Study design and area
The general design was a prospective study with the single net as the unit of observation and multiple cross-sectional surveys for evaluation of the primary outcomes, bio-assay and chemical residue analysis. The study protocol followed the WHO guidelines for phase III field trials  with minor modifications and compared the performance of the LLIN with that of a comparable mosquito net conventionally treated with the same insecticide as used in the LLIN. Study site was five villages in Kirongo Parish, Kyenjojo District, which have been described in detail previously . In short, this is an rural area in Western Uganda with moderate climate at altitudes of 1350-1550 m and a meso- to hyperendemic malaria situation.
Nets and net treatment
The LLIN Interceptor® was provided by BASF Corporation (Research Triangle Park, NC, USA). They were white, rectangular multifilament polyester nets of 75 denier and medium size (160 × 150 × 180 cm W × H × L). Long-lasting treatment was applied at production with FENDOZIN®, a mixture of the insecticide alpha-cypermethrin with a binding polymer at a target dose for the insecticide of 6.7 g/kg or 200 mg/m2.
Nets for the conventional treatment were also white, rectangular multifilament polyester nets of 75 denier (Siamdutch Mosquito Netting Co., Bangkok, Thailand) but of size 190 × 150 × 180 cm (W × H × L). Net treatment was done by a team of trained dippers and supervised by one of the authors (AK). Nets were treated individually in basins using one sachet of 6 ml alpha-cypermethrin 6% (FENDONA®, BASF, Midrand, South Africa) and a standard amount of water adequate for the size of net. Based on the content of the sachet of 360 mg of alpha-cypermethrin and the average size of the nets of 14.1 m2 the target dose was 24.9 mg/m2. Nets were dried lying flat on the ground without direct exposure to sunlight. The dipping team was provided with adequate protective gear.
A total of 200 LLIN and 100 conventionally treated nets were provided or prepared.
After treatment 10 of the conventionally treated nets were randomly selected for the baseline assessment. Similarly, 10 of the LLIN were also selected as baseline nets at this time. All nets for distribution to households (190 LLIN, 90 conventionally treated) were identified with a unique ID number written with wash resistant. The allocation of numbers to nets was random and only the principal investigator had the allocation list. In addition, each net was also marked with a water-soluble ink as a quality control for the assessment of washing.
From previous and on-going studies a complete household list for all 5 villages was available indicating the number of beds in the household and any study net already allocated. Based on these lists study nets were randomly allocated to households by the village health workers. The net allocation list was computerized and served as a net master list. Net allocation took place in May 2006.
A survey assessing the demographic and socio-economic characteristics of all households participating in the LLIN studies was undertaken in May 2006 before net distribution.
Net follow-up surveys were then undertaken every six months in September or October and April or May with a total of eight surveys, the last being in April 2010. During the net surveys all remaining nets were assessed for usage, dirtiness (clean, slightly dirty, dirty or very dirty as subjectively judged by the interviewer), washing frequency during the past six months, method of washing and drying as well as number and size of any holes in the net. Holes were categorized in three groups:
Size 1 (finger size): Any hole not larger than 2 cm in maximum diameter
Size 2 (hand size): Any hole larger than 2 cm and less than 10 cm in maximum diameter
Size 3 (head size): Any hole larger than 10 cm in maximum diameter
Any loss of nets was also recorded and the net master list updated accordingly.
Net collections and sample preparation
From the master list nets were randomly selected for outcome evaluation with 2-3 replacement numbers drawn in case the selected nets could not be traced on the day of sampling. These lists were communicated to the field team and nets were collected after the net follow-up surveys in order to ensure that washing information for the collected net was obtained. Households received a new LLIN as replacement but these nets were not part of the study. LLIN collections were done after six, 12, 24, 36 and 42 months of follow-up with a target sample size of 40 nets each except for the sample after 42 months. As the study was originally only planned for 36 months this collection comprised of all remaining LLIN, which was 21. Conventionally treated nets were sampled at six and 12 months with a target of 40 nets each and then all remaining nets at month 24.
Sampled nets were prepared in the laboratory in the following way: each net was carefully inspected and the general condition, presence of the wash-control mark and number and sizes of holes recorded. Using specially prepared templates netting material was cut always from the same location on the net, i.e. on the long side of the net below the label and half way between roof and lower border. The size of the sample was 30 × 30 cm for bio-assay samples and 10 × 10 cm for chemical residue samples. The chemical residue sample was cut directly next to the bio-assay sample. The labelled samples were packed in aluminium foil and kept in a fridge at 4-8°C before transport to the laboratory.
For each net one sample was taken for each laboratory test except for the baseline nets for which one bio-assay and three chemical residue samples were obtained. The chemical residue samples were taken from different sides of the net to allow assessment of within-net variability of insecticide distribution.
Bio-assays were carried out by the Centers of Disease Control, Atlanta, USA using WHO standardized procedures. For the tests 2-4 day old, unfed female Anopheles gambiae s.s. (Kisumu strain) were used. This species has been well established in culture for a long time and is known to be pyrethroid sensitive. Five mosquitoes were introduced into WHO cones at a time and four cones applied simultaneously onto the net sample with a three-minute exposure of the vectors. Tests were made at 25 ± 2°C under subdued light. After exposure, females were grouped into batches of 10 or 20 in 200 mL plastic cups and maintained at 28°C ± 2°C and 80% ± 10% relative humidity with honey solution provided. For each sample, a total of 40 mosquitoes were used. For each series a control was run with no exposure and results were only used if control mortality was less than 5%. Numbers of mosquitoes knocked down were recorded at 30 and 60 minutes and knock down rate at 60 minutes (KD60) calculated. Percentage mortalities were recorded after 24 hours using immediate and delayed mortality as defined by WHO guidelines , i.e. mosquitoes were scored as dead if they could not fly or stand upright on either the side or the bottom of the paper cups. Those that had lost one or more legs and could fly and stand upright without collapsing were considered to be alive.
In May 2009, the testing was shifted to the CDC partner Kenya Medical Research Institute in Kisumu using the same mosquito strain and methodology. However, due to the development of a resistance problem of the Kisumu strain the last bio-assays (42 months) were done at the Vector Control Reference Unit, National Institute for Communicable Diseases, Johannesburg, South Africa using the An. gambiae s.s. SUA strain. The methodology differed in that the WHO tubes for vector sensitivity testing were used as exposure device introducing the netting instead of the paper. Otherwise conditions were the same as in the CDC tests.
Chemical residue analysis was done at the Wallon Agricultural Research Centre (CRA-W), Gembloux, Belgium (WHO Collaborating Centre for Quality Control of Pesticides) using the ISO 17025 accredited analytical method RESMM002. The samples were measured and weighed and then introduced into a 100 mL Erlenmeyer flask. Alpha-cypermethrin was extracted from the sample by heating under reflux for 60 minutes with 40 mL xylene. After cooling to ambient temperature the extract was quantitatively transferred into a 50 mL volumetric flask. The flask was filled up to volume with xylene. A 10 times dilution was achieved in xylene. The final extract was then analysed for determination of alpha-cypermethrin by Capillary Gas Chromatography with 63Ni Electron Capture Detection (GC-ECD) using an external standard calibration. For each sample two chromatographic injections were performed and the mean reported as g/kg and mg/m2 of alpha-cypermethin. Before the analysis of samples, the analytical method was successfully validated on its specificity, linearity of detector response, repeatability, accuracy and limit of quantification. During the analysis of samples, the performance of the analytical method was checked in order to validate the analytical results.
Data entry and analysis
All data was entered in an EpiData 3.1 data and then transferred to Stata 11.0 statistical software (Stata Corp., College Station, USA) for management and analysis. For proportions (rates) exact binomial 95% confidence intervals were used. For continuous variables either the arithmetic or geometric mean or median was used depending on the distribution of values compared to a normal distribution. For the core outcomes multivariate analysis was applied using a logit or linear regression model with all potential co-variates. For analysis of net survey results with repeated observations on the same net generalized estimation equations (gee) were used.
The primary outcome of net effectiveness was based on the bio-assay results using the following criteria:
Optimal effectiveness: KD60 ≥ 95% or functional mortality ≥ 80%
Minimal effectiveness: KD60 ≥ 75% or functional mortality ≥ 50%
While the optimal effectiveness is equivalent to the WHOPES evaluation criteria , the minimal effectiveness criteria were taken from an unpublished recommendation by WHO (Pierre Guillet).
The physical integrity of the nets was evaluated by applying a proportionate holes index (pHI) based on the number of holes per category and weighted as follows:
pHI = size 1 holes + (size 2 holes × 9) + (size 3 holes × 56) and then a mean hole index calculated for the sample or sub-sample. The multiplication factors were chosen to reflect the approximate surface areas of the hole sizes (4, 36 and 225 cm2 respectively) resulting in one unit of the pHI being equivalent to 4 cm2 of hole surface. Data were then grouped into three categories of pHI 0-24 (or maximum 100 cm2 total hole surface) representing a net in good condition, pHI 25-299 (or maximum 0.1 m2 total hole surface) for a torn net and pHI 300 or above for a severely torn net.
Since previous studies had used a "simple" hole index (sHI) with weights 1, 2, 3 for the hole sizes this index was also calculated in order to allow comparisons:
Attrition rate for the net cohort was estimated by adjusting for the potential loss from sampled nets. For each group of sampled nets the number of nets that would have been lost was calculated by applying the observed attrition rates from the remaining nets. These were then added to the actually observed attrition and divided by the total of nets distributed.
The study was conducted according to the principles of the Declaration of Helsinki and the International Guidelines for Ethical Review of Epidemiological Studies and approved by the Uganda Council of Science and Technology.