Detection of insecticide resistance markers in Anopheles funestus from the Democratic Republic of the Congo using a targeted amplicon sequencing panel.

Vector control strategies have been successful in reducing the number of malaria cases and deaths globally, but the spread of insecticide resistance represents a significant threat to disease control. Insecticide resistance has been reported across Anopheles (An.) vector populations, including species within the An. funestus group. These mosquitoes are responsible for intense malaria transmission across sub-Saharan Africa, including in the Democratic Republic of the Congo (DRC), a country contributing > 12% of global malaria infections and mortality events. To support the continuous efficacy of vector control strategies, it is essential to monitor insecticide resistance using molecular surveillance tools. In this study, we developed an amplicon sequencing ("Amp-seq") approach targeting An. funestus, and using multiplex PCR, dual index barcoding, and next-generation sequencing for high throughput and low-cost applications. Using our Amp-seq approach, we screened 80 An. funestus field isolates from the DRC across a panel of nine genes with mutations linked to insecticide resistance (ace-1, CYP6P4, CYP6P9a, GSTe2, vgsc, and rdl) and mosquito speciation (cox-1, mtND5, and ITS2). Amongst the 18 non-synonymous mutations detected, was N485I, in the ace-1 gene associated with carbamate resistance. Overall, our panel represents an extendable and much-needed method for the molecular surveillance of insecticide resistance in An. funestus populations.

Investigating molecular mechanisms of insecticide resistance in the Eastern Democratic Republic of the Congo.

BACKGROUND: Malaria vector control in the Democratic Republic of the Congo is plagued by several major challenges, including inadequate infrastructure, lack of access to health care systems and preventative measures, and more recently the widespread emergence of insecticide resistance among Anopheles mosquitoes. Across 26 provinces, insecticide resistance has been reported from multiple sentinel sites. However, to date, investigation of molecular resistance mechanisms among Anopheles vector populations in DRC has been more limited. METHODS: Adult Anopheles gambiae sensu lato (s.l.) and Anopheles funestus s.l. were collected from two sites in Sud-Kivu province and one site in Haut-Uélé province and PCR-screened for the presence of 11 resistance mutations, to provide additional information on frequency of resistance mechanisms in the eastern DRC, and to critically evaluate the utility of these markers for prospective country-wide resistance monitoring. RESULTS: L1014F-kdr and L1014S-kdr were present in 75.9% and 56.7% of An. gambiae s.l. screened, respectively, with some individuals harbouring both resistant alleles. Across the three study sites, L43F-CYP4J5 allele frequency ranged from 0.42 to 0.52, with evidence for ongoing selection. G119S-ace1 was also identified in all sites but at lower levels. A triple mutant haplotype (comprising the point mutation CYP6P4-I236M, the insertion of a partial Zanzibar-like transposable element and duplication of CYP6AA1) was present at high frequencies. In An. funestus s.l. cis-regulatory polymorphisms in CYP6P9a and CYP6P9b were detected, with allele frequencies ranging from 0.82 to 0.98 and 0.65 to 0.83, respectively. CONCLUSIONS: This study screened the most up-to-date panel of DNA-based resistance markers in An. gambiae s.l. and An. funestus s.l. from the eastern DRC, where resistance data is lacking. Several new candidate markers (CYP4J5, G119S-ace1, the triple mutant, CYP6P9a and CYP6P9b) were identified, which are diagnostic of resistance to major insecticide classes, and warrant future, larger-scale monitoring in the DRC to inform vector control decisions by the National Malaria Control Programme.

Insecticide resistance profiles in malaria vector populations from Sud-Kivu in the Democratic Republic of the Congo.

BACKGROUND: Insecticide resistance has become a widespread problem causing a decline in the effectiveness of vector control tools in sub-Saharan Africa. In this situation, ongoing monitoring of vector susceptibility to insecticides is encouraged by the WHO to guide national malaria control programmes. Our study was conducted from April to November 2018 in Tchonka (Sud-Kivu, Democratic Republic of the Congo) and reported primary data on the resistance status of Anopheles funestus and Anopheles gambiae. METHODS: Insecticide susceptibility bioassays were performed on wild populations of A. funestus and A. gambiae using WHO insecticide-impregnated papers at discriminating concentration. In addition, PCR was performed to identify mosquito species and to detect kdr and ace-1R mutations involved in insecticide resistance. RESULTS: Bioassay results show resistance to all tested insecticides except pirimiphos-methyl, propoxur, fenitrothion and malathion with a mortality rate ranging from 95.48 to 99.86%. The addition of piperonyl butoxide (PBO) increased the susceptibility of vectors to deltamethrin and alpha-cypermethrin by exhibiting a mortality ranging from 91.50 to 95.86%. The kdr mutation was detected at high frequencies (approximately 0.98) within A. gambiae while ace-1R was not detected. CONCLUSIONS: This study provides useful data on the insecticide resistance profiles of malaria vector populations to better manage vector control. Our results highlight that, despite the high level of resistance, organophosphorus compounds and pyrethroids + PBO remain effective against the vectors.

Experimental hut evaluation of DawaPlus 3.0 LN and DawaPlus 4.0 LN treated with deltamethrin and PBO against free-flying populations of Anopheles gambiae s.l. in Vallée du Kou, Burkina Faso.

BACKGROUND: In view of widespread pyrethroid resistance in malaria vectors in Africa, two long-lasting insecticidal nets (LLINs) incorporated with a synergist, piperonyl butoxide (PBO), DawaPlus 3.0 (deltamethrin + PBO in the roof panel; deltamethrin alone in the side panels) and DawaPlus 4.0 (deltamethrin + PBO in all panels), were evaluated in an experimental hut trial in a rice growing irrigated area in Burkina Faso. Efficacy of nets was tested against free-flying malaria vector, Anopheles gambiae s.l., with high pyrethroid resistance involving L1014F kdr and CYP6P3P450 resistance mechanisms. METHODOLOGY: The efficacy of unwashed and 20-times washed DawaPlus 3.0 (polyethylene roof panel with 120 mg/m2 deltamethrin and 440 mg/m2 PBO; polyester side panels with deltamethrin 100 mg/m2) and DawaPlus 4.0 (same composition as roof of DawaPlus 3.0) was evaluated against DawaPlus 2.0 (80 mg/m2 deltamethrin; positive control). Volunteer sleepers and treatments were rotated in huts using a Latin square design on 63 consecutive nights during August-October 2016. Mortality, human blood-feeding inhibition, deterrence and exit rates of An. gambiae s.l. were monitored. PRINCIPAL FINDINGS: Significantly higher rates of mortality and blood-feeding inhibition were observed with unwashed DawaPlus 4.0 (36%; 47.5%) than unwashed DawaPlus 3.0 (11.8%; 33.3%), DawaPlus 2.0 (4.3%; 6.4%) or untreated net (P < 0.05). Washing reduced personal protective efficacy yet PBO-LLINs were more protective and both met the WHO criteria. CONCLUSIONS: The PBO-containing DawaPlus 4.0 significantly protected against An. gambiae s.l. in the study area. Unwashed DawaPlus 3.0 gave low to moderate protection against the positive control. PBO inhibits oxidase action; hence in areas with active malaria transmission having oxidase mechanisms, PBO nets could confer additional personal protection.

Effect of Bacillus thuringiensis var. israelensis Sugar Patches on Insecticide Resistant Anopheles gambiae s.l. Adults.

BACKGROUND: Large distribution of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) contributed to a significant decrease in malarial mortality. Unfortunately, large insecticide resistance in malaria vectors occurred and is a threat to the future use of these control approaches. The purpose of this study was to explore a new approach for vector control. Patches containing Bacillus thuringiensis var. israelensis (Bti) solubilized Cry toxins mixed with sugar were developed and tested in the laboratory with pyrethroid-resistant Anopheles gambiae s.l. using tunnel tests. METHODS: Mosquitoes were released at 6:00 p.m. into a large tunnel separated by a bed net, perforated with nine holes, from a smaller chamber with a guinea pig. Nine Bti sugar patches (BSPs) were attached to the bed net between the nine holes. Fourteen hours later (8:00 a.m.), mosquitoes were collected from the tunnel and the guinea pig chamber. Live females were kept in cups and were fed a sugar solution (5%) for 72 h and delayed mortality was followed. The results were reported as passing, blood fed and mortality rates. RESULTS: Mosquito populations that are resistant to the insecticides in the bed net, exhibited high mortality (60%) in the presence of the BSPs. Untreated bed nets with patches in the tunnel test killed 66-95% of the mosquitoes that landed and untreated bed nets were superior to treated bed nets. CONCLUSION: BSPs efficiently kill resistant mosquitoes that land on treated and untreated bed nets and thus could ultimately reduce the number of vector-borne malarial mosquitoes.

Evaluation of efficacy of Interceptor® G2, a long-lasting insecticide net coated with a mixture of chlorfenapyr and alpha-cypermethrin, against pyrethroid resistant Anopheles gambiae s.l. in Burkina Faso.

BACKGROUND: Malaria vectors have acquired widespread resistance throughout sub-Saharan Africa to many of the currently used insecticides. Hence, there is an urgent need to develop alternative strategies including the development of new insecticides for effective management of insecticide resistance. To maintain progress against malaria, it is necessary to identify other residual insecticides for mosquito nets. In the present WHOPES phase II analogue study, the utility of chlorfenapyr, a pyrrole class insecticide mixed with alpha-cypermethrin on a long-lasting mosquito bed net was evaluated against Anopheles gambiae s.l. METHODS: Bed nets treated with chlorfenapyr and alpha-cypermethrin and mixture of both compounds were tested for their efficacy on mosquitoes. Washed (20 times) and unwashed of each type of treated nets and were tested according to WHOPES guidelines. Efficacy of nets were expressed in terms of blood-feeding inhibition rate, deterrence, induced exophily and mortality rate. The evaluation was conducted in experimental huts of Vallée du Kou seven (VK7) in Burkina Faso (West Africa) following WHOPES phase II guidelines. In addition, a WHOPES phase I evaluation was also performed. RESULTS: Mixture treated nets killed significantly (P < 0.05) more mosquitoes than solo alpha-cypermethrin nets, unwashed and washed. Proportionally, this equated to mortalities of 78 and 76% (for mixture nets) compared to only 17 and 10% (for solo alpha-cypermethrin) to An. gambiae, respectively. In contrast mixture net proportions were not significantly (P > 0.05) different from nets treated with chlorfenapyr 200 mg/m2 unwashed (86%). The washed and unwashed nets treated with the mixtures resulted in personal protection against An. gambiae s.l. biting 34 and 44%. In contrast the personal protection observed for washed and unwashed alpha-cypermethrin treated nets generated (14 and 24%), and chlorfenapyr solo treated net was rather low (22%). CONCLUSION: Among all nets trialled, the combination of chlorfenapyr and alpha-cypermethrin on bed nets provided better mortality in phase II after 20 washes. Results suggest that this combination could be a potential insecticide resistance management tool for preventing malaria transmission in areas compromised by the spread of pyrethroid resistance.