When intensity of deltamethrin resistance in Anopheles gambiae s.l. leads to loss of Long Lasting Insecticidal Nets bio-efficacy : a case study in north Cameroon

Background: In Cameroon, insecticide resistance in Anopheles (An.) gambiae s.l. has been reported in several foci, prompting further investigations on associated patterns of Long-Lasting Insecticidal Nets (LLINs) bio-efficacy. The current study, conducted from June to August 2011, explored the intensity of deltamethrin resistance in An. gambiae s.l. from Pitoa and its impact on the residual bio-efficacy of LifeNet, a LLIN with deltamethrin incorporated into polypropylene nets (PND). Methods: Two-four days old females An. gambiae s.l. reared from larval collections in Pitoa were tested for susceptibility to DDT, permethrin and deltamethrin, using standard World Health Organization (WHO) tube assays. Intensity of deltamethrin resistance was explored using WHO tube assays, but across six working concentrations from 0.001 % to 0.5 %. Bio-efficacy of unwashed and washed PND was assessed using WHO cone test. Species identification and kdr 1014 genotyping were performed on mosquito samples that were not exposed to insecticides, using PCR-RFLP and HOLA methods respectively. The Kisumu reference susceptible strain of An. gambiae s.s. was used for comparisons. Results: A total of 1895 An. gambiae s.l. specimens from Pitoa were used for resistance and PND bio-efficacy testing. This mosquito population was resistant to DDT, permethrin and deltamethrin, with 18-40 min knockdown times for 50 % of tested mosquitoes and 59-77 % mortality. Deltamethrin Resistance Ratio compared with the Kisumu strain was estimated at >= 500 fold. LifeNets were effective against the susceptible Kisumu (100 % knockdown (KD60min) and mortality) and the resistant Pitoa samples (95 % KD60min, 83-95 % mortality). However, the bio-efficacy gradually dropped against the Pitoa samples when nets were washed (X-2 = 35.887, df = 8, p < 0.001), and fell under the WHO efficacy threshold (80 % mortality and/or 95 % KD60min) between 10 and 15 washes. The Pitoa samples were composed of three sibling species: An. arabiensis (132/154, 86 %), An. coluzzii (19/154, 12 %) and An. gambiae s.s. (3/154, 2 %). The kdr L1014F allele was found only in An. coluzzii (N-positive = 13/19), at 34 % frequency and heterozygote stage. No specimen carried the kdr L1014S allele. Conclusions: The current study showed that LifeNet might still offer some protection against the resistant An. gambiae s.l. population from Pitoa, provided appropriate dose of insecticide is available on the nets

Role of Anopheles (Cellia) rufipes (Gough, 1910) and other local anophelines in human malaria transmission in the northern savannah of Cameroon: a crosssectional survey

Background As part of a study to determine the impact of insecticide resistance on the effectiveness of longlasting insecticide treated nets (LLINs) in the north of Cameroon, the unexpectedly high density and anthropophilic behaviour of Anopheles rufipes lead us to investigate this species bionomics and role in human malaria parasite transmission. Methods For four consecutive years (2011–2014), annual cross-sectional sampling of adult mosquitoes was conducted during the peak malaria season (September-October) in three health districts in northern Cameroon. Mosquitoes sampled by human landing catch and pyrethrum spray catch methods were morphologically identified, their ovaries dissected for parity determination and Anopheles gambiae siblings were identified by molecular assay. Infection with P. falciparum and blood meal source in residual fauna of indoor resting anopheline mosquitoes were determined by enzyme-linked-immunosorbent assays. Results Anopheles gambiae (sensu lato) (s.l.) comprised 18.4% of mosquitoes collected with An. arabiensis representing 66.27% of the sibling species. The proportion of An. rufipes (2.7%) collected was high with a humanbiting rate ranging between 0.441 and 11.083 bites/person/night (b/p/n) and an anthropophagic rate of 15.36%. Although overall the members of An. gambiae complex were responsible for most of the transmission with entomological inoculation rates (EIR) reaching 1.221 infective bites/person/night (ib/p/n), An. arabiensis and An.coluzzii were the most implicated. The roles of An. funestus, An. pharoensis and An. paludis were minor. Plasmodium falciparum circumsporozoite protein rate in Anopheles rufipes varied from 0.6 to 5.7% with EIR values between 0.010 and 0.481 ib/p/n Conclusions The study highlights the epidemiological role of An. rufipes alongside the members of the An.gambiae complex, and several other sympatric species in human malaria transmission during the wet season in northern Cameroon. For the first time in Cameroon, An. rufipes has been shown to be an important local malaria vector, emphasising the need to review the malaria entomological profile across the country as pre-requisite to effective vector management strategies

Role of Anopheles (Cellia) rufipes (Gough, 1910) and other local anophelines in human malaria transmission in the northern savannah of Cameroon: a crosssectional survey

Background As part of a study to determine the impact of insecticide resistance on the effectiveness of longlasting insecticide treated nets (LLINs) in the north of Cameroon, the unexpectedly high density and anthropophilic behaviour of Anopheles rufipes lead us to investigate this species bionomics and role in human malaria parasite transmission. Methods For four consecutive years (2011–2014), annual cross-sectional sampling of adult mosquitoes was conducted during the peak malaria season (September-October) in three health districts in northern Cameroon. Mosquitoes sampled by human landing catch and pyrethrum spray catch methods were morphologically identified, their ovaries dissected for parity determination and Anopheles gambiae siblings were identified by molecular assay. Infection with P. falciparum and blood meal source in residual fauna of indoor resting anopheline mosquitoes were determined by enzyme-linked-immunosorbent assays. Results Anopheles gambiae (sensu lato) (s.l.) comprised 18.4% of mosquitoes collected with An. arabiensis representing 66.27% of the sibling species. The proportion of An. rufipes (2.7%) collected was high with a humanbiting rate ranging between 0.441 and 11.083 bites/person/night (b/p/n) and an anthropophagic rate of 15.36%. Although overall the members of An. gambiae complex were responsible for most of the transmission with entomological inoculation rates (EIR) reaching 1.221 infective bites/person/night (ib/p/n), An. arabiensis and An.coluzzii were the most implicated. The roles of An. funestus, An. pharoensis and An. paludis were minor. Plasmodium falciparum circumsporozoite protein rate in Anopheles rufipes varied from 0.6 to 5.7% with EIR values between 0.010 and 0.481 ib/p/n Conclusions The study highlights the epidemiological role of An. rufipes alongside the members of the An.gambiae complex, and several other sympatric species in human malaria transmission during the wet season in northern Cameroon. For the first time in Cameroon, An. rufipes has been shown to be an important local malaria vector, emphasising the need to review the malaria entomological profile across the country as pre-requisite to effective vector management strategies

Pyrethroid and etofenprox resistance in Anopheles gambiae and Anopheles coluzzii from vegetable farms in Yaounde, Cameroon: dynamics, intensity and molecular basis

Previous studies have indicated widespread insecticide resistance in malaria vector populations from Cameroon. However, the intensity of this resistance and underlying mechanisms are poorly known. Therefore, we conducted three cross-sectional resistance surveys between April 2018 and October 2019, using the revised World Health Organization protocol, which includes resistance incidences and intensity assessments. Field-collected Anopheles gambiae s.l. populations from Nkolondom, Nkolbisson and Ekie vegetable farms in the city of Yaounde were tested with deltamethrin, permethrin, alpha-cypermethrin and etofenprox, using 1x insecticide diagnostic concentrations for resistance incidence, then 5x and 10x concentrations for resistance intensity. Subsamples were analyzed for species identification and the detection of resistance-associated molecular markers using TaqMan(R) qPCR assays. In Nkolbisson, both An. coluzzii (96%) and An. gambiae s.s. (4%) were found together, whereas only An. gambiae s.s. was present in Nkolondom, and only An. coluzzii was present in Ekie. All three populations were resistant to the four insecticides (<75% mortality rates-MR1x), with intensity generally fluctuating over the time between mod-erate ( /=98%-MR10x) and high (76-97%-MR10x). The kdr L995F, L995S, and N1570Y, and the Ace-1 G280S-resistant alleles were found in An. gambiae from Nkolondom, at 73%, 1%, 16% and 13% frequencies, respectively, whereas only the kdr L995F was found in An. gambiae s.s. from Nkolbisson at a 50% frequency. In An. coluzzii from Nkolbisson and Ekie, we detected only the kdr L995F allele at 65% and 60% frequencies, respectively. Furthermore, expression levels of Cyp6m2, Cyp9k1, and Gste2 metabolic genes were highly upregulated (over fivefold) in Nkolondom and Nkolbisson. Pyrethroid and etofenprox-based vector control interventions may be jeopardized in the prospected areas, due to high resistance intensity, with multiple mechanisms in An. gambiae s.s. and An. coluzzii

Population genetic structure of the malaria vector Anopheles nili in sub-Saharan Africa

<p>Abstract</p> <p>Background</p> <p><it>Anopheles nili </it>is a widespread efficient vector of human malaria parasites in the humid savannas and forested areas of sub-Saharan Africa. Understanding <it>An. nili </it>population structure and gene flow patterns could be useful for the development of locally-adapted vector control measures.</p> <p>Methods</p> <p>Polymorphism at eleven recently developed microsatelitte markers, and sequence variation in four genes within the 28s rDNA subunit (ITS2 and D3) and mtDNA (COII and ND4) were assessed to explore the level of genetic variability and differentiation among nine populations of <it>An. nili </it>from Senegal, Ivory Coast, Burkina Faso, Nigeria, Cameroon and the Democratic Republic of Congo (DRC).</p> <p>Results</p> <p>All microsatellite loci successfully amplified in all populations, showing high and very similar levels of genetic diversity in populations from West Africa and Cameroon (mean Rs = 8.10-8.88, mean He = 0.805-0.849) and much lower diversity in the Kenge population from DRC (mean Rs = 5.43, mean He = 0.594). Bayesian clustering analysis of microsatellite allelic frequencies revealed two main genetic clusters in the dataset. The first one included only the Kenge population and the second grouped together all other populations. High Fst estimates based on microsatellites (Fst > 0.118, P < 0.001) were observed in all comparisons between Kenge and all other populations. By contrast, low Fst estimates (Fst < 0.022, P < 0.05) were observed between populations within the second cluster. The correlation between genetic and geographic distances was weak and possibly obscured by demographic instability. Sequence variation in mtDNA genes matched these results, whereas low polymorphism in rDNA genes prevented detection of any population substructure at this geographical scale.</p> <p>Conclusion</p> <p>Overall, high genetic homogeneity of the <it>An. nili </it>gene pool was found across its distribution range in West and Central Africa, although demographic events probably resulted in a higher level of genetic isolation in the marginal population of Kenge (DRC). The role of the equatorial forest block as a barrier to gene flow and the implication of such findings for vector control are discussed.</p

Urban malaria in sub-Saharan Africa: dynamic of the vectorial system and the entomological inoculation rate

Sub-Saharan Africa is registering one of the highest urban population growth across the world. It is estimated that over 75% of the population in this region will be living in urban settings by 2050. However, it is not known how this rapid urbanization will affect vector populations and disease transmission. The present study summarizes findings from studies conducted in urban settings between the 1970s and 2020 to assess the effects of urbanization on the entomological inoculation rate pattern and anopheline species distribution. Different online databases such as PubMed, ResearchGate, Google Scholar, Google were screened. A total of 90 publications were selected out of 1527. Besides, over 200 additional publications were consulted to collate information on anopheline breeding habitats and species distribution in urban settings. The study confirms high malaria transmission in rural compared to urban settings. The study also suggests that there had been an increase in malaria transmission in most cities after 2003, which could also be associated with an increase in sampling, resources and reporting. Species of the Anopheles gambiae complex were the predominant vectors in most urban settings. Anopheline larvae were reported to have adapted to different aquatic habitats. The study provides updated information on the distribution of the vector population and the dynamic of malaria transmission in urban settings. The study also highlights the need for implementing integrated control strategies in urban settings

High efficacy of microbial larvicides for malaria vectors control in the city of Yaounde Cameroon following a cluster randomized trial

The rapid expansion of insecticide resistance and outdoor malaria transmission are affecting the efficacy of current malaria control measures. In urban settings, where malaria transmission is focal and breeding habitats are few, fixed and findable, the addition of anti-larval control measures could be efficient for malaria vector control. But field evidences for this approach remains scarce. Here we provide findings of a randomized-control larviciding trial conducted in the city of Yaoundé that support the efficacy of this approach. A two arms random control trial design including 26 clusters of 2 to 4 km2 each (13 clusters in the intervention area and 13 in the non-intervention area) was used to assess larviciding efficacy. The microbial larvicide VectoMax combining Bacillus thuringiensis var israelensis (Bti) and Bacillus sphaericus in a single granule was applied every 2 weeks in all standing water collection points. The anopheline density collected using CDC light traps was used as the primary outcome, secondary outcomes included the entomological inoculation rate, breeding habitats with anopheline larvae, and larval density. Baseline entomological data collection was conducted for 17 months from March 2017 to July 2018 and the intervention lasted 26 months from September 2018 to November 2020. The intervention was associated with a reduction of 68% of adult anopheline biting density and of 79% of the entomological inoculation rate (OR 0.21; 95% CI 0.14–0.30, P < 0.0001). A reduction of 68.27% was recorded for indoor biting anophelines and 57.74% for outdoor biting anophelines. No impact on the composition of anopheline species was recorded. A reduction of over 35% of adult Culex biting densities was recorded. The study indicated high efficacy of larviciding for reducing malaria transmission intensity in the city of Yaoundé. Larviciding could be part of an integrated control approach for controlling malaria vectors and other mosquito species in the urban environment

Population structure of the malaria vector Anopheles moucheti in the equatorial forest region of Africa

<p>Abstract</p> <p>Background</p> <p><it>Anopheles moucheti </it>is a major malaria vector in forested areas of Africa. However, despite its important epidemiological role, it remains poorly known and insufficiently studied. Here, levels of genetic differentiation were estimated between different <it>A. moucheti </it>populations sampled throughout its distribution range in Central Africa.</p> <p>Methods</p> <p>Polymorphism at ten microsatellite markers was compared in mosquitoes sampled in Cameroon, the Democratic Republic of Congo and an island on Lake Victoria in Uganda. Microsatellite data were used to estimate genetic diversity within populations, their relative long-term effective population size, and the level of genetic differentiation between them.</p> <p>Results</p> <p>All specimens collected in Tsakalakuku (Democratic Republic of Congo) were identified as <it>A. m. bervoetsi </it>while other samples consisted of <it>A. m. moucheti</it>. Successful amplification was obtained at all microsatellite loci within all <it>A. m. moucheti </it>samples while only six loci amplified in <it>A. m. bervoetsi</it>. Allelic richness and heterozygosity were high for all populations except the island population of Uganda and <it>A. m. bervoetsi</it>. High levels of genetic differentiation were recorded between <it>A. m. bervoetsi </it>and each <it>A. m. moucheti </it>sample as well as between the island population of <it>A. m. moucheti </it>and mainland populations. Significant isolation by distance was evidenced between mainland populations.</p> <p>Conclusion</p> <p>High levels of genetic differentiation supports complete speciation of <it>A. m. bervoetsi </it>which should henceforth be recognized as a full species and named <it>A. bervoetsi</it>. Isolation by distance is the main force driving differentiation between mainland populations of <it>A. m. moucheti</it>. Genetically and geographically isolated populations exist on Lake Victoria islands, which might serve as relevant field sites for evaluation of innovative vector control strategies.</p

PLoS Pathog.

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission