The WHO assay is used on a global scale, including the Amazon basin [10
] and is a familiar and trusted tool. Its components are purchased from a single-source. This requisite exists to remove local error from the assay process, but it can compromise the flexibility and accessibility of the assay. The existing kit for the creation of a baseline characterization of susceptibility allows the assessment of five insecticide concentrations and a control and costs 60 USD, plus 18 USD for each set of impregnated papers [21
]. These sums can obviously spiral rapidly with expansions in the surveillance programme. In resource-poor environments, such as Peru, resistance monitoring campaigns are heavily centralized [22
], a consequence of the value and scarcity of WHO bioassay equipment, the need to plan far in advance in order to ensure a supply of valid insecticide-treated papers, and the lack of suitably trained technicians within local health authorities.
In comparison, the bottle assay has rarely been used to monitor resistance in South American countries [24
]. It is in common use elsewhere as a research aid [25
], but local health authorities around the world need comparative data on the ease and practicability of its implementation. The bottle assay requires generic 250 ml clear glass bottles, disposable plastic pipettes and a quantity of solvent. The original CDC protocol [9
] uses acetone to coat the bottles and technical insecticide as the source of active ingredient. Technical insecticide can be expensive to source (e.g. 1 g deltamethrin costs ca. 100 USD) although gram quantities are sufficient to prepare thousands of bottles. In the Amazon, acetone is expensive and its purchase restricted because of its role in the purification of cocaine.
This paper shows that ethanol (95% purity) can be used as an alternative to acetone, and that at least some formulations can be used in place of technical grade insecticides. It was also determined that once treated, pre-prepared bottles that had been capped and stored in the dark at ambient temperatures could be stored for at least 14 days and re-used on three occasions (after three uses they appear to lose effect, presumably due to the redistribution of insecticide caused by contact with mosquitoes, aspirators and moisture from the air or from mouth aspiration). Wherever bottles are to be pre-prepared and stored, similar tests should be used to define their effective shelf lifes.
Ethanol and formulated insecticides are readily available in local public health laboratories. The purchase of six clear 250 ml bottles (available in Peru from a number of suppliers), which will duplicate the capacity of the basic WHO kit outlined above, will cost less than 10 USD. The current study showed that the bottle assay characterized differences in deltamethrin susceptibility between Peruvian An. albimanus populations as effectively as the WHO assay. This suggests that it may be a valuable complementary or alternative tool under some conditions. It is important to note that, in our comparisons, a wettable powder formulation was associated with greater mortality than technical deltamethrin, and a suspension concentrate with less. These differences are unsurprising given that the uptake of insecticide is strongly affected by formulation, but it is clear that separate baselines will have to be defined for each one.
The identification of 10 μg a.i. technical deltamethrin as a diagnostic dose for both Ae. aegypti
and An. darlingi
in the bottle assay reflects the absence of pyrethroid resistance in either species in the Peruvian Amazon. This is confirmed by the results of the local public health laboratory, who undertook WHO assays in the same year that the bottle assays described in this paper were conducted. They used the same strain of Aedes, and populations of Anophelines from the villages of Libertad, Intuto and Zungarococha. They tested > 400 individuals from each locality and found no survivors at the WHO diagnostic dose [30
]. In the surveillance exercise using the bottle assay, a dose of 10 μg a.i./bottle was used to monitor susceptibility in Anophelines
at these same sites and also in the village of Ullpayacu. This surveillance exercise confirmed the deltamethrin-susceptible status of all Peruvian An. darlingi
populations. All bottle assays were conducted and completed on field-caught Anophelines, on the night of capture. In comparison, the usual procedure for the WHO assay is to collect the mosquitoes at night, maintain them at a health post or school house until the following day, and then expose them to WHO insecticide-treated papers for 1 hour. Mosquitoes are then transferred to clean holding tubes and held for a further 24 hours until the designated WHO assay endpoint [17
The total cost of the bottle assay surveillance exercise, coordinated by the technical team at the public health laboratory in Iquitos, and including all transport, materials and personnel costs, was 1,328 USD. Comparative costs for an identical exercise using the WHO kit are not available but might be considerably greater given the greater cost of the monitoring kits, the increased time needed to conduct the assays, and the centralised nature of the surveillance programme. The costs of a resistance monitoring programme utilising either method will, however, be negligible when contrasted with the economic impacts of vector borne disease and of mosquito control programmes overall. In Peru, 60% of the 87,500 malaria cases reported in 2005 occurred in the Amazon department of Loreto [22
]. Each case, including treatment and lost income, has been estimated to cost 250 USD [31
]. The total annual investment in vector control (insecticides and operational costs) in Peru is approximately 3.8 million USD (DIGESA, unpublished).