East Africa International Center of Excellence for Malaria Research: Impact on Malaria Policy in Uganda

Malaria is the leading cause of disease burden in sub-Saharan Africa. In 2010, the East Africa International Center of Excellence for Malaria Research, also known as the Program for Resistance, Immunology, Surveillance, and Modeling of Malaria (PRISM), was established to provide a comprehensive approach to malaria surveillance in Uganda. We instituted cohort studies and a robust malaria and entomological surveillance network at selected public health facilities that have provided a platform for monitoring trends in malaria morbidity and mortality, tracking the impact of malaria control interventions (indoor residual spraying of insecticide [IRS], use of long-lasting insecticidal nets [LLINs], and case management with artemisinin-based combination therapies [ACTs]), as well as monitoring of antimalarial drug and insecticide resistance. PRISM studies have informed Uganda's malaria treatment policies, guided selection of LLINs for national distribution campaigns, and revealed widespread pyrethroid resistance, which led to changes in insecticides delivered through IRS. Our continuous engagement and interaction with policy makers at the Ugandan Ministry of Health have enabled PRISM to share evidence, best practices, and lessons learned with key malaria stakeholders, participate in malaria control program reviews, and contribute to malaria policy and national guidelines. Here, we present an overview of interactions between PRISM team members and Ugandan policy makers to demonstrate how PRISM's research has influenced malaria policy and control in Uganda

Genome variation and population structure among 1142 mosquitoes of the African malaria vector species Anopheles gambiae and Anopheles coluzzii

Mosquito control remains a central pillar of efforts to reduce malaria burden in sub-Saharan Africa. However, insecticide resistance is entrenched in malaria vector populations, and countries with a high malaria burden face a daunting challenge to sustain malaria control with a limited set of surveillance and intervention tools. Here we report on the second phase of a project to build an open resource of high-quality data on genome variation among natural populations of the major African malaria vector species Anopheles gambiae and Anopheles coluzzii. We analyzed whole genomes of 1142 individual mosquitoes sampled from the wild in 13 African countries, as well as a further 234 individuals comprising parents and progeny of 11 laboratory crosses. The data resource includes high-confidence single-nucleotide polymorphism (SNP) calls at 57 million variable sites, genome-wide copy number variation (CNV) calls, and haplotypes phased at biallelic SNPs. We use these data to analyze genetic population structure and characterize genetic diversity within and between populations. We illustrate the utility of these data by investigating species differences in isolation by distance, genetic variation within proposed gene drive target sequences, and patterns of resistance to pyrethroid insecticides. This data resource provides a foundation for developing new operational systems for molecular surveillance and for accelerating research and development of new vector control tools. It also provides a unique resource for the study of population genomics and evolutionary biology in eukaryotic species with high levels of genetic diversity under strong anthropogenic evolutionary pressures

East Africa International Center of Excellence for Malaria Research: Summary of Key Research Findings

The Program for Resistance, Immunology, Surveillance, and Modeling of Malaria (PRISM) has been conducting malaria research in Uganda since 2010 to improve the understanding of the disease and measure the impact of population-level control interventions in the country. Here, we will summarize key research findings from a series of studies addressing routine health facility-based surveillance, comprehensive cohort studies, studies of the molecular epidemiology, and transmission of malaria, evaluation of antimalarial drug efficacy, and resistance across the country, and assessments of insecticide resistance. Among our key findings are the following. First, we found that in historically high transmission areas of Uganda, a combination of universal distribution of long-lasting insecticidal-treated nets (LLINs) and sustained indoor residual spraying (IRS) of insecticides lowered the malaria burden greatly, but marked resurgences occurred if IRS was discontinued. Second, submicroscopic infections are common and key drivers of malaria transmission, especially in school-age children (5–15 years). Third, markers of drug resistance have changed over time, with new concerning emergence of markers predicting resistance to artemisinin antimalarials. Fourth, insecticide resistance monitoring has demonstrated high levels of resistance to pyrethroids, appreciable impact of the synergist piperonyl butoxide to pyrethroid susceptibility, emerging resistance to carbamates, and complete susceptibility of malaria vectors to organophosphates, which could have important implications for vector control interventions. Overall, PRISM has yielded a wealth of information informing researchers and policy-makers on the malaria burden and opportunities for improved malaria control and eventual elimination in Uganda. Continued studies concerning all the types of surveillance discussed above are ongoing

Resistance to pirimiphos-methyl in West African Anopheles is spreading via duplication and introgression of the Ace1 locus

Vector population control using insecticides is a key element of current strategies to prevent malaria transmission in Africa. The introduction of effective insecticides, such as the organophosphate pirimiphos-methyl, is essential to overcome the recurrent emergence of resistance driven by the highly diverse Anopheles genomes. Here, we use a population genomic approach to investigate the basis of pirimiphos-methyl resistance in the major malaria vectors Anopheles gambiae and A. coluzzii. A combination of copy number variation and a single non-synonymous substitution in the acetylcholinesterase gene, Ace1, provides the key resistance diagnostic in an A. coluzzii population from Coˆte d’Ivoire that we used for sequence-based association mapping, with replication in other West African populations. The Ace1 substitution and duplications occur on a unique resistance haplotype that evolved in A. gambiae and introgressed into A. coluzzii, and is now common in West Africa primarily due to selection imposed by other organophosphate or carbamate insecticides. Our findings highlight the predictive value of this complex resistance haplotype for phenotypic resistance and clarify its evolutionary history, providing tools to for molecular surveillance of the current and future effectiveness of pirimiphos-methyl based interventions

Local selection in the presence of high levels of gene flow: Evidence of heterogeneous insecticide selection pressure across Ugandan Culex quinquefasciatus populations

Background: Culex quinquefasciatus collected in Uganda, where no vector control interventions directly targeting this species have been conducted, was used as a model to determine if it is possible to detect heterogeneities in selection pressure driven by insecticide application targeting other insect species. Methodology/Principal findings: Population genetic structure was assessed through microsatellite analysis, and the impact of insecticide pressure by genotyping two target-site mutations, Vgsc-1014F of the voltage-gated sodium channel target of pyrethroid and DDT insecticides, and Ace1-119S of the acetylcholinesterase gene, target of carbamate and organophosphate insecticides. No significant differences in genetic diversity were observed among populations by microsatellite markers with HE ranging from 0.597 to 0.612 and low, but significant, genetic differentiation among populations (FST = 0.019, P = 0.001). By contrast, the insecticide-resistance markers display heterogeneous allelic distributions with significant differences detected between Central Ugandan (urban) populations relative to Eastern and Southwestern (rural) populations. In the central region, a frequency of 62% for Vgsc-1014F, and 32% for the Ace1-119S resistant allele were observed. Conversely, in both Eastern and Southwestern regions the Vgsc-1014F alleles were close to fixation, whilst Ace1-119S allele frequency was 12% (although frequencies may be underestimated due to copy number variation at both loci). Conclusions/Significance: Taken together, the microsatellite and both insecticide resistance target-site markers provide evidence that in the face of intense gene flow among populations, disjunction in resistance frequencies arise due to intense local selection pressures despite an absence of insecticidal control interventions targeting Culex

Genome-wide transcriptional analyses in Anopheles mosquitoes reveal an unexpected association between salivary gland gene expression and insecticide resistance

Background To combat malaria transmission, the Ugandan government has embarked upon an ambitious programme of indoor residual spraying (IRS) with a carbamate class insecticide, bendiocarb. In preparation for this campaign, we characterized bendiocarb resistance and associated transcriptional variation among Anopheles gambiae s.s. mosquitoes from two sites in Uganda. Results Gene expression in two mosquito populations displaying some resistance to bendiocarb (95% and 79% An. gambiae s.l. WHO tube bioassay mortality in Nagongera and Kihihi, respectively) was investigated using whole-genome microarrays. Significant overexpression of several genes encoding salivary gland proteins, including D7r2 and D7r4, was detected in mosquitoes from Nagongera. In Kihihi, D7r4, two detoxification-associated genes (Cyp6m2 and Gstd3) and an epithelial serine protease were among the genes most highly overexpressed in resistant mosquitoes. Following the first round of IRS in Nagongera, bendiocarb-resistant mosquitoes were collected, and real-time quantitative PCR analyses detected significant overexpression of D7r2 and D7r4 in resistant mosquitoes. A single nucleotide polymorphism located in a non-coding transcript downstream of the D7 genes was found at a significantly higher frequency in resistant individuals. In silico modelling of the interaction between D7r4 and bendiocarb demonstrated similarity between the insecticide and serotonin, a known ligand of D7 proteins. A meta-analysis of published microarray studies revealed a recurring association between D7 expression and insecticide resistance across Anopheles species and locations. Conclusions A whole-genome microarray approach identified an association between novel insecticide resistance candidates and bendiocarb resistance in Uganda. In addition, a single nucleotide polymorphism associated with this resistance mechanism was discovered. The use of such impartial screening methods allows for discovery of resistance candidates that have no previously-ascribed function in insecticide binding or detoxification. Characterizing these novel candidates will broaden our understanding of resistance mechanisms and yield new strategies for combatting widespread insecticide resistance among malaria vectors

Parasite-based malaria diagnosis : are health systems in Uganda equipped enough to implement the policy?

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

Insecticide resistance in areas under investigation by the international centers of excellence for malaria research: a challenge for malaria control and elimination

Scale-up of the main vector control interventions, residual insecticides sprayed on walls or structures and/or impregnated in bed nets, together with prompt diagnosis and effective treatment, have led to a global reduction in malaria transmission. However, resistance in vectors to almost all classes of insecticides, particularly to the synthetic pyrethroids, is posing a challenge to the recent trend of declining malaria. Ten International Centers of Excellence for Malaria Research (ICEMR) located in the most malaria-endemic regions of the world are currently addressing insecticide resistance in the main vector populations, which not only threaten hope for elimination in malaria-endemic countries but also may lead to reversal where notable reductions in malaria have been documented. This communication illustrates the current status of insecticide resistance with a focus on the countries where activities are ongoing for 9 out of the 10 ICEMRs. Most of the primary malaria vectors in the ICEMR countries exhibit insecticide resistance, albeit of varying magnitude, and spanning all mechanisms of resistance. New alternatives to the insecticides currently available are still to be fully developed for deployment. Integrated vector management principles need to be better understood and encouraged, and viable insecticide resistance management strategies need to be developed and implemented

Insecticide Resistance in Areas Under Investigation by the International Centers of Excellence for Malaria Research: A Challenge for Malaria Control and Elimination.

Scale-up of the main vector control interventions, residual insecticides sprayed on walls or structures and/or impregnated in bed nets, together with prompt diagnosis and effective treatment, have led to a global reduction in malaria transmission. However, resistance in vectors to almost all classes of insecticides, particularly to the synthetic pyrethroids, is posing a challenge to the recent trend of declining malaria. Ten International Centers of Excellence for Malaria Research (ICEMR) located in the most malaria-endemic regions of the world are currently addressing insecticide resistance in the main vector populations, which not only threaten hope for elimination in malaria-endemic countries but also may lead to reversal where notable reductions in malaria have been documented. This communication illustrates the current status of insecticide resistance with a focus on the countries where activities are ongoing for 9 out of the 10 ICEMRs. Most of the primary malaria vectors in the ICEMR countries exhibit insecticide resistance, albeit of varying magnitude, and spanning all mechanisms of resistance. New alternatives to the insecticides currently available are still to be fully developed for deployment. Integrated vector management principles need to be better understood and encouraged, and viable insecticide resistance management strategies need to be developed and implemented

Strain Characterisation for Measuring Bioefficacy of ITNs Treated with Two Active Ingredients (Dual-AI ITNs): Developing a Robust Protocol by Building Consensus

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