The field test was carried out according to the guidelines of the WHO [3, 11]. The description of the test products and test procedures are given below.

### Test products

Bioefficacy and physical durability of Olyset® Plus and Olyset® Net, both manufactured by Sumitomo Chemical, were evaluated. The former is a polyethylene monofilament network with incorporated 2% permethrin corresponding to 20 g permethrin active ingredient (AI) / kg or 800 mg permethrin AI / m2 and 1% PBO (corresponding to 10 g PBO AI / kg or 400 mg PBO AI / m2). The latter is a polyethylene monofilament network into which 2% permethrin (w / w) are incorporated (corresponding to 800 mg permethrin AI / m2). Olyset® Net was recommended in 2009 by the WHO Pesticide Evaluation Scheme (WHOPES) for use in malaria control [12], while Olyset® Plus was issued a preliminary recommendation by the WHO in 2012 with the condition that the product be evaluated in a 3-year long-term study to confirm its long-term effectiveness in the field [13].

### Study design and area of ​​study

We conducted a prospective, randomized, controlled, large-scale 3-year field trial in a rice-irrigation area in Kirinyaga County, Kenya, from 2014 to 2017. The area is located about 100 km northeast of Nairobi at the foot of Mt altitude of about 1200 m above sea level [14]. It receives average annual rainfall of 1200–1600 mm with long rains from March to June and short rains from October to December. Due to the year-round high mosquito density, many locals use mosquito nets. Several mosquito net tests for national product registration have already been carried out here. Over 80% of the region’s residents have only primary education. Most of the houses in the area are made of adobe walls with corrugated iron roofs [14].

Four villages Maendeleo, Huruma, Kiratina and Kasarani were selected by a simple randomization, of which the first two villages randomly received Olyset® Plus and the other two villages Olyset® Net (Table 1). Households were selected at random in each of the selected villages.

Table 1 Summary of the networks distributed in the study villages

### Community awareness and net distribution

At the community level, meetings were organized with local leadership (chiefs, sub-chiefs and village chiefs) and health teams, including community health workers, at which the objectives of the study were clearly explained. Local public meetings (barazas) were later organized to raise awareness of the study in the community. Consent to participate in the study was obtained from the participating heads of household. A basic survey and a census were carried out in the selected households using a structured questionnaire. Information was collected on family size, level of education, occupations, average family income, type of house, number of sleeping places, the number of nets in each household, their usage behavior and washing practices. The old nets used in households were brought back and new ones were given. Each new network was given a unique identifier and a network master list was created. The nets were then distributed to the households according to the available sleeping places, ie one net per sleeping place.

### Net sampling scheme

The scheme of sampling networks at different times for bioassays and chemical assays is given in Table 2. The mesh pieces were made from the sampled meshes from positions 1-9 for bioassays and positions HP1-HP5 (at time 0) or positions HP2. cut –HP5 (at 12, 24 and 36 months) for chemical content analyzes (Fig. 1). All nets that were sampled and recovered at the specified investigation points were replaced with new ones.

Table 2 Number of samples from Olyset® Plus and Olyset® Net for assays at different times during the study Fig. 1

A rectangular net and its individual fields with positions for cutting pieces of net (positions 1–9 for bioassays; HP1 – HP5 for chemical assays)

### Bioassays

The insecticidal effect of the nets was assessed using cone bioassays and, if necessary, tunnel tests in accordance with WHO guidelines [3]. The bioassays were performed at time zero (baseline) and then every 6 months for up to 36 months. Standard WHO plastic cones were held in position with a plastic distributor on each of the pieces of mesh cut for bioassays and a total of 50 susceptible Kisumu strain Anopheles gambiae (without blood feeding, 2-5 days old) grown in the laboratory for 3 min (5 pieces per mesh × 5 mosquitoes per test × 2 repetitions). After exposure, the mosquitoes were carefully removed from the cones and kept separately in plastic cups with cotton wool moistened with 10% glucose solution. Knockdown (KD) was recorded 60 minutes and mortality 24 hours after exposure. Mosquitos exposed to untreated polyester mesh pieces were used as untreated controls. The bioassays were carried out at a temperature of 27 ± 2 ° C and 80 ± 10% relative humidity. If the mosquito knockdown rate was <95% and the mortality <80%, the mortality and the blood-sucking inhibitory effect of such nets were assessed in a tunnel test according to WHO guidelines [3].

For each net, only one piece of net was tested in the tunnel test, in which the mosquito mortality in the cone test came as close as possible to the average mortality [3]. One hundred, not blood-fed female An. gambiae Kisumu mosquitoes, 5-8 days old, were released at the longer end of glass tunnel equipment. At each end of the tunnel, a 25 cm square cage was covered with a polyester mesh. At the shorter end of the tunnel, a rabbit was placed that could not move but was available for mosquito bites. The mosquitos were released at 6:00 p.m. and recovered at 9:00 a.m. the next morning. During the test, the tunnel was kept in complete darkness in a place with a temperature of 27 ± 2 ° C and a relative humidity of 80% ± 10%. Another tunnel with an untreated network served as a control. The mean mortality rate and percent inhibition of bloodfeeding were recorded as follows:

1. I.

Mortality = This was measured by aggregating the death rates of mosquitoes from the two sections of the tunnel. If the mortality in the control was greater than 10%, the test was considered invalid.

$${ rm {Blood}} – { rm {Feeding , Inhibition}} left (% right) , = , [{1}00 times left( {{rm{Bfc }}-{rm{ Bft}}} right)] , / { rm {Bfc}}$$

where Bfc is the proportion of blood-fed mosquitoes in the control tunnel and Bft is the proportion of blood-fed mosquitoes in the tunnel with the treated net. The treated net was assumed to meet the WHO efficacy criteria if mosquito mortality was ≥ 80% and / or blood feeding inhibition was ≥ 90%.

### Analysis of the chemical content

The chemical content in the nets was determined at the beginning and in years 1, 2 and 3 using sampling nets according to the scheme described above. The pieces of net cut to determine the active ingredient content were individually rolled up and placed in new, clean and labeled aluminum foils and sent to the Phytopharmaceuticals Department of the Walloon Agricultural Research Center, Gembloux, Belgium, a WHO collaborating center, for chemical analysis.

### Assessment of the durability of cohort networks

In order to monitor the durability of nets (i.e. survival and changes in physical integrity) during their routine use, 250 Olyset® Plus and Olyset® Net were distributed to separate households (2 nets per household) in the same villages by simple randomization. From these network cohorts, all networks available in the households at the time of the survey were examined after 6, 12, 24 and 36 months for their presence and physical integrity. For this purpose, the nets were pulled in from the sleeping areas, hung individually on a rectangular frame outside the house and examined for possible holes in the side and roof panels. The holes were counted for each mesh, their diameter was measured and they were classified into the following categories according to WHO criteria for hole sizes: [3, 11]:

Size A1: 0.5–2 cm diameter, size A2: 2–10 cm diameter, size A3: 10–25 cm diameter, size A4:> 25 cm diameter.

The parameters for assessing the integrity of networks included the proportions of Olyset® Plus and Olyset® Net with holes or cracks of any size and the proportional hole index (pHI) for each network, which was calculated as follows [11]:

pHI = (1.23 × number of holes, size A1) + (28.28 × number of holes, size A2) + (240.56 × number of holes, size A3) + (706.95 × number of holes, size A4)

On the basis of the pHI values, Olyset® Plus and Olyset® Net were categorized as “good condition” (pHI: 0–64), nets in “acceptable” condition (pHI: 65–642) and torn nets, which probably do not offer any protective effect ( pHI> 642) [15]. The number of nets in “good” and “acceptable” condition together was used to estimate the survival time of the nets over time.

The sampled nets were also examined for repairs to holes or tears by households. After the inspection, the nets were returned to the same sleeping place in the same household.

### Household net washing practices

The net washing practices of the households were evaluated 6, 12, 24 and 36 months after the net distribution using a questionnaire.