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Background Methods used to sample mosquitoes are important to consider when estimating entomologic metrics. Human landing catches (HLCs) are considered the gold standard for collecting malaria vectors. However, HLCs are labour intensive, can expose collectors to transmission risk, and are difficult to implement at scale. This study compared alternative methods to HLCs for collecting Anopheles mosquitoes in eastern Uganda. Methods Between June and November 2021, mosquitoes were collected from randomly selected households in three parishes in Tororo and Busia districts. Mosquitoes were collected indoors and outdoors using HLCs in 16 households every 4 weeks. Additional collections were done indoors with prokopack aspirators, and outdoors with pit traps, in these 16 households every 2 weeks. CDC light trap collections were done indoors in 80 households every 4 weeks. Female Anopheles mosquitoes were identified morphologically and Anopheles gambiae sensu lato were speciated using PCR. Plasmodium falciparum sporozoite testing was done with ELISA. Results Overall, 4,891 female Anopheles were collected, including 3,318 indoors and 1,573 outdoors. Compared to indoor HLCs, vector density (mosquitoes per unit collection) was lower using CDC light traps (4.24 vs 2.96, density ratio [DR] 0.70, 95% CIs 0.63–0.77, p Anopheles funestus (75.0%) than indoor HLCs (25.8%), while pit traps collected a higher proportion of Anopheles arabiensis (84.3%) than outdoor HLCs (36.9%). Conclusion In this setting, the density and species of mosquitoes collected with alternative methods varied, reflecting the feeding and resting characteristics of the common vectors and the different collection approaches. These differences could impact on the accuracy of entomological indicators and estimates of malaria transmission, when using the alternative methods for sampling mosquitos, as compared to HLCs.

Durability monitoring of insecticide-treated nets (ITNs) containing a pyrethroid in combination with a second active ingredient (AI) must be adapted so that the insecticidal bioefficacy of each AI can be monitored independently. An effective way to do this is to measure rapid knock down of a pyrethroid-susceptible strain of mosquitoes to assess the bioefficacy of the pyrethroid component and to use a pyrethroid-resistant strain to measure the bioefficacy of the second ingredient. To allow robust comparison of results across tests within and between test facilities, and over time, protocols for bioefficacy testing must include either characterisation of the resistant strain, standardisation of the mosquitoes used for bioassays, or a combination of the two. Through a series of virtual meetings, key stakeholders and practitioners explored different approaches to achieving these goals. Via an iterative process we decided on the preferred approach and produced a protocol consisting of characterising mosquitoes used for bioefficacy testing before and after a round of bioassays, for example at each time point in a durability monitoring study. We present the final protocol and justify our approach to establishing a standard methodology for durability monitoring of ITNs containing pyrethroid and a second AI.

Insecticide resistance is a major impediment to the control of vectors and pests of public health importance and is a strongly selected trait capable of rapid spread, sometimes even between closely-related species. Elucidating the mechanisms generating insecticide resistance in mosquito vectors of disease, and understanding the spread of resistance within and between populations and species are vital for the development of robust resistance management strategies. Here we studied the mechanisms of resistance in two sympatric members of the Anopheles gambiae species complex – the major vector of malaria in sub-Saharan Africa – in order to understand how resistance has developed and spread in eastern Uganda, a region with some of the highest levels of malaria.\ud \ud In eastern Uganda, where the mosquitoes Anopheles arabiensis and An. gambiae can be found sympatrically, low levels of hybrids (0.4%) occur, offering a route for introgression of adaptively important variants between species. In independent microarray studies of insecticide resistance, Gste4, an insect-specific glutathione S-transferase, was among the most significantly up-regulated genes in both species. To test the hypothesis of interspecific introgression, we sequenced 2.3kbp encompassing Gste4. Whilst this detailed sequencing ruled out introgression, we detected strong positive selection acting on Gste4. However, these sequences, followed by haplotype-specific qPCR, showed that the apparent up-regulation in An. arabiensis is a result of allelic variation across the microarray probe binding sites which artefactually elevates the gene expression signal. Thus, facevalue acceptance of microarray data can be misleading and it is advisable to conduct a more detailed investigation of the causes and nature of such signal. The identification of positive selection acting on this locus led us to functionally express and characterise allelic variants of GSTE4. Although the in vitro data do not support a direct role for GSTE4 in metabolism, they do support a role for this enzyme in insecticide sequestration. Thus, the demonstration of a role for an up-regulated gene in metabolic resistance to insecticides should not be limited to simply whether it can metabolise insecticide; such a strict criterion would argue against the involvement of GSTE4 despite the weight of evidence to the contrary.

The sustainability of malaria control in Africa is threatened by rising levels of insecticide resistance, and new tools to prevent malaria transmission are urgently needed. To gain a better understanding of the mosquito populations that transmit malaria, we sequenced the genomes of 765 wild specimens ofAnopheles gambiaeandAnopheles coluzziisampled from 15 locations across Africa. The data reveal high levels of genetic diversity, with over 50 million single nucleotide polymorphisms across the 230 Mbp genome. We observe complex patterns of population structure and marked variations in local population size, some of which may be due at least in part to malaria control interventions. Insecticide resistance genes show strong signatures of recent selection associated with multiple independent mutations spreading over large geographical distances and between species. The genetic variability of natural populations substantially reduces the target space for novel gene-drive strategies for mosquito control. This large dataset provides a foundation for tracking the emergence and spread of insecticide resistance and developing new vector control tools.

Background An increase in effective malaria control since 2000 has contributed to a decline in global malaria morbidity and mortality. Knowing when and how existing interventions could be combined to maximise their impact on malaria vectors can provide valuable information for national malaria control programs in different malaria endemic settings. Here, we assess the effect of indoor residual spraying on malaria vector densities in a high malaria endemic setting in eastern Uganda as part of a cohort study where the use of long-lasting insecticidal nets (LLINs) was high. Methods Anopheles mosquitoes were sampled monthly using CDC light traps in 107 households selected randomly. Information on the use of malaria interventions in households was also gathered and recorded via a questionnaire. A Bayesian spatio-temporal model was then used to estimate mosquito densities adjusting for climatic and ecological variables and interventions. Results Anopheles gambiae (sensu lato) were most abundant (89.1%; n = 119,008) compared to An. funestus (sensu lato) (10.1%, n = 13,529). Modelling results suggest that the addition of indoor residual spraying (bendiocarb) in an area with high coverage of permethrin-impregnated LLINs (99%) was associated with a major decrease in mosquito vector densities. The impact on An. funestus (s.l.) (Rate Ratio 0.1508; 97.5% CI: 0.0144–0.8495) was twice as great as for An. gambiae (s.l.) (RR 0.5941; 97.5% CI: 0.1432–0.8577). Conclusions High coverage of active ingredients on walls depressed vector populations in intense malaria transmission settings. Sustained use of combined interventions would have a long-term impact on mosquito densities, limiting infectious biting. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1917-3) contains supplementary material, which is available to authorized users.

Background Malaria case management is a key strategy for malaria control. Effective coverage of parasite-based malaria diagnosis (PMD) remains limited in malaria endemic countries. This study assessed the health system's capacity to absorb PMD at primary health care facilities in Uganda. Methods In a cross sectional survey, using multi-stage cluster sampling, lower level health facilities (LLHF) in 11 districts in Uganda were assessed for 1) tools, 2) skills, 3) staff and infrastructure, and 4) structures, systems and roles necessary for the implementing of PMD. Results Tools for PMD (microscopy and/or RDTs) were available at 30 (24%) of the 125 LLHF. All LLHF had patient registers and 15% had functional in-patient facilities. Three months’ long stock-out periods were reported for oral and parenteral quinine at 39% and 47% of LLHF respectively. Out of 131 health workers interviewed, 86 (66%) were nursing assistants; 56 (43%) had received on-job training on malaria case management and 47 (36%) had adequate knowledge in malaria case management. Overall, only 18% (131/730) Ministry of Health approved staff positions were filled by qualified personnel and 12% were recruited or transferred within six months preceding the survey. Of 186 patients that received referrals from LLHF, 130(70%) had received pre-referral anti-malarial drugs, none received pre-referral rectal artesunate and 35% had been referred due to poor response to antimalarial drugs. Conclusion Primary health care facilities had inadequate human and infrastructural capacity to effectively implement universal parasite-based malaria diagnosis. The priority capacity building needs identified were: 1) recruitment and retention of qualified staff, 2) comprehensive training of health workers in fever management, 3) malaria diagnosis quality control systems and 4) strengthening of supply chain, stock management and referral systems.

Introduction Severe 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 facilities Methods and Findings We 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

Background In areas where the morphologically indistinguishable malaria mosquitoes Anopheles gambiae Giles and An. arabiensis Patton are sympatric, hybrids are detected occasionally via species-diagnostic molecular assays. An. gambiae and An. arabiensis exhibit both pre- and post-reproductive mating barriers, with swarms largely species-specific and male F1 (first-generation) hybrids sterile. Consequently advanced-stage hybrids (back-crosses to parental species), which would represent a route for potentially-adaptive introgression, are expected to be very rare in natural populations. Yet the use of one or two physically linked single-locus diagnostic assays renders them indistinguishable from F1 hybrids and levels of interspecific gene flow are unknown. Methods We used data from over 350 polymorphic autosomal SNPs to investigate post F1 gene flow via patterns of genomic admixture between An. gambiae and An. arabiensis from eastern Uganda. Simulations were used to investigate the statistical power to detect hybrids with different levels of crossing and to identify the hybrid category significantly admixed genotypes could represent. Results A range of admixture proportions were detected for 11 field-collected hybrids identified via single-locus species-diagnostic PCRs. Comparison of admixture data with simulations indicated that at least seven of these hybrids were advanced generation crosses, with backcrosses to each species identified. In addition, of 36 individuals typing as An. gambiae or An. arabiensis that exhibited outlying admixture proportions, ten were identified as significantly mixed backcrosses, and at least four of these were second or third generation crosses. Conclusions Our results show that hybrids detected using standard diagnostics will often be hybrid generations beyond F1, and that in our study area around 5% (95% confidence intervals 3%-9%) of apparently ‘pure’ species samples may also be backcrosses. This is likely an underestimate because of rapidly-declining detection power beyond the first two backcross generations. Post-F1 gene flow occurs at a far from inconsequential rate between An. gambiae and An. arabiensis, and, especially for traits under strong selection, could readily lead to adaptive introgression of genetic variants relevant for vector control. Electronic supplementary material The online version of this article (doi:10.1186/1756-3305-7-345) contains supplementary material, which is available to authorized users.