Exploiting Bacteriophage as a Diagnostic Tool

Tuberculosis (TB) is a fatal infectious disease that caused 1.5 million deaths worldwide in 2020. Developing countries are particularly affected and diagnosis, especially in children and adolescents, can be difficult. As well as infecting humans, the mycobacteria that cause TB can also infect cattle and have a major impact on farming. PBD Biotech has developed a diagnostic test for TB that leverages bacteriophage technology alongside qPCR to identify live, disease-causing pathogens in humans and cattle.

We spoke to Dr. Tomas Richardson, a research and development manager at PBD Biotech to learn more about what the development of Actiphage^® technology means for the future of TB diagnosis.

Katie Brighton (KB): Could you explain what bacteriophages are and their various uses?

Tomas Richardson (TR): Phage (or bacteriophage to give them their full name) are viruses that infect and replicate within bacteria as part of their natural lifecycle. During that process, the bacterial cell is typically destroyed and its contents and DNA are released.

In terms of their uses and applications, I think there’s a growing appreciation of phage, and viruses more generally, as both a research tool and in more applied settings. In recent years, phage have been used with increasing frequency to treat antibiotic-resistant bacterial infections. Phage have even been proposed as a tool for achieving tailored engineering of the gut microbiome, as researchers begin to gain a greater understanding of the roles of different kinds of bacteria found within gut flora. Viruses in general are also a rich source of components in synthetic biology, where they can be used to engineer orthologous circuits and systems.

KB: How has PBD Biotech exploited the use of bacteriophage technology to develop a novel diagnostic assay?

TR: Critically, phage exhibit host specificity and only infect live bacteria of a given species or group. What PBD Biotech has done with its Actiphage^® assay is combine phage technology (to detect only viable mycobacteria) with traditional qPCR methods (which are extremely sensitive and specific for target DNA) to create a highly sensitive and specific novel diagnostic assay that detects only live, disease-causing pathogens. The test is currently in clinical trials to assess its efficacy for the detection of Mycobacterium tuberculosis (TB) in humans. According to WHO figures, 1.5 million people died worldwide from TB in 2020 and millions more are carriers of the disease, so there is a significant need for a rapid and effective diagnostic.

In humans and other species, the causative agents of TB are called mycobacteria and are characterized by an unusually thick waxy cell wall. Unfortunately, this property renders mycobacteria recalcitrant to DNA extraction and PCR detection. Diagnosis of TB using culture-based methods is also extremely challenging: mainly due to the remarkably slow growth rates that they exhibit. For example, fast-growing bacteria like Escherichia coli grow and replicate with a doubling time of as little as 20 minutes, whereas mycobacteria doubling times can be in excess of 24 hours.

Current diagnostics for human TB also typically rely on sputum from the lungs to detect infection. However, almost half of all people with pulmonary TB are unable to produce sputum: particularly in early disease and in children. Actiphage^® is capable of detecting mycobacteria from blood samples, which are generally easier to collect. Hopefully, this should enable disease screening of whole populations for the first time. Because Actiphage^® can specifically detect live viable bacteria in the bloodstream, early data also suggests that the method may be particularly effective at determining when a human or animal is likely to become infectious or develop symptoms of TB.

KB: The Actiphage^® assay is also able to diagnose TB in cattle. What advantage does this combination carry over current testing methods?

TR: Over 40,000 cattle were culled due to bovine TB in the UK in 2021. The disease is a huge problem for farmers and a burden to taxpayers. The current primary screening test for bovine TB is the Single Intradermal Comparative Tuberculin Test (SICTT), which works by assessing the immune response of the cattle. To perform the test, a vet injects small amounts of avian and bovine tuberculin into the animal’s skin at two different sites to compare the relative immune responses. If the animal has previously been infected with bovine TB, a more pronounced localized swelling can be detected at the site of the bovine injection three days later.

The SICTT test is thought to have a sensitivity of 50–90 %. It takes three days to produce a result, requires at least two visits by a vet and cannot distinguish between infected and vaccinated cattle: thus preventing the use of vaccination as a tool for achieving biological control. Due to the relatively low sensitivity of the SICTT method, it also misses a significant proportion of carriers, leaving a reservoir of disease within herds.

Actiphage^® detects the presence of live pathogen: even at very low levels. It can therefore help to identify carriers of the disease before they become infectious so that they can be removed from the herd. The fact that Actiphage^® detects the pathogen, rather than the immune response, also means that it can be used as a DIVA assay to “Distinguish Infected from Vaccinated Animals”, which is potentially extremely useful in the fight to eradicate bovine TB.

KB: Does the Actiphage^® assay destroy as well as detect specific bacteria?

TR: Although phage are capable of infecting and destroying bacteria, there are currently no plans to develop Actiphage^® into an antibacterial treatment.  However, there is potential to use phage to target mycobacteria seen in so-called “incipient” disease; a phase of TB infection in which the previously latent disease gradually becomes active. According to a 2016 study published in PLOS Medicine, up to 1.7 billion people may be harboring latent TB infection worldwide. We also know that some people diagnosed with the latent disease have incipient, or even active, disease but are misclassified with existing diagnostic methods. If Actiphage^® can correctly identify incipient disease, that would be very exciting as it would potentially enable identification of “latent” TB infections that are in the process of progressing into active disease.

KB: Can you see the testing being applicable beyond its use in cattle? How about in diagnosing different diseases?

TR: Absolutely. Besides human TB, bovine TB, and Johne’s disease in cattle (which are caused by Mycobacterium tuberculosis, M. bovis and M. avium subspecies paratuberculosis, respectively), we have data to suggest that Actiphage^® can be used to detect over 20 species of mycobacteria. The technology has been successfully employed on blood samples from host species including bison and deer, as well as exotic animals such as lions, giraffes and camels. As mentioned earlier, Actiphage^® is also currently in clinical trials to assess its efficacy for detecting TB in humans.

KB: In addition to your goal to achieve World Organization for Animal Health (OIE) validation, what are the next steps/priorities for you and your team?

TR: Since bovine TB is a notifiable disease, OIE validation is certainly a critical step towards international acceptance of Actiphage^® as an effective diagnostic. However, the technology can also be used without OIE validation for the diagnosis of Johne’s disease. This is important as current diagnostic tests struggle to achieve sensitive detection of the causative agent (M. avium subspecies paratuberculosis).

Going forwards, I think PBD Biotech will be increasingly focussed on helping to diagnose and fight human tuberculosis too. Human TB is second only to COVID-19 as a fatal infectious disease worldwide. As such, working with partners to develop a highly effective human diagnostic with the potential to identify latent TB infections is going to be a key priority.

Tomas Richardson was speaking to Katie Brighton, Scientific Copywriter for Technology Networks.