Evaluating the attrition, fabric integrity and insecticidal durability of two dual active ingredient nets (Interceptor® G2 and Royal® Guard): methodology for a prospective study embedded in a cluster randomized controlled trial in Benin.

BACKGROUND: Following the World Health Organization (WHO) endorsement of dual active ingredient (AI) nets, an increased uptake of pyrethroid-chlorfenapyr and pyrethroid-pyriproxyfen nets is expected. Studies evaluating their physical and insecticidal durability are essential for making programmatic and procurement decisions. This paper describes the methodology for a prospective study to evaluate the attrition, fabric integrity, insecticidal durability of Interceptor® G2 (alpha-cypermethrin-chlorfenapyr) and Royal Guard® (alpha-cypermethrin-pyriproxyfen), compared to Interceptor® (alpha-cypermethrin), embedded in a 3-arm cluster randomized controlled trial (cRCT) in the Zou Department of Benin. METHODS: Ten clusters randomly selected from each arm of the cRCT will be used for the study. A total of 750 ITNs per type will be followed in 5 study clusters per arm to assess ITN attrition and fabric integrity at 6-, 12-, 24- and 36-months post distribution, using standard WHO procedures. A second cohort of 1800 nets per type will be withdrawn every 6 months from all 10 clusters per arm and assessed for chemical content and biological activity in laboratory bioassays at each time point. Alpha-cypermethrin bioefficacy in Interceptor® and Royal Guard® will be monitored in WHO cone bioassays and tunnel tests using the susceptible Anopheles gambiae Kisumu strain. The bioefficacy of the non-pyrethroid insecticides (chlorfenapyr in Interceptor® G2 and pyriproxyfen in Royal Guard®) will be monitored using the pyrethroid-resistant Anopheles coluzzii Akron strain. Chlorfenapyr activity will be assessed in tunnel tests while pyriproxyfen activity will be assessed in cone bioassays in terms of the reduction in fertility of blood-fed survivors observed by dissecting mosquito ovaries. Nets withdrawn at 12, 24 and 36 months will be tested in experimental hut trials within the cRCT study area against wild free-flying pyrethroid resistant An. gambiae sensu lato to investigate their superiority to Interceptor® and to compare them to ITNs washed 20 times for experimental hut evaluation studies. Mechanistic models will also be used to investigate whether entomological outcomes with each dual ITN type in experimental hut trials can predict their epidemiological performance in the cRCT. CONCLUSION: This study will provide information on the durability of two dual AI nets (Interceptor® G2 and Royal Guard®) in Benin and will help identify suitable methods for monitoring the durability of their insecticidal activity under operational conditions. The modelling component will determine the capacity of experimental hut trials to predict the epidemiological performance of dual AI nets across their lifespan.

Interceptor potential of C60 fullerene aqueous solution: a comparative analysis using the example of the antitumor antibiotic mitoxantrone.

We performed a qualitative and quantitative analysis of intermolecular interactions in aqueous solution between the antitumor antibiotic mitoxantrone and C60 fullerene in comparison with interactions between the antibiotic and well-known aromatic molecules such as caffeine and flavin mononucleotide, commonly referred to as interceptor molecules. For these purposes, we obtained equilibrium hetero-association constants of these interactions using a UV/Vis titration experiment. Special attention was paid to the interaction of C60 fullerene with mitoxantrone, which has been quantified for the first time. Based on the theory of interceptor-protector action and using a set of measured equilibrium constants we managed to estimate the relative biological effect of these mixtures in a model living system, taking human buccal epithelium cells as an example. We demonstrated that C60 fullerene is able to restore the functional activity of the buccal epithelium cell nucleus after exposure to mitoxantrone, which makes it possible to use C60 fullerene as regulator of medico-biological activity of the antibiotic.

Implementing OECD GLP principles for the evaluation of novel vector control tools: a case study with two novel LLINs, SafeNet® and SafeNet NF®.

BACKGROUND: To sustain high universal Long-Lasting Insecticidal Nets (LLINs) coverage, affordable nets that provide equivalent or better protection than standard LLINs, are required. Test facilities evaluating new LLINs require compliance to Good Laboratory Practice (GLP) standards to ensure the quality and integrity of test data. Following GLP principles allows for the reconstruction of activities during the conduct of a study and minimizes duplication of efficacy testing. This case study evaluated the efficacy of two LLINs: SafeNet NF® and SafeNet® LLIN. METHODS: The study was conducted according to GLP principles and followed World Health Organization guidelines for evaluating LLINs. The LLINs were assessed in experimental huts against wild, pyrethroid-resistant Anopheles arabiensis mosquitoes. Nets were either unwashed or washed 20 times and artificially holed to simulate a used torn net. Blood-feeding inhibition and mortality were compared with a positive control (Interceptor® LLIN) and an untreated net. RESULTS: Mosquito entry in the huts was reduced compared to negative control for the unwashed SafeNet NF, washed Safenet LLIN and the positive control arms. Similar exiting rates were found for all the treatment arms. Significant blood-feeding inhibition was only found for the positive control, both when washed and unwashed. All insecticide treatments induced significantly higher mortality compared to an untreated net. Compared to the positive control, the washed and unwashed SafeNet NF® resulted in similar mortality. For the SafeNet® LLINs the unwashed net had an equivalent performance, but the mortality for the washed net was significantly lower than the positive control. Internal audits of the study confirmed that all critical phases complied with Standard Operating Procedures (SOPs) and the study plan. The external audit confirmed that the study complied with GLP standards. CONCLUSIONS: SafeNet NF® and SafeNet® LLIN offered equivalent protection to the positive control (Interceptor® LLIN). However, further research is needed to investigate the durability, acceptability, and residual efficacy of these nets in the community. This study demonstrated that GLP-compliant evaluation of LLINs can be successfully conducted by African research institutions.

Durability of three types of dual active ingredient long-lasting insecticidal net compared to a pyrethroid-only LLIN in Tanzania: methodology for a prospective cohort study nested in a cluster randomized controlled trial.

BACKGROUND: Progress achieved by long-lasting insecticidal nets (LLINs) against malaria is threatened by widespread selection of pyrethroid resistance among vector populations. LLINs with non-pyrethroid insecticides are urgently needed. This study aims to assess the insecticide and textile durability of three classes of dual-active ingredient (A.I.) LLINs using techniques derived from established WHO LLIN testing methods to set new standards of evaluation. METHODS: A WHO Phase 3 active ingredients and textile durability study will be carried out within a cluster randomized controlled trial in 40 clusters in Misungwi district, Tanzania. The following treatments will be evaluated: (1) Interceptor®G2 combining chlorfenapyr and the pyrethroid alpha-cypermethrin, (2) Royal Guard® treated with pyriproxyfen and alpha-cypermethrin, (3) Olyset™ Plus which incorporates a synergist piperonyl butoxide and the pyrethroid permethrin, and (4) a reference standard alpha-cypermethrin only LLIN (Interceptor®). 750 nets will be followed in 5 clusters per intervention arm at 6, 12, 24 and 36 months post distribution for survivorship and hole index assessment. A second cohort of 1950 nets per net type will be identified in 10 clusters, of which 30 LLINs will be withdrawn for bio-efficacy and chemical analysis every 6 months up to 36 months and another 30 collected for experimental hut trials every year. Bio-efficacy will be assessed using cone bioassays and tunnel tests against susceptible and resistant laboratory strains of Anopheles gambiae sensu stricto. Efficacy of field-collected nets will be compared in six experimental huts. The main outcomes will be Anopheles mortality up to 72 h post exposure, blood feeding and egg maturation using ovary dissection to assess impact on fecundity. CONCLUSIONS: Study findings will help develop bio-efficacy and physical durability criteria for partner A.I., in relation to the cRCT epidemiological and entomological outcomes, and refine preferred product characteristics of each class of LLIN. If suitable, the bioassay and hut outcomes will be fitted to transmission models to estimate correlation with cRCT outcomes. TRIAL REGISTRATION NUMBER: NCT03554616.

Efficacy of interceptor® G2, a long-lasting insecticide mixture net treated with chlorfenapyr and alpha-cypermethrin against Anopheles funestus: experimental hut trials in north-eastern Tanzania.

BACKGROUND: The effectiveness of long-lasting insecticidal nets (LLIN), the primary method for preventing malaria in Africa, is compromised by evolution and spread of pyrethroid resistance. Further gains require new insecticides with novel modes of action. Chlorfenapyr is a pyrrole insecticide that disrupts mitochrondrial function and confers no cross-resistance to neurotoxic insecticides. Interceptor® G2 LN (IG2) is an insecticide-mixture LLIN, which combines wash-resistant formulations of chlorfenapyr and the pyrethroid alpha-cypermethrin. The objective was to determine IG2 efficacy under controlled household-like conditions for personal protection and control of wild, pyrethroid-resistant Anopheles funestus mosquitoes. METHODS: Experimental hut trials tested IG2 efficacy against two positive controls-a chlorfenapyr-treated net and a standard alpha-cypermethrin LLIN, Interceptor LN (IG1)-consistent with World Health Organization (WHO) evaluation guidelines. Mosquito mortality, blood-feeding inhibition, personal protection, repellency and insecticide-induced exiting were recorded after zero and 20 washing cycles. The trial was repeated and analysed using multivariate and meta-analysis. RESULTS: In the two trials held in NE Tanzania, An. funestus mortality was 2.27 (risk ratio 95% CI 1.13-4.56) times greater with unwashed Interceptor G2 than with unwashed Interceptor LN (p = 0.012). There was no significant loss in mortality with IG2 between 0 and 20 washes (1.04, 95% CI 0.83-1.30, p = 0.73). Comparison with chlorfenapyr treated net indicated that most mortality was induced by the chlorfenapyr component of IG2 (0.96, CI 0.74-1.23), while comparison with Interceptor LN indicated blood-feeding was inhibited by the pyrethroid component of IG2 (IG2: 0.70, CI 0.44-1.11 vs IG1: 0.61, CI 0.39-0.97). Both insecticide components contributed to exiting from the huts but the contributions were heterogeneous between trials (heterogeneity Q = 36, P = 0.02). WHO susceptibility tests with pyrethroid papers recorded 44% survival in An. funestus. CONCLUSIONS: The high mortality recorded by IG2 against pyrethroid-resistant An. funestus provides first field evidence of high efficacy against this primary, anthropophilic, malaria vector.

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

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

Assessing the efficacy of two dual-active ingredients long-lasting insecticidal nets for the control of malaria transmitted by pyrethroid-resistant vectors in Benin: study protocol for a three-arm, single-blinded, parallel, cluster-randomized controlled trial.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are currently the primary method of malaria control in sub-Saharan Africa and have contributed to a significant reduction in malaria burden over the past 15 years. However, this progress is threatened by the wide-scale selection of insecticide-resistant malaria vectors. It is, therefore, important to accelerate the generation of evidence for new classes of LLINs. METHODS: This protocol presents a three-arm superiority, single-blinded, cluster randomized controlled trial to evaluate the impact of 2 novel dual-active ingredient LLINs on epidemiological and entomological outcomes in Benin, a malaria-endemic area with highly pyrethroid-resistant vector populations. The study arms consist of (i) Royal Guard® LLIN, a net combining a pyrethroid (alpha-cypermethrin) plus an insect growth regulator (pyriproxyfen), which in the adult female is known to disrupt reproduction and egg fertility; (ii) Interceptor G2® LLIN, a net incorporating two adulticides (alpha-cypermethrin and chlorfenapyr) with different modes of action; and (iii) the control arm, Interceptor® LLIN, a pyrethroid (alpha-cypermethrin) only LLIN. In all arms, one net for every 2 people will be distributed to each household. Sixty clusters were identified and randomised 1:1:1 to each study arm. The primary outcome is malaria case incidence measured over 24 months through active case detection in a cohort of 25 children aged 6 months to 10 years, randomly selected from each cluster. Secondary outcomes include 1) malaria infection prevalence (all ages) and prevalence of moderate to severe anaemia in children under 5 years old, measured at 6 and 18 months post-intervention; 2) entomological indices measured every 3 months using human landing catches over 24 months. Insecticide resistance intensity will also be monitored over the study period. DISCUSSION: This study is the second cluster randomised controlled trial to evaluate the efficacy of these next-generation LLINs to control malaria transmitted by insecticide-resistant mosquitoes. The results of this study will form part of the WHO evidence-based review to support potential public health recommendations of these nets and shape malaria control strategies of sub-Saharan Africa for the next decade. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03931473 , registered on 30 April 2019.

Durability, household usage and washing pattern of DuraNet© and Interceptor® long-lasting insecticidal nets in long-term field trials in India.

BACKGROUND & OBJECTIVES: Universal coverage of population with long-lasting insecticidal nets (LLINs) living in endemic areas inhabited by ethnic tribal communities or in difficult to reach areas was found effective for disease control where other interventions such as indoor residual spraying have limited success because of operational issues. Evaluation of different LLINs with varied insecticides and fabrics are being evaluated to meet the demand of new products. This study was undertaken on two brands of LLINs, DuraNet© and Interceptor® that varied in fabric and manufacturing technologies to assess the usability in field conditions for atleast three years. METHODS: In large-scale field trials DuraNet©, alpha cypermethrin incorporated polyethylene net, was evaluated in Odisha state while, Interceptor® net, alpha cypermethrin coated polyester net was evaluated in Chhattisgarh and Gujarat states for a period of three years following WHO guidelines. Durability, usage pattern and washing behavior were monitored through periodic surveys and physical examination of nets. RESULTS: Survivorship of both the nets was 84-100% and every night usage rate was >62% in all seasons. Washing frequency was largely within the prescribed limits. The proportion of DuraNet© with holes was 26.7% in year one 74% in year three. In Gujarat, proportion of Interceptor® nets with holes at six months was 33.3% increased to 87% in year three and in Chhattisgarh, 6.7% after six months to 93.3% after three years of use respectively. INTERPRETATION & CONCLUSION: Both the nets revealed a useful life of three years under the field condition.

Which intervention is better for malaria vector control: insecticide mixture long-lasting insecticidal nets or standard pyrethroid nets combined with indoor residual spraying?

BACKGROUND: Malaria control today is threatened by widespread insecticide resistance in vector populations. The World Health Organization (WHO) recommends the use of a mixture of unrelated insecticides for indoor residual spraying (IRS) and long-lasting insecticidal nets (LNs) or as a combination of interventions for improved vector control and insecticide resistance management. Studies investigating the efficacy of these different strategies are necessary. METHODS: The efficacy of Interceptor® G2 LN, a newly developed LN treated with a mixture of chlorfenapyr (a pyrrole) and alpha-cypermethrin (a pyrethroid), was compared to a combined chlorfenapyr IRS and Interceptor® LN (a standard alpha-cypermethrin LN) intervention in experimental huts in Cove Southern Benin, against wild, free-flying, pyrethroid-resistant Anopheles gambiae s.l. A direct comparison was also made with a pyrethroid-only net (Interceptor® LN) alone and chorfenapyr IRS alone. RESULTS: WHO resistance bioassays performed during the trial demonstrated a pyrethroid resistance frequency of >90% in the wild An. gambiae s.l. from the Cove hut site. Mortality in the control (untreated net) hut was 5%. Mortality with Interceptor® LN (24%) was lower than with chlorfenapyr IRS alone (59%, P < 0.001). The combined Interceptor® LN and chlorfenapyr IRS intervention and the mixture net (Interceptor® G2 LN) provided significantly higher mortality rates (73 and 76%, respectively) and these did not differ significantly between both treatments (P = 0.15). Interceptor LN induced 46% blood-feeding inhibition compared to the control untreated net, while chlorfenapyr IRS alone provided none. Both mixture/combination strategies also induced substantial levels of blood-feeding inhibition (38% with combined interventions and 30% with Interceptor® G2 LN). A similar trend of improved mortality of pyrethroid-resistant An. gambiae s.l. from Cove was observed with Interceptor® G2 LN (79%) compared to Interceptor LN (42%, P < 0.001) in WHO tunnel tests. CONCLUSION: The use of chlorfenapyr and alpha-cypermethrin together as a mixture on nets (Interceptor® G2 LN) or a combined chlorfenapyr IRS and pyrethroid LN intervention provides improved control of pyrethroid-resistant malaria vectors by inducing significantly higher levels of mortality through the chlorfenapyr component and providing personal protection through the pyrethroid component. Both strategies are comparable in their potential to improve the control of malaria transmitted by pyrethroid resistant mosquito vectors.

Quantitative correlation of the in vitro biological effect with parameters of molecular complexation in mutagen-interceptor systems.

According to the theory of interceptor-protector action a quantitative link between the physico-chemical parameters of molecular complexation and in vitro biological effect in aromatic drug-interceptor systems must exist. In the present communication such link between relative change in mutagenicity of IQ-type aromatic mutagens on addition of aromatic interceptor molecules with equilibrium hetero-association constants of mutagen-interceptor complexation has been found using the published in vitro data in bacteria cell systems.