The WHO Phase II experimental hut trial demonstrated that the blood-feeding inhibition (the proportional reduction in biting / blood feeding) and percentage mosquito mortality induced by Interceptor LN washed 20 times was superior to that of the CTN washed to exhaustion, and therefore the LN fulfilled the WHOPES criterion of a long lasting insecticidal net . On the basis of these results, which formed part of an official WHOPES evaluation, Interceptor LN received interim recommendation as an approved LN . Earlier in 2013 the WHOPES guidelines for testing of LN were revised to include as a positive control a WHOPES-recommended LN with similar specifications to the candidate LN in terms of insecticide, treatment technique, netting material, and washing frequency (0 and 20 times) . The revised guidelines were issued after the current trial and, indeed, Interceptor now constitutes a LN appropriate to use as a positive control against new candidate LN. Because more brands of LN are being submitted to WHO for recommendation, one of the purposes of the revision is to demonstrate that new candidate LN match or exceed the standards set by previously approved LN such as Interceptor. Recent WHOPES trials have included both a reference LN washed 20 times and a CTN washed to exhaustion as comparison arms to ensure that equivalence or superiority of the reference LN to the CTN is being maintained.
The Phase I laboratory bio- and chemical assays confirmed that the Interceptor LN insecticide binding process imparts strong wash-retention characteristics. The laboratory washing regime stripped the alpha-cypermethrin from the conventionally treated net within a few washes (surface content falling from 200 to 10 mg/m2) and to levels undetectable by HPLC within 15 washes. However, in bioassays on the same CTN (washed 15 times) the median mosquito was knocked down after just 11 minutes exposure and mortality reached 28% in the 3 minute bioassays. A similar finding was observed in the Phase II experimental hut trials: the CTN washed 20 times had a surface concentration of only 1.2 mg/m2 and yet this net was still able to kill 44% of An. gambiae and 51% of An. funestus that entered and came into contact with it. The only explanation is that pyrethroids such as alpha-cypermethrin must have a strong affinity to the polyester netting fibres so that even after vigorous washing a thin layer of pyrethroid, virtually undetectable by HPLC yet sufficiently bioactive to induce knockdown and mortality, must still remain bound to the fibres.
The performance of the CTN and the level of mortality and knockdown it induced after washing, while being surprising, were still not comparable to those of the LN. Interceptor LN retained a surface concentration of over 40 mg/m2 after 20 washes (30 times greater than the CTN’s) and induced significantly higher levels of mortality than the CTN washed the same number of times. The comparison does, however, raise some issues and limitations concerning the WHOPES Phase II process. The Phase II preparation is designed to mimic the washing practices of net owning families; it cannot mimic the myriad ways in which insecticide is removed from the nets during a lifetime of use. An important source of removal during Phase III must be the abrasion a net is subjected to daily during 3 years of household use. In contrast, the only abrasion a net is subjected to during Phase II is the stirring and mashing during the 20 preparatory washes, and the 30–40 days of use during the hut trial. For this reason a WHOPES Phase II cannot anticipate or predict the outcome of evaluation after 3 years of household use. Only a WHOPES Phase III - in which nets are distributed to householders and re-gathered for testing after 3 years - can show whether a LN really does justify its WHO recommendation. For this reason the Phase II trial should only lead to an interim WHO recommendation. The WHO process is best seen as a series of gates with one phase setting a standard and leading to the next phase, rather than being predictive of the outcome of the next phase. The reality of this is demonstrated in the WHO report of the Phase III evaluation of Interceptor: it achieved the efficacy criteria of a true LN after 3 years of household use, whereas the CTN fell short of the efficacy criteria within just 1 year of use . The failure of the CTN within a year of Phase III would not be predicted by the relative mortality shown by the CTN washed to exhaustion and Interceptor LN during Phase II. This raises the question of whether the Phase II preparatory procedure should include an accelerated abrasion process between washes that better mimics the wear and tear that a net is subjected to during a lifetime of use.
Culex quinquefasciastus and Anopheles gambiae were fully capable of feeding through holed untreated nets but when the nets were treated with pyrethroid the proportion that fed was reduced substantially, from 50% to 10-20%. The level of personal protection from the LN was 75-80% for An. gambiae and 75-90% for Cx. quinquefasciatus. The results for An. gambiae were expected, the results for Cx. quinquefasciatus results were not. This is because Tanzanian Cx. quinquefasciatus are highly resistant to pyrethroids due to site insensitivity and oxidase mechanisms , less than 20% are killed by the LN or CTN, and yet very few succeeded in blood feeding. In West Africa too, pyrethroid resistant Cx quinquefasciatus struggle to feed through holed LNs or ITNs [18, 19]. In contrast, where An. gambiae has developed high level resistance due to a combination of kdr plus cytochrome P450 mechanisms  the proportion that manage to blood feed through holed LNs may increase to 60% or more [21, 22]. LN seem to lose their capacity to protect when anophelines become highly resistant yet seem to retain capacity to protect when Cx quinquefasciatus becomes resistant. The reason for the difference between genera is not clear but may be due to behavioural differences around the net. This is particularly relevant to East Africa because Cx. quinquefasciatus is an important vector of lymphatic filariasis there . The evidence from the present trial is that an LN will provide protection against Cx. quinquefasciatus-borne filariasis despite the species being resistant to pyrethroids.