and for An. funestus s.l. was 1.37 (0.70-2.68). Local calibration exercises are not reproducible, since only in a few studies did LT sample proportionally to HLC, and there was no geographical pattern or consistent trend with average density in the tendency for LT to either under- or over-sample., Conclusions: LT are a crude tool at best, but are relatively easy to deploy on a large scale. Spatial and temporal variation in mosquito densities and human malaria transmission exposure span several orders of magnitude, compared to which the inconsistencies of LT are relatively small. LT, therefore, remain an invaluable and safe alternative to HLC for measuring indoor malaria transmission exposure in Africa.

Background: Monitoring mosquito population dynamics is essential to guide selection and evaluation of malaria vector control interventions but is typically implemented by mobile, centrally-managed teams who can only visit a limited number of locations frequently enough to capture longitudinal trends. Community-based (CB) mosquito trapping schemes for parallel, continuous monitoring of multiple locations are therefore required that are practical, affordable, effective, and reliable., Methods: A CB surveillance scheme, with a monthly sampling and reporting cycle for capturing malaria vectors, using Centers for Disease Control and Prevention light traps (LT) and Ifakara Tent Traps (ITT), were conducted by trained community health workers (CHW) in 14 clusters of households immediately surrounding health facilities in rural south-east Zambia. At the end of the study, a controlled quality assurance (QA) survey was conducted by a centrally supervised expert team using human landing catch (HLC), LT and ITT to evaluate accuracy of the CB trapping data. Active surveillance of malaria parasite infection rates amongst humans was conducted by CHWs in the same clusters to determine the epidemiological relevance of these CB entomological surveys., Results: CB-LT and CB-ITT exhibited relative sampling efficiencies of 50 and 7%, respectively, compared with QA surveys using the same traps. However, cost per sampling night was lowest for CB-LT ($13.6), followed closely by CB-ITT ($18.0), both of which were far less expensive than any QA survey (HLC: $138, LT: $289, ITT: $269). Cost per specimen of Anopheles funestus captured was lowest for CB-LT ($5.3), followed by potentially hazardous QA-HLC ($10.5) and then CB-ITT ($28.0), all of which were far more cost-effective than QA-LT ($141) and QA-ITT ($168). Time-trends of malaria diagnostic positivity (DP) followed those of An. funestus density with a one-month lag and the wide range of mean DP across clusters was closely associated with mean densities of An. funestus caught by CB-LT (P < 0.001)., Conclusions: CB trapping schemes appear to be far more affordable, epidemiologically relevant and cost-effective than centrally supervised trapping schemes and may well be applicable to enhance intervention trials and even enable routine programmatic monitoring of vector population dynamics on unprecedented national scales.

Background: Active, population-wide mass screening and treatment (MSAT) for chronic Plasmodium falciparum carriage to eliminate infectious reservoirs of malaria transmission have proven difficult to apply on large national scales through trained clinicians from central health authorities., Methodology: Fourteen population clusters of approximately 1,000 residents centred around health facilities (HF) in two rural Zambian districts were each provided with three modestly remunerated community health workers (CHWs) conducting active monthly household visits to screen and treat all consenting residents for malaria infection with rapid diagnostic tests (RDT). Both CHWs and HFs also conducted passive case detection among residents who self-reported for screening and treatment., Results: Diagnostic positivity was higher among symptomatic patients self-reporting to CHWs (42.5%) and HFs (24%) than actively screened residents (20.3%), but spatial and temporal variations of diagnostic positivity were highly consistent across all three systems. However, most malaria infections (55.6%) were identified through active home visits by CHWs rather than self-reporting to CHWs or HFs. Most (62%) malaria infections detected actively by CHWs reported one or more symptoms of illness. Most reports of fever and vomiting, plus more than a quarter of history of fever, headache and diarrhoea, were attributable to malaria infection. The minority of residents who participated >12 times had lower rates of malaria infection and associated symptoms in later contacts but most residents were tested <4 times and high malaria diagnostic positivity (32%) in active surveys, as well as incidence (1.7 detected infections per person per year) persisted in the population. Per capita cost for active service delivery by CHWs was US$5.14 but this would rise to US$10.68 with full community compliance with monthly testing at current levels of transmission, and US$6.25 if pre-elimination transmission levels and negligible treatment costs were achieved., Conclusion: Monthly active home visits by CHWs equipped with RDTs were insufficient to eliminate the human infection reservoir in this typical African setting, despite reasonably high LLIN/IRS coverage. However, dramatic impact upon infection and morbidity burden might be attainable and cost-effective if community participation in regular testing could be improved and the substantial, but not necessarily prohibitive, costs are affordable to national programmes.

Background: Sampling malaria vectors and measuring their biting density is of paramount importance for entomological surveys of malaria transmission. Human landing catch (HLC) has been traditionally regarded as a gold standard method for surveying human exposure to mosquito bites. However, due to the risk of human participant exposure to mosquito-borne parasites and viruses, a variety of alternative, exposure-free trapping methods were compared in lowland, south-east Zambia., Methods: Centres for Disease Control and Prevention miniature light trap (CDC-LT), Ifakara Tent Trap model C (ITT-C), resting boxes (RB) and window exit traps (WET) were all compared with HLC using a 3 × 3 Latin Squares design replicated in 4 blocks of 3 houses with long lasting insecticidal nets, half of which were also sprayed with a residual deltamethrin formulation, which was repeated for 10 rounds of 3 nights of rotation each during both the dry and wet seasons., Results: The mean catches of HLC indoor, HLC outdoor, CDC-LT, ITT-C, WET, RB indoor and RB outdoor, were 1.687, 1.004, 3.267, 0.088, 0.004, 0.000 and 0.008 for Anopheles quadriannulatus Theobald respectively, and 7.287, 6.784, 10.958, 5.875, 0.296, 0.158 and 0.458, for An. funestus Giles, respectively. Indoor CDC-LT was more efficient in sampling An. quadriannulatus and An. funestus than HLC indoor (Relative rate [95% Confidence Interval] = 1.873 [1.653, 2.122] and 1.532 [1.441, 1.628], respectively, P < 0.001 for both). ITT-C was the only other alternative which had comparable sensitivity (RR = 0.821 [0.765, 0.881], P < 0.001), relative to HLC indoor other than CDC-LT for sampling An. funestus., Conclusions: While the two most sensitive exposure-free techniques primarily capture host-seeking mosquitoes, both have substantial disadvantages for routine community-based surveillance applications: the CDC-LT requires regular recharging of batteries while the bulkiness of ITT-C makes it difficult to move between sampling locations. RB placed indoors or outdoors and WET had consistently poor sensitivity so it may be useful to evaluate additional alternative methods, such as pyrethrum spray catches and back packer aspirators, for catching resting mosquitoes.