After bioassay the second piece cut from each net were individually labelled with the name of treatment group and number of washes and stored in the dark for subsequent residual insecticide quantification by high performance liquid chromatography (HPLC). For ease of identification the nets were marked with indelible ink using permanent markers before washing was started. The insecticide content of each net was determined by cutting from each net piece, smaller pieces of 2 by 2 cm then extracting the insecticide into a solution using a mixture of solvents. For deltamethrin and alphacypermethrin iso-octan plus 1, 4 dioxan with 0.15% HPLC grade water was used. Dibutyl phthalate was added as the internal standard. For permethrin acetone, 99.9% HPLC grade was used with 99.93% methyl alcohol as the internal standard. After extraction samples were thoroughly shaken to mix and then filtered by water pump suction on 0.45 micrometre membrane filter into a vial. An aliquot of 1 μL of the filtered solution was then injected onto a normal phase isocratic HPLC machine with a UV detector. The insecticide quantification was achieved using an internal calibration curve.
Statistical analysis
Statistical analyses were conducted with SAS, version 9.2. Insecticide residue on the nets after repeated washing was modelled with linear regression. Covariates which were included in this model were the number of washes, drying regimens (shade and spread on ground, sun and spread on ground, shade while hanged and sun while hanged). Mosquito mortalities were calculated using probit regression analysis with machine wash as the reference. Covariates which were included in the model were the number of washes and the washing procedures (washing by hand, machine washing and washing by beating on rocks).
Results
Determination of optimal drying regimen
It was observed that, in general, the rate at which LLINs lost insecticide when they were repeatedly washed and dried using four different drying regimens varied with the drying regimen and the net brand. Nets which were airdried hanging under the shade lost the least amount of insecticide when compared to those that were air-dried using other regimens. For example after 20 washes, the nets washed and air-dried while hanging under the shade significantly (p < 0.0001) retained more insecticide 62.5% compared to those dried hanging in direct sunlight 58.8%, spread on the ground under the shade 57.8% and spread on the ground in direct sunlight 56.3%. Among the four brands of LLINs used in the current study, Olyset brand of netting retained the highest amount of insecticide > 70% after wash 20. On this net the amount of insecticide retained was highest on the nets that were air-dried hanging under the shade, 80.7% compared to <70.8% that was recorded when dried hanging in direct sunlight. All the four brands of LLINs evaluated, with exception of TNT, recorded highest amount of insecticide on nets that were dried hanging under the shade. PermaNet recorded 27.5%, BASF 54.5%, and TNT 44%. Overall, after wash 20, PermaNet retained the least amount of insecticide between the four drying regimens 18%-27% compared to BASF and TNT of, 45 – 65% and 42 – 56% respectively; between the four wash drying regimens.