Comparison of different trapping methods to collect malaria vectors indoors and outdoors in western Kenya.

BACKGROUND: Vector surveillance is among the World Health Organization global vector control response (2017-2030) pillars. Human landing catches are a gold standard but difficult to implement and potentially expose collectors to malaria infection. Other methods like light traps, pyrethrum spray catches and aspiration are less expensive and less risky to collectors. METHODS: Three mosquito sampling methods (UV light traps, CDC light traps and Prokopack aspiration) were evaluated against human landing catches (HLC) in two villages of Rarieda sub-county, Siaya County, Kenya. UV-LTs, CDC-LTs and HLCs were conducted hourly between 17:00 and 07:00. Aspiration was done indoors and outdoors between 07:00 and 11:00 a.m. Analyses of mosquito densities, species abundance and sporozoite infectivity were performed across all sampling methods. Species identification PCR and ELISAs were done for Anopheles gambiae and Anopheles funestus complexes and data analysis was done in R. RESULTS: Anopheles mosquitoes sampled from 608 trapping efforts were 5,370 constituting 70.3% Anopheles funestus sensu lato (s.l.), 19.7% Anopheles coustani and 7.2% An. gambiae s.l. 93.8% of An. funestus s.l. were An. funestus sensu stricto (s.s.) and 97.8% of An. gambiae s.l. were Anopheles arabiensis. Only An. funestus were sporozoite positive with 3.1% infection prevalence. Indoors, aspiration captured higher An. funestus (mean = 6.74; RR = 8.83, P < 0.001) then UV-LT (mean = 3.70; RR = 3.97, P < 0.001) and CDC-LT (mean = 1.74; RR = 1.89, P = 0.03) compared to HLC. UV-LT and CDC-LT indoors captured averagely 0.18 An. arabiensis RR = 5.75, P = 0.028 and RR = 5.87, P = 0.028 respectively. Outdoors, UV-LT collected significantly higher Anopheles mosquitoes compared to HLC (An. funestus: RR = 5.18, P < 0.001; An. arabiensis: RR = 15.64, P = 0.009; An. coustani: RR = 11.65, P < 0.001). Anopheles funestus hourly biting indoors in UV-LT and CDC-LT indicated different peaks compared to HLC. CONCLUSIONS: Anopheles funestus remains the predominant mosquito species. More mosquitoes were collected using aspiration, CDC-LTs and UV-LTs indoors and UV-LTs and CD-LTs outdoors compared to HLCs. UV-LTs collected more mosquitoes than CDC-LTs. The varied trends observed at different times of the night suggest that these methods collect mosquitoes with diverse activities and care must be taken when interpreting the results.

Early morning anopheline mosquito biting, a potential driver of malaria transmission in Busia County, western Kenya.

BACKGROUND: Insecticide-treated nets (ITNs) contributed significantly to the decline in malaria since 2000. Their protective efficacy depends not only on access, use, and net integrity, but also location of people within the home environment and mosquito biting profiles. Anopheline mosquito biting and human location data were integrated to identify potential gaps in protection and better understand malaria transmission dynamics in Busia County, western Kenya. METHODS: Direct observation of human activities and human landing catches (HLC) were performed hourly between 1700 to 0700 h. Household members were recorded as home or away; and, if at home, as indoors/outdoors, awake/asleep, and under a net or not. Aggregated data was analysed by weighting hourly anopheline biting activity with human location. Standard indicators of human-vector interaction were calculated using a Microsoft Excel template. RESULTS: There was no significant difference between indoor and outdoor biting for Anopheles gambiae sensu lato (s.l.) (RR = 0.82; 95% CI 0.65-1.03); significantly fewer Anopheles funestus were captured outdoors than indoors (RR = 0.41; 95% CI 0.25-0.66). Biting peaked before dawn and extended into early morning hours when people began to awake and perform routine activities, between 0400-0700 h for An. gambiae and 0300-0700 h for An. funestus. The study population away from home peaked at 1700-1800 h (58%), gradually decreased and remained constant at 10% throughout the night, before rising again to 40% by 0600-0700 h. When accounting for resident location, nearly all bites within the peri-domestic space (defined as inside household structures and surrounding outdoor spaces) occurred indoors for unprotected people (98%). Using an ITN while sleeping was estimated to prevent 79% and 82% of bites for An. gambiae and An. funestus, respectively. For an ITN user, most remaining exposure to bites occurred indoors in the hours before bed and early morning. CONCLUSION: While use of an ITN was estimated to prevent most vector bites in this context, results suggest gaps in protection, particularly in the early hours of the morning when biting peaks and many people are awake and active. Assessment of additional human exposure points, including outside of the peri-domestic setting, are needed to guide supplementary interventions for transmission reduction.

Reducing Malaria Transmission through Reactive Indoor Residual Spraying: A Systematic Review.

In the final stages of malaria elimination, interventions to reduce malaria transmission are often centered around a confirmed case of malaria, as cases tend to cluster together at very low levels of transmission. The WHO commissioned a systematic review of the literature and synthesis of evidence for reactive indoor residual spraying (IRS) to develop official recommendations for countries. Several electronic databases were searched in November 2020. A total of 455 records were identified and screened; 20 full-text articles were assessed for eligibility. Two cluster-randomized trials met the inclusion criteria for epidemiological outcomes. Risk of bias was assessed using standard criteria. Because one study was a superiority trial in which the comparator included reactive case detection or mass drug administration and the other was a noninferiority trial in which the comparator was proactive, focal IRS, results could not be pooled. In the superiority trial, reactive IRS reduced malaria prevalence by 68% (risk ratio [RR]: 0.32; 95% CI: 0.13-0.80; certainty of evidence: HIGH) compared with no reactive IRS. No difference was observed for clinical malaria (RR: 0.65; 95% CI: 0.38-1.11; certainty of evidence: MODERATE). In the noninferiority study, the mean difference in incidence between reactive IRS and proactive IRS was 0.10 additional case per 1,000 person-years, which was within the prespecified noninferiority bound (95% CI: -0.38 to 0.58; certainty of evidence: MODERATE). The evidence indicates that reactive IRS may be a cost-effective tool for the prevention of malaria in elimination settings. As only two cluster-randomized controlled trials from sub-Saharan Africa were found, additional high-quality studies should be encouraged.

Development of Systematic Reviews to Inform WHO's Recommendations for Elimination and Prevention of Re-Establishment of Malaria: Methodology.

The basis for an evidence-based recommendation is a well-conducted systematic review that synthesizes the primary literature relevant to the policy or program question of interest. In 2020, the WHO commissioned 10 systematic reviews of potential interventions in elimination or post-elimination settings to summarize their impact on malaria transmission. This paper describes the general methods used to conduct this series of systematic reviews and notes where individual reviews diverged from the common methodology. The paper also presents lessons learned from conducting the systematic reviews to make similar future efforts more efficient, standardized, and streamlined.

Late morning biting behaviour of Anopheles funestus is a risk factor for transmission in schools in Siaya, western Kenya.

BACKGROUND: Children in Kenya spend a substantial amount of time at school, including at dawn and dusk when mosquitoes are active. With changing vector behaviour towards early morning biting, it is important to determine whether there is an additional risk of transmission in schools. This study sought to understand whether late morning biting by Anopheles funestus, previously documented in households in western Kenya, was replicated in schools. METHODS: From the 4th to the 6th of August 2023, human landing collections were conducted hourly in four schools in Alego Usonga sub-County, Siaya County. The collections were conducted in and outside five classrooms in each school and ran for 17 h, starting at 18:00 until 11:00 h the next morning. RESULTS: Anopheles funestus was the predominant species collected, forming 93.2% (N = 727) of the entire collection, with peak landing between 06:00 and 07:00 h and continuing until 11:00 h. More than half of the collected An. funestus were either fed or gravid, potentially indicative of multiple bloodmeals within each gonotrophic cycle, and had a sporozoite rate of 2.05%. CONCLUSION: School children spend up to 10 h of their daytime in schools, reporting between 06:00 and 07:00 h and staying in school until as late as 17:00 h, meaning that they receive potentially infectious mosquito bites during the morning hours in these settings. There is a need to consider vector control approaches targeting schools and other peridomestic spaces in the morning hours when An. funestus is active.

Detection of Anopheles stephensi Mosquitoes by Molecular Surveillance, Kenya.

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

Evaluation of community-based vector surveillance system for routine entomological monitoring under low malaria vector densities and high bed net coverage in western Kenya.

BACKGROUND: Entomological surveillance is traditionally conducted by supervised teams of trained technicians. However, it is expensive and limiting in the number of sites visited. Surveillance through community-based collectors (CBC) may be more cost-effective and sustainable for longitudinal entomological monitoring. This study evaluated the efficiency of CBCs in monitoring mosquito densities compared to quality-assured sampling conducted by experienced entomology technicians. METHODS: Entomological surveillance employing CBCs was conducted in eighteen clusters of villages in western Kenya using indoor and outdoor CDC light traps and indoor Prokopack aspiration. Sixty houses in each cluster were enrolled and sampled once every month. Collected mosquitoes were initially identified to the genus level by CBCs, preserved in 70% ethanol and transferred to the laboratory every 2 weeks. Parallel, collections by experienced entomology field technicians were conducted monthly by indoor and outdoor CDC light traps and indoor Prokopack aspiration and served as a quality assurance of the CBCs. RESULTS: Per collection, the CBCs collected 80% fewer Anopheles gambiae sensu lato (s.l.) [RR = 0.2; (95% CI 0.14-0.27)] and Anopheles coustani [RR = 0.2; (95% CI 0.06-0.53)] and 90% fewer Anopheles funestus [RR = 0.1; (95% CI 0.08-0.19)] by CDC light traps compared to the quality assured (QA) entomology teams. Significant positive correlations were however observed between the monthly collections by CBCs and QA teams for both An. gambiae and An. funestus. In paired identifications of pooled mosquitoes, the CBCs identified 4.3 times more Anopheles compared to experienced technicians. The cost per person-night was lower in the community-based sampling at $9.1 compared to $89.3 by QA per collection effort. CONCLUSION: Unsupervised community-based mosquito surveillance collected substantially fewer mosquitoes per trap-night compared to quality-assured collection by experienced field teams, while consistently overestimating the number of Anopheles mosquitoes during identification. However, the numbers collected were significantly correlated between the CBCs and the QA teams suggesting that trends observed by CBCs and QA teams were similar. Further studies are needed to evaluate whether adopting low-cost, devolved supervision with spot checks, coupled with remedial training of the CBCs, can improve community-based collections to be considered a cost-effective alternative to surveillance conducted by experienced entomological technicians.

Efficacy of partial spraying of SumiShield, Fludora Fusion and Actellic against wild populations of Anopheles gambiae s.l. in experimental huts in Tiassalé, Côte d'Ivoire.

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.

A comparison of the attractiveness of flowering plant blossoms versus attractive targeted sugar baits (ATSBs) in western Kenya.

Attractive Targeted Sugar Baits (ATSB) have been demonstrated to result in significant reductions in malaria vector numbers in areas of scarce vegetation cover such as in Mali and Israel, but it is not clear whether such an effect can be replicated in environments where mosquitoes have a wide range of options for sugar resources. The current study evaluated the attractiveness of the predominant flowering plants of Asembo Siaya County, western Kenya in comparison to an ATSB developed by Westham Co. Sixteen of the most common flowering plants in the study area were selected and evaluated for relative attractiveness to malaria vectors in semi-field structures. Six of the most attractive flowers were compared to determine the most attractive to local Anopheles mosquitoes. The most attractive plant was then compared to different versions of ATSB. In total, 56,600 Anopheles mosquitoes were released in the semi-field structures. From these, 5150 mosquitoes (2621 males and 2529 females) of An. arabiensis, An. funestus and An. gambiae were recaptured on the attractancy traps. Mangifera indica was the most attractive sugar source for all three species while Hyptis suaveolens and Tephrosia vogelii were the least attractive plants to the mosquitoes. Overall, ATSB version 1.2 was significantly more attractive compared to both ATSB version 1.1 and Mangifera indica. Mosquitoes were differentially attracted to various natural plants in western Kenya and ATSB. The observation that ATSB v1.2 was more attractive to local Anopheles mosquitoes than the most attractive natural sugar source indicates that this product may be able to compete with natural sugar sources in western Kenya and suggests this product may have the potential to impact mosquito populations in the field.

Residual insecticide surface treatment for preventing malaria: a systematic review protocol.

INTRODUCTION: Malaria presents a significant global public health burden, although substantial progress has been made, with vector control initiatives such as indoor residual surface spraying with insecticides and insecticide-treated nets. There now exists many different approaches to apply residual insecticide to indoor and outdoor surfaces in malaria-endemic settings, although no comprehensive systematic reviews exist evaluating these interventions. This manuscript outlines the protocol for a systematic review which aims to synthesise the best available evidence regarding full or partial indoor or outdoor residual insecticide surface treatment for preventing malaria. METHODS AND ANALYSIS: This review will comprehensively search the literature (both published and unpublished) for any studies investigating the effectiveness of residual insecticide surface treatment for malaria. Studies will be screened to meet the inclusion criteria by a minimum of two authors, followed by assessment of risk of bias (using appropriate risk-of-bias tools for randomised and non-randomised studies) and extraction of relevant information using structured forms by two independent authors. Meta-analysis will be carried out where possible for epidemiological outcomes such as malaria, anaemia, malaria-related mortality, all-cause mortality and adverse effects. Certainty in the evidence will be established with GRADE assessments. ETHICS AND DISSEMINATION: A full review report will be submitted to the Vector Control & Insecticide Resistance Unit, Global Malaria Program, WHO. A version of this report will be submitted for publication in an open access peer-reviewed journal. The report will inform the development of WHO recommendations regarding residual insecticide treatment for malaria. This systematic review does not require ethics approval as it is a review of primary studies. SYSTEMATIC REVIEW REGISTRATION: PROSPERO 293194.

Spatial repellents: The current roadmap to global recommendation of spatial repellents for public health use.

Spatial repellent (SR) products are envisioned to complement existing vector control methods through the continual release of volatile active ingredients (AI) providing: (i) protection against day-time and early-evening biting; (ii) protection in enclosed/semi-enclosed and peri-domestic spaces; (iii) various formulations to fit context-specific applications; and (iv) increased coverage over traditional control methods. SR product AIs also have demonstrated effect against insecticide-resistant vectors linked to malaria and Aedes-borne virus (ABV) transmission. Over the past two decades, key stakeholders, including World Health Organization (WHO) representatives, have met to discuss the role of SRs in reducing arthropod-borne diseases based on existing evidence. A key focus has been to establish a critical development path for SRs, including scientific, regulatory and social parameters that would constitute an outline for a SR target product profile, i.e. optimum product characteristics. The principal gap is the lack of epidemiological data demonstrating SR public health impact across a range of different ecological and epidemiological settings, to inform a WHO policy recommendation. Here we describe in brief trials that are designed to fulfill evidence needs for WHO assessment and initial projections of SR cost-effectiveness against malaria and dengue.

Screening eaves of houses reduces indoor mosquito density in rural, western Kenya.

BACKGROUND: Despite the scale-up of insecticide-treated nets and indoor residual spraying, the bulk of malaria transmission in western Kenya still occurs indoors, late at night. House improvement is a potential long-term solution to further reduce malaria transmission in the region. METHODS: The impact of eave screening on mosquito densities was evaluated in two rural villages in western Kenya. One-hundred-and-twenty pairs of structurally similar, neighbouring houses were used in the study. In each pair, one house was randomly selected to receive eave screening at the beginning of the study while the other remained unscreened until the end of the sampling period. Mosquito sampling was performed monthly by motorized aspiration method for 4 months. The collected mosquitoes were analysed for species identification. RESULTS: Compared to unscreened houses, significantly fewer female Anopheles funestus (RR = 0.40, 95% CI 0.29-0.55), Anopheles gambiae Complex (RR = 0.46, 95% CI 0.34-0.62) and Culex species (RR = 0.53, 95% CI 0.45-0.61) were collected in screened houses. No significant differences in the densities of the mosquitoes were detected in outdoor collections. Significantly fewer Anopheles funestus were collected indoors from houses with painted walls (RR = 0.05, 95% CI 0.01-0.38) while cooking in the house was associated with significantly lower numbers of Anopheles gambiae Complex indoors (RR = 0.60, 95% CI 0.45-0.79). Nearly all house owners (99.6%) wanted their houses permanently screened, including 97.7% that indicated a willingness to use their own resources. However, 99.2% required training on house screening. The cost of screening a single house was estimated at KES6,162.38 (US$61.62). CONCLUSION: Simple house modification by eave screening has the potential to reduce the indoor occurrence of both Anopheles and Culex mosquito species. Community acceptance was very high although education and mobilization may be needed for community uptake of house modification for vector control. Intersectoral collaboration and favourable government policies on housing are important links towards the adoption of house improvements for malaria control.

Natural sugar feeding rates of Anopheles mosquitoes collected by different methods in western Kenya.

Attractive targeted sugar baits (ATSBs) are a potential vector control tool that exploits the sugar-feeding behaviour of mosquitoes. We evaluated the sugar-feeding behaviour of Anopheles mosquitoes as part of baseline studies for cluster randomised controlled trials of ATSBs. Mosquitoes were collected indoors and outdoors from two villages in western Kenya using prokopack aspirations, malaise tent traps and ultraviolet (UV) light traps. Individual mosquitoes were subjected to the cold anthrone test to assess the presence of sugar. Overall, 15.7% of collected mosquitoes had fed on natural sugar sources. By species and sex, the proportion sugar-fed was 41.3% and 27.7% in male and female Anopheles funestus, 27.2% and 12.8% in male and female An. arabiensis, and 9.7% and 8.3% in male and female An. coustani, respectively. Sugar-feeding was higher in unfed than blood-fed mosquitoes and higher in male than gravid mosquitoes. Anopheles mosquitoes obtained sugar meals from natural sources during all physiological stages, whether they rest indoors or outdoors. These findings offer a potential avenue to exploit for the control of mosquitoes, particularly with the advent of ATSBs, which have been shown to reduce mosquito densities in other regions.

Temporal trends in molecular markers of drug resistance in Plasmodium falciparum in human blood and profiles of corresponding resistant markers in mosquito oocysts in Asembo, western Kenya.

BACKGROUND: Over the last two decades, the scale-up of vector control and changes in the first-line anti-malarial, from chloroquine (CQ) to sulfadoxine-pyrimethamine (SP) and then to artemether-lumefantrine (AL), have resulted in significant decreases in malaria burden in western Kenya. This study evaluated the long-term effects of control interventions on molecular markers of Plasmodium falciparum drug resistance using parasites obtained from humans and mosquitoes at discrete time points. METHODS: Dried blood spot samples collected in 2012 and 2017 community surveys in Asembo, Kenya were genotyped by Sanger sequencing for markers associated with resistance to SP (Pfdhfr, Pfdhps), CQ, AQ, lumefantrine (Pfcrt, Pfmdr1) and artemisinin (Pfk13). Temporal trends in the prevalence of these markers, including data from 2012 to 2017 as well as published data from 1996, 2001, 2007 from same area, were analysed. The same markers from mosquito oocysts collected in 2012 were compared with results from human blood samples. RESULTS: The prevalence of SP dhfr/dhps quintuple mutant haplotype C50I51R59N108I164/S436G437E540A581A613 increased from 19.7% in 1996 to 86.0% in 2012, while an increase in the sextuple mutant haplotype C50I51R59N108I164/H436G437E540A581A613 containing Pfdhps-436H was found from 10.5% in 2012 to 34.6% in 2017. Resistant Pfcrt-76 T declined from 94.6% in 2007 to 18.3% in 2012 and 0.9% in 2017. Mutant Pfmdr1-86Y decreased across years from 74.8% in 1996 to zero in 2017, mutant Pfmdr1-184F and wild Pfmdr1-D1246 increased from 17.9% to 58.9% in 2007 to 55.9% and 90.1% in 2017, respectively. Pfmdr1 haplotype N86F184S1034N1042D1246 increased from 11.0% in 2007 to 49.6% in 2017. No resistant mutations in Pfk13 were found. Prevalence of Pfdhps-436H was lower while prevalence of Pfcrt-76 T was higher in mosquitoes than in human blood samples. CONCLUSION: This study showed an increased prevalence of dhfr/dhps resistant markers over 20 years with the emergence of Pfdhps-436H mutant a decade ago in Asembo. The reversal of Pfcrt from CQ-resistant to CQ-sensitive genotype occurred following 19 years of CQ withdrawal. No Pfk13 markers associated with artemisinin resistance were detected, but the increased haplotype of Pfmdr1 N86F184S1034N1042D1246 was observed. The differences in prevalence of Pfdhps-436H and Pfcrt-76 T SNPs between two hosts and the role of mosquitoes in the transmission of drug resistant parasites require further investigation.

Strain Characterisation for Measuring Bioefficacy of ITNs Treated with Two Active Ingredients (Dual-AI ITNs): Developing a Robust Protocol by Building Consensus.

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.

Evaluation of the protective efficacy of a spatial repellent to reduce malaria incidence in children in western Kenya compared to placebo: study protocol for a cluster-randomized double-blinded control trial (the AEGIS program).

BACKGROUND: Spatial repellents are widely used for prevention of mosquito bites and evidence is building on their public health value, but their efficacy against malaria incidence has never been evaluated in Africa. To address this knowledge gap, a trial to evaluate the efficacy of Mosquito Shield™, a spatial repellent incorporating transfluthrin, was developed for implementation in Busia County, western Kenya where long-lasting insecticidal net coverage is high and baseline malaria transmission is moderate to high year-round. METHODS: This trial is designed as a cluster-randomized, placebo-controlled, double-blinded clinical trial. Sixty clusters will be randomly assigned in a 1:1 ratio to receive spatial repellent or placebo. A total of 6120 children aged ≥6 months to 10 years of age will be randomly selected from the study clusters, enrolled into an active cohort (baseline, cohort 1, and cohort 2), and sampled monthly to determine time to first infection by smear microscopy. Each cohort following the implementation of the intervention will be split into two groups, one to estimate direct effect of the spatial repellent and the other to estimate degree of diversion of mosquitoes and malaria transmission to unprotected persons. Malaria incidence in each cohort will be estimated and compared (primary indicator) to determine benefit of using a spatial repellent in a high, year-round malaria transmission setting. Mosquitoes will be collected monthly using CDC light traps to determine if there are entomological correlates of spatial repellent efficacy that may be useful for the evaluation of new spatial repellents. Quarterly human landing catches will assess behavioral effects of the intervention. DISCUSSION: Findings will serve as the first cluster-randomized controlled trial powered to detect spatial repellent efficacy to reduce malaria in sub-Saharan Africa where transmission rates are high, insecticide-treated nets are widely deployed, and mosquitoes are resistant to insecticides. Results will be submitted to the World Health Organization Vector Control Advisory Group for assessment of public health value towards an endorsement to recommend inclusion of spatial repellents in malaria control programs. TRIAL REGISTRATION: ClinicalTrials.gov NCT04766879 . Registered February 23, 2021.

Incidence and consequences of damage to insecticide-treated mosquito nets in Kenya.

BACKGROUND: Efforts to improve the impact of long-lasting insecticidal nets (LLINs) should be informed by understanding of the causes of decay in effect. Holes in LLINs have been estimated to account for 7-11% of loss in effect on vectorial capacity for Plasmodium falciparum malaria in an analysis of repeated cross-sectional surveys of LLINs in Kenya. This does not account for the effect of holes as a cause of net attrition or non-use, which cannot be measured using only cross-sectional data. There is a need for estimates of how much these indirect effects of physical damage on use and attrition contribute to decay in effectiveness of LLINs. METHODS: Use, physical integrity, and survival were assessed in a cohort of 4514 LLINs followed for up to 4 years in Kenya. Flow diagrams were used to illustrate how the status of nets, in terms of categories of use, physical integrity, and attrition, changed between surveys carried out at 6-month intervals. A compartment model defined in terms of ordinary differential equations (ODEs) was used to estimate the transition rates between the categories. Effects of physical damage to LLINs on use and attrition were quantified by simulating counterfactuals in which there was no damage. RESULTS: Allowing for the direct effect of holes, the effect on use, and the effect on attrition, 18% of the impact on vectorial capacity was estimated to be lost because of damage. The estimated median lifetime of the LLINs was 2.9 years, but this was extended to 5.7 years in the counterfactual without physical damage. Nets that were in use were more likely to be in a damaged state than unused nets but use made little direct difference to LLIN lifetimes. Damage was reported as the reason for attrition for almost half of attrited nets, but the model estimated that almost all attrited nets had suffered some damage before attrition. CONCLUSIONS: Full quantification of the effects of damage will require measurement of the supply of new nets and of household stocks of unused nets, and also of their impacts on both net use and retention. The timing of mass distribution campaigns is less important than ensuring sufficient supply. In the Kenyan setting, nets acquired damage rapidly once use began and the damage led to rapid attrition. Increasing the robustness of nets could substantially increase their lifetime and impact but the impact of LLIN programmes on malaria transmission is ultimately limited by levels of use. Longitudinal analyses of net integrity data from different settings are needed to determine the importance of physical damage to nets as a driver of attrition and non-use, and the importance of frequent use as a cause of physical damage in different contexts.

Comparison of four outdoor mosquito trapping methods as potential replacements for human landing catches in western Kenya.

INTRODUCTION: Longitudinal monitoring of outdoor-biting malaria vector populations is becoming increasingly important in understanding the dynamics of residual malaria transmission. However, the human landing catch (HLC), the gold standard for measuring human biting rates indoors and outdoors, is costly and raises ethical concerns related to increased risk of infectious bites among collectors. Consequently, routine data on outdoor-feeding mosquito populations are usually limited because of the lack of a scalable tool with similar sensitivity to outdoor HLC. METHODOLOGY: The Anopheles trapping sensitivity of four baited proxy outdoor trapping methods-Furvela tent trap (FTT), host decoy trap (HDT), mosquito electrocuting traps (MET) and outdoor CDC light traps (OLT)-was assessed relative to HLC in a 5 × 5 replicated Latin square conducted over 25 nights in two villages of western Kenya. Indoor CDC light trap (ILT) was run in one house in each of the compounds with outdoor traps, while additional non-Latin square indoor and outdoor HLC collections were performed in one of the study villages. RESULTS: The MET, FTT, HDT and OLT sampled approximately 4.67, 7.58, 5.69 and 1.98 times more An. arabiensis compared to HLC, respectively, in Kakola Ombaka. Only FTT was more sensitive relative to HLC in sampling An. funestus in Kakola Ombaka (RR = 5.59, 95% CI 2.49-12.55, P < 0.001) and Masogo (RR = 4.38, 95% CI 1.62-11.80, P = 0.004) and in sampling An. arabiensis in Masogo (RR = 5.37, 95% CI 2.17-13.24, P < 0.001). OLT sampled significantly higher numbers of An. coustani in Kakola Ombaka (RR = 3.03, 95% CI 1.65-5.56, P < 0.001) and Masogo (RR = 2.88, 95% CI 1.15-7.22, P = 0.02) compared to HLC. OLT, HLC and MET sampled mostly An. coustani, FTT had similar proportions of An. funestus and An. arabiensis, while HDT sampled predominantly An. arabiensis in both villages. FTT showed close correlation with ILT in vector abundance for all three species at both collection sites. CONCLUSION: FTT and OLT are simple, easily scalable traps and are potential replacements for HLC in outdoor sampling of Anopheles mosquitoes. However, the FTT closely mirrored indoor CDC light trap in mosquito indices and therefore may be more of an indoor mimic than a true outdoor collection tool. HDT and MET show potential for sampling outdoor host-seeking mosquitoes. However, the traps as currently designed may not be feasible for large-scale, longitudinal entomological monitoring. Therefore, the baited outdoor CDC light trap may be the most appropriate tool currently available for assessment of outdoor-biting and malaria transmission risk.

Development of a new barcode-based, multiplex-PCR, next-generation-sequencing assay and data processing and analytical pipeline for multiplicity of infection detection of Plasmodium falciparum.

BACKGROUND: Simultaneous infection with multiple malaria parasite strains is common in high transmission areas. Quantifying the number of strains per host, or the multiplicity of infection (MOI), provides additional parasite indices for assessing transmission levels but it is challenging to measure accurately with current tools. This paper presents new laboratory and analytical methods for estimating the MOI of Plasmodium falciparum. METHODS: Based on 24 single nucleotide polymorphisms (SNPs) previously identified as stable, unlinked targets across 12 of the 14 chromosomes within P. falciparum genome, three multiplex PCRs of short target regions and subsequent next generation sequencing (NGS) of the amplicons were developed. A bioinformatics pipeline including B4Screening pathway removed spurious amplicons to ensure consistent frequency calls at each SNP location, compiled amplicons by SNP site diversity, and performed algorithmic haplotype and strain reconstruction. The pipeline was validated by 108 samples generated from cultured-laboratory strain mixtures in different proportions and concentrations, with and without pre-amplification, and using whole blood and dried blood spots (DBS). The pipeline was applied to 273 smear-positive samples from surveys conducted in western Kenya, then providing results into StrainRecon Thresholding for Infection Multiplicity (STIM), a novel MOI estimator. RESULTS: The 24 barcode SNPs were successfully identified uniformly across the 12 chromosomes of P. falciparum in a sample using the pipeline. Pre-amplification and parasite concentration, while non-linearly associated with SNP read depth, did not influence the SNP frequency calls. Based on consistent SNP frequency calls at targeted locations, the algorithmic strain reconstruction for each laboratory-mixed sample had 98.5% accuracy in dominant strains. STIM detected up to 5 strains in field samples from western Kenya and showed declining MOI over time (q < 0.02), from 4.32 strains per infected person in 1996 to 4.01, 3.56 and 3.35 in 2001, 2007 and 2012, and a reduction in the proportion of samples with 5 strains from 57% in 1996 to 18% in 2012. CONCLUSION: The combined approach of new multiplex PCRs and NGS, the unique bioinformatics pipeline and STIM could identify 24 barcode SNPs of P. falciparum correctly and consistently. The methodology could be applied to field samples to reliably measure temporal changes in MOI.