Study location
HealthNet International (HNI) maintains a working insectary and testing site at one of their field stations, the Adizai refugee settlement. The insectary rears a fully susceptible strain of An. stephensi. The conditions are maintained at 26 +/- 2°C and 75 +/- 10% RH. Contact bioassays and overnight platform trials with insectary-reared mosquitoes took place at this site.
Overnight platform trials with wild-caught mosquitoes took place at the entomological field station in Azakhel refugee settlement. Both sites are located on the banks of the Kabul River, approximately 25 km from Peshawar. The camps have existed for 22 years.
The land in Azakhel is waterlogged and the rise in the water table during the spring snow melt and summer monsoon gives rise to innumerable mosquito breeding sites. Mosquito populations begin to rise in April with the majority being culicine species; anopheline densities increase from July. Peak mosquito density occurs in August and density declines in November. Cases of Vivax malaria occur from March to November and Falciparum malaria from August to December. Constructed on the Azakhel site are ten elevated platforms each measuring 6 m × 5 m and surrounded by water-filled 'moats' to exclude any scavenging ants.
Materials
The tents are made mostly of untreated canvas. Deltamethrin treated polyethylene threads (of the same material used to make Zerofly ®) are interwoven through the canvas fabric during manufacture. This composite material has a cream and blue striped appearance (figure 1). The tents have doors at both ends; each is made up of outer canvas door-flaps and inner mosquito mesh door-flaps. The mosquito mesh door-flaps are made from PermaNet™ polyester netting, which is pre-treated with deltamethrin.
Contact bioassays
World Health Organization (WHO) plastic bioassay cones were taped to the inner surface of the tents. Non-blood fed, insectary-reared, female, susceptible, An stephensi were exposed to the tent under the cones for three or ten minutes, after which they were held under insectary conditions (26 +/- 2°C and 75 +/- 10% RH) and given access to sugar solution. Knock-down was recorded after one hour and mortality after 24 hours.
Overnight platform trials with wild-caught mosquitoes
The method of outdoor, overnight evaluation carried out at the entomological field station has been used previously to evaluate the mortality and behavioural effects of treated tents [5] treated nets [12], and treated top sheets [13, 14].
Large trap-nets (length 6 m × height 2.5 m × width 5 m), made of untreated mosquito netting, were erected above ant-proof platforms upon which the tents were erected (figure 2). Four men slept in each tent in local dress (cotton shalwar-chemise), each covered by a woollen blanket. At one end of the tent the doors were securely closed, at the other end the canvas door flaps were tied open whilst the mesh doors hung loose. The closed end was alternated nightly between the two ends of the tent.
For the first half of the night wild, host-seeking mosquitoes were collected from the outside of the trap-nets and released within. Near to the test site calves were tethered under untreated nets to supplement the number of mosquitoes attracted to the site and available for release within the trap-nets. The following morning all mosquitoes were collected from within the trap-nets, separated into dead or alive, and kept in humidified cups with sugar solution for a further 12 hours before scoring delayed mortality. All mosquitoes were categorized as blood-fed or unfed, identified to genus level and the anophelines to species level.
End-point indicators used in analysis were: dawn mortality, 24 hour mortality and blood-feeding rate (feeding inhibition). Mortality analysis gives an indication of the potential mass effect on mosquito populations and blood-feeding rate an indication of personal protection. These tests took place during November 2003. All field staff gave informed consent and were given chloroquine and proguanil prophylaxis. The procedures used were approved by the Ethics Committee of the London School of Hygiene and Tropical Medicine.
Overnight platform trials with insectary-reared mosquitoes
Overnight tests were carried out with insectary-reared mosquitoes in January and February 2004, after the natural mosquito season had ended. Tents and trap-nets were erected as above. A cow was tethered within the tents in the place of sleepers. An stephensi and the other local vectors are highly zoophilic and cattle make a suitable alternative host.
Approximately 400 unfed, 5 – 7 day old, insectary-reared, An stephensi were released into the trap-nets at dusk, collected at dawn, put in holding cages with access to sugar solution and held in insectary conditions for 24 hours. Mosquitoes were sexed and categorized as blood-fed or unfed and dead or alive. Only female mosquitoes were included in the analysis. End-points for analysis were 24 hour mortality and blood-feeding rates.
Statistical analysis
Proportional data (mortality and blood-feeding) from the platform trials and the contact bioassays were analysed using blocked logistic regression (STATA 6 software). Comparisons between treatments were made by successively dropping treatments from the overall comparison. This process allows each treatment to be compared with every other. Means and confidence limits of the constant for each treatment were back-transformed for presentation as follows: