Long-lasting insecticide-treated nets (LLINs) are currently preferred to conventionally insecticide-treated nets (ITNs) for use in malaria control programmes [1–4]. Although LLINs offer an alternative solution to regular net re-treatment, their actual wash durability under field conditions is not known. For example the frequency of washing, the washing methods, and drying regimens that are used in the field are some of the factors which are likely to affect their efficacy and durability. The standard WHOPES recommended washing procedure is not applicable under field conditions and might be underestimating the abrasive washing practices that are used in the field and, therefore, overestimating the biological durability of LLINs in many countries [5, 6]. The manufacturers of LLINs, suggest that these kind of products do not require any further re-treatment throughout their lifetime, because they are treated with special binders which are wash durable [7]. Several studies undertaken under specific environmental conditions have since confirmed that LLINs offer longer time protection than conventional ITNs, [8–10]. The challenge is now to understand how these products will perform under various climatic conditions in the tropics where they are expected to be used.

Past studies have demonstrated different wash resistance of different brands of LLINs from one region to the other and even between laboratories [11, 12]. Due to these differences, WHOPES has provided guidelines on standardized procedures for washing and drying of LLINs for the purposes of comparison of similar products between different laboratories [13]. However, it may not be possible to standardize washing methods in every region or country due to local economics and general practices. It is, therefore, necessary to undertake studies like this, so as to understand and document how different washing and drying regimens can affect the performance of these new innovations and give guidelines on the best washing and drying procedures.

LLINs have been promoted for use in reducing human-vector contact for a long time. However, there is limited information on the effect of different drying regimens and washing methods and how they affect the efficacy of current brands of LLINs, especially under tropical conditions. A recent study in Iran showed that the effect of exposing PermaNet for a short period to direct sunlight during drying was much smaller, and that drying nets for long hours in the sun is not harmful [14]. It has also been demonstrated that direct sunlight is harmful to pyrethroid-based insecticide because uv rays break down pyrethrin molecules thereby rendering the insecticide ineffective [15]. However, the role played by sunlight in enhancing or rendering the pytherins ineffective still remains, controversial. Two studies conducted in India, [16] and [17], showed that exposing Olyset to sunlight for a few hours enhances efficacy. On the 4^th update report on LLINs, WHO after reviewing several studies on efficacy of Olyset, recommended placing the nets in polythene bags and exposing them to sunlight for a few hours to enhance heat assisted insecticide migration to the surface at the same time preventing the effect of UV light after washing [1]. It has also been documented that heat accelerates the rate of migration of permethrin molecules in the fibers, thereby increasing the bioavailability of insecticide on the net surface, especially if they are polyethylene based. In one laboratory study [18] Olyset and PermaNet were equally exposed to a controlled temperature of 60°C for 4 hrs after repeated washing, only Olyset, regenerated. Heat and not sunlight appears to be the main contributing factor in the regeneration of polyethylene based nets. In the current study, the washing durability and different drying regimens of four brands of LLINs under local conditions were evaluated. Two of the LLINs; BASF and TNT, have recently been approved by World Health Pesticide Evaluation Scheme (WHOPES) and there is no information about their performance under field conditions. The study also compared the traditional washing methods of hand rubbing and beating on rocks commonly used in local villages to the standard WHO recommended procedure that uses a standard washing machine and a specific soap

The current study found that the effectiveness and bio-durability of LLINs is influenced by washing methods and drying regimen that are used in the field. Overall, nets that were washed and dried hanging under the shade significantly retained more insecticide and were more effective compared to nets that were washed and dried using other regimens. Generally nets that were dried spread on the ground in direct sunlight lost the most amount of insecticide and were least effective. This finding suggests that contact between wet nets and the ground seem to facilitate transfer of insecticide from netting to the ground or to other surfaces. Unfortunately this is the most preferred method of drying nets in local villages where they are used for malaria vector contro1. The study also found that nets that were washed by the standard WHOPES washing protocol retained more insecticide and were generally more effective in killing mosquitoes than nets that were washed using a local method of beating on rocks. Among the factors that affect efficacy of LLINs, washing seem to be the most important and widely studied. In the current study, it was observed that generally, washing of LLINs gradually removed insecticides on all the four brands of that were evaluated regardless of washing method used. However, the rate at which the insecticide loss occurred was not uniform. It varied by washing method used, (abrasiveness) and the net type (brand). When the bio-efficacy was also measured, using the standard WHO cone bioassay method, mortalities of An. gambiae also varied with the number of washes, net brand and washing method. When the residual insecticide contents were compared using statistical procedures, it was also found that, the four drying regimens and the three washing procedures were significantly different and each net brand responded different to each drying and washing method. Nets that were washed by beating on rocks lost insecticide more rapidly and were generally less effective compared to nets washed by hand rubbing and by machine. The finding of the current study suggests that the standard WHOPES washing protocol of using specific machines and soap might be underestimates the actual insecticide loss on nets and overestimating their efficacy and durability under real field conditions where abrasive washing procedures are more abrasive. For example, after the 20^th wash, none of the nets achieved >80% mortality on the susceptible strain of An. gambiae ss by all the three washing methods.

There are very few studies that have evaluated the effect of different drying regimens on the above LLINs, two of which have been recently recommended as LLINs. The current study attempted to do so. In some unrelated earlier studies, it had been shown that direct sunlight was harmful to pyrethroids based insecticides because UV rays breaks down pyrethrin molecules thereby rendering the insecticide infective. On the basis of this earlier evidence, WHO recommended placing Olyset^® in sealed polythene bags and leaving them for a few hours in the sun after washing [1]. The idea was to facilitate insecticide migration to the surface by heating the net at the same time protecting it from uv rays. This recommendation was arbitrary withdrawn because it was cumbersome and the manufacturers of Olyset^® claimed that the incorporation of special binders, will, together with high temperatures in the tropics, facilitated automatic insecticide migration to the surface at the same time stabilize them against the effect of UV rays [2]. In the current study it was observed that overall, nets that were dried hanging on line under the shade, generally retained more insecticide. Individually Olyset and BASF brands of LLINs retained more insecticide when dried under the shade. While, the insecticide loss by drying PermaNet and TNT using the four regimens were not statistically different It would have been expected that the trend in insecticide retention by BASF and PermaNet, both of which are polyester based would be the same while that one of TNT and Olyset would also be the same, but this was not observed in the current study. The role played by sunlight in enhancing or rendering the pyrethrins ineffective still remains, controversial. Two studies conducted recently in India, [16, 17] showed that exposing Olyset^® to sunlight for a few hours enhanced efficacy. The current study also observed that Olyset generally retained more insecticide when it was dried in direct sunlight or hanging under the shade compared to other nets. The high insecticide retention by Olyset^® netting recorded in this study and its unavailability on the surface to cause corresponding mosquito mortality suggests that this requirement is still very important. This observation is supported by other studies which have documented that heat accelerates the rate of migration of permethrin molecules in the fibers, thereby increasing the bioavailability of insecticide on the net surface, especially if they are polyethylene based. In one such study Olyset^® and PermaNet^® were equally exposed to a controlled temperature of 30 and 60°C for 4 hrs after repeated washing, Olyset^®, regenerated at 60°C to achieve >90% of mosquitoes exposed and not PermaNet^®[7]. The current study also observed that even, though PermaNet retained a higher amount of insecticide when dried hanging under the shade compared to other drying regimens, the differences were not statistically significant. The same trend was observed on TNT. In contrast, there were differences in insecticide loss when BSF was air-dried hanging under the shade compared when it was dried hanging in direct sunlight. The findings of the current study, concurs with the results of a similar study conducted in Iran [14]. In the Iran study, Kayedi and others showed that drying PermaNet in direct sunlight for extended period of time of more than 3 hours was harmful. In the current study the four brands of LLINs used were left to dry for a specific period of 4 h.

The current study also compared the bio-efficacy of the above LLINs after repeated washing using WHOPES washing protocol and two local methods of hand rubbing and beating on rocks. The study found that in general nets that were washed using the WHOPES protocol remained effective longer than nets that were washed using local methods. When the differences were modelled by net type and washing method, it was observed that Olyset, PermaNet and BASF performed better when hand washed compared to machine wash. TNT and PermaNet performed better when washed by machine compared to washing on rocks. There was no difference between washing Olyset on rock and machine. The finding of the current study on some of the nets evaluated concurs with the results of a recent study by Sreehari [20]. The study, compared the bio-efficacy of three LLINs: PermaNet^®, Olyset^® and K-O Tab 1, 2, 3^® against An. culicifacies and An. stephensi after repeated washing using machine and hand wash and found that all the three LLINs that were evaluated, retained a significant amount of insecticide after repeated washing by both machine and hand. The study concluded that, nets washed by hand remained effective longer than nets washed by machine. In the current study it was observed that generally, PermaNet^® retained its efficacy longer with successive washes using all the three washing methods compared to other three brands of LLINs, but hand washing performed better on this net than machine washing while washing on rocks performed worst. This finding concurs with other studies conducted elsewhere, for example, in Iran Kayedi et al[14, 21] after evaluating wash resistance of 3 brands after repeated washing found that PermaNet^® was more effective. Elsewhere Gimnig et al[18] also compared wash resistant and biological activity of five brands of long lasting insecticide treated nets; (PermaNet^®, Olyset^®, Dawa^®, Insector^® and Athanor^®, with a conventionally treated net under laboratory, and found that PermaNet^® was the most biologically effective while Olyset^® was the most wash-resistant but biologically least biologically effective. The rest of the nets exhibited wide variation in insecticide retention and biological activity.

There are no comparable results for the two newer net brands that were evaluated, (BASF and TNT). But their performances in the current study suggest that they are significantly different in efficacy and durability compared to Olyset and PermaNet. The findings that nets dried spread on the ground generally lost more insecticide and were less effective compared to other drying regimens has major implication on LLINs long-term usage in the rural villages because it is the most preferred drying method. The results suggest that contact between wet net and ground accelerated the rate of insecticide loss from net surfaces. It is possible that insecticide molecules might be migrating from inside the netting to the ground or being denatured by exposure to sunlight. In the current study nets were left in place to dry for a defined period of four hours. In rural areas, it is a normal practice for washed nets to be left outdoor drying for an extended period, sometimes for the whole day. This means that LLINs used in the rural villages might be loosing insecticide at a faster rate depending on the washing frequency and subsequent drying using this regimen.

It is not possible to standardise washing procedures in the field, but there is need to educate rural people on the need to adopt gentle washing on LLINs. The washing of LLINs by beating on hard surfaces such as rocks is a commonly used procedure in the rural areas. This method has not been widely evaluated and there are no comparable studies. The use of this washing procedure is commonly applied on white nettings that have accumulated a lot of dirt. The use of alternative colours for LLINs can indirectly minimise the use of this abrasive method. There is need therefore, to conduct more research and document the effect of this washing method on the long-term use of LLINs. The newer LLINs; BASF^® and TNT^®, whose protective performance was found to be better than Olyset^® and lower than PermaNet^® have a greater potential of becoming important tools in malaria vector control programmes. These products have not yet undergone extensive evaluation, hence the need for more studies on these products.

The current study has demonstrated that LLINs that were washed and dried hanging on line under the shade generally, retained more insecticide and remained effective longer than those that were dried using other regimens. The rate of insecticide loss and subsequent reduction in efficacy was also dependant on the washing method used. Nets washed by beating on rocks lost insecticide faster and were least effective compared to nets washed by hand rubbing and washing by machine. The local method of washing LLINs by beating on rocks and air-drying by spreading on the ground in direct sunlight as commonly practiced local villages should be discouraged.