key: cord-0017654-dx2o1b6f authors: Gleave, Katherine; Lissenden, Natalie; Chaplin, Marty; Choi, Leslie; Ranson, Hilary title: Piperonyl butoxide (PBO) combined with pyrethroids in insecticide‐treated nets to prevent malaria in Africa date: 2021-05-24 journal: Cochrane Database Syst Rev DOI: 10.1002/14651858.cd012776.pub3 sha: 01299b6aed07833b957fc27f41b6129afe7b121a doc_id: 17654 cord_uid: dx2o1b6f BACKGROUND: Pyrethroid long‐lasting insecticidal nets (LLINs) have been important in the large reductions in malaria cases in Africa, but insecticide resistance in Anopheles mosquitoes threatens their impact. Insecticide synergists may help control insecticide‐resistant populations. Piperonyl butoxide (PBO) is such a synergist; it has been incorporated into pyrethroid‐LLINs to form pyrethroid‐PBO nets, which are currently produced by five LLIN manufacturers and, following a recommendation from the World Health Organization (WHO) in 2017, are being included in distribution campaigns. This review examines epidemiological and entomological evidence on the addition of PBO to pyrethroid nets on their efficacy. OBJECTIVES: To compare effects of pyrethroid‐PBO nets currently in commercial development or on the market with effects of their non‐PBO equivalent in relation to: 1. malaria parasite infection (prevalence or incidence); and 2. entomological outcomes. SEARCH METHODS: We searched the Cochrane Infectious Diseases Group (CIDG) Specialized Register, CENTRAL, MEDLINE, Embase, Web of Science, CAB Abstracts, and two clinical trial registers (ClinicalTrials.gov and WHO International Clinical Trials Registry Platform) up to 25 September 2020. We contacted organizations for unpublished data. We checked the reference lists of trials identified by these methods. SELECTION CRITERIA: We included experimental hut trials, village trials, and randomized controlled trials (RCTs) with mosquitoes from the Anopheles gambiae complex or the Anopheles funestus group. DATA COLLECTION AND ANALYSIS: Two review authors assessed each trial for eligibility, extracted data, and determined the risk of bias for included trials. We resolved disagreements through discussion with a third review author. We analysed data using Review Manager 5 and assessed the certainty of evidence using the GRADE approach. MAIN RESULTS: Sixteen trials met the inclusion criteria: 10 experimental hut trials, four village trials, and two cluster‐RCTs (cRCTs). Three trials are awaiting classification, and four trials are ongoing. Two cRCTs examined the effects of pyrethroid‐PBO nets on parasite prevalence in people living in areas with highly pyrethroid‐resistant mosquitoes (< 30% mosquito mortality in discriminating dose assays). At 21 to 25 months post intervention, parasite prevalence was lower in the intervention arm (odds ratio (OR) 0.79, 95% confidence interval (CI) 0.67 to 0.95; 2 trials, 2 comparisons; moderate‐certainty evidence). In highly pyrethroid‐resistant areas, unwashed pyrethroid‐PBO nets led to higher mosquito mortality compared to unwashed standard‐LLINs (risk ratio (RR) 1.84, 95% CI 1.60 to 2.11; 14,620 mosquitoes, 5 trials, 9 comparisons; high‐certainty evidence) and lower blood feeding success (RR 0.60, 95% CI 0.50 to 0.71; 14,000 mosquitoes, 4 trials, 8 comparisons; high‐certainty evidence). However, in comparisons of washed pyrethroid‐PBO nets to washed LLINs, we do not know if PBO nets had a greater effect on mosquito mortality (RR 1.20, 95% CI 0.88 to 1.63; 10,268 mosquitoes, 4 trials, 5 comparisons; very low‐certainty evidence), although the washed pyrethroid‐PBO nets did decrease blood‐feeding success compared to standard‐LLINs (RR 0.81, 95% CI 0.72 to 0.92; 9674 mosquitoes, 3 trials, 4 comparisons; high‐certainty evidence). In areas where pyrethroid resistance is moderate (31% to 60% mosquito mortality), mosquito mortality was higher with unwashed pyrethroid‐PBO nets compared to unwashed standard‐LLINs (RR 1.68, 95% CI 1.33 to 2.11; 751 mosquitoes, 2 trials, 3 comparisons; moderate‐certainty evidence), but there was little to no difference in effects on blood‐feeding success (RR 0.90, 95% CI 0.72 to 1.11; 652 mosquitoes, 2 trials, 3 comparisons; moderate‐certainty evidence). For washed pyrethroid‐PBO nets compared to washed standard‐LLINs, we found little to no evidence for higher mosquito mortality or reduced blood feeding (mortality: RR 1.07, 95% CI 0.74 to 1.54; 329 mosquitoes, 1 trial, 1 comparison, low‐certainty evidence; blood feeding success: RR 0.91, 95% CI 0.74 to 1.13; 329 mosquitoes, 1 trial, 1 comparison; low‐certainty evidence). In areas where pyrethroid resistance is low (61% to 90% mosquito mortality), studies reported little to no difference in the effects of unwashed pyrethroid‐PBO nets compared to unwashed standard‐LLINs on mosquito mortality (RR 1.25, 95% CI 0.99 to 1.57; 948 mosquitoes, 2 trials, 3 comparisons; moderate‐certainty evidence), and we do not know if there was any effect on blood‐feeding success (RR 0.75, 95% CI 0.27 to 2.11; 948 mosquitoes, 2 trials, 3 comparisons; very low‐certainty evidence). For washed pyrethroid‐PBO nets compared to washed standard‐LLINs, we do not know if there was any difference in mosquito mortality (RR 1.39, 95% CI 0.95 to 2.04; 1022 mosquitoes, 2 trials, 3 comparisons; very low‐certainty evidence) or on blood feeding (RR 1.07, 95% CI 0.49 to 2.33; 1022 mosquitoes, 2 trials, 3 comparisons; low‐certainty evidence). In areas where mosquito populations are susceptible to insecticides (> 90% mosquito mortality), there may be little to no difference in the effects of unwashed pyrethroid‐PBO nets compared to unwashed standard‐LLINs on mosquito mortality (RR 1.20, 95% CI 0.64 to 2.26; 2791 mosquitoes, 2 trials, 2 comparisons; low‐certainty evidence). This is similar for washed nets (RR 1.07, 95% CI 0.92 to 1.25; 2644 mosquitoes, 2 trials, 2 comparisons; low‐certainty evidence). We do not know if unwashed pyrethroid‐PBO nets had any effect on the blood‐feeding success of susceptible mosquitoes (RR 0.52, 95% CI 0.12 to 2.22; 2791 mosquitoes, 2 trials, 2 comparisons; very low‐certainty evidence). The same applies to washed nets (RR 1.25, 95% CI 0.82 to 1.91; 2644 mosquitoes, 2 trials, 2 comparisons; low‐certainty evidence). In village trials comparing pyrethroid‐PBO nets to LLINs, there was no difference in sporozoite rate (4 trials, 5 comparisons) nor in mosquito parity (3 trials, 4 comparisons). AUTHORS' CONCLUSIONS: In areas of high insecticide resistance, pyrethroid‐PBO nets have greater entomological and epidemiological efficacy compared to standard LLINs, with sustained reduction in parasite prevalence, higher mosquito mortality and reduction in mosquito blood feeding rates 21 to 25 months post intervention. Questions remain about the durability of PBO on nets, as the impact of pyrethroid‐PBO nets on mosquito mortality was not sustained over 20 washes in experimental hut trials, and epidemiological data on pyrethroid‐PBO nets for the full intended three‐year life span of the nets is not available. Little evidence is available to support greater entomological efficacy of pyrethroid‐PBO nets in areas where mosquitoes show lower levels of resistance to pyrethroids. Two cRCTs examined the e ects of pyrethroid-PBO nets on parasite prevalence in people living in areas with highly pyrethroid-resistant mosquitoes (< 30% mosquito mortality in discriminating dose assays). At 21 to 25 months post intervention, parasite prevalence was lower in the intervention arm (odds ratio (OR) 0.79, 95% confidence interval (CI) 0.67 to 0.95; 2 trials, 2 comparisons; moderate-certainty evidence). In highly pyrethroid-resistant areas, unwashed pyrethroid-PBO nets led to higher mosquito mortality compared to unwashed standard-LLINs (risk ratio (RR) 1.84, 95% CI 1.60 to 2.11; 14,620 mosquitoes, 5 trials, 9 comparisons; high-certainty evidence) and lower blood feeding success (RR 0.60, 95% CI 0.50 to 0.71; 14,000 mosquitoes, 4 trials, 8 comparisons; high-certainty evidence). However, in comparisons of washed pyrethroid-PBO nets to washed LLINs, we do not know if PBO nets had a greater e ect on mosquito mortality (RR 1.20, 95% CI 0.88 to 1.63; 10,268 mosquitoes, 4 trials, 5 comparisons; very low-certainty evidence), although the washed pyrethroid-PBO nets did decrease blood-feeding success compared to standard-LLINs (RR 0.81, 95% CI 0.72 to 0.92; 9674 mosquitoes, 3 trials, 4 comparisons; high-certainty evidence). In areas where pyrethroid resistance is moderate (31% to 60% mosquito mortality), mosquito mortality was higher with unwashed pyrethroid-PBO nets compared to unwashed standard-LLINs (RR 1.68, 95% CI 1.33 to 2.11; 751 mosquitoes, 2 trials, 3 comparisons; moderate-certainty evidence), but there was little to no di erence in e ects on blood-feeding success (RR 0.90, 95% CI 0.72 to 1.11; 652 mosquitoes, 2 trials, 3 comparisons; moderate-certainty evidence). For washed pyrethroid-PBO nets compared to washed standard-LLINs, we found little to no evidence for higher mosquito mortality or reduced blood feeding (mortality: RR 1.07, 95% CI 0.74 to 1.54; 329 mosquitoes, 1 trial, 1 comparison, low-certainty evidence; blood feeding success: RR 0.91, 95% CI 0.74 to 1.13; 329 mosquitoes, 1 trial, 1 comparison; low-certainty evidence). In areas where pyrethroid resistance is low (61% to 90% mosquito mortality), studies reported little to no di erence in the e ects of unwashed pyrethroid-PBO nets compared to unwashed standard-LLINs on mosquito mortality (RR 1.25, 95% CI 0.99 to 1.57; 948 mosquitoes, 2 trials, 3 comparisons; moderate-certainty evidence), and we do not know if there was any e ect on blood-feeding success (RR 0.75, 95% CI 0.27 to 2.11; 948 mosquitoes, 2 trials, 3 comparisons; very low-certainty evidence). For washed pyrethroid-PBO nets compared to washed standard-LLINs, we do not know if there was any di erence in mosquito mortality (RR 1.39, 95% CI 0.95 to 2.04; 1022 mosquitoes, 2 trials, 3 comparisons; very low-certainty evidence) or on blood feeding (RR 1.07, 95% CI 0.49 to 2.33; 1022 mosquitoes, 2 trials, 3 comparisons; low-certainty evidence). In areas where mosquito populations are susceptible to insecticides (> 90% mosquito mortality), there may be little to no di erence in the e ects of unwashed pyrethroid-PBO nets compared to unwashed standard-LLINs on mosquito mortality (RR 1.20, 95% CI 0.64 to 2.26; 2791 mosquitoes, 2 trials, 2 comparisons; low-certainty evidence). This is similar for washed nets (RR 1.07, 95% CI 0.92 to 1.25; 2644 mosquitoes, 2 trials, 2 comparisons; low-certainty evidence). We do not know if unwashed pyrethroid-PBO nets had any e ect on the blood-feeding success of susceptible mosquitoes (RR 0.52, 95% CI 0.12 to 2.22; 2791 mosquitoes, 2 trials, 2 comparisons; very low-certainty evidence). The same applies to washed nets (RR 1.25, 95% CI 0.82 to 1.91; 2644 mosquitoes, 2 trials, 2 comparisons; low-certainty evidence). In village trials comparing pyrethroid-PBO nets to LLINs, there was no di erence in sporozoite rate (4 trials, 5 comparisons) nor in mosquito parity (3 trials, 4 comparisons). In areas of high insecticide resistance, pyrethroid-PBO nets have greater entomological and epidemiological e icacy compared to standard LLINs, with sustained reduction in parasite prevalence, higher mosquito mortality and reduction in mosquito blood feeding rates 21 to 25 months post intervention. Questions remain about the durability of PBO on nets, as the impact of pyrethroid-PBO nets on mosquito mortality was not sustained over 20 washes in experimental hut trials, and epidemiological data on pyrethroid-PBO nets for the full intended three-year life span of the nets is not available. Little evidence is available to support greater entomological e icacy of pyrethroid-PBO nets in areas where mosquitoes show lower levels of resistance to pyrethroids. ⊕⊕⊕⊕ HIGH c Mosquito blood-feeding success is decreased with washed pyrethroid-PBO nets compared to standard washed LLINs in areas of high insecticide resistance *The risk in the intervention group (and its 95% CI) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI). CI: confidence interval; LLINs: long-lasting insecticidal nets; OR: odds ratio; PBO: pyrethroid-piperonyl butoxide; RR: risk ratio. High certainty: we are very confident that the true effect lies close to that of the estimate of the effect. Moderate certainty: we are moderately confident in the effect estimate: the true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different. Low certainty: our confidence in the effect estimate is limited: the true effect may be substantially different from the estimate of the effect. Very low certainty: we have very little confidence in the effect estimate: the true effect is likely to be substantially different from the estimate of effect. a Original numbers were used in this table; however in pooled analysis, events and total numbers were generated from cluster-adjusted results, which use the e ective sample size. Note that cluster adjustments do not change the point estimate of the e ect size -just the standard error. b Downgraded by one for inconsistency. c Not downgraded for imprecision: both best-and worst-case scenarios in this situation are important e ects. d Downgraded by one for imprecision due to wide CIs. e Downgraded by two for inconsistency due to unexplained qualitative heterogeneity. We do not know if washed pyrethroid-PBO nets have an effect on mosquito blood-feeding success in areas of no insecticide resistance *The risk in the intervention group (and its 95% CI) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI). CI: confidence interval; LLINs: long-lasting insecticidal nets; PBO: pyrethroid-piperonyl butoxide; RR: risk ratio. High certainty: we are very confident that the true effect lies close to that of the estimate of the effect. Moderate certainty: we are moderately confident in the effect estimate: the true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different. Low certainty: our confidence in the effect estimate is limited: the true effect may be substantially different from the estimate of the effect. Very low certainty: we have very little confidence in the effect estimate: the true effect is likely to be substantially different from the estimate of effect. a Original numbers are used in this table; however for the pooled analysis, events and total numbers were generated from cluster-adjusted results, which use the e ective sample size. Note that cluster adjustments do not change the point estimate of the e ect size, just the standard error. b Downgraded by two for imprecision due to extremely wide CIs. c Downgraded by one for inconsistency due to unexplained heterogeneity. Cochrane Database of Systematic Reviews Substantial progress has been made in reducing the burden of malaria in the 21st century. It is estimated that the clinical incidence of Plasmodium falciparum malaria in Africa dropped by 40% between 2000 and 2015, equating to prevention of 663 million cases (Bhatt 2015; WHO-GMP 2015) . However progress has stalled in recent years (WHO 2019a). Targeting the mosquito vector has proved to be the most e ective method of malaria prevention in Africa, with over two-thirds of malaria cases averted in the first 15 years of this century attributed to scale-up in the use of long-lasting insecticidal nets (LLINs) (Bhatt 2015) . This method of malaria prevention is particularly e ective in Africa, where the major malaria vectors Anopheles gambiae and Anopheles funestus are largely endophagic (feed indoors) and endophilic (rest indoors a er blood feeding). Currently all LLINs contain pyrethroids; pyrethroids have the required dual properties of low mammalian toxicity and rapid insecticidal activity (Zaim 2000), and their repellent or contact irritant e ects may enhance the personal protection of LLINs. Unfortunately, resistance to pyrethroids is now widespread in African malaria vectors (Ranson 2016). This may be the result of mutations in target-site proteins (target-site resistance) (Ranson 2011; Ridl 2008), which result in reduced sensitivity to the insecticide or increased activity of detoxification enzymes (metabolic resistance) (Mitchell 2012; Stevenson 2011), or other as yet poorly described resistance mechanisms, or a combination of all or some of these factors. The evolution of insecticide resistance and its continuing spread threaten the operational success of malaria vector control interventions. The current impact of this resistance on malaria transmission is largely unquantified and varies depending on level of resistance, malaria endemicity, and proportion of the human population using LLINs (Churcher 2016). A multi-country trial found no evidence that pyrethroid resistance reduced the personal protection provided by the use of LLINs (Kleinschmidt 2018). However, it is generally accepted that resistance will eventually erode the e icacy of pyrethroidonly LLINs, and that innovation in the LLIN market is essential to maintain the e icacy of this preventative measure (MPAC 2016). One way of controlling insecticide-resistant mosquito populations is through the use of insecticide synergists. Synergists are generally non-toxic and act by enhancing the potency of insecticides. Piperonyl butoxide (PBO) is a synergist that inhibits specific metabolic enzymes within mosquitoes and has been incorporated into pyrethroid-treated LLINs to form PBO-combination nets (herea er referred to as pyrethroid-PBO nets). Insecticide-synergist combination nets represent a new product class with the capacity to a ect insecticide-resistant populations. In 2017, the World Health Organization (WHO) gave pyrethroid-PBO nets an interim endorsement as a new vector control class and recommended that countries consider deploying these nets in areas where pyrethroid resistance has been confirmed among main malaria vectors (WHO-GMP 2017a). Currently six pyrethroid-PBO nets are in production: Olyset® Plus; PermaNet® 3.0; Veeralin® LN; Tsara Plus (previously DawaPlus 3.0); Tsara Boost (previously DawaPlus 4.0); and DuraNet Plus. Olyset Plus, which is manufactured by Sumitomo Chemical Company Ltd., is a polyethylene net treated with permethrin (20 g/kg ± 25%) and PBO (10 g/kg ± 25%) across the whole net (Sumitomo 2013). PermaNet 3.0, which is manufactured by Vestergaard Frandsen, is a mixed polyester (sides) polyethylene (roof) net treated with deltamethrin and PBO; PBO is found only on the roof of the net (25 g/kg ± 25%), and the concentration of deltamethrin varies depending on location (roof: 4.0 g/kg ± 25%) and yarn type (sides: 75-denier (thickness) yarn with 70-cm lower border 2.8 g/kg ± 25%, 100-denier yarn without border 2.1 g/kg ± 25%; Vestergaard 2015). Veeralin LN, manufactured by Vector Control Innovations Private Ltd., is a polyethylene net treated with alpha-cypermethrin (6.0 g/ kg) and PBO (2.2 g/kg) across the whole net (WHOPES 2016). Tsara Plus and Tsara Boost are manufactured by NRS Moon Netting FZE. Tsara Plus is treated with deltamethrin (3 g/kg) and PBO (11 g/ kg) on the roof, and with deltamethrin only (2.5 g/kg) on its sides. Tsara Boost is treated with deltamethrin (120 mg/m ) and PBO (440 mg/m ) on all panels. DuraNet Plus, manufactured by Shobikaa Impex Private Limited, is a polyethylene net treated with alphacypermethrin (6.0 g/kg) and PBO (2.2 g/kg) across the whole net. PBO inhibits metabolic enzyme families, in particular the cytochrome P450 enzymes that detoxify or sequester pyrethroids. Increased production of P450s is thought to be the most potent mechanism of pyrethroid resistance in malaria vectors, and pre-exposure to PBO has been shown to restore susceptibility to pyrethroids in laboratory bioassays on multiple pyrethroidresistant vector populations (Churcher 2016). All LLINs approved by the WHO Prequalification Team (formerly the WHO Pesticide Evaluation Scheme (WHOPES)) contain pyrethroids. Six bed nets that contain PBO have received WHO pre-qualification and have been recognized as a new product class by WHO (WHO-GMP 2017a). As pyrethroid-PBO nets are generally more expensive than conventional LLINs, it is important to determine if they are Library Trusted evidence. Informed decisions. Better health. Cochrane Database of Systematic Reviews superior to conventional LLINs, and under what circumstances, to enable cost-e ectiveness trials to be performed to inform procurement decisions. An Expert Review Group (ERG) commissioned by the WHO has recommended pyrethroid-PBO nets be considered for use in areas where the major malaria vectors are resistant to pyrethroids (WHO-GMP 2017a). This guidance has been adopted by some net providers, for example, the President's Malaria Initiative (PMI) (PMI 2018). The WHO recommendation was largely based on a single randomized controlled trial (RCT) of one pyrethroid-PBO net type conducted in Tanzania (Protopopo 2018), but it was also supported by a meta-analysis of performance of pyrethroid-PBO nets in experimental hut trials, which was used to parameterize a malaria transmission model to predict the public health benefit of pyrethroid-PBO nets (Churcher 2016). The WHO recommendation is that countries should consider deployment of this new product class in areas with intermediate levels of pyrethroid resistance, but it calls for further evidence, including data from a second clinical trial (WHO 2019b). Results of a second RCT evaluating the epidemiological impact of pyrethroid-PBO nets in Uganda were published in 2020, and this review has been updated to include these data (Staedke 2020). In an attempt to assess evidence of e ectiveness of pyrethroid-PBO nets against African malaria vectors in areas with di ering levels of insecticide resistance, we have conducted a systematic review of all relevant trials and examined both epidemiological and entomological endpoints. We appreciate that evaluation of PBO will depend on trials in which the background insecticide and dose are the same in both intervention and control groups; we are aware that most trials have evaluated pyrethroid-PBO nets against pyrethroid-only LLINs with di erent background insecticides and doses, which confounds the e ects. To compare e ects of pyrethroid-PBO nets currently in commercial development or on the market with e ects of their non-PBO equivalent in relation to: 1. malaria parasite infection (prevalence or incidence); and 2. entomological outcomes We included: 1. randomized trials that measured epidemiological outcomes, entomological outcomes, or both; and 2. experimental hut trials. See Table 1 for detailed WHOPES definitions. Anopheles gambiae complex or Anopheles funestus group. Included trials had to test a minimum of 50 mosquitoes per trial arm. We examined the insecticide resistance level (measured by phenotypic resistance) during data analysis. Adults and children living in malaria-endemic areas. Bed nets treated with both PBO and a pyrethroid insecticide. Nets must have received a minimum of interim-WHO approval ( Table 2) , and LLINs had to be treated with a WHO-recommended dose of pyrethroid (Table 3) . Conventional LLINs that contain pyrethroid only. Nets could be treated with the same insecticide at di erent doses from the intervention net to allow critical appraisal of all pyrethroid-PBO nets currently in development or on the market. For both intervention and control arms, nets could be unholed, holed, unwashed, or washed, provided the trials adhered to WHO guidelines (WHO 2013). Trials had to include at least one of the following primary outcomes to be eligible for inclusion. We identified all relevant trials regardless of language or publication status (published, unpublished, in press, and in progress). We have presented the search strategies in Appendix 1. Vittoria Lutje, the Cochrane Infectious Diseases Group (CIDG) Information Specialist, searched the following databases on 25 September 2020 using the search terms and strategy described in All analyses were stratified by trial design and mosquito insecticide resistance level when possible. We performed analyses for the primary outcomes stratified by follow-up time (4 to 6 months, 9 to 12 months, 16 to 18 months, and 21 to 25 months). We determined whether mosquito populations are susceptible or resistant to pyrethroid insecticides based on WHO definitions (WHO 2016; Table 4 ). We used 24-hour mosquito mortality to determine resistance status; however if this had been unavailable, we intended to use knock-down 60 minutes a er the end of the assay. We stratified resistant populations into low-, moderate-, and high-prevalence resistance groups (Table 5) , by dividing resistant mosquitoes (i.e. those with < 90% mortality) into three equal groups, with the lower third being most resistant and the upper third most susceptible. Two review authors (KG and NL or LC) independently screened titles and abstracts of all retrieved references based on the inclusion criteria (Table 6) . We resolved any inconsistencies between review authors' selections by discussion. If we were unable to reach an agreement, we consulted a third review author (HR). We retrieved full-text trial reports for all potentially relevant citations. Two review authors independently screened the full-text articles and identified trials for inclusion, and identified and recorded reasons for exclusion of ineligible trials in a Characteristics of excluded studies table. We resolved any disagreements through discussion or, if required, we consulted a third review author (HR). We identified and excluded duplicates and collated multiple reports of the same trial, so that each trial, rather than each report, was the unit of interest in the review. We recorded the selection process in su icient detail to complete a PRISMA flow diagram (Moher 2009). A er selection, we summarized all included trials according to the tables in Appendix 2. Two review authors (KG and NL or LC) independently extracted data from included trials using the predesigned data extraction form (Appendix 3). If data were missing from an included trial, we contacted the trial authors to ask for further information. We entered data into Review Manager 5 (RevMan 5) (Review Manager 2014). Two review authors (KG and NL or LC) independently assessed the risk of bias of each included trial using a set of predetermined criteria specific to each trial type adapted from Strode 2014 (Appendix 4). We assigned a classification of low, high, or unclear risk of bias for each component. For all included trials, we assessed whether any trial authors had submitted any conflicts of interest that may have biased trial methods or results. We assessed 12 criteria for village and RCTs: recruitment bias, comparability of mosquitoes between LLIN/pyrethroid-PBO net households (e.g. species composition), collectors blinded, household blinded, treatment allocation, allocation concealment, incomplete outcome data, raw data reported, clusters lost to follow-up, selective reporting, adjustment for data clustering, and trial authors' conflicting interests. For experimental hut trials, we assessed 11 criteria: comparability of mosquitoes between LLIN/pyrethroid-PBO net arms (e.g. species composition), collectors blinded, sleepers blinded, sleeper bias accounted for, treatment allocation, treatment rotation, standardized hut design, hut cleaning between treatments, incomplete outcome data, raw data reported, and trial authors' conflicting interests. For dichotomous data, we preferentially presented the risk ratio (RR). For the outcome of parasite prevalence from cRCTs, we used the odds ratio (OR) as the measure of e ect, as one study presented adjusted ORs that could not be converted to adjusted RRs using the standard formula presented in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011). We found no continuous or count data; however if we had, we would have used mean di erences (MDs) and rate ratios, respectively. We have presented all results with 95% confidence intervals (CIs). For trials randomized by hut or village, we used the adjusted measure of e ect reported in the paper if available. For the outcome of parasite prevalence from cRCTs, we converted adjusted RRs presented in one study -Staedke 2020 -to adjusted ORs using the standard formula presented in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011), so that this study could be pooled with Protopopo 2018. When adjusted measures of e ect were not reported, we used an intracluster correlation coe icient (ICC) and average cluster size to adjust the data ourselves (Higgins 2011 Section 16.3.4). If the included trial did not report the ICC value, we estimated the ICC value and performed sensitivity analyses to investigate the impact of estimating the ICC. When ICCs have been used to adjust results for clustering, forest plots for both hut and village trials show the e ective number of events and the number of mosquitoes a er adjustments for clustering. To adjust results of experimental hut trials for clustering, we treated each 'hut and night' combination as the unit of randomization, as each hut was tested with each type of net over a series of nights. Sleepers inside the huts were rotated each night, so by using "hut/night" as the unit of randomization, sleeper e ects were also accounted for. We calculated e ective sample sizes by estimating an ICC and a corresponding design e ect. We divided both the number of mosquitoes and the number experiencing the event by this design e ect. In the case of missing data, we contacted trial authors to request this information. If we had identified trials in which participants were lost to follow-up, we would have investigated the impact of missing data via imputation using a best/worst-case scenario analysis. When information on mosquito insecticide resistance was not collected at the time of the trial, review authors determined a suitable proxy. Proxy resistance data had to be taken from the same area and conducted within three years of the trial, and the same insecticide, dose, and mosquito species had to be used. More than 50 mosquitoes per insecticide should have been tested against an appropriate control. When no resistance data were available, we determined that resistance status was unclassified. We presented the results of included trials in forest plots, which we inspected visually, to assess heterogeneity (i.e. non-overlapping CIs generally signify statistical heterogeneity). We used the Chi test with a P value less than 0.1 to indicate statistical heterogeneity. We quantified heterogeneity by using the I statistic (Higgins 2003), and we interpreted a value greater than 75% to indicate considerable heterogeneity (Deeks 2017). To analyse the possibility of publication bias, we intended to use funnel plots if 10 trials with epidemiological endpoints were included in any of the meta-analysis. However, no analyses included 10 or more trials, so this plan was not applicable. When appropriate, we pooled the results of included trials using meta-analysis. We stratified results by type of trial, mosquito resistance status, and net type (i.e. by product, e.g. Olyset Plus). Four review authors (KG, NL, LC, and MC) analysed the data using RevMan 5 (Review Manager 2014), using the random-e ects model (if we detected heterogeneity; or if the I statistic value was greater than 75%) or the fixed-e ect model (for no heterogeneity; or if the I statistic value was less than 75%). The exception to this is that for the primary outcome of parasite prevalence from cluster trials, we pooled results using the fixed-e ect model, although heterogeneity between study results was substantial. For additional information, see 'E ects of Interventions: Epidemiological results'. We would have refrained from pooling trials in meta-analysis if it was not clinically meaningful to do so, due to clinical or methodological heterogeneity. We performed subgroup analyses according to whether nets were washed or unwashed. We intended to perform sensitivity analyses to determine the e ect of exclusion of trials that we considered to be at high risk of bias; however this approach was not applicable, as no trials were deemed at high risk. We would have performed a sensitivity analysis for missing data during imputation with best/worst-case scenarios, but again this was not applicable. We performed sensitivity analyses to investigate the impact of estimating an ICC to adjust trial results for clustering. We performed analyses using ICCs of 0.01, 0.05, and 0.1. Because results were robust to these adjustments, we used the most conservative ICC (0.1), and we adjusted all results from unadjusted cluster trials using this ICC. We have not presented analyses using the smaller ICCs (0.01 and 0.05). We assessed the certainty of evidence using the GRADE approach (Schünemann 2013). We constructed 'Summary of findings' tables using GRADEpro Guideline Development Tool (GDT) so ware (GRADEpro GDT 2015). We identified 389 records through our searches. We removed duplicates, leaving 347 records, and we screened all articles for possible inclusion. A er abstract and title screening, we excluded 322 ineligible trials. We assessed 25 full-text articles for eligibility and excluded nine articles for the following reasons: three trials did not share full data sets, two were laboratory studies, and four are ongoing. Sixteen trials met the inclusion criteria ( Figure 1 ). Trusted evidence. Informed decisions. Better health. Cochrane Database of Systematic Reviews We assessed 25 full-text articles for eligibility and excluded nine articles for the following reasons: three trials are awaiting classification because we were unable to obtain the full data sets a er we contacted trial authors (see Characteristics of studies awaiting classification table); four trials are ongoing (see Characteristics of ongoing studies section); and two trials included only laboratory data (Darriet 2011; Darriet 2013). We have provided a 'Risk of bias' assessment summary in Figure 2 . The criteria we used to assess risk of bias are provided in Appendix 5 (experimental hut trials) and in Appendix 6 (village trials). Trusted evidence. Informed decisions. Better health. Cochrane Database of Systematic Reviews Cochrane Database of Systematic Reviews We assessed all four village trials as having low risk of recruitment bias, as recruitment bias is related to human participants and so is not applicable to this review (Awolola 2014; Cisse 2017; Mzilahowa 2014; Stiles-Ocran 2013). We assessed the two cRCTs as having low risk, as no participants were recruited a er clusters had been randomized (Protopopo 2018; Staedke 2020). We judged all 10 experimental hut trials to be at low risk We assessed the 10 hut trials to be at unclear risk, as they did not specify whether observers, collectors and sleepers (hut trials) were blinded ( . This is not standard protocol for these trial designs and is thought unlikely to a ect the results. We judged four village trials to be at high risk of bias, as it was not stated whether collectors were blinded, and this may have a ected searching e orts during collection (Awolola 2014; Cisse 2017; Mzilahowa 2014; Stiles-Ocran 2013). We judged one cRCT as having high risk, as it was stated that LLIN allocation was not masked to collectors (Staedke 2020), and the other as having low risk because collectors were masked to treatment (Protopopo 2018). For household blinding, we judged all four village trials and both cRCTs to be at low risk of bias. Four village trials and one cRCT did not state whether households were blind to the intervention; however this was unlikely to influence the results (Awolola 2014; Cisse 2017; Mzilahowa 2014; Stiles-Ocran 2013; Staedke 2020). We judged one cRCT as having low risk, as inhabitants and field collectors were blinded to intervention arms (Protopopo 2018). We assessed the 10 hut trials to be at low risk for sleeper bias, as sleepers were rotated between huts according to a Latin square design ( We assessed the 10 hut trials to be at low risk for treatment allocation and rotation, as treatments were rotated between huts according to a We assessed all 10 hut trials to be at low risk of bias, as huts were built to standard West or East African specifications ( We assessed four hut trials to be at unclear risk, as they did not state whether huts were cleaned between treatment arms ( Staedke 2020 -to be at low risk for both incomplete outcome data and raw data reporting, as there were no incomplete outcome data, or missing data were later provided by trial authors. In cases when raw data were not reported, we were able to calculate them from the percentages and sample sizes given. When these data were not available, we did not include the trials. Staedke 2020 lost 14 clusters to follow-up at the latest time point and was therefore assessed as having unclear risk of bias. In the other village and cRCT trials, no clusters were lost to follow-up, and these trials were assessed as having low risk (Awolola 2014; Cisse 2017; Mzilahowa 2014; Protopopo 2018; Staedke 2020; Stiles-Ocran 2013). We assessed four village trials as having high risk of bias for statistical methods used, as they did not adjust for clustering (Awolola 2014; Cisse 2017; Mzilahowa 2014; Stiles-Ocran 2013). We assessed the two cRCTs as having low risk of bias, as they took clustering into account and adjusted for it in their statistical methods (Protopopo 2018; Staedke 2020). We assessed all village trials and cRCTs as having low risk of bias regarding selective reporting, as they appear to have reported all We We compared the e ects of pyrethroid-PBO nets currently in commercial development or on the market with their non-PBO equivalent in relation to malaria infection and entomological outcomes. This review is based on results from 16 trials. Two trials examined the e ects of pyrethroid-PBO nets (Olyset Plus and PermaNet 3.0) on parasite prevalence (Protopopo 2018; Staedke 2020). Pooling the latest endpoint a er the intervention from both trials revealed that parasite prevalence was decreased in the intervention arm (Olyset Plus and PermaNet 3.0) (OR 0.79, 95% CI 0.67 to 0.95; 2 trials, 2 comparisons; Analysis 1.1). There was little variation of e ect from the earliest time point (4 to 6 months a er: OR 0.74, 95% CI 0.62 to 0.89) to the latest time point (21 to 25 months a er: OR 0.79, 95% CI 0.67 to 0.95) (Analysis 1.2). We used a fixed-e ect model to pool data from the two studies. Although heterogeneity between study results was considerable, both studies demonstrated clear beneficial e ects with PBO nets. Performing random-e ects meta-analysis accounted for di erences between study results to the extent that identified benefits disappeared in the pooled analysis, indicating failure of the random-e ects model. Ten experimental hut trials (phase 2 trials) examined the e ects of pyrethroid-PBO nets on mosquito mortality, blood feeding, exophily, and deterrence ( . We pooled the results initially and then stratified them by insecticide resistance level and by net type. Two trials did not wash their nets and so did not report any data for the washed subgroup (Menze 2020 Toé 2018). One trial did not introduce holes into the nets and so did not report blood-feeding success data (Koudou 2011). (Table 7) . Heterogeneity in this pooled analysis was considerable, particularly for estimates of mortality. We therefore performed a pre-specified, stratified analysis, dividing the results into trials conducted in areas of low, moderate, or high resistance in the Anopheles population. We used WHO and Centers for Disease Control and Prevention (CDC) definitions of mosquito mortality from WHO tube assays or CDC bottle tests to classify mosquito resistance (Table 4 ). Both tests define mosquitoes as resistant when mortality is less than 90%. We further stratified resistance based on the following mortality levels: < 30%, high resistance; 31% to 60%, moderate resistance; and 61% to 90%, low resistance (Table 5 ). When resistance data were not collected at the time of the trial, we identified a suitable proxy based on previously described criteria (see Dealing with missing data section); when we could not identify a suitable proxy, we deemed the trial as 'unclassified' and did not include it in the resistance stratification. . It was not possible to stratify these data by resistance status due to the variability in resistance levels between villages within the same trial. Mosquito density was measured by a variety of methods and was summarized in di erent ways (e.g. mean number caught per house, mean number caught per village). When baseline data were collected, we calculated a percentage reduction. Higher reductions in mosquito densities were observed in pyrethroid-PBO net villages compared to LLIN villages (Table 8 ). See Summary of findings 1, Summary of findings 2, Summary of findings 3, and Summary of findings 4. Two cluster-randomized controlled trials (cRCTs) were performed on pyrethroid-piperonyl butoxide (PBO) nets. The first trial, which compared parasite prevalence in children using Olyset Plus nets with that in children using Olyset nets, in a region of Tanzania where mosquito vectors are highly resistant to pyrethroids, found that pyrethroid-PBO nets reduced parasite prevalence by 60% at the final time point (21 months) (Protopopo 2018). The second cRCT compared parasite prevalence in children using Olyset Plus or Permanet 3.0 nets with that in children using Olyset or Permanet 2.0 nets across East and West Uganda, where mosquito vectors are also highly resistant to pyrethroids, and found that pyrethroid-PBO nets reduced parasite prevalence by 17% at the latest time point (25 months) (Staedke 2020). All other trials included in this review measured entomological endpoints. Four village trials measured sporozoite rates in mosquitoes collected from houses using pyrethroid-PBO nets and standard pyrethroid long-lasting insecticidal nets (LLINs), but the results were highly heterogeneous and no evidence suggests that pyrethroid-PBO nets reduced the mosquito infection rate Tungu 2010), data showed improved performance of pyrethroid-PBO LLINs over standard LLINs in both increasing mosquito mortality and reducing blood feeding, but these results were highly heterogeneous. Stratifying experimental hut data by resistance levels in this population reduced heterogeneity. In areas where mosquitoes are highly resistant to pyrethroids, pyrethroid-PBO nets will reduce mosquito blood-feeding rates (i.e. users will be Library Trusted evidence. Informed decisions. Better health. Cochrane Database of Systematic Reviews better protected against mosquito bites by using pyrethroid-PBO nets). This impact on blood feeding is reduced when nets have been through the standard 20 washes recommended by the World Health Organization (WHO) to assess chemical durability, but it remains significant (high-certainty evidence). When resistance is high and new unwashed nets are used, mosquito mortality is substantially higher when the nets contain PBO compared to pyrethroid only (high-certainty evidence). However this e ect on mosquito mortality, which is important for the communitylevel protection a orded by LLIN usage (Hawley 2003; Maxwell 2002), is not sustained when nets have been washed multiple times. In this Cochrane Review, we classified mosquitoes as highly resistant if less than 30% were killed in a standard bioassay. When mortality rates exceeded 30%, we found little evidence to suggest that pyrethroid-PBO nets provided greater personal protection or resulted in greater mosquito mortality than standard pyrethroidonly nets. This result is not unexpected, given that in areas where resistance is uncommon or absent, exposure to pyrethroids alone would be expected to negatively a ect the mosquito; it is only in areas where the e icacy of pyrethroids has been eroded by the development of high levels of resistance that the addition of a synergist might be needed. We found no evidence for any di erence in the performance of pyrethroid-PBO nets from di erent manufacturers against highly pyrethroid-resistant mosquitoes. We stratified results by net type only for trials that were conducted in areas of high resistance. We have not reported comparisons for DawaPlus-PBO and Veeralin-PBO nets in this sub-analysis, as there was only a single data point for these net types. We did not stratify data from the cRCTs by net type, as one trial used only one net type (Protopopo 2018), and the second was not powered to detect di erences between nets from di erent manufacturers and assigned an uneven number of clusters to each net type (Staedke 2020). Unwashed Results from comparisons between pyrethroid-PBO nets from di erent manufacturers should be taken with great caution, given the very limited number of data points available, particularly for washed nets. Further trials, in which nets from di erent manufacturers are directly compared in the same trial, are needed to address the issue of equivalence between di erent pyrethroid-PBO nets. We appraised the certainty of evidence using the GRADE approach (Summary of findings 1 Summary of findings 2 Summary of findings 3 Summary of findings 4). The two cRCTs provided moderate-certainty evidence that pyrethroid-PBO nets reduced parasite prevalence for the duration of the trial (high-certainty evidence a er four to six months) (Protopopo 2018; Staedke 2020). This result was obtained from two independent studies, conducted in di erent locations and settings; therefore the evidence adheres to the WHO recommendation that at least two cRCTs must be completed to demonstrate public health value (WHO-GMP 2017b). The certainty of evidence from trials using entomological endpoints varied. Data from village trials were di icult to assess, as there was considerable heterogeneity in the level of pyrethroid resistance and presumably also in the resistance mechanisms, both within and between trials. Analysis of data from experimental hut trials yielded high-certainty evidence for superior performance of pyrethroid-PBO nets in areas of high resistance, but evidence from trials conducted in other settings was of low or very low certainty. All trials included in this review compared pyrethroid-PBO nets with the nearest equivalent pyrethroid-only LLINs. Further changes to net specifications were o en included when manufacturers incorporated the synergist. For example, the pyrethroid-PBO net manufactured by Vestergaard (PermaNet 3.0) contains higher levels of deltamethrin and yarn of a di erent denier (thickness) compared to the pyrethroid-only equivalent, PermaNet 2.0; the pyrethroid in Olyset Plus (Sumitomo Chemical Co. Ltd.) is released from the yarn at a di erent rate than that in the Olyset nets. These additional variations in chemical or physical composition, or both, of the nets make it di icult to directly assess the added value of the addition of PBO. Furthermore, the concentration of PBO and its site of application di er markedly between nets received from di erent manufacturers. Two of the currently available pyrethroid-PBO nets (PermaNet 3.0 and Tsara Plus 3.0) contain PBO only on the roof of the netting, exploiting the behavioural patterns of host-seeking mosquitoes to attempt to reach the net user by approaching from above (Parker 2015), whilst the remaining pyrethroid-PBO nets contain the synergist on all sides of the net. The amount of PBO contained within the net di ers by a factor of 25-fold. It is not known how net manufacturers selected the doses of PBO applied to the netting. With currently available data, it is not possible to draw any conclusions on which strategy for producing pyrethroid-PBO nets will prove the most e ective under field conditions. The optimum PBO:pyrethroid ratio will likely di er depending on the level of resistance in the mosquito and underpinning resistance mechanisms. Data from experimental hut trials suggest that the PBO component of pyrethroid-PBO nets is lost a er repeated washing, as enhanced mortality caused by the synergist nets is not maintained a er 20 washes. As the addition of PBO to pyrethroid LLINs is expected to enhance their performance only in areas where mosquitoes are resistant to pyrethroid insecticides, it was important to stratify the results by resistance status. To do this, we used the WHO definition of resistance as mosquito populations with less than 90% mortality in a discriminating dose assay (WHO 2016), and then we split the resistant populations into three groups, depending on the percentage of mortality observed. Discriminating dose assays provide an estimate of the prevalence of resistance in a population but do not indicate the strength of this resistance nor give any indication of the mechanism(s) underpinning the resistance. As PBO works primarily by inhibiting the metabolism of pyrethroids by cytochrome P450s, this synergist is likely to have had greatest impact in populations where resistance was primarily conferred by elevated P450 activity and further stratification according to resistance mechanisms might have proved informative. However, in reality, characterization of resistance in mosquitoes is still primarily performed by bioassays alone and the relevant contributions of di erent resistance mechanisms to the phenotype remain unknown. An exception to this is seen in An funestus, where pyrethroid resistance is almost entirely due to elevated P450 activity (Churcher 2016). Unfortunately, only one data set from experimental hut trials conducted where An funestus was the primary vector was made available to us at the time of this review. Other examples of missing data that may have influenced study results include the absence of data on resistance status in some settings. Three experimental hut trials did not measure resistance at the time of the trial (Moore 2016; N'Guessan 2010; Pennetier 2013). For two of these trials, we used proxies for resistance; however, no proxy data were available for An funestus in Moore 2016, and hence we did not include this population in the stratified analysis. Three trials did not share their data with the review authors; these included trials on nets from two of the more recent manufacturers to produce pyrethroid-PBO nets (N'Guessan 2016; Tungu 2017), which precluded stratified analysis for these net types. For clinical trials, both species composition and resistance level may vary between clusters and/or over the duration of the trial (e.g. the Uganda trial -Staedke 2020 -involved 104 clusters across the country as part of the national LLIN campaign). The population was classified as highly pyrethroid resistant based on data provided by the study authors (WHO tube bioassay conducted in Banangaizi East: deltamethrin 0.05%, 20.7% mosquito mortality, n = 163), but the resistance phenotype of the vector population is likely to vary considerably between clusters. One key finding of this trial was the decline in performance of pyrethroid-PBO nets a er washing. However, as discussed above, it is not clear how the standardized washing protocol employed in experimental hut trials of LLINs reflects the actual chemical retention of active ingredients under operational use. It is encouraging to note that the impact of pyrethroid-PBO nets in reducing parasite prevalence was sustained over two years, hence the policy implications of the loss in bio e icacy a er washing remain to be determined. This is an update of the first Cochrane Review of pyrethroid-PBO nets (Gleave 2018). An earlier meta-analysis of experimental hut data indicated that pyrethroid-PBO nets would have the greatest impact against mosquito populations with intermediate levels of resistance (Churcher 2016). Using transmission models to convert entomological outputs into estimates of public health benefit, the authors noted that the impact of pyrethroid-PBO nets would vary depending on mosquito species, resistance levels, parasite prevalence, and LLIN usage. The importance of taking these key parameters into account when predicting the public health impact of a switch to pyrethroid-PBO nets has been somewhat lost in policy documents and operational guidelines, which seek to provide a simple decision rule to aid net selection. Hence, in the WHO report from the 2017 Evidence Review Group on 'Conditions for deployment of mosquito nets treated with pyrethroid and piperonyl butoxide', it is recommended that "National malaria control programmes and their partners should consider deployment of pyrethroid-PBO nets in areas where pyrethroid resistance has been confirmed in the main malaria vectors" (WHO 2017). In technical guidelines from one of the major net distributors, the PMI, the conditions for deployment of PBO nets include "moderate levels of pyrethroid resistance (defined as 35% to 80% mortality), evidence that PBO restores pyrethroid susceptibility, and moderate to high malaria prevalence" (PMI 2018). The PMI definition of moderate resistance overlaps with our definitions of moderate and low resistance. However in our review, the best evidence for superior e icacy of pyrethroid-PBO nets is derived from areas with high resistance (< 30% mortality), and very little evidence suggests improved performance in areas with moderate or low levels of resistance. The di erences between these trials may have arisen from incorporation of a large data set of laboratory bioassays comparing mosquito mortality with or without pre-exposure to PBO in the modelling study. These laboratory bioassays rely on use of a single discriminating dose and identified multiple trials where highly resistant populations were not impacted by PBO. In the current review, the mosquito populations included were limited to sites in which experimental hut trials had been conducted, and this may not have fully captured the full diversity of resistance mechanisms in Anopheles mosquitoes. This again highlights the importance of further trials on the influence of resistance mechanisms on the impact of pyrethroid-PBO LLINs. The findings of this review support the recent WHO policy recommendation that pyrethroid-piperonyl butoxide (PBO) nets should be considered for deployment in areas where pyrethroid resistance has been confirmed in the main malaria vectors (WHO-GMP 2017a When evaluating these trials, it is important to remember that the PBO is an additive to the nets that is intended to increase their e icacy against pyrethroid-resistant mosquito populations. No evidence suggests that pyrethroid-PBO nets are less e ective than standard LLINs for inducing mosquito mortality in any setting. For personal protection, blood-feeding rates are similarly decreased under all resistance scenarios when unwashed PBO nets are used, although this has not been shown for washed nets in low-resistance or susceptible areas (low-certainty evidence). Hence if pyrethroid-PBO nets perform as well as, or better than, standard LLINs, the decision on whether to switch to nets incorporating the synergist is largely a question of economics. With fixed budgets, there is a risk that the target of universal coverage of LLINs may be more di icult to reach if more expensive pyrethroid-PBO nets are deployed. Indeed, the WHO clearly states that countries should consider deploying pyrethroid-PBO nets only in situations where coverage with standard vector-control interventions is not reduced (WHO-GMP 2017c). Trials of the cost-e ectiveness of pyrethroid-PBO nets have not yet been possible due to uncertainties over the price di erential between pyrethroid-PBO nets and LLINs. Experimental hut trials simultaneously comparing di erent pyrethroid-PBO nets in areas where mosquitoes have high levels of pyrethroid resistance are needed to demonstrate equivalency and to inform procurement decisions, particularly given the very di erent approaches used to incorporate PBO into LLINs employed by di erent manufacturers. The issue of durability of bioactive levels of the synergist on the nets also needs further study; current WHO protocols for measuring LLIN durability will need to be adjusted to utilize pyrethroid-resistant colonies of mosquitoes, so that the impact of PBO, and not just of the insecticide, can be measured over the net's intended life span. The issue of the value of entomological endpoints in estimating the public health value of new types of nets remains contentious (Killeen 2018; WHO-GMP 2017c). Performing experimental hut trials alongside future randomized controlled trials of nets containing synergists, or other novel active ingredients, would help resolve this issue. In relation to reporting trial results, study authors need to record the level of resistance in the local mosquito population at the time of the trial and should include this when reporting the results. Data on resistance mechanisms would also be of value toward a improved understanding of how this influences the performance of pyrethroid-PBO nets. One of the problems in this research field is that pyrethroid-PBO nets are commercial products. The pyrethroid-PBO nets currently undergoing RCTs have had additional alterations made to them, such as changing the concentration or rate at which the pyrethroid is released. However, these are the products for which policy decisions are needed that are based on evidence related to their relative e ectiveness. Thus, in this Cochrane Review, we examined the evidence concerning the e ectiveness of commercial products. During these comparisons, we considered other potential confounding factors. The Academic Editor of this review is Dr Hellen Gelband. We World Health Organization. World malaria report 2019. www.who.int/malaria/publications/world_malaria_report/en/. World Health Organization. Guidelines for malaria vector control. https://apps.who.int/iris/bitstream/ handle/10665/310862/9789241550499-eng.pdf?ua=1 (accessed prior to 13 May 2021). World Health Organization Global Malaria Programme. Conditions for use of long-lasting insecticidal nets treated with a pyrethroid and piperonyl butoxide. www.who.int/ malaria/areas/vector_control/use-of-pbo-treated-llins-report-nov2015.pdf (accessed 24 August 2018). World Health Organization. Conditions for deployment of mosquito nets treated with a pyrethroid and piperonyl butoxide. apps.who.int/iris/bitstream/handle/10665/258939/ WHO-HTM-GMP-2017.17-eng.pdf?sequence=1 (accessed 24 August 2018). World Health Organization. The evaluation process for vector control products. www.who.int/malaria/publications/atoz/ evaluation-process-vector-control-products/en/ (accessed 24 August 2018). Cluster-randomized controlled village trial Participants Households with at least 1 adult resident and 1 child aged 2 to 10 years, Anopheles species Study name Comparative evaluation of standard insecticide-treated bed nets and co-treated bed nets on malaria prevalence in Sud Ubangi, Democratic Republic of Congo: a cluster-randomised trial Outcomes Incidence rate of laboratory-confirmed clinical cases of malaria (time frame: participants will be actively followed up for 12 months, and any suspected case of clinical malaria will immediately lead to microscopy and RDT for confirmation). Microscopy to confirm the diagnosis of malaria sporozoite rate (time frame: Anopheles mosquitoes will be captured every 3 months during 1 year), sporozoite detection by ELISA to determine infectivity of Anopheles Cochrane Database of Systematic Reviews ELISA: enzyme-linked immunosorbent assay; PBO: piperonyl butoxide. Cone bioassays: these studies are conducted in the laboratory setting and use standard WHO protocols (WHO 2013, Section 2.2.1), when mosquitoes are exposed to a suitable LLIN (treated intervention or untreated control) for three minutes using a standard plastic WHO cone. Following net exposure, mosquitoes are transferred to a holding container and are maintained on a sugar solution diet while entomological outcomes (mosquitoes knocked down 1 hour post exposure, and mosquito mortality 24 hours post exposure) are measured. Tunnel tests: these studies are conducted in the laboratory setting and use standard WHO protocols (WHO 2013, Section 2.2.2). Mosquitoes are released into a glass tunnel covered at each end with untreated netting. The intervention or control LLIN net sample is placed one-third down the length of the tunnel, and the net contains 9 holes that enable mosquitoes to pass through. A suitable bait is immobilized in the shorter section of the tunnel, where it is available for mosquito biting. Mosquitoes are released into the opposite end of the tunnel and must make contact with the net and locate holes before they are able to feed on the bait. After 12 to 15 hours, mosquitoes are removed from both sections of the tunnel, and entomological outcomes (the number of mosquitoes in each section, mortality, and blood-feeding inhibition at the end of the assay and 24 hours post exposure) are recorded. Wire-ball bioassays: these studies are conducted in the laboratory setting, where mosquitoes are introduced into a wire-ball frame that has been covered with the intervention or control LLIN. Mosquitoes are exposed for 3 minutes, after which they are transferred to a holding container, and entomological outcomes (mosquitoes knocked down 1 hour post exposure, and mosquito mortality 24 hours post exposure) are measured. WHOPES Phase II. Experimental hut trials WHOPES Phase II experimental hut trials are field trials conducted in Africa where wild mosquito populations or local colonized populations are evaluated. Volunteers or livestock sleep in experimental huts under a purposefully holed LLIN, with 1 person or animal per hut. Huts are designed to resemble local housing based on a West or East African design (WHO 2013; Section 3.3.1-2). However these trials have identical design features, such as eave gaps or entry slits to allow mosquitoes to enter, and exit traps to capture exiting mosquitoes. LLINs and volunteers are randomly allocated to huts and are rotated in a Latin square to avoid bias, with huts cleaned between rotations to avoid contamination. Several nets, including an untreated control net, can be tested at the same time. Dead and live mosquitoes are collected each morning from inside the net, inside the hut, and inside the exit traps. They are then scored as blood-fed or non-blood-fed, and as alive or dead, and live mosquitoes are maintained for a further 24 hours to assess delayed mosquito mortality. WHOPES Phase III. Village trials WHOPES Phase III village trials are conducted in Africa where wild mosquito populations are evaluated. Villages chosen to be included in the study are similar in terms of size, housing structure, location, and data available on insecticide resistance status of local malaria vectors. Households are assigned as conventional LLINs or PBO-LLINs. Randomization can be done at the household or village level. Adult mosquitoes are collected from study houses, and mosquito density is measured. An indication of malaria transmission is measured at the study sites by recording infections in mosquitoes, parasite prevalence, or malaria incidence. Moderate resistance, An funestus An arabiensis (11) Akron, Permanet 3.0, Moderate resistance (12) Cote d'Ivoire, VEERALIN, Low resistance (13) Malanaville, Olyset Plus, High resistance (14) Tengrela, Olyset Plus High resistance (16) Tengrela Moderate resistance, An funestus An arabiensis (10) Akron, Permanet 3.0, Moderate resistance (11) Cote d'Ivoire, VEERALIN, Low resistance (12) Tengrela Olyset Plus, High resistance (16) Zeneti, Permanet 3.0, Susceptible (17) Malanville, Olyset Plus, High resistance PBO-LLIN Moderate resistance, An funestus Unclassified, An funestus (11) Akron, Permanet 3.0, Moderate resistance (12) Malanville, Olyset Plus, High resistance (13) Tengrela, Olyset Plus, High resistance (14) Tengrela We searched the Cochrane Infectious Diseases Group (CIDG) Specialized Register, CENTRAL, MEDLINE, Embase, Web of Science, CAB Abstracts, and two clinical trial registers (ClinicalTrials.gov and WHO International Clinical Trials Registry Platform) up to 25 September 2020. We contacted organizations for unpublished data. We checked the reference lists of trials identified by these methods. We included experimental hut trials, village trials, and randomized controlled trials (RCTs) with mosquitoes from the Anopheles gambiae complex or the Anopheles funestus group. Total events: Heterogeneity: Tau² = 0.08; Chi² = 84.86, df = 13 (P < 0.00001); I² = 85% Test for overall effect: Z = 5.46 (P < 0.00001) Test for subgroup differences: Chi² = 6.11, df = 1 (P = 0.01), I² = 83.6% Cochrane Database of Systematic Reviews 6 (Net* or bednet* or hammock* or curtain* or ITN* or LLIN* or "Insecticide-Treated Bednet*" or "Insecticide-Treated net*" Trial start/ end date Net washed Triallocation Cochrane Database of Systematic Reviews LLIN: long-lasting insecticidal nets; PBO: piperonyl butoxide. Unclear High Huts accessible to the same mosquito populationNo or unclear information reportedHuts not accessible to the same mosquito population If outcomes assessed were not blinded, but this is unlikely to influence the results, we will judge this to be low riskOutcomes assessed not blinded, and this is likely to influence the results If outcomes assessed were not blinded, but this is unlikely to influence the results, we will judge this to be low risk If outcomes assessed were not blinded, but this is unlikely to influence the results, we will judge this to be low riskOutcomes assessed not blinded, and this is likely to influence the results If outcomes assessed were not blinded, but this is unlikely to influence the results, we will judge this to be low risk We will update any references and background information Inclusion criteria We propose to remove objective 1 (evaluate whether adding PBO to pyrethroid LLINs increases the epidemiological and entomological effectiveness of the nets' and focus instead on comparing pyrethroid-PBO nets with their non-PBO equivalent (objective 2). As a result, laboratory studies will be excluded. We make this decision as we only identified two studies meeting the inclusion criteria for objective 1 in Gleave 2018, both of which were laboratory assays; results from these cannot readily be translated into public health outcomes. We will subgroup our analysis on epidemiological data by follow-up time.We will update the search strategy terms as one brand of bednet has changed name, and we will perform a new search to identify all possible trials.This Abstract amended. Authors' conclusions section: changed from "reduce mosquito mortality and blood feeding rates" to "increase mosquito mortality and reduce blood feeding rates" • Previously, PBO-nets were classified as PBO-LLINs; however as the durability of PBO on nets has not been classified as long-lasting, these were subsequently referred to as pyrethroid-PBO nets. As a result of this, our review title changed from 'Piperonyl butoxide (PBO) combined with pyrethroids in long-lasting insecticidal nets (LLINs) to prevent malaria in Africa' to 'Piperonyl butoxide (PBO) combined with pyrethroids in insecticide-treated nets to prevent malaria in Africa'.We added Leslie Choi as a review author.Additional criteria for assessing the risk of bias of village trials were added. These are in line with the Cochrane 'Risk of bias' tool (Higgins 2017), as well as the five additional criteria listed in Section 16.3.2 of the Cochrane Handbook for Systematic Reviews of Interventions that relate specifically to cluster-randomized trials (Higgins 2011).The published protocol stated all stratified analysis factors under subgroup analysis (Gleave 2017). We have corrected this to state that subgroup analysis was performed only on whether nets were unwashed or washed. The prespecified changes to the protocol (before the review update commenced) are given in Appendix 7. In brief, the published review included laboratory bioassay studies (n = 2) (Gleave 2018). We excluded studies using only laboratory assays from this review update due to the challenges in extrapolating public health value from laboratory bioassays alone. We amended the search strategy including di erent search terms due to a bed net brand name change. A new search was undertaken to capture all relevant trials for this update.