Laboratory tunnel tests with PermaNet 3.0 produced results consistent with experimental hut trials and help to explain the trends in blood feeding and mortality associated with PermaNet 3.0 and PermaNet 2.0 in the field. The tunnel tests demonstrated a synergistic interaction of PBO and deltamethrin on roof netting against susceptible An. gambiae and both susceptible and resistant Cx. quinquefasciatus relative to netting from side panels treated with deltamethrin alone. This synergy was manifested in higher mortality, reduced passage through the holes and reduced feeding rates with netting treated with PBO-deltamethrin. The synergy in tunnels against pyrethroid resistant Cx. quinquefasciatus was progressively lost over 10 washes and fully lost after 20 washes. Cone bioassays on resistant Cx. quinquefasciatus confirmed the loss of synergy over 20 washes.
In the experimental hut trial both PermaNet 2.0 and PermaNet 3.0 induced high rates of mortality against pyrethroid susceptible An. gambiae at 0 and 20 washes and both rates exceeded that of the CTN washed to just before cut off point. On the basis of this result PermaNet 3.0, like PermaNet 2.0 before it, warrants interim approval by WHO as a LLIN . It was encouraging that the An. gambiae blood feeding rate associated with the zero washed PermaNet 3.0 was lower than with the zero washed PermaNet 2.0. After 20 washes, however, the feeding rates between PermaNet 3.0 and PermaNet 2.0 no longer differed, indicating a loss of activity under field conditions.
It was initially encouraging that the mortality rate of pyrethroid resistant Cx. quinquefasciatus in huts with zero washed PermaNet 3.0 was higher than that with zero washed PermaNet 2.0. This indicated the PBO in PermaNet 3.0 was exerting a partial synergism against Cx. quinquefasciatus. As per tunnel test results the synergism in the huts was fully lost after 20 washes. Blood feeding rates in Cx. quinquefasciatus in the huts did not differ between PermaNet 2.0 and 3.0 either in unwashed or 20 washed nets. According to Khayrandish & Wood , WHO used synergists and nerve recordings to explore the resistance in Cx. quinquefasciatus from this region, enhanced oxidases and a nerve insensitivity mechanism, probably kdr, are responsible for pyrethroid resistance.
The roof netting showed little change in chemical content (either in deltamethrin or in PBO) after twenty washes. Certainly any small change observed was not sufficient to explain the large difference in efficacy (mortality) between unwashed and 20 times washed PermaNet 3.0 in cones, tunnel tests or experimental huts. This indicates that deltamethrin, the PBO or both compounds are depleted from the surface of the fibre after 20 washes and fail to migrate sufficiently from the core to the surface to allow full regeneration. It would seem that PBO rather than deltamethrin is the compound that remains locked in the fibre. The evidence for this stems from the zero difference in mortality in the tunnel tests between the 20 times washed PermaNet 3.0 and the 20 times washed PermaNet 2.0 against resistant Cx. quinquefasciatus taken together with the higher mortality with 20 times washed PermaNet 3.0 against susceptible Cx. quinquefasciatus (S): deltamethrin must still be present on the surface of both PermaNet 2.0 and 3.0 and causing some mortality of susceptible Cx. quinquefasciatus but there seems little or no PBO left on the surface of PermaNet 3.0 netting to allow synergy in resistant Cx. quinquefasciatus.
It is possible insufficient time was given between washing and tunnel testing for regeneration of PBO to occur. However, the evidence from the hut trial indicates this is not the reason since over the six weeks in the huts the PermaNet 3.0 washed 20 times showed no difference in performance to the PermaNet 2.0 washed 20 times, but during this six week interval there was plenty of time for the PBO to migrate to the surface of fibres. The no difference in Cx. quinquefasciatus mortality between PermaNet 3.0 washed 20 times and PermaNet 2.0 washed 20 times suggests a failure to regenerate.
It is possible that the higher mortality initially seen with PermaNet 3.0 relative to PermaNet 2.0 is due more to the higher loading dose of deltamethrin than to any contribution of PBO. An appropriate control to test this hypothesis - a 'PermaNet 3.0' loaded with the same dosage of deltamethrin but containing no PBO - was not available for testing. Such a control should always be considered in future testing of combination nets.
At present there is limited evidence that mosquitoes contact the roof of the net while seeking access to the host. This may not hold for all species, and more corroborative observations are required. Unless the majority of mosquitoes respond to host odour or convection plumes in this way, the 2-in-1 concept as a tactic for managing resistance management tactic is flawed. The higher mortality of Culex in huts with unwashed PermaNet 3.0 versus unwashed PermaNet 2.0 does, however, provide some support to the concept.
It is important to note that the laboratory tests and Phase II trials reported here refer to efficacy before and after standardized washing rather than to performance under long term household use. There is limited temporal dimension to this work because the interval between the start of washing and the completion of the trials was only three months. Because pyrethroids used on nets have low vapour pressure a pyrethroid LLIN that showed high efficacy after 20 Phase II washes might, quite reasonably, be expected to remain efficacious for at least three years of household use, as reported recently for PermaNet 2.0 and Olyset LLINs [41, 42]. We have no information on how long the PBO component of PermaNet 3.0 would remain effective in the field as the synergist incorporated into netting may have different physical characteristics to pyrethroids. By contrast there is reasonable expectation on the basis of current knowledge that the pyrethroid in conventional LLIN would last for three years or more .
The negligible difference in mortality between PermaNet 3.0 and 2.0 against An. gambiae in huts either before or after washing would seem unlikely to provide additional control of An. gambiae populations; besides, mortality with PermaNet 2.0 was already very high. With pyrethroid resistant Cx. quinquefasciatus almost half survived exposure to PermaNet 3.0 in the huts and this proportion increased to 64% after washing. As a combination net designed to control pyrethroid resistance mediated by mixed function oxidase mechanisms the capacity of PermaNet 3.0 to control pyrethroid multiple resistant mosquitoes or prevent selection of resistance appears limited.