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.

Evidence supporting deployment of next generation insecticide treated nets in Burkina Faso: bioassays with either chlorfenapyr or piperonyl butoxide increase mortality of pyrethroid-resistant Anopheles gambiae.

BACKGROUND: Pyrethroid resistance poses a major threat to the efficacy of insecticide-treated nets (ITNs) in Burkina Faso and throughout sub-Saharan Africa, particularly where resistance is present at high intensity. For such areas, there are alternative ITNs available, including the synergist piperonyl butoxide (PBO)-based ITNs and dual active ingredient ITNs such as Interceptor G2 (treated with chlorfenapyr and alpha-cypermethrin). Before deploying alternative ITNs on a large scale it is crucial to characterize the resistance profiles of primary malaria vector species for evidence-based decision making. METHODS: Larvae from the predominant vector, Anopheles gambiae sensu lato (s.l.) were collected from 15 sites located throughout Burkina Faso and reared to adults for bioassays to assess insecticide resistance status. Resistance intensity assays were conducted using WHO tube tests to determine the level of resistance to pyrethroids commonly used on ITNs at 1×, 5 × and 10 × times the diagnostic dose. WHO tube tests were also used for PBO synergist bioassays with deltamethrin and permethrin. Bottle bioassays were conducted to determine susceptibility to chlorfenapyr at a dose of 100 µg/bottle. RESULTS: WHO tube tests revealed high intensity resistance in An. gambiae s.l. to deltamethrin and alpha-cypermethrin in all sites tested. Resistance intensity to permethrin was either moderate or high in 13 sites. PBO pre-exposure followed by deltamethrin restored full susceptibility in one site and partially restored susceptibility in all but one of the remaining sites (often reaching mortality greater than 80%). PBO pre-exposure followed by permethrin partially restored susceptibility in 12 sites. There was no significant increase in permethrin mortality after PBO pre-exposure in Kampti, Karangasso-Vigué or Mangodara; while in Seguenega, Orodara and Bobo-Dioulasso there was a significant increase in mortality, but rates remained below 50%. Susceptibility to chlorfenapyr was confirmed in 14 sites. CONCLUSION: High pyrethroid resistance intensity in An. gambiae s.l. is widespread across Burkina Faso and may be a predictor of reduced pyrethroid ITN effectiveness. PBO + deltamethrin ITNs would likely provide greater control than pyrethroid nets. However, since susceptibility in bioassays was not restored in most sites following pre-exposure to PBO, Interceptor G2 may be a better long-term solution as susceptibility was recorded to chlorfenapyr in nearly all sites. This study provides evidence supporting the introduction of both Interceptor G2 nets and PBO nets, which were distributed in Burkina Faso in 2019 as part of a mass campaign.

Determination of the discriminating concentration of chlorfenapyr (pyrrole) and Anopheles gambiae sensu lato susceptibility testing in preparation for distribution of Interceptor® G2 insecticide-treated nets.

BACKGROUND: Following agricultural use and large-scale distribution of insecticide-treated nets (ITNs), malaria vector resistance to pyrethroids is widespread in sub-Saharan Africa. Interceptor® G2 is a new dual active ingredient (AI) ITN treated with alpha-cypermethrin and chlorfenapyr for the control of pyrethroid-resistant malaria vectors. In anticipation of these new nets being more widely distributed, testing was conducted to develop a chlorfenapyr susceptibility bioassay protocol and gather susceptibility information. METHODS: Bottle bioassay tests were conducted using five concentrations of chlorfenapyr at 12.5, 25, 50, 100, and 200 µg AI/bottle in 10 countries in sub-Saharan Africa using 13,639 wild-collected Anopheles gambiae sensu lato (s.l.) (56 vector populations per dose) and 4,494 pyrethroid-susceptible insectary mosquitoes from 8 colonized strains. In parallel, susceptibility tests were conducted using a provisional discriminating concentration of 100 µg AI/bottle in 16 countries using 23,422 wild-collected, pyrethroid-resistant An. gambiae s.l. (259 vector populations). Exposure time was 60 min, with mortality recorded at 24, 48 and 72 h after exposure. RESULTS: Median mortality rates (up to 72 h after exposure) of insectary colony mosquitoes was 100% at all five concentrations tested, but the lowest dose to kill all mosquitoes tested was 50 µg AI/bottle. The median 72-h mortality of wild An. gambiae s.l. in 10 countries was 71.5, 90.5, 96.5, 100, and 100% at concentrations of 12.5, 25, 50, 100, and 200 µg AI/bottle, respectively. Log-probit analysis of the five concentrations tested determined that the LC95 of wild An. gambiae s.l. was 67.9 µg AI/bottle (95% CI: 48.8-119.5). The discriminating concentration of 203.8 µg AI/bottle (95% CI: 146-359) was calculated by multiplying the LC95 by three. However, the difference in mortality between 100 and 200 µg AI/bottle was minimal and large-scale testing using 100 µg AI/bottle with wild An. gambiae s.l. in 16 countries showed that this concentration was generally suitable, with a median mortality rate of 100% at 72 h. CONCLUSIONS: This study determined that 100 or 200 µg AI/bottle chlorfenapyr in bottle bioassays are suitable discriminating concentrations for monitoring susceptibility of wild An. gambiae s.l., using mortality recorded up to 72 h. Testing in 16 countries in sub-Saharan Africa demonstrated vector susceptibility to chlorfenapyr, including mosquitoes with multiple resistance mechanisms to pyrethroids.

Intensity of pyrethroid resistance in Anopheles gambiae before and after a mass distribution of insecticide-treated nets in Kinshasa and in 11 provinces of the Democratic Republic of Congo.

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

Malaria vector species composition and entomological indices following indoor residual spraying in regions bordering Lake Victoria, Tanzania.

BACKGROUND: Vector control through long-lasting insecticidal nets (LLINs) and focal indoor residual spraying (IRS) is a major component of the Tanzania national malaria control strategy. In mainland Tanzania, IRS has been conducted annually around Lake Victoria basin since 2007. Due to pyrethroid resistance in malaria vectors, use of pyrethroids for IRS was phased out and from 2014 to 2017 pirimiphos-methyl (Actellic® 300CS) was sprayed in regions of Kagera, Geita, Mwanza, and Mara. Entomological surveillance was conducted in 10 sprayed and 4 unsprayed sites to determine the impact of IRS on entomological indices related to malaria transmission risk. METHODS: WHO cone bioassays were conducted monthly on interior house walls to determine residual efficacy of pirimiphos-methyl CS. Indoor CDC light traps with or without bottle rotator were hung next to protected sleepers indoors and also set outdoors (unbaited) as a proxy measure for indoor and outdoor biting rate and time of biting. Prokopack aspirators were used indoors to capture resting malaria vectors. A sub-sample of Anopheles was tested by PCR to determine species identity and ELISA for sporozoite rate. RESULTS: Annual IRS with Actellic® 300CS from 2015 to 2017 was effective on sprayed walls for a mean of 7 months in cone bioassay. PCR of 2016 and 2017 samples showed vector populations were predominantly Anopheles arabiensis (58.1%, n = 4,403 IRS sites, 58%, n = 2,441 unsprayed sites). There was a greater proportion of Anopheles funestus sensu stricto in unsprayed sites (20.4%, n = 858) than in sprayed sites (7.9%, n = 595) and fewer Anopheles parensis (2%, n = 85 unsprayed, 7.8%, n = 591 sprayed). Biting peaks of Anopheles gambiae sensu lato (s.l.) followed periods of rainfall occurring between October and April, but were generally lower in sprayed sites than unsprayed. In most sprayed sites, An. gambiae s.l. indoor densities increased between January and February, i.e., 10-12 months after IRS. The predominant species An. arabiensis had a sporozoite rate in 2017 of 2.0% (95% CI 1.4-2.9) in unsprayed sites compared to 0.8% (95% CI 0.5-1.3) in sprayed sites (p = 0.003). Sporozoite rates were also lower for An. funestus collected in sprayed sites. CONCLUSION: This study contributes to the understanding of malaria vector species composition, behaviour and transmission risk following IRS around Lake Victoria and can be used to guide malaria vector control strategies in Tanzania.

Susceptibility testing of Anopheles malaria vectors with the neonicotinoid insecticide clothianidin; results from 16 African countries, in preparation for indoor residual spraying with new insecticide formulations.

BACKGROUND: In 2017, more than 5 million house structures were sprayed through the U.S. President's Malaria Initiative, protecting more than 21 million people in sub-Saharan Africa. New IRS formulations, SumiShield™ 50WG and Fludora Fusion™ WP-SB, became World Health Organization (WHO) prequalified vector control products in 2017 and 2018, respectively. Both formulations contain the neonicotinoid active ingredient, clothianidin. The target site of neonicotinoids represents a novel mode of action for vector control, meaning that cross-resistance through existing mechanisms is less likely. In preparation for rollout of clothianidin formulations as part of national IRS rotation strategies, baseline susceptibility testing was conducted in 16 countries in sub-Saharan Africa. METHODS: While work coordinated by the WHO is ongoing to develop a suitable bottle bioassay procedure, there was no published guidance regarding clothianidin susceptibility procedures or diagnostic concentrations. Therefore, a protocol was developed for impregnating filter papers with 2% w/v SumiShield™ 50WG dissolved in distilled water. Susceptibility tests were conducted using insectary-reared reference Anopheles and wild collected malaria vector species. All tests were conducted within 24 h of treating papers, with mortality recorded daily for 7 days, due to the slow-acting nature of clothianidin against mosquitoes. Anopheles gambiae sensu lato (s.l.) adults from wild collected larvae were tested in 14 countries, with wild collected F0 Anopheles funestus s.l. tested in Mozambique and Zambia. RESULTS: One-hundred percent mortality was reached with all susceptible insectary strains and with wild An. gambiae s.l. from all sites in 11 countries. However, tests in at least one location from 5 countries produced mortality below 98%. While this could potentially be a sign of clothianidin resistance, it is more likely that the diagnostic dose or protocol requires further optimization. Repeat testing in 3 sites in Ghana and Zambia, where possible resistance was detected, subsequently produced 100% mortality. Results showed susceptibility to clothianidin in 38 of the 43 sites in sub-Saharan Africa, including malaria vectors with multiple resistance mechanisms to pyrethroids, carbamates and organophosphates. CONCLUSIONS: This study provides an interim diagnostic dose of 2% w/v clothianidin on filter papers which can be utilized by National Malaria Control Programmes and research organizations until the WHO concludes multi-centre studies and provides further guidance.

Nationwide insecticide resistance status and biting behaviour of malaria vector species in the Democratic Republic of Congo.

BACKGROUND: Globally, the Democratic Republic of Congo (DRC) accounted for 9% of malaria cases and 10% of malaria deaths in 2015. As part of control efforts, more than 40 million long-lasting insecticidal nets (LLINs) were distributed between 2008 and 2013, resulting in 70% of households owning one or more LLINs in 2014. To optimize vector control efforts, it is critical to monitor vector behaviour and insecticide resistance trends. Entomological data was collected from eight sentinel sites throughout DRC between 2013 and 2016 in Kingasani, Mikalayi, Lodja, Kabondo, Katana, Kapolowe, Tshikaji and Kalemie. Mosquito species present, relative densities and biting times were monitored using human landing catches (HLC) conducted in eight houses, three times per year. HLC was conducted monthly in Lodja and Kapolowe during 2016 to assess seasonal dynamics. Laboratory data included resistance mechanism frequency and sporozoite rates. Insecticide susceptibility testing was conducted with commonly used insecticides including deltamethrin and permethrin. Synergist bioassays were conducted with PBO to determine the role of oxidases in permethrin resistance. RESULTS: In Lodja, monthly Anopheles gambiae s.l. biting rates were consistently high at > 10 bites/person/night indoors and outdoors. In Kapolowe, An. gambiae s.l. dominated during the rainy season, and Anopheles funestus s.l. during the dry season. In all sites, An. gambiae and An. funestus biting occurred mostly late at night. In Kapolowe, significant biting of both species started around 19:00, typically before householders use nets. Sporozoite rates were high, with a mean of 4.3% (95% CI 3.4-5.2) for An. gambiae and 3.3% (95% CI 1.3-5.3) for An. funestus. Anopheles gambiae were resistant to permethrin in six out of seven sites in 2016. In three sites, susceptibility to deltamethrin was observed despite high frequency permethrin resistance, indicating the presence of pyrethroid-specific resistance mechanisms. Pre-exposure to PBO increased absolute permethrin-associated mortality by 24%, indicating that resistance was partly due to metabolic mechanisms. The kdr-1014F mutation in An. gambiae was present at high frequency (> 70%) in three sites (Kabondo, Kingasani and Tshikaji), and lower frequency (< 20%) in two sites (Lodja and Kapolowe). CONCLUSION: The finding of widespread resistance to permethrin in DRC is concerning and alternative insecticides should be evaluated.

A village level cluster-randomized entomological evaluation of combination long-lasting insecticidal nets containing pyrethroid plus PBO synergist in Southern Mali.

BACKGROUND: There is growing concern that malaria vector resistance to pyrethroid insecticides may reduce the effectiveness of long-lasting insecticidal nets (LLINs). Combination LLINs are designed to control susceptible and pyrethroid-resistant mosquito populations through a mixture of pyrethroid with piperonyl butoxide (PBO) synergist. A cluster randomized trial with entomology outcome measures was conducted in Mali to determine the added benefit over mono-treated pyrethroid predecessors. Four LLIN treatments; permethrin + PBO, permethrin, deltamethrin + PBO, and deltamethrin, were randomly allocated to four villages each (16 villages total) and distributed to cover every sleeping place. Entomological monitoring of indoor Anopheles resting densities, host preference, vector longevity, and sporozoite rates were monitored every 2 months over 2 years in 2014 and 2015. RESULTS: Bottle bioassays confirmed permethrin and deltamethrin resistance in Anopheles gambiae sensu lato (s.l.), (the predominant species throughout the study) with pre-exposure to PBO indicating partial involvement of oxidases. Between 2014 and 2015 the mean indoor resting density was greater in the deltamethrin + PBO LLIN arm than the deltamethrin LLIN arm at 3.05 (95% CI 3.00-3.10) An. gambiae s.l. per room per day compared with 1.9 (95% CI 1.87-1.97). There was no significant difference in sporozoite rate at 3.97% (95% CI 2.91-5.02) for the deltamethrin LLIN arm and 3.04% (95% CI 2.21-3.87) for deltamethrin + PBO LLIN arm (P = 0.17). However, when analysed by season there was some evidence that the sporozoite rate was lower in the deltamethrin + PBO LLIN arm than deltamethrin LLIN arm during the rainy/high malaria transmission seasons at 1.95% (95% CI 1.18-2.72) and 3.70% (95% CI 2.56-4.84) respectively (P = 0.01). CONCLUSIONS: While there was some evidence that An. gambiae s.l. sporozoite rates were lower in villages with deltamethrin + PBO LLINs during the high malaria transmission seasons of 2014-2015, there was no reduction in parity rates or indoor resting densities. There was also no evidence that permethrin + PBO LLINs provided any improved control when compared with permethrin LLINs. Combination nets may have a greater impact in areas where mixed function oxidases play a more important role in pyrethroid resistance.

Experimental hut evaluation of a novel long-lasting non-pyrethroid durable wall lining for control of pyrethroid-resistant Anopheles gambiae and Anopheles funestus in Tanzania.

BACKGROUND: A novel, insecticide-treated, durable wall lining (ITWL), which mimics indoor residual spraying (IRS), has been developed to provide prolonged vector control when fixed to the inner walls of houses. PermaNet® ITWL is a polypropylene material containing non-pyrethroids (abamectin and fenpyroximate) which migrate gradually to the surface. METHODS: An experimental hut trial was conducted in an area of pyrethroid-resistant Anopheles gambiae s.l. and Anopheles funestus s.s. to compare the efficacy of non-pyrethroid ITWL, long-lasting insecticidal nets (LLIN) (Interceptor®), pyrethroid ITWL (ZeroVector®), and non-pyrethroid ITWL + LLIN. RESULTS: The non-pyrethroid ITWL produced relatively low levels of mortality, between 40-50% for An. funestus and An. gambiae, across all treatments. Against An. funestus, the non-pyrethroid ITWL when used without LLIN produced 47% mortality but this level of mortality was not significantly different to that of the LLIN alone (29%, P = 0.306) or ITWL + LLIN (35%, P = 0.385). Mortality levels for An. gambiae were similar to An. funestus with non-pyrethroid ITWL, producing 43% mortality compared with 26% for the LLIN. Exiting rates from ITWL huts were similar to the control and highest when the LLIN was present. An attempt to restrict mosquito access by covering the eave gap with ITWL (one eave open vs four open) had no effect on numbers entering. The LLIN provided personal protection when added to the ITWL with only 30% blood-fed compared with 69 and 56% (P = 0.001) for ITWL alone. Cone bioassays on ITWL with 30 min exposure after the trial produced mortality of >90% using field An. gambiae. CONCLUSIONS: Despite high mortality in bioassays, the hut trial produced only limited mortality which was attributed to pyrethroid resistance against the pyrethroid ITWL and low efficacy in the non-pyrethroid ITWL. Hut ceilings were left uncovered and may have served as a potential untreated refuge. By analogy to IRS campaigns, which also do not routinely treat ceilings, high community coverage with ITWL may still reduce malaria transmission. Restriction of eave gaps by 75% proved an inadequate barrier to mosquito entry. The findings represent the first 2 months after installation and do not necessarily predict long-term efficacy.

Trends in US President's Malaria Initiative-funded indoor residual spray coverage and insecticide choice in sub-Saharan Africa (2008-2015): urgent need for affordable, long-lasting insecticides.

This article reports the changing pattern of US President's Malaria Initiative-funded IRS in sub-Saharan Africa between 2008 and 2015. IRS coverage in sub-Saharan Africa increased from <2 % of the at-risk population in 2005, to 11 % or 78 million people in 2010, mainly as a result of increased funding from PMI. The scaling up of IRS coverage in sub-Saharan Africa has been successful in several epidemiological settings and contributed to reduced malaria transmission rates. However, the spread and intensification of pyrethroid resistance in malaria vectors led many control programmes to spray alternative insecticides. Between 2009 and 2013, pyrethroid spraying decreased from 87 % (13/15) of PMI-funded countries conducting IRS to 44 % (7/16), while bendiocarb use increased from 7 % (1/15) to 56 % (9/16). Long-lasting pirimiphos-methyl CS received WHOPES recommendation in 2013 and was scheduled to be sprayed in 85 % (11/13) of PMI-funded countries conducting IRS in 2015. The gradual replacement of relatively inexpensive pyrethroids, firstly with bendiocarb (carbamate) and subsequently with pirimiphos methyl CS (organophosphate), has contributed to the downscaling of most PMI-funded IRS programmes. Overall, there was a 53 % decrease in the number of structures sprayed between years of peak coverage and 2015, down from 9.04 million to 4.26 million structures. Sizeable reductions in the number of structures sprayed were reported in Madagascar (56 %, 576,320-254,986), Senegal (64 %, 306,916-111,201), Tanzania (68 %, 1,224,095-389,714) and Zambia (63 %, 1,300,000-482,077), while in Angola, Liberia and Malawi PMI-funded spraying was suspended. The most commonly cited reason was increased cost of pesticides, as vector resistance necessitated switching from pyrethroids to organophosphates. There are worrying preliminary reports of malaria resurgence following IRS withdrawal in parts of Benin, Tanzania and Uganda. The increase in malaria cases following the end of the Global Malaria Eradication Programme in 1969 highlights the fragility of such gains when control efforts are weakened. At present there are several countries reliant on organophosphates and carbamates for IRS, and increasing incipient resistance is a serious threat that could result in IRS no longer being viable. A portfolio of new cost-effective insecticides with different modes of action is urgently needed.

The effectiveness of non-pyrethroid insecticide-treated durable wall lining to control malaria in rural Tanzania: study protocol for a two-armed cluster randomized trial.

BACKGROUND: Despite considerable reductions in malaria achieved by scaling-up long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), maintaining sustained community protection remains operationally challenging. Increasing insecticide resistance also threatens to jeopardize the future of both strategies. Non-pyrethroid insecticide-treated wall lining (ITWL) may represent an alternate or complementary control method and a potential tool to manage insecticide resistance. To date no study has demonstrated whether ITWL can reduce malaria transmission nor provide additional protection beyond the current best practice of universal coverage (UC) of LLINs and prompt case management. METHODS/DESIGN: A two-arm cluster randomized controlled trial will be conducted in rural Tanzania to assess whether non-pyrethroid ITWL and UC of LLINs provide added protection against malaria infection in children, compared to UC of LLINs alone. Stratified randomization based on malaria prevalence will be used to select 22 village clusters per arm. All 44 clusters will receive LLINs and half will also have ITWL installed on interior house walls. Study children, aged 6 months to 11 years old, will be enrolled from each cluster and followed monthly to estimate cumulative incidence of malaria parasitaemia (primary endpoint), time to first malaria episode and prevalence of anaemia before and after intervention. Entomological inoculation rate will be estimated using indoor CDC light traps and outdoor tent traps followed by detection of Anopheles gambiae species, sporozoite infection, insecticide resistance and blood meal source. ITWL bioefficacy and durability will be monitored using WHO cone bioassays and household surveys, respectively. Social and cultural factors influencing community and household ITWL acceptability will be explored through focus-group discussions and in-depth interviews. Cost-effectiveness, compared between study arms, will be estimated per malaria case averted. DISCUSSION: This protocol describes the large-scale evaluation of a novel vector control product, designed to overcome some of the known limitations of existing methods. If ITWL is proven to be effective and durable under field conditions, it may warrant consideration for programmatic implementation, particularly in areas with long transmission seasons and where pyrethroid-resistant vectors predominate. Trial findings will provide crucial information for policy makers in Tanzania and other malaria-endemic countries to guide resource allocations for future control efforts. TRIAL REGISTRATION: NCT02533336 registered on 13 July 2014.

A new class of insecticide for malaria vector control: evaluation of mosquito nets treated singly with indoxacarb (oxadiazine) or with a pyrethroid mixture against Anopheles gambiae and Culex quinquefasciatus.

BACKGROUND: Universal coverage with long-lasting insecticidal mosquito nets (LLIN) or indoor residual spraying (IRS) of houses remain the primary strategies for the control of mosquito vectors of malaria. Pyrethroid resistant malaria vectors are widespread throughout sub-Saharan Africa and new insecticides with different modes of action are urgently needed if malaria vector control is to remain effective. Indoxacarb is an oxadiazine insecticide that is effective as an oral and contact insecticide against a broad spectrum of agricultural pests and, due to its unique site of action, no cross-resistance has been detected through mechanisms associated with resistance to insecticides currently used in public health. METHODS: WHO tunnel tests of host seeking mosquitoes were carried out as a forerunner to experimental hut trials, to provide information on dosage-dependent mortality, repellency, and blood-feeding inhibition. A dosage range of indoxacarb treated netting (100-1000 mg/m(2)) was tested against a pyrethroid susceptible strain of Anopheles gambiae. In addition, efficacy of indoxacarb 500 mg/m(2) was compared with a standard pyrethroid formulation against pyrethroid susceptible and resistant Culex quinquefasciatus. Dosages between 25 and 300 mg/m(2) indoxacarb were tested in tunnel tests and in ball-frame bioassays as mixtures with alphacypermethrin 25 mg/m(2) and were compared with singly applied treatments against an insectary reared pyrethroid resistant strain of Cx. quinquefasciatus originally collected in Cotonou, Benin. RESULTS: There was a dosage-dependent response in terms of indoxacarb induced mortality, with dosages >100 mg/m(2) producing the best mortality response. In tunnel tests indoxacarb 500 mg/m(2) exceeded WHOPES thresholds with >80 % mortality of adult An. gambiae and blood-feeding inhibition of 75 %. No cross-resistance to indoxacarb was detected through mechanisms associated with resistance to pyrethroid insecticides and was equally effective against susceptible and resistant strains of Cx. quinquefasciatus. Indoxacarb 500 mg/m(2) killed 75 % of pyrethroid resistant Cx. quinquefasciatus compared with only 21 % mortality with alphacypermethrin 40 mg/m(2). Mixtures of indoxacarb with pyrethroid produced an additive response for both mortality and blood-feeding inhibition. The best performing mixture (indoxacarb 200 mg/m(2) + alphacypermethrin 25 mg/m(2)) killed 83 % of pyrethroid resistant Cx. quinquefasciatus and reduced blood-feeding by 88 %, while alphacypermethrin only killed 36 % and inhibited blood-feeding by 50 %. CONCLUSIONS: New insecticides with different modes of action to those currently used in mosquito vector control are urgently needed. Indoxacarb shows great promise as a mixture with a pyrethroid and should be evaluated in experimental hut trials to determine performance against wild free-flying, pyrethroid resistant An. gambiae and wash-resistant formulations developed.

The activity of the pyrrole insecticide chlorfenapyr in mosquito bioassay: towards a more rational testing and screening of non-neurotoxic insecticides for malaria vector control.

BACKGROUND: The rapid selection of pyrethroid resistance throughout sub-Saharan Africa is a serious threat to malaria vector control. Chlorfenapyr is a pyrrole insecticide which shows no cross resistance to insecticide classes normally used for vector control and is effective on mosquito nets under experimental hut conditions. Unlike neurotoxic insecticides, chlorfenapyr owes its toxicity to disruption of metabolic pathways in mitochondria that enable cellular respiration. A series of experiments explored whether standard World Health Organization (WHO) guidelines for evaluation of long-lasting insecticidal nets, developed through testing of pyrethroid insecticides, are suitable for evaluation of non-neurotoxic insecticides. METHODS: The efficacy of WHO recommended cone, cylinder and tunnel tests was compared for pyrethroids and chlorfenapyr. To establish bioassay exposure times predictive of insecticide-treated net (ITN) efficacy in experimental hut trials, standard three-minute bioassays of pyrethroid and chlorfenapyr ITNs were compared with longer exposures. Mosquito behaviour and response to chlorfenapyr ITN in bioassays conducted at night were compared to day and across a range of temperatures representative of highland and lowland transmission. RESULTS: Standard three-minute bioassay of chlorfenapyr produced extremely low levels of mortality compared to pyrethroids. Thirty-minute day-time bioassay produced mortality closer to hut efficacy of chlorfenapyr ITN but still fell short of the WHO threshold. Overnight tunnel test with chlorfenapyr produced 100% mortality and exceeded the WHO threshold of 80%. The endogenous circadian activity rhythm of anophelines results in inactivity by day and raised metabolism and flight activity by night. A model which explains improved toxicity of chlorfenapyr ITN when tested at night, and during the day at higher ambient temperature, is that activation of chlorfenapyr and disruption of respiratory pathways is enhanced when the insect is more metabolically and behaviourally active. CONCLUSIONS: Testing according to current WHO guidelines is not suitable for certain types of non-neurotoxic insecticide which, although highly effective in field trials, would be overlooked at the screening stage of evaluation through bioassay. Testing methods must be tailored to the characteristics and mode of action of each insecticide class. The WHO tunnel test on night-active anophelines is the most reliable bioassay for identifying the toxicity of novel insecticides.

Long-lasting control of Anopheles arabiensis by a single spray application of micro-encapsulated pirimiphos-methyl (Actellic® 300 CS).

BACKGROUND: Pyrethroid-resistant mosquitoes are an increasing threat to malaria vector control. The Global Plan for Insecticide Resistance Management (GPIRM) recommends rotation of non-pyrethroid insecticides for indoor residual spraying (IRS). The options from other classes are limited. The carbamate bendiocarb and the organophosphate pirimiphos-methyl (p-methyl) emulsifiable concentrate (EC) have a short residual duration of action, resulting in increased costs due to multiple spray cycles, and user fatigue. Encapsulation (CS) technology was used to extend the residual performance of p-methyl. METHODS: Two novel p-methyl CS formulations were evaluated alongside the existing EC in laboratory bioassays and experimental hut trials in Tanzania between 2008-2010. Bioassays were carried out monthly on sprayed substrates of mud, concrete, plywood, and palm thatch to assess residual activity. Experimental huts were used to assess efficacy against wild free-flying Anopheles arabiensis, in terms of insecticide-induced mortality and blood-feeding inhibition. RESULTS: In laboratory bioassays of An. arabiensis and Culex quinquefasciatus both CS formulations produced high rates of mortality for significantly longer than the EC formulation on all substrates. On mud, the best performing CS killed >80% of An. arabiensis for five months and >50% for eight months, compared with one and two months, respectively, for the EC. In monthly bioassays of experimental hut walls the EC was ineffective shortly after spraying, while the best CS formulation killed more than 80% of An. arabiensis for five months on mud, and seven months on concrete. In experimental huts both CS and EC formulations killed high proportions of free-flying wild An. arabiensis for up to 12 months after spraying. There was no significant difference between treatments. All treatments provided considerable personal protection, with blood-feeding inhibition ranging from 9-49% over time. CONCLUSIONS: The long residual performance of p-methyl CS was consistent in bioassays and experimental huts. The CS outperformed the EC in laboratory and hut bioassays but the EC longevity in huts was unexpected. Long-lasting p-methyl CS formulations should be more effective than both p-methyl EC and bendiocarb considering a single spray could be sufficient for annual malaria control. IRS with p-methyl 300 CS is a timely addition to the limited portfolio of long-lasting residual insecticides.

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.

Evaluation of a national universal coverage campaign of long-lasting insecticidal nets in a rural district in north-west Tanzania.

BACKGROUND: Insecticide-treated nets (ITN) are one of the most effective measures for preventing malaria. Mass distribution campaigns are being used to rapidly increase net coverage in at-risk populations. This study had two purposes: to evaluate the impact of a universal coverage campaign (UCC) of long-lasting insecticidal nets (LLINs) on LLIN ownership and usage, and to identify factors that may be associated with inadequate coverage. METHODS: In 2011 two cross-sectional household surveys were conducted in 50 clusters in Muleba district, north-west Tanzania. Prior to the UCC 3,246 households were surveyed and 2,499 afterwards. Data on bed net ownership and usage, demographics of household members and household characteristics including factors related to socio-economic status were gathered, using an adapted version of the standard Malaria Indicator Survey. Specific questions relating to the UCC process were asked. RESULTS: The proportion of households with at least one ITN increased from 62.6% (95% Confidence Interval (CI) = 60.9-64.2) before the UCC to 90.8% (95% CI = 89.0-92.3) afterwards. ITN usage in all residents rose from 40.8% to 55.7%. After the UCC 58.4% (95% CI = 54.7-62.1) of households had sufficient ITNs to cover all their sleeping places. Households with children under five years (OR = 2.4, 95% CI = 1.9-2.9) and small households (OR = 1.9, 95% CI = 1.5-2.4) were most likely to reach universal coverage. Poverty was not associated with net coverage. Eighty percent of households surveyed received LLINs from the campaign. CONCLUSIONS: The UCC in Muleba district of Tanzania was equitable, greatly improving LLIN ownership and, more moderately, usage. However, the goal of universal coverage in terms of the adequate provision of nets was not achieved. Multiple, continuous delivery systems and education activities are required to maintain and improve bed net ownership and usage.

An evidence synthesis approach for combining different data sources illustrated using entomological efficacy of insecticides for indoor residual spraying.

BACKGROUND: Prospective malaria public health interventions are initially tested for entomological impact using standardised experimental hut trials. In some cases, data are collated as aggregated counts of potential outcomes from mosquito feeding attempts given the presence of an insecticidal intervention. Comprehensive data i.e. full breakdowns of probable outcomes of mosquito feeding attempts, are more rarely available. Bayesian evidence synthesis is a framework that explicitly combines data sources to enable the joint estimation of parameters and their uncertainties. The aggregated and comprehensive data can be combined using an evidence synthesis approach to enhance our inference about the potential impact of vector control products across different settings over time. METHODS: Aggregated and comprehensive data from a meta-analysis of the impact of Pirimiphos-methyl, an indoor residual spray (IRS) product active ingredient, used on wall surfaces to kill mosquitoes and reduce malaria transmission, were analysed using a series of statistical models to understand the benefits and limitations of each. RESULTS: Many more data are available in aggregated format (N = 23 datasets, 4 studies) relative to comprehensive format (N = 2 datasets, 1 study). The evidence synthesis model had the smallest uncertainty at predicting the probability of mosquitoes dying or surviving and blood-feeding. Generating odds ratios from the correlated Bernoulli random sample indicates that when mortality and blood-feeding are positively correlated, as exhibited in our data, the number of successfully fed mosquitoes will be under-estimated. Analysis of either dataset alone is problematic because aggregated data require an assumption of independence and there are few and variable data in the comprehensive format. CONCLUSIONS: We developed an approach to combine sources from trials to maximise the inference that can be made from such data and that is applicable to other systems. Bayesian evidence synthesis enables inference from multiple datasets simultaneously to give a more informative result and highlight conflicts between sources. Advantages and limitations of these models are discussed.

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.

Systematic review of the entomological impact of insecticide-treated nets evaluated using experimental hut trials in Africa.

Resistance of anopheline mosquitoes to pyrethroid insecticides is spreading rapidly across sub-Saharan Africa, diminishing the efficacy of insecticide-treated nets (ITNs) - the primary tool for preventing malaria. The entomological efficacy of indoor vector control interventions can be measured in experimental hut trials (EHTs), where hut structures resemble local housing, but allow the collection of mosquitoes that entered, exited, blood-fed and/or died. There is a need to understand how the spread of resistance changes ITN efficacy and to elucidate factors influencing EHT results, including differences in experimental hut design, to support the development of novel vector control tools. A comprehensive database of EHTs was compiled following a systematic review to identify all known trials investigating ITNs or indoor residual spraying across sub-Saharan Africa. This analysis focuses on EHTs investigating ITNs and uses Bayesian statistical models to characterise the complex interaction between ITNs and mosquitoes, the between-study variability, and the impact of pyrethroid resistance. As resistance rises, the entomological efficacy of ITNs declines. They induce less mortality and are less likely to deter mosquitoes from entering huts. Despite this, ITNs continue to offer considerable personal protection by reducing mosquito feeding until resistance reaches high levels. There are clear associations between the different entomological impacts of ITNs, though there is still substantial variability between studies, some of which can be accounted for by hut design. The relationship between EHT outcomes and the level of resistance (as measured by discriminating dose bioassays) is highly uncertain. The meta-analyses show that EHTs are an important reproducible assay for capturing the complex entomological efficacy of ITNs on blood-feeding mosquitoes. The impact of pyrethroid resistance on these measures appears broadly consistent across a wide geographical area once hut design is accounted for, suggesting results can be extrapolated beyond the sites where the trials were conducted. Further work is needed to understand factors influencing EHT outcomes and how the relationship between outcomes and resistance varies when different methods are used to assess the level of resistance in wild mosquito populations. This will allow more precise estimates of the efficacy of these important vector control tools.