Evaluation of pesticide residues in commercial Swiss beeswax collected in 2019 using ultra-high performance liquid chromatographic analysis

The aim of this study was to determine residue levels of pesticides in Swiss commercial beeswax. Foundation samples were collected in 2019 from nine commercial manufacturers for analysis of 21 pesticides using ultra-high performance liquid chromatography. Individual samples showed the variability and residue ranges and pooled samples represented the average annual residue values of the Swiss production. In total, 17 pesticides were identified and 13 pesticides were quantified. They included 13 acaricides and/or insecticides, two fungicides as well as a synergist and a repellent. The means calculated from individual samples were similar to the average annual residue values for most tested pesticides. Mean values of 401, 236, 106 and 3 μg·kg−1 were obtained for the beekeeping-associated contaminants coumaphos, tau-fluvalinate, bromopropylate and N-(2,4-Dimethylphenyl)-formamide (DMF; breakdown product of amitraz), respectively. For the other pesticides, the mean values were 203 μg·kg−1 (synergist piperonyl butoxide), 120 μg·kg−1 (repellent N,N-Diethyl-3-methylbenzamide, DEET), 19 μg·kg−1 (chlorfenvinphos) and 4 μg·kg−1 ((E)-fenpyroximate), while the means for acrinathrin, azoxystrobin, bendiocarb, boscalid, chlorpyrifos, flumethrin, permethrin, propoxur and thiacloprid were below the limit of quantification (< LOQ). Individual samples contained from seven to 14 pesticides. The ranges of values for coumaphos and piperonyl butoxide (from 14 to 4270 μg·kg−1; from 6 to 1555 μg·kg−1, respectively) were larger as compared to the ranges of values for DEET and tau-fluvalinate (from < LOQ to 585 μg·kg−1; from 16 to 572 μg·kg−1, respectively). In conclusion, the most prominent contaminants were the pesticides coumaphos and tau-fluvalinate, which are both acaricides with previous authorization for beekeeping in Switzerland, followed by piperonyl butoxide, a synergist to enhance the effect of insecticides. Graphical abstract

A quasi-experimental study estimating the impact of long-lasting insecticidal nets with and without piperonyl butoxide on pregnancy outcomes

Background Long-lasting insecticidal nets (LLINs) are the main vector control tool for pregnant women, but their efficacy may be compromised, in part, due to pyrethroid resistance. In 2017, the Ugandan Ministry of Health embedded a cluster randomized controlled trial into the national LLIN campaign, where a random subset of health subdistricts (HSDs) received LLINs treated with piperonyl butoxide (PBO), a chemical synergist known to partially restore pyrethroid sensitivity. Using data from a small, non-randomly selected subset of HSDs, this secondary analysis used quasi-experimental methods to quantify the overall impact of the LLIN campaign on pregnancy outcomes. In an exploratory analysis, differences between PBO and conventional (non-PBO) LLINs on pregnancy outcomes were assessed. Methods Birth registry data (n = 39,085) were retrospectively collected from 21 health facilities across 12 HSDs, 29 months before and 9 months after the LLIN campaign (from 2015 to 2018). Of the 12 HSDs, six received conventional LLINs, five received PBO LLINs, and one received a mix of conventional and PBO LLINs. Interrupted time-series analyses (ITSAs) were used to estimate changes in monthly incidence of stillbirth and low birthweight (LBW; <2500 g) before-and-after the campaign. Poisson regression with robust standard errors modeled campaign effects, adjusting for health facility-level differences, seasonal variation, and time-varying maternal characteristics. Comparisons between PBO and conventional LLINs were estimated using difference-in-differences estimators. Results ITSAs estimated the campaign was associated with a 26% [95% CI: 7–41] reduction in stillbirth incidence (incidence rate ratio (IRR) = 0.74 [0.59–0.93]) and a 15% [-7, 33] reduction in LBW incidence (IRR=0.85 [0.67–1.07]) over a 9-month period. The effect on stillbirth incidence was greatest for women delivering 7–9 months after the campaign (IRR=0.60 [0.41–0.87]) for whom the LLINs would have covered most of their pregnancy. The IRRs estimated from difference-in-differences analyses comparing PBO to conventional LLINs was 0.78 [95% CI: 0.52, 1.16] for stillbirth incidence and 1.15 [95% CI: 0.87, 1.52] for LBW incidence. Conclusions In this region of Uganda, where pyrethroid resistance is high, this study found that a mass LLIN campaign was associated with reduced stillbirth incidence. Effects of the campaign were greatest for women who would have received LLINs early in pregnancy, suggesting malaria protection early in pregnancy can have important benefits that are not necessarily realized through antenatal malaria services. Results from the exploratory analyses comparing PBO and conventional LLINs on pregnancy outcomes were inconclusive, largely due to the wide confidence intervals that crossed the null. Thus, future studies with larger sample sizes are needed.

Bioefficacy and durability of Olyset® Plus, a permethrin and piperonyl butoxide-treated insecticidal net in a 3-year long trial in Kenya

Background Long-lasting insecticide nets (LLINs) are a core malaria intervention. LLINs should retain efficacy against mosquito vectors for a minimum of three years. Efficacy and durability of Olyset® Plus, a permethrin and piperonyl butoxide (PBO) treated LLIN, was evaluated versus permethrin treated Olyset® Net. In the absence of WHO guidelines of how to evaluate PBO nets, and considering the manufacturer’s product claim, Olyset® Plus was evaluated as a pyrethroid LLIN. Methods This was a household randomized controlled trial in a malaria endemic rice cultivation zone of Kirinyaga County, Kenya between 2014 and 2017. Cone bioassays and tunnel tests were done against Anopheles gambiae Kisumu. The chemical content, fabric integrity and LLIN survivorship were monitored. Comparisons between nets were tested for significance using the Chi-square test. Exact binomial distribution with 95% confidence intervals (95% CI) was used for percentages. The WHO efficacy criteria used were ≥ 95% knockdown and/or ≥ 80% mortality rate in cone bioassays and ≥ 80% mortality and/or ≥ 90% blood-feeding inhibition in tunnel tests. Results At 36 months, Olyset® Plus lost 52% permethrin and 87% PBO content; Olyset® Net lost 24% permethrin. Over 80% of Olyset® Plus and Olyset® Net passed the WHO efficacy criteria for LLINs up to 18 and 12 months, respectively. At month 36, 91.2% Olyset® Plus and 86.4% Olyset® Net survived, while 72% and 63% developed at least one hole. The proportionate Hole Index (pHI) values representing nets in good, serviceable and torn condition were 49.6%, 27.1% and 23.2%, respectively for Olyset® Plus, and 44.9%, 32.8% and 22.2%, respectively for Olyset® Net but were not significantly different. Conclusions Olyset® Plus retained efficacy above or close to the WHO efficacy criteria for about 2 years than Olyset® Net (1–1.5 years). Both nets did not meet the 3-year WHO efficacy criteria, and showed little attrition, comparable physical durability and survivorship, with 50% of Olyset® Plus having good and serviceable condition after 3 years. Better community education on appropriate use and upkeep of LLINs is essential to ensure effectiveness of LLIN based malaria interventions. Graphical Abstract

A Review on Non-target Toxicity of Deltamethrin and Piperonyl Butoxide: Synergist

Deltamethrin and piperonyl butoxide two synthetic pyrethroids, when used in a combination it produces synergistic effect. This two insecticide has found to be widely used in the management of mosquito, housefly and other insects to control the various vector born diseases. In this review we assessed the toxic effect of deltamethrin and piperonyl butoxide on beneficial organisms commonly available in the ecosystem. It was found to be toxic to fish, honey bees the prime pollinators of crop plant; earthworm is also susceptible at a lethal concentration for a particular exposure. As far the birds are concerned, they have a less toxic risk in lower concentration of exposure. The alterations obtained in the hematological, biochemical and histopathological studies, further conclude that it can cause environment hazards and toxic to the non-targeted organisms. This investigation gives an insight into the combined toxicological profile of deltamethrin and PBO for better risk assessment and safe use of pyrethroids and their synergist in non-targeted organisms.

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

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

Emerging mosquito resistance to piperonyl butoxide-synergized pyrethroid insecticide and its mechanism

Piperonyl butoxide (PBO)-synergized pyrethroid products are widely available for the control of pyrethroid-resistant mosquitoes. To date, no study has formally examined mosquito resistance to PBO-synergized insecticides. We used Culex quinquefasciatus as a model mosquito examined the insecticide resistance mechanisms of mosquitoes to PBO-synergized pyrethroid using modified World Health Organization tube bioassays and biochemical analysis of metabolic enzyme expressions prior- and post-PBO exposure. We measured mosquito mortalities and metabolic enzyme expressions in mosquitoes with/without pre-exposure to different PBO concentrations and exposure durations. We found that field Culex quinquefasciatus mosquitoes were resistant to all insecticides tested, including PBO-synergized pyrethroids (mortality ranged from 3.7±4.7% to 66.7±7.7%), except malathion. Field mosquitoes had elevated levels of carboxylesterase (COE, 3.8-fold) and monooxygenase (P450, 2.1-fold) but not glutathione S-transferase (GST) compared to susceptible mosquitoes. When the field mosquitoes were pre-exposed to 4% PBO, the 50% lethal concentration of deltamethrin was reduced from 0.22% to 0.10%, compare to 0.02% for susceptible mosquitoes. Knockdown resistance gene mutation (L1014F) rate was 62% in field mosquitoes. PBO pre-exposure suppressed P450 enzyme expression levels by 25~34%, GST by 11%, and had no impact on COE enzyme expression. Even with the optimal PBO concentration and exposure duration, field mosquitoes had significantly higher P450 enzyme expression levels after PBO exposure compared to laboratory controls. These results demonstrate that PBO alone may not be enough to control highly pyrethroid resistant mosquitoes due to the multiple resistance mechanisms. Mosquito resistance to PBO-synergized insecticide should be closely monitored.

Long-Lasting Insecticidal Nets Incorporating Piperonyl Butoxide Reduce the Risk of Malaria in Children in Western Kenya: A Cluster Randomized Controlled Trial

Malaria vectors have acquired an enzyme that metabolizes pyrethroids. To tackle this problem, we evaluated long-lasting insecticidal nets incorporating piperonyl butoxide (PBO-LLINs) with a community-based cluster randomized control trial in western Kenya. The primary endpoints were anopheline density and Plasmodium falciparum polymerase chain reaction (PCR)-positive prevalence (PCRpfPR) of children aged 7 months to 10 years. Four clusters were randomly selected for each of the treatment and control arms (eight clusters in total) from 12 clusters, and PBO-LLINs and standard LLINs were distributed in February 2011 to 982 and 1,028 houses for treatment and control arms, respectively. Entomological surveys targeted 20 houses in each cluster, and epidemiological surveys targeted 150 children. Cluster-level permutation tests evaluated the effectiveness using the fitted values from individual level regression models adjusted for baseline. Bootstrapping estimated 95% confidence intervals (CIs). The medians of anophelines per house were 1.4 (interquartile range [IQR]: 2.3) and 3.4 (IQR: 3.7) in the intervention and control arms after 3 months, and 0.4 (IQR: 0.2) and 1.6 (IQR: 0.5) after 10 months, respectively. The differences were –2.5 (95% CI: –6.4 to –0.6) and –1.3 (95% CI: –2.0 to –0.7), respectively. The datasets of 861 and 775 children were analyzed in two epidemiological surveys. The median PCRpfPRs were 25% (IQR: 11%) in the intervention arm and 52% (IQR: 11%) in the control arm after 5 months and 33% (IQR: 11%) and 45% (IQR: 5%) after 12 months. The PCRpfPR ratios were 0.67 (95% CI: 0.38, 0.91) and 0.74 (95% CI: 0.53, 0.90), respectively. We confirmed the superiority of PBO-LLINs.

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

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