Nanoparticle based rapid diagnostics for TB disease

Tuberculosis (TB) caused by the Mycobacterium tuberculosis (M. tb) bacterium is one the three major causes of infectious disease deaths worldwide. Approximately one quarter of the global population is infected, with annually 10 million new cases and 1.4 million deaths. While TB is treatable with suitable antibiotic combinations, disease symptoms are variable and easily confused with other conditions, meaning that individuals with active disease and capable of infecting others are not always quickly identified, allowing continued transmission. Fast, accurate identification of these patients is key to achieving the prompt antibiotic administration necessary to prevent infected individuals spreading disease. The World Health Organisation (WHO) has identified new diagnostic technologies to detect TB disease as one research focus for their End TB Strategy designed to reduce TB incidence by 80 % by 2030. This proposal aims to develop and evaluate tools to capture and detect M. tb in samples from patients with possible TB disease: specifically sputum coughed up by infected adults. The long term goal is to create a rapid diagnostic method capable of identifying TB disease and that is able to be used in resource-poor settings (i.e. the low and middle-income countries where the TB disease burden is greatest and medical infrastructure is limited).
Our method combines three complementary technologies to capture and identify M. tb: 1. generation of specific sugar molecules able to bind selectively and tightly to the M. tb cell; and their attachment to 2. magnetic and 3. fluorescent particles. This will create two types of material able to bind M. tb - a magnetic particle, making the bacterium magnetic and enabling its capture in a magnetic field; and a fluorescent particle making the bacterium fluorescent and able to be detected using a microscope. Combining these particles will enable us to capture, concentrate and visualise M. tb from patient samples. Our experimental approach divides into three workpackages (WPs): WP1 focuses on using a range of innovative approaches to synthesise sugars able to bind M. tb; WP2 focuses on attaching these sugars to magnetic and fluorescent particles and investigating the physical properties (size, shape, magnetic and optical characteristics) of the resulting materials; and WP3 on evaluating the binding of these materials to bacteria. We will first test binding to the weakened BCG (vaccine) strain of M. tb in laboratory media mimicking the composition of sputum, comparing this with binding to other bacteria that may be present in these samples to establish their selectivity for M. tb over other species. We will extend these experiments to study M. tb strains isolated from patients. Finally we will evaluate our method on samples from patients presenting with possible TB infection, comparing our results with parallel tests carried out on the same samples using traditional methods (growth in culture, sputum microscopy or the GeneXpert system that detects M. tb genetic material). The results will determine whether this new approach can detect bacteria in the quantities present in patient samples and is able to discriminate between M. tb and other bacteria.
Workpackages 1 and 2 will take place at the University of Bristol (UoB), workpackage 3 at UoB and the Kenya Medical Research Institute (KEMRI) where all experiments with patient-derived bacteria and samples will take place. Kenya is a WHO High Burden TB country; KEMRI has the expertise and infrastructure necessary to evaluate TB detection methods. Our proposal combines novel science (development of new materials that selectively bind M. tb) with support for posts, infrastructure and bidirectional exchange visits that will develop research capacity in Kenya. The diversity of our research team and the strong links between KEMRI, healthcare practitioners and policymakers will ensure delivery of impact in both the research community and potential end users.

We aim to develop sensitive, specific detection of tuberculosis (TB) disease based upon glycan-functionalisation of two types of nanoparticles: superparamagnetic iron oxide nanoparticles (SPIONs) and fluorescent carbon dots (FCDs). In combination these will capture and label Mycobacterium tuberculosis (M. tb) bacteria from solubilized sputum samples, providing the basis for a rapid, robust tool for point-of-care TB detection in resource-limited settings.

Potential beneficiaries include UoB and KEMRI researchers, the wider academic community researching M. tb biology and TB detection, and researchers in a broader range of fields interested in methodologies for glycan and nanoparticle chemistry. In the longer term we aim for public health and societal impact in Kenya and adjoining countries through improved early diagnosis of TB disease, so reducing the TB burden in these and other low and middle-income countries.

Local impact will arise from support for TB research at KEMRI, building capacity by funding an early career scientist who will benefit from training and networking opportunities during their visit to UoB, and the opportunity to work in a multidisciplinary environment with facilities unavailable at KEMRI. Wider impact across research communities will accrue from our application of new synthetic methodologies to generate novel glycans and glycan-functionalised nanoparticles (FCDs, SPIONs) and our evaluation of binding of these materials to M. tb and other bacteria. These findings will impact understanding of M. tb - glycan interactions, with implications extending through capture and detection to identification of candidate vaccine targets. In addition, methodological advances that improve and streamline glycan synthesis will find wider applications in carbohydrate chemistry and glycobiology. Success of this project will justify funding applications to support both larger scale evaluation of the methodology and its development as a point-of-care diagnostic device for resource-limited settings, requiring further development of TB research capacity at KEMRI.

Public health benefits will accrue from application of a technology to reliably identify M. tb infection in patients presenting with early signs of disease. Early, accurate identification reduces the TB burden by effectively targeting treatment, maximizing use of resources (medicines), reducing the healthcare burden and interrupting the chain of transmission by reducing numbers of infectious individuals with active but untreated disease. Existing point-of-care diagnostics (e.g. GeneXpert) are limited by cost and infrastructure dependence; a low cost point-of-care diagnostic requiring no substantial infrastructure would particularly improve TB treatment in isolated communities where healthcare access is most limited.

In addition to the burden upon healthcare provision, TB disease exerts substantial societal and economic impacts whose costs may be greater than those directly associated with treatment. TB patients and their families/carers lose income (3 - 4 months work; 20 - 30 % annual household income) through inability to work; in case of premature death approximately 15 years income is lost. Where women undertake most household tasks additional impacts accrue from loss of activities (eg cooking, childcare) that support other family members. Patients may also suffer social rejection, loss of employment and other forms of discrimination leading to reduced quality of life and depression and exacerbating indirect disease impact. Effective diagnostics enabling early targeting of TB treatment will reduce all of these economic and societal.

Lastly, public engagement activities, in concert with those of eg BristolAMR and the Bristol Institute for Biodesign, will also inform the wider public, especially the next generation of researchers, on the global importance of TB and wider issues around infectious disease and development in a global public health context.