Efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr nets on entomological indicators of malaria transmission: third year of a randomised controlled trial in Benin.

The present cluster-randomised control trial aims to assess the entomological efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr LLINs compared to the standard pyrethroid-only LLINs, in their third year of community usage. Adult mosquito collections were performed every 3 months, in 4 randomly selected houses in each of the 60 trial clusters, using human landing catches. Adult mosquitoes were morphologically identified and Anopheles vectors were molecularly speciated and screened for the presence of the L1014F kdr mutation using PCR. Plasmodium falciparum sporozoite infection was assessed using ELISA. A subset of An. gambiae s.l. was also dissected to examine parity and fertility rates across study arms. There was no evidence of a significant reduction in indoor vector density and entomological inoculation rate by the pyrethroid-pyriproxyfen [DR 0.94 (95% CI 0.46-1.88), p = 0.8527; and RR 1.10 (95% CI 0.44-2.72), p = 0.8380], and pyrethroid-chlorfenapyr [DR 0.74 (95% CI 0.37-1.48), p = 0.3946; and RR 1.00 (95% CI 0.40-2.50), p = 0.9957] LLINs, respectively. The same trend was observed outdoors. Frequencies of the L1014F kdr mutation, as well as parous and fertility rates, were similar between study arms. In the third year after net distribution, entomological indicators show that the two dual active-ingredients nets performed similarly to the standard pyrethroid-only LLIN. To maintain malaria gains, it is crucial that net distribution cycles fit with their operational lifespan.

Detection of DENV-1, DENV-3, and DENV-4 Serotypes in Aedes aegypti and Aedes albopictus, and Epidemic Risk in the Departments of Oueme and Plateau, South-Eastern Benin.

Background: This study conducted in the departments of Oueme and Plateau aims to assess the presence of the dengue virus and its different serotypes in Aedes aegypti and Aedes albopictus, as well as the epidemic risk incurred by the populations. Methods: Collections of adult mosquitoes using human landing catches (HLC) were carried out in six communes, three (Porto-Novo, Adjarra, and Avrankou) in the Oueme department and the rest (Ifangni, Kétou, and Pobè) in the Plateau department. Pools of ten Aedes mosquitoes were formed, and stored at -80°C in RNA later. RT-PCR was used to detect dengue virus, and conventional PCR for the different serotypes. Inspection of water containers and collection of Aedes larvae was performed inside and around each house to calculate the stegomyan indices. Results: In the six communes, the dengue virus was present both in Ae. aegypti and Ae. albopictus. Combined data of the two Aedes species at the communes level revealed infection rates ranging from 80.00% (95% CI: 61.43-92.29) to 96.67% (95% CI: 82.78-99.92). In all the communes, the values of stegomyan indices reached the WHO threshold, which indicates the existence of the risk of an arbovirus epidemic. In addition, the infection rates were similar for Ae. aegypti [88.19% (95% CI: 81.27-93.24)] and Ae. albopictus [86.79% (95% CI: 74.66-94.52)]. The three virus serotypes detected in the pools of Aedes were DENV-1, DENV-3, and DENV-4, with a high prevalence for the first two. Conclusion: This study revealed that three serotypes (DENV-1, DENV-3, and DENV-4) of dengue virus circulate in Ae. aegypti and Ae. albopictus in the departments of Oueme and Plateau. Moreover, the risk of transmission of arboviruses was globally high and variable from commune to commune. This information is essential for informed decision-making in the preventive control of the disease.

Did the prolonged residual efficacy of clothianidin products lead to a greater reduction in vector populations and subsequent malaria transmission compared to the shorter residual efficacy of pirimiphos-methyl?

BACKGROUND: The residual activity of a clothianidin + deltamethrin mixture and clothianidin alone in IRS covered more than the period of malaria transmission in northern Benin. The aim of this study was to show whether the prolonged residual efficacy of clothianidin-based products resulted in a greater reduction in vector populations and subsequent malaria transmission compared with the shorter residual efficacy of pirimiphos-methyl. METHODS: Human bait mosquito collections by local volunteers and pyrethrum spray collections were used in 6 communes under IRS monitoring and evaluation from 2019 to 2021. ELISA/CSP and species PCR tests were performed on Anopheles gambiae sensu lato (s.l.) to determine the infectivity rate and subspecies by commune and year. The decrease in biting rate, entomological inoculation rate, incidence, inhibition of blood feeding, resting density of An. gambiae s.l. were studied and compared between insecticides per commune. RESULTS: The An. gambiae complex was the major vector throughout the study area, acounting for 98.71% (19,660/19,917) of all Anopheles mosquitoes collected. Anopheles gambiae s.l. collected was lower inside treated houses (45.19%: 4,630/10,245) than outside (54.73%: 5,607/10,245) after IRS (p < 0.001). A significant decrease (p < 0.001) in the biting rate was observed after IRS in all departments except Donga in 2021 after IRS with clothianidin 50 WG. The impact of insecticides on EIR reduction was most noticeable with pirimiphos-methyl 300 CS, followed by the clothianidin + deltamethrin mixture and finally clothianidin 50 WG. A reduction in new cases of malaria was observed in 2020, the year of mass distribution of LLINs and IRS, as well as individual and collective protection measures linked to COVID-19. Anopheles gambiae s.l. blood-feeding rates and parous were high and similar for all insecticides in treated houses. CONCLUSION: To achieve the goal of zero malaria, the optimal choice of vector control tools plays an important role. Compared with pirimiphos-methyl, clothianidin-based insecticides induced a lower reductions in entomological indicators of malaria transmission.

Bio-efficacy of Olyset® Plus, PermaNet® 3.0 and Interceptor® G2 on pyrethroid-resistant populations of Anopheles gambiae s.l. prior to the June 2023 net distribution campaign in Benin, West Africa.

BACKGROUND: This study investigates the effectiveness of new-generation mosquito nets, like Olyset® Plus and PermaNet® 3.0, and dual-action nets such as Interceptor® G2, against pyrethroid-resistant Anopheles gambiae mosquitoes following the 2023 mass distribution of long-lasting insecticidal nets in Benin. METHODS: We tested wild mosquito populations from six communes in Benin against various pyrethroid (permethrin 0.75%, alphacypermethrin 0.05%, and deltamethrin 0.05%) using WHO tube tests. Additionally, we exposed mosquitoes to chlorfenapyr 100 µg/ml using the CDC bottle bioassay method. A subset of mosquitoes underwent biochemical and PCR tests to check the overexpression of metabolic enzymes and the Kdr L1014F mutation. We evaluated the effectiveness of Olyset® Plus, PermaNet® 3.0, and Interceptor® G2 nets using cone and tunnel tests on both laboratory and field populations of An. gambiae. RESULTS: Overall, the highest mortality rate was 60% with pyrethroid and 98 to100% with chlorfenapyr. In cone tests, all three types of nets induced mortality rates above 80% in the susceptible laboratory strain of An. gambiae. Notably, Olyset® Plus showed the highest mortality rates for pyrethroid-resistant mosquitoes in cone tests, ranging from 81.03% (95% CI: 68.59-90.13) in Djougou to 96.08% (95% CI: 86.54-99.52) in Akpro-Missérété. PermaNet® 3.0 had variable rates, from 42.5% (95% CI: 27.04-59.11) in Djougou to 58.54% (95% CI: 42.11-73.68) in Porto-Novo. However, revealed good results for Interceptor® G2, with 94% (95% CI: 87.40-97.77) mortality and 89.09% blood sampling inhibition in local populations of An. gambiae. In comparison, Interceptor® had lower rates of 17% (95% CI: 10.23-25.82) and 60%, respectively. CONCLUSION: These results suggest that tunnel tests are effective for evaluating dual-active ingredient nets. Additionally, Interceptor® G2 and PBO nets like Olyset® Plus could be considered as alternatives against pyrethroid-resistant mosquitoes.

The frequency of kdr and ace-1 alleles in Anopheles gambiae s.l. before and during indoor residual spraying (IRS) implementation and four years after IRS withdrawal in three districts in Atacora, Benin.

BACKGROUND: Indoor residual spraying (IRS) was first implemented in the Atacora department, Benin from 2011 to 2012 using bendiocarb (carbamate) followed by annual spraying with pirimiphos-methyl (organophosphate) from 2013 to 2018. Before and after IRS implementation in Atacora, standard pyrethroid insecticide-treated bed nets were the main method of vector control in the area. This study investigated the knockdown resistance (kdr) gene (L1014F) and the acetylcholinesterase (ace-1) gene (G119S), before and during IRS implementation, and 4-years after IRS withdrawal from Atacora. This was done to assess how changes in insecticide pressure from indoor residual spraying may have altered the genotypic resistance profile of Anopheles gambiae s.l. METHOD: Identification of sibling species of An. gambiae s.l. and detection of the L1014F mutation in the kdr gene and G119S mutation in ace-1 genes was done using molecular analysis. Allelic and genotypic frequencies were calculated and compared with each other before and during IRS implementation and 4 years after IRS withdrawal. The Hardy-Weinberg equilibrium and genetic differentiation within and between populations were assessed. RESULTS: Prevalence of the L1014F mutation in all geographic An. gambiae s.l. (An. gambiae s.s., Anopheles. coluzzii, Anopheles. arabiensis, and hybrids of "An. gambiae s.s. and An. coluzzii") populations increased from 69% before IRS to 87% and 90% during and after IRS. The G119S allele frequency during IRS (20%) was significantly higher than before IRS implementation (2%). Four years after IRS withdrawal, allele frequencies returned to similar levels as before IRS (3%). Four years after IRS withdrawal, the populations showed excess heterozygosity at the ace-1 gene and deficit heterozygosity at the kdr gene, whereas both genes had excess heterozygosity before and during IRS (FIS < 0). No genetic differentiation was observed within the populations. CONCLUSIONS: This study shows that the withdrawal of IRS with bendiocarb and pirimiphos-methyl may have slowed down the selection of individual mosquitoes with ace-1 resistance alleles in contrast to populations of An. gambiae s.l. with the L1014F resistance allele of the kdr gene. This may suggest that withdrawing the use of carbamates or organophosphates from IRS or rotating alternative insecticides with different modes of action may slow the development of ace-1 insecticide-resistance mutations. The increase in the prevalence of the L1014F mutation of the kdr gene in the population, despite the cessation of IRS, could be explained by the growing use of pyrethroids and DDT in agriculture and for other domestic use. More observational studies in countries where carbamates or organophosphates are still being used as public health insecticides may provide additional insights into these associations.

Distribution and Abundance of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Benin, West Africa.

Updated information on the distribution and abundance of Aedes aegypti and Aedes albopictus is crucial to prepare African countries, such as Benin, for possible arboviral disease outbreaks. This study aims to evaluate the geographical distribution, abundance and biting behaviour of these two vectors in Benin. Three sampling techniques were used in this study. The collection of Aedes spp. adults were made through human landing catch (HLC), immatures were captured with the use of ovitraps, and a dipping technique was used for the collection of Aedes spp. in 23 communes located along the North-South and East-West transect of Benin. Adult Aedes mosquitoes were collected indoors and outdoors using HLC. Mosquito eggs, larvae and pupae were collected from containers and ovitraps. The adult mosquitoes were morphologically identified, then confirmed using a polymerase chain reaction (PCR). Overall, 12,424 adult specimens of Aedes spp. were collected, out of which 76.53% (n = 9508) and 19.32% (n = 2400) were morphologically identified as Ae. aegypti and Ae. albopictus, respectively. Geographically, Ae. aegypti was found across the North-South transect unlike Ae. albopictus, which was only encountered in the southern part of the country, with a great preponderance in Avrankou. Furthermore, an exophagic behaviour was observed in both vectors. This updated distribution of Aedes mosquito species in Benin will help to accurately identify areas that are at risk of arboviral diseases and better plan for future vector control interventions.

Assessing the susceptibility and efficacy of traditional neurotoxic (pyrethroid) and new-generation insecticides (chlorfenapyr, clothianidin, and pyriproxyfen), on wild pyrethroid-resistant populations of Anopheles gambiae from southern Benin.

BACKGROUND: The objective of this study was to determine the susceptibility of wild Anopheles gambiae sensu lato (s.l.) from southern Benin to the new insecticides (chlorfenapyr (CFP), pyriproxyfen (PPF), and clothianidin (CTD)) and assess the efficacy of insecticide-treated bed nets (ITNs) that contain these new products. METHODS: Wild An. gambiae from the Benin communes of Allada, Ifangni, Akpro-Missérété, and Porto-Novo were tested for their susceptibility to CFP and PPF using the WHO bottle tests, and pyrethroids (alpha-cypermethrin, deltamethrin, and permethrin) and CTD using WHO tube tests. WHO cone tests were used to evaluate the efficacy of Interceptor® (which contains alpha-cypermethrin (ACM) only), Interceptor® G2, (CFP + ACM), and Royal Guard® nets (PPF + ACM). The ovaries of blood-fed An. gambiae from Ifangni exposed to a new PPF net were dissected, and egg development status was examined using Christopher's stages to determine the fertility status of the mosquitoes. Using a standardized protocol, the oviposition rate and oviposition inhibition rate were calculated from live blood-fed An. gambiae placed in oviposition chambers after exposure to PPF. RESULTS: In all four mosquito populations, pyrethroid mortality ranged from 5 to 80%, while chlorfenapyr and clothianidin mortality ranged from 98 to 100%. At Ifangni, all mosquitoes exposed to Royal Guard® nets were infertile (100%) while the majority (74.9%) of mosquitoes exposed to Interceptor® nets had fully developed their eggs to Christopher's stage V. The oviposition inhibition rate after exposure of the mosquitoes to the PPF was 99% for the wild population of An. gambiae s.l. and the susceptible laboratory strain, An. gambiae sensu stricto (Kisumu). CONCLUSIONS: The results of this study suggest that pyrethroid-resistant An. gambiae from the selected communes in southern Benin are susceptible to chlorfenapyr, clothianidin, and pyriproxyfen. In addition, based on bioassay results, new and unused Interceptor® G2 and Royal Guard® nets were effective on Ifangni's mosquito populations. Despite the availability of new effective insecticides, continued vigilance is needed in Benin. Therefore, monitoring of resistance to these insecticides will continue to periodically update the Benin national insecticide resistance database and management plan.

What can be learned from the residual efficacy of three formulations of insecticides (pirimiphos-methyl, clothianidin and deltamethrin mixture, and clothianidin alone) in large-scale in community trial in North Benin, West Africa?

BACKGROUND: In Alibori and Donga, two departments of high malaria incidence of Northern Benin, pirimiphos-methyl, mixture deltamethrin + clothianidin, as well as clothianidin were used at large scale for IRS. The present study aimed to assess the residual efficacy of these products. METHODS: Immatures of Anopheles gambiae sensu lato (s.l.) collected in the communes of Kandi and Gogounou (Department of Alibori), Djougou and Copargo (Department of Donga) were reared until adulthood. Females aged 2-5 days were used for susceptibility tube tests following the WHO protocol. The tests were conducted with deltamethrin (0.05%), bendiocarb (0.1%), pirimiphos-methyl (0.25%) and clothianidin (2% weight per volume). For cone tests performed on cement and mud walls, the An. gambiae Kisumu susceptible strain was used. After the quality control of the IRS performed 1-week post-campaign, the evaluation of the residual activity of the different tested insecticides/mixture of insecticides was conducted on a monthly basis. RESULTS: Over the three study years, deltamethrin resistance was observed in all the communes. With bendiocarb, resistance or possible resistance was observed. In 2019 and 2020, full susceptibility to pirimiphos-methyl was observed, while possible resistance to the same product was detected in 2021 in Djougou, Gogounou and Kandi. With clothianidin, full susceptibility was observed 4-6 days post-exposure. The residual activity lasted 4-5 months for pirimiphos-methyl, and 8-10 months for clothianidin and the mixture deltamethrin + clothianidin. A slightly better efficacy of the different tested products was observed on cement walls compared to the mud walls. CONCLUSION: Overall, An. gambiae s.l. was fully susceptible to clothianidin, while resistance/possible resistance was observed the other tested insecticides. In addition, clothianidin-based insecticides showed a better residual activity compared to pirimiphos-methyl, showing thus their ability to provide an improved and prolonged control of pyrethroid resistant vectors.

Insecticide resistance status of Aedes aegypti and Aedes albopictus mosquitoes in southern Benin, West Africa.

BACKGROUND: The emergence of insecticide resistance in Aedes mosquitoes could undermine efforts to control arboviruses. The present study aims to assess in some communes of Southern Benin, the susceptibility level of Aedes aegypti (Linnaeus, 1762) and Aedes albopictus (Skuse, 1894) to insecticides commonly used in public health, as well as mechanisms involved. METHODS: Females Ae. albopictus and Ae. aegypti collected in Ifangni, Porto-Novo, Avrankou, Adjarra and Kétou from June 2021 to October 2022, were exposed to: deltamethrin 0.05%, permethrin 0.75%, alpha-cypermethrin 0.05%, pirimiphos methyl 0.25% and bendiocarb 0.1%, following the standard WHO susceptibility tube test protocol. In some sites, pre-exposure to the synergist PBO was used to verify if pyrethroid resistance of populations of Aedes was mediated by oxidases. RESULTS: Full susceptibility to deltamethrin and permethrin was observed in all tested populations of Ae. albopictus. However, with alphacypermethrin, a suspected resistance was observed in Adjarra (94.67%), Ifangni (93%) and Porto-Novo (94%), and a resistance in Avrankou (83%). The PBO-alphacypermethrin tests performed, led to a full susceptibility (100%) in all four sites, which confirms the full involvement of oxidases in resistance of all tested populations of Ae. albopictus to alphacypermethrin. At the opposite, Aedes aegypti was either resistant or suspected of being resistant to all tested pyrethroids in all four sites, except in Ifangni where a full susceptibility to alphacypermethrin was observed. The full susceptibility of Ae. aegypti to bendiocarb and pirimiphos-methyl in all communes suggests that these two insecticides can be good candidates for an effective control of pyrethroid-resistant Aedes vector populations. Use of permethrin and deltamethrin could also be considered for controlling populations of Ae. albopictus. CONCLUSION: Results of the present study will help guide strategy to implement for an effective control of Aedes vector populations in Benin.

Trophic preferences of Anopheles coluzzii (Diptera: Culicidae): what implications for malaria vector control in Benin?

The main objective of the present study is to assess the preferences in terms of vertebrate hosts of Anopheles coluzzii, the main malaria vector in the pastoral area of Malanville, Benin, where rice cultivation and livestock are the main source of income for the populations. Adult mosquitoes were collected through pyrethrum spray catch, and human landing catch in two communes in Benin: Malanville, a pastoral area, and Porto-Novo, a nonpastoral area. Molecular species identification was performed through PCR within the Anopheles gambiae complex. Blood meal origin and P. falciparum sporozoite infection were determined using ELISA blood meal and circumsporozoite protein tests, respectively. Overall, 97% of females of An. gambiae s.l. were An. coluzzii, with biting behavior more pronounced outdoors in the pastoral area. In Malanville, the main vertebrate hosts on which females An. coluzzii blood fed were goats (44%), humans (24.29%), bovines (22%), and pigs (1.4%). Our results also showed that single-host blood meals (human: 24.29% or animal: 68%) were mostly observed compared to mixed blood meals (8.58%). The human biting rate (HBR) and P. falciparum sporozoite rate (SR) of An. coluzzii were 66.25 bites/man/night and 0.77%, respectively. However, in the nonpastoral zone (Porto-novo), 93.98% of samples were An. coluzzii. The latter blood-fed mostly (86.84%) on humans, with an estimated HBR of 21.53 b/m/n and SR of 5.81%. The present study revealed an opportunistic and zoophagic behavior of An. coluzzii in the Malanville area with an overall low mean SR.

Assessing Insecticide Susceptibility and Resistance Intensity of Anopheles gambiae s.l. Populations From Some Districts of Benin Republic, West Africa.

Pyrethroid resistance is widespread in sub-Saharan Africa. The objective of this study was to assess the insecticide resistance intensity in Anopheles gambiae s.l. (Diptera: Culicidae) in four districts of Benin in order to better understand how pyrethroid-only nets are likely to be effective. Thus, adult females of An. gambiae s.l., reared from field-collected larvae were used for assessing resistance intensity to permethrin and deltamethrin. They were tested at 1×, 5×, and 10× the diagnostic dose, using both WHO susceptibility tube testing and CDC bottle bioassays. Identification of molecular species, as well as of L1014F Kdr and Ace-1R mutations was performed using the PCR. The level of expression of biochemical enzymes was also evaluated. Overall, moderate to high resistance intensity to permethrin and deltamethrin was observed, irrespective of the testing method. While the L1014F Kdr frequency was high (>75%), Ace-1R was low (≤6%) in An. gambiaes.s. and Anopheles coluzzii, the two predominant species [52% (95% CI: 44.8-59.1) and 45% (95% CI: 38.0-52.2), respectively]. Anopheles arabiensis was found at very low frequency (3%, 95%CI: 1.1-6.4). For Biochemical analyses, α and ß-esterases were over-expressed in all four districts, while mixed-function oxidases (MFOs) were over-expressed in only one. Overall, the two testing methods led to comparable conclusions, though there were a few inconsistencies between them. The moderate-high resistance intensity observed in the study area suggests that dual active-ingredient (AI) long-lasting insecticidal nets (LLINs) may provide better control of insecticide-resistant mosquitoes.

Pyrethroid Resistance Intensity in Anopheles gambiae s.l. from Different Agricultural Production Zones in Benin, West Africa.

Agricultural production activities usually occur in Benin with the use of a huge amount of insecticides including pyrethroids for pest control. It is therefore important to regularly monitor pyrethroid resistance intensity in Anopheles gambiae s.l., the main malaria vector. This study was conducted in cereal, cotton, rice growing, and urban market gardening areas throughout the country in 2018 and 2019. Females An. gambiae s.l. field-collected as larvae were exposed to deltamethrin 1 × (0.05%), 2 × (0.1%), 5 × (0.25%), and 10 × (0.5%) and permethrin 1 × (0.75%), 2 × (1.5%), 5 × (3.75%), and 10 × (7.5%). Synergist assays were also performed using World Health Organization articles combining piperonyl butoxide (PBO) (4%) + deltamethrin 1 × and, PBO (4%) + Permethrin 1 × . Molecular species and L1014F kdr mutation were identified using PCR. Expression of metabolic enzymes was also assessed through biochemical tests. After exposure to permethrin and deltamethrin 10 × , An. gambiae s.l. displayed mortality rates <98%. Synergist assays induced significantly higher mortality rates than pyrethroids alone (p < 0.05). An. gambiae s.l. complex was composed of An. gambiae s.s., Anopheles coluzzii, and Anopheles arabiensis, with mean frequency of the L1014F kdr mutation >75%. Overexpression of nonspecific α and ß esterases was observed in the cereal, cotton, and urban market gardening areas, while an overexpression of mixed function oxidases was observed in the cotton and rice growing areas. Overall, An. gambiae s.l. showed high resistance intensity to both deltamethrin and permethrin. The synergist and biochemical tests performed suggest that PBO long-lasting insecticidal nets may provide a greater control of pyrethroid-resistant mosquitoes.

Coverage, Usage, Physical Integrity, and Bio-efficacy of Olyset Nets in the Plateau Region, South Benin Following the 2011 Nationwide Distribution.

The present study investigated in 8 villages of the Plateau region the coverage, usage, physical integrity, and bio-efficacy of the Olyset nets distributed nationwide by the Benin's National Malaria Control Programme in July 2011. The questionnaire administered as well as the observations made in the households allowed estimating the coverage and usage rates of the 2011 Olyset nets. While their physical integrity was assessed through standard WHO methodology, their bio-efficacy was evaluated through gas chromatography, and WHO cone testing performed with the Kisumu susceptible strain. Mosquito collections through human landing catches (HLCs) were also performed in torn nets to assess if a loss of protection of sleepers occurred as the nets fabric integrity got more damaged. Nine months postdistribution, the coverage and usage rates of the 2011 Olyset nets were 67.4% (95% CI: 65.8-68.9) and 73.3% (95% CI: 70.7-75.8) respectively. About 28% of the 2011 Olyset nets were torn. A drastic drop of the insecticide quantity on the fibers of the nets [from 7.08 µg (95% CI: 5.74-8.42) to 0.2 µg (95% CI: 0.01-0.38)] as well as mortality rates <80% were observed with most nets evaluated. Moreover, the biting rates of An. gambiae s.l. (Diptera: Culicidae) inside torn nets increased in line with their fabric integrity loss. These data support the conclusion that future deployment of nets in the field must be strengthened by community sensitization on their correct use in order to postpone as much as possible appearance of holes and loss of insecticidal activity and encourage repairing of torn nets.

Characterization of resistance profile (intensity and mechanisms) of Anopheles gambiae in three communes of northern Benin, West Africa.

BACKGROUND: The selection and the spread of insecticide resistance in malaria vectors to the main classes of insecticides used in vector control tools are a major and ongoing challenge to malaria vector control programmes. This study aimed to determine the intensity of vector resistance to insecticides in three regions of Benin with different agro-ecological characteristics. METHODS: Larvae of Anopheles gambiae sensu lato (s.l.) were collected from September to November 2017 in different larval sites in three northern Benin communes: Parakou, Kandi and Malanville. Two to five-day-old, non-blood-fed, female mosquitoes were exposed to papers impregnated with deltamethrin, permethrin and bendiocarb at dosages of 1 × the diagnostic dose, 5 × and 10 × to determine the intensity of resistance in these vectors. Molecular frequencies of the kdr L1014F and ace-1R G119S insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled at each study site. RESULTS: Resistance to pyrethroids (permethrin and deltamethrin) was recorded in all three communes with mortality rates below 60% using the diagnostic dose (1x). The results obtained after exposure of An. gambiae to permethrin 10 × were 99% in Kandi, 98% in Malanville and 99% in Parakou. With deltamethrin 10x, mortality rates were 100% in Kandi, 96% in Malanville and 73% in Parakou. For the diagnostic dose of bendiocarb, suspected resistance was recorded in the communes of Malanville (97%) and Kandi (94%) while sensitivity was observed in Parakou (98%).Using the 10 × dose, mortality was 98% in Kandi, 100% in Malanville and 99% in Parakou. The frequencies of the kdr L1014F allele varied between 59 and 83% depending on the sites and species of the An. gambiae complex, while the frequency of the ace-1R G119S gene varied between 0 and 5%. Biochemical tests showed high levels of oxidase and esterase activity compared to the susceptible colony strain of An. gambiae sensu stricto (Kisumu strain). CONCLUSION: Anopheles gambiae showed a generalized loss of susceptibility to permethrin and deltamethrin but also showed moderate to high intensity of resistance in different regions of Benin. This high intensity of resistance is a potential threat to the effectiveness of vector control.

Intensity and mechanisms of deltamethrin and permethrin resistance in Anopheles gambiae s.l. populations in southern Benin.

BACKGROUND: Insecticide resistance is threatening the effectiveness of efforts to control malaria vectors in Benin. This study explores the levels and mechanisms of insecticide resistance in An. gambiae s.l. to pyrethroids. METHODS: Larvae were collected from August 2017 to July 2018 in five communes in southern Benin (Adjohoun, Allada, Bohicon, Cotonou, and Porto-Novo) representing diverse ecological regions, and were reared in Benin's insectary. Two- to five-day-old female mosquitoes from each district were exposed to multiple doses of deltamethrin and permethrin (1×, 2×, 5×, and 10×) using the WHO insecticide resistance intensity bioassay. The effect of pre-exposure to the synergist, piperonyl butoxide (PBO), was also tested at different pyrethroid doses. Molecular allele frequencies of kdr (1014F) and ace-1R (119S) insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled from each study area. RESULTS: An. gambiae s.l. were resistant to pyrethroid-only exposure up to 10× the diagnostic doses in all the study sites for both deltamethrin and permethrin. Mortality was significantly higher in An. gambiae s.l. pre-exposed to PBO followed by exposure to deltamethrin or permethrin compared to mosquitoes exposed to deltamethrin or permethrin only (p < 0.001). The difference in mortality between deltamethrin only and PBO plus deltamethrin was the smallest in Cotonou (16-64%) and the greatest in Bohicon (12-93%). The mortality difference between permethrin only and PBO plus permethrin was the smallest in Cotonou (44-75%) and the greatest in Bohicon (22-72%). In all the study sites, the kdr resistance allele (1014F) frequency was high (75-100%), while the ace-1 resistance allele (G119S) frequency was low (0-3%). Analysis of the metabolic enzymatic activity of An. gambiae s.l. showed overexpression of nonspecific esterases and glutathione S-transferases (GST) in all study sites. In contrast to the PBO results, oxidase expression was low and was similar to the susceptible An. gambiae s.s. Kisumu strain in all sites. CONCLUSION: There is high-intensity resistance to pyrethroids in southern Benin. However, pre-exposure to PBO significantly increased susceptibility to the pyrethroids in the different An. gambiae s.l. populations sampled. The use of PBO insecticide-treated bed nets may help maintain the gains in An. gambiae (s.l.) control in southern Benin.

Entomological assessment of the risk of dengue outbreak in Abomey-Calavi Commune, Benin.

BACKGROUND: In May 2019, a confirmed dengue fever case was detected at the local hospital of Abomey-Calavi Commune in southern Benin. In Benin, there remains a dearth of literature concerning the distribution and biology of Aedes aegypti, the principal vector of dengue fever. This study was initiated by the Ministry of Health to partially fill this gap. The findings allowed us to assess the arboviral transmission risk incurred by the population of Abomey-Calavi to support programmatic decision-making. METHODS: Entomological assessments were conducted in 5% of the houses, meaning 314 houses selected from 11 boroughs in Abomey-Calavi Centre district and 9 villages in Hêvié district. The surveyed breeding sites were water containers located in (domestic) and around (peri-domestic) the dwellings. When a container was positive (housing larvae), a portion of the immature population was sampled with a larval dipper and poured into labeled jars. Immatures were then reared to adulthood at the Centre de Recheche Entomologique de Cotonou (CREC) insectary. Adult mosquitoes were morphologically identified to species level by site and, a subsample of the collected Ae. aegypti mosquitoes were used for WHO susceptibility tube tests. RESULTS: Of the 1372 adult Aedes specimens which emerged from the collected larvae and pupae, 1356 Ae. aegypti (98.83%), 10 Ae. luteocephalus, and 4 Ae. vittatus were identified. The Breteau indices were 160.2 in Abomey-Calavi Centre and 150 in Hêvié, whereas the House indices were 58.5% and 61.6% in the respective districts. WHO insecticide susceptibility tube tests showed that the mortality rates were 38.71% in Abomey-Calavi Centre and 85.71% in Hêvié for permethrin, and 72.22% in Abomey-Calavi Centre and 100% in Hêvié for deltamethrin. CONCLUSION: The two districts were highly infested by Ae. aegypti whose breeding sites were mostly man-made. Considering this, human behavioral change to substantially reduce the number of larval habitats is necessary to control the vector populations. As Aedes mosquitoes are day biters, the use of repellents such as ointments and smoke coils can also be useful.

Physical integrity and survivorship of long-lasting insecticidal nets distributed to households of the same socio-cultural community in Benin, West Africa.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are designed to survive and sustain their physical barrier for 3 years in household conditions. However, studies have shown that most of these nets are usually torn or no longer present in the households in less than 3 years. This study was initiated in Benin to compare the survivorship and physical integrity of seven types of LLINs in a same socio-geographic area. METHODS: In August 2017, 1890 households were selected in 9 villages in the municipality of Zagnanado in central Benin. Each one of the selected households received one of the seven LLIN products: Aspirational®, DawaPlus® 2.0, OlysetNet®, PermaNet® 2.0, PermaNet® 3.0, Royal Sentry® and Yorkool®. Overall, 270 LLINs of each type were freely distributed in Zagnanado, at a rate of 30 LLINs per type per village. These bed nets have been monitored and evaluated every 6 months to identify the most resilient and preferred LLINs in the community. Net survivorship was assessed using the rate of net loss and physical condition. RESULTS: The survivorship of all types of LLIN was estimated at 92% (95% CI 90.33-92.96) after 6 months and 70% (95% CI 67.25-71.81) after a year of use. At 12 months, all bed nets monitored were below the NetCalc model threshold of 92.8% for an LLIN with a lifespan of 3 years. Only 1.73% of all types of LLIN had a visible loss of integrity after 6 months with a median proportionate hole index (PHI) estimated at zero. The percentage significantly increased after 12 months with 10.41% of damaged nets (all types of LLINs). The median PHI for each brand of net was 23, 196, 141, 23, 23, 121 and 72, respectively for Aspirational®, DawaPlus® 2.0, OlysetNet®, PermaNet® 2.0, PermaNet® 3.0, Royal Sentry® and Yorkool®. A significant difference was noted between the PHI at 6 and 12 months (p < 0.0001). After 12 months, the DawaPlus®2.0, OlysetNet® and Royal Sentry® suffered significantly more damage compared to the others (p < 0.001). CONCLUSION: The results of this study showed that after a year of use, the survivorship of the 7 LLIN products in households was lower than expected. However, all the LLIN products successfully met WHO standards for physical integrity after 12 months of use. The monitoring continues. The next steps will help to identify the most sustainable LLINs.

Resistance status of Anopheles gambiae s.l. to insecticides following the 2011 mass distribution campaign of long-lasting insecticidal nets (LLINs) in the Plateau Department, south-eastern Benin.

BACKGROUND: In 2011, Benin's National Malaria Control Programme (NMCP) organized a nationwide mass distribution campaign of LLINs throughout the country. Following this intervention, it was important to assess whether the level of susceptibility of malaria vectors to insecticides had remained the same as compared to the pre-intervention period. The current study investigated this. METHODS: Larval collections were conducted in Ifangni, Sakété, Pobè and Kétou districts located in Plateau department, Southeastern Benin before (2009) and after (2012-2013) LLIN distribution. Anopheles gambiae sensu lato (s.l.) larvae from the 4 study districts were reared to adulthood and WHO susceptibility tests were conducted. The insecticides tested were deltamethrin (0.05%), permethrin (0.75%), bendiocarb (0.1%) and DDT (4%). Molecular species identification as well as, the characterization of the kdr L1014F mutation were also performed in the An. gambiae s.l. complex using PCR method. RESULTS: Overall, a significant decrease in mortality rates of An. gambiae s.l. to deltamethrin (0.05%), permethrin (0.75%) and DDT (4%) was observed post-LLIN distribution, respectively: (100% vs 80.9%, p < 0.0001), (77.5% vs 70%, p = 0.01) and, (47.8% vs 4.4%, p < 0.0001). By contrast, susceptibility of vectors to bendiocarb (0.1%) remained the same (100% mortality in the WHO susceptibility tube tests) pre- and post-intervention. An increase in the kdr L1014F frequency was observed post-LLIN distribution [F(kdr) = 0.91)] compared to the pre-intervention period [F(kdr) = 0.56], p < 0.0001. Anopheles coluzzii and An. gambiae were the two molecular species identified in the study area. CONCLUSION: The decrease susceptibility to pyrethroids and DDT as well as, the increase in the frequency of the kdr L1014F mutation after the intervention stressed at the time, the need for the development and implementation of effective insecticide resistance management strategies. At present, an update of the vectors resistance status in the area is also necessary for decision-making.

Lessons learned, challenges and outlooks for decision-making after a decade of experience monitoring the impact of indoor residual spraying in Benin, West Africa.

BACKGROUND: Since 2008, Indoor Residual Spraying (IRS) has been performed in Benin in 19 districts, including 4 in southern Benin, 9 in Atacora, and 8 in Atacora, Alibori and Donga in northern Benin. However, Benin still struggles with questions about IRS cost-benefit and epidemiological impact. Lessons learned and challenges from 10 years of IRS in Benin to be shared with the stakeholders involved in vector control implementation for decision-making. METHODS: Entomological parameters have been assessed entomological parameters in IRS communes since 2008. In all IRS intervention communes, decreases in human biting rate (HBR) of Anopheles gambiae, blood feeding inhibition and entomological inoculation rate (EIR) as compared to control district have been measured. RESULTS: EIR was reduced by 80-90%, which is encouraging, but should be observed with caution because: (i) the reduction may be insufficient to decrease epidemiological indicators given that the residual EIR in IRS districts is still higher than it is in some regions of stable malaria; (ii) the reduction in EIR is based on comparisons with control communes, but it is difficult to select control areas with the same environmental characteristics as intervention areas; (iii) despite the reduction, half of all mosquitoes that entered IRS-treated houses succeeded in taking human blood meals. Further, there are behaviours among Benin's population that limit IRS efficacy, including recent data showing that > 90% of people are not protected by IRS between 7 and 10 p.m. This is due to the fact that they remain outdoors and that most people are not protected from mosquito bites after 10 p.m. because they either sleep outdoors without IRS protection or indoors without an ITN. Moreover, people have large amounts of clothing hanging on walls where mosquitoes can rest instead of IRS-treated walls. Finally, other components are important to consider in implementing IRS among which: (i) Vector resistance management strategies are sometimes poorly understood; this is actually different from the need to replace one insecticide with another after the emergence of resistance; (ii) African countries should prepare to finance IRS themselves. CONCLUSION: To curtail residual malaria transmission, additional interventions able to target vectors escaping IRS should be prioritized.

Population Dynamics of Anopheles gambiae s.l. and Culex quinquefasciatus in Rural and Urban Settings Before an Indoor Residual Spraying Campaign in Northern Benin.

Background: The purpose of this report is to provide information on Culicidae diversity; biting behavior and spatio-seasonal variation of abundance of Anopheles gambiae s.l. and Culex quinquefasciatus in rural and urban settings of the Alibori and Donga regions, Northern Benin, where an indoor residual spraying (IRS) campaign to control malaria is planned. Methods: Both human landing catches, associated with pyrethrum spray catches were used to monitor the mosquito populations in 12 sites with 1 urban and 1 rural located in each of the 6 districts randomly selected in the two targeted regions. After morphological identification of all mosquito specimens, biting behavior and density of An. gambiae s.l. and Cx quinquefasciatus were studied. PCR was also performed on An. gambiae s.l., to identify sibling species and its seasonal variation. Results: A total of 10,367 mosquitoes were captured, related to 14 species of the genera, Anopheles, Aedes, Culex and Mansonia. Of the total species collection, 40.39% were An. gambiae s.l. and 56.85% were Cx. quinquefasciatus. An. gambiae s.l. was more abundant in Donga (2521 specimens) compared with Alibori (1666 specimens). The opposite trend was observed with Cx. quinquefasciatus (2162 specimens in Donga against 4028 in Alibori). An. gambiae s.l. was predominant and displayed a higher blood feeding rate in rural areas, whereas Cx. quinquefasciatus was in majority in urban areas. An. gambiae s.l. was more endophagic, whereas Cx. quinquefasciatus showed similar indoor and outdoor biting behavior. An. gambiae s.l. was composed of An. coluzzii found in majority in the drought, and An. gambiae, which was predominant in the rainy season. Conclusion: The predominance of the malaria vector, An. gambiae s.l. and their higher blood feeding rate and their significantly high endophagy in rural areas indicate that these areas should be primarily targeted with the IRS operations to have a substantial impact on malaria transmission. Endophagy, characteristic of An. gambiae s.l. in our study area, suggests that IRS will have a positive impact on vector control if implemented 1 week before June that is the onset of the rainy season.