Study area and population
Approval for this study was obtained from the ethical committees of the Ifakara Health Research and Development Centre and the National Institute of Medical Research in Tanzania (Ref: NIMR/HQ/R.8a/Vol. VII/114), and of the Swiss Tropical Institute in Basel, Switzerland.
Two villages where Olyset™ nets had been distributed from 1994 to 1995 were selected for the study. In both settings the weather pattern is characterized by a dry season and a rainy season from December to April.
Mbwawa is a rural village located about 60 km West of Dar es Salaam and 8 km north of the main Tanzanian-Zambia highway. The population belongs to the Zaramo tribe and consists of 1690 individuals in 399 households whose main occupation is small-scale farming of maize, plantains, tomatoes and other fruits. Parasite rate in children under 10 years was 75% (caused nearly always by Plasmodium falciparum) and the entomological inoculation rate peaked at 7.4 infective bites per night during the main transmission season [12].
Mvumi Mission is a large rural village situated 40 km south-east of Tanzania's capital Dodoma with a current population of about 11,576 distributed among 2,315 households. Inhabitants of Mvumi are mainly Wagogo who are small-scale farmers. Crops in this area are sorghum, millet, maize, groundnuts and grapes. There is a Mission Hospital in Mvumi [14]. In a cross-sectional survey carried out in July 1994 the malaria prevalence was found to be 57% in children under 10 years, with Plasmodium falciparum accounting for 63% of positive slides [13].
Mosquito net distribution
Mbwawa
The only source of Olyset™ nets produced by Sumitomo Chemicals Co. Ltd. (Japan) has been the distribution of free nets in December 1994 in the frame of a scientific trial [12]. In total 1,300 nets were distributed, with one to three nets being given to each household. The nets were of blue colour and had a size of 1.1 × 1.8 × 1.5 metres. Since the expansion of a large-scale social marketing project (SMITN) to the Coastal Region in the late 1990s and the general expansion of the commercial market for nets, polyester mosquito nets became largely available for sale in the area. No coverage figures were available before the present study.
Mvumi
As a response to the increase in malaria cases in the region a total of 3,900 Olyset™ nets of the same size as above were distributed by the District Malaria Control Unit to villagers between January and March 1995. A further 450 nets were released between January and April 1996 [13, 15]. In the present study only the nets distributed in early 1995 were followed up. Nets were given out at the highly subsidised price of TSh. 800 (approx. USD 1.5 in 1995) per net. In 1998, three years later, the SMITN social marketing project was launched in Dodoma Region and Mvumi was one of the pilot villages where different distribution strategies were tried out. Two types of polyester nets, bundled with deltamethrin tablets branded Ngao, were sold in the village through the project. One of the nets (brand Lea Mwana) was sold by the Hospital's mother and child clinic at a subsided price of TSh 2500 (USD 3 in 1998) to pregnant women and children under 5 years. This net had a blue colour similar to the Olyset™ net and measured 1.5 × 1.8 × 1.8 metres. The second net (brand Njozi Njema) was smaller (1.2 × 1.8 × 1.8 metres), green and sold for TSh 3500 (USD 4.2 in 1998) at the hospital pharmacy and local shops to the general population. From 1998 onwards there was, therefore, good access to polyester nets.
Household questionnaire
In Mvumi a list of households which had received Olyset™ nets in 1995 was provided by the Village Health Officer. Households belonging to eight different parts of the village were randomly selected from the list until a total of 50 households were obtained in which the occupants still possessed at least one Olyset™ net. Tracking fifty nets allowed us to assess basic net parameters with an acceptable precision, while not over-stretching field capacity in this labour-intensive follow-up. Households which had been given an Olyset™ net but did not have it anymore were also interviewed using a shorter version of the questionnaire in order to find out why this was so. A similar two-step random sampling approach was used in Mbwawa.
Once permission had been secured from the village authorities and consent obtained from a senior household member, a structured questionnaire was applied. The first part of the questionnaire assessed the presence, current state and use of the Olyset™ net(s), as well as its(their) re-treatment with insecticide.
The second part of the questionnaire assessed the opinion of people with regard to the advantages and disadvantages of Olyset™ nets compared to ordinary polyester nets, the performance of the nets when new and at the time of study.
Finally, the preference for Olyset™ nets or other nets marketed in the village was assessed with the following hypothetical question: if you had to buy a new net today and had the money for it, would you prefer to buy an Olysef™ net (Olyset™ net shown to the person) or such a net (two different polyester nets shown)? All these nets cost the same price. Please explain your choice. In order to exclude net cost as a factor influencing the choice of net, interviewers made a strong point in stating that all nets presented to the respondent had the same price. In the case of Olyset™ nets this was possible since Olyset™ nets were not on sale at that time. Two of the most frequently sold polyester mosquito nets available on the market on each study site were presented along with a new Olyset™ net, all in their original packaging. Since the polyester nets were not pre-treated but sold with an insecticide treatment kit, the Olyset™ net was offered with an insecticide kit as well.
Olyset™ net sampling and testing
Every tenth household interviewed was offered a new large, white polyester insecticide-treated net plus three insecticide sachets for re-treatment in exchange for their used Olyset™ net. Hence, five Olyset™ nets were collected in each site. These nets were individually packaged in plastic bags and kept at room temperature until shipment for testing. Keeping them in a refrigerator or freezer was not deemed necessary since the nets had already been left for seven years at room temperature.
A first set of 50 × 50 cm samples from each of the 10 nets was sent to the manufacturer (Sumitomo Chemicals Co. Ltd.) for permethrin content assessment. Testing was performed following standard procedures outlined in a WHO document (WHO/IS/NI/331/2002). Standard three minutes exposure bioassays were also conducted on these nets, but unfortunately only with Aedes aegypti and results are not reported here.
A second set of samples was sent to the Agricultural Research Centre in Gembloux, Belgium, a WHO Collaborating Centre. In order to measure permethrin content on the surface of the yarn a 10 × 10 cm piece (corresponding to about 600 mg) was cut from each Olyset™ net sample, accurately weighed and introduced into a 100 ml conical flask. Thirty ml of acetone were added and the flask was shaken for one minute. The acetone extract was quantitatively transferred into a 50 ml volumetric flask. Two ml of an internal standard solution (diethylhexyl adipate) were added and the flask was filled up to volume with acetone. The extract was put into an injection vial and analysed by capillary gas chromatography with flame ionisation detection (GC-FID) for determination of permethrin using the internal standard calibration. In order to determine the permethrin content inside the Olyset™ yarn after the first acetone wash, the sample was cut into 2–3 mm pieces and homogenised. A sub-sample of 400 mg was accurately weighed and introduced into a 100 ml conical flask. Thirty ml of xylene were added and permethrin was extracted by heating under reflux for 60 minutes. The extract was allowed to reach ambient temperature and quantitatively transferred into a 50 ml volumetric flask. Two ml of an internal standard solution (diethylhexyl adipate) were added and the flask was filled up to volume with xylene. The extract was put into an injection vial and analysed as above.
Finally, bioassay results for Anopheles sp. were obtained from 20 × 20 cms samples sent to a WHO Collaborating Centre (Laboratoire de Lutte contre les Insectes Nuisibles) in Montpellier, France. Tests were done using standard WHO cones with a three minutes exposure time. In order to have an accurate exposure, batches of only five female mosquitoes (An. gambiae, Kisumu susceptible strain) were introduced in cones at a time. Ten repeats were done for each net sample (50 mosquitoes total). After testing, mosquitoes were grouped in plastic cups covered with netting with honey solution provided and maintained for 24 h at 30°C and 80% humidity. Percentage mortality was noted after 24 hours according to two criteria: "effective" mortality (i.e. mosquito looks dead and does not move any more, the endpoint in the standard WHO methodology) and "functional" mortality (mosquito still moves but in such a feeble and uncoordinated way that it would not survive in a natural environment). Knock-down (KD) rates were noted 60 minutes after exposure.
Data analysis
Quantitative data was summarized using proportions and means. Comparison of proportions between categorical variables was performed by a chi-squared test using STATA (STATA version 7, TX, USA). Fisher's exact tests of significance were obtained where appropriate. Comparison between quantitative variables (such as insecticide content) was done using simple linear regression. Means were compared by the non-parametric Kruskall-Wallis test. Significance was determined at the 5% level. Data was stratified by village when significant differences were found.