Using spatial statistics and genomics to develop epidemiologically relevant definitions of insecticide resistance in African Malaria Vectors

Lead Research Organisation: Liverpool School of Tropical Medicine
Department Name: Vector Biology

Abstract

In the last 15 years 260 million clinical cases of malaria have been prevented in Sub Saharan Africa, overwhelmingly through the use of insecticides that target the mosquito vector. Insecticide resistance is therefore a major threat to the sustained control of malaria. Whole genome sequencing of malaria mosquitoes has the power to revolutionise our understanding of the evolution of insecticide resistance; allowing us to design new insecticides and to develop strategies that delay the onset of resistance. However genomic technologies will only reach their full potential with improved sampling design and when extensive individual and population level metadata (collection location, infection/ disease state etc) accompany each sequenced sample. For the problem of insecticide resistance in the major malaria vector Anopheles gambiae a deep understanding of the biology lies with SSA scientists whilst the skills to sequence and analyse the data are held by centres in the North. This foundation proposal is designed to draw together these two groups with support from a world-leading spatial statistics group around the key issue of insecticide resistance evolution. Our vision is to further develop the programme to a Pan-African level, eventually devolving leadership to our SSA partners who will have the skills and experience to sustain the network.

Technical Summary

Malaria is a major cause of mortality and morbidity in Sub-Saharan Africa (SSA) and one of the biggest impediments to economic development. Malaria control is threatened by insecticide resistance which demands improved insecticide resistance management (IRM). Molecular markers could greatly aid improved IRM, but are currently underused. Our proposal will develop and train control programme linked SSA vector biologists to design and analyse genomic data to assess Anopheles population structure and to identify resistance-linked genetic variation. The overarching aim is to develop a nucleus of SSA scientists best able to exploit the power of whole genome sequencing to address questions of public health importance.

Using the key phenotype of insecticide resistance as an exemplar we will:
1. develop and implement standardised, server-based pipelines for the analysis of whole genome sequence data to identify patterns of population structure, genetic diversity and insecticide resistance candidate genes/variants
2. develop spatially- and temporally-explicit sampling frameworks for malaria vectors to permit local-scale genetic tracking and prediction of (i) population connectivity, (ii) temporal stability and (iii) insecticide resistance.
3. test extant and novel DNA markers for prediction of resistance in females of malaria transmission age

Planned Impact

Pathways to impact
The proposed foundation award is directly aligned to the 2014-2019 MRC Strategic Plan, specifically Strategic Aim 3: Global Health. To "deliver improved health outcomes in infectious diseases, which impact heavily on the health and wellbeing of populations in resource-poor settings" and to "develop investment in the translation of research findings into health systems to ensure that the outcomes of research can be better implemented into practice."

Moreover in line with the specific MRC Global Challenges Research Fund Foundation call the proposal encompasses:Early phase discovery and translation research
-Extending scope of existing research to wider global settings
-Population studies, sample/data collection, data integration
-New research tools and techniques
-Development of collaborations, partnerships, teams
-Creating pathways to impact in LMICs

Translating the potential of genomic science from early phase discovery into translational 'real-world' public health impacts is reliant upon cohesive interdisciplinary research within effective partnerships. Thus far the Low and Middle Income Country partners who work at the interface between applied and translational science have not been able to adequately shape the basic science studies that are needed to address public health priorities. The key to this foundation award is that we will engage beneficiaries from the outset of the project so that they can help us develop the research agenda to meet their needs.

Impact Pathway 1: Inception network meeting. Target Audience: Key stakeholders in malaria control programmes
Year 1 meetings will be held in all countries to present our objectives and long term goals to local stakeholders. These key stakeholders will include NMCP representatives, WHO-Regional Officers, CDC country representatives, President's Malaria Initiative representatives, Abt associates managers. Our objective is to solicit feedback on how best our activities can dovetail with the requirements of implementers to provide timely and relevant information for control programme design and monitoring. In addition we will establish collaborative links with a stakeholder network which can be maintained throughout the course of the foundation and beyond.

Impact Pathway 2: Capacity strengthening and resource provision for regional skill dissemination. Target Audience: Vector control scientists in SSA
As part of Aim 1 of the project we will train staff from our network, and/or members of their groups to analyse genomic data within a supported Galaxy pipeline framework. This will become a semi-devolved self-supporting dissemination mechanism for knowledge and skills, providing impact to in-country scientists beyond that of the immediate network in a sustainable way.

Impact Pathway 3: Integration of knowledge and tools into malaria control programme decision making. Target Audience: National malaria control programme managers and practitioners.
From the links established at the inception meetings we will be able to disseminate findings directly, and in an adaptive manner to control programme managers and practitioners, sharing data and soliciting feedback, e.g. on geographical areas of interest which could be incorporated into our sampling. In addition in year 2 we will present findings directly to in-country NMCPs and communicate findings via international conference presentation and publication in open access journals. Lay summaries will also be produced with translations into French and Kiswahili.
 
Description The key achievements were:
1. Identification of a number of new insecticide resistance molecular markers that may be used by disease control programmes to inform policy and practice
2. The development of a rigorous vector sampling framework which takes into account environmental conditions obtained from open data (i.e. remote sensing and meteorological stations) thereby allowing control programmes to move away from haphazard/purposive sampling.
3. Development of thriving north:south and south:south partnerships between research groups with interests in the application of genomic technologies to malaria vector surveillance and control.

Our objectives were
1. To develop and implement standardised, server-based pipelines for the analysis of whole genome sequence data to identify patterns of population structure, genetic diversity and insecticide resistance candidate genes/variants

The project team developed and applied a number of analytical pipelines. One key analysis was the development of approaches to identify variation in the number of copies if genes in the mosquito genome. This led to the discovery of how ubiquitous gene duplication events are in the malaria mosquito genome and how they are over represented around key insecticide resistance- associated gene families. Using the whole-genome sequencing data from 1142 samples in the Anopheles gambiae1000 genomes project, we identified 250 gene-containing copy number variants (CNVs), encompassing a total of 267 genes of which 28 were in gene families linked to metabolic insecticide resistance, representing significant enrichment of these families. The five major gene clusters for metabolic resistance all contained CNVs, with 44 different CNVs being found across these clusters and multiple CNVs frequently covering the same genes. These 44 CNVs are widespread (45% of individuals carry at least one of them) and have been spreading through positive selection, indicated by their high local frequencies and extended haplotype homozygosity. Our results demonstrate the importance of CNVs in the response to selection, highlighting the urgent need to identify the contribution of each CNV to insecticide resistance and to track their spread as the use of insecticides in malaria endemic countries intensifies and as the operational deployment of next-generation bed nets targeting metabolic resistance gathers pace

2. To develop spatially- and temporally-explicit sampling frameworks for malaria vectors to permit local-scale genetic tracking and prediction of (i) population connectivity, (ii) temporal stability and (iii) insecticide resistance.

Vector-borne disease control and monitoring rely on vector surveillance, mostly carried out using trap-based indices. Trap-based indices (density, population changes, distribution, etc.) are calculated from mosquito catches and require a system of traps dispersed in the field in sufficient numbers to represent mosquito population ecology and dynamics. However much of the sampling conducted in vector surveillance studies is opportunistic and lacks a rigorous sampling framework. Often, ecological and entomological sampling designs rely solely on resource availability rather than aiming to maximize representativeness and precision of
the variable of interest, e.g. collectors target locations where disease vectors are known to be abundant. We developed and implemented an approach which takes into account environmental conditions obtained from open data (i.e. remote sensing and meteorological stations). Notably, we showed that a configuration of 30 locations with four households each (120 samples) will have a similar accuracy in the predictions of mosquito abundance as 200 random samples. Our approach Our analysis provides an example of how to fully describe the assumptions, conditions and constraints of sampling strategies.

3. To test extant and novel DNA markers for prediction of resistance in females of malaria transmission age

Obtaining phenotypic measurements of insecticide resistance is laborious and error-prone. High-throughput genotyping offers the prospect of quick and repeatable estimates of resistance, while also allowing resistance markers to be tracked and studied. To demonstrate the potential of highly-mulitplexed genotypic screening for measuring resistance-association of mutations and tracking their spread, we developed a panel of 28 known or putative resistance markers in the major malaria vector Anopheles gambiae, which we used to screen mosquitoes from a wide swathe of Sub-Saharan Africa (Burkina Faso, Ghana, Democratic Republic of Congo (DRC) and Kenya). This work is ongoing with molecular characterization of resistance ongoing in SSA partner labs.
Exploitation Route There is renewed in interest in vector surveillance and a number of groups are deploying the spatially-explicit sampling frameworks for vector monitoring and evaluation in a variety of disease systems. Partners in Kisumu, Kenya are also working on applying the methodology to a cluster randomised control trial of a new anti-vector intervention- an attractive sugar bait (ATSB).
Sectors Healthcare

 
Description Participated in national and African region meetings which influence malaria vector control policy.
Geographic Reach Africa 
Policy Influence Type Participation in a advisory committee
 
Description Anopheles gambiae and Anopheles arabiensis genetic diversity and association with insecticide resistance in Kenya
Amount $100,000 (USD)
Funding ID OPP1210319 
Organisation Bill and Melinda Gates Foundation 
Sector Charity/Non Profit
Country United States
Start 04/2019 
End 04/2020
 
Description NIHR-Wellcome Partnership for Global Health Research Collaborative Award, 'Controlling emergent Anopheles stephensi in Ethiopia and Sudan (CEASE)'
Amount £3,552,923 (GBP)
Funding ID 220870/Z/20/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2021 
End 12/2024
 
Description New advances in insecticide resistance genomics: using Machine Learning to predict resistance phenotype from large-scale genomic data
Amount £507,681 (GBP)
Funding ID MR/T001070/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 12/2019 
End 11/2022
 
Description Royal Society Wolfson Fellowship in Translational Genomics of Malaria Vectors
Amount £150,000 (GBP)
Funding ID RSWF\FT\180003 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2019 
End 12/2025
 
Description The East-South Africa Anopheles funestus Consortium: Building the capacity for An. funestus entomological surveillance, insecticide resistance and genomic diversity studies in Malawi, Kenya, Uganda and Tanzania
Amount $100,000 (USD)
Funding ID OPP1210327 
Organisation Bill and Melinda Gates Foundation 
Sector Charity/Non Profit
Country United States
Start 04/2019 
End 04/2020
 
Description Wellcome Trust Seed Award in Science
Amount £94,334 (GBP)
Funding ID 212501/Z/18/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2019 
End 09/2020
 
Title Algorithms 
Description Set of algorithms for ecological stratification of the sampling area and for allocation of the sampling locations. 
Type Of Material Computer model/algorithm 
Year Produced 2018 
Provided To Others? Yes  
Impact Used by external (non-Lancaster) PhDs and researchers for teaching and research. 
URL https://lancaster.box.com/v/GAARDIANstats
 
Title Environmental data 
Description This link contains all the data used to produce the sampling locations for GAARDIAN project 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Used by external (non-Lancaster) PhDs and researchers for teaching and research. 
URL https://lancaster.box.com/v/GAARDIANstats
 
Description African Vector Bioinformatics Network (AVBN) 
Organisation Ifakara Health Institute
Country Tanzania, United Republic of 
Sector Charity/Non Profit 
PI Contribution Under this partnerships we developed a research proposal on "African Vector Bioinformatics Network: Translating genomic data to public health application" and submitted to PAMCA for funding. The proposal passed through the initial review process, however it was not funded.
Collaborator Contribution African Vector Bioinformatics Network (AVBN) was established by vector biologists from 6 Francophone and Anglophone African countries namely; Benin, Ivory-Coast, Ghana, Kenya, Tanzania and Senegal. Continued with the collaboration with the National Malaria Control Programme (NMCP) and Ifakara Health Institute (IHI) of Tanzania.
Impact Grant application to PAMCA Continued collaboration with the National Malaria Control Programme (NMCP) and Ifakara Health Institute (IHI) of Tanzania.
Start Year 2018
 
Description African Vector Bioinformatics Network (AVBN) 
Organisation National Malaria Control Programme (NMCP)
Country Ghana 
Sector Public 
PI Contribution Under this partnerships we developed a research proposal on "African Vector Bioinformatics Network: Translating genomic data to public health application" and submitted to PAMCA for funding. The proposal passed through the initial review process, however it was not funded.
Collaborator Contribution African Vector Bioinformatics Network (AVBN) was established by vector biologists from 6 Francophone and Anglophone African countries namely; Benin, Ivory-Coast, Ghana, Kenya, Tanzania and Senegal. Continued with the collaboration with the National Malaria Control Programme (NMCP) and Ifakara Health Institute (IHI) of Tanzania.
Impact Grant application to PAMCA Continued collaboration with the National Malaria Control Programme (NMCP) and Ifakara Health Institute (IHI) of Tanzania.
Start Year 2018
 
Description Bilateral Partnership 
Organisation AngloGold Ashanti Malaria Control Ltd
Country Ghana 
Sector Charity/Non Profit 
PI Contribution A collaboration with industry partner to provide technical support to their vector control programme
Collaborator Contribution The award has result in a bilateral partnership culminating in the signing of a Memorandum of Understanding between UCC/LSTM and AngloGold Ashanti Malaria Control Ltd, Ghana for collaborations in malaria vector research including joint publications.
Impact Not as yet
Start Year 2018
 
Description Bilateral Partnership 
Organisation University of Cape Coast
Country Ghana 
Sector Academic/University 
PI Contribution A collaboration with industry partner to provide technical support to their vector control programme
Collaborator Contribution The award has result in a bilateral partnership culminating in the signing of a Memorandum of Understanding between UCC/LSTM and AngloGold Ashanti Malaria Control Ltd, Ghana for collaborations in malaria vector research including joint publications.
Impact Not as yet
Start Year 2018
 
Description Farming entomofauna 
Organisation Swiss Center for Scientific Research in Ivory Coast
Country Cote d'Ivoire 
Sector Charity/Non Profit 
PI Contribution Applied for the GCRF Agri systems research to enhance livelihoods in developing countries.
Collaborator Contribution Supported conceptual and methodological framework.
Impact Application number BB/S014659/1 (unsuccessful)
Start Year 2018
 
Description Farming entomofauna 
Organisation University of Abobo-Adjamé
Country Cote d'Ivoire 
Sector Academic/University 
PI Contribution Applied for the GCRF Agri systems research to enhance livelihoods in developing countries.
Collaborator Contribution Supported conceptual and methodological framework.
Impact Application number BB/S014659/1 (unsuccessful)
Start Year 2018
 
Description Joint grant award 
Organisation Kenyan Institute for Medical Research (KEMRI)
Country Kenya 
Sector Public 
PI Contribution Dr Eric Odhiambo (KEMRI, Kenya), Dr. Luc Djogbenou (University of Abomey-Calavi, Benin) applied for and were awarded a grant to build the the Capacity for the utility of Next Generation Sequencing in Insecticide Resistance Management by African National Malaria Control programs.
Collaborator Contribution The three research groups worked together contributing towards the project's aim.
Impact None as yet
Start Year 2018
 
Description Joint grant award 
Organisation University of Abomey-Calavi
Country Benin 
Sector Academic/University 
PI Contribution Dr Eric Odhiambo (KEMRI, Kenya), Dr. Luc Djogbenou (University of Abomey-Calavi, Benin) applied for and were awarded a grant to build the the Capacity for the utility of Next Generation Sequencing in Insecticide Resistance Management by African National Malaria Control programs.
Collaborator Contribution The three research groups worked together contributing towards the project's aim.
Impact None as yet
Start Year 2018
 
Description Geospatial/sampling teaching 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact The course was designed to provide spatial statistical skills (from sampling to mapping) to a group of 30 people of different background (Academics, technicians, health researchers, public health managers) in Riyadh, Saudi Arabia.

A PhD student as been funded by the National Health Laboratory of Saudi Arabia to work on vector borne diseases in Saudi Arabia (Luigi Sedda main supervisor). The student is currently doing the PhD in Spatial Epidemiology at Lancaster University.
Year(s) Of Engagement Activity 2018
URL http://chicas.lancaster-university.uk/people/omar.html
 
Description International invited speaker Italy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact This was a seminar for Academics at the University of Salento in Italy. I am now collaborating with them in order to improve bio-geostatistical models for distribution of vectors and diseases.
Year(s) Of Engagement Activity 2018
 
Description Invited Session International conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was an invited and funded (by the Entomological Society of America) session within the 2018 ESA, ESC and ESBC Joint Annual Meeting: Crossing Borders: Entomology in a Changing World. The session was well attended and debate carried over after the session.

The interest in this session produce requests from two international scientists to join the framework of the current award.
Year(s) Of Engagement Activity 2018
URL https://www.entsoc.org/event-calendar/2018-esa-esc-and-esbc-joint-annual-meeting-entomology-2018
 
Description Symposium 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Organised symposium at the Pan African Mosquito Control Association meeting in Zimababwe
Year(s) Of Engagement Activity 2018