Study site
The study was carried out in Van Duc A village, Bac Lieu province (Mekong Delta), southern Vietnam (9.18353 N, 105.30339 E). In previous studies An. epiroticus, Anopheles sinensis, Anopheles subpictus, Anopheles nimpe, Anopheles campestris and Anopheles vagus were collected in the study site [15]. The main species of interest is An. epiroticus, which is also the most abundant. This population of An. epiroticus is resistant against deltamethrin, alpha-cypermethrin, etofenprox and cyfluthrin but susceptible to DDT [9]. Additional WHO tube bioassays [16] were done using propoxur 0.1% and malathion 5% at discriminative dosage to obtain a full picture of the insecticide resistance status of the species. In 2008 bioassays were done with deltamethrin 0.05% to assess differences in resistance status between 2005 and 2008.
Hut construction
Six experimental huts adapted from the huts used in West Africa were built using local construction materials (Figure 1) [17]. The huts were built on a concrete floor and had a wooden structure. The outside walls were covered with nipa-palm leaves. The lower parts of the inside walls were made of leaves and the upper part of plastic hessian sheeting. The roof was covered with palm tree leaves and inside with plastic sheeting. Each hut was surrounded by a water filled gutter to prevent the entry of scavengers such as ants. The entry side of the huts faced a large brackish water swamp. Entry slits were available in the front panel of the hut: two entry slits (0.75 m long) at each side of the door and one large slit above the door over the entire width of the front panel (3 m). These slits were designed to prevent mosquitoes from escaping once they entered the hut. The slits were covered by a plastic curtain from 5 a.m. till 6 p.m. Each hut was provided with a full screened veranda trap for collecting exophilic mosquitoes. The escape rate from the huts was evaluated by releasing 100 mosquitoes in each hut at 9 p.m. and re-capturing the mosquitoes in the morning from 6 till 8 a.m. The huts were adapted till at least 75% of mosquitoes were re-captured.
Treatment arms
Unwashed and washed Permanet 2.0 and 3.0 were compared to a negative control (Table 1). Permanet 2.0 is a polyester LLIN coated with deltamethrin. PermaNet 3.0 is a complex long-lasting insecticidal net designed for the control of insecticide resistant mosquito populations. It comprises deltamethrin-coated-polyester side panels and a deltamethrin plus piperonyl butoxide (PBO) incorporated-polyethylene roof. The multifilament polyester (75 denier) yarns of the side panels have a deltamethrin load of 85 mg AI/m2. The fabric of the lower part (70 cm) is denser compared to the higher part resulting in deltamethrin concentrations of 115 mg AI/m2. The roof is made of 100 denier polyethylene monofilament containing 4 g AI/kg deltamethrin and 25 g/kg (± 25%) PBO by incorporation. The safety of PermaNet 3.0 has been reviewed by WHO, following the WHO generic risk assessment model for insecticide treatment of mosquito nets and their subsequent use and concluded that use and washing of these treated nets do not pose an undue hazard to the user [18].
Six holes each 4 cm × 4 cm were made in each mosquito net, two in each long side and one at each end, to simulate the conditions of a torn net and to put the emphasis on testing whether the insecticidal treatment, rather than the net, effectively prevents biting of the sleepers.
Study design
The design was adapted from the WHO guidelines [19]. A base line study was conducted in December 2006 to evaluate the attractiveness of the six huts. The trial lasted for six weeks from 23 September till 28 October 2008 and treatment arms rotated weekly among the six experimental huts. Treatments were assigned randomly among huts in such a way that each treatment was tested once, during one week, in each hut according to a Latin Square Design. Six nets were used per treatment arm and each net was tested during one night of the week. Each morning the nets were removed and stored in their corresponding plastic bag. At the end of the week huts were cleaned and ventilated for one day before moving to another treatment. In each hut, a team of two adult volunteers slept under one net from 7 p.m. to 5 a.m. The six teams of volunteers rotated daily according to the Latin square design described above. Before the volunteers left the net at 5 a.m. they collected the dead and live mosquitoes inside the net. At 6 a.m., live and dead mosquitoes were collected from the hut (room) and from the veranda. Mosquitoes were identified using a standardized key for anophelines of Southeast Asia [20], counted and scored by hut and collection place as dead or alive, blood-fed, unfed or gravid. Live females were put in cups supplied with 10% sugar solution for 24 hours, after which any delayed mortality was recorded.
Ethical consideration: The protocol was approved by the ethical committees of the University Hospital of Antwerp (EC Nr 6/45/221) and of NIMPE Hanoi Vietnam. Volunteers gave written informed consent.
Bioassay
Cone bioassays, following the WHO procedures [19], were carried out on one net per arm before washing the nets, just before the trial in case the nets were washed and after the trial. Bioassays were done at NIMPE-Hanoi using 2-5 days old unfed females of a full susceptible An. dirus s.s. colony strain kept at NIMPE-Hanoi for ten years which allowed to evaluate the bio-availability of the insecticides on the net. For each tested net 10 pieces (2 × 4 side panels, 2 × 1 roof) of net were tested resulting in 50 mosquitoes per net. Mosquitoes were exposed during three minutes. Knockdown was recorded 60 minutes after exposure and mortality 24 h post exposure and compared to the WHO criteria for LLIN i.e. > 80% mortality and/or > 95% knockdown [21]. For PermaNet 3.0 washed 20 times, 30 sections were tested (10 on the lower side panels below 70 cm, 10 on the higher side panels, 10 on the roof).
Chemical analysis
Net pieces were analysed before washing, after washing before the trial and after the trial. The side panels and roof were tested separately. The chromatographic determination of deltamethrin, deltamethrin R-isomer and piperonyl butoxide was based on the CIPAC method 32+33+345/TK/(M)/3. The chemical analyses were performed by a WHO reference centre (Pesticides Research Department of the Walloon Agricultural Centre, Gembloux, Belgium).
Statistical analysis
The outcomes measured in experimental huts were: (1) the entry rate or total number of mosquitoes found in the huts. This is used to measure the deterrent effect which is the reduction in hut entry relative to the control huts, (2) the exit rate measured as the proportion of mosquitoes found in the veranda, (3) blood feeding or proportion of blood fed female mosquitoes, (4) the mortality rate calculated as the proportion of dead mosquitoes, immediate and delayed observations combined, found in the huts. The percentage personal protection was calculated as (BFC-BFT)/(BFC) * 100, where BFC is the total number of blood fed females in the control hut and BFT the total number of blood-fed female mosquitoes in the treated hut. The overall killing effect of a treatment was calculated as (DT-DC)/(TC) * 100, where DT is the total number of dead mosquitoes in the treated hut, DC the total number of dead mosquitoes in the control hut and TC is the total number of mosquitoes collected in the control hut [19]. Statistical analysis of numbers (entry rates) was done by means of a negative binomial regression. The entry, blood feeding and mortality rates were compared between control arm and intervention arms by logistic regression (Stata 9).