Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes coronavirus disease 2019 (Covid-19), a disease which first disease emerged in China in December 2019. The virus has now spread across the world, with all but a very few countries now having cases. Although the disease is fatal in a small number of individuals, the majority of Covid-19 cases either are asymptomatic or result in only mild disease. Internationally there have been a variety of different strategies to reduce the burden of the disease including vaccine development, the search for new medicines and finally the identification (or diagnosis) and isolation of those infected (to prevent the disease spreading throughout communities). Over the last 9 months there has been a huge effort in developing diagnostic sensors, using a range of strategies including those for laboratory-based testing and those can be used within communities. Within these sensors developed, there are sensors that can tell if you have previously been infected (based upon determining antibodies in the blood) and those that tell whether you are currently infected (by measuring the viruses' genetic material from a throat swab). This proposal is concerned with detecting those people who are currently infected in a format that can be readily used by GPs in primary care, care homes and schools. One significant challenge is that many of the those infected with the SARS-CoV-2 virus have symptoms that are similar either to those with a cold (commonly caused by the Respiratory Syncytial Virus in children and adults) and flu (caused by the influenza virus). We have therefore chosen to make a low-cost, disposable sensor, that can not only be used in the community without the need for a centralised testing laboratory, but which also tells the GP if the individual being tested has COVID-19, flu or a cold. The proposal aims to simplify the method of sample testing that it can be caried out simply, giving a test result that is easily read by eye, in a manner similar to a pregnancy test (as a band, on a strip of paper).

Point-of-care rapid testing devices, particularly paper-based analytical devices, are powerful diagnostic tools both in developed countries (e.g., 2020 UK Operation Moonshot) and in deprived regions of the world (e.g., World Health Organisation's Global Malaria Programme). These devices can enable rapid, affordable, and widely accessible diagnosis, but compared to laboratory-based analytical techniques, have limited sensitivity, making them inappropriate for the diagnosis of early-stage diseases. Improving the sensitivity of point-of-care rapid tests substantially without sacrificing their advantages, is an ongoing technological challenge. To address this challenge, this proposal aims to introduce a new paradigm for the rapid preconcentration of biomarkers (i.e., the molecules associated with a specific disease) in paper-based analytical devices by harvesting the energy associated with salty water solutions. This radically new concept will be tested and validated in colorimetric lateral flow devices, potentially leading to orders of magnitude improvement in device sensitivity, without relying on auxiliary power sources, or compromising the device portability, simplicity, and ease of fabrication and use. Electrokinetic techniques, where the biomarkers are dragged by an external electric field, have been successfully used to improve the sensitivity of paper-based analytical systems by preconcentrating the biomarkers at the detection region of the devices. However, the use of electrical components (e.g., batteries, integrated circuits) and the requirement for voltage supply and regulation severely compromise device simplicity and introduce additional challenges around sustainable manufacturing and disposal of the devices, especially in low-resource settings. Our proposed strategy will exploit the spontaneous local electric field generated at the interface between electrolyte solutions to rapidly direct and accumulate the biomarkers at the detection region without using any power supply or auxiliary equipment. Combined experimental and numerical studies will be conducted to gain a quantitative understanding of the mass transport mechanisms governing the proposed preconcentration process. A mathematical modelling-guided approach will be used to design proof-of-principle lateral flow devices that will be feasibility tested and validated for ultrasensitive detection of model analytes and HIV and malaria biomarkers. Validating our novel paradigm for biomarker preconcentration will allow the development of breakthrough rapid diagnostics technologies, through ultrasensitive and yet simple and low-cost lateral flow tests, dipsticks, and microfluidic paper-based analytical devices, for early and affordable diagnosis of chronic and infectious diseases. These innovative, cheap, rapid and highly sensitive diagnostic tools, operated by unskilled users in the community, will support the delivery of the NHS Long Term Plan for a more sustainable diagnosis-led and community-based healthcare. These technologies will also contribute to the global democratisation of diagnostics, which is imperative in delivering health and economic sustainability in the developing world.

Diseases caused by a range of parasites have a devastating impact on the health, welfare and productivity of sheep and cattle. Infections affect milk production, weight gain of young animals and in some cases, can cause acute disease and rapid death. To avoid the devastating consequences of parasitic infection, farmers use medicines to prevent disease. However resistance to many of these medicines is developing, meaning they are becoming less effective on many farms. To try and prevent further development and spread of resistance, through industry advisory bodies such as Control of Parasites Sustainably (COWS) and Sustainable Control of Parasites in Sheep (SCOPS) recommend diagnosing infection before animals are treated. However in many cases we lack diagnostic tests that can provide rapid results to guide treatment. The aim of this project is to develop and deliver pen-side tests, that farmers can use and that provide diagnostic information within 10 minutes. We will build on existing work, in which we have produced a lateral flow test to diagnose infection with the parasite, liver fluke. We will work with farmers, vets and animal health advisors, to ensure that pen-side tests are produced and delivered to meet farmers' needs and empower them to take decisions about treatment, supported by decision support systems. In addition to developing a lateral flow test for liver fluke, we will also work on a similar lateral flow test for bovine lungworm, a devastating and acute disease of calves and increasingly adult milking cows. We will evaluate the use of recombinant proteins as the basis for both lateral flow tests and working with a leading biotechnology we will produce proof of principal lateral flow tests, based on recombinant proteins. The architecture of both tests will be based on our existing, first generation liver fluke test. The project will deliver a better understanding of how to encourage the industry to adopt rapid diagnostic tests as they become available. It will demonstrate how results from those tests can be interpreted in a manner that suits most farmers and finally it will deliver a second generation liver fluke lateral flow test and a lungworm diagnostic test to a point where a commercial partner can consider taking them to full commercialisation. The results from the project will be delivered t project partticipants in the first instance and then rolled out to the industry through KE programmes supported by our project partners.

Up to one third of all foods is spoiled by fungi. This is a major concern for global food security. Spoilage of food by fungi renders the food inedible, and potentially toxic due to formation of substances termed mycotoxins. Fungal spores are abundant in the environment and prolific contaminants, being able to develop and grow in many conditions. Despite a variety of strategies to preserve foods, spoilage microorganisms fight back and some develop resistance to particular preservatives. Soft drinks are typically acidic and these conditions tend to inhibit other organisms like bacteria. However, many species of fungi (especially the moulds) are able to grow in acidic conditions and cause spoilage. A main preservative strategy is to include weak acids like sorbic acid to inhibit growth under these conditions. At permitted levels, sorbic acid inhibits most yeasts and fungi, but a number can still grow and some can degrade the sorbic acid to non-toxic products which alter flavour. Consequently, a chronic level of mould spoilage occurs in the manufacture of soft drinks. Our work has shown that most spoilage fungi produce a small subpopulation of spores that are highly resistant to sorbic acid. Spores produced by other fungi seem to be more uniformly sensitive to the preservative. It is this ability to produce a subset of spores that are hyper-resistant, responsible for fungal growth at higher sorbic acid concentrations, which is the focus of this project. We will apply the latest genetic technologies to single spores to find out what defines these hyper-resistant spores. We will then exploit that information to develop novel methods for tracking these spores in laboratory media and beverages. Finally, we will apply these new tools to find alternative agents that can inhibit the sorbic acid-resistant spores. This approach could offer solutions for reformulating preservatives to give more complete inhibition of spoilage moulds, an area of particular interest to our industrial partners supporting this project. While soft drinks and sorbic acid resistance provide the exemplar for this work, the knowledge generated will help develop strategies for preventing fungal food spoilage more broadly.

We shall develop a rapid, sensitive, cost-effective on-farm diagnostic test capable of detecting the organisms responsible for calf pneumonia to inform herd management and reduce the unnecessary use of antibiotics. Early diagnosis of pneumonia will allow the farmer to administer treatment in a proportionate and timely way. Calf pneumonia is a complex disease caused by a variety of infectious agents. Typically, the clinical disease is caused by certain species of bacteria that may normally cause no adverse effects to the lungs but do so when the animal is compromised in some way, such as a specific viral infection in combination with other stress factors, such as weaning, changes of feed, variation in ambient temperature and humidity. The estimated lifetime economic cost of a case of pneumonia in a dairy heifer is £772, highlighting the potential returns from investing in reducing the impact of this disease. Despite the substantial cost that calf pneumonia causes the UK cattle industry, most farmers prefer to reduce the likelihood of the disease occurring or rely on detecting pneumonia through non-specific means. That is not to say that diagnostic tests are not available, but the high cost of tests and the fact that the results are delayed and therefore cannot inform treatment decisions are likely reasons why these are not used routinely. We intend to understand the impediments to livestock diagnostic test use in more detail in this project through engagement with farmers, vets, and calf rearers. Recent developments in rapid molecular diagnostics (many by the project team) offer the possibility of delivering a test that is considerably cheaper, quicker and more sensitive than existing commercial tests, opening up the opportunity to use such a test for routine surveillance on-farm. This would enable early intervention and the development of specific treatment protocols, thus reducing antimicrobial resistance and improving calf welfare. Our project sits at the intersections between policy change, economic opportunity, changing practice, public perception of the industry, and even antibiotic stewardship. We are a new and focused partnership that combines working knowledge of dairy farming, expertise in veterinary infectious disease, diagnostic test development and stakeholder engagement methods. The test will be based on simultaneous detection of any of the six most common infections associated with calf pneumonia using a simple swab of the nasal passages, not unlike the current lateral flow tests for COVID-19. The swab will be placed in a novel device developed by the project partners that gives a simple final readout of the test result using a lateral flow device. Proof of principle has been demonstrated for the technology we intend to adopt for the detection of at least some of the pathogens associated with calf pneumonia, and three of the partners have experience of the development of rapid tests using this technology for pathogens of veterinary importance, and respiratory infectious disease testing in humans (incl. COVID-19). However, developing a test is only half the story, and we must ensure the test is co-developed with those who will ultimately use it. One of the partners is a dairy farmer, another a livestock veterinarian, and two others have worked with calf rearers for the last five years on pneumonia management and antibiotic resistance. Using methods, such as interviews/workshops with calf rearers and vets, we shall ask questions such as, how do you currently control pneumonia, how quick does the test need to be from start to result, how much should it cost, which sales channel is most attractive, and what will you do differently in response to the test result? This will be vital information to ensure the test we develop has the best chance of being used on farm to the benefit of animal health and welfare, the GB cattle industry, and beyond.