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.

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.

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.

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

BACKGROUND: As part of a study to determine the impact of insecticide resistance on the effectiveness of long-lasting 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 human-biting 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.

Anopheles ziemanni a locally important malaria vector in Ndop health district, north west region of Cameroon.

BACKGROUND: Malaria transmission in Cameroon is mediated by a plethora of vectors that are heterogeneously distributed across the country depending on the biotope. To effectively guide malaria control operations, regular update on the role of local Anopheles species is essential. Therefore, an entomological survey was conducted between August 2010 and May 2011 to evaluate the role of the local anopheline population in malaria transmission in three villages of the Ndop health district in the northwest region of Cameroon where malaria is holoendemic, as a means to acquiring evidence based data for improved vector intervention. METHODS: Mosquitoes were sampled both indoor and outdoor for four consecutive nights in each locality during each month of survey. Sampling was done by the human landing catch method on volunteers. Anopheles species were identified morphologically and their ovaries randomly dissected for parity determination. Infection with Plasmodium falciparum was detected by Circumsporozoite protein ELISA. Members of An. gambiae complex were further identified to molecular level by PCR and RFLP PCR. RESULTS: An. ziemanni was the main malaria vector and whether outdoor or indoor. The man biting rate for the vectors ranged from 6.75 to 8.29 bites per person per night (b/p/n). The entomological inoculation rate for this vector species was 0.0278 infectious bites per person per night (ib/p/n) in Mbapishi, 0.034 ib/p/n in Mbafuh, and 0.063 ib/p/n in Backyit. These were by far greater than that for An. gambiae. No difference was observed in the parity rate of these two vectors. PCR analysis revealed the presence of only An. colluzzi (M- form). CONCLUSIONS: An. ziemanni is an important local malaria vector in Ndop health district. The findings provide useful baseline information on the anopheles species composition, their distribution and role in malaria transmission that would guide the implementation of integrated vector management strategies in the locality.

A preliminary investigation of the relationship between water quality and Anopheles gambiae larval habitats in Western Cameroon.

BACKGROUND: Water quality and anopheline habitat have received increasing attention due to the possibility that challenges during larval life may translate into adult susceptibility to malaria parasite infection and/or insecticide resistance. METHODS: A preliminary study of Anopheles gambiae s.s. larval habitats in the north-west and south-west regions of Cameroon was conducted in order to detect associations between An. gambiae s.s. molecular form and 2La inversion distributions with basic water quality parameters. Water quality was measured by temperature, pH, conductivity, total dissolved solids (TDS) at seven sites in Cameroon and one site in Selinkenyi, Mali. RESULTS: Principal components and correlation analyses indicated a complex relationship between 2La polymorphism, temperature, conductivity and TDS. Cooler water sites at more inland locations yielded more S form larvae with higher 2La inversion polymorphism while warmer water sites yielded more M form larvae with rare observations of the 2La inversion. DISCUSSION: More detailed studies that take into account the population genetics but also multiple life stages, environmental data relative to these life stages and interactions with both humans and the malaria parasite may help us to understand more about how and why this successful mosquito is able to adapt and diverge, and how it can be successfully managed.

Pyrethroid resistance in Anopheles gambiae from the rubber cultivated area of Niete, South Region of Cameroon.

Anti-vector intervention remains the most effective way of controlling malaria. Although in Cameroon and elsewhere focus is on the use of long-lasting insecticidal nets and indoor residual spraying, the efficacy of both methods greatly depends on the continuing susceptibility of the vectors to the insecticides used. The emergence and spread of insecticide resistance in the major malaria vectors constitute a huge challenge to control programmes. Consequently, routine monitoring and evaluation of vector resistance status to insecticides are mandatory for early detection of resistance should it arise, and effectively planning future anti-vector interventions especially in areas reputed for routine application in agriculture. The WHO bioassay kit was used to determine the susceptibility status of Anopheles gambiae s.l. populations to seven insecticides belonging to four classes (organochlorine, organophosphate, carbamate and pyrethroids) in Niete, an area of intense rubber cultivation in southern forested Cameroon. Species and molecular forms of An. gambiae s.l. as well as the presence of knock down resistance (kdr) mutations were determined using polymerase chain reaction (PCR) techniques. All Anopheles tested was identified as An. gambiae s.s. and of the M molecular form. Based on WHO classification, while the mosquitoes were fully (100%) susceptible to malathion and bendiocarb, resistance was confirmed to DDT and the pyrethroids, permethrin and lambda-cyhalothrin. The other pyrethroids (deltamethrin and cyfluthrin) showed signs of developing resistance. Resistance to DDT and pyrethroids is indicative of existing cross resistance mechanisms between these insecticides. The increase in knockdown times was greater than twofold that of the reference susceptible strain, suggesting the possible involvement of kdr mutations, also confirmed in this study. The findings highlight the need for constant evaluation, re-evaluation and monitoring of the insecticides for malaria vector control in Cameroon. However, bendiocarb and malathion can be used and may require alternation or combination with insecticides of other classes to better manage the occurrence and spread of resistance in Niete.

Seasonal prevalence of malaria vectors and entomological inoculation rates in the rubber cultivated area of Niete, South Region of Cameroon.

BACKGROUND: Development of large scale agro-industries are subject to serious environmental modifications. In malaria endemic areas this would greatly impact on the transmission paradigm. Two cross-sectional entomological surveys to characterize the Anopheles fauna and their entomological inoculation rates were conducted during May 2010 (peak rainy season) and December 2010 (peak dry season) in the intense rubber cultivated area of Niete in southern forested Cameroon. METHODS: Mosquitoes were sampled by night collections on human volunteers, identified morphologically and members of the Anopheles gambiae complex further identified to species and molecular form. Parity status was determined following the dissection of the ovaries. Plasmodium falciparum circumsporozoite antigen indices were estimated after the identification of CS antigen by ELISA and the average entomological inoculation rates determined. RESULTS: A total of 1187 Anopheles was collected, 419 (35.3%) in the rainy season and 768 (64.7%) in the dry season. Species found were the M molecular form of An. gambiae s.s (66.8%), An. ziemanni (28.3%), An. paludis (4.7%), An. smithii (0.2%). An. gambiae M-form was the principal species in the dry (56.2%) and wet (86.2%) seasons. Average overall entomological inoculation rate for the malaria vectors varied between the dry season (1.09 ib/p/n) and the rainy season (2.30 ib/p/n). CONCLUSIONS: Malaria transmission in Niete occurs both in the dry and rainy season with the intensities peaking in the dry season. This is unlike previous studies in other areas of southern forested Cameroon where transmission generally peaks in the rainy season. Environmental modifications due to agro-industrial activities might have influenced vector distribution and the dynamics of malaria transmission in this area. This necessitates the possible implementation of control strategies that are related to the eco-geography of the area.

Anopheline fauna and malaria transmission in four ecologically distinct zones in Cameroon.

Knowledge of baseline malaria transmission intensity in a given environment is important to guide malaria control interventions. In Cameroon, recent information on malaria transmission intensity is insufficient. Therefore, an entomological study was conducted in four ecologically different sites throughout the country to assess the seasonal patterns in malaria transmission intensity. Anopheles arabiensis was the main vector in six of the nine study sites, while An. gambiae sensu stricto was the most important vector in the other three sites. Clear differences in entomological inoculation rates (EIR) were observed between the study sites, ranging from 0.1 infective bites per person per night in the sahelian zone of the country to 5.5 infective bites per person per night in the forest zone. Based on the observed behaviour of the vectors, insecticide-treated bed nets will be highly effective in controlling malaria. However, in the high transmission areas, additional measures will be needed to reduce the malaria burden to acceptable levels.

The distribution of insecticide resistance in Anopheles gambiae s.l. populations from Cameroon: an update.

Insecticides are a key component of vector-based malaria control programmes in Cameroon. As part of ongoing resistance surveillance efforts, Anopheles gambiae s.l. female mosquitoes were exposed to organochlorine (DDT), a carbamate (bendiocarb), an organophosphate (malathion), and three pyrethroids (deltamethrin, lambda-cyhalothrin and permethrin) in WHO bioassay test kits. Results indicated a higher level of resistance (reduced mortality and knockdown effect) to DDT and pyrethroids in populations of A. gambiae s.s. than in A. arabiensis. The West and East African knockdown resistance (kdr) mutations were found in both species but at much higher frequencies in A. gambiae s.s. The West Africa kdr mutant was also more frequent in the A. gambiae S form than in the M form. No resistance to bendiocarb and malathion was found. Carbamate and organophosphorous compounds could thus be used as alternatives in locations in Cameroon where pyrethroid-resistant populations are found.

Relationship between kdr mutation and resistance to pyrethroid and DDT insecticides in natural populations of Anopheles gambiae.

The spread of insecticide resistance genes in Anopheles gambiae Giles sensu stricto threatens to compromise vector-based malaria control programs. Two mutations at the same locus in the voltage-gated sodium channel gene are known to confer knockdown resistance (kdr) to pyrethroids and DDT. Kdr-e involves a leucine-serine substitution, and it was until recently thought to be restricted to East Africa, whereas kdr-w, which involves a leucine-phenylalanine substitution, is associated with resistance in West Africa. In this study, we analyze the frequency and relationship between the kdr genotypes and resistance to type I and type II pyrethroids and DDT by using WHO test kits in both the Forest-M and S molecular forms of An. gambiae in Cameroon. Both kdr-w and kdr-e polymorphisms were found in sympatric An. gambiae, and in many cases in the same mosquito. Kdr-e and kdr-w were detected in both forms, but they were predominant in the S form. Both kdr-e and kdr-w were closely associated with resistance to DDT and weakly associated with resistance to type II pyrethroids. Kdr-w conferred greater resistance to permethrin than kdr-e. We also describe a modified diagnostic designed to detect both resistant alleles simultaneously.