Metabolic resistance to insecticides such as pyrethroids in mosquito vectors threatens control of malaria in Africa. Unless it is managed, recent gains in reducing malaria transmission could be lost. To improve monitoring and assess the impact of insecticide resistance on malaria control interventions, we elucidated the molecular basis of pyrethroid resistance in the major African malaria vector, Anopheles funestus. We showed that a single cytochrome P450 allele (CYP6P9a_R) in A. funestus reduced the efficacy of insecticide-treated bednets for preventing transmission of malaria in southern Africa. Expression of key insecticide resistance genes was detected in populations of this mosquito vector throughout Africa but varied according to the region. Signatures of selection and adaptive evolutionary traits including structural polymorphisms and cis-regulatory transcription factor binding sites were detected with evidence of selection due to the scale-up of insecticide-treated bednet use. A cis-regulatory polymorphism driving the overexpression of the major resistance gene CYP6P9a allowed us to design a DNA-based assay for cytochrome P450–mediated resistance to pyrethroid insecticides. Using this assay, we tracked the spread of pyrethroid resistance and found that it was almost fixed in mosquitoes from southern Africa but was absent from mosquitoes collected elsewhere in Africa. Furthermore, a field study in experimental huts in Cameroon demonstrated that mosquitoes carrying the resistance CYP6P9a_R allele survived and succeeded in blood feeding more often than did mosquitoes that lacked this allele. Our findings highlight the need to introduce a new generation of insecticide-treated bednets for malaria control that do not rely on pyrethroid insecticides.

Metabolic resistance to insecticides is threatening malaria control in Africa. However, the extent to which it impacts malaria transmission remains unclear. Here, we investigated the association between a marker of glutathione S-transferase mediated metabolic resistance and Plasmodium infection in field population of Anopheles funestus s.s. in comparison to the A296S-RDL target site mutation. The 119F-GSTe2 resistant allele was present in southern (Obout) (56%) and central (Mibellon) (25%) regions of Cameroon whereas the 296S-RDL resistant allele was detected at 98.5% and 15% respectively. The whole mosquito Plasmodium and sporozoite infection rates were 57% and 14.8% respectively in Obout (n = 508) and 19.7% and 5% in Mibellon (n = 360). No association was found between L119F-GSTe2 genotypes and whole mosquito infection status. However, when analyzing oocyst and sporozoite infection rates separately, the resistant homozygote 119F/F genotype was significantly more associated with Plasmodium infection in Obout than both heterozygote (OR = 2.5; P = 0.012) and homozygote susceptible (L/L119) genotypes (OR = 2.10; P = 0.013). In contrast, homozygote RDL susceptible mosquitoes (A/A296) were associated more frequently with Plasmodium infection than other genotypes (OR = 4; P = 0.03). No additive interaction was found between L119F and A296S. Sequencing of the GSTe2 gene showed no association between the polymorphism of this gene and Plasmodium infection. Glutathione S-transferase metabolic resistance is potentially increasing the vectorial capacity of resistant An . funestus mosquitoes. This could result in a possible exacerbation of malaria transmission in areas of high GSTe2-based metabolic resistance to insecticides.

BackgroundImmunochromatographic card test (ICT) is a tool to map the distribution of Wuchereria bancrofti. In areas highly endemic for loaisis in DRC and Cameroon, a relationship has been envisaged between high L. loa microfilaria (Mf) loads and ICT positivity. However, similar associations have not been demonstrated from other areas with contrasting levels of L. loa endemicity. This study investigated the cross-reactivity of ICT when mapping lymphatic filariasis (LF) in areas with contrasting endemicity levels of loiasis and mansonellosis in Cameroon.Methodology/Principal FindingsA cross-sectional study to assess the prevalence and intensity of W. bancrofti, L. loa and M. perstans was carried out in 42 villages across three regions (East, North-west and South-west) of the Cameroon rainforest domain. Diurnal blood was collected from participants for the detection of circulating filarial antigen (CFA) by ICT and assessment of Mf using a thick blood smear. Clinical manifestations of LF were also assessed. ICT positives and patients clinically diagnosed with lymphoedema were further subjected to night blood collection for the detection of W. bancrofti Mf. Overall, 2190 individuals took part in the study. Overall, 24 individuals residing in 14 communities were tested positive by ICT, with prevalence rates ranging from 0% in the South-west to 2.1% in the North-west. Lymphoedema were diagnosed in 20 individuals with the majority of cases found in the North-west (11/20), and none of them were tested positive by ICT. No Mf of W. bancrofti were found in the night blood of any individual with a positive ICT result or clinical lymphoedema. Positive ICT results were strongly associated with high L. loa Mf intensity with 21 subjects having more than 8,000 L. loa Mf ml/blood (Odds ratio = 15.4; 95%CI: 6.1–39.0; p < 0.001). Similarly, a strong positive association (Spearman’s rho = 0.900; p = 0.037) was observed between the prevalence of L. loa and ICT positivity by area: a rate of 1% or more of positive ICT results was found only in areas with an L. loa Mf prevalence above 15%. In contrast, there was no association between ICT positivity and M. perstans prevalence (Spearman’s rho = - 0.200; p = 0.747) and Mf density (Odds ratio = 1.8; 95%CI: 0.8–4.2; p = 0.192).Conclusions/SignificanceThis study has confirmed the strong association between the ICT positivity and L. loa intensity (Mf/ml of blood) at the individual level. Furthermore, the study has demonstrated that ICT positivity is strongly associated with high L. loa prevalence. These results suggest that the main confounding factor for positive ICT test card results are high levels of L. loa. The findings may indicate that W. bancrofti is much less prevalent in the Central African region where L. loa is highly endemic than previously assumed and accurate re-mapping of the region would be very useful for shrinking of the map of LF distribution.

BackgroundThe immunochromatographic test (ICT) for lymphatic filariasis is a serological test designed for unequivocal detection of circulating Wuchereria bancrofti antigen. It was validated and promoted by WHO as the primary diagnostic tool for mapping and impact monitoring for disease elimination following interventions. The initial tests for specificity and sensitivity were based on samples collected in areas free of loiasis and the results suggested a near 100 % specificity for W. bancrofti. The possibility of cross-reactivity with non-Wuchereria bancrofti antigens was not investigated until recently, when false positive results were observed in three independent studies carried out in Central Africa. Associations were demonstrated between ICT positivity and Loa loa microfilaraemia, but it was not clearly established if these false positive results were due to L. loa or can be extended to other filarial nematodes. This study brought further evidences of the cross-reactivity of ICT card with L. loa and Onchocerca ochengi (related to O. volvulus parasite) using in vivo and in vitro systems.MethodsTwo filarial/host experimental systems (L. loa-baboon and O. ochengi-cattle) and the in vitro maintenance of different stages (microfilariae, infective larvae and adult worm) of the two filariae were used in three experiments per filarial species. First, whole blood and sera samples were prepared from venous blood of patent baboons and cattle, and applied on ICT cards to detect circulating filarial antigens. Secondly, larval stages of L. loa and O. ochengi as well as O. ochengi adult males were maintained in vitro. Culture supernatants were collected and applied on ICT cards after 6, 12 and 24 h of in vitro maintenance. Finally, total worm extracts (TWE) were prepared using L. loa microfilariae (Mf) and O. ochengi microfilariae, infective larvae and adult male worms. TWE were also tested on ICT cards. For each experiment, control assays (whole blood and sera from uninfected babon/cattle, culture medium and extraction buffer) were performed.ResultsPositive ICT results were obtained with whole blood and sera of L. loa microfilaremic baboons, culture supernatants of L. loa Mf and infective larvae as well as with L. loa Mf protein extracts. In contrast, negative ICT results were observed with whole blood and sera from the O. ochengi-cattle system. Surprisingly, culture supernatant of O. ochengi adult males and total worm extracts (Mf, infective larvae and adult worm) were positive to the test.ConclusionsThis study has provided further evidence of L. loa cross-reactivity for the ICT card. All stages of L. loa seem capable of inducing the cross-reactivity. Onchocerca ochengi. can also induce cross-reactivity in vitro, but this is less likely in vivo due to the location of parasite. The availability of the parasite proteins in the blood stream determines the magnitude of the cross-reactivity. The cross-reactivity of the ICT card to these non-W. bancrofti filariae poses some doubts to the reliability and validity of the curren...