Affordable near-patient diagnostics to distinguish infectious diseases in the Philippines (AND2ID in Ph)
Lead Research Organisation:
University of Cambridge
Department Name: Chemical Engineering and Biotechnology
Abstract
Acute fever may be caused by a range of pathogens, but clinical symptoms may be too non-specific to differentiate the causative organism, so correct diagnosis requires pathogen-specific diagnostic tests. These diagnostics are too expensive for routine use in resource-limited settings. This means that many patients are treated empirically with broad spectrum antibiotics, which may be unecessary, toxic, and increase the risk of antimicrobial resistance. Conversely delayed diagnosis and treatment may lead to poor outcomes.
A barrier to low-cost diagnostics in the Philippines, arises from a value chain that spans the world, without Purchasing Power Parity (PPP). If we could use technologies that can be manufactured locally, using local resources, then we have the first step to providing affordable diagnostics in resource poor areas, and delivering a sustained improvement in healthcare, while also developing the local economy.
We have made an enzyme (BOON-enzyme) that can do these tests and can be produced almost anywhere without special facilities. We have made the enzyme pink, so that you can see that it has been produced and we have a way of making it stick to sand so that it is very stable. We are going to use one of these enzymes - BOON-Taq in polymerase chain reaction (PCR) - to perform a clinical study in patients with suspected dengue and leptospirosis at the University of Santo Tomas in Manila, to assess the impact of the use of diagnostics on the patient pathway and the disease burden. As a result of these trials we will design a diagnostic kit that can be taken out to rural clinics and we will undertake clinical trials in such a clinic. We will support the study by developing a healthcare economics model of the impact of diagnostics on the patient pathway.
A barrier to low-cost diagnostics in the Philippines, arises from a value chain that spans the world, without Purchasing Power Parity (PPP). If we could use technologies that can be manufactured locally, using local resources, then we have the first step to providing affordable diagnostics in resource poor areas, and delivering a sustained improvement in healthcare, while also developing the local economy.
We have made an enzyme (BOON-enzyme) that can do these tests and can be produced almost anywhere without special facilities. We have made the enzyme pink, so that you can see that it has been produced and we have a way of making it stick to sand so that it is very stable. We are going to use one of these enzymes - BOON-Taq in polymerase chain reaction (PCR) - to perform a clinical study in patients with suspected dengue and leptospirosis at the University of Santo Tomas in Manila, to assess the impact of the use of diagnostics on the patient pathway and the disease burden. As a result of these trials we will design a diagnostic kit that can be taken out to rural clinics and we will undertake clinical trials in such a clinic. We will support the study by developing a healthcare economics model of the impact of diagnostics on the patient pathway.
Technical Summary
Infectious diseases are a major cause of morbidity and mortality in the Philippines. Patients may present with fever and a wide range of non-specific symptoms, which are difficult to diagnose without specialist laboratory tests. Dengue is a major public health problem in the Philippines, and is endemic in all regions of the country. Leptospirosis is also endemic and may be misdiagnosed as dengue due to similarity in early clinical manifestations. Current diagnostic tests that rely on culture of pathogens or detection of raised antibody titres can take days to weeks to provide actionable results, and are too slow to guide initial patient management. In the first week of infection, nucleic acid testing (NAT) is the most reliable method to identify the causative agent, but these are expensive and usually require well-resourced laboratories.
The aim of this project is to create innovative low cost diagnostics, using local resources and show whether reduction in cost of a diagnostic has an impact on the ability to target treatment (including antibiotics) and lessen the impact of febrile illness on the patient, society and the economy.
The project has three steams: (1) Technological Innovation, (2) Clinical study, (3) Economic modelling and will result in
- Laboratory evaluation of a new NAT reagent in UK and Philippines
- Clinical trial of of the new test in Philippines with impact analysis of the clinical pathway.
- Development of an affordable diagnostic NAT platform design with prototype production for trials in a rural clinical site in Philippines for dengue and leptospirosis.
- Cost-effectiveness analysis and healthcare economics model of diagnostic platforms.
- Impact analysis of diagnostics on patient pathway.
The aim of this project is to create innovative low cost diagnostics, using local resources and show whether reduction in cost of a diagnostic has an impact on the ability to target treatment (including antibiotics) and lessen the impact of febrile illness on the patient, society and the economy.
The project has three steams: (1) Technological Innovation, (2) Clinical study, (3) Economic modelling and will result in
- Laboratory evaluation of a new NAT reagent in UK and Philippines
- Clinical trial of of the new test in Philippines with impact analysis of the clinical pathway.
- Development of an affordable diagnostic NAT platform design with prototype production for trials in a rural clinical site in Philippines for dengue and leptospirosis.
- Cost-effectiveness analysis and healthcare economics model of diagnostic platforms.
- Impact analysis of diagnostics on patient pathway.
Planned Impact
Who will potentially benefit from this research?
Infectious diseases are a major cause of morbidity and mortality in the Philippines. The diagnosis of the causative agent is often limited by the lack of availability and/or the high cost of laboratory diagnostic tests. Patients are therefore at risk of being over-treated with unnecessary antibiotics (which can drive antimicrobial resistance) or under-treated (resulting in poor clinical outcome. The development of a high-tech low-cost diagnostic that is able to diagnose infections such as dengue and leptospirosis would represent a major advance in the field and may be generalizable to other infections / countries.
The following groups of people are likely to benefit from this research:
- Study participants / patients
- Medical practitioners
- Medical practitioners
- Study investigators
- The wider scientific community
- Public health officials and policy makers
- The commercial sector
- The wider health economy
How will we maximize the impact of this research?
We will seek to actively identify and engage all of the relevant stakeholders at various stages of the research project i.e. during study preparation and set-up, during the course of the research studies, and when we present and disseminate the research findings.
This project will establish new collaborations between a multi-disciplinary group of researchers at leading UK and Philippine institutions. The investigators involved in the research will acquire new knowledge and expertise in development, manufacture, evaluation of a new high-tech low-cost diagnostic and its performance in a low-income setting
The research will result in capacity building through training of Philippine scientists, and enable the translation of a cutting-edge technology into a developing country setting, to enable local investigation and management of infectious diseases. We will organize / provide training to the study investigators, drawing on the existing expertise and infrastructure available in Cambridge Public Health England and the University of Santo Tomas. This will occur via training workshops and personal supervision of research assistants / clinical researchers.
The results of the studies will be presented at national and international meetings and published in peer-reviewed journals. It will therefore benefit the scientific community both in the Philippines and beyond. We will also disseminate the findings via institutional press releases / websites, standard media outlets (websites, newspaper, television and radio interviews) and social media (e.g. Twitter).
The information gained from these studies will be used to infom local policies for the diagnosis and management of infectious diseases during the course of the project. We will engage with local clinicians and policy makers to develop appropriate policies and procedures for use in hospital and rural settings. These may also be applicable to other low- or middle-income countries that have similarly high rates of dengue and leptospirosis.
In terms of beneficiaries within the commercial private sector these are likely to include companies that currently manufacture commercially available rapid diagnostic tests. The challenge will be for them to reduce the costs of consumables and manufacture so that these platforms can be used in low- and middle-income settings.
Finally, the research training, laboratory skills and data analysis skills obtained during the project are potentially transferable to other sectors.
Infectious diseases are a major cause of morbidity and mortality in the Philippines. The diagnosis of the causative agent is often limited by the lack of availability and/or the high cost of laboratory diagnostic tests. Patients are therefore at risk of being over-treated with unnecessary antibiotics (which can drive antimicrobial resistance) or under-treated (resulting in poor clinical outcome. The development of a high-tech low-cost diagnostic that is able to diagnose infections such as dengue and leptospirosis would represent a major advance in the field and may be generalizable to other infections / countries.
The following groups of people are likely to benefit from this research:
- Study participants / patients
- Medical practitioners
- Medical practitioners
- Study investigators
- The wider scientific community
- Public health officials and policy makers
- The commercial sector
- The wider health economy
How will we maximize the impact of this research?
We will seek to actively identify and engage all of the relevant stakeholders at various stages of the research project i.e. during study preparation and set-up, during the course of the research studies, and when we present and disseminate the research findings.
This project will establish new collaborations between a multi-disciplinary group of researchers at leading UK and Philippine institutions. The investigators involved in the research will acquire new knowledge and expertise in development, manufacture, evaluation of a new high-tech low-cost diagnostic and its performance in a low-income setting
The research will result in capacity building through training of Philippine scientists, and enable the translation of a cutting-edge technology into a developing country setting, to enable local investigation and management of infectious diseases. We will organize / provide training to the study investigators, drawing on the existing expertise and infrastructure available in Cambridge Public Health England and the University of Santo Tomas. This will occur via training workshops and personal supervision of research assistants / clinical researchers.
The results of the studies will be presented at national and international meetings and published in peer-reviewed journals. It will therefore benefit the scientific community both in the Philippines and beyond. We will also disseminate the findings via institutional press releases / websites, standard media outlets (websites, newspaper, television and radio interviews) and social media (e.g. Twitter).
The information gained from these studies will be used to infom local policies for the diagnosis and management of infectious diseases during the course of the project. We will engage with local clinicians and policy makers to develop appropriate policies and procedures for use in hospital and rural settings. These may also be applicable to other low- or middle-income countries that have similarly high rates of dengue and leptospirosis.
In terms of beneficiaries within the commercial private sector these are likely to include companies that currently manufacture commercially available rapid diagnostic tests. The challenge will be for them to reduce the costs of consumables and manufacture so that these platforms can be used in low- and middle-income settings.
Finally, the research training, laboratory skills and data analysis skills obtained during the project are potentially transferable to other sectors.
Publications
Henderson CJ
(2019)
Gene to diagnostic: Self immobilizing protein for silica microparticle biosensor, modelled with sarcosine oxidase.
in Biomaterials
Henderson C
(2021)
Design and model for 'falling particle' biosensors
in Sensors and Actuators B: Chemical
Wong JJY
(2022)
Electrochemically induced in vitro focal hypoxia in human neurons.
in Frontiers in cell and developmental biology
Bird AR
(2023)
Biocatalytic synthesis of 2'-deoxynucleotide 5'-triphosphates from bacterial genomic DNA: Proof of principle.
in Biotechnology and bioengineering
| Description | A key finding is that production of the biological reagents for nucleic acid testing can be (and has been) set up de novo in the Philippines using the technology developed by the Cambridge team, and be used for nucleic acid testing. However, considerable training is required in laboratory procedure to develop quality outcomes. As a result of the work done, a business plan is being developed as part of the project for start up of a local biotech business to produce the material required at much lower cost, compared with imported materials. The model that will emerge will become a template for adaption in other ODA countries. We know that acute fever does not distinguish the disease. In the absence of diagnostics, doctors adopt a 'likelihood' model on the basis of what is expected in the environment. This leads to misdiagnosis and incorrect treatment and spillover events that are likely to continue undetected without suitable diagnostics. Affordability is one of the prime barriers to access to diagnostics. In the Philippines for example, a PCR test costs about the same as in the UK, yet the base salary is 10% of the UK. It isn't enough to set up diagnostics facilities in low income countries, if the materials are still coming from high income countries. 60 to 90% of the cost of these diagnostics can be related to the cost of the biological reagent, so the impact of local production of these materials may become significant. There is also a synergy with the review of NHS diagnostics capacity (NHS Long Term Plan) commissioned from Professor Sir Mike Richards: "Diagnostics: Recovery and Renewal", which recommends "the need for a new diagnostics model, where more facilities are created in free standing locations away from main hospital sites, including on the high street and in retail locations, providing quicker and easier access to a range of tests on the same day, supporting earlier diagnosis, greater convenience to patients and the drive to reduce health inequalities." |
| Exploitation Route | Enabling diagnostics to be produced and used locally improves access and affordability in low income countries. It potentially has a socioeconomic impact is terms of job creation, healthcare and education, through the model of distributed local manufacturing with high community involvement. |
| Sectors | Creative Economy Healthcare Manufacturing including Industrial Biotechology |
| Description | 1. Societal and economic impact on DAC countries. WHO proposed that neglected tropical diseases (NTDs) act as a tracer of inequality in access to health systems. Data collected from different socio-economic backgrounds suggest that eradication of infectious causes of death in rural low income countries would increase in life expectancy (6.8 years at age 15). The technology from this project is still in early days and is being prepared for publication and further development. However, in addition to the Philippines it has had particular impact in Ghana, where we have forged long term collaborations and have gained more traction in going forward. Even in well supported laboratories, equipped by donor and NGO investment, instrumentation sits idle once reagent costs go beyond annual budget. In Ghana the technology is beginning to be used to enable testing to be performed beyond the testing budget. 2. The work undertaken has left a legacy that is enabling Increasing testing that could greatly improve management of the spread of (re)emerging infectious disease in the future, as well as easing pressure on clinical health facilities. Addressing affordability and availability is crucial to meeting Sustainable Development Goal 3 (SDG3, "Good health and well-being"). Furthermore, establishing local production of diagnostic technologies would be a significant boost to the industrial sector (SDG9, "Industry, innovation and infrastructure"), and create skilled jobs (SDG8, "Decent work and economic growth"). This is well aligned with national development strategies, eg. Ghana Beyond Aid which identifies increased manufacturing capability as key to economic development. 3. Gender inequalities in allocation of resources, eg income, education, healthcare, nutrition and political voice, are strongly associated with morbidity and mortality in ODA countries and tend to worsen adverse effects on women of low socio-economic status. In certain circumstances, women are required to seek their husband's permission before accessing treatment or seeking interventions. Reduction of maternal mortality is a Ghanaian Government priority: Community-based health planning and services (CHPS) have regional and rural facilities, and a step to inclusion and cultural change comes from the engagement with Community-Based Health Volunteers (CBHVs). Our programme is contributing to the impact on these traditional equality barriers, and give voice to women in designing maternal IVD facilities, disrupting the differential access to and control over health resources, both within and outside families that are influenced by cultural gendered norms. One area of testing where we have already done work in Ghana is malaria. Malaria is recorded as the highest cause of death (53.6%) among pregnant women in Ghana, followed by HEV (13.1%). Malaria also accounts for ~3-15% of anaemia and 25% of severe anaemia in pregnant women from malaria-endemic areas. It contributes to maternal death, still births, low birth weights, and impairment of foetal development. Co-infection can contribute to an increased likelihood of dying during pregnancy. HEV is also an indicated risk factor with poor maternal and foetal outcomes. It is often asymptomatic and mild in men and non-pregnant women, but has a high fatality rate (40%) among pregnant women. HEV is commonly acquired through consumption of faecally contaminated drinking water. |
| First Year Of Impact | 2023 |
| Sector | Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Societal |
| Title | Research data supporting: "Biocatalytic Synthesis of 2'-Deoxynucleotide 5'-Triphosphates from Bacterial Genomic DNA: Proof of Principle" |
| Description | Zip folder containing the raw data and analysis to support the discovery of a novel synthesis pathway for 2'-deoxynucleotide 5'-triphosphates. The .zip file contains the following subfolders. (1) DNA-gels: 15 image files (.tif) of agarose gel electrophoresis of extracted E. coli genomic DNA and polymerase chain reaction (PCR) results. The README file contains detailed descriptions of the samples in each gel image. (2) Enzyme-kinetics: Microsoft excel files containing the raw absorbance measurements at 340 nm output by TECAN plate reader. The activity of nucleotide kinase enzymes was characterized using the double coupled pyruvate kinase-lactate dehydrogenase assay described by Agarwal et al (1978). This assay measures the decrease in absorbance at 340 nm as NADH is converted to NAD+ at a 1:1 molar ratio as the product of the enzyme reaction is generated. Each experiment contains a well plate layout file, a NADH standard curve, and the analyzed reaction velocity files. The initial velocity (mol product/s) is determined by the linear region of the product vs. time curve. Product concentration is determined from the A340 nm standard curve. To determine the Michaelis-Menten parameters, initial velocity was plotted against substrate concentration and fit with a nonlinear regression, generating the KM (uM) and Vmax (umol product/s) values. The specific activity is determined by normalizing the Vmax against the mass of enzyme. Detailed calculations are included in enzyme-kinetics-files.xlsx. (3) HPLC: High pressure liquid chromatography data for commercial nucleotide standards and nucleotides produced via the synthesis pathway proposed in this work. The dataset includes two types of .csv files output by Agilent ChemStation software. Signal files include time (s) and the DAD signal at 259 nm. Integration files contain analysis of the peak signal as calculated by the ChemStation algorithm. See the README file for a detailed description of columns in the integration file. The peak integration of commercial standards at varying concentration was used to generate a standard curve. The signature elution time of commercial standards was used to determine molecular identity of nucleotides in the in-house produced samples. See the Files.xlsx sheet for detailed calculations and sample descriptions. (4) PicoGreen: Microsoft excel files containing the raw fluorescence readings from PicoGreen assays of PCR reactions. Lambda genome DNA diluted in 1X Tris-EDTA buffer was used to generate a standard curve (concentrations in ng/mL). (5) Prism-data-analysis: GraphPad Prism files containing the nonlinear regression analysis of enzyme kinetics (.pzfx). The results of the analysis are also included in enzyme-kinetics-files.xlsx. (6) Protein-gels: sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) images for visualization of recombinantly expressed proteins. The README file contains detailed descriptions of the samples in each gel image. Four (.tif) files, one (.jpg) file, and one Microsoft excel file with image quantification data from analysis in ImageJ software. See the main manuscript for more details. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2023 |
| Provided To Others? | Yes |
| URL | https://www.repository.cam.ac.uk/handle/1810/347866 |
| Description | USTH |
| Organisation | University of Santo Tomas Hospital |
| Country | Philippines |
| Sector | Hospitals |
| PI Contribution | Contribution to nucleic acid testing in clinical trial |
| Collaborator Contribution | Clinical trial of patients recruited with febrile disease. |
| Impact | Data on incidence of febrile disease in the Philippines and identity of the disease. |
| Start Year | 2020 |
| Title | NAD |
| Description | A nucleic acid amplification test has been developed that can be produced in low income countries at low cost. The production process has been tested in the Philippines and Malaysia and Ghana. A clinical trial is being started in the Philippines. |
| Type | Diagnostic Tool - Non-Imaging |
| Current Stage Of Development | Initial development |
| Year Development Stage Completed | 2020 |
| Development Status | Under active development/distribution |
| Impact | none yet |
| Description | Schools workshop: Low cost diagnostics in low resource areas |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Schools |
| Results and Impact | 2 workshops were delivered in Manila to pupils from 6 schools to explain how molecular biology can be used to produce reagents for nucleic acid diagnostics. The students learned about for the nucleic acid testing works, what the components of the test do and how to detect and interpret the outcome. These were hands on workshops and the pupils made nucleic acid tests and then used them to amplify DNA. They loaded electrophoresis gels and determined for the pattern of bands what the disease was. |
| Year(s) Of Engagement Activity | 2023 |
| Description | Virtual laboratory training to transfer polymerase production protocol and scale-up to collaborators and train members of laboratories in LMIC's |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Study participants or study members |
| Results and Impact | As a result of the Covid Pandemic, we were not able to travel to the Philippines to train our collaborators in the fundamentals of molecular biology and enzyme production. We have therefore created 4 hours of training through a virtual laboratory. The laboratory has been delivered in 3 parts as a pre-recorded video (with manual) and live contextual presentation and opportunity for questions. As a result of this training, our collaborators in the Philippines have been able to set up and produce their own BST polymerases de novo. This enzyme is now being used in isothermal nucleic acid testing for the project. Leading from the first virtual presentation, we have received further invitations to offer the virtual lab training for in house DNA polymerase production. For example, at the Nanotechnology Research and Innovation Bootcamp 2021, arranged by the African MRS in collaboration with the UN Economic Commission for Africa. This has led to further discussions with Samuel Chignome (BITRI, Botswana) and Victor Konde (UNECA) over establishing centres across Africa where the virtual training can be turned into in person training and capability can be built. We have also delivered the virtual lab in other Asian countries and are scheduled to deliver it for UKM Malaysia in April 2022. |
| Year(s) Of Engagement Activity | 2021,2022 |
| Description | Workshop/ Pop-up Laboratory: From Gene to Diagnostics. International Conference on Chemistry Chemistry, lnnovations for World Needs |
| 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 | Participants attended the "pop-up" laboratory to learn about the production of enzymes for nucleic acid amplification assays. The activities included plasmid design. Low cost production of enzyme. Isolation and downstream strategies. Use in nucleic acid amplification. Detection and identification of different infectious diseases. The abstract is below: This workshop looks at the production of lower cost enzymes for in vitro diagnostic (IVD) tests or for research with minimum resources. We call these enzymes the "Boon-Enzymes". The Boon-Enzymes are heterogenous recombinant proteins that have been designed to immobilise onto silica (component: R5 silaffin-tag) or paper (cellulose-component: cellulose binding domain (CBD)) or other material and provide a visual indication of protein expression and allow for protein tracking (component: mCherry). They also contain a functional protein component. For example, in a nucleic acid amplification test, an enzyme is required to amplify targeted DNA/RNA (component: BST 2.0 DNA polymerase, reverse transcriptase X), or for immunoassay, a protein to specifically bind to antigen targets is needed (component: single-chain variable fragment (scFv)) or in other IVDs the active enzyme reagent needs to be provided, that is able to target the required analyte as a substrate (eg component:sarcosine oxidase). The workshop will introduce the principles of protein engineering and protein expression. We will discuss the encoding of the genetic information for the required protein into an in vitro expression vector and then the induction of the RNA synthesis for protein expression in E. coli. We will take the participants through the production of the enzyme, tracking the development of the pink protein in the expression system. Using the R5 silaffin tag as an example, we will give the participants the opportunity to collect the protein from the cell culture. The affinity between the R5 silaffin-tag and the silica is electrostatic. Under the slightly basic conditions that the user will be operating in (pH 7.2) the silica is negatively charged, whereas, the silaffin-tag is positively charged due to the presence of both lysine and arginine amino acids (sequence: SSKKSGSYSGSKGSKRRIL). The binding of the protein to the silica can be followed as the silica turns pink. Finally, the "pink silica enzyme" will be used in a diagnostic test and the outcome of the reaction measured visually. |
| Year(s) Of Engagement Activity | 2023 |