The sustainability of malaria control in Africa is threatened by the rise of insecticide resistance in Anopheles mosquitoes that transmit the disease1. To gain a deeper understanding of how mosquito populations are evolving, we sequenced the genomes of 765 specimens of Anopheles gambiae and Anopheles coluzzii sampled from 15 locations across Africa, identifying over 50 million single nucleotide polymorphisms within the accessible genome. These data revealed complex population structure and patterns of gene flow, with evidence of ancient expansions, recent bottlenecks, and local variation in effective population size. Strong signals of recent selection were observed in insecticide resistance genes, with multiple sweeps spreading over large geographical distances and between species. The design of novel tools for mosquito control using gene drive will need to take account of high levels of genetic diversity in natural mosquito populations.

BackgroundLong-lasting insecticidal nets (LLINs) and indoor residual spraying of insecticide (IRS) are the primary vector control interventions used to prevent malaria in Africa. Although both interventions are effective in some settings, high-quality evidence is rarely available to evaluate their effectiveness following deployment by a national malaria control program. In Uganda, we measured changes in key malaria indicators following universal LLIN distribution in three sites, with the addition of IRS at one of these sites.Methods and FindingsComprehensive malaria surveillance was conducted from October 1, 2011, to March 31, 2016, in three sub-counties with relatively low (Walukuba), moderate (Kihihi), and high transmission (Nagongera). Between 2013 and 2014, universal LLIN distribution campaigns were conducted in all sites, and in December 2014, IRS with the carbamate bendiocarb was initiated in Nagongera. High-quality surveillance evaluated malaria metrics and mosquito exposure before and after interventions through (a) enhanced health-facility-based surveillance to estimate malaria test positivity rate (TPR), expressed as the number testing positive for malaria/number tested for malaria (number of children tested for malaria: Walukuba = 42,833, Kihihi = 28,790, and Nagongera = 38,690); (b) cohort studies to estimate the incidence of malaria, expressed as the number of episodes per person-year [PPY] at risk (number of children observed: Walukuba = 340, Kihihi = 380, and Nagongera = 361); and (c) entomology surveys to estimate household-level human biting rate (HBR), expressed as the number of female Anopheles mosquitoes collected per house-night of collection (number of households observed: Walukuba = 117, Kihihi = 107, and Nagongera = 107). The LLIN distribution campaign substantially increased LLIN coverage levels at the three sites to between 65.0% and 95.5% of households with at least one LLIN. In Walukuba, over the 28-mo post-intervention period, universal LLIN distribution was associated with no change in the incidence of malaria (0.39 episodes PPY pre-intervention versus 0.20 post-intervention; adjusted rate ratio [aRR] = 1.02, 95% CI 0.36–2.91, p = 0.97) and non-significant reductions in the TPR (26.5% pre-intervention versus 26.2% post-intervention; aRR = 0.70, 95% CI 0.46–1.06, p = 0.09) and HBR (1.07 mosquitoes per house-night pre-intervention versus 0.71 post-intervention; aRR = 0.41, 95% CI 0.14–1.18, p = 0.10). In Kihihi, over the 21-mo post-intervention period, universal LLIN distribution was associated with a reduction in the incidence of malaria (1.77 pre-intervention versus 1.89 post-intervention; aRR = 0.65, 95% CI 0.43–0.98, p = 0.04) but no significant change in the TPR (49.3% pre-intervention versus 45.9% post-intervention; aRR = 0.83, 95% 0.58–1.18, p = 0.30) or HBR (4.06 pre-intervention versus 2.44 post-intervention; aRR = 0.71, 95% CI 0.30–1.64, p = 0.40). In Nagongera, over the 12-mo post-intervention period, universal LLIN distribution was associated with a reduction in the TPR (45...

Insecticide resistance in the malaria vector An. gambiae s.l. threatens insecticide-based control efforts, necessitating regular monitoring. We assessed resistance in field-collected An. gambiae s.l. from Jinja, Uganda using WHO biosassays. Only An. gambiae s.s. and An. arabiensis (≈70%) were present. Female An. gambiae exhibited extremely high pyrethroid resistance (permethrin LT50 >2h; deltamethrin LT50 >5h). Female An. arabiensis were resistant to permethrin and exhibited reduced susceptibility to deltamethrin. However, whilst An. gambiae were DDT resistant, An. arabiensis were fully susceptible. Both species were fully susceptible to bendiocarb and fenitrothion. Kdr 1014S has increased rapidly in the Jinja population of An. gambiae s.s. and now approaches fixation (≈95%), consistent with insecticide-mediated selection, but is currently at low frequency in An. arabiensis (0.07%). Kdr 1014F was also at low frequency in An. gambiae. These frequencies preclude adequately-powered tests for association with phenotypic resistance. PBO synergist bioassays resulted in near complete recovery of pyrethroid susceptibility suggesting involvement of CYP450s in resistance. A small number (0.22%) of An. gambiae s.s. x An. arabiensis hybrids were found, suggesting the possibility of introgression of resistance alleles between species. The high levels of pyrethroid resistance encountered in Jinja threaten to reduce the efficacy of vector control programmes which rely on pyrethroid impregnated bednets or indoor spraying of pyrethroids.

Metabolic resistance to pyrethroid insecticides is widespread in Anopheles mosquitoes and is a major threat to malaria control. DNA markers would aid predictive monitoring of resistance, but few mutations have been discovered outside of insecticide-targeted genes. Isofemale family pools from a wild Ugandan Anopheles gambiae population, from an area where operational pyrethroid failure is suspected, were genotyped using a candidate-gene enriched SNP array. Resistance-associated SNPs were detected in three genes from detoxification superfamilies, in addition to the insecticide target site (the Voltage Gated Sodium Channel gene, Vgsc). The putative associations were confirmed for two of the marker SNPs, in the P450 Cyp4j5 and the esterase Coeae1d by reproducible association with pyrethroid resistance in multiple field collections from Uganda and Kenya, and together with the Vgsc-1014S (kdr) mutation these SNPs explained around 20% of variation in resistance. Moreover, the >20 Mb 2La inversion also showed evidence of association with resistance as did environmental humidity. Sequencing of Cyp4j5 and Coeae1d detected no resistance-linked loss of diversity, suggesting selection from standing variation. Our study provides novel, regionally-validated DNA assays for resistance to the most important insecticide class, and establishes both 2La karyotype variation and humidity as common factors impacting the resistance phenotype.

IntroductionSevere malaria is a life-threatening medical emergency and requires prompt and effective treatment to prevent death. There is paucity of published information on current practices of severe malaria case management in sub-Saharan Africa; we evaluated the management practices for severe malaria in Ugandan health facilitiesMethods and FindingsWe did a cross sectional survey, using multi-stage sampling methods, of health facilities in 11 districts in the eastern and mid-western parts of Uganda. The study instruments were adapted from the WHO hospital care assessment tools. Between June and August 2009, 105 health facilities were surveyed and 181 health workers and 868 patients/caretakers interviewed. None of the inpatient facilities had all seven components of a basic care package for the management of severe malaria consistently available during the 3 months prior to the survey. Referral practices were appropriate for <10% (18/196) of the patients. Prompt care at any health facility was reported by 29% (247/868) of patients. Severe malaria was correctly diagnosed in 27% of patients (233).Though the quinine dose and regimen was correct in the majority (611/868, 70.4%) of patients, it was administered in the correct volumes of 5% dextrose in only 18% (147/815). Most patients (80.1%) had several doses of quinine administered in one single 500 ml bottle of 5% dextrose. Medications were purchased by 385 (44%) patients and medical supplies by 478 patients (70.6%).ConclusionsManagement of severe malaria in Ugandan health facilities was sub-optimal. These findings highlight the challenges of correctly managing severe malaria in resource limited settings. Priority areas for improvement include triage and emergency care, referral practises, quality of diagnosis and treatment, availability of medicines and supplies, training and support supervision.