High vector diversity and malaria transmission dynamics in five sentinel sites in Cameroon.

BACKGROUND: Malaria remains one of the main causes of morbidity and mortality in Cameroon. To inform vector control intervention decision making, malaria vector surveillance was conducted monthly from October 2018 to September 2020 in five selected sentinel sites (Gounougou and Simatou in the North, and Bonabéri, Mangoum and Nyabessang in the South). METHODS: Human landing catches (HLCs), U.S. Centers for Disease Control and Prevention (CDC) light traps, and pyrethrum spray catches (PSCs) were used to assess vector density, species composition, human biting rate (HBR), endophagic index, indoor resting density (IRD), parity, sporozoite infection rates, entomological inoculation rate (EIR), and Anopheles vectorial capacity. RESULTS: A total of 139,322 Anopheles mosquitoes from 18 species (or 21 including identified sub-species) were collected across all sites. Out of the 18 species, 12 were malaria vectors including Anopheles gambiae sensu lato (s.l.), Anopheles funestus s.l.., Anopheles nili, Anopheles moucheti, Anopheles paludis, Anopheles demeilloni, Anopheles. pharoensis, Anopheles ziemanni, Anopheles multicinctus, Anopheles tenebrosus, Anopheles rufipes, and Anopheles marshallii. Anopheles gambiae s.l. remains the major malaria vector (71% of the total Anopheles) collected, though An. moucheti and An. paludis had the highest sporozoite rates in Nyabessang. The mean indoor HBR of Anopheles ranged from 11.0 bites/human/night (b/h/n) in Bonabéri to 104.0 b/h/n in Simatou, while outdoors, it varied from 24.2 b/h/n in Mangoum to 98.7 b/h/n in Simatou. Anopheles gambiae s.l. and An. moucheti were actively biting until at least 8:00 a.m. The mean Anopheles IRD was 17.1 females/room, and the parity rate was 68.9%. The mean EIRs for each site were 55.4 infective bites/human/month (ib/h/m) in Gounougou, 99.0 ib/h/m in Simatou, 51.2 ib/h/m in Mangoum, 24.4 ib/h/m in Nyabessang, and 18.1 ib/h/m in Bonabéri. Anopheles gambiae s.l. was confirmed as the main malaria vector with the highest vectorial capacity in all sites based on sporozoite rate, except in Nyabessang. CONCLUSION: These findings highlight the high malaria transmission occurring in Cameroon and will support the National Malaria Control Program to design evidence-based malaria vector control strategies, and deployment of effective and integrated vector control interventions to reduce malaria transmission and burden in Cameroon, where several Anopheles species could potentially maintain year-round transmission.

Pyrethroid and Etofenprox Resistance in Anopheles gambiae and Anopheles coluzzii from Vegetable Farms in Yaoundé, 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 Ekié vegetable farms in the city of Yaoundé were tested with deltamethrin, permethrin, alpha-cypermethrin and etofenprox, using 1× insecticide diagnostic concentrations for resistance incidence, then 5× and 10× concentrations for resistance intensity. Subsamples were analyzed for species identification and the detection of resistance-associated molecular markers using TaqMan® 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 Ekié. All three populations were resistant to the four insecticides (<75% mortality rates-MR1×), with intensity generally fluctuating over the time between mod-erate (<98%-MR5×; ≥98%-MR10×) and high (76-97%-MR10×). 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 Ekié, 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.

Burden of mosquito-borne diseases across rural versus urban areas in Cameroon between 2002 and 2021: prospective for community-oriented vector management approaches.

BACKGROUND: Over the past two decades, Cameroon has recorded one of the highest rates of urban population growth in sub-Saharan Africa. It is estimated that more than 67% of Cameroon's urban population lives in slums, and the situation is far from improving as these neighbourhoods are growing at an annual rate of 5.5%. However, it is not known how this rapid and uncontrolled urbanization affects vector populations and disease transmission in urban versus rural areas. In this study, we analyse data from studies conducted on mosquito-borne diseases in Cameroon between 2002 and 2021 to determine the distribution of mosquito species and the prevalence of diseases they transmit with regards to urban areas versus rural areas. METHODS: A search of various online databases, such as PubMed, Hinari, Google and Google Scholar, was conducted for relevant articles. A total of 85 publications/reports were identified and reviewed for entomological and epidemiological data from the ten regions of Cameroon. RESULTS: Analysis of the findings from the reviewed articles revealed 10 diseases transmitted by mosquitoes to humans across the study regions. Most of these diseases were recorded in the Northwest Region, followed by the North, Far North and Eastern Regions. Data were collected from 37 urban and 28 rural sites. In the urban areas, dengue prevalence increased from 14.55% (95% confidence interval [CI] 5.2-23.9%) in 2002-2011 to 29.84% (95% CI 21-38.7%) in 2012-2021. In rural areas, diseases such as Lymphatic filariasis and Rift valley fever, which were not present in 2002-2011, appeared in 2012-2021, with a prevalence of 0.4% (95% CI 0.0- 2.4%) and 10% (95% CI 0.6-19.4%), respectively. Malaria prevalence remained the same in urban areas (67%; 95% CI 55.6-78.4%) between the two periods, while it significantly decreased in rural areas from 45.87% (95% CI 31.1-60.6%) in 2002-2011 to 39% (95% CI 23.7-54.3%) in the 2012-2021 period (*P = 0.04). Seventeen species of mosquitoes were identified as involved in the transmission of these diseases, of which 11 were involved in the transmission of malaria, five in the transmission of arboviruses and one in the transmission of malaria and lymphatic filariasis. The diversity of mosquito species was greater in rural areas than in urban areas during both periods. Of the articles reviewed for the 2012-2021 period, 56% reported the presence of Anopheles gambiae sensu lato in urban areas compared to 42% reported in 2002-2011. The presence of Aedes aegypti increased in urban areas in 2012-2021 but this species was absent in rural areas. Ownership of long-lasting insecticidal nets varied greatly from one setting to another. CONCLUSIONS: The current findings suggest that, in addition to malaria control strategies, vector-borne disease control approaches in Cameroon should include strategies against lymphatic filariasis and Rift Valley fever in rural areas, and against dengue and Zika viruses in urban areas.

Patterns of Kdr-L995F Allele Emergence Alongside Detoxifying Enzymes Associated with Deltamethrin Resistance in Anopheles gambiae s.l. from North Cameroon.

Understanding how multiple insecticide resistance mechanisms occur in malaria vectors is essential for efficient vector control. This study aimed at assessing the evolution of metabolic mechanisms and Kdr L995F/S resistance alleles in Anopheles gambiae s.l. from North Cameroon, following long-lasting insecticidal nets (LLINs) distribution in 2011. Female An. gambiae s.l. emerging from larvae collected in Ouro-Housso/Kanadi, Be-Centre, and Bala in 2011 and 2015, were tested for susceptibility to deltamethrin + piperonyl butoxide (PBO) or SSS-tributyl-phosphoro-thrithioate (DEF) synergists, using the World Health Organization's standard protocol. The Kdr L995F/S alleles were genotyped using Hot Ligation Oligonucleotide Assay. Tested mosquitoes identified using PCR-RFLP were composed of An. arabiensis (68.5%), An. coluzzii (25.5%) and An. gambiae (6%) species. From 2011 to 2015, metabolic resistance increased in Ouro-Housso/Kanadi (up to 89.5% mortality to deltametnrin+synergists in 2015 versus <65% in 2011; p < 0.02), while it decreased in Be-Centre and Bala (>95% mortality in 2011 versus 42-94% in 2015; p < 0.001). Conversely, the Kdr L995F allelic frequencies slightly decreased in Ouro-Housso/Kanadi (from 50% to 46%, p > 0.9), while significantly increasing in Be-Centre and Bala (from 0-13% to 18-36%, p < 0.02). These data revealed two evolutionary trends of deltamethrin resistance mechanisms; non-pyrethroid vector control tools should supplement LLINs in North Cameroon.

Epidemiological and entomological studies of malaria transmission in Tibati, Adamawa region of Cameroon 6 years following the introduction of long-lasting insecticide nets.

BACKGROUND: Malaria remains a serious public health problem in Cameroon. Implementation of control interventions requires prior knowledge of the local epidemiological situation. Here we report the results of epidemiological and entomological surveys carried out in Tibati, Adamawa Region, Cameroon, an area where malaria transmission is seasonal, 6 years after the introduction of long-lasting insecticidal bed nets. METHODS: Cross-sectional studies were carried out in July 2015 and 2017 in Tibati. Thick blood smears and dried blood spots were collected from asymptomatic and symptomatic individuals in the community and at health centers, respectively, and used for the molecular diagnosis of Plasmodium species. Adult mosquitoes were collected by indoor residual spraying and identified morphologically and molecularly. The infection status of Plasmodium spp. was determined by quantitative PCR, and positivity of PCR-positive samples was confirmed by Sanger sequencing. RESULTS: Overall malaria prevalence in our study population was 55.0% (752/1367) and Plasmodium falciparum was the most prevalent parasite species (94.3%), followed by P. malariae (17.7%) and P. ovale (0.8%); 92 (12.7%) infections were mixed infections. Infection parameters varied according to clinical status (symptomatic/asymptomatic) and age of the sampled population and the collection sites. Infection prevalence was higher in asymptomatic carriers (60.8%), but asexual and sexual parasite densities were lower. Prevalence and intensity of infection decreased with age in both the symptomatic and asymptomatic groups. Heterogeneity in infections was observed at the neighborhood level, revealing hotspots of transmission. Among the 592 Anopheles mosquitoes collected, 212 (35.8%) were An. gambiae, 172 (29.1%) were An. coluzzii and 208 (35.1%) were An. funestus (s.s.). A total of 26 (4.39%) mosquito specimens were infected by Plasmodium sp. and the three Anopheles mosquitoes transmitted Plasmodium at equal efficiency. Surprisingly, we found an An. coluzzii specimen infected by Plasmodium vivax, which confirms circulation of this species in Cameroon. The positivity of all 26 PCR-positive Plasmodium-infected mosquitoes was successively confirmed by sequencing analysis. CONCLUSION: Our study presents the baseline malaria parasite burden in Tibati, Adamawa Region, Cameroon. Our results highlight the high malaria endemicity in the area, and hotspots of disease transmission are identified. Parasitological indices suggest low bednet usage and that implementation of control interventions in the area is needed to reduce malaria burden. We also report for the first time a mosquito vector with naturally acquired P. vivax infection in Cameroon.

Bionomics of Anopheline species and malaria transmission dynamics along an altitudinal transect in Western Cameroon.

BACKGROUND: Highland areas of Africa are mostly malaria hypoendemic, due to climate which is not appropriate for anophelines development and their reproductive fitness. In view of designing a malaria control strategy in Western Cameroon highlands, baseline data on anopheline species bionomics were collected. METHODS: Longitudinal entomological surveys were conducted in three localities at different altitudinal levels. Mosquitoes were captured when landing on human volunteers and by pyrethrum spray catches. Sampled Anopheles were tested for the presence of Plasmodium circumsporozoite proteins and their blood meal origin with ELISA. Entomological parameters of malaria epidemiology were assessed using Mac Donald's formula. RESULTS: Anopheline species diversity and density decreased globally from lowland to highland. The most aggressive species along the altitudinal transect was Anopheles gambiae s.s. of S molecular form, followed in the lowland and on the plateau by An. funestus, but uphill by An. hancocki. An. gambiae and An. ziemanni exhibited similar seasonal biting patterns at the different levels, whereas different features were observed for An. funestus. Only indoor resting species could be captured uphill; it is therefore likely that endophilic behaviour is necessary for anophelines to climb above a certain threshold. Of the ten species collected along the transect, only An. gambiae and An. funestus were responsible for malaria transmission, with entomological inoculation rates (EIR) of 90.5, 62.8 and zero infective bites/human/year in the lowland, on the plateau and uphill respectively. The duration of gonotrophic cycle was consistently one day shorter for An. gambiae as compared to An. funestus at equal altitude. Altitudinal climate variations had no effect on the survivorship and the subsequent life expectancy of the adult stage of these malaria vectors, but most probably on aquatic stages. On the contrary increasing altitude significantly extended the duration of gonotrophic cycle and reduced: the EIR, their preference to human blood and consequently the malaria stability index. CONCLUSION: Malaria epidemiological rooting in the outskirts of Western Cameroon highlands evolves with increasing altitude, gradually from stable to unstable settings. This suggests a potential risk of malaria epidemic in highlands, and the need for a continuous epidemiological surveillance.

Polymorphism of intron-1 in the voltage-gated sodium channel gene of Anopheles gambiae s.s. populations from Cameroon with emphasis on insecticide knockdown resistance mutations.

Sequence variation at the intron-1 of the voltage-gated sodium channel gene in Anopheles gambiae M- and S-forms from Cameroon was assessed to explore the number of mutational events originating knockdown resistance (kdr) alleles. Mosquitoes were sampled between December 2005 and June 2006 from three geographical areas: (i) Magba in the western region; (ii) Loum, Tiko, Douala, Kribi, and Campo along the Atlantic coast; and (iii) Bertoua, in the eastern continental plateau. Both 1014S and 1014F kdr alleles were found in the S-form with overall frequencies of 14% and 42% respectively. Only the 1014F allele was found in the M-form at lower frequency (11%). Analysis of a 455 bp region of intron-1 upstream the kdr locus revealed four independent mutation events originating kdr alleles, here named MS1 -1014F, S1-1014S and S2-1014S kdr-intron-1 haplotypes in S-form and MS3-1014F kdr-intron-1 haplotype in the M-form. Furthermore, there was evidence for mutual introgression of kdr 1014F allele between the two molecular forms, MS1 and MS3 being widely shared by them. Although no M/S hybrid was observed in analysed samples, this wide distribution of haplotypes MS1 and MS3 suggests inter-form hybridizing at significant level and emphasizes the rapid diffusion of the kdr alleles in Africa. The mosaic of genetic events found in Cameroon is representative of the situation in the West-Central African region and highlights the importance of evaluating the spatial and temporal evolution of kdr alleles for a better management of insecticide resistance.

Trends in DDT and pyrethroid resistance in Anopheles gambiae s.s. populations from urban and agro-industrial settings in southern Cameroon.

BACKGROUND: Pyrethroid insecticides are widely used for insect pest control in Cameroon. In certain insect species, particularly the malaria vector Anopheles gambiae, resistance to this class of insecticides is a source of great concern and needs to be monitored in order to sustain the efficacy of vector control operations in the fields. This study highlights trends in DDT and pyrethroid resistance in wild An. gambiae populations from South Cameroon. METHODS: Mosquitoes were collected between 2001 and 2007 in four sites in South Cameroon, where insecticides are used for agricultural or personal protection purposes. Insecticide use was documented in each site by interviewing residents. Batches of 2-4 days old adult female mosquitoes reared from larval collections were tested for susceptibility to DDT, permethrin and deltamethrin using standard WHO procedures. Control, dead and survivors mosquitoes from bioassays were identified by PCR-RFLP and characterized for the kdr mutations using either the AS-PCR or the HOLA method. RESULTS: Four chemical insecticide groups were cited in the study sites: organochlorines, organophosphates, carbamates and pyrethroids. These chemicals were used for personal, crop or wood protection. In the four An. gambiae populations tested, significant variation in resistance levels, molecular forms composition and kdr frequencies were recorded in the time span of the study. Increases in DDT and pyrethroid resistance, as observed in most areas, were generally associated with an increase in the relative frequency of the S molecular form carrying the kdr mutations at higher frequencies. In Mangoum, however, where only the S form was present, a significant increase in the frequency of kdr alleles between 2003 to 2007 diverged with a decrease of the level of resistance to DDT and pyrethroids. Analyses of the kdr frequencies in dead and surviving mosquitoes showed partial correlation between the kdr genotypes and resistance phenotypes, suggesting that the kdr mechanism may act with certain co-factors to be identified. CONCLUSION: These results demonstrate the ongoing spread of kdr alleles in An. gambiae in Central Africa. The rapid evolution of insecticide resistance in this highly dynamic and genetically polymorphic species remains a challenge for its control.

Malaria Transmission around the Memve'ele Hydroelectric Dam in South Cameroon: A Combined Retrospective and Prospective Study, 2000⁻2016.

Dam constructions are considered a great concern for public health. The current study aimed to investigate malaria transmission in the Nyabessan village around the Memve'ele dam in South Cameroon. Adult mosquitoes were captured by human landing catches in Nyabessan before and during dam construction in 2000-2006 and 2014-2016 respectively, as well as in the Olama village, which was selected as a control. Malaria vectors were morphologically identified and analyzed for Plasmodium falciparum circumsporozoite protein detection and molecular identification of Anopheles (A.) gambiae species. Overall, ten malaria vector species were identified among 12,189 Anopheles specimens from Nyabessan (N = 6127) and Olama (N = 6062), including A. gambiae Giles (1902), A. coluzzii Coetzee (2013), A. moucheti Evans (1925), A. ovengensis Awono (2004), A. nili Theobald (1903), A. paludis Theobald (1900), A. zieanni, A. marshallii Theobald (1903), A. coustani Laveran (1900), and A. obscurus Grünberg (1905). In Nyabessan, A. moucheti and A. ovengensis were the main vector species before dam construction (16-50 bites/person/night-b/p/n, 0.26-0.71 infective bites/person/night-ib/p/n) that experienced a reduction of their role in disease transmission in 2016 (3-35 b/p/n, 0-0.5 ib/p/n) (p < 0.005). By contrast, the role of A. gambiae s.l. and A. paludis increased (11-38 b/p/n, 0.75-1.2 ib/p/n) (p < 0.01). In Olama, A. moucheti remained the main malaria vector species throughout the study period (p = 0.5). These findings highlight the need for a strong vector-borne disease surveillance and control system around the Memve'ele dam.

Spatial distribution of Anopheles gambiae sensu lato larvae in the urban environment of Yaoundé, Cameroon.

BACKGROUND: The rapid and unplanned urbanization of African cities is considered to increase the risk of urban malaria transmission. The present study objective was to assess factors influencing the spatio-temporal distribution of Anopheles gambiae s.l. larvae in the city of Yaoundé, Cameroon. METHODS: All water bodies were checked once every 2 months for the presence of mosquito larvae from March 2017 to May 2018 in 32 districts of Yaoundé. Physico-chemical characteristics including the size, depth, turbidity, pH, temperature, conductivity, sulfates, organophosphates, hydrogen peroxide (H2O2), conductivity, iron and calcium were recorded and analyzed according to anopheline larvae presence or absence. High resolution satellite images from landsat sentinel Enhanced Thematic Mapper were used for spatial mapping of both field and environmental variables. Bivariate and multivariate logistic regression models were used to identify variables closely associated with anopheline larvae distribution. RESULTS: A total of 18 696 aquatic habitats were checked and only 2942 sites (15.7%) contained anopheline larvae. A high number of sites with anopheline larvae (≥ 69%) presented late instar larvae (L3, L4 and pupae). Anopheline mosquito larvae were sampled from a variety of breeding sites including puddles (51.6%), tire prints (12.9%), wells (11.7%) and drains (11.3%). Bivariate logistic regression analyses associated anopheline larvae presence with the absence of predators, absence of algae, absence of vegetation and depth of less than 1 m. Conductivity, turbidity, organophosphates, H2O2 and temperature were significantly high in breeding sites with anopheline larvae than in breeding sites without these larvae (P <  0.1). Anopheline species collected included An. coluzzii (91.1%) and An. gambiae s.s. (8.9%). GIS mapping indicated a heterogeneous distribution of anopheline breeding habitats in the city of Yaoundé. Land cover analysis indicated high variability of the city of Yaoundé's landscape. CONCLUSIONS: The data confirms adaptation of An. gambiae s.l. to the urban domain in the city of Yaoundé and calls for urgent actions to improve malaria vector control.

Spatial and temporal development of deltamethrin resistance in malaria vectors of the Anopheles gambiae complex from North Cameroon.

The effectiveness of insecticide-based malaria vector control interventions in Africa is threatened by the spread and intensification of pyrethroid resistance in targeted mosquito populations. The present study aimed at investigating the temporal and spatial dynamics of deltamethrin resistance in An. gambiae s.l. populations from North Cameroon. Mosquito larvae were collected from 24 settings of the Garoua, Pitoa and Mayo Oulo Health Districts (HDs) from 2011 to 2015. Two to five days old female An. gambiae s.l. emerging from larval collections were tested for deltamethrin resistance using the World Health Organization's (WHO) standard protocol. Sub samples of test mosquitoes were identified to species using PCR-RFLP and genotyped for knockdown resistance alleles (Kdr 1014F and 1014S) using Hot Ligation Oligonucleotide Assay (HOLA). All the tested mosquitoes were identified as belonging to the An. gambiae complex, including 3 sibling species mostly represented by Anopheles arabiensis (67.6%), followed by Anopheles coluzzii (25.4%) and Anopheles gambiae (7%). Deltamethrin resistance frequencies increased significantly between 2011 and 2015, with mosquito mortality rates declining from 70-85% to 49-73% in the three HDs (Jonckheere-Terstra test statistic (JT) = 5638, P< 0.001), although a temporary increase of mortality rates (91-97%) was seen in the Pitoa and Mayo Oulo HDs in 2012. Overall, confirmed resistance emerged in 10 An. gambiae s.l. populations over the 24 field populations monitored during the study period, from 2011 to 2015. Phenotypic resistance was mostly found in urban settings compared with semi-urban and rural settings (JT = 5282, P< 0.0001), with a spatial autocorrelation between neighboring localities. The Kdr 1014F allelic frequencies in study HDs increased from 0-30% in 2011 to 18-61% in 2014-2015 (JT = 620, P <0.001), especially in An. coluzzii samples. The overall frequency of the Kdr 1014S allele was 0.1%. This study revealed a rapid increase and widespread deltamethrin resistance frequency as well as Kdr 1014F allelic frequencies in An. gambiae s.l. populations over time, emphasizing the urgent need for vector surveillance and insecticide resistance management strategies in Cameroon.

Patterns of anopheline feeding/resting behaviour and Plasmodium infections in North Cameroon, 2011-2014: implications for malaria control.

BACKGROUND: Effective malaria control relies on evidence-based interventions. Anopheline behaviour and Plasmodium infections were investigated in North Cameroon, following long-lasting insecticidal net (LLIN) distribution in 2010. METHODS: During four consecutive years from 2011 to 2014, adult mosquitoes were collected indoors, outdoors and in exit traps across 38 locations in the Garoua, Pitoa and Mayo-Oulo health districts. Anophelines were morphologically and molecularly identified, then analysed for blood meal origins and Plasmodium falciparum circumsporozoite protein (Pf-CSP). Blood from children under 5 years-old using LLINs was examined for Plasmodium infections. RESULTS: Overall, 9376 anophelines belonging to 14 species/sibling species were recorded. Anopheles gambiae (s.l.) [An. arabiensis (73.3%), An. coluzzii (17.6%) and An. gambiae (s.s.) (9.1%)] was predominant (72%), followed by An. funestus (s.l.) (20.5%) and An. rufipes (6.5%). The recorded blood meals were mainly from humans (28%), cattle (15.6%) and sheep (11.6%) or mixed (45%). Pf-CSP rates were higher indoors (3.2-5.4%) versus outdoors (0.8-2.0%), and increased yearly (χ2 < 18, df = 10, P < 0.03). Malaria prevalence in children under 5 years-old, in households using LLINs was 30% (924/3088). CONCLUSIONS: The present study revealed the variability of malaria vector resting and feeding behaviour, and the persistence of Plasmodium infections regardless the use of LLINs. Supplementary interventions to LLINs are therefore needed to sustain malaria prevention in North Cameroon.

High malaria transmission sustained by Anopheles gambiae s.l. occurring both indoors and outdoors in the city of Yaoundé, Cameroon.

Background: Malaria remains a major public health problem in Cameroon; however, despite reports on the adaptation of anopheline species to urban habitats, there is still not enough information on malaria transmission pattern in urban settings. In the frame of a larval control trial in the city of Yaoundé, we conducted baseline surveys to assess malaria transmission dynamics in this city. Methods: Adult mosquitoes were collected indoors and outdoors using CDC light traps and human landing catches from March 2017 to March 2018 in 30 districts of Yaoundé, Cameroon. Mosquitoes were sorted by genus and identified to the species level using PCR. The TaqMan method and ELISA were used to determine mosquito infection status to Plasmodium. Bioassays were conducted to assess female Anopheles gambiae susceptibility to insecticides. Results: A total of 218,991 mosquitoes were collected. The main malaria vectors were An. gambiae s.l. (n=6154) and An. funestus s.l. (n=229). Of the 1476 An. gambiae s.l. processed by PCR, 92.19% were An. coluzzii and 7.81% An. gambiae. An. funestus s.l. was composed of 93.01% (173/186) An. funestus and 4.84% (13/186) An. leesoni. The average biting rate of anopheline was significantly high outdoor than indoor (P=0.013). Seasonal variation in mosquito abundance and biting rate was recorded. The infection rate by Plasmodium falciparum was 2.13% (104/4893 mosquitoes processed). The annual entomological inoculation rate was found to vary from 0 to 92 infective bites/man/year (ib/m/y). Malaria transmission risk was high outdoor (66.65 ib/m/y) compared to indoor (31.14 ib/m/y). An. gambiae s.l. was found highly resistant to DDT, permethrin and deltamethrin. High prevalence of the West Africa kdr allele 1014F was recorded and this was not found to influence An. gambiae s.l. infection status.   Conclusion: The study suggests high malaria transmission occurring in the city of Yaoundé and call for immediate actions to improve control strategies.

The bionomics of the malaria vector Anopheles rufipes Gough, 1910 and its susceptibility to deltamethrin insecticide in North Cameroon.

BACKGROUND: Following the recent discovery of the role of Anopheles rufipes Gough, 1910 in human malaria transmission in the northern savannah of Cameroon, we report here additional information on its feeding and resting habits and its susceptibility to the pyrethroid insecticide deltamethrin. METHODS: From 2011 to 2015, mosquito samples were collected in 38 locations across Garoua, Mayo Oulo and Pitoa health districts in North Cameroon. Adult anophelines collected using outdoor clay pots, window exit traps and indoor spray catches were checked for feeding status, blood meal origin and Plasmodium circumsporozoite protein. The susceptibility of field-collected An. rufipes to deltamethrin was assessed using WHO standard procedures. RESULTS: Of 9327 adult Anopheles collected in the 38 study sites, An. rufipes (6.5%) was overall the fifth most abundant malaria vector species following An. arabiensis (52.4%), An. funestus (s.l.) (20.8%), An. coluzzii (12.6%) and An. gambiae (6.8%). This species was found outdoors (51.2%) or entering houses (48.8%) in 35 suburban and rural locations, together with main vector species. Apart from human blood with index of 37%, An. rufipes also fed on animals including cows (52%), sheep (49%), pigs (16%), chickens (2%) and horses (1%). The overall parasite infection rate of this species was 0.4% based on the detection of P. falciparum circumsporozoite proteins in two of 517 specimens tested. Among the 21 An. rufipes populations assessed for deltamethrin susceptibility, seven populations were classified as "susceptible" (mortality ≥ 98%) , ten as "probable resistant" with a mortality range of 90-97% and four as "resistant" with a mortality range of 80-89%. CONCLUSIONS: This study revealed changeable resting and feeding behaviour of An. rufipes, as well as further evidence on its ability to carry human malaria parasites in North Cameroon. Besides, this species is developing physiological resistance to deltamethrin insecticide which is used in treated nets and agriculture throughout the country, and should be regarded as one of potential targets for the control of residual malaria parasite transmission in Africa.

A preliminary test of the protective efficacy of permethrin-treated bed nets in an area of Anopheles gambiae metabolic resistance to pyrethroids in north Cameroon.

A trial of permethrin-treated nets (PTNs) versus untreated nets (UTNs) was conducted in Pitoa (north Cameroon), where the main malaria vectors, Anopheles gambiae s.l. and Anopheles arabiensis, show metabolic-based permethrin resistance. The deterrent effect of permethrin greatly reduced A. gambiae biting rate inside rooms where PTNs were installed. After 3 months of net use, malaria reinfection rate was significantly lower in children sleeping under a PTN, but no such effect was observed after 6 months. Parasitaemia was not significantly different between the two arms. These findings suggest good, although transitory, personal protection against malaria conferred by PTNs in an area of metabolic-based permethrin resistance.

Spectrum of metabolic-based resistance to DDT and pyrethroids in Anopheles gambiae s.l. populations from Cameroon.

Some populations of Anopheles gambiae s.l. from Cameroon were reported to develop resistance to DDT or pyrethroids but were free of the kdr mutation "Leucine-Phenylalanine" (Leu-Phe). This study reports on the metabolic activity of non-specific esterases (NSEs), mixed function oxidases (MFOs), and glutathione S-transferases (GSTs), three enzyme systems commonly involved in insecticide resistance. Biochemical assays were performed in DDT or pyrethroid-resistant populations of An. gambiae s.l. from Douala, Mbalmayo, Pitoa, and Simatou neighborhoods. Enzyme activity was compared to the Kisumu-susceptible reference strain using the Mann-Whitney test. Most of the tested samples had elevated NSE activity (P < 0.02). The Douala sample evenly displayed elevated GST activity (P < 0.001), while high MFO level was recorded in the Pitoa sample (P < 0.001). MFO or GST levels were sometimes lower or similar to that of the Kisumu strain. These results suggest metabolic detoxification is a major DDT or pyrethroid resistance mechanism and emphasize the need for further investigations on An. gambiae s.l. resistance mechanisms in Cameroon.

High susceptibility of wild Anopheles funestus to infection with natural Plasmodium falciparum gametocytes using membrane feeding assays.

BACKGROUND: Anopheles funestus is a major vector of malaria in sub-Saharan Africa. However, because it is difficult to colonize, research on this mosquito species has lagged behind other vectors, particularly the understanding of its susceptibility and interactions with the Plasmodium parasite. The present study reports one of the first experimental infections of progeny from wild-caught An. funestus with the P. falciparum parasite providing a realistic avenue for the characterisation of immune responses associated with this infection. METHODS: Wild-fed resting An. funestus females were collected using electric aspirators and kept in cages for four days until they were fully gravid and ready to oviposit. The resulting eggs were reared to adults F1 mosquitoes under insectary conditions. Three to five day-old An. funestus F1 females were fed with infected blood taken from gametocyte carriers using an artificial glass-parafilm feeding system. Feeding rate was recorded and fed mosquitoes were dissected at day 7 to count oocysts in midguts. Parallel experiments were performed with the known Plasmodium-susceptible An. coluzzii Ngousso laboratory strain, to monitor our blood handling procedures and infectivity of gametocytes. RESULTS: The results revealed that An. funestus displays high and similar level of susceptibility to Plasmodium infection compared to An. coluzzii, and suggest that our methodology produces robust feeding and infection rates in wild An. funestus progeny. The prevalence of infection in An. funestus mosquitoes was 38.52 % (range 6.25-100 %) and the median oocyst number was 12.5 (range 1-139). In parallel, the prevalence in An. coluzzii was 39.92 % (range 6.85-97.5 %), while the median oocyst number was 32.1 (range 1-351). CONCLUSIONS: Overall, our observations are in line with the fact that both species are readily infected with P. falciparum, the most common and dangerous malaria parasite in sub-Saharan Africa, and since An. funestus is widespread throughout Africa, malaria vector control research and implementation needs to seriously address this vector species too. Additionally, the present work indicates that it is feasible to generate large number of wild F1 infected An. funestus mosquitoes using membrane feeding assays, which can be used for comprehensive study of interactions with the Plasmodium parasite.

Malaria vectors and urbanization in the equatorial forest region of south Cameroon.

Entomological surveys were carried out in the town of Mbalmayo and in the nearby rural village of Olama, within the equatorial forest zone of Cameroon. Mosquitoes were captured when landing on human volunteers and by pyrethrum spray catches. Malaria vectors captured were Anopheles gambiae Giles (M and S forms) and A. moucheti Evans in both areas, together with A. funestus Giles in Mbalmayo. One A. marshallii (Theobald) specimen infected by Plasmodium falciparum was found in Olama. Anopheles moucheti was the most abundant anopheline species caught in Olama, while A. gambiae was the most abundant in Mbalmayo. All these vectors were highly anthropophilic as indicated by the fact that only 5 of 201 blood meals analysed had been taken from non-human hosts. Plasmodium falciparum was the only malaria parasite species found in Mbalmayo, while P. malariae was also found in Olama. The annual entomological inoculation rate was estimated at 129 infective bites/person/year in Mbalmayo and 322 in Olama. Comparison with data published in 1955 from Mbalmayo, before expansion of the town, showed the impact of urbanization on the composition of the vector system and malaria transmission dynamics. Such changes should be considered when implementing sustainable control measures.

Geographic distribution and breeding site preference of Aedes albopictus and Aedes aegypti (Diptera: culicidae) in Cameroon, Central Africa.

Presence in Cameroon of the recently introduced Aedes (Stegomyia) albopictus (Skuse) in association with the indigenous Aedes aegypti (L.) raises public heath concerns because it might alter the risk of arbovirus transmission. The breeding site and distribution of the two Stegomyia species are updated and reported following entomological surveys carried out in 22 localities throughout Cameroon, with a total of 1,353 containers with water visited. Ae. aegypti was found in every location sampled, showing higher infestation rates in northern Cameroon. Breeding populations of Ae. albopictus were observed in all 19 southern localities, up to the Adamaoua mountains, but the species was not recorded further north. In the area where both species are present, they were often sampled in the same larval developmental sites, suggesting convergent habitat segregation. The most frequently encountered artificial and natural breeding sites were used tires, discarded tins and plastic containers, abandoned car parts, brick holes, dead leaves on the ground, tree holes, and rock pools. Further monitoring of the demographic as well as geographic expansion of Ae. albopictus in this Afrotropical environment and its relationships with indigenous Ae. aegypti should provide insight into the biology of this highly invasive species and help to implement arboviruses surveillance programs in the area.