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BackgroundThe World Malaria Report 2021 revealed that Nigeria accounted for 27% of 241 million malaria cases worldwide and 31% of 602,000 malaria deaths. This WHO report is in sharp contrast to the ambitious goal of the National Malaria Strategic Plan (NMSP) 2014-2020, which aimed to transition Nigeria from malaria control to malaria elimination status by 2020. In this study, we combined contact bioassay with HPLC analysis to investigate how the end users’ treatment of Long-Lasting Insecticide Treated Nets (LLINs) inadvertently contributes to the unabated rise in malaria cases.MethodsWe randomly selected a few LLINs used under normal household conditions in Port-Harcourt and one on sale in Lagos, Nigeria. The continued potency of these LLINs to protect the users against malaria vectors, assuming the local vectors are insecticide-susceptible, was evaluated by exposing laboratory-susceptibleAnopheles gambiaKisumu strain to the nets according to the WHO cone bioassay protocol. After the exposure, the active ingredients (AI) in the LLINs were extracted and analysed using reverse phase HPLC to establish the quantity of the AIs and correlate it to bio-efficacy. The AI loss per wash was also computed based on the information provided by the users.ResultsThe labels on the LLINs revealed them to be Royal Sentry, PermaNet 2.0 and DuraNet. The bio-efficacy of these LLINs under household usage is less than two years. AI lost per wash was higher by 2.5-fold in LLIN made of polyethylene nets than polyester nets: 0.35g/Kg/wash and 0.14g/Kg/wash, respectively. The AI lost per wash increased by 31% for net exposed longer to the harsh environmental conditions of this region. The LLIN (ParmaNet) purchased in the open market has the highest AI concentration but only achieved 70.62% mortality, far below the acceptable standard.ConclusionThe AI in the LLIN bought from the open market was the highest, but of poor quality. This bed net was a counterfeit product, not approved by WHOPES. More importantly, this study revealed that field examination of LLINs under normal household use should be examined per region for policy makers to have informed knowledge of the duration of the bio-efficacy of the LLINs being distributed.

Vaccination prepares the body’s immune system ready to recognise, destroy and remember foreign disease-causing agents when it encounters them. It is the most effective way of averting communicable diseases if fully accepted and demanded by the population at risk of spreading contagious diseases. Social mobilisation has been a critical way of encouraging demands, as well as acceptance and compliance, by the general public for immunisation services. Vaccination campaign against poliovirus by WHO since 1988 was a huge success across the globe except in three countries: Nigeria, Afghanistan and Pakistan. Up until 3 years ago, polio eradication remained a challenge in Nigeria due mainly to non-acceptance of the vaccination by the hard-to-reach (HtoR) group. In this study, carried out at the height of the epidemic in Nigeria, we investigated the effects that social mobilisation on polio vaccination had on the hard-to-reach group. The participants were selected based on three criteria: age of the child, religion and social status. Semi-structured interview was employed and data analysed using the thematic content analysis. The results revealed that religion, service factor-distance to health facility and health staff behaviour are the key factors limiting the social mobilisation effort among the HtoR group. Nigeria began to make success only when one of the most respected Muslim leaders took ownership of the campaign for the elimination of polio, which has now placed Nigeria among the polio-free countries. Therefore, effective social mobilisation for vaccination programs of the HtoR group could be achieved faster with the involvement of religious leaders because of the enormous influence they have on their followers. This revelation is very important to note for the success of the upcoming vaccination campaign against COVID-19 disease, now that effective vaccines against the disease have been developed and ready for distribution. Keywords: Poliovirus, COVID-19, vaccination, social mobilisation, hard-to-reach population.

Background Malaria control relies mainlyon insecticide-based tools. However, the effectiveness of these tools is threatened by widespread insecticide resistance in malaria vectors, highlighting the need for alternative control approaches. The endosymbiont Asaia has emerged as a promising candidate for paratransgenic control of malaria, but its biology and genetics still need to be further analyzed across Africa. Here, we investigated the prevalence of Asaia and its maternal transmission in the natural population of Anopheles mosquitoes in Cameroon. Methods Indoor-resting adult mosquitoes belonging to four species (An. coluzzii, An. arabiensis, An. funestus and An. gambiae) were collected from eight localities across Cameroon from July 2016 to February 2020. PCR was performed on the Asaia-specific 16S ribosomal RNA gene, and samples positive by PCR for Asaia were confirmed by Sanger sequencing and phylogenetic analysis. The vertical transmission of Asaia was investigated by screening F1 mosquitoes belonging to F0Asaia-positive females. Results A total of 895 mosquitoes were screened. We found 43% (384) Asaia infection prevalence in four mosquito species. Phylogenetic analysis revealed that Asaia from Cameroon clustered together with the strains of Asaia isolated from other parts of the world. In addition, seven nucleotide sequence variants were found with low genetic diversity (π = 0.00241) and nucleotide sequence variant diversity (Hd = 0.481). Asaia was vertically transmitted with high frequency (range from 42.5 to 100%). Conclusions This study provides field-based evidence of the presence of Asaia in Anopheles mosquitoes in Cameroon for exploitation as a symbiont in the control of malaria in sub-Saharan Africa. Graphical abstract

The current National Malaria Strategy Plan (NMSP) 2014 to 2020, aims to transit Nigeria from malaria control to malaria elimination status by 2020. However, the most recent WHO Malaria Report 2018 revealed that Nigeria accounted for 25% of 219 million of the global malaria cases and 19% of 435,000 malaria deaths in 2017. The strategies of NMSP among others are: To significantly scale up Indoor Residual Spray (IRS); universal coverage of Long Lasting Insecticide Net (LLIN) and emphasis on parasite confirmation before malaria treatment. This study was carried out to determine how these core strategies have impacted pregnant and nursing mothers in the proper use of these malaria control tools. We surveyed 183 nursing and pregnant mothers with a total of 560 children, most under five years, and employed quantitative questionnaire and interviewed the mothers on how their perceptions of the malaria strategies are limiting the effort of malaria elimination in Nigeria. Our findings revealed that LLIN is still low: 1 bed net to 4 persons instead of the recommended 1 to 2 persons by WHO. Over 90% believed that sleeping under LLINs produce heat and nightmares and therefore reluctant to use them. IRS is unpopular and rarely used with just 3% applying IRS for malaria bite prevention. All the women claimed to be familiar with malaria symptoms and by implication, 76% use the antimalarial drugs without parasite confirmation. The Nigerian government needs to support strategies with awareness campaign to educate on the proper use of vector control tools and the risk of drug resistance due to indiscriminate use of antimalarial drugs. It can be concluded that the war against malaria in Nigeria can only be won when the target beneficiaries are made to understand and take ownership of strategies to eliminate the malaria burden. Key words: Malaria, parasite, vector control tools, malaria elimination, insecticide resistance, environmental pollution, malaria vaccines.

This study investigated parents’ and children’s perception of the challenges of acquiring primary and post-primary education in Tai and Etche communities of Rivers State. Qualitative data were obtained via individual interviews and Focus Group Discussions (FGD). Parents within the age range of 16 to 45 years and children in the secondary school within the ages of 14 to 18 years participated in this study. One hundred questionnaires were administered to the adults while a focus group discussion was conducted among thirty four children. The findings revealed lack of infrastructure, lack of funds/poverty and long distance walk to school as the three major barriers to acquiring education in these communities; which could also be said to cut across several schools in Rivers State. Over 60% of the population are peasant farmers with meagre earnings and less than 8% are civil servants. About 10% of the children are fully responsible for the funding of their education while the remaining also hawk goods to support their parents’ meagre earnings paying for their education. This study recommends full implementation of the Universal Basic Education (UBE) which makes education free and compulsory for all children. Philanthropic aids and governmental or non-governmental interventions would equally be needed to create enabling environments for learning and motivation for the children of these communities. Key words: Universal Basic Education, illiteracy, rural community, poverty, children, parents, perception, child labour.

Resistance is threatening the effectiveness of insecticide-based interventions in use for malaria control. Pinpointing genes associated with resistance is crucial for evidence-based resistance management targeting the major malaria vectors. Here, a combination of RNA-seq based genome-wide transcriptional analysis and RNA-silencing in vivo functional validation were used to identify key insecticide resistance genes associated with DDT and DDT/permethrin cross-resistance across Africa. A cluster of glutathione-S-transferase from epsilon group were found to be overexpressed in resistant populations of Anopheles funestus across Africa including GSTe1 [Cameroon (fold change, FC: 2.54), Ghana (4.20), Malawi (2.51)], GSTe2 [Cameroon (4.47), Ghana (7.52), Malawi (2.13)], GSTe3 [Cameroon (2.49), Uganda (2.60)], GSTe4 in Ghana (3.47), GSTe5 [Ghana (2.94), Malawi (2.26)], GSTe6 [Cameroun (3.0), Ghana (3.11), Malawi (3.07), Uganda (3.78)] and GSTe7 (2.39) in Ghana. Validation of GSTe genes expression profiles by qPCR confirmed that the genes are differentially expressed across Africa with a greater overexpression in DDT-resistant mosquitoes. RNAi-based knock-down analyses supported that five GSTe genes are playing a major role in resistance to pyrethroids (permethrin and deltamethrin) and DDT in An. funestus, with a significant recovery of susceptibility observed when GSTe2, 3, 4, 5 and GSTe6 were silenced. These findings established that GSTe3, 4, 5 and 6 contribute to DDT resistance and should be further characterized to identify their specific genetic variants, to help design DNA-based diagnostic assays, as previously done for the 119F-GSTe2 mutation. This study highlights the role of GSTes in the development of resistance to insecticides in malaria vectors and calls for actions to mitigate this resistance.

Pyrethroid resistance in the malaria vector Anopheles albimanus presents an obstacle to malaria elimination in the Americas. Here, An. albimanus CYP6P5 (the most overexpressed P450 in a Peruvian population) was functionally characterized. Recombinant CYP6P5 metabolized the type II pyrethroids, deltamethrin and α-cypermethrin with comparable affinities (K

The overexpression and overactivity of key cytochrome P450s (CYP450) genes are major drivers of metabolic resistance to insecticides in African malaria vectors such as Anopheles funestus s.s. Previous RNAseq-based transcription analyses revealed elevated expression of CYP325A specific to Central African populations but its role in conferring resistance has not previously been demonstrated. In this study, RT-qPCR consistently confirmed that CYP325A is highly over-expressed in pyrethroid-resistant An. funestus from Cameroon, compared with a control strain and insecticide-unexposed mosquitoes. A synergist bioassay with PBO significantly recovered susceptibility for permethrin and deltamethrin indicating P450-based metabolic resistance. Analyses of the coding sequence of CYP325A Africa-wide detected high-levels of polymorphism, but with no predominant alleles selected by pyrethroid resistance. Geographical amino acid changes were detected notably in Cameroon. In silico homology modelling and molecular docking simulations predicted that CYP325A binds and metabolises type I and type II pyrethroids. Heterologous expression of recombinant CYP325A and metabolic assays confirmed that the most-common Cameroonian haplotype metabolises both type I and type II pyrethroids with depletion rate twice that the of the DR Congo haplotype. Analysis of the 1 kb putative promoter of CYP325A revealed reduced diversity in resistant mosquitoes compared to susceptible ones, suggesting a potential selective sweep in this region. The establishment of CYP325A as a pyrethroid resistance metabolising gene further explains pyrethroid resistance in Central African populations of An. funestus. Our work will facilitate future efforts to detect the causative resistance markers in the promoter region of CYP325A to design field applicable DNA-based diagnostic tools.

Growing insecticide resistance in malaria vectors is threatening the effectiveness of insecticide-based interventions, including Long Lasting Insecticidal Nets (LLINs). However, the impact of metabolic resistance on the effectiveness of these tools remains poorly characterized. Using experimental hut trials and genotyping of a glutathione S-transferase resistance marker (L119F-GSTe2), we established that GST-mediated resistance is reducing the efficacy of LLINs against Anopheles funestus. Hut trials performed in Cameroon revealed that Piperonyl butoxide (PBO)-based nets induced a significantly higher mortality against pyrethroid resistant An. funestus than pyrethroid-only nets. Blood feeding rate and deterrence were significantly higher in all LLINs than control. Genotyping the L119F-GSTe2 mutation revealed that, for permethrin-based nets, 119F-GSTe2 resistant mosquitoes have a greater ability to blood feed than susceptible while the opposite effect is observed for deltamethrin-based nets. For Olyset Plus, a significant association with exophily was observed in resistant mosquitoes (OR = 11.7; p < 0.01). Furthermore, GSTe2-resistant mosquitoes (cone assays) significantly survived with PermaNet 2.0 (OR = 2.1; p < 0.01) and PermaNet 3.0 (side) (OR = 30.1; p < 0.001) but not for Olyset Plus. This study shows that the efficacy of PBO-based nets (e.g., blood feeding inhibition) against pyrethroid resistant malaria vectors could be impacted by other mechanisms including GST-mediated metabolic resistance not affected by the synergistic action of PBO. Mosaic LLINs incorporating a GST inhibitor (diethyl maleate) could help improve their efficacy in areas of GST-mediated resistance.

Despite the highest global burden of malaria, information on bionomics and insecticide resistance status of malaria vectors is grossly lacking in the densely populated Sahelo-Sudanian region of Nigeria. To support evidence-based vector control we characterised transmission and resistance profiles of Anopheles coluzzii populations from three sites in northern Nigeria. High sporozoite infection (~19.51%) was found in the An. coluzzii populations. A high pyrethroid resistance was observed with only 1% mortality against deltamethrin, a high LD50 (96.57 µg/ml), and a high LT50 (170.27 min, resistance ratio of ~51 compared with the fully susceptible Ngoussou colony). Moderate carbamate resistance was observed. Synergist bioassays significantly recovered deltamethrin susceptibility implicating CYP450s (mortality = 85%, χ2 = 134.04, p

Background: The arbovirus vector, Aedes albopictus, originating from Asia, has recently invaded African countries, including the Republic of the Congo, where it was associated with a chikungunya outbreak. Up until now, little was known about its distribution in relation to the native Aedes aegypti and how the invasion will modify the epidemiology of arboviral diseases. Here, we assessed the current distribution of Ae. albopictus and Ae. aegypti in the Republic of the Congo and explored the genetic diversity of the invading species, Ae. albopictus. Methods: Immature stages of Aedes were collected in nine locations in the Republic of the Congo in 2017 following a north-south transect and reared to adult stage. Adults were morphologically identified, counted and grouped according to species and location. Genetic diversity of Ae. albopictus was assessed by analyzing the cytochrome oxidase I ( COI) gene. Results: Ae. albopictus and Ae. aegypti were found together across the country in all the locations investigated. The invasive species is predominant over the native species in all locations except Brazzaville, suggesting that Ae. albopictus is displacing Ae. aegypti across Congo. When comparing the species distributions across the two largest cities, Brazzaville and Pointe Noire, Ae. albopictus was more prevalent than Ae. aegypti in the suburbs whereas the opposite situation was reported in the city centre. Mitochondrial DNA analysis revealed very low genetic diversity of Ae. albopictus with only three haplotypes recorded across the country supporting the recent introduction of this species in the Republic of the Congo. Phylogenetic tree analysis revealed that Ae. albopictus from Congo originated from other tropical Asian countries such as China, likely as a result of increasing trade links. Conclusion: These findings are important for the implementation of vector control strategies and can serve as a foundation for further research on these vectors in the country.

Background:The arbovirus vector,Aedes albopictus,originating from Asia, has recently invaded African countries, including the Republic of the Congo, where it was associated with a chikungunya outbreak. Up until now, little was known about its distribution in relation to the nativeAedes aegyptiand how the invasion will modify the epidemiology of arboviral diseases. Here, we assessed the current distribution ofAe. albopictusandAe. aegyptiin the Republic of the Congo and explored the genetic diversity of the invading species,Ae. albopictus.Methods:Immature stages ofAedeswere collected in nine locations in the Republic of the Congo in 2017 following a north-south transect and reared to adult stage. Adults were morphologically identified, counted and grouped according to species and location. Genetic diversity ofAe. albopictuswas assessed by analyzing the cytochrome oxidase I (COI) gene.Results:Ae.albopictusandAe. aegyptiwere found together across the country in all the locations investigated. The invasive species is predominant over the native species in all locations except Brazzaville, suggesting thatAe. albopictusis displacingAe. aegyptiacross Congo. When comparing the species distributions across the two largest cities, Brazzaville and Pointe Noire,Ae. albopictuswas more prevalent thanAe. aegyptiin the suburbs whereas the opposite situation was reported in the city centre. Mitochondrial DNA analysis revealed very low genetic diversity ofAe. albopictuswith only three haplotypes recorded across the country supporting the recent introduction of this species in the Republic of the Congo. Phylogenetic tree analysis revealed thatAe. albopictusfrom Congo originated from other tropical Asian countries such as China, likely as a result of increasing trade links.Conclusion:These findings are important for the implementation of vector control strategies and can serve as a foundation for further research on these vectors in the country.

Background. Malaria remains an important public health issue in Benin, with Anopheles gambiae s.l. and Anopheles funestus s.s being the predominant vectors. This study was designed to generate information on An. funestus distribution, molecular speciation, Plasmodium infection rate and insecticide susceptibility status across Benin. Methods. Mosquito samples were collected from December 2014 to January 2016 in 46 localities in Benin. These samples were mapped and An. funestus collected were speciated to the molecular level. Plasmodium infection rate was determined using a Taqman assay and susceptibility to insecticides was assessed using the WHO guidelines. The genotyping of the L119F- Gste2 mutation was also carried out. Results. An. funestus was found in 8 out of the 46 localities surveyed with a high presence in Tanongou (wet Sudanese ecological zone), Kpome, Doukonta and Pahou (sub-equatorial ecological zone). Molecular identifications revealed that only An. funestus s.s was present in southern Benin, whereas in Tanongou (northern Benin) An. funestus s.s. and An. leesoni were found in sympatry at proportions of 77.7% and 22.3% respectively. Plasmodium infection rate of An. funestus was higher in southern Benin at a range of 13 to 18% compared to 5.6% recorded in Tanongou. High DDT (8±0.5%) and permethrin (11±0.5%) resistance were observed in Doukonta, Kpome and Pahou, contrasting with relatively low resistance profiles: mortality-DDT=90±3.18% and mortality-permethrin=100% in Tanongou. Genotyping analysis revealed high frequency of the resistant 119F allele in the South (Kpome and Doukonta) compared to the North (Tanongou). Discussion and Conclusion. The high presence of An. funestus in the South compared to the North could be due to favorable environmental and climatic conditions found in both regions. A significant Plasmodium infection rate was recorded across the country. A high resistance profile was recorded in the southern Benin; this raises the need for further investigations on resistance selection factors.