Mycobacteria are very difficult to culture and can take weeks to grow. The aim of this project is to is to use novel patented bacteriophage technology to rapidly detect and diagnose infections caused by mycobacteria, such as Tuberculosis and Johne’s disease.

Challenge

Mycobacterial pathogens are responsible for a range of diseases in both humans and animals. TB in humans is primarily caused by M. tuberculosis and TB in cattle is primarily caused by M. bovis. Both organisms are members of the Mycobacterium tuberculosis complex group of organisms. Culture of these organisms is generally seen as the gold standard diagnostic, however both M. tuberculosis and M. bovis are slow growing mycobacteria taking up to 12 weeks to form colonies on solid media. Slow growth and low sensitivity makes the use of culture as a diagnostic for TB infections both impractical and inefficient and hunting, locomotor strategies in response to limited resources (water, food).

Many of the current diagnostics for TB infections are immunologically-based, where the host response to infection is used to diagnose infection. A major problem with this approach is that mycobacteria are generally characterised by their ability to avoid their host’s immune system, which can result in inconsistent detection of infected individuals, particularly where the pathogen is effectively evading immunity. In other words, the methods may fail to detect infection due to the pathogen’s success against the host. Molecular methods, such as PCR exist to detect mycobacterial pathogens to overcome the reliance on immune response. However, the widely used PCR assays are expensive and tend not to have the required sensitivity to detect TB in a range of matrices due to the inefficient lysis of mycobacteria as well as potential inhibitors that are often found in samples being tested.

Solution

The phage-assay is a patented tool developed by Dr Ben Swift and Dr Cath Rees at the University of Nottingham, for the detection of mycobacteria from blood in a range of species including cattle and humans. This invention uses bacteriophage (viruses that infect bacteria) as a lysis reagent to release DNA from viable mycobacteria for detection by any other nucleic acid amplification method. The phage have evolved enzymes to lyse mycobacteria efficiently at the end of their replication cycle, so that release of genomic DNA from the infected mycobacteria is ensured. This lysis event is efficient and robust compared to standard mechanical or chemical lysis methods for mycobacteria. Phage replication and lysis will only occur in actively growing cells, so that the detection event only measures viable cells. We have demonstrated that phage assay can be used in combination with different PCR assays to detect with high degree of specificity different mycobacteria, including M. bovis and M. tuberculosis from clinical blood samples demonstrating an extremely low limit of detection and greater sensitivity than culture. The phage-assay is developed as TB diagnostic and has the potential for much wider applications in studying fundamental aspects if infection. The phage-assay combines the strengths of culture, the sensitivity of phage lysis and the specificity of PCR, providing a powerful tool to detect TB.

Impact

 Our research has a wide ranging impact in both the animal and human world.

In animals bovine TB affects not only cattle, but farmers, vets and policy makers. The social and economic impact of bovine TB is huge. Other mycobacterial diseases in animals such as Johne’s disease cause economic hardship for millions of farmers as well as negatively impacting animal health. With all these diseases, novel, accurate diagnostics are needed.

In humans, 10 million people very year catch TB and around 1.4 million people die, including 240,000 children. It is estimated that 3 million people have TB but have not been diagnosed. The World Health Organisation as has said that timely accurate diagnosis is the cornerstone for eradicating TB and here we hope to develop the tools to help achieve this goal.

Partners

This project is funded by a Bloomsbury Studentship

Publications/Presentations

For full list of publications visit: https://scholar.google.co.uk/citations?user=Ywkl5LAAAAAJ&hl=en