A review of selective indoor residual spraying for malaria control.

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

Occupational exposure to malaria, leishmaniasis and arbovirus vectors in endemic regions: A systematic review.

Vector-borne diseases, including dengue, leishmaniasis and malaria, may be more common among individuals whose occupations or behaviours bring them into frequent contact with these disease vectors outside of their homes. A systematic review was conducted to ascertain at-risk occupations and situations that put individuals at increased risk of exposure to these disease vectors in endemic regions and identify the most suitable interventions for each exposure. The review was conducted in accordance with PRISMA guidelines on articles published between 1945 and October 2021, searched in 16 online databases. The primary outcome was incidence or prevalence of dengue, leishmaniasis or malaria. The review excluded ecological and qualitative studies, abstracts only, letters, commentaries, reviews, and studies of laboratory-acquired infections. Studies were appraised, data extracted, and a descriptive analysis conducted. Bite interventions for each risk group were assessed. A total of 1170 articles were screened and 99 included. Malaria, leishmaniasis and dengue were presented in 47, 41 and 24 articles, respectively; some articles presented multiple conditions. The most represented populations were soldiers, 38% (43 of 112 studies); refugees and travellers, 15% (17) each; migrant workers, 12.5% (14); miners, 9% (10); farmers, 5% (6); rubber tappers and missionaries, 1.8% (2) each; and forest workers, 0.9% (1). Risk of exposure was categorised into round-the-clock or specific times of day/night dependent on occupation. Exposure to these vectors presents a critical and understudied concern for outdoor workers and mobile populations. When devising interventions to provide round-the-clock vector bite protection, two populations are considered. First, mobile populations, characterized by their high mobility, may find potential benefits in insecticide-treated clothing, though more research and optimization are essential. Treated clothing offers personal vector protection and holds promise for economically disadvantaged individuals, especially when enabling them to self-treat their clothing to repel vectors. Secondly, semi-permanent and permanent settlement populations can receive a combination of interventions that offer both personal and community protection, including spatial repellents, suitable for extended stays. Existing research is heavily biased towards tourism and the military, diverting attention and resources from vulnerable populations where these interventions are most required like refugee populations as well as those residing in sub-Saharan Africa.

Field evaluation of a volatile pyrethroid spatial repellent and etofenprox treated clothing for outdoor protection against forest malaria vectors in Cambodia.

Cambodia's goal to eliminate malaria by 2025 is challenged by persistent transmission in forest and forest fringe areas, where people are exposed to Anopheles mosquito bites during the day and night. Volatile pyrethroid spatial repellents (VPSRs) and insecticide-treated clothing (ITC) could address these gaps. This study evaluated the outdoor application of one passive transfluthrin-based VPSR, four etofenprox-ITCs paired with a picaridin topical repellent, and a combination of VPSR and ITC against wild Anopheles landing in Cambodia. A 7 × 7 Latin-square study was conducted over 49 collection nights in temporary open structures in Mondulkiri Province. All interventions substantially reduced Anopheles landing, with protective efficacy ranging from 61 to 95%. Mathematical modeling showed significant reductions in vectoral capacity, especially with the combined ITC and VPSR and VPSR alone, albeit with decreased effectiveness over time. These interventions have the potential to reduce outdoor and daytime Anopheles biting, offering valuable contributions to malaria elimination efforts in Cambodia and the Greater Mekong Subregion, contingent upon achieving effective coverage and adherence.

Additional blood meals increase sporozoite infection in Anopheles mosquitoes but not Plasmodium falciparum genetic diversity.

The availability of nutrients from mosquito blood meals accelerates the development of Plasmodium falciparum laboratory strains in artificially infected Anopheles gambiae mosquitoes. The impact of multiple blood meals on the number of P. falciparum genotypes developing from polyclonal natural human malaria infections (field-isolates) remains unexplored. Here, we experimentally infect An. gambiae with P. falciparum field-isolates and measure the impact of an additional non-infectious blood meal on parasite development. We also assess parasite genetic diversity at the blood stage level of the parasite in the human host and of the sporozoites in the mosquito. Additional blood meals increase the sporozoite infection prevalence and intensity, but do not substantially affect the genetic diversity of sporozoites in the mosquito. The most abundant parasite genotypes in the human blood were transmitted to mosquitoes, suggesting that there was no preferential selection of specific genotypes. This study underlines the importance of additional mosquito blood meals for the development of parasite field-isolates in the mosquito host.

Comparison of the Trapping Efficacy of Locally Modified Gravid Aedes Trap and Autocidal Gravid Ovitrap for the Monitoring and Surveillance of Aedes aegypti Mosquitoes in Tanzania.

The study assessed the trapping efficacy of locally modified (1) Gravid Aedes Trap (GAT) lined with insecticide-treated net (ITN) as a killing agent and (2) Autocidal Gravid Ovitrap (AGO) with sticky board in the semi-field system (SFS) and field setting. Fully balanced Latin square experiments were conducted to compare GAT lined with ITN vs. AGO, both with either yeast or grass infusion. Biogent-Sentinel (BGS) with BG-Lure and no CO2 was used as a standard trap for Aedes mosquitoes. In the SFS, GAT outperformed AGO in collecting both nulliparous (65% vs. 49%, OR = 2.22, [95% CI: 1.89-2.60], p < 0.001) and gravid mosquitoes (73% vs. 64%, OR = 1.67, [95% CI: 1.41-1.97], p < 0.001). Similar differences were observed in the field. Yeast and grass infusion did not significantly differ in trapping gravid mosquitoes (OR = 0.91, [95% CI: 0.77-1.07], p = 0.250). The use of ITN improved mosquito recapture from 11% to 70% in the SFS. The same trend was observed in the field. Yeast was chosen for further evaluation in the optimized GAT due to its convenience and bifenthrin net for its resistance management properties. Mosquito density was collected when using 4× GATs relative to BGS-captured gravid mosquitoes 64 vs. 58 (IRR = 0.82, [95% CI: 0.35-1.95], p = 0.658) and showed no density dependence. Deployment of multiple yeast-baited GAT lined with bifenthrin net is cost-effective (single GAT < $8) compared to other traps such as BGS ($160).

Screening of malaria infections in human blood samples with varying parasite densities and anaemic conditions using AI-Powered mid-infrared spectroscopy.

BACKGROUND: Effective testing for malaria, including the detection of infections at very low densities, is vital for the successful elimination of the disease. Unfortunately, existing methods are either inexpensive but poorly sensitive or sensitive but costly. Recent studies have shown that mid-infrared spectroscopy coupled with machine learning (MIRs-ML) has potential for rapidly detecting malaria infections but requires further evaluation on diverse samples representative of natural infections in endemic areas. The aim of this study was, therefore, to demonstrate a simple AI-powered, reagent-free, and user-friendly approach that uses mid-infrared spectra from dried blood spots to accurately detect malaria infections across varying parasite densities and anaemic conditions. METHODS: Plasmodium falciparum strains NF54 and FCR3 were cultured and mixed with blood from 70 malaria-free individuals to create various malaria parasitaemia and anaemic conditions. Blood dilutions produced three haematocrit ratios (50%, 25%, 12.5%) and five parasitaemia levels (6%, 0.1%, 0.002%, 0.00003%, 0%). Dried blood spots were prepared on Whatman™ filter papers and scanned using attenuated total reflection-Fourier Transform Infrared (ATR-FTIR) for machine-learning analysis. Three classifiers were trained on an 80%/20% split of 4655 spectra: (I) high contrast (6% parasitaemia vs. negative), (II) low contrast (0.00003% vs. negative) and (III) all concentrations (all positive levels vs. negative). The classifiers were validated with unseen datasets to detect malaria at various parasitaemia levels and anaemic conditions. Additionally, these classifiers were tested on samples from a population survey in malaria-endemic villages of southeastern Tanzania. RESULTS: The AI classifiers attained over 90% accuracy in detecting malaria infections as low as one parasite per microlitre of blood, a sensitivity unattainable by conventional RDTs and microscopy. These laboratory-developed classifiers seamlessly transitioned to field applicability, achieving over 80% accuracy in predicting natural P. falciparum infections in blood samples collected during the field survey. Crucially, the performance remained unaffected by various levels of anaemia, a common complication in malaria patients. CONCLUSION: These findings suggest that the AI-driven mid-infrared spectroscopy approach holds promise as a simplified, sensitive and cost-effective method for malaria screening, consistently performing well despite variations in parasite densities and anaemic conditions. The technique simply involves scanning dried blood spots with a desktop mid-infrared scanner and analysing the spectra using pre-trained AI classifiers, making it readily adaptable to field conditions in low-resource settings. In this study, the approach was successfully adapted to field use, effectively predicting natural malaria infections in blood samples from a population-level survey in Tanzania. With additional field trials and validation, this technique could significantly enhance malaria surveillance and contribute to accelerating malaria elimination efforts.

Evaluating human landing catches as a measure of mosquito biting and the importance of considering additional modes of action.

Entomological evaluations of vector control tools often use human landing catches (HLCs) as a standard measure of a direct human-vector contact. However, some tools have additional characteristics, such as mortality, and HLCS are not sensitive for measuring other effects beyond landing inhibition. Therefore, additional measures may need to be considered when evaluating these tools for public health use. This study has two main aims (1) the evaluate the accuracy of HLCs as a proxy for feeding and (2) to compare the predicted reduction in vectorial capacity when we do and do not consider these additional characteristics. To achieve this, we analyse previously published semi-field data from an experiment which used HLCs and another where mosquitoes were allowed to feed in the presence of different dosages of the volatile pyrethroid spatial repellent, transfluthrin. We compare results for two mathematical models: one which only considers the reduction in feeding effect and one which also considers mortality before and after feeding (using data gathered by the aspiration of mosquitoes after the semi-field feeding/landing period and 24 h survival monitoring). These Bayesian hierarchical models are parameterised using Bayesian inference. We observe that, for susceptible mosquitoes, reduction in landing is underestimated by HLCs. For knockdown resistant mosquitoes the relationship is less clear; with HLCs sometimes appearing to overestimate this characteristic. We find HLCs tend to under-predict the relative reduction in vectorial capacity in susceptible mosquitoes while over-predicting this impact in knockdown-resistant mosquitoes. Models without secondary effects have lower predicted relative reductions in vectorial capacities. Overall, this study highlights the importance of considering additional characteristics to reduction in biting of volatile pyrethroid spatial repellents. We recommend that these are considered when evaluating novel vector control tools.

Inference for entomological semi-field experiments: Fitting a mathematical model assessing personal and community protection of vector-control interventions.

The effectiveness of vector-control tools is often assessed by experiments as a reduction in mosquito landings using human landing catches (HLCs). However, HLCs alone only quantify a single characteristic and therefore do not provide information on the overall impacts of the intervention product. Using data from a recent semi-field study which used time-stratified HLCs, aspiration of non-landing mosquitoes, and blood feeding, we suggest a Bayesian inference approach for fitting such data to a stochastic model. This model considers both personal protection, through a reduction in biting, and community protection, from mosquito mortality and disarming (prolonged inhibition of blood feeding). Parameter estimates are then used to predict the reduction of vectorial capacity induced by etofenpox-treated clothing, picaridin topical repellents, transfluthrin spatial repellents and metofluthrin spatial repellents, as well as combined interventions for Plasmodium falciparum malaria in Anopleles minimus. Overall, all interventions had both personal and community effects, preventing biting and killing or disarming mosquitoes. This led to large estimated reductions in the vectorial capacity, with substantial impact even at low coverage. As the interventions aged, fewer mosquitoes were killed; however the impact of some interventions changed from killing to disarming mosquitoes. Overall, this inference method allows for additional modes of action, rather than just reduction in biting, to be parameterised and highlights the tools assessed as promising malaria interventions.

Sub-lethal exposure to chlorfenapyr reduces the probability of developing Plasmodium falciparum parasites in surviving Anopheles mosquitoes.

BACKGROUND: Pyrethroid resistance in the key malaria vectors threatens the success of pyrethroid-treated nets. To overcome pyrethroid resistance, Interceptor® G2 (IG2), a 'first-in-class' dual insecticidal net that combines alpha-cypermethrin with chlorfenapyr, was developed. Chlorfenapyr is a pro-insecticide, requiring bio-activation by oxidative metabolism within the insect's mitochondria, constituting a mode of action preventing cross-resistance to pyrethroids. Recent epidemiological trials conducted in Benin and Tanzania confirm IG2's public health value in areas with pyrethroid-resistant Anopheles mosquitoes. As chlorfenapyr might also interfere with the metabolic mechanism of the Plasmodium parasite, we hypothesised that chlorfenapyr may provide additional transmission-reducing effects even if a mosquito survives a sub-lethal dose. METHODS: We tested the effect of chlorfenapyr netting to reduce Plasmodium falciparum transmission using a modified WHO tunnel test with a dose yielding sub-lethal effects. Pyrethroid-resistant Anopheles gambiae s.s. with L1014F and L1014S knockdown resistance alleles and expression levels of pyrethroid metabolisers CYP6P3, CYP6M2, CYP4G16 and CYP6P1 confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) prior to conducting experiments were exposed to untreated netting and netting treated with 200 mg/m3 chlorfenapyr for 8 h overnight and then fed on gametocytemic blood meals from naturally infected individuals. Prevalence and intensity of oocysts and sporozoites were determined on day 8 and day 16 after feeding. RESULTS: Both prevalence and intensity of P. falciparum infection in the surviving mosquitoes were substantially reduced in the chlorfenapyr-exposed mosquitoes compared to untreated nets. The odds ratios in the prevalence of oocysts and sporozoites were 0.33 (95% confidence interval; 95% CI 0.23-0.46) and 0.43 (95% CI 0.25-0.73), respectively, while only the incidence rate ratio for oocysts was 0.30 (95% CI 0.22-0.41). CONCLUSION: We demonstrated that sub-lethal exposure of pyrethroid-resistant mosquitoes to chlorfenapyr substantially reduces the proportion of infected mosquitoes and the intensity of the P. falciparum infection. This will likely also contribute to the reduction of malaria in communities beyond the direct killing of mosquitoes.

A randomized, double-blind placebo-control study assessing the protective efficacy of an odour-based 'push-pull' malaria vector control strategy in reducing human-vector contact.

Novel malaria vector control strategies targeting the odour-orientation of mosquitoes during host-seeking, such as 'attract-and-kill' or 'push-and-pull', have been suggested as complementary tools to indoor residual spraying and long-lasting insecticidal nets. These would be particularly beneficial if they can target vectors in the peri-domestic space where people are unprotected by traditional interventions. A randomized double-blind placebo-control study was implemented in western Kenya to evaluate: a 'push' intervention (spatial repellent) using transfluthrin-treated fabric strips positioned at open eave gaps of houses; a 'pull' intervention placing an odour-baited mosquito trap at a 5 m distance from a house; the combined 'push-pull' package; and the control where houses contained all elements but without active ingredients. Treatments were rotated through 12 houses in a randomized-block design. Outdoor biting was estimated using human landing catches, and indoor mosquito densities using light-traps. None of the interventions provided any protection from outdoor biting malaria vectors. The 'push' reduced indoor vector densities dominated by Anopheles funestus by around two thirds. The 'pull' device did not add any benefit. In the light of the high Anopheles arabiensis biting densities outdoors in the study location, the search for efficient outdoor protection and effective pull components needs to continue.

A semi-field evaluation in Thailand of the use of human landing catches (HLC) versus human-baited double net trap (HDN) for assessing the impact of a volatile pyrethroid spatial repellent and pyrethroid-treated clothing on Anopheles minimus landing.

BACKGROUND: The mosquito landing rate measured by human landing catches (HLC) is the conventional endpoint used to evaluate the impact of vector control interventions on human-vector exposure. Non-exposure based alternatives to the HLC are desirable to minimize the risk of accidental mosquito bites. One such alternative is the human-baited double net trap (HDN), but the estimated personal protection of interventions using the HDN has not been compared to the efficacy estimated using HLC. This semi-field study in Sai Yok District, Kanchanaburi Province, Thailand, evaluates the performance of the HLC and the HDN for estimating the effect on Anopheles minimus landing rates of two intervention types characterized by contrasting modes of action, a volatile pyrethroid spatial repellent (VSPR) and insecticide-treated clothing (ITC). METHODS: Two experiments to evaluate the protective efficacy of (1) a VPSR and (2) ITC, were performed. A block randomized cross-over design over 32 nights was carried out with both the HLC or HDN. Eight replicates per combination of collection method and intervention or control arm were conducted. For each replicate, 100 An. minimus were released and were collected for 6 h. The odds ratio (OR) of the released An. minimus mosquitoes landing in the intervention compared to the control arm was estimated using logistic regression, including collection method, treatment, and experimental day as fixed effects. RESULTS: For the VPSR, the protective efficacy was similar for the two methods: 99.3%, 95% CI (99.5-99.0) when measured by HLC, and 100% (100, Inf) when measured by HDN where no mosquitoes were caught (interaction test p = 0.99). For the ITC, the protective efficacy was 70% (60-77%) measured by HLC but there was no evidence of protection when measured by HDN [4% increase (15-27%)] (interaction test p < 0.001). CONCLUSIONS: Interactions between mosquitoes, bite prevention tools and the sampling method may impact the estimated intervention protective efficacy. Consequently, the sampling method must be considered when evaluating these interventions. The HDN is a valid alternative trapping method (relative to the HLC) for evaluating the impact of bite prevention methods that affect mosquito behaviour at a distance (e.g. VPSR), but not for interventions that operate through tarsal contact (e.g., ITC).

Malaria rapid diagnostic tests reliably detect asymptomatic Plasmodium falciparum infections in school-aged children that are infectious to mosquitoes.

BACKGROUND: Asymptomatic malaria infections (Plasmodium falciparum) are common in school-aged children and represent a disease transmission reservoir as they are potentially infectious to mosquitoes. To detect and treat such infections, convenient, rapid and reliable diagnostic tools are needed. In this study, malaria rapid diagnostic tests (mRDT), light microscopy (LM) and quantitative polymerase chain reaction (qPCR) were used to evaluate their performance detecting asymptomatic malaria infections that are infectious to mosquitoes. METHODS: One hundred seventy asymptomatic school-aged children (6-14 years old) from the Bagamoyo district in Tanzania were screened for Plasmodium spp. infections using mRDT (SD BIOLINE), LM and qPCR. In addition, gametocytes were detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR) for all qPCR-positive children. Venous blood from all P. falciparum positive children was fed to female Anopheles gambiae sensu stricto mosquitoes via direct membrane feeding assays (DMFAs) after serum replacement. Mosquitoes were dissected for oocyst infections on day 8 post-infection. RESULTS: The P. falciparum prevalence in study participants was 31.7% by qPCR, 18.2% by mRDT and 9.4% by LM. Approximately one-third (31.2%) of asymptomatic malaria infections were infectious to mosquitoes in DMFAs. In total, 297 infected mosquitoes were recorded after dissections, from which 94.9% (282/297) were derived from infections detected by mRDT and 5.1% (15/297) from subpatent mRDT infections. CONCLUSION: The mRDT can be used reliably to detect children carrying gametocyte densities sufficient to infect high numbers of mosquitoes. Subpatent mRDT infections contributed marginally to the pool of oocyts-infected mosquitoes.

Semi-field evaluation of a volatile transfluthrin-based intervention reveals efficacy as a spatial repellent and evidence of other modes of action.

Presently, the most common malaria control tools-i.e., long lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS)-are limited to targeting indoor biting and resting behaviors of Anopheles mosquito species. Few interventions are targeted towards malaria control in areas where transmission is driven or persists due to outdoor biting behaviors. This study investigated a volatile pyrethroid-based spatial repellent (VPSR) designed to bridge this gap and provide protection from mosquito bites in outdoor spaces. Southern Province, Zambia, is one such environment where outdoor biting is suspected to contribute to malaria transmission, where people are active in the evening in open-walled outdoor kitchens. This study assessed the VPSR in replica kitchens within a controlled semi-field environment. Endpoints included effects on mosquito host seeking, immediate and delayed mortality, deterrence, blood feeding inhibition, and fertility. Host-seeking was reduced by approximately 40% over the course of nightly releases in chambers containing VPSR devices. Mosquito behavior was not uniform throughout the night, and the modeled effect of the intervention was considerably higher when hourly catch rates were considered. These two observations highlight a limitation of this overnight semi-field design and consideration of mosquito circadian rhythms is recommended for future semi-field studies. Additionally, deterrence and immediate mortality were both observed in treatment chambers, with evidence of delayed mortality and a dose related response. These results demonstrate a primarily personal protective mode of action with possible positive and negative community effects. Further investigation into this primary mode of action will be conducted through a field trial of the same product in nearby communities.

Multi-country evaluation of the durability of pyrethroid plus piperonyl-butoxide insecticide-treated nets: study protocol.

BACKGROUND: Mass distributions of long-lasting insecticidal nets (LLINs) have contributed to large reductions in the malaria burden. However, this success is in jeopardy due in part to the increasing pyrethroid-resistant mosquito population as well as low LLINs coverage in various areas because the lifespan of LLINs is often shorter than the interval between replenishment campaigns. New insecticide-treated nets (ITNs) containing pyrethroid and piperonyl-butoxide (PBO) have shown a greater reduction in the incidence of malaria than pyrethroid LLINs in areas with pyrethroid-resistant mosquitoes. However, the durability (attrition, bio-efficacy, physical integrity and chemical retainment) of pyrethroid-PBO ITNs under operational settings has not been fully characterized. This study will measure the durability of pyrethroid-PBO ITNs to assess whether they meet the World Health Organization (WHO) three years of operational performance criteria required to be categorized as "long-lasting". METHODS: A prospective household randomized controlled trial will be conducted simultaneously in Tanzania, India and Côte d'Ivoire to estimate the field durability of three pyrethroid-PBO ITNs (Veeralin®, Tsara® Boost, and Olyset® Plus) compared to a pyrethroid LLIN: MAGNet®. Durability monitoring will be conducted up to 36 months post-distribution and median survival in months will be calculated. The proportion of ITNs: (1) lost (attrition), (2) physical integrity, (3) resistance to damage score, (4) meeting WHO bio-efficacy (≥ 95% knockdown after 1 h or ≥ 80% mortality after 24 h for WHO cone bioassay, or ≥ 90% blood-feeding inhibition or ≥ 80% mortality after 24 h for WHO Tunnel tests) criteria against laboratory-reared resistant and susceptible mosquitoes, and insecticidal persistence over time will be estimated. The non-inferiority of Veeralin® and Tsara® Boost to the first-in-class, Olyset® Plus will additionally be assessed for mortality, and the equivalence of 20 times washed ITNs compared to field aged ITNs will be assessed for mortality and blood-feeding inhibition endpoints in the Ifakara Ambient Chamber Test, Tanzania. CONCLUSION: This will be the first large-scale prospective household randomized controlled trial of pyrethroid-PBO ITNs in three different countries in East Africa, West Africa and South Asia, simultaneously. The study will generate information on the replenishment intervals for PBO nets.

Modified World Health Organization (WHO) Tunnel Test for Higher Throughput Evaluation of Insecticide-Treated Nets (ITNs) Considering the Effect of Alternative Hosts, Exposure Time, and Mosquito Density.

The standard World Health Organization (WHO) tunnel test is a reliable laboratory bioassay used for "free-flying" testing of insecticide-treated nets (ITNs) bio-efficacy where mosquitoes pass through a ITN sample to reach a live animal bait. Multiple parameters (i.e., bait, exposure time, and mosquito density) may affect the outcomes measured in tunnel tests. Therefore, a comparison was conducted of alternative hosts, exposure time, and lower mosquito density against the current gold standard test (100 mosquitoes, animal bait, and 12-h exposure) as outlined in the WHO ITN evaluation guideline. This was done with the aim to make the tunnel test cheaper and with higher throughput to meet the large sample sizes needed for bio-efficacy durability monitoring of chlorfenapyr ITNs that must be evaluated in "free-flying" bioassays. Methods: A series of experiments were conducted in the WHO tunnel test to evaluate the impact of the following factors on bio-efficacy endpoints of mosquito mortality at 24-h (M24) and 72-h (M72) and blood-feeding success (BFS): (1) baits (rabbit, membrane, human arm); (2) exposure time in the tunnel (1 h vs. 12 h); and (3) mosquito density (50 vs. 100). Finally, an alternative bioassay using a membrane with 50 mosquitoes (membrane-50) was compared to the gold standard bioassay (rabbit with 100 mosquitoes, rabbit-100). Pyrethroid-resistant Anopheles arabiensis and pyrethroid susceptible Anopheles gambiae were used to evaluate Interceptor® and Interceptor® G2 ITNs. Results: Using a human arm as bait gave a very different BFS, which impacted measurements of M24 and M72. The same trends in M24, M72 and BFS were observed for both Interceptor® ITN and Interceptor® G2 unwashed and washed 20 times measured using the gold standard WHO tunnel test (rabbit-100) or rabbit with 50 mosquitoes (rabbit-50). M24, M72 and BFS were not statistically different when either 50 or 100 mosquitoes were used with rabbit bait in the tunnel bioassay for either the susceptible or resistant strains. No systematic difference was observed between rabbit-50 and rabbit-100 in the agreement by the Bland and Altman method (B&A). The mean difference was 4.54% (-22.54-31.62) in BFS and 1.71% (-28.71-32.12) in M72 for rabbit-50 versus rabbit-100. Similar M24, M72 and lower BFS was measured by membrane-50 compared to rabbit-100. No systematic difference was observed in the agreement between membrane-50 and rabbit-100, by B&A. The mean difference was 9.06% (-11.42-29.64) for BSF and -5.44% (-50.3-39.45) for M72. Both membrane-50, rabbit-50 and rabbit-100 predicted the superiority of Interceptor® G2 over Interceptor® ITN for the resistant strain on M72. Conclusion: These results demonstrate that WHO tunnel tests using rabbit bait may be run with 50 mosquitoes to increase sample sizes needed for bio-efficacy durability monitoring of ITNs in "free-flying" bioassays. Using a membrane feeder with 50 mosquitoes is a potential replacement for the WHO tunnel bioassay with animal bait if control blood feeding rates can be improved to 50% because blood feeding impacts mosquito survival after exposure to insecticides.

Comparison of cone bioassay estimates at two laboratories with different Anopheles mosquitoes for quality assurance of pyrethroid insecticide-treated nets.

BACKGROUND: Quality assurance (QA) of insecticide-treated nets (ITNs) delivered to malaria-endemic countries is conducted by measuring physiochemical parameters, but not bioefficacy against malaria mosquitoes. This study explored utility of cone bioassays for pre-delivery QA of pyrethroid ITNs to test the assumption that cone bioassays are consistent across locations, mosquito strains, and laboratories. METHODS: Double-blinded bioassays were conducted on twenty unused pyrethroid ITNs of 4 brands (100 nets, 5 subsamples per net) that had been delivered for mass distribution in Papua New Guinea (PNG) having passed predelivery inspections. Cone bioassays were performed on the same net pieces following World Health Organization (WHO) guidelines at the PNG Institute of Medical Research (PNGIMR) using pyrethroid susceptible Anopheles farauti sensu stricto (s.s.) and at Ifakara Health Institute (IHI), Tanzania using pyrethroid susceptible Anopheles gambiae s.s. Additionally, WHO tunnel tests were conducted at IHI on ITNs that did not meet cone bioefficacy thresholds. Results from IHI and PNGIMR were compared using Spearman's Rank correlation, Bland-Altman (BA) analysis and analysis of agreement. Literature review on the use of cone bioassays for unused pyrethroid ITNs testing was conducted. RESULTS: In cone bioassays, 13/20 nets (65%) at IHI and 8/20 (40%) at PNGIMR met WHO bioefficacy criteria. All nets met WHO bioefficacy criteria on combined cone/tunnel tests at IHI. Results from IHI and PNGIMR correlated on 60-min knockdown (KD60) (rs = 0.6,p = 0.002,n = 20) and 24-h mortality (M24) (rs = 0.9,p < 0.0001,n = 20) but BA showed systematic bias between the results. Of the 5 nets with discrepant result between IHI and PNGIMR, three had confidence intervals overlapping the 80% mortality threshold, with averages within 1-3% of the threshold. Including these as a pass, the agreement between the results to predict ITN failure was good with kappa = 0.79 (0.53-1.00) and 90% accuracy. CONCLUSIONS: Based on these study findings, the WHO cone bioassay is a reproducible bioassay for ITNs with > 80% M24, and for all ITNs provided inherent stochastic variation and systematic bias are accounted for. The literature review confirms that WHO cone bioassay bioefficacy criteria have been previously achieved by all pyrethroid ITNs (unwashed), without the need for additional tunnel tests. The 80% M24 threshold remains the most reliable indicator of pyrethroid ITN quality using pyrethroid susceptible mosquitoes. In the absence of alternative tests, cone bioassays could be used as part of pre-delivery QA.

The Centres for Disease Control light trap (CDC-LT) and the human decoy trap (HDT) compared to the human landing catch (HLC) for measuring Anopheles biting in rural Tanzania.

BACKGROUND: Vector mosquito biting intensity is an important measure to understand malaria transmission. Human landing catch (HLC) is an effective but labour-intensive, expensive, and potentially hazardous entomological surveillance tool. The Centres for Disease Control light trap (CDC-LT) and the human decoy trap (HDT) are exposure-free alternatives. This study compared the CDC-LT and HDT against HLC for measuring Anopheles biting in rural Tanzania and assessed their suitability as HLC proxies. METHODS: Indoor mosquito surveys using HLC and CDC-LT and outdoor surveys using HLC and HDT were conducted in 2017 and in 2019 in Ulanga, Tanzania in 19 villages, with one trap/house/night. Species composition, sporozoite rates and density/trap/night were compared. Aggregating the data by village and month, the Bland-Altman approach was used to assess agreement between trap types. RESULTS: Overall, 66,807 Anopheles funestus and 14,606 Anopheles arabiensis adult females were caught with 6,013 CDC-LT, 339 indoor-HLC, 136 HDT and 195 outdoor-HLC collections. Indoors, CDC-LT caught fewer An. arabiensis (Adjusted rate ratio [Adj.RR] = 0.35, 95% confidence interval [CI]: 0.27-0.46, p < 0.001) and An. funestus (Adj.RR = 0.63, 95%CI: 0.51-0.79, p < 0.001) than HLC per trap/night. Outdoors, HDT caught fewer An. arabiensis (Adj.RR = 0.04, 95%CI: 0.01-0.14, p < 0.001) and An. funestus (Adj.RR = 0.10, 95%CI: 0.07-0.15, p < 0.001) than HLC. The bias and variability in number of mosquitoes caught by the different traps were dependent on mosquito densities. The relative efficacies of both CDC-LT and HDT in comparison to HLC declined with increased mosquito abundance. The variability in the ratios was substantial for low HLC counts and decreased as mosquito abundance increased. The numbers of sporozoite positive mosquitoes were low for all traps. CONCLUSIONS: CDC-LT can be suitable for comparing mosquito populations between study arms or over time if accuracy in the absolute biting rate, compared to HLC, is not required. CDC-LT is useful for estimating sporozoite rates because large numbers of traps can be deployed to collect adequate mosquito samples. The present design of the HDT is not amenable for use in large-scale entomological surveys. Use of HLC remains important for estimating human exposure to mosquitoes as part of estimating the entomological inoculation rate (EIR).

"In starvation, a bone can also be meat": a mixed methods evaluation of factors associated with discarding of long-lasting insecticidal nets in Bagamoyo, Tanzania.

BACKGROUND: Between 2000 and 2019, more than 1.8 billion long-lasting insecticidal nets (LLINs) were distributed in Africa. While the insecticidal durability of LLINs is around 3 years, nets are commonly discarded 2 years post distribution. This study investigated the factors associated with the decision of users to discard LLINs. METHODS: A mixed-method sequential explanatory approach using a structured questionnaire followed by focus group discussions (FGDs) to collect information on experiences, views, reasons, how and when LLINs are discarded. Out of 6,526 households that responded to the questionnaire of LLINs durability trial, 160 households were randomly selected from the households in four villages in Bagamoyo Tanzania for FGDs but only 155 households participated in the FGDs. Five of the household representatives couldn't participate due to unexpected circumstances. A total of sixteen FGDs each comprising of 8-10 adults were conducted; older women (40-60 years), older men (40-60 years), younger women (18-39 years), younger men (18-39 years). During the FGDs, participants visually inspected seven samples of LLINs that were "too-torn" based on Proportionate Hole Index recommended by the World Health Organization (WHO) guidelines on LLIN testing, the nets were brought to the discussion and participants had to determine if such LLINs were to be kept or discarded. The study assessed responses from the same participants that attended FGD and also responded to the structured questionnaire, 117 participants fulfilled the criteria, thus data from only 117 participants are analysed in this study. RESULTS: In FGDs, integrity of LLIN influenced the decision to discard or keep a net. Those of older age, women, and householders with lower income were more likely to classify a WHO "too-torn" net as "good". The common methods used to discard LLINs were burning and burying. The findings were seen in the quantitative analysis. For every additional hole, the odds of discarding a WHO "too-torn" LLIN increased [OR = 1.05 (95%CI (1.04-1.07)), p < 0.001]. Younger age group [OR = 4.97 (95%CI (3.25-7.32)), p < 0.001], male-headed households [OR = 6.85 (95%CI (4.44 -10.59)), p < 0.001], and wealthy households [OR = 3.88 (95%CI (2.33-6.46)), p < 0.001] were more likely to discard LLINs. CONCLUSION: Integrity of LLIN was the main determinant for discarding or keeping LLINs and the decision to discard the net is associated with socioeconomic status of the household, and the age and gender of respondents. WHO "too torn" nets are encouraged to be used instead of none until replacement, and disposal of nets should be based on recommendation.

Hitting the right note at the right time: Circadian control of audibility in Anopheles mosquito mating swarms is mediated by flight tones.

Mating swarms of malaria mosquitoes form every day at sunset throughout the tropical world. They typically last less than 30 minutes. Activity must thus be highly synchronized between the sexes. Moreover, males must identify the few sporadically entering females by detecting the females' faint flight tones. We show that the Anopheles circadian clock not only ensures a tight synchrony of male and female activity but also helps sharpen the males' acoustic detection system: By raising their flight tones to 1.5 times the female flight tone, males enhance the audibility of females, specifically at swarm time. Previously reported "harmonic convergence" events are only a random by-product of the mosquitoes' flight tone variance and not a signature of acoustic interaction between males and females. The flight tones of individual mosquitoes occupy narrow, partly non-overlapping frequency ranges, suggesting that the audibility of individual females varies across males.

Effect of interventions to reduce malaria incidence among military personnel on active duty: study protocol for a cluster randomised controlled trial of the impact of etofenprox-treated uniforms, permethrin-treated uniforms and DEET insect repellent.

BACKGROUND: While there is strong evidence that bite protection methods such as permethrin-treated clothing and topical insect repellents are protective against insect bites, there are few studies assessing the impact on malaria infection. This study will estimate the protective efficacy of treated uniforms and DEET insect repellent on the incidence of malaria infection among military personnel in an operational setting. Permethrin-treated uniforms used with DEET lotion will be compared to etofenprox-treated uniforms with DEET lotion. The effect of DEET lotion will be estimated by comparing permethrin-treated uniforms with DEET or placebo lotion. METHOD: A cluster randomised double-blind placebo-controlled trial is planned to evaluate the effectiveness of the interventions on preventing malaria infections in soldiers on active duty at Mgambo National Service Camp in Tanga, Tanzania. The arms are (1) permethrin-treated uniform with 30% DEET liposome formula; (2) permethrin-treated uniform with placebo lotion; (3) candidate insect repellent system, i.e. etofenprox-treated uniform with 30% DEET liposome formula; and (4) placebo, i.e. untreated uniforms with placebo lotion. The primary outcome is the incidence of Plasmodium falciparum malaria infection detected by polymerase chain reaction (PCR) by active case detection using surveys every 2 weeks for 12 months. Rapid diagnostic tests will be used for the diagnosis of participants with symptoms. The unit of randomisation will be combania: companies formed by recruits aged 18 to 25 years; combania do activities together and sleep in the same dormitory. Unequal randomisation will be used to optimise statistical power for the primary comparison between permethrin-treated uniforms with DEET and etofenprox-treated uniforms with DEET. DISCUSSION: This trial will provide the estimate of the effects of permethrin with DEET compared to those of the new fabric treatment etofenprox with DEET and any additional effect of using DEET. The results will inform strategies to protect military personnel and civilians who have more outdoor or occupational malaria exposure than the general public. TRIAL REGISTRATION: ClinicalTrials.gov NCT02938975 .