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Long-lasting insecticide-treated nets (LLINs) play a crucial role in preventing malaria transmission. LLINs should remain effective for at least three years, even after repeated washings. Currently, monitoring insecticides in LLINs is cumbersome, costly, and requires specialized equipment and hazardous solvents. Our aim was to develop a simple, high-throughput and low-resource method for measuring insecticides in LLINs. To extract insecticides, polyethylene-LLIN samples were heated at 85 °C for 45 min in a non-hazardous solvent mix containing dicyclohexylphthalate as an internal standard. The extraction solvent was reduced from 50 to 5 ml using a 0.2 g sample, 90% smaller than the recommended sample size. By optimizing HPLC chromatography, we simultaneously detected pyrethroid and pyriproxyfen insecticides with high sensitivity in LLIN's extract. The method can quantify levels ≥ 0.0015% permethrin, 0.00045% alpha-cypermethrin and 0.00025% pyriproxyfen (w/w) in polyethylene, allowing for insecticide tracking before and after the use of LLINs. This method can be used to assess LLINs with 1% pyriproxyfen (pyriproxyfen-LLIN) or 2% permethrin (Olyset® Net), 1% pyriproxyfen and 2% permethrin (Olyset® Duo), or 0.55% pyriproxyfen and 0.55% alpha-cypermethrin (Royal Gaurd®). One can run 120 samples (40 nets) simultaneously with high precision and accuracy, improving throughput and reducing labour, costs, and environmental impact.

Additional file 2. Median alpha-cypermethrin a.i. concentrations on filter papers fitted to houses at different wall heights (n = 158 at 0.2 m; n = 157 at 1.2 m; n = 165 at 2 m). The horizontal lines represent the alpha-cypermethrin a.i. target concentration range (50 mg �� 20% a.i./m2). Error bars represents the lower and upper adjacent values to the median; points represent the outside values.

Additional file 5. (A) Median spray rates (m2/min) in IRS-treated houses with (+ F.P.) and without (��� F.P.) filter papers fitted to the walls pre-spraying, stratified by the level of householder compliance with the request to empty their houses in preparation for IRS application (n = 55 houses); (B) Median insecticide a.i. concentrations (mg/m2) delivered to houses stratified by householder compliance (n = 25 houses).

Additional file 1. Protocol of laboratory assays.

Additional file 4. Variation in the spray rates by individual health workers (A) median observed spray rates (m2/min); and (B) observed/expected spray rate ratio where the expected ratio is 19 m2/min for the spraying equipment calibration.

Additional file 3. Frequency distribution of the observed/expected spray rates in IRS-treated houses (n = 80 houses).

Background Indoor residual spraying (IRS) of DDT is used to control visceral leishmaniasis (VL) in India. However, the quality of spraying is severely compromised by a lack of affordable field assays to monitor target doses of insecticide. Our aim was to develop a simple DDT insecticide quantification kit (IQK) for monitoring DDT levels in an operational setting. Methodology/ principle findings DDT quantification was based on the stoichiometric release of chloride from DDT by alkaline hydrolysis and detection of the released ion using Quantab chloride detection strips. The assay was specific for insecticidal p,p`-DDT (LoQ = 0.082 g/m2). Bostik discs were effective in post spray wall sampling, extracting 25–70% of active ingredient depending on surface. Residual DDT was sampled from walls in Bihar state in India using Bostik adhesive discs and DDT concentrations (g p,p`-DDT/m2) were determined using IQK and HPLC (n = 1964 field samples). Analysis of 161 Bostik samples (pooled sample pairs) by IQK and HPLC produced excellent correlation (R2 = 0.96; Bland-Altman bias = −0.0038). IQK analysis of the remaining field samples matched HPLC data in identifying households that had been under sprayed, in range or over sprayed. Interpretation A simple dipstick assay has been developed for monitoring DDT spraying that gives comparable results to HPLC. By making laboratory-based analysis of DDT dosing accessible to field operatives, routine monitoring of DDT levels can be promoted in low- and middle- income countries to maximise the effectiveness of IRS., Author Summary Visceral leishmaniasis (VL) is a major parasitic disease on the Indian Subcontinent, with 85% of the disease incidence in India. DDT is being used to control the sandfly vector of VL, but the quality of indoor residual spraying (IRS) is not routinely assessed due to a lack of practical assays. Here we have developed a simple DDT insecticide quantification kit (IQK) for measuring the amounts of DDT being applied to surfaces during large scale vector control operations. We demonstrate that the IQK provides equivalent data to the gold standard high performance liquid chromatography (HPLC) assay. Being truly point-of-care, this test is able to immediately inform the management of spray quality. Integration of the IQK will allow non-laboratory staff such as spray team supervisors to establish robust quality assurance procedures for DDT dosing that are vital to support the South Asian VL elimination efforts.

In spite of the recent increase in endoperoxide antimalarials under development, it remains unclear if all these chemotypes share a common mechanism of action. This is important since it will influence cross‐resistance risks between the different classes. Here we investigate this proposition using novel clickable 1,2,4‐trioxolane activity based protein‐profiling probes (ABPPs). ABPPs with potent antimalarial activity were able to alkylate protein target(s) within the asexual erythrocytic stage of Plasmodium falciparum (3D7). Importantly, comparison of the alkylation fingerprint with that generated from an artemisinin ABPP equivalent confirms a highly conserved alkylation profile, with both endoperoxide classes targeting proteins in the glycolytic, hemoglobin degradation, antioxidant defence, protein synthesis and protein stress pathways, essential biological processes for plasmodial survival. The alkylation signatures of the two chemotypes show significant overlap (ca. 90 %) both qualitatively and semi‐quantitatively, suggesting a common mechanism of action that raises concerns about potential cross‐resistance liabilities.