The ecology and behaviour of insecticide resistant malaria vectors and implications for control in Burkina Faso

Long-Lasting Insecticide-Treated Nets (LLINs) and Indoor Residual Spraying (IRS) are the most common and successful methods for malaria vector control in Africa. There is growing evidence of shifts in mosquito vector biting and resting behaviours in several African settings where high LLIN coverage has been achieved. These changes, combined with growing insecticide resistance, may reduce intervention success by decreasing the contact between vectors and insecticide-treated surfaces. While insecticide resistance in malaria vectors has been widely investigated, less is known about the implications of mosquito behavioural changes to malaria control. In recent years, LLIN programmes appear to have a reducing impact in a small number of high burden African countries including Burkina Faso. This reducing effectiveness is hypothesized to be the result of insecticide resistance, but the potential additional contribution of mosquito behavioural avoidance strategies has not yet been investigated in Burkina Faso. The aim of this PhD was to investigate the contribution of insecticide resistance and mosquito behaviours to the persistence of malaria transmission in southwestern Burkina Faso following a national LLIN-distribution campaign. Specific objectives were to (i) evaluate the performance of a new mosquito sampling method, the Mosquito Electrocuting Trap (MET) to measure spatial and temporal variation in human exposure to malaria vectors; and characterize the spatial, seasonal and longer-term trends in (ii) vector ecology and behaviours, (iii) insecticide resistance within Anopheles gambiae sensu lato (s.l.) and (iv) malaria vector survival and transmission potential in rural Burkina Faso. A two-year programme of longitudinal mosquito vector surveillance was initiated within 12 villages of south-western Burkina Faso in 2016, shortly after completion of a mass LLIN distribution. Host seeking malaria vectors were sampled monthly using Human Landing Catches (HLC) and METs conducted inside houses and in the surrounding outdoor area (911 households in total). Resting bucket traps (RBTs) were used to sample indoor and outdoor resting vectors. In an initial study (Chapter 2), I evaluated the performance of the MET relative to the HLC for sampling host-seeking malaria vectors over 15 months in 12 villages. Overall, the MET caught proportionately fewer An. gambiae s.l. than the HLC (mean estimated number of 0.78 versus 1.82 indoors, and 1.05 versus 2.04 outdoors). However provided a consistent representation of vector species composition, seasonal and spatial dynamics, biting behaviour (e.g. location and time) and malaria infection rates relative. The MET slightly underestimated the proportion of bites that could be prevented by LLINs relative to the HLC (5%). However, given the major advantage of the MET of reducing human infection risk during sampling, I conclude these limitations are acceptable and that the MET presents a promising and safer alternative for monitoring human exposure to malaria vectors in outdoor environments. Vector sampling was extended (using HLCs and RBTs) to investigate longer-term temporal changes in vector ecology and behaviour (Chapter 3). Analysis of a subset (20%) of the An. gambiae s.l. (N= 7852) indicated that An. coluzzii (53.82%) and An. gambiae (45.9%) were the main vector species. There was substantial variation in vector abundance between sites and seasons, with a predicted ~23% reduction in An. gambiae s.l. biting density from start to end of study. A higher proportion of outdoor biting (~54%) was detected than expected from previous studies; but there was no evidence of spatial, seasonal or longer-term changes in exophagy. Species level analyses indicated that revealed moderate but statistically significant different in the exophagy and biting time between An. coluzzii and An. gambiae. Combining information on biting times and location (indoors versus outdoors), I estimated that ~85% of exposure could be prevented using good quality and effective LLINs during standard sleeping hours (10 pm – 5 am). Bioassays were conducted on the An. gambiae s.l. population at 9 out of the original 12 study villages to estimate spatial, seasonal and longer-term variation in insecticide resistance (IR) over the study period. Overall, only 23% of An. gambiae s.l. exposed to a diagnostic dose of deltamethrin were killed within 24 hours; indicating that all surveyed populations are resistant. Furthermore, IR increased over the study period, with significant reduction in mortality after exposure to deltamethrin in bioassays. There was no evidence of variation in IR between An. gambiae and An. coluzzii. Finally, the transmission potential of An. gambiae s.l. in this area was investigated through assessment of mosquito parity rates (a proxy of survival), malaria infection rates and estimation of annual Entomological Inoculation Rates (EIR; Chapter 5). The daily survival rate of malaria vectors in this area was > 90%), but with variation between villages and seasons. After controlling for this spatial and seasonal variation, there was evidence of a longer-term increase in vector survival over the study period. In contrast, both mosquito vector biting densities and their malaria infection rates declined over the study period. This resulted in a drop in the predicted EIR from 320 to 105 infective bites per person/year respectively in year 1 and 2. Considering the proportion of exposure estimated to be preventable by effective LLIN use (~85%, Chapter 2 &3), I estimated that residents in this area are still exposed to ~32 infective bites per person per year even when this intervention is used. This confirms that even with 100% coverage and usage of highly effective LLINs, high levels of transmission will persist in this setting. Taking the case of Burkina Faso as an example, results obtained here confirm that both IR and outdoor biting by malaria vectors are contributing to the persistence of transmission in high burden African countries. Consequently, a successful vector control programme in this context need a clear insecticide resistance management plan and supplementary tools that target vectors feeding and resting outdoors

The sticky resting box, a new tool for studying resting behaviour of Afrotropical malaria vectors

Background: Monitoring densities of adult mosquito populations is a major challenge in efforts to evaluate the epidemiology of mosquito-borne diseases, and their response to vector control interventions. In the case of malaria, collection of outdoor-resting Anophelines is rarely incorporated into surveillance and control, partially due to the lack of standardized collection tools. Such an approach, however, is increasingly important to investigate possible changes in mosquito behaviour in response to the scale up of Insecticide Treated Nets and Indoor Residual Spraying. In this study we evaluated the Sticky Resting Box (SRB) - i.e. a sticky variant of previously investigated mosquito Resting Box, which allows passive collection of mosquitoes entering the box – and compared its performance against traditional methods for indoor and outdoor resting mosquito sampling.<p></p> Methods: Daily collections were carried out in two neighbouring villages of Burkina Faso during rainy season 2011 and dry season 2012 by SRB located indoors and outdoors, and by Back-Pack aspiration inside houses (BP) and in ad hoc built outdoor pit-shelters (PIT).<p></p> Results: Overall, almost 20,000 Culicidae specimens belonging to 16 species were collected and morphologically identified. Malaria vectors included Anopheles coluzzii (53%), An. arabiensis (12%), An. gambiae s.s. (2.0%) and An. funestus (4.5%). The diversity of species collected in the two villages was similar for SRB and PIT collections outdoors, and significantly higher for SRB than for BP indoors. The population dynamics of An. gambiae s.l. mosquitoes, as obtained by SRB-collections was significantly correlated with those obtained by the traditional methods. The predicted mean estimates of An. gambiae s.l. specimens/sampling-unit/night-of-collections was 6- and 5-times lower for SRB than for BP indoors and PIT outdoors, respectively.<p></p> Conclusions: Overall, the daily performance of SRB in terms of number of malaria vectors/trap was lower than that of traditionally used approaches for in- and outdoor collections. However, unlike these methods, SRB could be set up to collect mosquitoes passively over at least a week. This makes SRB a promising tool for passively monitoring anopheline resting populations, with data presented here providing guidance for how to set up SRB-based collections to acquire information comparable to those obtained with other methods.<p></p&gt

Cytochrome P450associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae.

The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of 14C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An. gambiae.Fil: Balabanidou, Vasileia. Foundation for Research and Technology-Hellas; Grecia. Universidad de Creta; GreciaFil: Kampouraki, Anastasia. Universidad de Creta; GreciaFil: Mac Lean, Marina. University of Nevada; Estados UnidosFil: Blomquist, Gary J.. University of Nevada; Estados UnidosFil: Tittiger, Claus. University of Nevada; Estados UnidosFil: Juarez, Marta Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Mijailovsky, Sergio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Chalepakis, George. Universidad de Creta; GreciaFil: Anthousi, Amalia. Universidad de Creta; GreciaFil: Lynd, Amy. Liverpool School of Tropical Medicine; Reino UnidoFil: Antoine, Sanou. Liverpool School of Tropical Medicine; Reino UnidoFil: Hemingway, Janet. Liverpool School of Tropical Medicine; Reino UnidoFil: Ranson, Hilary. Liverpool School of Tropical Medicine; Reino UnidoFil: Lycett, Gareth J.. Liverpool School of Tropical Medicine; Reino UnidoFil: Vontas, John. Foundation for Research and Technology-Hellas; Grecia. Agricultural University of Athens; Greci

Quantifying individual variability in exposure risk to mosquito bites in the Cascades region, Burkina Faso

Background The Cascades region, Burkina Faso, has a high malaria burden despite reported high insecticide-treated mosquito net (ITN) use. Human and vector activities outside the hours when indoor interventions offer direct protection from infectious bites potentially increase exposure risk to bites from malaria-transmitting Anopheles mosquitoes. This work investigated the degree of variation in human behaviour both between individuals and through time (season) to quantify how it impacts exposure to malaria vectors. Methods Patterns in human overnight activity (18:00–06:00) to quantify time spent using an ITN across 7 successive nights in two rural communities, Niakore (N = 24 participants) and Toma (71 participants), were observed in the dry and rainy seasons, between 2017 and 2018. Hourly human landing Anopheles mosquito catches were conducted in Niakore specifically, and Cascades region generally, between 2016 and 2017. Data were statistically combined to estimate seasonal variation in time spent outdoors and Anopheles bites received per person per night (bpppn). Results Substantial variability in exposure to outdoor Anopheles bites was detected within and between communities across seasons. In October, when Anopheles densities are highest, an individual’s risk of Anopheles bites ranged from 2.2 to 52.2 bites per person per night (bpppn) within the same week with variable risk dependent on hours spent indoors. Comparably higher outdoor human activity was observed in April and July but, due to lower Anopheles densities estimated, bpppn were 0.2–4.7 and 0.5–32.0, respectively. Males and people aged over 21 years were predicted to receive more bites in both sentinel villages. Conclusion This work presents one of the first clear descriptions of the degree of heterogeneity in time spent outdoors between people and across the year. Appreciation of sociodemographic, cultural and entomological activities will help refine approaches to vector control

Surveillance of Transmitted Drug-Resistant HIV Among Young Pregnant Women in Ouagadougou, Burkina Faso

Burkina Faso began rapid antiretroviral therapy (ART) scale-up in 2003 and by December 2009, 26 448 individuals were on treatment. With rapid scale-up of ART, some degree of human immunodeficiency virus transmitted drug resistance (TDR) is inevitable. Following World Health Organization methods, between June 2008 and July 2009, Burkina Faso assessed TDR in primigravid pregnant women aged <25 years attending antenatal care clinics in Ouagadougou, Burkina Faso. TDR was classified as moderate (5%-15%) for both nucleoside reverse-transcriptase inhibitors and nonnucleoside reverse-transcriptase inhibitors. The observed moderate TDR in Ouagadougou is a cause for concern and calls for closer monitoring of Burkina Faso's ART progra

Evaluation of mosquito electrocuting traps as a safe alternative to the human landing catch for measuring human exposure to malaria vectors in Burkina Faso

Background: Measuring human exposure to mosquito bites is a crucial component of vector-borne disease surveillance. For malaria vectors, the human landing catch (HLC) remains the gold standard for direct estimation of exposure. This method, however, is controversial since participants risk exposure to potentially infected mosquito bites. Recently an exposure-free mosquito electrocuting trap (MET) was developed to provide a safer alternative to the HLC. Early prototypes of the MET performed well in Tanzania but have yet to be tested in West Africa, where malaria vector species composition, ecology and behaviour are different. The performance of the MET relative to HLC for characterizing mosquito vector population dynamics and biting behaviour in Burkina Faso was evaluated. Methods: A longitudinal study was initiated within 12 villages in Burkina Faso in October 2016. Host-seeking mosquitoes were sampled monthly using HLC and MET collections over 14 months. Collections were made at 4 households on each night, with METs deployed inside and outside at 2 houses, and HLC inside and outside at another two. Malaria vector abundance, species composition, sporozoite rate and location of biting (indoor versus outdoor) were recorded. Results: In total, 41,800 mosquitoes were collected over 324 sampling nights, with the major malaria vector being Anopheles gambiae sensu lato (s.l.) complex. Overall the MET caught fewer An. gambiae s.l. than the HLC (mean predicted number of 0.78 versus 1.82 indoors, and 1.05 versus 2.04 outdoors). However, MET collections gave a consistent representation of seasonal dynamics in vector populations, species composition, biting behaviour (location and time) and malaria infection rates relative to HLC. As the relative performance of the MET was somewhat higher in outdoor versus indoor settings, this trapping method slightly underestimated the proportion of bites preventable by LLINs compared to the HLC (MET = 82.08%; HLC = 87.19%). Conclusions: The MET collected proportionately fewer mosquitoes than the HLC. However, estimates of An. gambiae s.l. density in METs were highly correlated with HLC. Thus, although less sensitive, the MET is a safer alternative than the HLC. Its use is recommended particularly for sampling vectors in outdoor environments where it is most sensitive

Anopheline species composition and the 1014F-genotype in different ecological settings of Burkina Faso in relation to malaria transmission

Background: A three-year longitudinal study was conducted in four sentinel sites from different ecological settings in Burkina Faso, between 2008 and 2010 to identify longitudinal changes in insecticide resistance within Anopheles gambiae complex species based on larval collection. During this study, adult mosquitoes were also collected indoor and outdoor using several methods of collection. The present study reports the diversity of malaria vectors and the 1014F-genotype from this adult collection and investigates the association between this 1014F-genotype and sporozoite rate. Methods: Adult mosquitoes were collected from July to August (corresponding to the start of rainy season) and October to November (corresponding to the end of rainy season) over 3 years (2008–2010) at four sites across the country, using pyrethrum spray catches (PSC), exit traps and pit shelters. Anopheles gambiae complex mosquitoes were identified to species and genotyped for the L1014F kdr mutation by PCR using genomic DNA. The circumsporozoite antigen of Plasmodium falciparum was detected in mosquitoes using sandwich ELISA. Results: Overall 9212 anopheline mosquitoes were collected during the study period. Of those, 6767 mosquitoes were identified as Anopheles gambiae sensu lato (s.l.). Anopheles arabiensis, Anopheles coluzzii, Anopheles gambiae and or Anopheles funestus were incriminated as vectors of P. falciparum in the study area with an average sporozoite rate of 5%, (95% CI 4.14–5.99%). The kdr1014F-genotype frequencies were 11.44% (95% CI 2.5–39.85%), 19.2% (95% CI 4.53–53.73%) and 89.9 (95% CI 63.14–97.45%), respectively for An. arabiensis, An. coluzzii and An. gambiae. The proportion of the 1014F-genotype varied between sporozoite-infected and uninfected An. gambiae s.l. group. There was no significant difference in the 1014F-genotype frequency between infected and uninfected mosquitoes. Conclusion: The current study shows the diversity of malaria vectors and significant interaction between species composition and kdr1014F-genotype in An. gambiae complex mosquitoes from Burkina Faso. In this study, no associations were found between the 1014F-genotype and P. falciparum infection in the major malaria vector An. gambiae s.l

A cohort study to identify risk factors for Plasmodium falciparum infection in Burkinabe children: implications for other high burden high impact countries

Background: Progress in controlling malaria has stalled in recent years. Today the malaria burden is increasingly concentrated in a few countries, including Burkina Faso, where malaria is not declining. A cohort study was conducted to identify risk factors for malaria infection in children in southwest Burkina Faso, an area with high insecticide-treated net (ITN) coverage and insecticide-resistant vectors. Methods: Incidence of Plasmodium falciparum infection was measured in 252 children aged 5 to 15 years, using active and passive detection, during the 2017 transmission season, following clearance of infection. Demographic, socio-economic, environmental, and entomological risk factors, including use of ITNs and insecticide resistance were monitored. Results: During the six-month follow-up period, the overall incidence of P. falciparum infection was 2.78 episodes per child (95% CI = 2.66–2.91) by microscopy, and 3.11 (95% CI = 2.95–3.28) by polymerase chain reaction (PCR). The entomological inoculation rate (EIR) was 80.4 infective bites per child over the six-month malaria transmission season. At baseline, 80.6% of children were reported as sleeping under an ITN the previous night, although at the last survey, 23.3% of nets were in poor condition and considered no longer protective. No association was found between the rate of P. falciparum infection and either EIR (incidence rate ratio (IRR): 1.00, 95% CI: 1.00–1.00, p = 0.08) or mortality in WHO tube tests when vectors were exposed to 0.05% deltamethrin (IRR: 1.05, 95% CI: 0.73–1.50, p = 0.79). Travel history (IRR: 1.52, 95% CI: 1.45–1.59, p < 0.001) and higher socio-economic status were associated with an increased risk of P. falciparum infection (IRR: 1.05, 95% CI: 1.00–1.11, p = 0.04). Conclusions: Incidence of P. falciparum infection remains overwhelmingly high in the study area. The study findings suggest that because of the exceptionally high levels of malaria transmission in the study area, malaria elimination cannot be achieved solely by mass deployment of ITNs and additional control measures are needed

Insecticide resistance and behavioural adaptation as a response to long-lasting insecticidal net deployment in malaria vectors in the Cascades region of Burkina Faso

The decline in malaria across Africa has been largely attributed to vector control using long-lasting insecticidal nets (LLINs). However, this intervention has prompted widespread insecticide resistance (IR) and been associated with changes in mosquito behaviour that reduce their contact with LLINs. The relative importance and rate at which IR and behavioural adaptations emerge are poorly understood. We conducted surveillance of mosquito behaviour and IR at 12 sites in Burkina Faso to assess the magnitude and temporal dynamics of insecticide, biting and resting behaviours in vectors in the 2-year period following mass LLIN distribution. Insecticide resistance was present in all vector populations and increased rapidly over the study period. In contrast, no longitudinal shifts in LLIN-avoidance behaviours (earlier or outdoor biting and resting) were detected. There was a moderate but statistically significant shift in vector species composition from Anopheles coluzzii to Anopheles gambiae which coincided with a reduction in the proportion of bites preventable by LLINs; possibly driven by between-species variation in behaviour. These findings indicate that adaptations based on insecticide resistance arise and intensify more rapidly than behavioural shifts within mosquito vectors. However, longitudinal shifts in mosquito vector species composition were evident within 2 years following a mass LLIN distribution. This ecological shift was characterized by a significant increase in the exophagic species (An. gambiae) and coincided with a predicted decline in the degree of protection expected from LLINs. Although human exposure fell through the study period due to reducing vector densities and infection rates, such ecological shifts in vector species along with insecticide resistance were likely to have eroded the efficacy of LLINs. While both adaptations impact malaria control, the rapid increase of the former indicates this strategy develops more quickly in response to selection from LLINS. However, interventions targeting both resistance strategies will be needed

Strengthening adult mosquito surveillance in Africa for disease control: Learning from the present

Mosquito surveillance is essential to successfully control and eliminate mosquito-borne diseases. Yet, it is often done by numerous organizations with little collaboration, incomplete understanding of existing gaps, and limited long-term vision. There is a clear disconnect between entomological and epidemiological indices, with entomological data informing control efforts inadequately. Here we discuss current mosquito surveillance practises across the heterogenous disease landscape in Africa. We advocate for the development of mosquito surveillance strategic plans to increase the impact and functionality of mosquito surveillance. We urge for a proactive approach to set up centralized mosquito data systems under custodian of national governments, focus on epidemiologically relevant mosquito data and increase robustness of mosquito surveillance using a more spatially explicit sampling design