Brands of LLIN
A total of five brands of LLINs were distributed to households: NetProtect®, PermaNet® 2.0, DawaPlus® 2.0, Olyset Net® and Life Net® (Table 1).
NetProtect® mosquito nets were white, rectangular (length = 190, width = 180 and height = 150 cm) made of 118 denier polyethylene mono filaments and 136 holes/in2 mesh. PermaNet® 2.0 mosquito nets were white, rectangular (length = 190, width = 180 and height= 200 cm), made with 75 denier polyester filament tulle and 156 holes/inch2 mesh. DawaPlus®2.0 were white, rectangular (length = 200, width =160 and height =180 cm), made of polyester multi-filament tulle, 75 deniers and 156 holes/inch2 mesh. Olyset Net® were white, conical (circumference 1250 cm, H= 250 cm), made with a denier polyethylene mono filament tulle greater than 150 cm and 56 holes/inch2 mesh. Life Net® nets were the only mosquito nets made from polypropylene, they were white, rectangular (length =190, width =180 and height =150 cm) made with multi-filament tulle with 110 denier and 156 holes/inch2 mesh.
Aside from the Olyset Net® mosquito nets which were treated with permethrin, all other nets distributed were impregnated with deltamethrin. Olyset Net® and Life Net® were donated by OMVS (Senegal River Basin Development Organization) and BAYER AG (Germany), respectively, while the three other brands (PermaNet® 2.0, DawaPlus® 2.0 and NetProtect®) were purchased from vendors approved by manufacturers.
Study areas and LLINs distribution
The study was carried out in 2011 to 2014 in two different bio climatic areas of Senegal (Mbagame: 15° 46’59.68’’W; 16° 29’18.23” N; Thiès: 14°44’23.076” N; 16°50’6.901” W): one in the north, in the Senegal River Valley and the other in the west, in the region of Thiès (Figure 1).
Senegal River Valley
Olyset Net® mosquito nets were distributed in the village of Mbagame, a semi-urban site located in the Sahelian area in the Richard-Toll health district. The average annual temperatures are around 36-38°C during the rainy season which lasts two to three months July-September.
This area is characterized by rice crop irrigation, which is a favorable environment for Anopheles pharoensis, a species that is present year-round. Its density varies in relation to the water release cycles of the irrigated perimeters. The sporozoite rate in An. pharoensis is zero or low [19-21], however, in some locations the biting rate exceeds 100 bites per night (b/p/n). The very high use of mosquito nets throughout the district, particularly in the Walo area (an area at risk of flooding), has been attributed to be in response to this high biting rate .
Anopheles arabiensis was the most abundant vector in the area, and other species in the An. gambiaecomplex, including An. gambiae s.s., and An. melas were also identified but these were detected in very low numbers. Malaria transmission was very low in 2008 with zero prevalence in children less than five years age . In recent years, this has been designated a pre-elimination area by the NMCP, the only one in the country.
The four other types of LLINs (NetProtect®, PermaNet® 2.0, Dawaplus®2.0 and Life Net®) were distributed in five villages of the Thiès region, in the Sudano-Sahelian area in western Senegal. Four villages are located in the catchment area of the dispensary of Hanène, a village 16.3 km away from the regional capital Thiès, while the fifth is within the catchment of area of the dispensary of Notto Diobass, 6 km from Thiès. All the villages are within the health district of Thiès, located 70 km north of Dakar.
The climate in the Thiès area is Sudano-Sahelian, with a very long dry season from November to July, characterized by low rainfall, low relative humidity (15% on average), warm winds and average annual temperatures ranging from 27 ° C to 32 ° C, with a temperature range between 5°C and 10°C. The rainy season was short, lasting three to four months (July/August-October). Average annual rainfall was low and varies widely from year to year (578.3mm on average) .
Thiès is characterized by low malaria endemicity, with an incidence of cases of between 5 and 15 per 1000 inhabitants . Malaria transmission is seasonal and short. It takes place from September to December. The main vector in the area is An. arabiensis, whose larval sites are generally created by the rain , but shallow wells used for market gardening in certain villages are also very favorable larval habitats.
Mild malaria cases are treated at the household level or at the dispensary in Hanène which four of the study villages depend on. The use of insecticide impregnated mosquito nets remains the main intervention for vector control in the area.
Identification of sleeping places, estimation of the number of LLINs to be distributed and household survey
Prior to net distribution, a household survey including all residents who had spent the previous night in the household and all sleeping points was conducted in study villages. The gender of the head of the household and any schooling were noted during the survey. Material goods and standard of living indicators (presence of electricity, cooking facilities, building structure description, etc.) were noted during the survey.
Community agents were recruited in each village and after training and orientation, the agents field tested the survey sheet for the identification of sleeping spaces before data collection commenced. For each household, the number of sleeping places to be covered by LLINs prior to distribution was identified and recorded on a survey sheet and then entered in a database. Bedrooms that already had a mosquito net during the survey were not included in the study. Before the distribution of mosquito nets, households participated in an awareness day and coupons containing the number of LLINs to be received were given to future recipients of each village.
Distribution and use of LLINs
At the distribution in 2011, each LLIN was labeled with a unique identifier and recorded in a database. The labeling was done by concession number, number of households in the concession, as well as the number and rank type(s) of each LLIN introduced. The types of net distributed were randomized to obtain one-third of each type in the households of Hanène and the villages annexed to the health post. Life Net and other types of nets whose durability was not monitored due to disagreement with the local representative, were distributed to households in Ngollar.
At each household visit, information on the use, mode of drying and washing of each net was collected through a questionnaire. Use of net was confirmed in situ by the surveyor. Effective use was defined as use on the night prior to the survey visit.
Sampling of LLINs for analysis
Every six months during the three-year study period, 28-30 LLINs of each type were randomly selected in the field and replaced with new LLINs, for a total of 900 over the length of the study. The removed LLINs were assessed for physical integrity, residual insecticidal activity and chemical insecticide content analysis as described below.
Physical integrity of LLINs
The condition of each LLIN was checked every six months. Physical integrity of sampled LLINs was assessed in the laboratory by searching for holes or tears on 170 Dawa Plus® 2.0, 171 NetProtect®, 143 Life Net®, 178 Olyset Net® and 177 PermaNet® 2.0. Holes were classified as size 1 (0.5-2 cm diameter), size 2 (2-10 cm diameter) or size 3 (diameter > 10 cm) . The proportional hole index (pHI) value was calculated on each LLIN inspected per WHO guidelines [25, 26]. It is the sum of the proportional indexes by categories. The proportional index for a category was the product of the number of holes in category and the index attributed to that size: pHI= # size 1 holes + (# size 2 holes × 23) + (# size 3 holes × 196) + (# size 4 holes × 576)
The pHI allowed for the classification of nets into three categories according to established thresholds:
Good: 0 ≤ pHI ≤ 64; Damaged: 65 ≤ pHI ≤642; Torn: pHI> 642 . Size 4 holes were not considered in this study.
Residual insecticide biological efficacy
Each semester, bioassays were performed on each of the sampled mosquito nets to determine the residual insecticide effectiveness.
Bioassays were performed with an An. coluzzii strain  susceptible to deltamethrin and permethrin, the insecticides present in the different types of distributed LLINs. For each mosquito net, the bioassays were carried out on five pieces of 30cm x 30cm tulle (taken from the four sides and on the roof) and kept in aluminum foil at 4 ° C at least one month until testing.
Each section of the mosquito net was inserted between two superimposed rectangular transparent locally produced Plexiglas plates (45 cm x 25 cm). The upper plate used to hold the WHO cones  in place had four holes with a diameter equal to the internal diameter of the cones.
With a mouth aspirator, five female mosquitoes aged 3 to 5 days were introduced into the first cone, which was then immediately plugged with a cotton ball. The mosquito exposure time was three minutes per cone. To maintain exact exposure time, mosquitoes were introduced in the second, third, and forth cones at one-minute intervals after the first cone.
An independent timer measured the duration of exposure of female mosquitoes per cone. The experiment was carried out on all five sections of mosquito net, with two cones per piece (5 females tested/per cone) and a total of 50 females tested per mosquito net.
Fifty mosquitoes from the same An. coluzzii colony exposed to an untreated mosquito net served as a negative control each test day. For positive controls, three new unused nets of each type of LLIN were tested using the same method: fifty females per mosquito net, at a rate of 50 females per net. The cones used on one type of net were also used on the positive controls. At the end of the exposure, the mosquitoes were transferred into a paper cup covered with non-insecticide treated mosquito net, fed with 10% sucrose, and maintained in coolers at 28 ° C ± 2 and 80% ± 10% relative humidity. Mosquitoes were then observed at 60 min to determine the knock down (KD 60) and then after 24 hours to assess mortality.
Chemical insecticide content analysis
The residual insecticide content analysis of the removed LLINs at various time points specified in the study was carried out using either high performance liquid chromatography (HPLC) or gas chromatography at the US Centers for Disease Prevention (CDC) in Atlanta, Georgia, USA. Specimens were cut from each bed net using the sampling pattern recommended by WHOPES . For all the net samples, insecticide from five swatches of all the four sides and one from the top were extracted and analyzed. Each specimen was trimmed to a 10 cm x10 cm square (0.010m2) using a die cutting in a pneumatic press. During the cutting operation, each specimen was sandwiched between layers of aluminum foil to prevent cross-contamination. The specimen set from each net was analyzed as a group to yield an average value of insecticide concentration for the whole net. Chemical analysis was based on methods published by the Collaborative International Pesticides Analytical Council (CIPAC). Deltamethrin analysis of PermaNet®2.0 and Dawa Plus® samples was based on CIPAC Method 333 [29-31].
Extraction of deltamethrin
PermaNet® 2.0 and Dawa Plus® 2.0
Each specimen set was weighed and placed in a 125 ml screw capped Erlenmeyer flask and 50 ml of the extraction solvent mixture was added making sure that the net was completely submerged in the mixture. The flask was tightly capped and sealed with paraffin film before placing in an ultrasonic bath for 15 min. Later the flask was shaken in a bath at 25 °C for 30 min at a frequency of 155 cycles per min. The extract was transferred into a chromatographic vial after filtering through a glass syringe fitted with a 0.45μm reconstituted cellulose syringe filter.
Life Net® and NetProtect ®
Each specimen set was weighed and placed in a 250ml round-bottom boiling flask followed by the addition of 95 ml of xylene and 5 ml of a known concentration of internal standard dipropyl phthalate, (2mg/ml). The flask was fitted with a reflux condenser and heated to boiling for 30 min. After cooling, approximately 2.5ml of the extract was filtered and transferred to a glass tube and evaporated to dryness under a stream of dry nitrogen for 30 min at 60°C. A known volume (0.75ml) of the mobile phase (94/6 (v/v) isooctane/1, 4-dioxane) was used to reconstitute the residue and was transferred to a sample vial using a glass syringe fitted with a 0.45μm reconstituted cellulose syringe filter. The filtrate was centrifuged (500g) for 2min and transferred into a chromatographic vial after filtering through a glass syringe fitted with a 0.45μm reconstituted cellulose syringe filter.
High performance liquid chromatography (HPLC) analysis
The deltamethrin content was analyzed using Agilent 1200 HPLC equipped with a UV detector set at 230nm and a 150X4.6mm (i.d.) Ascentis Si 5μm column held at 40°C. The mobile phase was 94/6 (v/v) isooctane/1, 4-dioxane with a flow rate of 1.5ml/min. For each extract, three injections of 20μl were made. Injections, calibrations, and quantification of the deltamethrin content were followed as per the procedure in the Collaborative International Pesticides Analytical Council (CIPAC) 333 [28-31].
Extraction of permethrin for Olyset Net®
Permethrin analysis of Olyset® Net samples was based on CIPAC Method 331 . Each specimen set was weighed and placed in a 100ml round-bottom boiling flask, followed by heptane (45ml) and triphenyl phosphate internal standard (5.0ml of known concentration in heptane). The flask was fitted with a reflux condenser and heated to boiling for 45min. After cooling, approximately 1.5ml of the extract was transferred to a chromatographic sample vial using a glass syringe fitted with a 0.45μm reconstituted cellulose syringe filter
Gas chromatography analysis
The extracts were analyzed using an Agilent 6890N chromatograph fitted with a 30m x 0.25mm (i.d.) fused silica DB-1 capillary column coated with 0.25μm cross linked polydimethylsiloxane stationary phase. Ultra-high purity nitrogen (1.2ml/min) was used as the carrier gas. Injector port, column oven, and detector temperatures were 265°C, 240°C, and 300°C, respectively. Flame ionization (FID) was used for analyte detection. Two injections were used for each sample and the results averaged. Permethrin concentration was calculated by comparing permethrin/triphenyl phosphate peak area ratios against a calibration curve generated from solutions containing known permethrin /triphenyl phosphate mass ratios.
Data Entry and statistical analysis
Household survey data were entered on EPI data Entry 3.0, exported to Excel Microsoft office 2010 and analyzed with R software. The calculation of proportions and averages was done according to the normal law by 95% Confidence Intervals (CI). Comparisons and proportions were made by Chi-squared tests (χ2) of homogeneity. Generalized linear models were performed to understand the relationship between mosquito mortality and LLIN washing and mortality bioassay results and insecticide chemical content. The analysis of the condition of the mosquito net, the values obtained for pHI, the torn surface and its shape was used to establish one of the criteria of durability ; a mosquito net was in good condition when the pHI was between 0-64, is usable when pHI was in 65-642 and was too torn when the pHI was greater than 643 . The acceptable residual efficacy of each type of LLIN was determined based on WHO criteria .
Residual efficacy was optimal if the KD 60 or the mortality rate (MR), defined as the number of dead females relative to the total number of mosquitoes exposed per mosquito net, was ≥ 95% or ≥ 80%, respectively, after 20 washes in the laboratory or after three years of use . The proportion of mosquito nets meeting optimal bio efficacy levels of KD60 and mortality rates were determined for each type of LLINs by performing cone bioassays in the laboratory. A minimal bio-efficacy criteria of ≥75% for KD or ≥50% mortality rate was used to determine the efficacy status of LLINs . The MR was corrected by the Abbott formula  if the mortality rate for controls was less than 20% and the bioassay was redone if it was greater than 20%. Generalized linear regression models were run to estimate the relationship in mortality rate between washed and non-washed mosquito nets
The retention rate was calculated for each type of LLIN at each follow up period. It was defined as the ratio of the number of the original study mosquito nets available in households to the total number of mosquito nets initially distributed. The mosquito nets declared lost but found in the following survey were included in the calculation of the retention rate of the previous semester.
The study was approved by National Committee of Ethics and Health Research in Senegal (CNERS) under number 175 and the Office of the WHO in Senegal.