Efficacy of an insecticide paint against insecticide-susceptible and resistant mosquitoes - Part 1: Laboratory evaluation

Every year, 300-500 million clinical episodes of malaria occur, resulting in about one million deaths [1]. A vast majority of these deaths involve children less than 5 years old in sub-Saharan Africa [2, 3]. The fighting of malaria in sub-Saharan Africa is mainly focused on vector control through the use of insecticide-treated nets (ITNs) and indoor residual insecticide spraying (IRS) [4, 5]. At present, pyrethroids are the only insecticides recommended for treatment of mosquito nets [6]. Despite the great value of pyrethroid-treated nets in malaria vector control, their efficacy may be threatened by resistance of major malaria vectors to this class of insecticides [7]. IRS is the main method of attacking adult mosquitoes in houses, but the technique requires teams of trained personnel and special equipment to be transported to where they are needed [8].

A present recommendation towards resistance management is alternating or using in combination different insecticides or novel strategies in the framework of an integrated vector management [9] while respecting the Stockholm Convention on Persistent Organic Pollutants (POPs) of finding sustainable alternatives to POPs in integrated pest management practices where possible [10].

Insecticide paint Inesfly 5A IGR™ is composed of two organophosphates (OPs), chlorpyriphos (1.5%) and diazinon (1.5%) and an insect growth regulator (IGR), pyriproxyfen (0,063%). The product is a white vinyl paint with an aqueous base. Active ingredients reside within Ca CO³ + resin microcapsules. The formulation allows a gradual release of active ingredients, increasing its durability. Microcapsules range from one to several hundred micrometers in size. Toxicology studies performed so far support the paint's safety in terms of irritancy (ocular, dermal and systemic), cytotoxicity and mutagenicity [11] and allergenicity [12]. Acute inhalation toxicity studies classified this paint as Category III (according to WHO) and category IV (according to EPA) - no warning label required in either case [12]. Analysis on cholinesterase levels showed no variations before/after treatment. Values were within reference values for all subjects [13].

The efficacy of Inesfly 5A IGR™ was studied under laboratory conditions for over 12 months at the Laboratoire de Lutte contre les Insectes Nuisibles/Institut de Recherche pour le Développement (LIN/IRD), the WHO reference laboratory for insecticide testing, in Montpellier, France. These highly-controlled evaluations against mosquitoes specifically resistant to the paint's insecticides were triggered by the encouraging results obtained during preliminary testing in malaria-endemic areas in Benin and Côte d'Ivoire, West Africa, against local populations of Anopheles gambiae the main malaria vector in sub-Saharan Africa. Resistance to OPs has been described in vector and pest mosquitoes in various parts of the world, including West Africa [14, 15]. The paint's efficacy was tested against laboratory strains of the urban pest Culex quinquefasciatus susceptible and resistant to OPs. At the time of the study, there was no laboratory strain of An. gambiae specifically resistant to OPs. Efficacy was studied in two ways: delayed mortality and effect of the IGR on fecundity, fertility and larval development.

After treatment with insecticide paint Inesfly 5A IGR™, 100% of OP-susceptible females died after 24-hours on all surfaces, porous and non-porous at both doses, 1 kg/6 m² and 1 kg/12 m². Killing was significant (87-100%) even against OP-resistant females on all surfaces except cement treated at the lower dose, 1 kg/12 m².

One year after initial treatment, mortality rates were still quite high, 93-100%, on non-porous surfaces (softwood and hard plastic) at both doses and against both, OP-resistant and susceptible females. On the other hand, the lethal effect on porous surfaces like cement had disappeared by six months after treatment against resistant and susceptible mosquitoes.

Long-term efficacy was an issue of porosity of materials rather than the pH of materials or the dose applied: active principles are kept in an acid pH within its microcapsule, making it more resistant to alkalinity than other conventional paints. To study whether efficacy hinged more on porosity than dose, a parallel study was performed. Cement-made surfaces painted with a control layer and an insecticide paint layer at 1 kg/6 m², performed as well as two insecticide paint layers at 1 kg/6 m², even though the latter had twice the dose (Mosqueira et al., unpublished data). Hence, the first layer acted as a screen (even if it did not have insecticide) that allowed the bioavailability of the insecticide on the second layer. Porosity is also an issue for IRS. DDT may last for six months on cement surfaces, though it usually leaves walls stained [19].

A Phase II field study on this same paint, Inesfly 5A IGR™, was performed in Benin, West Africa for one year against local An. gambiae and Cx. quinquefasciatus populations resistant to pyrethroids. Experimental houses were built with locally-made cement. Long-term efficacy tests included 30 minute-WHO bioassay cones using the insecticide susceptible reference strain Cx. quinquefasciatus S-Lab. Six months after treatment, mortality rates in the Phase II study on cement-made surfaces treated with one layer at 1 Kg/6 m² were still very high, 98-100% [20] compared to the 3% observed in the Phase I study. The difference observed in the long-term efficacy may be due to the type of cement used in Phase I and II, ready-mixed cement versus traditionally made cement, respectively. The greater the proportion of water to cement, the more porous the hardened cement will be. To test this hypothesis, Phase I surfaces with locally made cement were made in Benin. Surfaces were kept away from light when not tested. Temperature and humidity were the same to Phase II experimental houses. Mortality rates were lower on the Phase I Benin surfaces but differences were not significant compared to Phase II cement houses (Mosqueira et al., unpublished data) as opposed to the mortality rates obtained on Phase I mixed-cement surfaces.

Another recent study has tested the efficacy and the residual effect of Inesfly 5A IGR™ insecticide paint against the main vector of Chagas disease in South America, Triatoma infestans, on different surfaces (wood, cement block and adobe bricks). Insecticide paint yielded longer and higher mortality rates in triatomines than other conventional products [21], and porosity also seemed to be an issue - cement surfaces performed worse than wood and even adobe-made surfaces. Insecticide paints have been used for some time concomitantly with home improvement as a control method for Chagas disease with good results [22, 23].

Pyriproxyfen is toxic to a broad spectrum of insects during their developmental stages. Research on the dengue vector, Aedes aegypti, shows that contaminated adults can render oviposition sites unproductive by horizontal dissemination of pyriproxyfen even at small concentrations [24–26]. A study performed by Itoh et al [24] showed pyriproxyfen had a larger impact on fecundity when females were exposed to pyriproxyfen before blood feeding. Inversely, pyriproxyfen's effect on egg-hatching [24, 27, 28] and adult emergence [24, 28] seems to be higher when females have blood fed before being exposed to treatment.

In the present study, the effect of pyriproxyfen was studied on OP-resistant Cx quinquefasciatus females that survived a 30-minute exposition to cement-treated surfaces. Cement surfaces were chosen because, being the most porous, they were the only ones that left enough females alive to follow their offspring. Females were exposed to treated surfaces about 36 hours before blood feeding, a timing that would favour a reduction in fecundity over fertility and adult emergence. This is in fact the observation made. For the first nine months, a reduction in fecundity was observed at both doses. A reduction in adult emergence was observed also for nine months but only at the higher dose. An effect on fertility was only observed after treatment and not after nine months. In a recent Phase I evaluation on adult Anopheles stephensi females exposed to pyriproxyfen 2% one day after blood feeding, results were the opposite. A reduction in fertility in treatment groups compared to control, whereas fecundity was also reduced but differences failed to be significant [29]. The potential application of horizontal dissemination in malaria vector control needs to be studied [30]. Could the pyriproxyfen that was picked up by females that have survived a prolonged contact with painted walls be then transferred to the oviposition sites of malaria vectors? A project is in progress on different surfaces and blood feeding timing.

Results on non-porous surfaces are satisfying. There is a need to look for ways to deal with the porosity of surfaces like cement. Possible options may include two layers of paint, as discussed above, applying a coating resin, or even natural oil sealers first. The way surfaces are made also makes a difference: cement surfaces can be made less porous depending on the proportion of substances used. Hardwood is more porous than softwood. What would seem clear is that solutions need to be "user-friendly" and appealing in keeping with one of the paint's operational advantages.

There may be a reason to be optimistic about the potential that the insecticide paint may have as an additional tool in malaria and pest control: 1) High long-term killing rates against OP-resistant mosquitoes, 2) IGR's effect on fecundity, fertility and adult emergence and, 3) operational advantages: users can apply the paint themselves and take responsibility for their home improvement.

Laboratory assays against OP-resistant Cx. quinquefasciatus point at the paint's potential in attaining high mortality rates for up to 12 months despite resistance status. Ways to deal with the porosity of certain materials need to be explored. Pyriproxyfen's effect on the fecundity, fertility and adult emergence of exposed adult females affords an added tool in reducing overall pest and malaria vector population densities when the lethal effect of OPs diminishes over time. The paint is easily applied and improves communities' homes. A semi-field study performed following WHOPES Phase II procedures in Benin, West Africa against local populations of pyrethroid-resistant An. gambiae and Cx. quinquefasciatus populations has confirmed the product's promising profile. Future goals include performing a large-scale entomological, epidemiological and community acceptability study in West Africa.