High insecticide resistance intensity of Anopheles gambiae (s.l.) and low efficacy of pyrethroid LLINs in Accra, Ghana.

BACKGROUND: Insecticide resistance of Anopheles gambiae (s.l.) against public health insecticides is increasingly reported in Ghana and need to be closely monitored. This study investigated the intensity of insecticide resistance of An. gambiae (s.l.) found in a vegetable growing area in Accra, Ghana, where insecticides, herbicides and fertilizers are massively used for plant protection. The bioefficacy of long-lasting insecticidal nets (LLINs) currently distributed in the country was also assessed to delimitate the impact of the insecticide resistance intensity on the effectiveness of those nets. METHODS: Three- to five-day-old adult mosquitoes that emerged from collected larvae from Opeibea, Accra (Ghana), were assayed using CDC bottle and WHO tube intensity assays against different insecticides. The Vgsc-L1014F and ace-1 mutations within the population were also characterized using PCR methods. Furthermore, cone bioassays against different types of LLINs were conducted to evaluate the extent and impact of the resistance of An. gambiae (s.l.) from Opeibea. RESULTS: Anopheles gambiae (s.l.) from Opeibea were resistant to all the insecticides tested with very low mortality observed against organochlorine, carbamates and pyrethroid insecticides using WHO susceptibility tests at diagnostic doses during three consecutive years of monitoring. The average frequencies of Vgsc-1014F and ace-1 in the An. gambiae (s.l.) population tested were 0.99 and 0.76, respectively. The intensity assays using both CDC bottle and WHO tubes showed high resistance intensity to pyrethroids and carbamates with survivals at 10× the diagnostic doses of the insecticides tested. Only pirimiphos methyl recorded a low resistance intensity with 100% mortality at 5× the diagnostic dose. The bioefficacy of pyrethroid LLINs ranged from 2.2 to 16.2% mortality while the PBO LLIN, PermaNet® 3.0, was 73%. CONCLUSIONS: WHO susceptibility tests using the diagnostic doses described the susceptibility status of the mosquito colony while CDC bottle and WHO tube intensity assays showed varying degrees of resistance intensity. Although both methods are not directly comparable, the indication of the resistance intensity showed the alarming insecticide resistance intensity in Opeibea and its surroundings, which could have an operational impact on the efficacy of vector control tools and particularly on pyrethroid LLINs.

Synergist bioassays: A simple method for initial metabolic resistance investigation of field Anopheles gambiae s.l. populations.

Metabolic resistance and the potential role of permeability-glycoprotein (P-gp) efflux pumps were investigated in a pyrethroid-resistant wild Anopheles gambiae s.l. Tiassalé population, using WHO susceptibility assays with deltamethrin (0.05%), with and without pre-exposure to synergists. The synergists used included an inhibitor of P-glycoprotein efflux pumps (verapamil), an inhibitor of esterases (EN 16-5), and an inhibitor of P450s and esterases (piperonyl butoxide). Pre-exposure to verapamil followed by deltamethrin led to a slight but non-significant (P=0.59) increase in mortality relative to exposure to deltamethrin alone (64.5% versus 69.2%). Similarly, pre-exposure to EN 16-5 yielded a non-significant increase in mortality (to 76.6%; P=0.85) but a significant increase in the knock down rate (from 48.3% to 78.7%; P<0.01). Pre-exposure with PBO caused a significant increase in mortality (to 93.1%; P<0.001) and knockdown rate (100%; P<0.001), which related to a 2.9 fold decrease in the resistance level. The results provide evidence that metabolic resistance mechanisms are present within the assessed mosquito population. The decrease in time to knock down of this population with deltamethrin following exposure to EN16-5 and PBO is of particular relevance to vector control, where quick knock down is a highly desired characteristic. The suspected resistance mechanisms present in this population merit further investigation through biochemical and molecular analyses for full resistance profile characterization. Bioassays with synergists can provide a quick and easy basis for initial characterization of resistant mosquito populations, without the need of preserved specimens, expensive equipment and substrates or specialized expertise.

Can piperonyl butoxide enhance the efficacy of pyrethroids against pyrethroid-resistant Aedes aegypti?

BACKGROUND: Pyrethroid resistance can be considered the main threat to the continued control of many mosquito vectors of disease. Piperonyl butoxide (PBO) has been used as a synergist to help increase the efficacy of certain insecticides. This enhancement stems from its ability to inhibit two major metabolic enzyme systems, P450s and non-specific esterases, and to enhance cuticular penetration of the insecticide. OBJECTIVE: To compare the mortality of a characterized resistant Aedes aegypti strain, Nha Trang, from Vietnam and the susceptible laboratory strain Bora Bora on netting with the pyrethroid deltamethrin (DM) alone and in combination with PBO. METHODS: Resistance mechanisms were characterized using molecular and bioassay techniques; standard PCR was used to test for the kdr target site mutation. Potential genes conferring metabolic resistance to DM were identified with microarray analysis using the Ae. aegypti 'detox chip'. These data were analysed alongside results from WHO susceptibility tests. P450, CYP9J32, was significantly overexpressed in the DM-resistant strain compared with the susceptible Bora Bora strain. Another five genes involved with oxidative stress responses in mosquitoes were also significantly overexpressed. The Nha Trang strain was homozygous for two kdr mutations. WHO cone bioassays were used to investigate mortality with incorporated DM-treated nets with and without PBO. PBO used in combination with DM resulted in higher mortality than DM alone. CONCLUSION: Synergists may have an important role to play in the future design of vector control products in an era when alternatives to pyrethroids are scarce.