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Comparative efficacy of two pyrethroid-piperonyl butoxide nets (Olyset Plus and PermaNet 3.0) against pyrethroid resistant malaria vectors: a non-inferiority assessment

Comparative efficacy of two pyrethroid-piperonyl butoxide nets (Olyset Plus and PermaNet 3.0)... Background: Pyrethroid-PBO nets were conditionally recommended for control of malaria transmitted by mosqui- toes with oxidase-based pyrethroid-resistance based on epidemiological evidence of additional protective effect with Olyset Plus compared to a pyrethroid-only net (Olyset Net). Entomological studies can be used to assess the compara- tive performance of other brands of pyrethroid-PBO ITNs to Olyset Plus. Methods: An experimental hut trial was performed in Cové, Benin to compare PermaNet 3.0 (deltamethrin plus PBO on roof panel only) to Olyset Plus (permethrin plus PBO on all panels) against wild pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) following World Health Organization ( WHO) guidelines. Both nets were tested unwashed and after 20 standardized washes compared to Olyset Net. Laboratory bioassays were also performed to help explain find- ings in the experimental huts. Results: With unwashed nets, mosquito mortality was higher in huts with PermaNet 3.0 compared to Olyset Plus (41% vs. 28%, P < 0.001). After 20 washes, mortality declined significantly with PermaNet 3.0 (41% unwashed vs. 17% after washing P < 0.001), but not with Olyset Plus (28% unwashed vs. 24% after washing P = 0.433); Olyset Plus induced significantly higher mortality than PermaNet 3.0 and Olyset Net after 20 washes. PermaNet 3.0 showed a higher wash retention of PBO compared to Olyset Plus. A non-inferiority analysis performed with data from unwashed and washed nets together using a margin recommended by the WHO, showed that PermaNet 3.0 was non-inferior to Olyset Plus in terms of mosquito mortality (25% with Olyset Plus vs. 27% with PermaNet 3.0, OR = 1.528, 95%CI = 1.02– 2.29) but not in reducing mosquito feeding (25% with Olyset Plus vs. 30% with PermaNet 3.0, OR = 1.192, 95%CI = 0.77–1.84). Both pyrethroid-PBO nets were superior to Olyset Net. Conclusion: Olyset Plus outperformed PermaNet 3.0 in terms of its ability to cause greater margins of improved mos- quito mortality compared to a standard pyrethroid net, after multiple standardized washes. However, using a margin of non-inferiority defined by the WHO, PermaNet 3.0 was non-inferior to Olyset Plus in inducing mosquito mortal- ity. Considering the low levels of mortality observed and increasing pyrethroid-resistance in West Africa, it is unclear whether either of these nets would demonstrate the same epidemiological impact observed in community trials in East Africa. *Correspondence: corine.ngufor@lshtm.ac.uk London School of Hygiene and Tropical Medicine (LSHTM), London, UK Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ngufor et al. Malaria Journal (2022) 21:20 Page 2 of 13 Keywords: Experimental huts, Piperonyl butoxide, PBO, Olyset plus, PermaNet 3.0, Olyset, Mixture, LLIN, Insecticide resistance, Pyrethroid-PBO, Cove, Benin, Pyrethroid resistance, Insecticide-treated nets, Long-lasting insecticidal nets, Next generation nets, Anopheles, Cove Benin Background World Health Organization (WHO) list of prequali- Long-lasting insecticidal nets (LLINs) remain one of fied vector control products: PermaNet 3.0, Veeralin, the most powerful tools to reduce malaria transmission Tsara Boost, Tsara Plus and very recently DuraNet in a community and provide personal protection to the Plus [7]. These nets have all demonstrated superiority user [1, 2]. They have contributed significantly to recent over pyrethroid-only nets in terms of mosquito mor- reductions in malaria burden [3]. Their efficacy is how - tality and blood-feeding inhibition in multiple experi- ever threatened by increasing resistance to pyrethroids mental hut trials across Africa [11–17]. However, they [4, 5]; the insecticide of choice used on bed-nets owing differ from Olyset Plus in their design and specifica - to its safety, low cost and rapid activity on vector mos- tions; typically, the location of PBO on the net (i.e., all quitoes [6]. To maintain the effectiveness of insecticide panels vs. roof panel only), the type and dose of pyre- treated nets for malaria control, new types of LLINs throid used, bioavailability and retention of PBO after treated with alternative insecticides and compounds washing and could, therefore, differ in entomological which can either replace or complement pyrethroids on and epidemiological impact. A recent large cluster- bed-nets are urgently needed. randomized trial in Uganda, evaluating the efficacy of A new class of insecticide treated nets (ITNs) com- Olyset Plus and PermaNet 3.0 compared to pyrethroid- bining pyrethroids and piperonyl butoxide (PBO) (pyre- only nets in a setting of high pyrethroid resistance, throid-PBO ITNs) have been developed [7]. PBO is a also demonstrated better protection against malaria synergist that inhibits specific metabolic enzymes such with pyrethroid-PBO nets compared to pyrethroid- as mixed-function oxidases within mosquitoes that only nets for up to 18  months confirming the findings detoxify or sequester insecticides before they can have of the Tanzanian trial [18]. While the Ugandan trial a toxic effect on the mosquito. Pyrethroid-PBO nets was not powered to directly compare between the dif- can therefore induce increased mortality of pyrethroid- ferent ITN brands tested, the results showed that the resistant malaria vectors that express mixed function additional protective effect of the pyrethroid-PBO net oxidase based pyrethroid resistance mechanisms that compared to the pyrethroid-only net was initially large are inhibited by the PBO in the net. These nets were with PermaNet 3.0 at 6  months post-distribution but given an interim endorsement as a new WHO class of lasted only up to 12 months whereas additional protec- vector control products in 2017 based on epidemiologi- tive effect with Olyset Plus appeared to have a delayed cal data from a cluster randomized controlled trial in onset which was not observed at 6 months but became North Eastern Tanzania [8], that demonstrated addi- evident at 12 months and lasted up to 18 months. There tional malaria control with one prototype pyrethroid- are major differences in design between Olyset Plus and PBO net (Olyset Plus) compared to a pyrethroid-only PermaNet 3.0, which could have implications on their net (Olyset Net), against pyrethroid resistant malaria epidemiological impact; Olyset Plus is a polyethylene vectors of moderate intensity, partly conferred by net incorporated with permethrin and PBO on all pan- monooxygenase-based resistance mechanism. Pyre- els while PermaNet 3.0 is a polyester net coated with throid-PBO ITNs are conditionally recommended for deltamethrin with PBO restricted to the roof of the net. malaria vector control instead of pyrethroid-only ITNs To generate assurance of comparative performance in areas of confirmed intermediate levels of resistance of new candidate products within an established WHO mediated by monooxygenase-based resistance mecha- vector control product class, without the need for epi- nism [9]. This endorsement has been followed by an demiological evidence for each new product, the WHO increasing uptake of pyrethroid-PBO nets worldwide has developed new experimental hut study guidelines [10]; in sub-Saharan Africa for example, the proportion for assessing their non-inferiority to a first in class prod - of pyrethroid-PBO nets of all nets delivered increased uct for which evidence of public health value has already from 3% in 2018 to 35% in 2021. been generated [19]. These provisional guidelines are to While Olyset Plus was the first in class pyrethroid- be piloted with pyrethroid-PBO nets by comparing other PBO net to demonstrate public health value as WHO/PQ-listed pyrethroid-PBO nets with the first in observed in the Tanzanian trial [8], there are currently class product, Olyset Plus. This study compared the effi - five additional types of pyrethroid-PBO ITNs on the cacy and wash resistance of Olyset Plus and PermaNet Nguf or et al. Malaria Journal (2022) 21:20 Page 3 of 13 3.0 and assessed the non-inferiority of PermaNet 3.0 to to PBO and to insecticides lasted 1  h, knockdown was Olyset Plus in experimental huts against wild free-flying recorded after 60 min and mortality after 24 h. pyrethroid resistant malaria vectors in Southern Benin. Experimental hut treatments Methods Olyset Plus and PermaNet 3.0 were compared in the Experimental hut trial experimental huts when unwashed and after 20 stand- Experimental hut site ardized washes. A WHO-recommended pyrethroid-only Experimental huts are small standardized human habita- long-lasting net (Olyset Net) was included to demon- tions approved by the WHO for the controlled evalua- strate the added effect of PBO on the insecticide-resist - tion of indoor vector control tools against wild free-flying ant local vector species. Nets were washed using savon mosquitoes. Mosquitoes enter the huts freely at night de Marseilles and rinsed twice following WHO proce- to interact with the human host and the vector control dures for washing nets for experimental hut studies [22]. intervention and in the morning on each day of the trial, The following seven (7) treatments were thus tested in they are collected from the different compartments of seven experimental huts: each hut and scored for entomological outcomes. The experimental hut study was performed at the CREC/ 1. Untreated polyethylene net LSHTM experimental hut station situated in a large 2. Olyset Net unwashed (permethrin only) rice growing area in Cové, Southern Benin, where the 3. Olyset Net washed 20 times. local mosquito population has been shown to be resist- 4. PermaNet 3.0 unwashed (Roof: deltamethrin ant to pyrethroids [20]. The rice paddies provide exten - plus PBO; sides: deltamethrin only) sive breeding sites for Anopheles gambiae throughout the 5. PermaNet 3.0 washed 20 times. year. The huts are built on concrete plinths surrounded 6. Olyset Plus unwashed (permethrin plus PBO on all by water-filled moats to prevent entry of scavenging ants panels) and have veranda traps to capture the exiting mosqui- 7. Olyset Plus washed 20 times. toes. The walls are made of brick plastered with cement on the inside, with a corrugated iron roof. The huts have a ceiling of palm thatch and four window slits (1 cm gap) Hut trial procedure on the walls through which mosquitoes enter. The local Treatments were allocated to the experimental huts on a vector population in Cove is resistant to pyrethroids and weekly basis using a randomized Latin square design to DDT and consists of a mixture of Anopheles coluzzii and adjust for any variation in hut attractiveness and mini- Anopheles gambiae sensu stricto (s.s.), with the latter mize any carry over effect between treatments. Three occurring at lower proportions (23%) and only in the dry replicate nets of each type were prepared, and these were season [20]. Molecular analysis revealed a L1014F kdr rotated every 2 days on each week (6 days) of the trial. To allele frequency of 89%. Microarray studies also found simulate wear and tear, each net was intentionally holed CYP6P3, a P450 validated as an efficient metabolizer of with six 16 cm holes (two holes on each side and one on pyrethroids [21], to be overexpressed in Cove [20]. each end). The trial ran for 42 nights between February and April Insecticide resistance bioassays of 2017. Consenting human volunteer sleepers slept in To assess the frequency of pyrethroid resistance and the huts from 9:00 p.m. to 5:00 a.m. each night and were presence of mixed function oxidases in the Cové vec- rotated daily through the huts to account for individual tor population during the trial, adult mosquitoes that attractiveness to mosquitoes. At dawn, the volunteer emerged from larvae collected from breeding sites close sleepers collected mosquitoes in the room of the hut and to experimental huts were tested in WHO cylinder bio- under the bed nets and the veranda using torches and assays with and without pre-exposure to PBO. A total aspirators. The mosquitoes were then transferred to the of ~ 100 mosquitoes of the pyrethroid resistant An. laboratory for processing where they were identified and gambiae s.l. Cove strain and the pyrethroid susceptible scored for their blood feeding status, mortality and hut An. gambiae Kisumu strain were exposed to treated fil - position. Mosquitoes were held at 27 ± 2  °C during the ter papers in WHO cylinder bioassays in batches of 25. observations. Tests were performed with papers treated with perme- The following outcome measures were used to assess thrin 0.75%, alpha-cypermethrin 0.05% and deltamethrin the efficacy of each treatment in the experimental huts: 0.05%. To assess presence of MFO, some mosquitoes were also pre-exposed to papers treated with 4% PBO 1. Deterrence—the proportional reduction in number prior to exposure to insecticide-treated papers. Exposure of mosquitoes entering huts with treated nets. Ngufor et al. Malaria Journal (2022) 21:20 Page 4 of 13 2. Exiting rates estimated from the proportions of mos- natural host-seeking behaviour of mosquitoes at night in quitoes collected from the verandas of treatment and the presence of a net. It consists of a square glass cylin- control huts. der (25  cm high, 25  cm wide, 60  cm in length) divided 3. Mortality—the proportion of mosquitoes killed into two sections by means of a netting frame fitted into (immediate plus delayed) relative to the total col- a slot across the tunnel. An anesthetized guinea pig was lected. housed unconstrained in a small cage in one section, and 4. Blood-feeding—the proportion of blood-fed mosqui- mosquitoes were released in the other section at dusk toes relative to the total collected. and left overnight. The net samples were holed with nine 5. Blood-feeding inhibition—the proportional reduc- 1-cm diameter holes to allow host-seeking mosquitoes to tion in blood feeding in huts with insecticide treated penetrate the baited chamber; an untreated net sample nets relative to controls with untreated nets. served as the control. The tunnels were kept overnight 6. Personal protection—the proportional reduction in in a dark room at 25–29  °C and 75–85% RH. The next mosquito biting by insecticide treated nets relative to morning, the numbers found alive or dead, fed, or unfed, untreated nets. in each section were recorded. Live mosquitoes were pro- vided with sugar solution and delayed mortality recorded after 24 h. The guinea pigs used in this study were kept in accordance with institutional guidelines for animal care. Supplementary laboratory bioassays To help further explain the results obtained in the experi- mental huts, WHO cone bioassays and tunnel tests were Chemical analysis performed on samples of netting (30 × 30  cm) obtained At the end of the experimental hut trial, five pieces of from Olyset Net and Olyset Plus when unwashed and netting (25 × 25  cm) obtained from the panels of repli- after 10 and 20 washes. Washing was performed in the cate nets of each net type (before and after washing) used laboratory following WHO guidelines [22]. PermaNet 3.0 in the huts were assessed for deltamethrin, permethrin was not tested in the laboratory bioassays owing to the and PBO content using HPLC. Insecticide was extracted restricted application of PBO to the roof of the net pre- from each net piece with an area of 48  sq cm collected venting a realistic direct comparison with Olyset Plus in from the five net samples (25 × 25  cm) obtained from bioassays especially tunnel tests. Net samples from each each whole net. The insecticide content of each sample ITN type and each wash point were tested against the fol- was determined by injecting ten μl aliquots of the extract lowing strains: on a reverse-phase Hypersil GOLD C18 column (75  Å, 250 × 4.6  mm, 5-μm particle size; Thermo Scientific) at 1. An. gambiae sensu lato (s.l.) strains from Cove, Benin room temperature. A mobile phase of 70% acetonitrile in (Cove strain) which is highly pyrethroid resistant. It −1 water was used at a flow rate of 1  ml  min   to separate originates from the experimental hut station in Cove the target analyte. Chromatographic peaks of the insecti- and has shown > 200-fold resistance compared to the cides and internal standard were detected at a wavelength susceptible Kisumu strain in susceptibility bioassays. of 232 nm with the Ultimate 3000 UV detector and ana- Resistance is mediated by elevated levels of P450s lysed with Dionex Chromeleon 6.8 Chromatography and high frequencies of kdr [20]. Data System software. Quantities of insecticide were 2. An. gambiae VKPer strain, which originated from calculated from standard curves established by known the Kou Valley in Burkina Faso. VKPer has moderate concentrations of the insecticide authenticated standards levels of pyrethroid resistance mediated only by high and corrected by internal standard readings in each sam- frequencies of kdr. ple relative to control. 3. An. gambiae s.s. Kisumu strain, a reference suscepti- Data from chemical analysis was used to calculate the ble strain which originated from Kisumu Kenya. percentage retention of each active ingredient after 20 washes relative to the unwashed net and the wash reten- Approximately two hundred 2–5  days old mosquitoes tion index. Wash-resistance index was calculated accord- of each strain were exposed for 3  min in cone bioassays ing to WHO guidelines [22] as indicated below: to four net samples of each net type in cohorts of 5 mos- quitoes per cone. Knock down in cone bioassays was Wash resistance index = 100 × n (tn/t0) recorded after 1 h and mortality after 24 h. free migration stage behaviour Two to three hundred 5–8  days old mosquitoes of each strain were also exposed to each net type in tun- where tn = total active ingredient content after n washing nel tests in replicates of 50 mosquitoes per net sample. cycles, t0 = total active ingredient content before wash- The tunnel test is a laboratory assay designed to simulate ing, n = number of washes. Nguf or et al. Malaria Journal (2022) 21:20 Page 5 of 13 Data analysis nurse; any sleepers testing positive for malaria were with- Proportional outcomes (blood-feeding, exiting and drawn from the study and treated properly. mortality) related to each experimental hut treatment (unwashed and washed 20 times) were assessed using Results binomial generalized linear mixed models (GLMMs) Insecticide resistance in malaria vectors in Cove with a logit link function, fitted using the ‘lme4’ package Mortality with permethrin and alpha-cypermethrin for R (version 3.5.3). A separate model was fitted for each treated papers was 100% with the laboratory-maintained outcome. In addition to the fixed effect of each treat - pyrethroid-susceptible An. gambiae Kisumu strain. With ment, each model included random effects to account wild pyrethroid resistant An. gambiae s.l. from Cove, for the following sources of variation: between the huts; mortality rates were < 50% with all three pyrethroid insec- between the sleepers; between the weeks of the trial; and ticides tested (Table 1) thus confirming the high levels of finally, an observation-level random effect to account for pyrethroid resistance in this vector population. Mortality variation not explained by the other terms in the model however increased from 19.2 to 52.5% with permethrin, (over dispersion). 41.7% to 69.7% with deltamethrin and 0% to 82.4% with alphacypermethrin after pre-exposure of the Cove strain Ethical considerations to PBO synergist (Table 1). This result demonstrated that This study received ethical approval from the Ministry of mixed function oxidases are overexpressed in the wild Health in Benin and from the Ethics Review Committee Cove vector population and their effect can be effectively of the London School of Hygiene & Tropical Medicine. inhibited by the PBO synergist. Informed consent was obtained from each human volun- teer sleeper who slept in the huts to attract mosquitoes Experimental hut trial results prior to their participation. Sleepers were also offered Mosquito entry and exiting rates in experimental huts chemoprophylaxis. Through the course of the study, they A total of 6711 pyrethroid resistant female An. gambiae were examined regularly for signs of fever by a stand-by s.l. were collected during the experimental hut trial. The entry and exiting rates of wild pyrethroid resistant An. gambiae s.l. from the experimental huts with the differ - Table 1 Mortality of pyrethroid resistant Anopheles gambiae ent ITN types tested in the trial are presented in Table 2. s.l. from Cove in WHO cylinder bioassays with and without pre- Compared to the control, Olyset Net did not deter mos- exposure to PBO quitoes from entering the experimental huts (0% when unwashed and after 20 washes). Mosquito deterrence N exposed N dead % Dead was significantly higher with PermaNet 3.0 compared Control 101 0 0.0 to Olyset Plus both unwashed (49% vs. 28%, P < 0.001) PBO 4% only 103 3 2.9 and after 20 washes (36% vs. 0%, P < 0.001). Neverthe- Permethrin 0.75% 104 20 19.2 less, early exiting of mosquitoes from the experimen- Deltamethrin 0.05% 91 38 41.7 tal huts into the veranda trap did not differ significantly Alpha-cypermethrin 0.05% 102 0 0.0 between both pyrethroid-PBO net types both when PBO 4% then permethrin 0.75% 99 52 52.5 unwashed (65% with PermaNet 3.0 vs 68% with Olyset PBO 4% then deltamethrin 0.05% 99 69 69.7 Plus, P = 0.232) and after 20 washes (60% with PermaNet PBO 4% then alpha-cypermethrin 102 84 82.4 3.0 vs. 56% with Olyset Plus, P = 0.053). 0.05% Table 2 Entry and exiting rates of wild pyrethroid resistant Anopheles gambiae s.l. in experimental huts in Cove, Benin Net type Control Olyset Net PermaNet 3.0 Olyset Plus Number of washes 0 0 20 0 20 0 20 N collected 962 1135 1546 489 616 688 1275 a ab b c d d ab N females/night 23 27 37 12 15 16 30 % deterrence – 0* 0* 49 36 28 0 N exiting 429 639 669 319 370 468 712 a b a cd ce d be % exiting 45 56 43 65 60 68 56 95% conf. limits 41.5–47.7 53.4–59.2 40.8–45.7 61.0–69.5 56.2–63.9 64.4–71.4 53.1–58.6 Values along a row bearing the same letter label are not significantly different (P > 0.05) Value set to zero as more mosquitoes caught in Olyset Net huts than control huts Ngufor et al. Malaria Journal (2022) 21:20 Page 6 of 13 Mortality of wild pyrethroid‑resistant Anopheles gambiae s.l. vs. 12%). After 20 washes, mortality with PermaNet 3.0 in experimental huts declined to the same level as the unwashed Olyset Net, Mortality rates of wild pyrethroid resistant mosqui- (17% vs. 18%, P = 0.061) but remained significantly higher toes that entered the experimental huts are presented in with Olyset Plus (24% vs. 17%, P = 0.036). Fig.  1 with further details provided in Table  3. The low - est mortality was achieved with Olyset Net (18% before Mosquito blood‑feeding rates in experimental huts washing and 12% after 20 washes). Percentage mortality The blood-feeding rates of wild pyrethroid-resistant An. with unwashed pyrethroid-PBO nets was highest with gambiae s.l that entered the experimental huts are pre- PermaNet 3.0 (41%) but this declined significantly after sented in Fig. 2 with further details on mosquito feeding 20 washes (17%, P < 0.001). Mortality with unwashed provided in Table  4. The percentage blood-feeding was Olyset Plus was 28% and while this value was significantly lower in huts with the unwashed pyrethroid-PBO ITNs lower than the mortality shown by unwashed PermaNet and was lowest of all with unwashed Olyset Plus as com- 3.0 (P < 0.001), it did not decrease significantly after 20 pared to unwashed PermaNet 3.0 (8% vs 19%, P < 0.001). washes (28% vs. 24%, P = 0.433) whereas for PermaNet For all net types, the data showed an overall increase in 3.0 it declined. Hence, Olyset Plus induced higher mor- blood feeding with washed nets compared to unwashed tality rates than PermaNet 3.0 after 20 washes (24% vs nets and a decrease in blood-feeding inhibition relative 17%, P < 0.001). With respect to the pyrethroid-only ITN, to the untreated net. Percentage blood-feeding when both pyrethroid-PBO nets induced significantly higher washed 20 times did not differ significantly between the mortality rates than Olyset Net with nets washed 20 pyrethroid-PBO types (38% with PermaNet 3.0 vs. 35% times (P < 0.001) though the difference was higher with with Olyset Plus, P = 0.708). The proportions of mosqui - Olyset Plus (24% vs. 12%), than with PermaNet 3.0 (17% toes collected resting in the nets were lowest with the Unwashed Washed 20X 30 e 20 b Control (untreated net) Olyset NetPermaNet 3.0Olyset Plus Fig. 1 Mortality of wild pyrethroid-resistant Anopheles gambiae s.l. in experimental huts in Cove, Benin evaluating different net types. Vertical lines indicate 95% confidence interval estimates whilst bars with the same letter label are not significantly different (P > 0.05) Table 3 Mortality of wild pyrethroid resistant Anopheles gambiae s.l. in experimental huts in Cove, Benin Net type Control Olyset Net PermaNet 3.0 Olyset Plus Number of washes 0 0 20 0 20 0 20 Total collected 962 1135 1546 489 616 688 1275 Total dead 12 209 284 198 103 190 297 a b c d b e e Mortality (%) 1 18 12 41 17 28 24 95% conf. limits 0.6–2.0 16.2–20.1 10.3–13.5 36.1–44.8 13.8–19.8 24.4–31.1 21.0–25.6 Corrected for control (%) – 17 11 41 17 27 22 Values along a row bearing the same letter label are not significantly different (P > 0.05) % Mortality Nguf or et al. Malaria Journal (2022) 21:20 Page 7 of 13 Unwashed Washed 20X Control (untreated net) Olyset NetPermaNet 3.0Olyset Plus Fig. 2 Blood-feeding rates of wild pyrethroid-resistant Anopheles gambiae s.l. in experimental huts in Cove, Benin evaluating different net types. Vertical lines indicate 95% confidence interval estimates whilst bars with the same letter label are not significantly different (P > 0.05) Table 4 Blood-feeding rates of wild pyrethroid resistant Anopheles gambiae s.l. in experimental huts in Cove, Benin Net type Control Olyset Net PermaNet 3.0 Olyset Plus Number of washes 0 0 20 0 20 0 20 Total collected 962 1135 1546 489 616 688 1275 Total blood fed 496 324 807 95 231 56 442 a b a c d e d % Blood fed 52 29 52 19 38 8 35 95% conf intervals 48.4–54.7 25.9–31.2 49.7–54.7 15.9–22.9 33.7–41.3 6.1–10.2 32.1–37.3 % Blood-feeding inhibition – 44 0 63 27 85 33 inside net 294 184 542 28 130 17 241 a bc a d b e c inside net (%) 31 16 35 6 21 2 19 Personal protection (%) – 35 0* 81 53 89 11 Values along a row bearing the same letter label are not significantly different (P > 0.05) Value set to zero as more blood fed mosquitoes were caught in washed Olyset Net huts than control hut unwashed pyrethroid-PBO nets (2–6%), which is con- the difference in mosquito mortality between the candi - sistent with the higher toxicity observed with this type of date and active comparator product is greater than 0.7. net. Personal protection with both types of pyrethroid- and (2) The upper 95% confidence interval estimate of the PBO ITNs were > 80% when unwashed but this declined odds ratio describing the difference in mosquito blood- after 20 washes to 53% with PermaNet 3.0 and 11% with feeding between the candidate and active comparator Olyset Plus. product is less than 1.43. Following the WHO guidelines, both unwashed and washed data of each product were Non‑inferiority assessment analysed together to generate single estimates of efficacy According to recent provisional WHO guidelines [19], representative of the overall performance over the life- for a candidate pyrethroid-PBO ITN product to be time of the product in the field. included in this new WHO intervention class without the Each primary endpoint for non-inferiority (mortal- need for epidemiological evidence, it must demonstrate ity and blood-feeding rate for unwashed and washed non-inferiority to the first in class product which has nets combined), was assessed using binomial general- already demonstrated public health value (Olyset Plus) ized linear mixed models (GLMMs) with a logit link and superiority to a pyrethroid-only LLIN in experimen- function fitted using the ‘lme4’ package of R version tal hut trials [19]. Briefly, the candidate pyrethroid-PBO 3.5.3 for Windows as described earlier. Results from the product is deemed non-inferior if: (1) The lower 95% non-inferiority assessment of PermaNet 3.0 to Olyset confidence interval estimate of the odds ratio describing Plus are presented in Table  5 below. The odds ratio for % Blood-fed Ngufor et al. Malaria Journal (2022) 21:20 Page 8 of 13 Table 5 Results from the non-inferiority assessment of PermaNet 3.0 to Olyset Plus against wild pyrethroid-resistant Anopheles gambiae s.l. in experimental huts in Cove, Benin Control Net Olyset Net Olyset Plus PermaNet 3.0 Total collected 962 2681 1963 1105 Mortality Total dead 12 393 488 301 Mortality (%) 1 15 25 27 Odds ratio – – – 1.528 Std. err (on log odds scale) – – – 0.206 95% conf. interval – – – 1.021–2.289 WHO non-inferiority margin – – – Lower 95%CI > 0.7 Conclusion – – – Non-inferior Blood-feeding rate Total blood-fed 496 1131 498 326 Blood-feeding (%) 52 42 25 30 Blood-feeding inhibition – 19 52 42 Odds ratio – – – 1.192 Std. err (on log odds scale) – – – 0.222 95% conf. interval – – – 0.772–1.841 WHO non-inferiority margin – – – Upper 95%CI < 1.43 Conclusion – – – Not non-inferior Combined data for washed and unwashed nets of each net type the difference in mosquito mortality between PermaNet bioassay mortality however dropped significantly with 3.0 and Olyset Plus was 1.528 (95% confidence interval: washed net samples. Mortality with the untreated net 1.021–2.289) while the odds ratio for the difference in samples did not exceed 5% with any strain tested. mosquito blood feeding was 1.192 (95% confidence inter - val: 0.772–1.841). Following the WHO criteria described Tunnel test results above, PermaNet 3.0 was non-inferior to Olyset Plus in The results from the tunnel tests comparing Olyset Plus terms of its ability to kill wild pyrethroid- resistant An. and Olyset Net unwashed and after 10 and 20 washes gambiae s.l. in the experimental hut trial in Cove Benin. against all three strains are presented in Figs.  4 and 5 In contrast PermaNet 3.0 was not non-inferior to Olyset for mortality and blood-feeding inhibition respectively. Plus in terms of proportions of mosquitoes that blood- Mortality rates were generally higher in the tunnels fed and, therefore, fails to demonstrate non-inferiority tests compared to the cone bioassays and decreased as for blood feeding inhibition in this trial. The results also the strain become more pyrethroid-resistant (Fig.  4). showed superiority of both pyrethroid-PBO net types to Mortality rates with the Kisumu strain were very high Olyset Net both in terms of mosquito mortality (25–27% with both Olyset Net and Olyset Plus (> 95%). With the vs. 15%, P < 0.001) and reducing blood-feeding (25%-30% VKPer strain, mortality remained > 80% after 20 washes vs. 42%, P < 0.001). with both ITN types. With the Cove strain, mortality was significantly higher with Olyset Plus compared to Supplementary laboratory bioassays results Olyset Net at 0 and 10 washes but about the same after Cone bioassay results 20 washes. With unwashed nets, blood-feeding inhi- The 3-min cone bioassay mortality results for all 3 mos - bition in the tunnel tests was consistently higher with quito strains and wash points tested are presented in Olyset Plus (> 90%) compared to Olyset Net (27–75%) for Fig.  3. Unwashed Olyset Net induced very low mortality all three strains tested (Fig.  5). After 10 and 20 washes, rates against the susceptible Kisumu strain (17–20%) and blood-feeding inhibition of the Kisumu and VKPer strain even lower mortality rates against the pyrethroid resist- remained > 80% with Olyset Plus and Olyset Net. With ant strains (< 5%). Cone bioassay mortality rates with Cove strain, blood-feeding inhibition was also higher unwashed Olyset Plus were higher across all 3 strains with Olyset Plus at 0 and 10 washes but declined to compared to Olyset Net though mortality decreased as about the same level as Olyset Net after 20 washes (56%). the strain tested became more pyrethroid-resistant. Cone Nguf or et al. Malaria Journal (2022) 21:20 Page 9 of 13 0 wash 10 washes 20 washes Olyset NetOlyset Plus Olyset NetOlyset Plus Olyset NetOlyset Plus Kisumu - SVKPer - RCove - R Fig. 3 Mortality (%) of susceptible and resistant strains of An. gambiae s.l. in cone bioassays with Olyset Plus and Olyset Net. S = susceptible, R = resistant 0 wash 10 washes 20 washes Olyset NetOlyset Plus Olyset NetOlyset Plus Olyset NetOlyset Plus Kisumu - SVKPer - RCove - R Fig. 4 Tunnel test mortality (%) of susceptible and resistant strains of Anopheles gambiae s.l. exposed to Olyset Plus vs. Olyset Net Mortality in the untreated control tunnel was < 10% with in both types of pyrethroid-PBO ITNs after 20 washes all three strains. (58.7% vs. 25% with Olyset Plus and 80.9% vs. 68.8% with PermaNet 3.0, P < 0.05). The decrease in PBO content after washing was more evident in Olyset Plus Chemical analysis results than in PermaNet 3.0 netting, hence the wash retention For PermaNet 3.0, PBO was only recorded from index of PBO was higher with PermaNet 3.0 compared the roof panel of the nets; for Olyset Plus, PBO was to Olyset Plus (98.1% vs. 93.3%). recorded on all 5 panels (Table  6). Compared to the pyrethroid component, much less PBO was  retained % Mortality % Mortality Ngufor et al. Malaria Journal (2022) 21:20 Page 10 of 13 0 wash 10 washes 20 washes Olyset NetOlyset Plus Olyset NetOlyset Plus Olyset NetOlyset Plus Kisumu - SVKPer - RCove - R Fig. 5 Blood-feeding inhibition (%) of susceptible and resistant strains of An. gambiae s.l. in tunnel tests with Olyset Plus and Olyset Net. Blood-feeding inhibition was calculated relative to the control tunnel. S = susceptible, R = Resistant Table 6 Chemical analysis of net samples after experimental hut trial at Cove, Benin LN type Active ingredient AI content (g/kg) Retention, % Wash retention Unwashed Washed 20× index (%) Olyset Net Permethrin 12.0 12.0 100.0 100.0 Olyset Plus Permethrin 15.5 9.1 58.7 97.4 PBO 8.0 2.0 25.0 93.3 PermaNet 3.0 Deltamethrin (roof of net) 4.2 3.4 80.9 98.9 PBO (roof of net) 16.0 11.0 68.8 98.1 by increased mono-oxygenase activity. The experimen - Discussion tal hut trial demonstrated improved levels of mortality Following the interim endorsement of pyrethroid-PBO and blood-feeding inhibition with both pyrethroid-PBO nets by the WHO [23], malaria vector control pro- ITN types compared to a standard pyrethroid-only grammes are faced with additional choice of different LLIN against a vector population that was very resistant brands of prequalified pyrethroid-PBO nets [7]. Consid - to pyrethroids; this was supported by results from the ering the wide variations in design of the available brands laboratory assays which compared Olyset Plus to Olyset of these nets, studies generating the required assurance Net against pyrethroid-resistant mosquito strains. These of comparative performance to Olyset Plus (the first in observations are partly attributable to the synergistic class pyrethroid-PBO net to demonstrate empirical evi- effect of the PBO on pyrethroid resistance and are con - dence of entomological and epidemiological impact) are sistent with other experimental hut trials across Africa necessary. This study compared the efficacy and assessed and epidemiological trials performed in Tanzania [8] and the non-inferiority of PermaNet 3.0 to Olyset Plus in Uganda [18]. experimental huts against pyrethroid resistant malaria Twenty washes in experimental hut studies are indi- vectors in a highly endemic country of West Africa, fol- cated by WHO as a proxy for the ability of an ITN to lowing WHO guidelines [19, 22]. withstand multiple washes under operational use over The WHO susceptibility bioassays confirmed the high a 3-year life span [19, 22]. With unwashed nets, the lev- levels of pyrethroid resistance in the vector population at els of improved mortality relative to the standard pyre- the experimental hut site during the trial, corroborating throid-only net were higher with PermaNet 3.0 than previous findings [20]. The increased mortality achieved with Olyset Plus. However, unlike with Olyset Plus, this in bioassays with pre-exposure to PBO showed that effect was lost with PermaNet 3.0 after twenty washes; pyrethroid-resistance was indeed at least partly mediated % Blood-feeding Inhibition Nguf or et al. Malaria Journal (2022) 21:20 Page 11 of 13 PermaNet 3.0 killed significantly lower proportions of to the upper panel and pyrethroid to side panels versus mosquitoes than Olyset Plus and same proportions as an ITN with all 5 panels treated with pyrethroid-PBO. an unwashed pyrethroid-only ITN. Though the RCT in Behavioural studies of mosquitoes around nets indicate Uganda was not powered to assess differences between that mosquitoes may first make multiple contacts with the pyrethroid-PBO ITN brands tested (PermaNet 3.0 vs. the roof panel in response to odour plumes [26]; how- Olyset Plus) [18], the results from our trial appear con- ever, experimental hut trials comparing restricted versus sistent with some of the differences in epidemiological full PBO coverage on nets are too few to be definitive on effect observed between both brands: (1) The high exper - the question of efficacy. imental hut mortality with the unwashed PermaNet 3.0 Despite the differences in performance observed supports the higher initial protective effect observed with between both pyrethroid-PBO net types with regards the net in the Ugandan trial at the 6 months epidemiolog- to their impact after 20 standardized washes, the non- ical survey which was not seen with Olyset Plus. (2) The inferiority analysis performed in accordance with recent higher rate of decline in experimental hut mortality after WHO guidelines [19] showed that PermaNet 3.0 was washing with PermaNet 3.0 compared to Olyset Plus is non-inferior to Olyset Plus in terms of mosquito mor- consistent with the shorter-lived epidemiological effect in tality but not with blood-feeding inhibition. The higher the Ugandan trial with PermaNet 3.0 (up to 12  months) blood-feeding inhibition observed with Olyset Plus could compared to Olyset Plus which remained more protec- be due to the high excito-repellency of permethrin in tive than the pyrethroid-only net at 18 months. However, Olyset Plus compared to deltamethrin in PermaNet 3.0 care should be taken to not over-interpret the compari- [27]. Alternatively, the study may not have had sufficient sons between results from our hut trial and Ugandan power to demonstrate non-inferiority of PermaNet 3.0 RCT considering the different geographical settings and to Olyset Plus for both endpoints; further studies are the lack of sufficient power to differentiate between the on-going to help guide power calculations for ITN non- epidemiological impact of both pyrethroid-PBO net type inferiority studies. The non-inferiority margin used for in the Ugandan trial. the analysis was defined by WHO as an odds ratio of 0.7 The difference in hut performance between both pyre - in mosquito mortality and feeding between a candidate throid-PBO ITNs can be attributed to differences in the net and the first in class net considered acceptable for retention and movement of PBO across the polymer fibre both products to be in the same policy class. According in Olyset Plus compared to PermaNet 3.0 and/or dif- to these guidelines, if non-inferiority is demonstrated ferences in design and specification. Retention of bioef - in two independent experimental hut trials in different ficacy of pyrethroid-PBO nets is a fine balance between geographical locations representative of where the prod- migration and replenishment of PBO from the core to ucts will be deployed, then PermaNet 3.0 will be placed the surface of fibres and the maintenance of an internal in the same WHO vector control product class as Olyset reservoir sufficient to last the lifespan of the LLIN, which Plus [19]. It is however not clear whether non-inferiority is typically set at 3 years of use [24]. The chemical analy - must be demonstrated for both endpoints (mortality and sis results showed a faster release of PBO in the Olyset blood-feeding) for a second-in class product to become Plus netting after 20 washes compared to PermaNet 3.0 part of an intervention class. While the guidelines were though it is unclear whether this may have increased the developed more like a compromise between the risk of bioavailability of the PBO on the surface of Olyset Plus accepting an inferior product and the feasibility of con- after washing. Whether sufficient PBO would remain ducting epidemiological trials, the findings from our within the fibres of both pyrethroid-PBO nets after trial show that non-inferiority experimental hut trials 3 years of household use is presently unknown and is the are complex, and results must be interpreted with care. subject of ongoing WHO durability trials of Olyset Plus Comparative performance between products may also and PermaNet 3.0 which are not yet completed [18, 25]. depend on other location-specific factors, such as the Another factor which may contribute to the discrepan- intensity of insecticide resistance and behaviour of the cies in efficacy of the two pyrethroid-PBO ITNs are dif - target vector population which should be taken into con- ferences in design and specification: Olyset Plus is treated sideration when choosing between products of the same with the pyrethroid permethrin while PermaNet 3.0 is class. treated with deltamethrin, and Olyset Plus contains PBO While the present hut trial in Benin and earlier hut tri- on every panel whereas in PermaNet 3.0, PBO is available als in Benin, Tanzania, Cameroon, Burkina Faso, Côte only on the top panel of the net. It is not clear whether d’Ivoire and Vietnam where the vectors were also resist- the restricted application of PBO to the roof of the net ant have shown some additional effect of pyrethroid- would affect bioefficacy. This would require comparative PBO nets over a standard pyrethroid net [12, 13, 15–17], trials of the PermaNet 3.0 with pyrethroid-PBO restricted the margin appears to vary depending on the level of Ngufor et al. Malaria Journal (2022) 21:20 Page 12 of 13 Authors’ contributions pyrethroid-resistance encountered [28]. The absolute CN designed and supervised the study, analysed the data and drafted the increase in hut trial mortality with Olyset Plus compared manuscript. JF and AA performed the hut trial and laboratory bioassays. to Olyset Net in the present study (24–28% vs. 12–18%) JDC and TSC performed the non-inferiority analysis and contributed to data interpretation and manuscript revision. HI performed the chemical analysis. conducted in an area of intense pyrethroid-resistance MR contributed to study design, data interpretation and manuscript revision. [20] is lower than what has been previously reported with All authors read and approved the final manuscript. Olyset Plus in another area in Northern Benin where Funding pyrethroid resistance was less prevalent (67–81% vs. This project was funded by the LSHTM ITD Athena Swan Career restart fellow- 36–42%) [16]. Compared to East Africa, West Africa has ship and an independent research grant from Sumitomo Chemical Company shown historically higher intensity of pyrethroid resist- awarded to Corine Ngufor. Funding covered research costs and operational expenses. The funders had no role in study design, data collection and analy- ance in malaria vectors [29] mediated by complex and sis, decision to publish, or preparation of the manuscript. multiple insecticide resistance mechanisms which may not be effectively tackled by the synergistic effects of Availability of data and materials The datasets used and/or analysed during the current study are available from PBO in pyrethroid-PBO ITNs [4, 23]. It is therefore not the corresponding author on reasonable request. clear whether a diminishment in experimental hut mor- tality with pyrethroid-PBO nets due to increasing inten- Declarations sity of pyrethroid-resistance would translate to a reduced epidemiological effect of pyrethroid-PBO ITNs in West Ethics approval and consent to participate The study was approved by the Ethics Review Committee of LSHTM and the Africa compared to East Africa which has been the site of Ministry of Health, Benin. Informed consent was obtained from each human the only epidemiological trials so far. volunteer sleeper who slept in the huts to attract mosquitoes, prior to their participation. Sleepers were also offered chemoprophylaxis. Through the course of the study, they were examined regularly for signs of fever; any sleep- ers testing positive for malaria were withdrawn from the study and treated in Conclusion accordance with country guidelines. Olyset Plus outperformed PermaNet 3.0 in terms of its Consent for publication ability to induce improved levels of mosquito mortality Not applicable. compared to a standard pyrethroid LLIN after multiple standardized washes. Nevertheless, a non-inferiority Competing interests The authors declare that they have no competing interests. analysis of both ITN types following recent WHO guide- lines showed that they were comparable in their ability Author details to kill mosquitoes. Compared to the situation existing London School of Hygiene and Tropical Medicine (LSHTM), London, UK. Centre de Recherches Entomologiques de Cotonou (CREC), Cotonou, Benin. in Benin several years ago, both pyrethroid-PBO ITNs Pan African Malaria Vector Research Consortium (PAMVERC), Cotonou, Benin. showed less impact against the mosquito vector due to 4 5 Liverpool School of Tropical Medicine, Liverpool L3 5QA, UK. MRC Centre increased levels of resistance. West Africa constitutes for Global Infectious Disease Analysis, Infectious Disease Epidemiology, Impe- rial College London, Norfolk Place, London W2 1PG, UK. a different environment and ecology from East Africa, with historically higher intensity of pyrethroid resistance Received: 20 May 2021 Accepted: 3 January 2022 in malaria vectors. It is not clear whether either of these nets would have the same epidemiological impact against malaria demonstrated with pyrethroid-PBO nets in trials in East Africa. A cluster randomized trial of pyrethroid- References 1. Maxwell CA, Msuya E, Sudi M, Njunwa KJ, Carneiro IA, Curtis CF. Eec ff t of PBO nets with epidemiological outcome indicators is community-wide use of insecticide-treated nets for 3–4 years on malarial urgently required in West Africa to establish its effective - morbidity in Tanzania. Trop Med Int Health. 2002;7:1003–8. ness against malaria. 2. Levitz L, Janko M, Mwandagalirwa K, Thwai KL, Likwela JL, Tshefu AK, et al. Eec ff t of individual and community-level bed net usage on malaria prevalence among under-fives in the Democratic Republic of Congo. Malar J. 2018;7:39. Abbreviations 3. WHO. World Malaria Report 2020. Geneva: World Health Organization; ITN: Insecticide treated nets; LLIN: Long-lasting insecticidal nets; PBO: Pipero- nyl butoxide; WHO: World Health Organization; PQ: Prequalification team; 4. WHO. Global report on insecticide resistance in malaria vectors: WHOPES: WHO Pesticide Evaluation Scheme; CREC: Centre de Recherche 2010–2016. Geneva: World Health Organization; 2018. Entomologique de Cotonou; LSHTM: London School of Hygiene & Tropical 5. Ranson H, Lissenden N. Insecticide resistance in African Anopheles Medicine; HPLC: High Performance Liquid Chromatography; Kdr: Knock down mosquitoes: a worsening situation that needs urgent action to maintain resistance. malaria control. Trends Parasitol. 2016;32:187–96. 6. Zaim M, Aitio A, Nakashima N. Safety of pyrethroid-treated mosquito Acknowledgements nets. Med Vet Entomol. 2000;14:1–5. We thank John Lucas and Dr. John Invest of Sumitomo for providing the nets. 7. WHO. List of WHO prequalified vector control products. Geneva: World We also thank the technical staff of CREC (Abibatou Odjo, Estelle Vigninou, Health Organization; 2021. https:// www. who. int/ pq- vector- contr ol/ prequ Laurette Kiki, Augustin Fongnikin etc.) for their assistance. We appreciate Prof alifi ed- lists/ Prequ alifi edPro ducts 27Jan uary2 020. pdf? ua=1. Accessed 27 Martin Akogbeto for administrative support. We are grateful to the rice farmers Nov 2021. at Cove and the volunteer sleepers for their participation in the hut study. Nguf or et al. Malaria Journal (2022) 21:20 Page 13 of 13 8. Protopopoff N, Mosha JF, Lukole E, Charlwood JD, Wright A, Mwalimu 27. Achee NL, Sardelis MR, Dusfour I, Chauhan KR, Grieco JP. Characteriza- CD, et al. Eec ff tiveness of a long-lasting piperonyl butoxide-treated tion of spatial repellent, contact irritant, and toxicant chemical actions insecticidal net and indoor residual spray interventions, separately and of standard vector control compounds. J Am Mosq Control Assoc. together, against malaria transmitted by pyrethroid-resistant mosquitoes: 2009;25:156–67. a cluster, randomised controlled, two-by-two factorial design trial. Lancet. 28. Churcher TS, Lissenden N, Griffin JT, Worrall E, Ranson H. The impact of 2018;391:1577–88. pyrethroid resistance on the efficacy and effectiveness of bednets for 9. WHO. Guidelines for malaria vector control. Geneva: World Health Organi- malaria control in Africa. Elife. 2016;5:e16090. zation; 2019. 29. Hancock PA, Hendriks CJM, Tangena J-A, Gibson H, Hemingway J, Cole- 10. AMP: Alliance for malaria prevention. 2021. https:// netma pping proje ctall man M, et al. Mapping trends in insecticide resistance phenotypes in iance forma laria preve ntion. com/. Accessed 04 Mar 2021. African malaria vectors. PLoS Biol. 2020;18:e3000633. 11. Corbel V, Chabi J, Dabiré RK, Etang J, Nwane P, Pigeon O, et al. 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Tungu P, Magesa S, Maxwell C, Malima R, Masue D, Sudi W, et al. Evalu- ation of PermaNet 3.0 a deltamethrin-PBO combination net against Anopheles gambiae and pyrethroid resistant Culex quinquefasciatus mosquitoes: an experimental hut trial in Tanzania. Malar J. 2010;9:21. 15. Oumbouke WA, Rowland M, Koffi AA, Alou LPA, Camara S, N’Guessan R. Evaluation of an alpha-cypermethrin + PBO mixture long-lasting insec- ticidal net VEERALIN LN against pyrethroid resistant Anopheles gambiae s.s.: an experimental hut trial in M’bé, central Côte d’Ivoire. Parasit Vectors. 2019;12:544. 16. Pennetier C, Bouraima A, Chandre F, Piameu M, Etang J, Rossignol M, et al. Efficacy of Olyset Plus, a new long-lasting insecticidal net incorporating permethrin and piperonyl-butoxide against multi-resistant malaria vec- tors [corrected]. PLoS ONE. 2013;8:e75134. 17. Kweka EJ, Lyaruu LJ, Mahande AM. 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Comparative efficacy of two pyrethroid-piperonyl butoxide nets (Olyset Plus and PermaNet 3.0) against pyrethroid resistant malaria vectors: a non-inferiority assessment

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Abstract

Background: Pyrethroid-PBO nets were conditionally recommended for control of malaria transmitted by mosqui- toes with oxidase-based pyrethroid-resistance based on epidemiological evidence of additional protective effect with Olyset Plus compared to a pyrethroid-only net (Olyset Net). Entomological studies can be used to assess the compara- tive performance of other brands of pyrethroid-PBO ITNs to Olyset Plus. Methods: An experimental hut trial was performed in Cové, Benin to compare PermaNet 3.0 (deltamethrin plus PBO on roof panel only) to Olyset Plus (permethrin plus PBO on all panels) against wild pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) following World Health Organization ( WHO) guidelines. Both nets were tested unwashed and after 20 standardized washes compared to Olyset Net. Laboratory bioassays were also performed to help explain find- ings in the experimental huts. Results: With unwashed nets, mosquito mortality was higher in huts with PermaNet 3.0 compared to Olyset Plus (41% vs. 28%, P < 0.001). After 20 washes, mortality declined significantly with PermaNet 3.0 (41% unwashed vs. 17% after washing P < 0.001), but not with Olyset Plus (28% unwashed vs. 24% after washing P = 0.433); Olyset Plus induced significantly higher mortality than PermaNet 3.0 and Olyset Net after 20 washes. PermaNet 3.0 showed a higher wash retention of PBO compared to Olyset Plus. A non-inferiority analysis performed with data from unwashed and washed nets together using a margin recommended by the WHO, showed that PermaNet 3.0 was non-inferior to Olyset Plus in terms of mosquito mortality (25% with Olyset Plus vs. 27% with PermaNet 3.0, OR = 1.528, 95%CI = 1.02– 2.29) but not in reducing mosquito feeding (25% with Olyset Plus vs. 30% with PermaNet 3.0, OR = 1.192, 95%CI = 0.77–1.84). Both pyrethroid-PBO nets were superior to Olyset Net. Conclusion: Olyset Plus outperformed PermaNet 3.0 in terms of its ability to cause greater margins of improved mos- quito mortality compared to a standard pyrethroid net, after multiple standardized washes. However, using a margin of non-inferiority defined by the WHO, PermaNet 3.0 was non-inferior to Olyset Plus in inducing mosquito mortal- ity. Considering the low levels of mortality observed and increasing pyrethroid-resistance in West Africa, it is unclear whether either of these nets would demonstrate the same epidemiological impact observed in community trials in East Africa. *Correspondence: corine.ngufor@lshtm.ac.uk London School of Hygiene and Tropical Medicine (LSHTM), London, UK Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ngufor et al. Malaria Journal (2022) 21:20 Page 2 of 13 Keywords: Experimental huts, Piperonyl butoxide, PBO, Olyset plus, PermaNet 3.0, Olyset, Mixture, LLIN, Insecticide resistance, Pyrethroid-PBO, Cove, Benin, Pyrethroid resistance, Insecticide-treated nets, Long-lasting insecticidal nets, Next generation nets, Anopheles, Cove Benin Background World Health Organization (WHO) list of prequali- Long-lasting insecticidal nets (LLINs) remain one of fied vector control products: PermaNet 3.0, Veeralin, the most powerful tools to reduce malaria transmission Tsara Boost, Tsara Plus and very recently DuraNet in a community and provide personal protection to the Plus [7]. These nets have all demonstrated superiority user [1, 2]. They have contributed significantly to recent over pyrethroid-only nets in terms of mosquito mor- reductions in malaria burden [3]. Their efficacy is how - tality and blood-feeding inhibition in multiple experi- ever threatened by increasing resistance to pyrethroids mental hut trials across Africa [11–17]. However, they [4, 5]; the insecticide of choice used on bed-nets owing differ from Olyset Plus in their design and specifica - to its safety, low cost and rapid activity on vector mos- tions; typically, the location of PBO on the net (i.e., all quitoes [6]. To maintain the effectiveness of insecticide panels vs. roof panel only), the type and dose of pyre- treated nets for malaria control, new types of LLINs throid used, bioavailability and retention of PBO after treated with alternative insecticides and compounds washing and could, therefore, differ in entomological which can either replace or complement pyrethroids on and epidemiological impact. A recent large cluster- bed-nets are urgently needed. randomized trial in Uganda, evaluating the efficacy of A new class of insecticide treated nets (ITNs) com- Olyset Plus and PermaNet 3.0 compared to pyrethroid- bining pyrethroids and piperonyl butoxide (PBO) (pyre- only nets in a setting of high pyrethroid resistance, throid-PBO ITNs) have been developed [7]. PBO is a also demonstrated better protection against malaria synergist that inhibits specific metabolic enzymes such with pyrethroid-PBO nets compared to pyrethroid- as mixed-function oxidases within mosquitoes that only nets for up to 18  months confirming the findings detoxify or sequester insecticides before they can have of the Tanzanian trial [18]. While the Ugandan trial a toxic effect on the mosquito. Pyrethroid-PBO nets was not powered to directly compare between the dif- can therefore induce increased mortality of pyrethroid- ferent ITN brands tested, the results showed that the resistant malaria vectors that express mixed function additional protective effect of the pyrethroid-PBO net oxidase based pyrethroid resistance mechanisms that compared to the pyrethroid-only net was initially large are inhibited by the PBO in the net. These nets were with PermaNet 3.0 at 6  months post-distribution but given an interim endorsement as a new WHO class of lasted only up to 12 months whereas additional protec- vector control products in 2017 based on epidemiologi- tive effect with Olyset Plus appeared to have a delayed cal data from a cluster randomized controlled trial in onset which was not observed at 6 months but became North Eastern Tanzania [8], that demonstrated addi- evident at 12 months and lasted up to 18 months. There tional malaria control with one prototype pyrethroid- are major differences in design between Olyset Plus and PBO net (Olyset Plus) compared to a pyrethroid-only PermaNet 3.0, which could have implications on their net (Olyset Net), against pyrethroid resistant malaria epidemiological impact; Olyset Plus is a polyethylene vectors of moderate intensity, partly conferred by net incorporated with permethrin and PBO on all pan- monooxygenase-based resistance mechanism. Pyre- els while PermaNet 3.0 is a polyester net coated with throid-PBO ITNs are conditionally recommended for deltamethrin with PBO restricted to the roof of the net. malaria vector control instead of pyrethroid-only ITNs To generate assurance of comparative performance in areas of confirmed intermediate levels of resistance of new candidate products within an established WHO mediated by monooxygenase-based resistance mecha- vector control product class, without the need for epi- nism [9]. This endorsement has been followed by an demiological evidence for each new product, the WHO increasing uptake of pyrethroid-PBO nets worldwide has developed new experimental hut study guidelines [10]; in sub-Saharan Africa for example, the proportion for assessing their non-inferiority to a first in class prod - of pyrethroid-PBO nets of all nets delivered increased uct for which evidence of public health value has already from 3% in 2018 to 35% in 2021. been generated [19]. These provisional guidelines are to While Olyset Plus was the first in class pyrethroid- be piloted with pyrethroid-PBO nets by comparing other PBO net to demonstrate public health value as WHO/PQ-listed pyrethroid-PBO nets with the first in observed in the Tanzanian trial [8], there are currently class product, Olyset Plus. This study compared the effi - five additional types of pyrethroid-PBO ITNs on the cacy and wash resistance of Olyset Plus and PermaNet Nguf or et al. Malaria Journal (2022) 21:20 Page 3 of 13 3.0 and assessed the non-inferiority of PermaNet 3.0 to to PBO and to insecticides lasted 1  h, knockdown was Olyset Plus in experimental huts against wild free-flying recorded after 60 min and mortality after 24 h. pyrethroid resistant malaria vectors in Southern Benin. Experimental hut treatments Methods Olyset Plus and PermaNet 3.0 were compared in the Experimental hut trial experimental huts when unwashed and after 20 stand- Experimental hut site ardized washes. A WHO-recommended pyrethroid-only Experimental huts are small standardized human habita- long-lasting net (Olyset Net) was included to demon- tions approved by the WHO for the controlled evalua- strate the added effect of PBO on the insecticide-resist - tion of indoor vector control tools against wild free-flying ant local vector species. Nets were washed using savon mosquitoes. Mosquitoes enter the huts freely at night de Marseilles and rinsed twice following WHO proce- to interact with the human host and the vector control dures for washing nets for experimental hut studies [22]. intervention and in the morning on each day of the trial, The following seven (7) treatments were thus tested in they are collected from the different compartments of seven experimental huts: each hut and scored for entomological outcomes. The experimental hut study was performed at the CREC/ 1. Untreated polyethylene net LSHTM experimental hut station situated in a large 2. Olyset Net unwashed (permethrin only) rice growing area in Cové, Southern Benin, where the 3. Olyset Net washed 20 times. local mosquito population has been shown to be resist- 4. PermaNet 3.0 unwashed (Roof: deltamethrin ant to pyrethroids [20]. The rice paddies provide exten - plus PBO; sides: deltamethrin only) sive breeding sites for Anopheles gambiae throughout the 5. PermaNet 3.0 washed 20 times. year. The huts are built on concrete plinths surrounded 6. Olyset Plus unwashed (permethrin plus PBO on all by water-filled moats to prevent entry of scavenging ants panels) and have veranda traps to capture the exiting mosqui- 7. Olyset Plus washed 20 times. toes. The walls are made of brick plastered with cement on the inside, with a corrugated iron roof. The huts have a ceiling of palm thatch and four window slits (1 cm gap) Hut trial procedure on the walls through which mosquitoes enter. The local Treatments were allocated to the experimental huts on a vector population in Cove is resistant to pyrethroids and weekly basis using a randomized Latin square design to DDT and consists of a mixture of Anopheles coluzzii and adjust for any variation in hut attractiveness and mini- Anopheles gambiae sensu stricto (s.s.), with the latter mize any carry over effect between treatments. Three occurring at lower proportions (23%) and only in the dry replicate nets of each type were prepared, and these were season [20]. Molecular analysis revealed a L1014F kdr rotated every 2 days on each week (6 days) of the trial. To allele frequency of 89%. Microarray studies also found simulate wear and tear, each net was intentionally holed CYP6P3, a P450 validated as an efficient metabolizer of with six 16 cm holes (two holes on each side and one on pyrethroids [21], to be overexpressed in Cove [20]. each end). The trial ran for 42 nights between February and April Insecticide resistance bioassays of 2017. Consenting human volunteer sleepers slept in To assess the frequency of pyrethroid resistance and the huts from 9:00 p.m. to 5:00 a.m. each night and were presence of mixed function oxidases in the Cové vec- rotated daily through the huts to account for individual tor population during the trial, adult mosquitoes that attractiveness to mosquitoes. At dawn, the volunteer emerged from larvae collected from breeding sites close sleepers collected mosquitoes in the room of the hut and to experimental huts were tested in WHO cylinder bio- under the bed nets and the veranda using torches and assays with and without pre-exposure to PBO. A total aspirators. The mosquitoes were then transferred to the of ~ 100 mosquitoes of the pyrethroid resistant An. laboratory for processing where they were identified and gambiae s.l. Cove strain and the pyrethroid susceptible scored for their blood feeding status, mortality and hut An. gambiae Kisumu strain were exposed to treated fil - position. Mosquitoes were held at 27 ± 2  °C during the ter papers in WHO cylinder bioassays in batches of 25. observations. Tests were performed with papers treated with perme- The following outcome measures were used to assess thrin 0.75%, alpha-cypermethrin 0.05% and deltamethrin the efficacy of each treatment in the experimental huts: 0.05%. To assess presence of MFO, some mosquitoes were also pre-exposed to papers treated with 4% PBO 1. Deterrence—the proportional reduction in number prior to exposure to insecticide-treated papers. Exposure of mosquitoes entering huts with treated nets. Ngufor et al. Malaria Journal (2022) 21:20 Page 4 of 13 2. Exiting rates estimated from the proportions of mos- natural host-seeking behaviour of mosquitoes at night in quitoes collected from the verandas of treatment and the presence of a net. It consists of a square glass cylin- control huts. der (25  cm high, 25  cm wide, 60  cm in length) divided 3. Mortality—the proportion of mosquitoes killed into two sections by means of a netting frame fitted into (immediate plus delayed) relative to the total col- a slot across the tunnel. An anesthetized guinea pig was lected. housed unconstrained in a small cage in one section, and 4. Blood-feeding—the proportion of blood-fed mosqui- mosquitoes were released in the other section at dusk toes relative to the total collected. and left overnight. The net samples were holed with nine 5. Blood-feeding inhibition—the proportional reduc- 1-cm diameter holes to allow host-seeking mosquitoes to tion in blood feeding in huts with insecticide treated penetrate the baited chamber; an untreated net sample nets relative to controls with untreated nets. served as the control. The tunnels were kept overnight 6. Personal protection—the proportional reduction in in a dark room at 25–29  °C and 75–85% RH. The next mosquito biting by insecticide treated nets relative to morning, the numbers found alive or dead, fed, or unfed, untreated nets. in each section were recorded. Live mosquitoes were pro- vided with sugar solution and delayed mortality recorded after 24 h. The guinea pigs used in this study were kept in accordance with institutional guidelines for animal care. Supplementary laboratory bioassays To help further explain the results obtained in the experi- mental huts, WHO cone bioassays and tunnel tests were Chemical analysis performed on samples of netting (30 × 30  cm) obtained At the end of the experimental hut trial, five pieces of from Olyset Net and Olyset Plus when unwashed and netting (25 × 25  cm) obtained from the panels of repli- after 10 and 20 washes. Washing was performed in the cate nets of each net type (before and after washing) used laboratory following WHO guidelines [22]. PermaNet 3.0 in the huts were assessed for deltamethrin, permethrin was not tested in the laboratory bioassays owing to the and PBO content using HPLC. Insecticide was extracted restricted application of PBO to the roof of the net pre- from each net piece with an area of 48  sq cm collected venting a realistic direct comparison with Olyset Plus in from the five net samples (25 × 25  cm) obtained from bioassays especially tunnel tests. Net samples from each each whole net. The insecticide content of each sample ITN type and each wash point were tested against the fol- was determined by injecting ten μl aliquots of the extract lowing strains: on a reverse-phase Hypersil GOLD C18 column (75  Å, 250 × 4.6  mm, 5-μm particle size; Thermo Scientific) at 1. An. gambiae sensu lato (s.l.) strains from Cove, Benin room temperature. A mobile phase of 70% acetonitrile in (Cove strain) which is highly pyrethroid resistant. It −1 water was used at a flow rate of 1  ml  min   to separate originates from the experimental hut station in Cove the target analyte. Chromatographic peaks of the insecti- and has shown > 200-fold resistance compared to the cides and internal standard were detected at a wavelength susceptible Kisumu strain in susceptibility bioassays. of 232 nm with the Ultimate 3000 UV detector and ana- Resistance is mediated by elevated levels of P450s lysed with Dionex Chromeleon 6.8 Chromatography and high frequencies of kdr [20]. Data System software. Quantities of insecticide were 2. An. gambiae VKPer strain, which originated from calculated from standard curves established by known the Kou Valley in Burkina Faso. VKPer has moderate concentrations of the insecticide authenticated standards levels of pyrethroid resistance mediated only by high and corrected by internal standard readings in each sam- frequencies of kdr. ple relative to control. 3. An. gambiae s.s. Kisumu strain, a reference suscepti- Data from chemical analysis was used to calculate the ble strain which originated from Kisumu Kenya. percentage retention of each active ingredient after 20 washes relative to the unwashed net and the wash reten- Approximately two hundred 2–5  days old mosquitoes tion index. Wash-resistance index was calculated accord- of each strain were exposed for 3  min in cone bioassays ing to WHO guidelines [22] as indicated below: to four net samples of each net type in cohorts of 5 mos- quitoes per cone. Knock down in cone bioassays was Wash resistance index = 100 × n (tn/t0) recorded after 1 h and mortality after 24 h. free migration stage behaviour Two to three hundred 5–8  days old mosquitoes of each strain were also exposed to each net type in tun- where tn = total active ingredient content after n washing nel tests in replicates of 50 mosquitoes per net sample. cycles, t0 = total active ingredient content before wash- The tunnel test is a laboratory assay designed to simulate ing, n = number of washes. Nguf or et al. Malaria Journal (2022) 21:20 Page 5 of 13 Data analysis nurse; any sleepers testing positive for malaria were with- Proportional outcomes (blood-feeding, exiting and drawn from the study and treated properly. mortality) related to each experimental hut treatment (unwashed and washed 20 times) were assessed using Results binomial generalized linear mixed models (GLMMs) Insecticide resistance in malaria vectors in Cove with a logit link function, fitted using the ‘lme4’ package Mortality with permethrin and alpha-cypermethrin for R (version 3.5.3). A separate model was fitted for each treated papers was 100% with the laboratory-maintained outcome. In addition to the fixed effect of each treat - pyrethroid-susceptible An. gambiae Kisumu strain. With ment, each model included random effects to account wild pyrethroid resistant An. gambiae s.l. from Cove, for the following sources of variation: between the huts; mortality rates were < 50% with all three pyrethroid insec- between the sleepers; between the weeks of the trial; and ticides tested (Table 1) thus confirming the high levels of finally, an observation-level random effect to account for pyrethroid resistance in this vector population. Mortality variation not explained by the other terms in the model however increased from 19.2 to 52.5% with permethrin, (over dispersion). 41.7% to 69.7% with deltamethrin and 0% to 82.4% with alphacypermethrin after pre-exposure of the Cove strain Ethical considerations to PBO synergist (Table 1). This result demonstrated that This study received ethical approval from the Ministry of mixed function oxidases are overexpressed in the wild Health in Benin and from the Ethics Review Committee Cove vector population and their effect can be effectively of the London School of Hygiene & Tropical Medicine. inhibited by the PBO synergist. Informed consent was obtained from each human volun- teer sleeper who slept in the huts to attract mosquitoes Experimental hut trial results prior to their participation. Sleepers were also offered Mosquito entry and exiting rates in experimental huts chemoprophylaxis. Through the course of the study, they A total of 6711 pyrethroid resistant female An. gambiae were examined regularly for signs of fever by a stand-by s.l. were collected during the experimental hut trial. The entry and exiting rates of wild pyrethroid resistant An. gambiae s.l. from the experimental huts with the differ - Table 1 Mortality of pyrethroid resistant Anopheles gambiae ent ITN types tested in the trial are presented in Table 2. s.l. from Cove in WHO cylinder bioassays with and without pre- Compared to the control, Olyset Net did not deter mos- exposure to PBO quitoes from entering the experimental huts (0% when unwashed and after 20 washes). Mosquito deterrence N exposed N dead % Dead was significantly higher with PermaNet 3.0 compared Control 101 0 0.0 to Olyset Plus both unwashed (49% vs. 28%, P < 0.001) PBO 4% only 103 3 2.9 and after 20 washes (36% vs. 0%, P < 0.001). Neverthe- Permethrin 0.75% 104 20 19.2 less, early exiting of mosquitoes from the experimen- Deltamethrin 0.05% 91 38 41.7 tal huts into the veranda trap did not differ significantly Alpha-cypermethrin 0.05% 102 0 0.0 between both pyrethroid-PBO net types both when PBO 4% then permethrin 0.75% 99 52 52.5 unwashed (65% with PermaNet 3.0 vs 68% with Olyset PBO 4% then deltamethrin 0.05% 99 69 69.7 Plus, P = 0.232) and after 20 washes (60% with PermaNet PBO 4% then alpha-cypermethrin 102 84 82.4 3.0 vs. 56% with Olyset Plus, P = 0.053). 0.05% Table 2 Entry and exiting rates of wild pyrethroid resistant Anopheles gambiae s.l. in experimental huts in Cove, Benin Net type Control Olyset Net PermaNet 3.0 Olyset Plus Number of washes 0 0 20 0 20 0 20 N collected 962 1135 1546 489 616 688 1275 a ab b c d d ab N females/night 23 27 37 12 15 16 30 % deterrence – 0* 0* 49 36 28 0 N exiting 429 639 669 319 370 468 712 a b a cd ce d be % exiting 45 56 43 65 60 68 56 95% conf. limits 41.5–47.7 53.4–59.2 40.8–45.7 61.0–69.5 56.2–63.9 64.4–71.4 53.1–58.6 Values along a row bearing the same letter label are not significantly different (P > 0.05) Value set to zero as more mosquitoes caught in Olyset Net huts than control huts Ngufor et al. Malaria Journal (2022) 21:20 Page 6 of 13 Mortality of wild pyrethroid‑resistant Anopheles gambiae s.l. vs. 12%). After 20 washes, mortality with PermaNet 3.0 in experimental huts declined to the same level as the unwashed Olyset Net, Mortality rates of wild pyrethroid resistant mosqui- (17% vs. 18%, P = 0.061) but remained significantly higher toes that entered the experimental huts are presented in with Olyset Plus (24% vs. 17%, P = 0.036). Fig.  1 with further details provided in Table  3. The low - est mortality was achieved with Olyset Net (18% before Mosquito blood‑feeding rates in experimental huts washing and 12% after 20 washes). Percentage mortality The blood-feeding rates of wild pyrethroid-resistant An. with unwashed pyrethroid-PBO nets was highest with gambiae s.l that entered the experimental huts are pre- PermaNet 3.0 (41%) but this declined significantly after sented in Fig. 2 with further details on mosquito feeding 20 washes (17%, P < 0.001). Mortality with unwashed provided in Table  4. The percentage blood-feeding was Olyset Plus was 28% and while this value was significantly lower in huts with the unwashed pyrethroid-PBO ITNs lower than the mortality shown by unwashed PermaNet and was lowest of all with unwashed Olyset Plus as com- 3.0 (P < 0.001), it did not decrease significantly after 20 pared to unwashed PermaNet 3.0 (8% vs 19%, P < 0.001). washes (28% vs. 24%, P = 0.433) whereas for PermaNet For all net types, the data showed an overall increase in 3.0 it declined. Hence, Olyset Plus induced higher mor- blood feeding with washed nets compared to unwashed tality rates than PermaNet 3.0 after 20 washes (24% vs nets and a decrease in blood-feeding inhibition relative 17%, P < 0.001). With respect to the pyrethroid-only ITN, to the untreated net. Percentage blood-feeding when both pyrethroid-PBO nets induced significantly higher washed 20 times did not differ significantly between the mortality rates than Olyset Net with nets washed 20 pyrethroid-PBO types (38% with PermaNet 3.0 vs. 35% times (P < 0.001) though the difference was higher with with Olyset Plus, P = 0.708). The proportions of mosqui - Olyset Plus (24% vs. 12%), than with PermaNet 3.0 (17% toes collected resting in the nets were lowest with the Unwashed Washed 20X 30 e 20 b Control (untreated net) Olyset NetPermaNet 3.0Olyset Plus Fig. 1 Mortality of wild pyrethroid-resistant Anopheles gambiae s.l. in experimental huts in Cove, Benin evaluating different net types. Vertical lines indicate 95% confidence interval estimates whilst bars with the same letter label are not significantly different (P > 0.05) Table 3 Mortality of wild pyrethroid resistant Anopheles gambiae s.l. in experimental huts in Cove, Benin Net type Control Olyset Net PermaNet 3.0 Olyset Plus Number of washes 0 0 20 0 20 0 20 Total collected 962 1135 1546 489 616 688 1275 Total dead 12 209 284 198 103 190 297 a b c d b e e Mortality (%) 1 18 12 41 17 28 24 95% conf. limits 0.6–2.0 16.2–20.1 10.3–13.5 36.1–44.8 13.8–19.8 24.4–31.1 21.0–25.6 Corrected for control (%) – 17 11 41 17 27 22 Values along a row bearing the same letter label are not significantly different (P > 0.05) % Mortality Nguf or et al. Malaria Journal (2022) 21:20 Page 7 of 13 Unwashed Washed 20X Control (untreated net) Olyset NetPermaNet 3.0Olyset Plus Fig. 2 Blood-feeding rates of wild pyrethroid-resistant Anopheles gambiae s.l. in experimental huts in Cove, Benin evaluating different net types. Vertical lines indicate 95% confidence interval estimates whilst bars with the same letter label are not significantly different (P > 0.05) Table 4 Blood-feeding rates of wild pyrethroid resistant Anopheles gambiae s.l. in experimental huts in Cove, Benin Net type Control Olyset Net PermaNet 3.0 Olyset Plus Number of washes 0 0 20 0 20 0 20 Total collected 962 1135 1546 489 616 688 1275 Total blood fed 496 324 807 95 231 56 442 a b a c d e d % Blood fed 52 29 52 19 38 8 35 95% conf intervals 48.4–54.7 25.9–31.2 49.7–54.7 15.9–22.9 33.7–41.3 6.1–10.2 32.1–37.3 % Blood-feeding inhibition – 44 0 63 27 85 33 inside net 294 184 542 28 130 17 241 a bc a d b e c inside net (%) 31 16 35 6 21 2 19 Personal protection (%) – 35 0* 81 53 89 11 Values along a row bearing the same letter label are not significantly different (P > 0.05) Value set to zero as more blood fed mosquitoes were caught in washed Olyset Net huts than control hut unwashed pyrethroid-PBO nets (2–6%), which is con- the difference in mosquito mortality between the candi - sistent with the higher toxicity observed with this type of date and active comparator product is greater than 0.7. net. Personal protection with both types of pyrethroid- and (2) The upper 95% confidence interval estimate of the PBO ITNs were > 80% when unwashed but this declined odds ratio describing the difference in mosquito blood- after 20 washes to 53% with PermaNet 3.0 and 11% with feeding between the candidate and active comparator Olyset Plus. product is less than 1.43. Following the WHO guidelines, both unwashed and washed data of each product were Non‑inferiority assessment analysed together to generate single estimates of efficacy According to recent provisional WHO guidelines [19], representative of the overall performance over the life- for a candidate pyrethroid-PBO ITN product to be time of the product in the field. included in this new WHO intervention class without the Each primary endpoint for non-inferiority (mortal- need for epidemiological evidence, it must demonstrate ity and blood-feeding rate for unwashed and washed non-inferiority to the first in class product which has nets combined), was assessed using binomial general- already demonstrated public health value (Olyset Plus) ized linear mixed models (GLMMs) with a logit link and superiority to a pyrethroid-only LLIN in experimen- function fitted using the ‘lme4’ package of R version tal hut trials [19]. Briefly, the candidate pyrethroid-PBO 3.5.3 for Windows as described earlier. Results from the product is deemed non-inferior if: (1) The lower 95% non-inferiority assessment of PermaNet 3.0 to Olyset confidence interval estimate of the odds ratio describing Plus are presented in Table  5 below. The odds ratio for % Blood-fed Ngufor et al. Malaria Journal (2022) 21:20 Page 8 of 13 Table 5 Results from the non-inferiority assessment of PermaNet 3.0 to Olyset Plus against wild pyrethroid-resistant Anopheles gambiae s.l. in experimental huts in Cove, Benin Control Net Olyset Net Olyset Plus PermaNet 3.0 Total collected 962 2681 1963 1105 Mortality Total dead 12 393 488 301 Mortality (%) 1 15 25 27 Odds ratio – – – 1.528 Std. err (on log odds scale) – – – 0.206 95% conf. interval – – – 1.021–2.289 WHO non-inferiority margin – – – Lower 95%CI > 0.7 Conclusion – – – Non-inferior Blood-feeding rate Total blood-fed 496 1131 498 326 Blood-feeding (%) 52 42 25 30 Blood-feeding inhibition – 19 52 42 Odds ratio – – – 1.192 Std. err (on log odds scale) – – – 0.222 95% conf. interval – – – 0.772–1.841 WHO non-inferiority margin – – – Upper 95%CI < 1.43 Conclusion – – – Not non-inferior Combined data for washed and unwashed nets of each net type the difference in mosquito mortality between PermaNet bioassay mortality however dropped significantly with 3.0 and Olyset Plus was 1.528 (95% confidence interval: washed net samples. Mortality with the untreated net 1.021–2.289) while the odds ratio for the difference in samples did not exceed 5% with any strain tested. mosquito blood feeding was 1.192 (95% confidence inter - val: 0.772–1.841). Following the WHO criteria described Tunnel test results above, PermaNet 3.0 was non-inferior to Olyset Plus in The results from the tunnel tests comparing Olyset Plus terms of its ability to kill wild pyrethroid- resistant An. and Olyset Net unwashed and after 10 and 20 washes gambiae s.l. in the experimental hut trial in Cove Benin. against all three strains are presented in Figs.  4 and 5 In contrast PermaNet 3.0 was not non-inferior to Olyset for mortality and blood-feeding inhibition respectively. Plus in terms of proportions of mosquitoes that blood- Mortality rates were generally higher in the tunnels fed and, therefore, fails to demonstrate non-inferiority tests compared to the cone bioassays and decreased as for blood feeding inhibition in this trial. The results also the strain become more pyrethroid-resistant (Fig.  4). showed superiority of both pyrethroid-PBO net types to Mortality rates with the Kisumu strain were very high Olyset Net both in terms of mosquito mortality (25–27% with both Olyset Net and Olyset Plus (> 95%). With the vs. 15%, P < 0.001) and reducing blood-feeding (25%-30% VKPer strain, mortality remained > 80% after 20 washes vs. 42%, P < 0.001). with both ITN types. With the Cove strain, mortality was significantly higher with Olyset Plus compared to Supplementary laboratory bioassays results Olyset Net at 0 and 10 washes but about the same after Cone bioassay results 20 washes. With unwashed nets, blood-feeding inhi- The 3-min cone bioassay mortality results for all 3 mos - bition in the tunnel tests was consistently higher with quito strains and wash points tested are presented in Olyset Plus (> 90%) compared to Olyset Net (27–75%) for Fig.  3. Unwashed Olyset Net induced very low mortality all three strains tested (Fig.  5). After 10 and 20 washes, rates against the susceptible Kisumu strain (17–20%) and blood-feeding inhibition of the Kisumu and VKPer strain even lower mortality rates against the pyrethroid resist- remained > 80% with Olyset Plus and Olyset Net. With ant strains (< 5%). Cone bioassay mortality rates with Cove strain, blood-feeding inhibition was also higher unwashed Olyset Plus were higher across all 3 strains with Olyset Plus at 0 and 10 washes but declined to compared to Olyset Net though mortality decreased as about the same level as Olyset Net after 20 washes (56%). the strain tested became more pyrethroid-resistant. Cone Nguf or et al. Malaria Journal (2022) 21:20 Page 9 of 13 0 wash 10 washes 20 washes Olyset NetOlyset Plus Olyset NetOlyset Plus Olyset NetOlyset Plus Kisumu - SVKPer - RCove - R Fig. 3 Mortality (%) of susceptible and resistant strains of An. gambiae s.l. in cone bioassays with Olyset Plus and Olyset Net. S = susceptible, R = resistant 0 wash 10 washes 20 washes Olyset NetOlyset Plus Olyset NetOlyset Plus Olyset NetOlyset Plus Kisumu - SVKPer - RCove - R Fig. 4 Tunnel test mortality (%) of susceptible and resistant strains of Anopheles gambiae s.l. exposed to Olyset Plus vs. Olyset Net Mortality in the untreated control tunnel was < 10% with in both types of pyrethroid-PBO ITNs after 20 washes all three strains. (58.7% vs. 25% with Olyset Plus and 80.9% vs. 68.8% with PermaNet 3.0, P < 0.05). The decrease in PBO content after washing was more evident in Olyset Plus Chemical analysis results than in PermaNet 3.0 netting, hence the wash retention For PermaNet 3.0, PBO was only recorded from index of PBO was higher with PermaNet 3.0 compared the roof panel of the nets; for Olyset Plus, PBO was to Olyset Plus (98.1% vs. 93.3%). recorded on all 5 panels (Table  6). Compared to the pyrethroid component, much less PBO was  retained % Mortality % Mortality Ngufor et al. Malaria Journal (2022) 21:20 Page 10 of 13 0 wash 10 washes 20 washes Olyset NetOlyset Plus Olyset NetOlyset Plus Olyset NetOlyset Plus Kisumu - SVKPer - RCove - R Fig. 5 Blood-feeding inhibition (%) of susceptible and resistant strains of An. gambiae s.l. in tunnel tests with Olyset Plus and Olyset Net. Blood-feeding inhibition was calculated relative to the control tunnel. S = susceptible, R = Resistant Table 6 Chemical analysis of net samples after experimental hut trial at Cove, Benin LN type Active ingredient AI content (g/kg) Retention, % Wash retention Unwashed Washed 20× index (%) Olyset Net Permethrin 12.0 12.0 100.0 100.0 Olyset Plus Permethrin 15.5 9.1 58.7 97.4 PBO 8.0 2.0 25.0 93.3 PermaNet 3.0 Deltamethrin (roof of net) 4.2 3.4 80.9 98.9 PBO (roof of net) 16.0 11.0 68.8 98.1 by increased mono-oxygenase activity. The experimen - Discussion tal hut trial demonstrated improved levels of mortality Following the interim endorsement of pyrethroid-PBO and blood-feeding inhibition with both pyrethroid-PBO nets by the WHO [23], malaria vector control pro- ITN types compared to a standard pyrethroid-only grammes are faced with additional choice of different LLIN against a vector population that was very resistant brands of prequalified pyrethroid-PBO nets [7]. Consid - to pyrethroids; this was supported by results from the ering the wide variations in design of the available brands laboratory assays which compared Olyset Plus to Olyset of these nets, studies generating the required assurance Net against pyrethroid-resistant mosquito strains. These of comparative performance to Olyset Plus (the first in observations are partly attributable to the synergistic class pyrethroid-PBO net to demonstrate empirical evi- effect of the PBO on pyrethroid resistance and are con - dence of entomological and epidemiological impact) are sistent with other experimental hut trials across Africa necessary. This study compared the efficacy and assessed and epidemiological trials performed in Tanzania [8] and the non-inferiority of PermaNet 3.0 to Olyset Plus in Uganda [18]. experimental huts against pyrethroid resistant malaria Twenty washes in experimental hut studies are indi- vectors in a highly endemic country of West Africa, fol- cated by WHO as a proxy for the ability of an ITN to lowing WHO guidelines [19, 22]. withstand multiple washes under operational use over The WHO susceptibility bioassays confirmed the high a 3-year life span [19, 22]. With unwashed nets, the lev- levels of pyrethroid resistance in the vector population at els of improved mortality relative to the standard pyre- the experimental hut site during the trial, corroborating throid-only net were higher with PermaNet 3.0 than previous findings [20]. The increased mortality achieved with Olyset Plus. However, unlike with Olyset Plus, this in bioassays with pre-exposure to PBO showed that effect was lost with PermaNet 3.0 after twenty washes; pyrethroid-resistance was indeed at least partly mediated % Blood-feeding Inhibition Nguf or et al. Malaria Journal (2022) 21:20 Page 11 of 13 PermaNet 3.0 killed significantly lower proportions of to the upper panel and pyrethroid to side panels versus mosquitoes than Olyset Plus and same proportions as an ITN with all 5 panels treated with pyrethroid-PBO. an unwashed pyrethroid-only ITN. Though the RCT in Behavioural studies of mosquitoes around nets indicate Uganda was not powered to assess differences between that mosquitoes may first make multiple contacts with the pyrethroid-PBO ITN brands tested (PermaNet 3.0 vs. the roof panel in response to odour plumes [26]; how- Olyset Plus) [18], the results from our trial appear con- ever, experimental hut trials comparing restricted versus sistent with some of the differences in epidemiological full PBO coverage on nets are too few to be definitive on effect observed between both brands: (1) The high exper - the question of efficacy. imental hut mortality with the unwashed PermaNet 3.0 Despite the differences in performance observed supports the higher initial protective effect observed with between both pyrethroid-PBO net types with regards the net in the Ugandan trial at the 6 months epidemiolog- to their impact after 20 standardized washes, the non- ical survey which was not seen with Olyset Plus. (2) The inferiority analysis performed in accordance with recent higher rate of decline in experimental hut mortality after WHO guidelines [19] showed that PermaNet 3.0 was washing with PermaNet 3.0 compared to Olyset Plus is non-inferior to Olyset Plus in terms of mosquito mor- consistent with the shorter-lived epidemiological effect in tality but not with blood-feeding inhibition. The higher the Ugandan trial with PermaNet 3.0 (up to 12  months) blood-feeding inhibition observed with Olyset Plus could compared to Olyset Plus which remained more protec- be due to the high excito-repellency of permethrin in tive than the pyrethroid-only net at 18 months. However, Olyset Plus compared to deltamethrin in PermaNet 3.0 care should be taken to not over-interpret the compari- [27]. Alternatively, the study may not have had sufficient sons between results from our hut trial and Ugandan power to demonstrate non-inferiority of PermaNet 3.0 RCT considering the different geographical settings and to Olyset Plus for both endpoints; further studies are the lack of sufficient power to differentiate between the on-going to help guide power calculations for ITN non- epidemiological impact of both pyrethroid-PBO net type inferiority studies. The non-inferiority margin used for in the Ugandan trial. the analysis was defined by WHO as an odds ratio of 0.7 The difference in hut performance between both pyre - in mosquito mortality and feeding between a candidate throid-PBO ITNs can be attributed to differences in the net and the first in class net considered acceptable for retention and movement of PBO across the polymer fibre both products to be in the same policy class. According in Olyset Plus compared to PermaNet 3.0 and/or dif- to these guidelines, if non-inferiority is demonstrated ferences in design and specification. Retention of bioef - in two independent experimental hut trials in different ficacy of pyrethroid-PBO nets is a fine balance between geographical locations representative of where the prod- migration and replenishment of PBO from the core to ucts will be deployed, then PermaNet 3.0 will be placed the surface of fibres and the maintenance of an internal in the same WHO vector control product class as Olyset reservoir sufficient to last the lifespan of the LLIN, which Plus [19]. It is however not clear whether non-inferiority is typically set at 3 years of use [24]. The chemical analy - must be demonstrated for both endpoints (mortality and sis results showed a faster release of PBO in the Olyset blood-feeding) for a second-in class product to become Plus netting after 20 washes compared to PermaNet 3.0 part of an intervention class. While the guidelines were though it is unclear whether this may have increased the developed more like a compromise between the risk of bioavailability of the PBO on the surface of Olyset Plus accepting an inferior product and the feasibility of con- after washing. Whether sufficient PBO would remain ducting epidemiological trials, the findings from our within the fibres of both pyrethroid-PBO nets after trial show that non-inferiority experimental hut trials 3 years of household use is presently unknown and is the are complex, and results must be interpreted with care. subject of ongoing WHO durability trials of Olyset Plus Comparative performance between products may also and PermaNet 3.0 which are not yet completed [18, 25]. depend on other location-specific factors, such as the Another factor which may contribute to the discrepan- intensity of insecticide resistance and behaviour of the cies in efficacy of the two pyrethroid-PBO ITNs are dif - target vector population which should be taken into con- ferences in design and specification: Olyset Plus is treated sideration when choosing between products of the same with the pyrethroid permethrin while PermaNet 3.0 is class. treated with deltamethrin, and Olyset Plus contains PBO While the present hut trial in Benin and earlier hut tri- on every panel whereas in PermaNet 3.0, PBO is available als in Benin, Tanzania, Cameroon, Burkina Faso, Côte only on the top panel of the net. It is not clear whether d’Ivoire and Vietnam where the vectors were also resist- the restricted application of PBO to the roof of the net ant have shown some additional effect of pyrethroid- would affect bioefficacy. This would require comparative PBO nets over a standard pyrethroid net [12, 13, 15–17], trials of the PermaNet 3.0 with pyrethroid-PBO restricted the margin appears to vary depending on the level of Ngufor et al. Malaria Journal (2022) 21:20 Page 12 of 13 Authors’ contributions pyrethroid-resistance encountered [28]. The absolute CN designed and supervised the study, analysed the data and drafted the increase in hut trial mortality with Olyset Plus compared manuscript. JF and AA performed the hut trial and laboratory bioassays. to Olyset Net in the present study (24–28% vs. 12–18%) JDC and TSC performed the non-inferiority analysis and contributed to data interpretation and manuscript revision. HI performed the chemical analysis. conducted in an area of intense pyrethroid-resistance MR contributed to study design, data interpretation and manuscript revision. [20] is lower than what has been previously reported with All authors read and approved the final manuscript. Olyset Plus in another area in Northern Benin where Funding pyrethroid resistance was less prevalent (67–81% vs. This project was funded by the LSHTM ITD Athena Swan Career restart fellow- 36–42%) [16]. Compared to East Africa, West Africa has ship and an independent research grant from Sumitomo Chemical Company shown historically higher intensity of pyrethroid resist- awarded to Corine Ngufor. Funding covered research costs and operational expenses. The funders had no role in study design, data collection and analy- ance in malaria vectors [29] mediated by complex and sis, decision to publish, or preparation of the manuscript. multiple insecticide resistance mechanisms which may not be effectively tackled by the synergistic effects of Availability of data and materials The datasets used and/or analysed during the current study are available from PBO in pyrethroid-PBO ITNs [4, 23]. It is therefore not the corresponding author on reasonable request. clear whether a diminishment in experimental hut mor- tality with pyrethroid-PBO nets due to increasing inten- Declarations sity of pyrethroid-resistance would translate to a reduced epidemiological effect of pyrethroid-PBO ITNs in West Ethics approval and consent to participate The study was approved by the Ethics Review Committee of LSHTM and the Africa compared to East Africa which has been the site of Ministry of Health, Benin. Informed consent was obtained from each human the only epidemiological trials so far. volunteer sleeper who slept in the huts to attract mosquitoes, prior to their participation. Sleepers were also offered chemoprophylaxis. Through the course of the study, they were examined regularly for signs of fever; any sleep- ers testing positive for malaria were withdrawn from the study and treated in Conclusion accordance with country guidelines. Olyset Plus outperformed PermaNet 3.0 in terms of its Consent for publication ability to induce improved levels of mosquito mortality Not applicable. compared to a standard pyrethroid LLIN after multiple standardized washes. Nevertheless, a non-inferiority Competing interests The authors declare that they have no competing interests. analysis of both ITN types following recent WHO guide- lines showed that they were comparable in their ability Author details to kill mosquitoes. Compared to the situation existing London School of Hygiene and Tropical Medicine (LSHTM), London, UK. Centre de Recherches Entomologiques de Cotonou (CREC), Cotonou, Benin. in Benin several years ago, both pyrethroid-PBO ITNs Pan African Malaria Vector Research Consortium (PAMVERC), Cotonou, Benin. showed less impact against the mosquito vector due to 4 5 Liverpool School of Tropical Medicine, Liverpool L3 5QA, UK. MRC Centre increased levels of resistance. West Africa constitutes for Global Infectious Disease Analysis, Infectious Disease Epidemiology, Impe- rial College London, Norfolk Place, London W2 1PG, UK. a different environment and ecology from East Africa, with historically higher intensity of pyrethroid resistance Received: 20 May 2021 Accepted: 3 January 2022 in malaria vectors. It is not clear whether either of these nets would have the same epidemiological impact against malaria demonstrated with pyrethroid-PBO nets in trials in East Africa. 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Journal

Malaria Journal – Springer Journals

Published: Jan 11, 2022

Keywords: Experimental huts; Piperonyl butoxide; PBO; Olyset plus; PermaNet 3.0; Olyset; Mixture; LLIN; Insecticide resistance; Pyrethroid-PBO; Cove; Benin; Pyrethroid resistance; Insecticide-treated nets; Long-lasting insecticidal nets; Next generation nets; Anopheles; Cove Benin

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