Development of a phage-based molecular assay to detect human Mycobacterium tuberculosis infection in blood

Supervisors:

• Dr Pranabashis Haldar
• Professor Galina Mukamolova

Project description:

Tuberculosis (TB) remains a serious threat to global health and is the leading cause of death from an infectious disease. In 2019, more than 10 million people fell ill with TB and 1.3 million died. Most cases of TB arise following a period of latent infection. It is estimated that one quarter of the entire global population is latently infected with Mycobacterium tuberculosis (Mtb), representing an enormous potential reservoir of future disease.

Efforts to eradicate TB focus on early diagnosis and treatment of TB to prevent onward transmission of the infection. In parallel, identifying and treating people with high risk latent infection can help prevent TB. To be effective, new tools are needed to support TB diagnosis and risk stratification of latent infection. Although TB primarily affects the lungs, recent studies in animals, including non-human primates suggest some dissemination of Mtb infection into the blood stream is common prior to containment of Mtb within granulomas. Mtb is usually transported in blood intracellularly, within circulating peripheral blood mononuclear cells. These observations invite the possibility for developing a blood based TB diagnostic. A blood test to diagnose TB would help overcome the limitations of existing diagnostic tools which require sampling of tissue and fluid from the site of infection. For lung disease, this usually requires a sample of sputum to be provided, however a significant proportion of people cannot provide a sample which necessitates the use of invasive procedures and delays diagnosis. 

This exciting, state-of-the-art project will explore the application of a recently developed mycobacteriophage-based assay (Actiphage® assay) that utilises a mycobacteriophage (D29) to infect viable and metabolically active mycobacteria located within peripheral blood mononuclear cells. The mycobacterial DNA released after phage infection can be detected with PCR. The assay has yielded promising results in a recent clinical pilot study of patients with TB at Leicester., identifying Mtb in the blood of a high proportion of people with lung TB and also some people with latent infection that were at higher risk of developing TB. Although promising, our data indicate that the assay is currently suboptimal and will require further development to improve reliability and standardisation. The assay is also limited by the requirement for fresh blood for testing. Ideally, the ability to test blood samples which have been stored will greatly enhance the scope of the assay.

The project will focus on establishing protocols for effectively using this assay on cryopreserved samples and improving detection of Mtb from fresh patient samples. Using our knowledge and experience of Mtb resuscitation, we aim to develop experimental protocols to improve resuscitation of mycobacteria from infected cell lines and clinical samples. 

The project will also address fundamental aspects of TB and Mtb persistence in latent TB and improve our understanding of host-microbial interactions that underpin clinical Mtb infection phenotypes.

References:

1. Swift BMC, Meade N, Barron ES, Bennett M, Perehenic T, Hughes V, Stevenson K, Rees CED (2020). The development and use of Actiphage® to detect viable mycobacteria from bovine tuberculosis and Johne's disease-infected animals. Microb Biotechnol;13(3):738-746. doi: 10.1111/1751-7915.13518.

2. Verma R, Swift BMC, Handley-Hartill W, Lee JK, Woltmann G, Rees CED, Haldar P (2020). A novel, high-sensitivity, bacteriophage-based assay identifies low-level Mycobacterium tuberculosis bacteremia in immunocompetent patients with active and incipient tuberculosis. Clin Infect Dis. 70(5):933-936. doi: 10.1093/cid/ciz548.

3. Rosser A, Stover C, Pareek M, Mukamolova GV. (2017) Resuscitation-promoting factors are important determinants of the pathophysiology in Mycobacterium tuberculosis infection. Crit Rev Microbiol 17:1-10.