Can the use of larviciding with biological compounds contribute in increasing Anopheles gambiae s.l. susceptibility to pyrethroid in a population expressing high resistance intensity?

BACKGROUND: Larviciding using non-insecticide compounds is considered appropriate for controlling outdoor biting mosquitoes and for managing insecticide resistance. However, there is still not enough information on the influence of larviciding in managing pyrethroid resistance. In the present study, we checked whether the introduction of larviciding using the biolarvicide VectoMax G in the city of Yaoundé is contributing in restoring the susceptibility of An. coluzzii populations to pyrethroids. METHODOLOGY: The susceptibility status of field An. coluzzii population was evaluated at different time points before and during larviciding treatments. In addition, An. coluzzii larvae collected in the city of Yaoundé, were split into four groups and exposed to different selection regimes for many generations as follow; (i): deltamethrin 0.05%_only, (ii): Vectomax_only, (iii): Vectomax+deltamethrin 0.05%, (iv): VectoMax+deltamethrin 0.05% + susceptible. Life traits parameters were measured in the progeny and compared between colonies. The control was the susceptible laboratory strain "Ngousso". Kdr allele frequency and the profile of expression of different detoxification genes and oxidative stress genes was checked using qPCR analysis. Gene's expression was compared between the first and the last generation of each colony and in field populations collected before and during larviciding. RESULTS: An increase in mosquito susceptibility to deltamethrin and permethrin was recorded for the field populations after larviciding implementation. Resistance intensity to deltamethrin was found to decrease from high to low in field populations. Only the colony vectomax+deltamethrin+susceptible presented a high susceptibility to deltamethrin after 21 generations. The kdr gene frequency was found to be unchanged in the field population and laboratory colonies. A significant decrease in the overexpression profile of Gste2 was detected in field population after larviciding implementation. Other genes showing a similar pattern though not significant were Cyp6z1, Cyp6p1 and Cyp6g16. Concerning fitness only the colony vectomax+deltamethrin+susceptible was found to display a fitness profile similar to the susceptible colony with high fecundity, high hatching rate, short development time and long adult survival rate. CONCLUSION: The profile of the field population supported reversal of phenotypic resistance to pyrethroids however no reduction in the frequency of the kdr allele was recorded. Some detoxification genes were detected less overexpressed. The study suggest that reversal may take longer to achieve in a population expressing a very high resistance profile and under continuous insecticide selection pressure.

Malaria prevention in the city of Yaoundé: knowledge and practices of urban dwellers.

BACKGROUND: Malaria prevention in Cameroon mainly relies on the use of ITNs. Although several free distribution campaigns of treated nets have been conducted across the country, bed net usage remains very low. A household survey was conducted to assess knowledge of the population and practices affecting treated net usage in the city of Yaoundé. METHODS: A community-based descriptive cross-sectional survey was conducted in January 2017 in 32 districts of the city of Yaoundé. Parents (household head, spouse or an elder representative) who consented to the study, were interviewed using a structured pre-tested questionnaire. Interviews were conducted in French or English. A questionnaire consisting of 22 questions was administered to know (i) people's knowledge and attitude on preventive measures; and, (ii) attitudes concerning the treatment of malaria and estimated amount spent for malaria prevention and treatment. RESULTS: A total of 1643 household heads were interviewed. Over 94% of people interviewed associated malaria transmission to mosquito bites. The main methods used against mosquito bites were: treated bed nets (94%; n = 1526) and insecticide spray or coils (32.2%; n = 523). The majority of people interviewed reported using bed nets mainly to prevent from mosquito bites (84.4%, n = 1257), rather than for malaria prevention (47.3%). Knowledge and attitude analysis revealed that people with university or secondary level of education have better knowledge of malaria, prevention and treatment measures compared to those with the primary level (OR = 7.03; P < 0.001). Also, wealthy households were more aware of good practices concerning malaria prevention and treatment compared to poor ones. In the majority of districts of Yaoundé, over 50% of people interviewed per district, had good knowledge of malaria and prevention measures but less than 50% applied good practices concerning malaria treatment and prevention. The amount spent annually by a household for vector control was CFAF 11,589 ± 1133 (US$21.87 ± 2.14) and CFAF 66,403 ± 4012 (US$125.29 ± 7.57) for malaria treatment. CONCLUSION: The study indicated that, despite good knowledge of malaria and prevention measures, few people apply good practices. More sensitization needs to be done to improve adherence to good practices concerning malaria prevention and treatment.

Correction to: Malaria prevention in the city of Yaoundé: knowledge and practices of urban dwellers.

Following publication of the original article [1], the authors flagged an error concerning Table 3.

Larviciding intervention targeting malaria vectors also affects Culex mosquito distribution in the city of Yaoundé, Cameroon.

Although Culex species are considered to be equally affected by control measures targeting malaria vectors, there is still not enough evidence of the impact of interventions such as larviciding on the distribution of these mosquito species. The present study assessed the impact of a larviciding trial targeting malaria vectors on Culex mosquito species in the city of Yaoundé, Cameroon. A cluster randomized trial comparing 13 treated clusters and 13 untreated clusters was implemented. Data were collected at baseline and during the larviciding intervention, from March 2017 to November 2020. The microbial larvicide VectoMax G was applied once every 2 weeks in the intervention areas. Adult mosquitoes were collected using CDC light traps in both intervention and non-intervention areas and compared between arms. Globally, larviciding intervention was associated with 69% reduction in aquatic habitats with Culex larvae and 36.65% reduction of adult Culex densities in houses. Adult Culex densities were reduced both indoors (35.26%) and outdoors (42.37%). No change in the composition of Culex species was recorded. The study suggests a high impact of larviciding on Culex mosquito species distribution. The impact of the intervention can be improved if typical Culex breeding habitats including pit latrines are targeted.

Rapid evolution of insecticide resistance and patterns of pesticides usage in agriculture in the city of Yaoundé, Cameroon.

BACKGROUND: The practice of agriculture in urban settings contributes to the rapid expansion of insecticide resistance in malaria vectors. However, there is still not enough information on pesticide usage in most urban settings. The present study aims to assess the evolution of Anopheles gambiae (s.l.) population susceptibility to insecticides and patterns of pesticide usage in agriculture in the city of Yaoundé, Cameroon. METHODS: WHO susceptibility tests and synergist PBO bioassays were conducted on adult An. gambiae (s.l.) mosquitoes aged 3 to 5 days emerging from larvae collected from the field. Seven insecticides (deltamethrin, permethrin, DDT, bendiocarb, propoxur, fenitrothion and malathion) were evaluated. The presence of target site mutation conferring knockdown (kdr) resistance was investigated using TaqMan assay, and mosquito species were identified using SINE-PCR. Surveys on 81 retailers and 232 farmers were conducted to assess general knowledge and practices regarding agricultural pesticide usage. RESULTS: High resistance intensity to pyrethroids was observed with a high frequency of the kdr allele 1014F and low frequency of the kdr 1014S allele. The level of susceptibility of An. gambiae (s.l.) to pyrethroids and carbamates was found to decrease with time (from > 34% in 2017 to < 23% in 2019 for deltamethrin and permethrin and from 97% in 2017 to < 86% in 2019 for bendiocarb). Both An. gambiae (s.s.) and An. coluzzii were recorded. Over 150 pesticides and fertilizers were sold by retailers for agricultural purposes in the city of Yaoundé. Most farmers do not respect safety practices. Poor practices including extensive and inappropriate application of pesticides as well as poor management of perished pesticides and empty pesticide containers were also documented. CONCLUSIONS: The study indicated rapid evolution of insecticide resistance and uncontrolled usage of pesticides by farmers in agriculture. There is an urgent need to address these gaps to improve the management of insecticide resistance.

Knowledge, practices and perceptions of communities during a malaria larviciding randomized trial in the city of Yaoundé, Cameroon.

BACKGROUND: Urban malaria is becoming a major public health concern in major cities in Cameroon. To improve malaria vector control, a pilot larviciding trial was conducted to assess its impact on mosquito density and malaria transmission intensity in Yaoundé. The present study investigated perceptions and practices of communities on malaria control during the larviciding trial implemented in Yaoundé. METHODS: Quantitative and qualitative data were collected in non-intervention and intervention areas. Quantitative data were collected during three cross-sectional surveys using a structured pre-tested questionnaire while qualitative data were obtained through interviews. A total of 26 in-depth interviews and eight focus group discussions with community members were performed. A binary logistic regression model was used to assess the perception of the community on larviciding impact on some malaria or bed nets use indicators. RESULTS: People living in intervention areas were 2.64 times more likely to know the mode of malaria transmission (95% CI: 1.82-3.84; p<0.001) and 1.3 time more likely to know mosquito breeding habitats (95% CI: 1.06-1.56; p = 0.009) compared to those living in non-intervention areas. In intervention areas, interviewee opinions on larviciding were generally good i.e. most interviewees reported having noticed a reduction in mosquito nuisance and malaria cases following larviciding implementation; whereas in non-intervention areas no report of reduction of mosquito nuisance was recorded. LLINs were regularly used by the population despite the implementation of larviciding treatments. There was high interest in larviciding program and demand for continuation, even if this needs the community involvement. CONCLUSION: The larviciding program in the city of Yaoundé did not negatively affected community members' behaviour and practices concerning the use of treated nets. The study indicated the acceptance of larviciding program by the population. This positive environment could favour the implementation of future antilarval control activities in the city of Yaoundé.

Fitness cost of insecticide resistance on the life-traits of a Anopheles coluzzii population from the city of Yaoundé, Cameroon.

Background: Pyrethroid resistance is rapidly expanding in An. gambiae s.l. populations across Sub-Saharan Africa. Yet there is still not enough information on the fitness cost of insecticide resistance . In the present study, the fitness cost of insecticide resistance on Anopheles coluzzii population from the city of Yaoundé was investigated.   Methods: A resistant An. coluzzii colony was established from field collected mosquitoes resistant to both DDT and pyrethroid and selected for 12 generations with deltamethrin 0.05%. The Ngousso laboratory susceptible strain was used as control. A total of 100 females of each strain were blood fed and allowed for individual eggs laying, and then different life traits parameters such as fecundity, fertility, larval development time, emergence rate and longevity were measured. The TaqMan assay was used to screen for the presence of the L1014F and L1014S kdr mutations. Results:  Field collected mosquitoes from the F0 generation had a mortality rate of 2.05% for DDT, 34.16% for permethrin and 50.23% for deltamethrin. The mortality rate of the F12 generation was 30.48% for deltamethrin, 1.25% for permethrin  and 0% for DDT. The number of eggs laid per female was lower in the resistant colony compared to the susceptible (p <0.0001). Insecticide resistant larvae were found with a significantly long larval development time (10.61±0.33 days) compare to susceptible (7.57±0.35 days). The number of emerging females was significantly high in the susceptible group compared to the resistant . The adults lifespan was also significantly high for susceptible (21.73±1.19 days) compared to resistant (14.63±0.68 days). Only the L1014F- kdr allele was detected in resistant population.. Conclusion: The study suggests that pyrethroid resistance is likely associated with a high fitness cost on An.coluzzii populations. The addition of new tools targeting specifically larval stages could improve malaria vectors control and insecticide resistance management.

Status of Insecticide Resistance and Its Mechanisms in Anopheles gambiae and Anopheles coluzzii Populations from Forest Settings in South Cameroon.

A key factor affecting malaria vector control efforts in Cameroon is the rapid expansion of insecticide resistance in Anopheles gambiae s.l (An. gambiae) populations; however, mechanisms involved in insecticide resistance in forest mosquito populations are still not well documented yet. The present study was conducted to screen molecular mechanisms conferring insecticide resistance in An. gambiae s.l. populations from the South Cameroon forest region. WHO bioassays were conducted with F0 An. gambiae females aged three to four days from forest (Sangmelima, Nyabessan, and Mbandjock) and urban sites (Yaoundé (Bastos and Nkolondom)), against pyrethroids (permethrin 0.75% and deltamethrin 0.05%) and carbamates (bendiocarb 0.1%). Members of the An. Gambiae s.l. species complex were identified using molecular diagnostic tools. TaqMan assays were used to screen for target site mutations. The expression profiles of eight genes implicated in insecticide resistance were assessed using RT-qPCR. Cuticle hydrocarbon lipids were measured to assess their potential implication in insecticide resistance. Both An. Gambiae and An. coluzzii were detected. An. gambiae was highly prevalent in Sangmelima, Nyabessan, Mbandjock, and Nkolondom. An. coluzzii was the only species found in the Yaoundé city center (Bastos). Low mortality rate to both pyrethroids and bendiocarb was recorded in all sites. High frequency of L1014F allele (75.32-95.82%) and low frequencies of L1014S (1.71-23.05%) and N1575Y (5.28-12.87%) were recorded. The G119S mutation (14.22-35.5%) was detected for the first time in An. gambiae populations from Cameroon. This mutation was rather absent from An. coluzzii populations. The detoxification genes Cyp6m2, Cyp9k1, Cyp6p4, Cyp6z1, as well as Cyp4g16 which catalyzes epicuticular hydrocarbon biosynthesis, were found to be overexpressed in at least one population. The total cuticular hydrocarvbon content, a proxy of cuticular resistance, did not show a pattern associated with pyrethroid resistance in these populations. The rapid emergence of multiple resistance mechanisms in An. Gambiae s.l. population from the South Cameroon forest region is of big concern and could deeply affect the sustainability of insecticide-based interventions strategies in this region.

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.

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.