The current study evaluated the feeding success and survival rates of An. gambiae through four brands of LLINs namely Olyset, PermaNet BASF and TNT before and after repeated washing under laboratory condition. The study has shown that the LLINs which were evaluated were very effective in preventing mosquitoes from feeding through them before washing. However, as the washing progressed it was observed that the number of mosquitoes feeding gradually increased. The increase in feeding and survival rates with subsequent washing was net brand dependant. The study also observed that among the four brands of LLINs evaluated the feeding rates and survival after feeding were lower among the mosquitoes that fed through PermaNet and were highest among the mosquitoes that fed through Olyset, compared to other brands of LLINs.
There are no studies directly comparing the feeding and survival success of An. gambiae on the above brands of LLINs. However, there are related studies, which have evaluated the effect of ITNs on blood feeding and house exit behavior. In one such study Mathenge and others  evaluated the effect of permethrin-impregnated nets on exiting, blood feeding success, and time of feeding of malaria vectors in western Kenya. The study found that the number of An. gambiae s.l. entering houses was unaffected by the presence of bed nets, but An. gambiae s.s. and Anopheles funestus were more likely to exit bed-netted houses. The study also found that although there was a small shift in biting time, no differences were detected in Anopheles arabiensis rates of blood feeding and exiting. The current study seems to confirm that An. gambiae s.l. has the ability to adapt to some extend to the presence of insecticide-treated nets. The repellency effect that was observed which deterred field mosquitoes from landing and feeding was strongest before washing and appeared to diminish with repeated washing, hence the observed increase in feeding and survival rates with subsequent increase in the number of washes. This study also observed that all the LLINs evaluated were equally effective in preventing mosquitoes from feeding when unwashed. In general, repeated washing resulted in reduced mortality and an increase in survival rate. It is important to note that nets were not washed according to WHOPES protocol which uses specific soap and machine. The washing procedure and soap used in the current study is similar to the one commonly used in the local villages in rural areas. The short time interval between washes might not have accorded enough time for polyethylene based LLINs to regenerate, hence might have resulted in the observed low feeding deterrent rates observed on Olyset nets. However, in a field situation where people live and cook in single roomed houses nets accumulate dirt from soot and over-handling within a short time which often results in increase in washing frequency. The observed increase in the feeding success and survival rates also varied with net brand. The highest mortality was recorded on vectors that fed through PermaNet, and the least mortality was recorded on mosquitoes that fed on Olyset. Polyester based LLINs might have performed better than Polyethylene based LLINs due to shorter time interval required for regeneration. Olyset has been reported to take up to 15 days for self regeneration to occur , but the self regeneration period of this net is still controversial. Elsewhere after the same period of time, Olyset nets that were held at 30°C did not regenerate . The current study raises an important question on what might happen in a field situation between washes of such kind of netting. Even if the washing interval is increased to allow for the regeneration, sleepers will be exposed to field mosquitoes within the regeneration period.
The results of the current study have shown that there could be a relationship between the progressive increase in feeding success and reduction in mortality with successive increase in the number of washes. This finding concurs with the findings of other studies which have reported that the effectiveness of LLINs is affected by the number of washes. In one laboratory based study carried out at CDC in Atlanta USA, comparing wash resistant of six types of LLINs, mortality of less than 10% was recorded on Olyset brand of LLIN after only six washes using susceptible laboratory reared An. gambiae s.s. in cone bioassay tests . The current study found that among the four brands of LLINs evaluated, PermaNet was more effective. These finding concurs with another related study carried out in Iran, which compared the bio-efficacy of three brands of LLINs PermaNet, Yorkool and A-Z nets. The study found that PermaNet was more wash resistant and bio-effective . Elsewhere in a related study also carried out in Iran, evaluating the effect of washing on the bio-efficacy of Olyset using cone bioassays, Rafinejad and others observed a 97% mortality on unwashed Olyset nets and 9% mortality after 20 washes . In the current study, it was observed that more vectors succeeded in feeding through Olyset compared to BASF and TNT and those that attempted to feed through PermaNet were least successful.
Among the four LLINs, which were evaluated, Olyset and PermaNet have been on the market longest and are the most studied. There is an accumulation of field use data on the above two brands which has enabled them utilize the feed back for product improvement. PermaNet for example has evolved from PermaNet 1.0 to 2.0 and it is still undergoing improvement. However, most of the studies carried out comparing the bio-efficacy of the two markets dominated LLINs [7–9] have consistently shown that Olyset is more wash durable and less bio-effective compared to PermaNet may be because of the treatment technology used and netting material . The current study adds more evidence on the findings of the above earlier studies. Repeated washing generally affected the ability of all the LLINs to prevent mosquitoes from feeding. It is also evident that Olyset with its superior wash durability was least effective in killing and preventing mosquitoes from feeding may be because of the longer time required for insecticide to migrate from the inside of the fibres to the net surface. The current study was conducted under laboratory condition using adult mosquitoes reared from larvae and F1 generation of field collected mosquitoes from an area where PermaNet and Olyset brands of LLINs have been in use for a period of over two years. Based on these findings, it is necessary that this kind of study be expanded and carried out in a field setting using free-flying wild mosquitoes
The expected protection by LLINs against malaria vectors is based on the assumption that the products will remain effective for a longer time, killing or repelling mosquitoes in a real world situation regardless of the washing methods used. WHOPES set criteria for LLINs approval [2, 3], is only a guideline and cannot be expected to be applicable in a field situation. Based on WHOPES recommendation, it is generally believed that washing of LLINs below 20 washes has no effect on their efficacy. These assumptions are sustained by the fact that laboratory studies using susceptible strain of mosquitoes have repeatedly shown that LLINs offer long time protection . More over, previous studies had shown that sleeping under untreated net offers limited protection because when a sleeper comes in contact with the untreated netting mosquitoes can bite through. The introduction of ITNs was an improvement because they offered both physical and chemical barrier . The challenges that were encountered then were the unscheduled frequent washing by users to keep nets clean unaware of insecticide loses and re-treatment compliance was very low . The introduction of LLINs was to enable users wash their nets as needed without compromising their effectiveness. In this regard LLINs had a triple effect of combining physical and chemical barriers to washing durability of the insecticide . This study has shown that the short frequency used in washing LLINs using a local method and detergent, could cause the protective efficacy against wild mosquitoes to diminish and therefore should be avoided.
Given the current finding, it might be feasible to reduce malaria cases by half as projected at the Africa summit meeting in 2000 on Roll Back Malaria , by using LLINs in focal groups, but it might not be possible to reduce these cases beyond the above projections, even though universal net coverage and use in malaria endemic zone of western Kenya is a real possibility . Currenly, LLINs are abundantly available at subsidized prices in government and public health facilities . Before the adoption of LLINs, impressive results had been achieved in lowering malaria cases using ITNs. For example, in one control program conducted in western Kenya, Ter Kuile et al associated a 60% reduction in both clinical malaria and severe anaemia to ITNs use . In a similar study also conducted in western Kenya, Phillips-Howard et al observed a 23% protective efficacy of ITNs for children under 5 years . In the same study, a 59% drop in entomological indices and sporozoite rate was associated with ITNs use in western Kenya . In all the above studies, ITN coverage and use was under strict supervision. But it is curious to note that reduction in malaria cases did not correspond to the massive net coverage. Similar results were also recorded in Ghana, and Tanzania, [28, 29] among other sub-Sahara African countries. Because of the above successes, it was projected that the introduction of LLINs will consolidated gains that had been made and drive further down malaria transmission rates to bellow national disease burden levels among developing countries in sub-Sahara African countries like Kenya . To date this has not happened six years after official adoption of LLINs and malaria is still the leading cause of infant and childhood mortality .
From the current study, it can be speculated that there are other complex factors related to vector behavior that are not fully understood. Mosquitoes can quickly adapt to the presence of treated nets by momentarily avoiding contact when nets are freshly introduced but quickly rebound in numbers and start feeding once washing of the nets starts taking place. Validation of efficacy of new LLINs products is derived from bioassay data of directly exposing susceptible vectors to the nets without the alterative of offering them a chance to obtain a blood meal and then assessing their survival rates. The current study attempted this approach. This approach was used based on published evidence that vectors have inherent intrinsic behavior of circumventing lethal exposures to insecticide-treated materials, thereby perpetuating malaria transmission at low levels and maintaining their survival [12–14]. This can also be explained from the fact that village-wide coverage does not immediately translate into interrupting the vector human contact circle sufficiently enough to stop transmission [25, 26]. Indeed from studies conducted in western Kenya using ITNs, it was observed that despite mass distribution and evidence of daily use, which were strictly monitored by research assistants, transmission still occurred. Various explanations were then advanced. One of them was that vectors were biting earlier before people went to bed . But evidence has also been shown of community wide mass effect of treated nets [32, 33]. The observations reported by the current study possess challenges to malaria control programs of what might be happening in the filed. If wild vectors can feed through washed LLINs because of the shortened interval between washes or longer periods required after washing for some LLINs to regenerate, then it might be time for vector control experts to think on stop gap measures to use for protecting LLIN users between the regeneration periods. This might be a challenge to the whole strategy of using LLINs as a key component of controlling malaria vectors.