Background There is an urgent need for insecticides with novel modes of action against mosquito vectors. Broflanilide is a meta-diamide, discovered and named Tenebenal™ by Mitsui Chemicals Agro, Inc., which has been identified as a candidate insecticide for use in public health products. Methods To evaluate its potential for use in public health, Tenebenal™ was screened using an array of methodologies against Anopheles and Aedes strains. Initially it was assessed for intrinsic efficacy by topical application. Tarsal contact bioassays were then conducted to further investigate its efficacy, as well as its potency and speed of action. The potential of the compound for use in indoor residual spray (IRS) applications was investigated by testing the residual efficacy of a prototype IRS formulation on a range of typical house building substrates, and its potential for use in long-lasting insecticidal nets (LLIN) was tested using dipped net samples. Finally, bioassays using well-characterized insecticide-resistant mosquito strains and an in silico screen for mutations in the insecticide’s target site were performed to assess the risk of cross-resistance to Tenebenal™. Results Tenebenal™ was effective as a tarsal contact insecticide against both Aedes and Anopheles mosquitoes, with no apparent cross-resistance caused by mechanisms that have evolved to insecticides currently used in vector control. Topical application showed potent intrinsic activity against a Kisumu reference strain and an insecticide-resistant strain of Anopheles gambiae. Applied to filter paper in a WHO tube bioassay, Tenebenal™ was effective in killing 100% of susceptible and resistant strains of An. gambiae and Aedes aegypti at a concentration of 0.01%. The discriminating concentration of 11.91 µg/bottle shows it to be very potent relative to chemistries previously identified as having potential for vector control. Mortality occurs within 24 h of exposure, 80% of this mortality occurring within the first 10 h, a speed of kill somewhat slower than seen with pyrethroids due to the mode of action. The potential of Tenebenal™ for development in LLIN and IRS products was demonstrated. At least 12 months residual efficacy of a prototype IRS formulation applied at concentrations up to 200 mg of AI/sq m was demonstrated on a range of representative wall substrates, and up to 18 months on more inert substrates. A dipped net with an application rate of around 2 g/sq m Tenebenal™ killed 100% of exposed mosquitoes within a 3-min exposure in a WHO cone test. Conclusions Tenebenal™ is a potent insecticide against adult Aedes and Anopheles mosquitoes, including strains resistant to classes of insecticide currently used in vector control. The compound has shown great potential in laboratory assessment and warrants further investigation into development for the control of pyrethroid-resistant mosquitoes.

Defining the pathogenic threat of envenoming by south african shield-nosed and coral snakes (genus aspidelaps), and revealing the likely efficacy of available antivenom. Jprot (2018), 1 Defining the pathogenic threat of envenoming by south african shield-nosed and coral snakes (genus aspidelaps), and revealing the likely efficacy of available antivenom ABSTRACT While envenoming by the southern African shield-nosed or coral snakes (genus Aspidelaps) has caused fatalities, bites are uncommon. Consequently, this venom is not used in the mixture of snake venoms used to immunise horses for the manufacture of regional SAIMR (South African Institute for Medical Research) polyvalent antivenom.Aspidelaps species are even excluded from the manufacturer's list of venomous snakes that can be treated by this highly effective product. This leaves clinicians, albeit rarely, in a therapeutic vacuum when treating envenoming by these snakes. This is a significantly understudied small group of nocturnal snakes and little is known about their venom compositions and toxicities. Using a murine preclinical model, this study determined that the paralysing toxicity of venoms from Aspidelaps scutatus intermedius, A. lubricus cowlesi and A. l. lubricus approached that of venoms from highly neurotoxic African cobras and mambas. This finding was consistent with the cross-genus dominance of venom three-finger toxins, including numerous isoforms which showed extensive interspecific variation. Our comprehensive analysis of venom proteomes showed that the three Aspidelaps species possess highly similar venom proteomic compositions. We also revealed that the SAIMR polyvalent antivenom crossreacted extensively in vitro with venom proteins of the three Aspidelaps. Importantly, this cross-genus venom-IgG binding translated to preclinical (in a murine model) neutralisation of A. s. intermedius venom-induced lethality by the SAIMR polyvalent antivenom, at doses comparable with those that neutralise venom from the cape cobra (Naja nivea), which the antivenom is directed against. Our results suggest a wider than anticipated clinical utility of the SAIMR polyvalent antivenom, and here we seek to inform southern African clinicians that this readily available antivenom is likely to prove effective for victims of Aspidelaps envenoming.

Fenazaquin, pyridaben, tolfenpyrad and fenpyroximate are Complex I inhibitors offering a new mode of action for insecticidal malaria vector control. However, extended exposure to pyrethroid based products such as long-lasting insecticidal nets (LLINs) has created mosquito populations that are largely pyrethroid-resistant, often with elevated levels of P450s that can metabolise and neutralise diverse substrates. To assess cross-resistance liabilities of the Complex I inhibitors, we profiled their susceptibility to metabolism by P450s associated with pyrethroid resistance in Anopheles gambiae (CYPs 6M2, 6P3, 6P4, 6P5, 9J5, 9K1, 6Z2) and An. funestus (CYP6P9a). All compounds were highly susceptible. Transgenic An. gambiae overexpressing CYP6M2 or CYP6P3 showed reduced mortality when exposed to fenpyroximate and tolfenpyrad. Mortality from fenpyroximate was also reduced in pyrethroid-resistant strains of An. gambiae (VK7 2014 and Tiassalé 13) and An. funestus (FUMOZ-R). P450 inhibitor piperonyl butoxide (PBO) significantly enhanced the efficacy of fenpyroximate and tolfenpyrad, fully restoring mortality in fenpyroximate-exposed FUMOZ-R. Overall, results suggest that in vivo and in vitro assays are a useful guide in the development of new vector control products, and that the Complex I inhibitors tested are susceptible to metabolic cross-resistance and may lack efficacy in controlling pyrethroid resistant mosquitoes.

Insect-specific flaviviruses (ISFs), are a group of nonpathogenic flaviviruses that only infect insects. ISFs can have a high prevalence in mosquito populations, but their transmission routes are not well understood.

Background: Quality control of indoor residual spraying (IRS) is necessary to ensure that spray operators (SOs) deposit the correct concentration of insecticide on sprayed structures, while also confirming that spray records are not being falsified. Methods: Using high-performance liquid chromatography (HPLC), this study conducted quality control of the organophosphate insecticide pirimiphos-methyl (Actellic 300CS), during the 2018 IRS round on Bioko Island, Equatorial Guinea. Approximately 60 SOs sprayed a total of 67,721 structures in 16,653 houses during the round. Houses that were reportedly sprayed were randomly selected for quality control testing. The SOs were monitored twice in 2018, an initial screening in March followed by sharing of results with the IRS management team and identification of SOs to be retrained , and a second screening in June to monitor the effectiveness of training. Insecticide samples were adhesive-lifted from wooden and cement structures and analysed using HPLC. Results: The study suggests that with adequate quality control measures and refresher training, suboptimal spraying was curtailed, with a significant increased concentration delivered to the bedroom (difference = 0.36, P < 0.001) and wooden surfaces (difference 0.41, P = 0.001). Additionally, an increase in effective coverage by SOs was observed, improving from 80.7% in March to 94.7% in June after retraining (McNemar's test; P = 0.03). Conclusions: The ability to randomly select, locate, and test houses reportedly sprayed within a week via HPLC has led to improvements in the performance of SOs on Bioko Island, enabling the project to better evaluate its own performance.

BackgroundIndoor Residual Spraying (IRS) of insecticides is a key method to reduce transmission by Triatoma infestans, vector of Chagas disease in a large part of South America. However, the successes of IRS in the Gran Chaco region straddling Bolivia, Argentina and Paraguay, have not equalled those in other Southern Cone countries.AimsThis study evaluated routine IRS practises and insecticide quality control in a typical endemic community in the Bolivian Chaco.MethodsAlpha-cypermethrin active ingredient (a.i.) concentrations captured onto filter papers fitted to sprayed wall surfaces, and in prepared spray tank solutions, were measured using an adapted Insecticide Quantification Kit (IQK™). The results were analysed by negative binomial GLM regression in relation to the time (minutes) spray teams spent treating houses, spray rates (surface area to spray [m2/minute]), householder compliance to empty houses for IRS delivery, and the visual presence/absence of filter papers. The IQK™ assays developed for these samples were validated against HPLC quantification methods. Results: Substantial variations in the delivered a.i. concentrations were observed; only 10.4% (50/480) of filter papers, and 8.8% (5/57) of houses received the target concentration. The delivered concentrations were not related to those in the matched spray tank solutions. The sedimentation of a.i. in the surface solution of prepared spray tanks was rapid, resulting in a 29% loss of a.i. content within 5 minutes, and 48.5% after 15 minutes. The delivered concentrations were positively associated with the time spent spraying the house, and inversely related to the spray rate, but showed weak correlations in both cases. The influence of householder compliance on spray rates were significant, though associated differences in delivered concentrations were not detected. No differences were observed in spray rates between houses fitted with filter papers and houses without.ConclusionsSuboptimal delivery of IRS is partially attributed to the insecticide physical characteristics, and the need for revision of insecticide delivery methods, which includes training of IRS teams and community education to encourage compliance. The IQK™ is a necessary field-friendly tool to improve IRS quality, and to facilitate health worker training and decision making by the Chagas disease vector control managers.

BackgroundSnake venoms contain many proteinaceous toxins that can cause severe pathology and mortality in snakebite victims. Interestingly, mRNA encoding such toxins can be recovered directly from venom, although yields are low and quality is unknown. It also remains unclear whether such RNA contains information about toxin isoforms and whether it is representative of mRNA recovered from conventional sources, such as the venom gland. Answering these questions will address the feasibility of using venom-derived RNA for future research relevant to biomedical and antivenom applications.Methodology/Principal FindingsVenom was extracted from several species of snake, including both members of the Viperidae and Elapidae, and either lyophilized or immediately added to TRIzol reagent. TRIzol-treated venom was incubated at a range of temperatures (4–37°C) for a range of durations (0–48 hours), followed by subsequent RNA isolation and assessments of RNA quantity and quality. Subsequently, full-length toxin transcripts were targeted for PCR amplification and Sanger sequencing. TRIzol-treated venom yielded total RNA of greater quantity and quality than lyophilized venom, and with quality comparable to venom gland-derived RNA. Full-length sequences from multiple Viperidae and Elapidae toxin families were successfully PCR amplified from TRIzol-treated venom RNA. We demonstrated that venom can be stored in TRIzol for 48 hours at 4–19°C, and 8 hours at 37°C, at minimal cost to RNA quality, and found that venom RNA encoded multiple toxin isoforms that seemed homologous (98–99% identity) to those found in the venom gland.Conclusions/SignificanceThe non-invasive experimental modifications we propose will facilitate the future investigation of venom composition by using venom as an alternative source to venom gland tissue for RNA-based studies, thus obviating the undesirable need to sacrifice snakes for such research purposes. In addition, they expand research horizons to rare, endangered or protected snake species and provide more flexibility to performing fieldwork on venomous snakes in tropical conditions.

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