Jason Hendry - Mobile Malaria Project
London Calling 2019
Despite reductions in malaria prevalence in the last two decades, the World Health Organization still reported an estimated 435 thousand deaths in 2017, the majority occurring in children under the age of five. Moreover, continued progress is threatened by emerging drug and insecticide resistance. Our team won the 2019 Land Rover Bursary, supported by the Royal Geographic Society, on a proposal to convert a 2019 Land Rover Discovery into a mobile sequencing lab and drive it 6300km across Africa, from the Atlantic to Indian Ocean. During our journey, we met with local research teams and policy makers striving to combat malaria, and produced materials aiming to raise public awareness and keep malaria on the global development agenda. My role in the project was to develop and pilot the mobile lab which, with local collaborators, we used to sequence antimalarial resistance genes in Zambia, and whole mosquito genomes in Kenya. We hope our project promotes the feasibility of a decentralized approach to pathogen and vector sequencing and marks the beginning of long-term collaborations incorporating in-country nanopore sequencing with policy-directed malaria research.
Opening the Field sequencing breakout, we were delighted to to be joined by the University of Oxford’s Jason Hendry who had just returned from a six-week research expedition to Africa, utilising nanopore sequencing to investigate drug resistance in malaria. Jason revealed that, despite significant reductions in malaria prevalence over the last 20 years, the World Health Organization still reported 219 million cases of malaria infection and 439,000 associated deaths in 2017 alone.
The current treatment for malarial infection is Artemisinin-based Combination Therapies (ACTs), which comprise of the fast-acting artemisinin-based compounds combined with one of a number of companion drugs. However, resistance to ACTs is on the rise and has been seen in Asia and small pockets of Africa. Jason commented, that the spread of malarial resistance from Asia to Africa is a familiar pattern and has been observed in the past with resistance to both chloroquine (QC) – the original antimalarial drug – and its successor sulfadoxine-pyrimethamine (SP). The primary concern is that this same situation is repeating and that soon resistance to ACTs will have spread throughout the African continent.
Sequencing could provide an accurate method of monitoring the spread of ACT-resistant malaria and support the implementation of more effective control strategies. However, Jason commented that, despite substantial reductions in the cost of sequencing over the past several years, traditional sequencing technology is still too expensive and requires significant expertise in molecular biology and data analysis. For this reason, the majority of sequencing is confined to large sequencing centres.
The team at the University of Oxford became aware of a grant from Land Rover and the Royal Geographic Society which would provide the winning proposal with £30,000 and a Land Rover vehicle with which to make a ‘challenging journey’ that would inspire and engage others. Their proposal was to convert the vehicle into a mobile sequencing laboratory and trial in-country genetic surveillance of drug resistance in Africa. At the core of this proposal was the use of the portable MinION device. Having successfully won the grant, the “Lab Rover” and ‘Mobile Malaria Project’ were born.
Jason described how they perhaps took the ‘challenging journey’ element of the proposal too seriously by plotting a 7,351 km route though Namibia, Zambia, Tanzania, and Kenya — stopping along the way to perform sample analyses.
The first experiment was carried out at the National Malaria Elimination Centre in Lusaka, Zambia, where they sequenced 6 samples (4 cases and 2 controls) targeting 4 key resistance genes that confer resistance to ACT, CQ, and SP.
The data showed a single read accuracy of 97.4% and consensus accuracy of 99.54–99.88% across the four genes studied; however, the latter could be increased to 100% by removing a small number of deletions – allowing confident calling of single nucleotides. By downsampling their data, it was revealed that they could call SNVs in the 4 key resistance genes studied at 100% accuracy from just 100 reads. Based on the fact that they obtained 7 million reads in total, this would allow a staggering 70,000 samples to be sequenced on a single flow cell. Alternatively, they could sequence a single sample to sufficient depth in just 3 seconds (based on generating 4,000 reads in approximately 2 minutes). Interestingly, the Zambian case sample revealed sensitivity to CQ, which corresponds to the lack of use of this drug for many years.
Unfortunately, Jason didn’t have time to review the full data set obtained from their epic journey; however, he did reveal how another element of the project was to support, educate, and inspire local researchers. They achieved this through running training workshops across the length and duration of their journey. One specific example took place in the small village of Busia in Kenya, where they set up the mobile lab on a plastic table at the side of the road. Here, a student from the Kenya Medical Research Institute, who had never before undertaken nanopore sequencing, successfully performed library preparation and flow cell loading for 12 local malaria samples, whilst under the watchful eyes of his supervisor and, according to Jason, ‘most of the village’. They drove with the sequencing in progress back to the research institute and arrived three hours later with 3 Gb of data, which mapped to two of the major malaria vectors known to be present in the area.