Improvements in genetic and genomic technology have enabled field-deployable molecular laboratories and these have been deployed in a variety of epidemics that capture headlines. In this editorial, we highlight the importance of building physical and personnel capacity in low and middle income countries to deploy these technologies to improve diagnostics, understand transmission dynamics and provide feedback to endemic communities on actionable timelines. We describe our experiences with molecular field research on schistosomiasis, trypanosomiasis and rabies and urge the wider tropical medicine community to embrace these methods and help build capacity to benefit communities affected by endemic infectious diseases

Adriko, Moses

Ajambo, Diana

Brunker, Kirstyn

Faust, Christina L.

Guma, Andrew

Lamberton, Poppy H.L.

Moses, Arinaitwe

Mugenyi, Albert

Rowel, Candia

Ryan, Mary

Tukahebwa, Edridah M.

Waiswa, Charles

Wampande, Eddie M.

Wangoola, Robert Mandela

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LESSONSFROMTHEFIELDTrans R Soc Trop Med Hyg 2021; 115: 3–5doi:10.1093/trstmh/traa086 Advance Access publication 18 September 2020Harnessing technology and portability to conduct molecularepidemiology of endemic pathogens in resource-limited settingsChristina L. Faust a,b,∗,†, Kirstyn Brunkera, Diana Ajamboc, Mary Ryand, Arinaitwe Mosesc, Candia Rowelc,Robert Mandela Wangoolae, Eddie M. Wampandef, Andrew Gumac, Moses Adrikoc, Poppy H. L. Lambertona,b,Edridah M. Tukahebwac, Albert Mugenyie, and Charles WaiswaeaInstitute of Biodiversity, Animal Health and Comparative Medicine, University of Glasgow, Glasgow G12 8QQ, UK; bWellcome Centre forIntegrative Parasitology, University of Glasgow, Glasgow G12 8QQ, UK; cVector Control Division, Ministry of Health, Kampala, Uganda;dGlasgow Centre for International Development, University of Glasgow, Glasgow G12 8QQ, UK; eCoordinating Office for the Control ofTrypanosomiasis in Uganda, Kampala, Uganda; fDepartment of Biotechnical and Diagnostic Sciences, Makerere University, Kampala,Uganda∗Corresponding author: Tel: +00 44 (0) 7931 292792; E-mail: christina.faust@glasgow.ac.uk†Present address: Department of Biology, Center for Infectious Disease Dynamics, Penn State University, University Park, PA, 16802, USA.Received 20 March 2020; revised 10 August 2020; editorial decision 21 August 2020; accepted 21 August 2020Improvements in genetic and genomic technology have enabled field-deployable molecular laboratories andthese have been deployed in a variety of epidemics that capture headlines. In this editorial, we highlight theimportance of building physical and personnel capacity in low and middle income countries to deploy thesetechnologies to improve diagnostics, understand transmission dynamics and provide feedback to endemic com-munities on actionable timelines. We describe our experiences with molecular field research on schistosomiasis,trypanosomiasis and rabies and urge the wider tropical medicine community to embrace these methods andhelp build capacity to benefit communities affected by endemic infectious diseases.Ebola, yellow fever, Zika and COVID-19 outbreaks in the last fewyears have benefitted from real-time sequencing of pathogensto understand the transmission and evolution of these emergingviruses.1 The portability and low cost of MinION sequencers havecaptured the imagination of researchers and the public, but alsomade ‘lab in a suitcase’ sequencing a possibility. Studying genet-ics no longer requires a laboratory facility with bright lights andhefty equipment: the technology and creative solutions facilitat-ing PCR product-free and sterile environments nearly anywherehave enabled a rapid response to emerging pathogens. Althoughthese epidemics dominate headlines, we argue that these oppor-tunities should also be extended to improve diagnostics, under-standing of transmission dynamics and to design more effectiveintervention strategies to target endemic infectious pathogensin low and middle income countries (LMICs). Moreover, build-ing core capacity and routine molecular surveillance in the areasmost burdened by infectious disease is critical to ensure epidemicpreparedness.There is immense potential to use new technologies and low-ered equipment costs to move capacity into those regions thatare most impacted by infectious diseases. Countless molecularwork currently relies on shipping samples to complete process-ing, often with long delays and removed from those who collectsamples and deal with pathogens on a daily basis. The decliningcost of portable PCR machines, quantitative PCR and sequencersmake them a more feasible option and several are robust tofield conditions. These portable pieces of equipment mean thatmachines can stay in country and be moved on site to studyspecific endemic diseases in real time. They can even be usedin stationary laboratories to build scalable molecular capacity atreference laboratories. Consumables are a real challenge, as wediscuss below, but newprotocols, tools and reagents are loweringhurdles for conducting molecular work.A key bottleneck to developing molecular capacity in the field,or in areas with intermittent supply, is access to reliable electric-ity. Power is required to run machines, heat samples during lysisand to maintain a cold chain for essential reagents and unsta-ble nucleic acids. Several creative solutions have removed theneed for electricity, ranging from hand-powered centrifuges totemperature-stable polymerases. A range of commercially avail-able reagents now no longer need refrigeration, require a ‘hot-start’ to enable PCR set-up at room temperature, or both. Theseadvances reduce cross-amplification and enable accuratemolec-ular work to be carried out in the field. In our work diagnosingzoonotic human African trypanosomiasis (rHAT; causative agentTrypanosoma brucei rhodesiense), we found that results obtainedin the field with a modified extraction protocol and temperature-stable reagents were consistent with results later obtained with© The Author(s) 2020. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. This is an Open Accessarticle distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permitsunrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.3Downloaded from https://academic.oup.com/trstmh/article/115/1/3/5908315 by guest on 11 February 2021C. L. Faust et al.Figure 1. Field deployment of molecular laboratories in Uganda and Tanzania (A) for diagnosing Trypanosoma brucei rhodesiense (a causative agentof human African trypanosomiasis), (B) zoonotic schistosomes and (C) rabies genomic surveillance.the same samples in a molecular lab at Makerere University(Figure 1A). If cold chains are necessary, liquid nitrogen, dry iceand reusable ice packs can work for short-term options (daysto weeks). When we could not avoid power altogether, we usedsolar panels and lithium batteries to run low wattage centrifugesand PCR machines. Combining resources with the CoordinatingOffice for the Control of Trypanosomiasis in Uganda and the Vec-tor Control Division, Ministry of Health, Uganda, allowed purchaseof the equipment and enabled a field-deployable molecular lab.The constraints of nucleic acid extraction can also be a majorhindrance to molecular work. New methods allow DNA extrac-tion from plants, animals and environmental samples at roomtemperature, or nearly room temperature. We have been usingFTATM cards to lyse cells, inactivate pathogens and bind DNA tothe matrix in field conditions. After 1 h of drying, DNA can beeluted from tissue samples and whole organisms (i.e. parasites)using modified solutions then used directly in PCR reactions(Figure 1B). Expensive commercially available kits can rapidlyextract DNA within minutes in the field and possess single-usecontained columns to prevent cross-contamination. If moreextensive lysis steps are required before sample extraction, thereare portable tissue homogenisers and water baths that can bepowered by low wattage. In a pinch, toothpicks, thermometers,candles and charcoal stoves have all been used successfully assubstitutes.An additional challenge of conducting molecular work in thefield is maintaining a safe working space. The ARTIC network(https://artic.network/ebov) has developed accessible protocolsto inactivate, amplify and sequence viral samples in the fieldin a robust, sensitive and safe manner. The development ofportable laboratories has inspired many of the kit and processingpipelines described here. These researchers have used portableglove boxes to help contain infectious material before they areinactivated, andmade their packing lists and operating protocolsopen for others to learn from. Portable glove boxes help main-tain safety, keep the space sterile, and can be lightweight andpackable (Figure 1C). PortableUV lights and bleach are both cheapand effective in sterilising and preventing cross-contamination ofworkspaces. However, the scope of work will be constrained bythe level of containment required for specific pathogens.Field laboratories are increasingly being used for in-depthstudies that go beyond diagnosis, even generating wholegenomes. Our published and validated pipeline for rabiesgenomic research can be applied in low-resource settings andwill be an important resource for informing elimination cam-paigns.2 The Cassava Virus Action Project has developed genomicpipelines to improve food security in Africa, using whole genomesequencing to characterise the diversity of cassava viruses infect-ing a farmer’s field and provide recommendations of whichstrains of cassava would be ideal for replanting.3 This providesincreases in yield for the individual farmer but also providesimportant information on how these cassava viruses are evolv-ing in response to human control interventions.The application of these new technologies must also be metwith a commitment to training local researchers. Funding forsome of our training work has come through the Global Chal-lenges Research Fund, part of theUK’s Official Development Assis-tance commitment. Funding initiatives that support exchange4Downloaded from https://academic.oup.com/trstmh/article/115/1/3/5908315 by guest on 11 February 2021Transactions of the Royal Society of Tropical Medicine and Hygieneand training opportunities are crucial for developing capacity andwe hope that such important financial support will continue. Ingeneral, LMICs suffer from a lack of bioinformatic expertise, butdedicated new initiatives such as the Pan African BioinformaticsNetwork show promising growth in this area.4One of the most beneficial aspects of field laboratories is thespeed at which samples can be collected and analysed. Thesefast pipelines are revolutionising how we study outbreaks andcontrol important animal, plant and human diseases. Deployablerabies virus sequencing has lowered costs and reduced sample-to-sequence lag times from up to 6months (with sample export)to a matter of days, providing rapid, actionable epidemiologicalinsights for rabies control efforts.2 The nearly instantaneous feed-back to affected communities can help avoid fatigue that com-munities experience when they must wait months or even yearsfor results. Whole genome sequencing of bacteria in the Philip-pines is informing antimicrobial resistancemanagement and pol-icy in real time.5 Field molecular laboratories can help identifyinfected animals using pen-side tests and make informed deci-sions on culling and treatment to limit future human cases andeconomic losses.6Despite these obvious benefits, we believe one of the biggestchallenges in conducting molecular work is sourcing and pay-ing for consumables. Local suppliers in LMICs offer a limitedrange of molecular reagents, items are often out of stock or takemonths to deliver and are prohibitively expensive. Importationregulations exacerbate the delays and expense of acquiring nec-essary reagents, and customs delays compromise the integrityof reagents. In our experience, airway bills, including duty taxand in-transit cold storage costs, often exceed the price of thereagents themselves. A secure, affordable supply chain will ulti-mately ensure the sustainability of molecular research in LMICs.While global companies continue to monopolise prices this willremain a major challenge to research implementation in theseareas. Companies simply must do more to improve supply chainsand the accessibility of their products. The wider research com-munity is also beginning to develop low-cost ‘do-it-yourself’ alter-natives for some reagents, which provide alternatives to some ofthese consumable needs (i.e. https://bomb.bio/about/).By bringing the lab to the sample, it is possible to avoid delayscaused by shipping samples and long lag times in communicat-ing results back to the affected communities. It is essential tobuild physical and human capital to carry out this work withinendemic settings and ensure that consumables are not a limit-ing factor in conducting this work. Helicopter science limits theperspective of an endemic disease, and building confidence in theapplication ofmolecularmethods throughout academic and gov-ernment human and animal health institutions will only benefitfrom these advances.Authors’ contributions: CLF, KB, EMT, AM and CW conceived the study.CLF, KB, DA, MR, AM, CR, RMW, EMW, AG and MA implemented fieldworkand assessments. CLF and KB drafted the manuscript. All authors readand approved the final manuscript.Acknowledgements: A special thanks to all the communities that hostedvarious aspects of testing and trialling field molecular laboratories.Thanks to O. Ericsson for many hours of dedicated molecular training inUganda.Funding: The work in Uganda was supported by the University of Glas-gow, the Scottish Funding Council and the Global Challenges ResearchFund to principal investigator CLF [grant number EP/S51584X/1]. Rabiesworkwas supported by theWellcome Trust through a Senior Research Fel-lowship to principal investigator Katie Hampson [grant number 207569],a Training Fellowship in Public Health and Tropical Medicine to collabora-tor S. M. Thumbi [grant number 110330] and Institutional Strategic Sup-port Fund grants to the University of Glasgow [grant number 204820].KB also received a Royal Society of Tropical Medicine and Hygiene SmallGrant 2017 [grant number GR000892] as well as funding from the GlobalChallenges Research Fund and Scottish Funding Council through a grantawarded to theUniversity of Glasgow [grant number EP/S51584X/1]. PHLLand CLF were also funded by an European Research Council starting grantto PHLL [SCHISTO_PERSIST grant number 680088].Competing interests: None declared.Ethical approval: The methods and surveys used for these studies havebeen reviewed and approved by the Vector Control Division ResearchEthics Committee (VCDREC/095), the Uganda National Council of Scienceand Technology (UNCST-NS 633) and the University of Glasgow VeterinarySciences Research Ethics Committee (Ref27a/18).References1 Quick J, Loman NJ, Duraffour S, et al. Real-time, portablegenome sequencing for Ebola surveillance. Nature. 2016;530:228.2 Brunker K, Jaswant G, Thumbi SM, et al. Rapid in-country sequenc-ing of whole virus genomes to inform rabies elimination programmes.Wellcome Open Research. 2020;5:3.3 Boykin LM, Sseruwagi P, Alicai T, et al. Tree Lab: Portable genomics forearly detection of plant viruses and pests in Sub-Saharan Africa. Genes.2019;10:632.4 Baichoo S, Souilmi Y, Panji S, et al. Developing reproducible bioin-formatics analysis workflows for heterogeneous computing environ-ments to support African genomics. BMC Bioinformatics. 2018;19(1):457.5 Argimón S, MasimMAL, Gayeta JM, et al. See and Sequence: IntegratingWhole-Genome Sequencing Within the National Antimicrobial Resis-tance Surveillance Program in the Philippines. BioRxiv https://doi.org/10.1101/808378). Accessed 20 March 2020.6 Rambo-Martin BL, Keller MW, Wilson MM, et al. Influenza: a virusfield surveillance at a swine-human interface. mSphere. 2020;5:e00822–19.5Downloaded from https://academic.oup.com/trstmh/article/115/1/3/5908315 by guest on 11 February 2021

Harnessing technology and portability to conduct molecular epidemiology of endemic pathogens in resource-limited settings

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Harnessing technology and portability to conduct molecular epidemiology of endemic pathogens in resource-limited settings

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