We have previously focused on the importance of diagnostic collaboration in combating TB. This time, PBD Biotech’s Ben Swift explains the impact of his company’s work with Leicester University in developing phage-based diagnostics in an era of growing antimicrobial resistance.
As the threat of Covid-19 declines, tuberculosis (TB) has once again become the world’s most deadly infectious disease, killing 1.6 million people in 2021. Now a new type of diagnostic test based on bacteriophages is offering the potential to revolutionise the detection and treatment of this disease.
Detection of Mycobacterium tuberculosis (Mtb), the pathogen that causes TB, is problematic. Culture of the bacterium is the gold standard diagnosis; however Mtb is extremely slow-growing, taking up to eight weeks to culture from sputum. Mycobacteria are also characterised by an unusually thick cell wall, making it difficult to extract DNA for PCR.
Additionally, Mtb has evolved to avoid the host’s immune system and can remain dormant for many years (termed latent TB infection, or LTBI), only progressing to full disease when the body is immunocompromised or weakened through stress or malnutrition. It is estimated that a quarter of the world’s population has LTB and show no symptoms of the disease.
This study takes us closer to a sensitive and specific molecular diagnostic for tuberculosis disease progression that allows the rapid detection, control and early treatment of this fatal disease
Treatment of TB is also challenging, requiring months of multiple and combined antibiotics, and growing anti-microbial resistance (AMR) is a major concern.
The lack of effective diagnostics has been highlighted by the World Health Organisation (WHO) [see Laboratory News, Issue 3, 2023].
Research to improve therapies and to gain early diagnosis of disease progression is at the heart of its End TB campaign.
Potential of bacteriophages
The therapeutic use of bacteriophages (viruses that infect bacteria) has been known for many decades. They are used in Eastern European medicine for the treatment of common bacterial infections, but there is limited knowledge of their clinical use for mycobacteria.
The potential of phages for treatment and diagnosis has recently been demonstrated, and the increasing interest was evidenced by the launch of the first UK Centre for Phage Research earlier this year.
Lytic phages infect, replicate and break open their host bacterium, and this mechanism has been harnessed by a new diagnostic for tuberculosis, Actiphage TB, which uses a bacteriophage specific to Mtb to detect the bacterium in a blood sample. Once lysed, the bacterial DNA is released for identification with qPCR, enabling a diagnosis within hours.
Bacteriophages therefore offer great potential, as only viable bacilli are detected, and the specificity is defined by the phage’s host range. They are produced at low cost, are easy to handle and the rapid rate of infection drastically reduces reporting times.
Differentiating latent TB from TB disease
The mostly widely used diagnostic for latent TB is currently Interferon-Gamma Release Assays (IGRAs) which are whole-blood tests that detect if a person has been exposed to Mtb and developed an immune response.
This response can help to diagnose Mtb infection, but IGRAs do not differentiate latent tuberculosis infection from active tuberculosis infection. As such they cannot distinguish those patients that are at risk of progression from LTBI to full tuberculosis disease.
The detection of patients most likely to progress to active TB infection is therefore crucial both to the control of infection and the targeting of treatment to those at risk.
TB disease progression
A number of disease states are possible following infection, and a person can migrate from one to another dependent on the health of their immune system.
Infection with Mtb, usually by inhaling droplets containing the bacilli, triggers the protective immune response which acts to contain the infection to the lung or local lymph nodes.
First, the innate immune response stimulates alveolar macrophages, which ingest the mycobacteria to kill it – which is not always effective. Secondly, T-cells are activated and move to the site of infection to attempt to wall-off the bacteria within granulomas.
From here, the pathogen tries to replicate and leave the granuloma, while the immune response attempts to destroy it. If the immune system is weakened the Mtb can escape and spread within the body. Traditionally it was thought this was through the airways, but more recently research has demonstrated that Mtb may disseminate throughout the body from the lungs into the bloodstream or lymphatic system to other organs, as Verma et al., 2019 demonstrated. The presence of Mtb in the blood is therefore an indication of disease progression.
Can a phage-based diagnostic support risk stratification?
Clinical studies of the phage-based diagnostic at University Hospitals of Leicester NHS Trust involved 36 persons that shared a household with 15 patients known to have pulmonary TB. The results, presented at ECCMID 2022 (European Congress of Clinical Microbiology & Infectious Diseases), showed for the first time that live bacteria can be detected in the blood of people with incipient TB infection, at almost three times greater specificity than the gold standard IGRA test for this state of latent infection.
The trial demonstrated the potential for a rapid, non-invasive biomarker for early screening of asymptomatic individuals.
All the participants were tested with Actiphage TB, which uses a phage to detect bacteria in blood, and IGRA QuantiFERON-TB Gold Plus (QFT), which measures the immune response to infection. The individuals were followed prospectively over 12 months according to whether or not they developed evidence of progressive TB infection based on clinical assessment and highly sensitive imaging with PET-CT scans. Those with evidence of infectionrelated activity on PET-CT had further invasive sampling using bronchoscopic methods.
Dr Pranab Haldar from the University of Leicester was the lead clinician on the trial. He explained that the study used imaging and microbiological characteristics to provide a clinical definition of incipient TBI and the health of the participants was monitored after 12 months.
At that time, he said: “Our findings demonstrate the potential utility of Actiphage TB as a pathogen directed biomarker for improving risk stratification of LTBI that can potentially complement the evolving panel of host-directed immune biomarkers.”
Entering new clinical trial
The University of Leicester in collaboration with PBD Biotech, developers of Actiphage TB, have just announced a larger clinical trial with more than 100 patients. The study will assess Actiphage TB’s performance against the WHO minimum reference target product profile thresholds of 75% sensitivity and 80% specificity for a community TB triage test.
This study takes us closer to a sensitive and specific molecular diagnostic for tuberculosis disease progression that allows the rapid detection, control and early treatment of this fatal disease.
Dr Ben Swift is Co- Founder and Director of R&D at PBD Biotech. Currently Lecturer in Antimicrobial Resistance at the Royal Veterinary College, his work, with Professor Cath Rees, Professor in Microbiology at the University of Nottingham, led to the creation of the Actiphage TB diagnostic. He continues to be involved in developing rapid detection methods for mycobacterial diseases, with a special interest in the transmission of mycobacterial pathogens between humans and animals