Whole genome sequence analysis of population structure and insecticide resistance markers in Anopheles melas from the Bijagós Archipelago, Guinea-Bissau.

BACKGROUND: Anopheles melas is an understudied malaria vector with a potential role in malaria transmission on the Bijagós Archipelago of Guinea-Bissau. This study presents the first whole-genome sequencing and population genetic analysis for this species from the Bijagós. To our knowledge, this also represents the largest population genetic analysis using WGS data from non-pooled An. melas mosquitoes. METHODS: WGS was conducted for 30 individual An. melas collected during the peak malaria transmission season in 2019 from six different islands on the Bijagós Archipelago. Bioinformatics tools were used to investigate the population structure and prevalence of insecticide resistance markers in this mosquito population. RESULTS: Insecticide resistance mutations associated with pyrethroid resistance in Anopheles gambiae s.s. from the Bijagós were absent in the An. melas population, and no signatures of selective sweeps were identified in insecticide resistance-associated genes. Analysis of structural variants identified a large duplication encompassing the cytochrome-P450 gene cyp9k1. Phylogenetic analysis using publicly available mitochondrial genomes indicated that An. melas from the Bijagós split into two phylogenetic groups because of differentiation on the mitochondrial genome attributed to the cytochrome C oxidase subunits COX I and COX II and the NADH dehydrogenase subunits 1, 4, 4L and 5. CONCLUSIONS: This study identified an absence of insecticide-resistant SNPs common to An. gambiae in the An. melas population, but did identify structural variation over insecticide resistance-associated genes. Furthermore, this study presents novel insights into the population structure of this malaria vector using WGS analysis. Additional studies are required to further understand the role of this vector in malaria transmission.

Mosquitocidal effect of ivermectin-treated nettings and sprayed walls on Anopheles gambiae s.s.

Ivermectin (IVM) has been proposed as a new tool for malaria control as it is toxic on vectors feeding on treated humans or cattle. Nevertheless, IVM may have a direct mosquitocidal effect when applied on bed nets or sprayed walls. The potential for IVM application as a new insecticide for long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) was tested in this proof-of-concept study in a laboratory and semi-field environment. Laboratory-reared, insecticide-susceptible Kisumu Anopheles gambiae were exposed to IVM on impregnated netting materials and sprayed plastered- and mud walls using cone bioassays. The results showed a direct mosquitocidal effect of IVM on this mosquito strain as all mosquitoes died by 24 h after exposure to IVM. The effect was slower on the IVM-sprayed walls compared to the treated nettings. Further work to evaluate possibility of IVM as a new insecticide formulation in LLINs and IRS will be required.

Household-level effects of seasonal malaria chemoprevention in the Gambia.

BACKGROUND: In 2022 the WHO recommended the discretionary expansion of the eligible age range for seasonal malaria chemoprevention (SMC) to children older than 4 years. Older children are at lower risk of clinical disease and severe malaria so there has been uncertainty about the cost-benefit for national control programmes. However, emerging evidence from laboratory studies suggests protecting school-age children reduces the infectious reservoir for malaria and may significantly impact on transmission. This study aimed to assess whether these effects were detectable in the context of a routinely delivered SMC programme. METHODS: In 2021 the Gambia extended the maximum eligible age for SMC from 4 to 9 years. We conducted a prospective population cohort study over the 2021 malaria transmission season covering 2210 inhabitants of 10 communities in the Upper River Region, and used a household-level mixed modelling approach to quantify impacts of SMC on malaria transmission. RESULTS: We demonstrate that the hazard of clinical malaria in older participants aged 10+ years ineligible for SMC decreases by 20% for each additional SMC round per child 0-9 years in the same household. Older inhabitants also benefit from reduced risk of asymptomatic infections in high SMC coverage households. Spatial autoregression tests show impacts are highly localised, with no detectable spillover from nearby households. CONCLUSIONS: Evidence for the transmission-reducing effects of extended-age SMC from routine programmes implemented at scale has been previously limited. Here we demonstrate benefits to the entire household, indicating such programmes may be more cost-effective than previously estimated.

Seasonal malaria chemoprevention (SMC) is the provision of monthly, preventative, anti-malaria medication to young children at times when they are most at risk of severe disease. Recently the World Health Organisation recommended expanding SMC to children older than 4 years. Older children with malaria typically remain symptomless so the advantages were unclear. However, laboratory evidence suggests this group continues to transmit malaria to others. We conducted a population study in 2021 in 10 communities in the Gambia where SMC was extended to all children up to 9 years of age for the first time. We found household members aged over 9 years were less likely to get clinical disease when most young children in the same household did receive SMC. This suggests an added protection of SMC for those who do not receive it, potentially increasing cost-effectiveness.

Genomic surveillance of Anopheles mosquitoes on the Bijagós Archipelago using custom targeted amplicon sequencing identifies mutations associated with insecticide resistance.

BACKGROUND: Insecticide resistance is reducing the efficacy of vector control interventions, consequently threatening efforts to control vector-borne diseases, including malaria. Investigating the prevalence of molecular markers of resistance is a useful tool for monitoring the spread of insecticide resistance in disease vectors. The Bijagós Archipelago (Bijagós) in Guinea-Bissau is a region of stable malaria transmission where insecticide-treated nets are the mainstay for malaria control. However, the prevalence of molecular markers of insecticide resistance in malaria vectors is not well understood. METHODS: A total of 214 Anopheles mosquitoes were analysed from 13 islands across the Bijagós. These mosquitoes were collected using CDC light traps in November 2019, during the peak malaria transmission season. High-throughput multiplex amplicon sequencing was used to investigate the prevalence of 17 different molecular markers associated with insecticide resistance in four genes: vgsc, rdl, ace1 and gste2. RESULTS: Of the 17 screened mutations, four were identified in mosquitoes from the Bijagós: vgsc L995F (12.2%), N1570Y (6.2%) and A1746S (0.7%) and rdl A269G (1.1%). This study is the first to report the L995F knock-down resistance (kdr)-west allele in Anopheles melas on the Archipelago. An additional eight non-synonymous single-nucleotide polymorphisms were identified across the four genes which have not been described previously. The prevalences of the vgsc L995F and N1570Y mutations were higher on Bubaque Island than on the other islands in this study; Bubaque is the most populous island in the archipelago, with the greatest population mobility and connection to continental Guinea-Bissau. CONCLUSIONS: This study provides the first surveillance data for genetic markers present in malaria vectors from islands across the Bijagós Archipelago. Overall prevalence of insecticide resistance mutations was found to be low. However, the identification of the vgsc L995F and N1570Y mutations associated with pyrethroid resistance warrants further monitoring. This is particularly important as the mainstay of malaria control on the islands is the use of pyrethroid insecticide-treated nets.

Validation of a method for the dry preservation and rehydration of Anopheles gambiae sensu lato for parity analysis to assess the impact of vector control measures in the field.

BACKGROUND: As the control of malaria remains heavily dependent on vector management interventions, it is important to understand the impact of these on mosquito populations. Age-grading is a valuable tool for this; however, logistical challenges in remote, resource-poor areas make current methodologies difficult to incorporate into clinical trials and routine surveillance. Our aim was to validate a methodology that could be easily implemented in such settings. Using dried mosquito specimens instead of freshly killed ones, we validated the commonly used ovarian tracheation technique for assessing population age structure. METHODS: Laboratory-reared Anopheles coluzzii mosquitoes with known parity status were dry preserved in silica gel for up to 12 weeks and rehydrated prior to parity assessment. The results were compared to parity results for freshly killed mosquitoes from the same colony. Preserved, field-caught Anopheles gambiae sensu lato (s.l.) from Guinea-Bissau were assessed by three different assessors blinded to each other's scores. An overall index of agreement was calculated using inter-rater reliability of all assessor pairings. The impact of preservation time was investigated using a one-way ANOVA to look for differences in assessor agreement over three time periods. RESULTS: The parity status was correctly identified for 90% of dry preserved and rehydrated insectary-reared An. coluzzii and for 98% of freshly killed insectary-reared An. coluzzii. The inter-rater reliability was highest (0.94) for freshly killed An. coluzzii. The results for all time points showed excellent strength of agreement between assessors. For field-caught An. gambiae s.l., the overall index of agreement between all three assessors was 0.86 (95% confidence interval 0.78-0.93), indicating almost perfect agreement. There was no significant difference between assessor agreement between time frames. CONCLUSIONS: Dry preserving and rehydrating Anopheles mosquitoes provides an alternative to using freshly killed mosquitoes to assess the efficacy of a control intervention in remote settings where it is logistically difficult to dissect fresh specimens. This method also provides the flexibility required for parity assessment to be done on larger scales over bigger areas.

Protocol for a cluster randomised placebo-controlled trial of adjunctive ivermectin mass drug administration for malaria control on the Bijagós Archipelago of Guinea-Bissau: the MATAMAL trial.

INTRODUCTION: As malaria declines, innovative tools are required to further reduce transmission and achieve elimination. Mass drug administration (MDA) of artemisinin-based combination therapy (ACT) is capable of reducing malaria transmission where coverage of control interventions is already high, though the impact is short-lived. Combining ACT with ivermectin, an oral endectocide shown to reduce vector survival, may increase its impact, while also treating ivermectin-sensitive co-endemic diseases and minimising the potential impact of ACT resistance in this context. METHODS AND ANALYSIS: MATAMAL is a cluster-randomised placebo-controlled trial. The trial is being conducted in 24 clusters on the Bijagós Archipelago, Guinea-Bissau, where the peak prevalence of Plasmodium falciparum (Pf) parasitaemia is approximately 15%. Clusters have been randomly allocated to receive MDA with dihydroartemisinin-piperaquine and either ivermectin or placebo. The primary objective is to determine whether the addition of ivermectin MDA is more effective than dihydroartemisinin-piperaquine MDA alone in reducing the prevalence of P. falciparum parasitaemia, measured during peak transmission season after 2 years of seasonal MDA. Secondary objectives include assessing prevalence after 1 year of MDA; malaria incidence monitored through active and passive surveillance; age-adjusted prevalence of serological markers indicating exposure to P. falciparum and anopheline mosquitoes; vector parous rates, species composition, population density and sporozoite rates; prevalence of vector pyrethroid resistance; prevalence of artemisinin resistance in P. falciparum using genomic markers; ivermectin's impact on co-endemic diseases; coverage estimates; and the safety of combined MDA. ETHICS AND DISSEMINATION: The trial has been approved by the London School of Hygiene and Tropical Medicine's Ethics Committee (UK) (19156) and the Comite Nacional de Eticas de Saude (Guinea-Bissau) (084/CNES/INASA/2020). Results will be disseminated in peer-reviewed publications and in discussion with the Bissau-Guinean Ministry of Public Health and participating communities. TRIAL REGISTRATION NUMBER: NCT04844905.

Distribution of Anopheles gambiae thioester-containing protein 1 alleles along malaria transmission gradients in The Gambia.

BACKGROUND: Thioester-containing protein 1 (TEP1) is a highly polymorphic gene playing an important role in mosquito immunity to parasite development and associated with Anopheles gambiae vectorial competence. Allelic variations in TEP1 could render mosquito either susceptible or resistant to parasite infection. Despite reports of TEP1 genetic variations in An. gambiae, the correlation between TEP1 allelic variants and transmission patterns in malaria endemic settings remains unclear. METHODS: TEP1 allelic variants were characterized by PCR from archived genomic DNA of > 1000 An. gambiae mosquitoes collected at 3 time points between 2009 and 2019 from eastern Gambia, where malaria transmission remains moderately high, and western regions with low transmission. RESULTS: Eight common TEP1 allelic variants were identified at varying frequencies in An. gambiae from both transmission settings. These comprised the wild type TEP1, homozygous susceptible genotype, TEP1s; homozygous resistance genotypes: TEP1rA and TEP1rB, and the heterozygous resistance genotypes: TEP1srA, TEP1srB, TEP1rArB and TEP1srArB. There was no significant disproportionate distribution of the TEP1 alleles by transmission setting and the temporal distribution of alleles was also consistent across the transmission settings. TEP1s was the most common in all vector species in both settings (allele frequencies: East = 21.4-68.4%. West = 23.5-67.2%). In Anopheles arabiensis, the frequency of wild type TEP1 and susceptible TEP1s was significantly higher in low transmission setting than in high transmission setting (TEP1: Z = - 4.831, P < 0.0001; TEP1s: Z = - 2.073, P = 0.038). CONCLUSIONS: The distribution of TEP1 allele variants does not distinctly correlate with malaria endemicity pattern in The Gambia. Further studies are needed to understand the link between genetic variations in vector population and transmission pattern in the study settings. Future studies on the implication for targeting TEP1 gene for vector control strategy such as gene drive systems in this settings is also recommended.

Entomological impact of mass administration of ivermectin and dihydroartemisinin-piperaquine in The Gambia: a cluster-randomized controlled trial.

BACKGROUND: Vector control interventions in sub-Saharan Africa rely on insecticide-treated nets and indoor residual spraying. Insecticide resistance, poor coverage of interventions, poor quality nets and changes in vector behavior threaten the effectiveness of these interventions and, consequently, alternative tools are needed. Mosquitoes die after feeding on humans or animals treated with ivermectin (IVM). Mass drug administration (MDA) with IVM could reduce vector survival and decrease malaria transmission. The entomological impact of MDA of combined IVM and dihydroartemisinin-piperaquine was assessed in a community-based, cluster-randomized trial. METHODS: A cluster-randomized trial was implemented in 2018 and 2019 in 32 villages in the Upper River Region, The Gambia. The with the inhabitants of 16 intervention villages eligible to receive three monthly rounds of MDA at the beginning of the malaria transmission season. Entomological surveillance with light traps and human landing catches (HLC) was carried out during a 7- to 14-day period after each round of MDA, and then monthly until the end of the year. The mosquitocidal effect of IVM was determined by direct membrane feeding assays. RESULTS: Of the 15,017 mosquitoes collected during the study period, 99.65% (n = 14,965) were Anopheles gambiae sensu lato (An. gambiae s.l.), comprising Anopheles arabiensis (56.2%), Anopheles coluzzii (24.5%), Anopheles gambiae sensu stricto (An. gembiae s.s.; 16.0%) and Anopheles funestus sensu lato (An. funestus s.l.; 0.35%). No effect of the intervention on vector parity was observed. Vector density determined on light trap collections was significantly lower in the intervention villages in 2019 (adjusted incidence rate ratio: 0.39; 95% confidence interval [CI]: 0.20, 0.74; P = 0.005) but not in 2018. However, vector density determined in HLC collections was similar in both the intervention and control villages. The entomological inoculation rate was significantly lower in the intervention villages than in the control villages (odds ratio: 0.36, 95% CI: 0.19, 0.70; P = 0·003). Mosquito mortality was significantly higher when blood fed on IVM-treated individuals up to 21 days post-treatment, particularly in adults and individuals with a higher body mass index. CONCLUSION: Mass drug administration with IVM decreased vector density and the entomological inoculation rate while the effect on vector parity was less clear. Survival of mosquitoes fed on blood collected from IVM-treated individuals was significantly lower than that in mosquitoes which fed on controls. The influence of host characteristics on mosquito survivorship indicated that dose optimization could improve IVM efficacy. Future detailed entomological evaluation trials in which IVM is administered as stand-alone intervention may elucidate the contribution of this drug to the observed reduction in transmission.

A report on preparation, expansion and future outlook of COVID-19 testing in Gambia.

Background: The outbreak of COVID-19 disease and rapid spread of the virus outside China led to its declaration as a Public Health Emergency of International Concern (PHEIC) in January 2020. Key elements of the early intervention strategy focused on laboratory diagnosis and screening at points of entry and imposition of restrictions in crossborder activities. Objective: We report the role the Medical Research Council Unit, The Gambia (MRCG) played in the early implementation of molecular testing for COVID-19 in The Gambia as part of the national outbreak response. Methods: Laboratory staff members, with experience in molecular biology assays, were identified and trained on COVID-19 testing at the Africa CDC training workshop in Dakar, Senegal. Thereafter risks assessments, drafting of standard operating procedures (SOPs) and inhouse training enabled commencement of testing using commercial RTPCR kits. Subsequently, testing was expanded to the National Public Health Laboratroy and also implemented across field sites for rapid response across the country. Results: Capacity for COVID-19 testing at MRCG was developed and can process aproximately 350 tests per day, which can be further scaled up as the demand for testing increases. Conclusion: The long presence of the Unit in The Gambia and strong collaborative relationship with the National Health Ministry, allowed for a synergistc approach in mounting an effective response that contributed in delaying the establishment of community transmission in the country.

Efficacy, Safety and Tolerability of Pyronaridine-artesunate in Asymptomatic Malaria-infected Individuals: a Randomized Controlled Trial.

BACKGROUND: Pyronaridine-artesunate (PA) is a registered artemisinin-based combination therapy, potentially useful for mass drug administration campaigns. However, further data are needed to evaluate its efficacy, safety and tolerability as full or incomplete treatment in asymptomatic Plasmodium falciparum-infected individuals. METHODS: This phase II, multi-center, open label, randomized clinical trial was conducted in The Gambia and Zambia. Participants with microscopically confirmed asymptomatic P. falciparum infection were randomly assigned (1:1:1) to receive a 3-day, 2-day, or 1-day treatment regimen of PA (180:60 mg), dosed according to bodyweight. The primary efficacy outcome was polymerase chain reaction (PCR)-adjusted adequate parasitological response (APR) at day 28 in the per-protocol population. RESULTS: A total of 303 participants were randomized. Day 28 PCR-adjusted APR was 100% for both the 3-day (98/98) and 2-day regimens (96/96), and 96.8% (89/94) for the 1-day regimen. Efficacy was maintained at 100% until day 63 for the 3-day and 2-day regimens but declined to 94.4% (84/89) with the 1-day regimen. Adverse event frequency was similar between the 3-day (51.5% [52/101]), 2-day (52.5% [52/99]), and 1-day (54.4% [56/103]) regimens; the majority of adverse events were of grade 1 or 2 severity (85% [136/160]). Asymptomatic, transient increases (>3 times the upper limit of normal) in alanine aminotransferase/aspartate aminotransferase were observed for 6/301 (2.0%) participants. CONCLUSIONS: PA had high efficacy and good tolerability in asymptomatic P. falciparum-infected individuals, with similar efficacy for the full 3-day and incomplete 2-day regimens. Although good adherence to the 3-day regimen should be encouraged, these results support the further investigation of PA for mass drug administration campaigns. CLINICAL TRIALS REGISTRATION: NCT03814616.

Mass drug administration of ivermectin and dihydroartemisinin-piperaquine against malaria in settings with high coverage of standard control interventions: a cluster-randomised controlled trial in The Gambia.

BACKGROUND: Although the malaria burden has substantially decreased in sub-Saharan Africa, progress has stalled. We assessed whether mass administration of ivermectin (a mosquitocidal drug) and dihydroartemisinin-piperaquine (an antimalarial treatment) reduces malaria in The Gambia, an area with high coverage of standard control interventions. METHODS: This open-label, cluster-randomised controlled trial was done in the Upper River region of eastern Gambia. Villages with a baseline Plasmodium falciparum prevalence of 7-46% (all ages) and separated from each other by at least 3 km to reduce vector spillover were selected. Inclusion criteria were age and anthropometry (for ivermectin, weight of ≥15 kg; for dihydroartemisinin-piperaquine, participants older than 6 months); willingness to comply with trial procedures; and written informed consent. Villages were randomised (1:1) to either the intervention (ivermectin [orally at 300-400 µg/kg per day for 3 consecutive days] and dihydroartemisinin-piperaquine [orally depending on bodyweight] plus standard control interventions) or the control group (standard control interventions) using computer-based randomisation. Laboratory staff were masked to the origin of samples. In the intervention group, three rounds of mass drug administration once per month with ivermectin and dihydroartemisinin-piperaquine were given during two malaria transmission seasons from Aug 27 to Oct 31, 2018, and from July 15 to Sept 30, 2019. Primary outcomes were malaria prevalence by qPCR at the end of the second intervention year in November 2019, and Anopheles gambiae (s l) parous rate, analysed in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, NCT03576313. FINDINGS: Between Nov 20 and Dec 7, 2017, 47 villages were screened for eligibility in the study. 15 were excluded because the baseline malaria prevalence was less than 7% (figure 1). 32 villages were enrolled and randomised to either the intervention or control group (n=16 in each group). The study population was 10 638, of which 4939 (46%) participants were in intervention villages. Coverage for dihydroartemisinin-piperaquine was between 49·0% and 58·4% in 2018, and between 76·1% and 86·0% in 2019; for ivermectin, coverage was between 46·9% and 52·2% in 2018, and between 71·7% and 82·9% in 2019. In November 2019, malaria prevalence was 12·8% (324 of 2529) in the control group and 5·1% (140 of 2722) in the intervention group (odds ratio [OR] 0·30, 95% CI 0·16-0·59; p<0·001). A gambiae (s l) parous rate was 83·1% (552 of 664) in the control group and 81·7% (441 of 540) in the intervention group (0·90, 0·66-1·25; p=0·537). In 2019, adverse events were recorded in 386 (9·7%) of 3991 participants in round one, 201 (5·4%) of 3750 in round two, and 168 (4·5%) of 3752 in round three. None of the 11 serious adverse events were related to the intervention. INTERPRETATION: The intervention was safe and well tolerated. In an area with high coverage of standard control interventions, mass drug administration of ivermectin and dihydroartemisinin-piperaquine significantly reduced malaria prevalence; however, no effect of ivermectin on vector parous rate was observed. FUNDING: Joint Global Health Trials Scheme. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Evolution of the Pyrethroids Target-Site Resistance Mechanisms in Senegal: Early Stage of the Vgsc-1014F and Vgsc-1014S Allelic Frequencies Shift.

The evolution and spread of insecticide resistance mechanisms amongst malaria vectors across the sub-Saharan Africa threaten the effectiveness and sustainability of current insecticide-based vector control interventions. However, a successful insecticide resistance management plan relies strongly on evidence of historical and contemporary mechanisms circulating. This study aims to retrospectively determine the evolution and spread of pyrethroid resistance mechanisms among natural Anopheles gambiae s.l. populations in Senegal. Samples were randomly drawn from an existing mosquito sample, collected in 2013, 2017, and 2018 from 10 sentinel sites monitored by the Senegalese National Malaria Control Programme (NMCP). Molecular species of An. gambiae s.l. and the resistance mutations at the Voltage-gated Sodium Channel 1014 (Vgsc-1014) locus were characterised using PCR-based assays. The genetic diversity of the Vgsc gene was further analyzed by sequencing. The overall species composition revealed the predominance of Anopheles arabiensis (73.08%) followed by An. gambiae s.s. (14.48%), Anopheles coluzzii (10.94%) and Anopheles gambiae-coluzii hybrids (1.48%). Both Vgsc-1014F and Vgsc-1014S mutations were found in all studied populations with a spatial variation of allele frequencies from 3% to 90%; and 7% to 41%, respectively. The two mutations have been detected since 2013 across all the selected health districts, with Vgsc-L1014S frequency increasing over the years while Vgsc-1014F decreasing. At species level, the Vgsc-1014F and Vgsc-1014S alleles were more frequent amongst An. gambiae s.s. (70%) and An. arabiensis (20%). The Vgsc gene was found to be highly diversified with eight different haplotypes shared between Vgsc-1014F and Vgsc-1014S. The observed co-occurrence of Vgsc-1014F and Vgsc-1014S mutations suggest that pyrethroid resistance is becoming a widespread phenomenon amongst malaria vector populations, and the NMCP needs to address this issue to sustain the gain made in controlling malaria.

Intense and Mild First Epidemic Wave of Coronavirus Disease, The Gambia.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is evolving differently in Africa than in other regions. Africa has lower SARS-CoV-2 transmission rates and milder clinical manifestations. Detailed SARS-CoV-2 epidemiologic data are needed in Africa. We used publicly available data to calculate SARS-CoV-2 infections per 1,000 persons in The Gambia. We evaluated transmission rates among 1,366 employees of the Medical Research Council Unit The Gambia (MRCG), where systematic surveillance of symptomatic cases and contact tracing were implemented. By September 30, 2020, The Gambia had identified 3,579 SARS-CoV-2 cases, including 115 deaths; 67% of cases were identified in August. Among infections, MRCG staff accounted for 191 cases; all were asymptomatic or mild. The cumulative incidence rate among nonclinical MRCG staff was 124 infections/1,000 persons, which is >80-fold higher than estimates of diagnosed cases among the population. Systematic surveillance and seroepidemiologic surveys are needed to clarify the extent of SARS-CoV-2 transmission in Africa.

Influence of insecticide resistance on the biting and resting preferences of malaria vectors in the Gambia.

BACKGROUND: The scale-up of indoor residual spraying and long-lasting insecticidal nets, together with other interventions have considerably reduced the malaria burden in The Gambia. This study examined the biting and resting preferences of the local insecticide-resistant vector populations few years following scale-up of anti-vector interventions. METHOD: Indoor and outdoor-resting Anopheles gambiae mosquitoes were collected between July and October 2019 from ten villages in five regions in The Gambia using pyrethrum spray collection (indoor) and prokopack aspirator from pit traps (outdoor). Polymerase chain reaction assays were performed to identify molecular species, insecticide resistance mutations, Plasmodium infection rate and host blood meal. RESULTS: A total of 844 mosquitoes were collected both indoors (421, 49.9%) and outdoors (423, 50.1%). Four main vector species were identified, including An. arabiensis (indoor: 15%, outdoor: 26%); An. coluzzii (indoor: 19%, outdoor: 6%), An. gambiae s.s. (indoor: 11%, outdoor: 16%), An. melas (indoor: 2%, outdoor: 0.1%) and hybrids of An. coluzzii-An. gambiae s.s (indoors: 3%, outdoors: 2%). A significant preference for outdoor resting was observed in An. arabiensis (Pearson X2 = 22.7, df = 4, P<0.001) and for indoor resting in An. coluzzii (Pearson X2 = 55.0, df = 4, P<0.001). Prevalence of the voltage-gated sodium channel (Vgsc)-1014S was significantly higher in the indoor-resting (allele freq. = 0.96, 95%CI: 0.78-1, P = 0.03) than outdoor-resting (allele freq. = 0.82, 95%CI: 0.76-0.87) An. arabiensis population. For An. coluzzii, the prevalence of most mutation markers was higher in the outdoor (allele freq. = 0.92, 95%CI: 0.81-0.98) than indoor-resting (allele freq. = 0.78, 95%CI: 0.56-0.86) mosquitoes. However, in An. gambiae s.s., the prevalence of Vgsc-1014F, Vgsc-1575Y and GSTe2-114T was high (allele freq. = 0.96-1), but did not vary by resting location. The overall sporozoite positivity rate was 1.3% (95% CI: 0.5-2%) in mosquito populations. Indoor-resting An. coluzzii had mainly fed on human blood while indoor-resting An. arabiensis fed on animal blood. CONCLUSION: In this study, high levels of resistance mutations were observed that could be influencing the mosquito populations to rest indoors or outdoors. The prevalent animal-biting behaviour demonstrated in the mosquito populations suggest that larval source management could be an intervention to complement vector control in this setting.

Gametocyte carriage after seasonal malaria chemoprevention in Plasmodium falciparum infected asymptomatic children.

BACKGROUND: Treatment of clinical Plasmodium falciparum malaria with sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) is associated with increased post-treatment gametocyte carriage. The effect of seasonal malaria chemoprevention (SMC) with SP and AQ on gametocyte carriage was assessed in asymptomatic P. falciparum infected children. METHODS: The study was carried out in eastern Gambia. Asymptomatic P. falciparum malaria infected children aged 24-59 months old who were eligible to receive SMC (SMC group) and children 5-8 years that were not eligible to receive SMC (comparison group) were recruited. Gametocytaemia was determined by molecular methods before and after SMC administration. Gametocyte carriage between the groups was compared using the chi-squared test and within-person using conditional logistic regression. RESULTS: During the 2017 and 2018 malaria transmission seasons, 65 and 75 children were recruited in the SMC and comparison groups, respectively. Before SMC administration, gametocyte prevalence was 10.7% (7/65) in the SMC group and 13.3% (10/75) in the comparison group (p = 0.64). At day 13 (IQR 12, 13) after SMC administration, this was 9.4% (5/53) in children who received at least the first dose of SMC treatment and 12.7% (9/71) for those in the comparison group (p = 0.57). Similarly, there was no difference in prevalence of gametocytes between children that adhered to all 3-day doses of SMC treatment 15.6% (5/32) and those in the comparison group (p = 0.68). In the SMC group, within-group gametocyte carriage was similar before and after SMC administration in children that received at least the first dose of SMC treatment (OR 0.6, 95% CI 0.14-2.51; p = 0.48) and in those that adhered to all 3-day doses of SMC treatment (OR 1.0, 95% CI 0.20-4.95; p = 1.0). CONCLUSION: In this study with relative low gametocyte prevalence prior to SMC treatment, no evidence was observed that SMC treatment increased gametocyte carriage in asymptomatic P. falciparum malaria infected children.

Differential contribution of Anopheles coustani and Anopheles arabiensis to the transmission of Plasmodium falciparum and Plasmodium vivax in two neighbouring villages of Madagascar.

BACKGROUND: Malaria is still a heavy public health concern in Madagascar. Few studies combining parasitology and entomology have been conducted despite the need for accurate information to design effective vector control measures. In a Malagasy region of moderate to intense transmission of both Plasmodium falciparum and P. vivax, parasitology and entomology have been combined to survey malaria transmission in two nearby villages. METHODS: Community-based surveys were conducted in the villages of Ambohitromby and Miarinarivo at three time points (T1, T2 and T3) during a single malaria transmission season. Human malaria prevalence was determined by rapid diagnostic tests (RDTs), microscopy and real-time PCR. Mosquitoes were collected by human landing catches and pyrethrum spray catches and the presence of Plasmodium sporozoites was assessed by TaqMan assay. RESULTS: Malaria prevalence was not significantly different between villages, with an average of 8.0% by RDT, 4.8% by microscopy and 11.9% by PCR. This was mainly due to P. falciparum and to a lesser extent to P. vivax. However, there was a significantly higher prevalence rate as determined by PCR at T2 ([Formula: see text] = 7.46, P = 0.025). Likewise, mosquitoes were significantly more abundant at T2 ([Formula: see text] = 64.8, P < 0.001), especially in Ambohitromby. At T1 and T3 mosquito abundance was higher in Miarinarivo than in Ambohitromby ([Formula: see text] = 14.92, P < 0.001). Of 1550 Anopheles mosquitoes tested, 28 (1.8%) were found carrying Plasmodium sporozoites. The entomological inoculation rate revealed that Anopheles coustani played a major contribution in malaria transmission in Miarinarivo, being responsible of 61.2 infective bites per human (ib/h) during the whole six months of the survey, whereas, it was An. arabiensis, with 36 ib/h, that played that role in Ambohitromby. CONCLUSIONS: Despite a similar malaria prevalence in two nearby villages, the entomological survey showed a different contribution of An. coustani and An. arabiensis to malaria transmission in each village. Importantly, the suspected secondary malaria vector An. coustani, was found playing the major role in malaria transmission in one village. This highlights the importance of combining parasitology and entomology surveys for better targeting local malaria vectors. Such study should contribute to the malaria pre-elimination goal established under the 2018-2022 National Malaria Strategic Plan.

A survey of Anopheles species composition and insecticide resistance on the island of Bubaque, Bijagos Archipelago, Guinea-Bissau.

BACKGROUND: Bubaque is the most populous island of the Bijagos archipelago, a group of malaria-endemic islands situated off the coast of Guinea-Bissau, West Africa. Malaria vector control on Bubaque relies almost exclusively on the use of long-lasting insecticidal nets (LLINs). However, there is little information on local vector bionomics and insecticide resistance. METHODS: A survey of mosquito species composition was performed at the onset of the wet season (June/July) and the beginning of the dry season (November/December). Sampling was performed using indoor adult light-traps and larval dipping. Anopheles mosquitoes were identified to species level and assessed for kdr allele frequency by TaqMan PCR. Females were analysed for sporozoite positivity by CSP-ELISA. Resistance to permethrin and α-cypermethrin was measured using the CDC-bottle bioassay incorporating the synergist piperonyl-butoxide. RESULTS: Several Anopheles species were found on the island, all belonging to the Anopheles gambiae sensu lato (s.l.) complex, including An. gambiae sensu stricto, Anopheles coluzzii, Anopheles melas, and An. gambiae/An. coluzzii hybrids. Endophagic Anopheles species composition and abundance showed strong seasonal variation, with a majority of An. gambiae (50% of adults collected) caught in June/July, while An. melas was dominant in November/December (83.9% of adults collected). Anopheles gambiae had the highest sporozoite rate in both seasons, with infection rates of 13.9% and 20% in June/July and November/December, respectively. Moderate frequencies of the West African kdr allele were found in An. gambiae (36%), An. coluzzii (35%), An. gambiae/An. coluzzii hybrids (42%). Bioassays suggest moderate resistance to α-cypermethrin, but full susceptibility to permethrin. CONCLUSIONS: The island of Bubaque maintained an An. gambiae s.l. population in both June/July and November/December. Anopheles gambiae was the primary vector at the onset of the wet season, while An. melas is likely to be responsible for most dry season transmission. There was moderate kdr allele frequency and synergist assays suggest likely metabolic resistance, which could reduce the efficacy of LLINs. Future control of malaria on the islands should consider the seasonal shift in mosquito species, and should employ continuous monitoring for insecticide resistance.

Insecticides and Insecticide Resistance.

Vector control has significantly reduced malaria morbidity in many regions of the world where the disease was endemic and is now moving toward malaria elimination. Among the tools available for vector control, the use of long-lasting insecticidal bed nets (LLINs) and indoor residual spraying (IRS) has proved most effective. However, Anopheles mosquitoes are becoming increasingly resistant to insecticides. In this chapter, we describe the main aspects of vector control-with a particular focus on insecticidal products commonly used in vector control as well as on mechanisms of insecticide resistance. We also discuss the impact of insecticide resistance on malaria transmission.

Evidence of Insecticide Resistance to Pyrethroids and Bendiocarb in Anopheles funestus from Tsararano, Marovoay District, Madagascar.

INTRODUCTION: In Madagascar, malaria control relies on the countrywide use of long lasting insecticide treated bed nets (LLINs) and on indoor residual spraying (IRS) in the central highland area as well as a small area on the eastern coast. We tested insecticide resistance mechanisms of Anopheles funestus from Tsararano, a malaria endemic village in the coastal health district of Marovoay. METHODS: Insecticide susceptibility bioassays were done in July 2017 on first-generation Anopheles funestus (F1) to assess (i) the susceptibility to permethrin (0.05%), deltamethrin (0.05%), DDT (4%), malathion (5%), fenitrothion (1%), and bendiocarb (0.1%); (ii) the effect of preexposure to the piperonyl butoxide (PBO) synergist; and (iii) the enzymatic activities of cytochrome P450, esterases, and glutathione S-transferases (GST). RESULTS: Our results demonstrated that An. funestus was phenotypically resistant to pyrethroids and bendiocarb, with a mortality rate (MR) of 33.6% (95%CI: 24.5-43.7%) and 86% (95%CI: 77.6-92.1%), respectively. In contrast, An. funestus were 100% susceptible to DDT and organophosphates (malathion and fenitrothion). Preexposure of An. funestus to PBO synergist significantly restored the susceptibility to bendiocarb (MR=100%) and increased the MR in the pyrethroid group, from 96% (95%CI: 90.0-98.9%) to 100% for deltamethrin and permethrin, respectively (χ 2 = 43, df = 3, P< 0.0001). Enzymatic activities of cytochrome P450 and α-esterases were significantly elevated among An. funestus compared with the IPM reference strain (Mann-Whitney U= 30, P<0.0001; U = 145.5, P <0.0001, respectively). No significant differences of ß-esterases activities compared to the IPM reference strain were observed (Mann-Whitney U = 392.5, P = 0.08). CONCLUSION: In Tsararano, despite the absence of an IRS programme, there is evidence of high levels of insecticide resistance to pyrethroids and bendiocarb in An.

Evidence of multiple insecticide resistance mechanisms in Anopheles gambiae populations in Bangui, Central African Republic.

BACKGROUND: Knowledge of insecticide resistance status in the main malaria vectors is an essential component of effective malaria vector control. This study presents the first evaluation of the status of insecticide resistance in Anopheles gambiae populations from Bangui, the Central African Republic. METHODS: Anopheles mosquitoes were reared from larvae collected in seven districts of Bangui between September to November 2014. The World Health Organisation's bioassay susceptibility tests to lambda-cyhalothrin (0.05%), deltamethrin (0.05%), DDT (4%), malathion (5%), fenitrothion (1%) and bendiocarb (0.1%) were performed on adult females. Species and molecular forms as well as the presence of L1014F kdr and Ace-1 R mutations were assessed by PCR. Additional tests were conducted to assess metabolic resistance status. RESULTS: After 1 h exposure, a significant difference of knockdown effect was observed between districts in all insecticides tested except deltamethrin and malathion. The mortality rate (MR) of pyrethroids group ranging from 27% (CI: 19-37.5) in Petevo to 86% (CI: 77.6-92.1) in Gbanikola; while for DDT, MR ranged from 5% (CI: 1.6-11.3) in Centre-ville to 39% (CI: 29.4-49.3) in Ouango. For the organophosphate group a MR of 100% was observed in all districts except Gbanikola where a MR of 96% (CI: 90-98.9) was recorded. The mortality induced by bendiocarb was very heterogeneous, ranging from 75% (CI: 62.8-82.8) in Yapele to 99% (CI: 84.5-100) in Centre-ville. A high level of kdr-w (L1014F) frequency was observed in all districts ranging from 93 to 100%; however, no kdr-e (L1014S) and Ace-1 R mutation were found in all tested mosquitoes. Data of biochemical analysis showed significant overexpression activities of cytochrome P450, GST and esterases in Gbanikola and Yapele (χ 2 = 31.85, df = 2, P < 0.001). By contrast, esterases activities using α and ß-naphthyl acetate were significantly low in mosquitoes from PK10 and Ouango in comparison to Kisumu strain (χ 2 = 17.34, df = 2, P < 0.005). CONCLUSIONS: Evidence of resistance to DDT and pyrethroids as well as precocious emergence of resistance to carbamates were detected among A. gambiae mosquitoes from Bangui, including target-site mutations and metabolic mechanisms. The co-existence of these resistance mechanisms in A. gambiae may be a serious obstacle for the future success of malaria control programmes in this region.