Study site and experimental huts
The experimental hut trials (EHTs) took place at the Dala Suna site, beautifully located along the shores of Lake Kanyaboli, coordinates 0° 02′ 08.5″ N, 34° 11′ 05.0″ E, within the Alego Usonga sub-County of Siaya County, in the heart of western Kenya. This strategic location offers an optimal environment for malaria vector breeding due to its proximity to swamps, which teem with high populations of the malaria carriers Anopheles funestus throughout the year, alongside seasonal surges of Anopheles arabiensis, averaging over 300 and more than 20 mosquitoes per household nightly, respectively.
The region is characterized by two distinct rainy seasons from March to May and October to November, ensuring a consistent high rate of malaria transmission all year long. The local populace primarily engages in subsistence farming, livestock rearing, fishing, and small-scale trading activities, all vital for their livelihoods.
The experimental huts were meticulously constructed to reflect a typical household in Kenya, featuring mosquito entry and exit points including eaves, windows, and doors. Each hut was fitted with mosquito exit traps on all four windows—two on the front and two on the back. The walls were built from sturdy blocks, lined with mud on the interior, while the floors were elegantly tiled with white tiles for efficient collection of knocked-down mosquitoes. With corrugated iron roofs and a 10-cm gap at the eaves, wood baffles were strategically installed to hinder mosquito exit while facilitating entry. Each hut sat elevated on a concrete base, surrounded by a moat filled with water to effectively deter ants.
Baseline evaluation of insecticide resistance profile
Larvae of the Anopheles gambiae sensu lato (s.l.) species were carefully gathered using a standard dipper (Model 320) from natural breeding sites. Meanwhile, blood-fed adults of An. funestus were collected with the Prokopack (model 1419) after obtaining explicit consent from the households closest to the experimental hut site. This collection process occurred over the months of August through October 2022. The collected specimens were delivered to the KEMRI-CGHR insectary, where An. gambiae s.l. larvae were nurtured into adults, and the adult An. funestus were encouraged to lay eggs, raising their first filial (F1) generation to 3–5 days old for in-depth insecticide resistance testing.
The larvae thrived in rainwater, nourished on high-quality Koi premium fish food under rigorously controlled conditions (27 ± 2 ºC, 80 ± 10% RH, and a 12:12 light-darkness cycle). Adult mosquitoes were kept on a 10% sugar solution until the time of the bioassay. Simultaneously, the Kisumu strain of An. gambiae, known for its sensitivity to insecticides, was also reared under the same parameters to serve as a reliable bioassay control.
Susceptibility testing was conducted to evaluate the response of both An. gambiae s.l. and An. funestus from the Lake Kanyaboli site to the active ingredients found in the Long-Lasting Insecticidal Nets (LLINs) to be evaluated: PermaNet 3.0 (deltamethrin + PBO), Interceptor® G2 (alpha-cypermethrin + chlorfenapyr), and PermaNet® Dual (deltamethrin + chlorfenapyr). The WHO tube assay and CDC bottle tests were meticulously performed on 3 to 5-day-old F0 An. gambiae s.l. and F1 An. funestus adults, in line with WHO protocols.
The WHO tube tests involved exposing mosquitoes to filter papers impregnated with varying concentrations of insecticides such as 0.05% alpha-cypermethrin, 0.75% permethrin, or 0.05% deltamethrin for one hour, where knockdown rates were documented every ten minutes, and mortality was recorded 24 hours post-exposure. To delve deeper into the resistance, diagnostic concentrations were increased to 5X and 10X to ascertain the intensity of insecticide resistance to pyrethroids. Non-insecticide-impregnated filter paper served as a control.
Net treatments and treatment arms
The nets used in this trial, including PermaNet® 3.0 and PermaNet® Dual, were supplied by Vestergaard Sarl based in Lausanne, Switzerland, while the Interceptor® G2 was provided by BASF from Ludwigshafen, Germany. PermaNet® 3.0 was a pivotal component of this study, as it represents the first dual-active insecticide-treated bed net that incorporates PBO on the top panel alongside deltamethrin on the side panels, currently serving as the standard of care in the region to combat the growing issue of pyrethroid resistance. In contrast, the untreated nets were constructed of basic polyester fabric without any insecticidal treatment.
The Interceptor® G2 is notably fabricated from polyester fabric, with added coatings of 2.4 g/kg (100 mg/m2) of alpha-cypermethrin and 4.8 g/kg (200 mg/m2) of chlorfenapyr. The composition of PermaNet® 3.0 features polyester fabric coated with 2.1 g/kg (84 mg/m2) of deltamethrin on the sides, in addition to polyethylene that incorporates 4.0 g/kg (120 mg/m2) of deltamethrin and an impressive 25.0 g/kg (800 mg/m2) of PBO on the roof.
Net washing
For each study arm, seven nets were randomly selected from a cohort of 21 nets of each production batch and subjected to twenty meticulous washes following the WHO washing criteria. Each type of LLIN was washed separately in its designated washing station to avoid any risk of contamination. The washing process involved immersing each net individually in a 16-L aluminum basin filled with 10 L of clean groundwater, which had a neutral pH of 7.0 and a hardness rating of 5 degrees, to which 20 g of soap was added and fully dissolved just prior to the washing.
Every net was washed for ten minutes with a three-minute agitation, followed by a four-minute soaking period and another three-minute stirring session. After washing, samples were rinsed twice in 10 L of clean groundwater, employing the same method, then dried in the shade and stored at room temperature between each washing. To emulate the wear-and-tear of the nets during actual use, all LLINs intended for the hut trials, both for treatment wash points and control nets, were strategically given six holes measuring 4 × 4 cm, in line with WHO guidelines.
Hut trial procedure
The experimental hut trials utilized a 7 by 7 Latin square design (LSD) aimed at evaluating the entomological efficacy of PermaNet® Dual, Interceptor® G2, and PermaNet® 3.0 LLINs, both washed 20 times and unwashed, against freely flying pyrethroid-resistant An. funestus. Each wash point compared the efficacy of these LLINs to an untreated net, serving as a critical negative control. A total of 49 nets were employed, comprised of seven replicates of each LLIN type (seven washed and seven unwashed), while the untreated/control net had seven nets.
Throughout the trial, seven human volunteer sleepers consented to stay in the experimental huts from 8:30 PM to 6:30 AM each night, helping attract hungry mosquitoes. To account for varying individual attractiveness, the sleepers were rotated among the huts daily, adhering to a simple 7*7 LSD system. The net installations inside the experimental huts were achieved by expertly tying the edges of the roof panel to nails fixed at the upper corners of the hut wall using string. The treatments were rotated weekly among the experimental huts, precisely according to a Latin square design, to control the effect of hut positioning.
Each round of mosquito collection lasted for seven days, with the eighth day allocated for cleaning the huts to prevent contamination and eliminate any carry-over effects before the subsequent rotation cycle.
The treatment arms evaluated during each experimental hut trial included:
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1.
Untreated net (control)—7 replicates of nets unwashed.
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2.
PermaNet® Dual—7 replicates of nets washed 20 times.
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3.
PermaNet® Dual—7 replicates of unwashed nets.
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4.
Interceptor® G2—7 replicates of nets washed 20 times.
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5.
Interceptor® G2—7 replicates of unwashed nets.
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6.
PermaNet® 3.0—7 replicates of nets washed 20 times.
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7.
PermaNet® 3.0—7 replicates of unwashed nets.
Mosquito collections and processing
Seven consenting human volunteers accommodated in experimental huts from 8:30 PM to 06:30 AM sought to attract wild, free-flying mosquitoes for research. Weekly malaria prophylaxis (mefloquine) was administered, with participants instructed to report any side effects encountered during the evaluation. Starting at 6:30 AM, trained personnel used mouth aspirators to collect mosquitoes until 08:00 each morning. The volunteers gathered both dead and alive mosquitoes from within the huts and from window exit traps, utilizing mouth aspiration to ensure a thorough collection process.
The collected specimens were scored upon their collection sites—wall, roof, floor, net, under-bed, and from the window exit traps. Once securely collected, the mosquitoes were placed into clean, netted paper cups and given access to a 10% sugar solution for nourishment during transportation to the field insectary laboratory. Inside the laboratory, the mosquitoes were categorized by status (alive or dead, blood-fed or unfed, gravid or half-gravid) and identified morphologically to species, following the established taxonomical key. Observations for knockdown were made on live mosquitoes within one hour of collection, while mortality data was meticulously recorded every 24 hours for up to 72 hours post-collection.
Supplementary laboratory assays
Cone test
Cone testing was executed using net pieces (25 cm × 25 cm) derived from both pre-test and post-trial samples, across all wash points of each LLIN utilized in this comprehensive evaluation. Each net piece had four cones attached, where five non-blood-fed female mosquitoes were carefully aspirated into each cone and exposed for three minutes. Both An. gambiae, representing the Kisumu strain, and An. funestus F1 of 3–5 days were demonstrated within each cone. Throughout this experiment, 100 mosquitoes were deployed per net/species to ensure statistical validity. Subsequent to exposure, the mosquitoes were transferred into clean paper cups, provided with access to a 10% sugar solution, and monitored, recording knockdown at 60 minutes post-exposure, with mortality fully documented at 24 hours, 48 hours, and 72 hours.
Tunnel test
The tunnel test was meticulously conducted to observe host-seeking mosquitoes’ mortality and blood-feeding success within an experimental chamber. This study branched out to give further insights into the efficacy of unwashed versus washed nets utilized in the experimental huts. It aimed specifically at the pyrethroid-resistant An. funestus F1, employing the same net samples of Interceptor® G2 and PermaNet® Dual previously assessed in the cone assay.
The tunnel test chamber emulated real-world interactions between free-flying mosquitoes and nets during host-seeking. A rabbit bait, enticingly exposed for feeding, was held in a cage situated in a short section of the square glass tunnel, while in the expansive portion, 100 5–8-day-old mosquitoes were released and monitored overnight under controlled conditions (27 ± 2 ºC temperature, 80 ± 10% RH). Each net piece was specifically fitted with nine small holes, measuring 1 cm in diameter, to facilitate mosquito entry into the baited chamber. Upon collection from the tunnel each morning, captured mosquitoes were examined to assess mortality and blood-feeding success, with delayed mortality recorded every 24 hours for a maximum of 72 hours.
Chemical assays
Two nets were systematically selected from all wash points across every arm, both before and after the hut trials, yielding five pieces from each net (with three pieces from the top and one each from the sides of PermaNet 3.0, totaling seven pieces) in adherence to WHO guidelines. These cut net pieces were dispatched, carefully wrapped in aluminum foil, to the Vestergaard ISO/IEC 17025 accredited Vector Control Laboratories situated in Vietnam for advanced testing to verify the wash retention of active ingredients contained within the net pieces, employing validated analytical methods as established by the Collaborative International Pesticides Analytical Council (CIPAC).
Extraction of deltamethrin from the roof of PermaNet 3.0 (roof) involved a heating process under reflux for 30 minutes using xylene, coupled with dicyclohexyl phthalate as an internal standard. Post-evaporation, residue dissolving in hexane was conducted. Interest in other active ingredients included the meticulous extraction of alpha-cypermethrin in Interceptor® G2 along with the chlorfenapyr in both Interceptor® G2 and PermaNet® Dual, utilizing sonication with heptane and dicyclohexyl phthalate. Lastly, PBO from PermaNet 3.0 roof samples was extracted through heating under reflux, similarly utilizing xylene and followed by gas chromatography with flame ionization detection (GC-FID).
Data analysis
The primary outcomes measured focused on comparing the treatments against control experimental huts covering blood-feeding inhibition, immediate, and delayed mortality. Additionally evaluated were induced exophily, determining the proportion of mosquitoes found in exit traps, alongside deterrence, assessing the proportional reduction in the total mosquito count collected in treated huts relative to control huts with untreated nets.
The analysis of proportional outcomes (mortality, blood feeding, and exophily) between treatments and controls across all wash points was conducted using a blocked logistic regression model, as the numerical outcomes (entry) were assessed via a negative binomial regression model. Non-inferiority testing between PermaNet® Dual and Interceptor® G2 for both mortality and blood feeding adhered strictly to WHO protocols. This comprehensive analysis encompassed both washed and unwashed nets, including an independent variable in the washing model.
Ethical considerations and compliance with GLP
The trial received ethical approval from the Scientific and Ethical Review Unit of KEMRI (SERU 4536) for the involvement of human participants and animals. This study had also been thoroughly reviewed by the CDC and confirmed to meet the definition of research involving human subjects, maintaining ethical rigor throughout its processes. Prior to recruitment, explicit informed consent was acquired from all volunteer sleepers, who were provided a weekly regimen of malaria prophylaxis (Mefloquine) to safeguard their health.
This site has achieved accreditation from the Kenya Pest Control Products Board (PCPB), enabling the national evaluation of vector control products essential for registration purposes. The study was conducted in strict accordance with WHO non-inferiority guidelines tailored for assessing second-in-class LLINs. Additionally, the site took initial steps towards GLP accreditation, ensuring that all study protocols adhered meticulously to GLP requirements.
Welcome to the Mosquito Circus: Spectacular Trials in Kenya!
Ladies and Gentlemen, gather around! You’re about to experience a rollercoaster ride of science! We’re deep in the heart of western Kenya, near the enchanting shores of Lake Kanyaboli, where mosquitoes are the real stars of the show. I mean, who needs Hollywood when you have a bounty of Anopheles funestus and Anopheles arabiensis buzzing around like they own the place? Let’s go behind the scenes of the Experimental Hut Trials (EHTs) and see what makes these little bloodsuckers tick!
The Stage Setup: Experimental Huts at Dala Suna
Our first act takes place at the Dala Suna experimental hut site, a place designed to mimic your average Kenyan household—minus the late-night drama and family bickering. These huts boast strategically placed mosquito exit traps, all brilliantly fitted to ensure that these winged beasts enter but struggle to leave. It’s a bit like inviting friends over for a party and sealing the doors once they arrive—except, well, these guests have a taste for blood!
Made from blocks and lined with mud (not exactly the Ritz, but hey, we’re fighting malaria here!), these huts have tiles that make cleaning up after a mosquito party just a little bit easier. And let’s not forget about the moat! That’s right; we’ve got a moat to keep the ants at bay—because what’s worse than a mosquito? A mosquito with an army of ants, obviously!
Gathering the Mosquito Misfits
This isn’t just your average bug hunt! No, this is meticulous science! Scientists are collecting An. gambiae sensu lato (that’s a fancy way of saying “various types of Anopheles”) from their natural breeding sites. Standard dipper in hand, they’re diving into the wetlands to fish out larvae, while blood-fed adults are coaxed indoors using the Prokopack; it’s basically the mosquito equivalent of a five-star buffet, and let me tell you, these mosquitoes did not RSVP!
The Insecticide Smorgasbord
Next up, it’s time for the insecticide buffet! Researchers had a delightful selection to test: PermaNet 3.0 (the ‘Miguelito’ of mosquito nets, featuring deltamethrin and PBO), the Interceptor G2 (that stylish duo of alpha-cypermethrin and chlorfenapyr), and finally, the PermaNet Dual. Don’t let the names fool you; this is serious business! The mosquitoes might not appreciate the cocktails being mixed up for them, but it’s a crucial aspect of this EHT production!
Using rigorous methods like WHO tube assays and CDC bottle tests, researchers ensure that the mosquitoes get their fill of insecticides—relax, it’s not as harsh as it sounds. They just want to know which net gets the most ‘knockouts’ and which ones leave the mosquitoes more energized!
It’s All in the Wash: Testing Bed Net Durability
Ever tried washing your bedsheets and mistakenly shrunk them? Well, the scientists at this trial took it to the next level. They subjected each type of net to not just a rinse but a full-on washing marathon—20 washes to be precise! Talk about overkill—but when you’re dealing with pyrethroid-resistant mosquitoes, there’s no room for error! They strategically poked holes in them to simulate real-life wear and tear because, let’s face it, how many of you have a pristine net after a month of use? Exactly!
The Star of the Show: Human Volunteers
Here’s the kicker: real human volunteers, sacrificing sleep from 8:30 PM to 6:30 AM! These brave souls are like the unsung heroes in this mosquito drama. Equipping them with mefloquine to ward off malaria, they sleep in the huts as living bait, rotating like clockwork each day to keep the mosquitoes guessing. Just imagine the pillow talk! “Did you see how many mosquitoes I attracted last night?” “Pfft, I had way more!” The competition is fierce, folks.
And the Results Are In! (Well, Sort Of)
After much anticipation (and a few too many slapped mosquitoes), researchers will analyze the results based on various measures. Blood-feeding inhibition, immediate and delayed mortality, and the enticing lure for our little winged pals will be measured. Anticipation builds to see which net reigns supreme in this fierce battle of wills between humans and malaria vectors!
Wrap Up: Ethical Waves and the Future
And let’s not forget the ethical side of this performance! With all the proper permissions, informed consent (no one likes a surprise blood donation, ahem), and protocols in place, rest assured that this is more than just a show—it’s a serious step towards conquering malaria.
Final Thoughts
So, there we have it, folks! A fascinating glimpse into the life of mosquito trials at Dala Suna. From experimental huts and heroic volunteers to the generous buffet of insecticides, this is where science meets the wild world of mosquitoes. It’s a courageous effort towards understanding and combating malaria—a fervent fight in the battle of brains versus bugs. Stay tuned for the next act; who knows what surprises await in this thrilling saga!
Chegg
3 and H4 Trials
As the curtain rises on the H3 and H4 trials, the experts conduct a personalized performance with our mosquito volunteers. Using a specially designed tunnel test chamber, these researchers simulate genuine interactions between free-flying mosquitoes and the treated nets. Picture it: a rabbit, enticingly exposed as the bait, sits securely in one section of the tunnel while 100 eager mosquitoes are released in the other half. It’s a delicate balancing act of science, where the fate of each mosquito hinges on their interaction with the nets. Researchers monitor their nocturnal escapades, assessing mortality and blood-feeding rates, and even record any delayed effects over the course of three days. As with any grand performance, data analysis is where the real magic happens! The researchers delve into the primary outcomes, scrutinizing everything from blood-feeding inhibition to delayed mortality. They even evaluate ‘exophily’—a fancy term that determines how many mosquitoes decide to exit the treated huts. It’s a real-life game of survival, and the researchers utilize advanced statistical models to uncover important insights. Non-inferiority tests ensure that no net gets left behind, following strict WHO protocols, ensuring results are both ethical and scientifically sound! Of course, behind every spectacular show is a commitment to ethical standards. The trial doesn’t just seek to unveil the effectiveness of these nets but does so while ensuring that all involved—both human participants and our mosquito friends—are treated with respect and care. Following WHO guidelines and with the approval of ethical review boards, participants receive malaria prophylaxis, ensuring their well-being during the trials. As we close the curtains on our Mosquito Circus, it’s clear that this isn’t just a show—it’s a vital step towards combating malaria and enhancing public health. Each net tested, each mosquito monitored plays a part in eradicating this disease. So, while the mosquitoes may be the stars today, the true heroes are the researchers dedicated to making our world a safer place!The Behind-the-Scenes Data Analysis
Ethical Considerations: The Real MVPs
In Conclusion: The Legacy of the Mosquito Circus
