Although the use of bacteriophages for therapy is becoming more widely accepted, the application of phages for disease diagnosis is still in development.

Diagnosis is more exacting and requires the phage to be purified and stored ready for use. As this knowledge can be applied to other areas, we thought it would be of interest to share our experiences of preparing a bacteriophage for the diagnosis of tuberculosis, the world’s most lethal infectious disease.

Use of phages for diagnosis

Phages are specific to a particular bacterial host. This capability can be leveraged for the detection and identification of live pathogens, as the phage will only infect and replicate within viable bacteria.

Phage-based diagnostics have proven to be rapid, specific, and often cost-effective solutions for bacterial detection within clinical diagnostics, food safety testing, environmental monitoring, veterinary medicine, and biodefense.

There are a number of ways that engineered or naturally occurring phages are used for diagnostic applications:

• Phage amplification assay: phages replicate within the target bacteria in the sample, leading to the amplification of phage particles. These particles can be measured by changes in turbidity, fluorescence, or other quantifiable signals.
• Phage display technology: detection is based on the binding affinity between phage-displayed molecules and bacterial surface antigens.
• Phage typing: a known bacterial strain is tested for susceptibility to a panel of phages, creating a unique phage sensitivity profile.
• Phage-induced bacterial lysis: the release of cellular contents that can be measured using, for example, turbidity, colorimetry, or fluorescence.
• Phage-based biosensors: binding of the phage to a target bacteria triggers a signal. This is transduced into a measurable output, such as an electrical or optical signal.
• Phage-derived proteins: the activity of certain phage-derived proteins can be measured, such as endolysins which specifically lyse the bacterial cell wall.

Production of phages for diagnostics

The phage need to be propagated to create a sufficient volume for use as a diagnostic.

1. Phage-induced lysate

First the relevant bacterium is cultured until it reaches the optimal growth phase.

The phage is then introduced to infect and replicate within the bacterial host. This leads to the phage-induced lysis and the release of free phages.

After successful replication, the phage lysates are harvested and purified to remove bacterial debris and contaminants.

2. Purification

Phage purification and concentration may involve centrifugation, filtration using specific pore-sized filters, ultrafiltration, precipitation using polyethylene glycol, and dialysis or buffer exchange techniques.

The phage titre is determined by measuring the concentration of phage particles.

Finally, it is necessary to carry out the appropriate quality control tests to assess the purity of the phage preparation and its performance.

3. Storage

Purified phages are stored at low temperatures until they are ready for use in diagnostic assays.

To preserve their viability during storage and transport, cryoprotectants can be added to phage suspensions.

Alternatively, lyophilization of phages into a dry powder form can enhance stability and enable transport at ambient temperatures. Once lyophilized, phages can be reconstituted with sterile water or the desired media at the destination.

4. Transport

Phages in liquid or lyophilised form should be packaged in airtight containers, protecting them from moisture, light, and physical damage during transit. It is crucial to transport phages at controlled ambient temperatures, avoiding extremes of heat or cold, and if possible, aim for shorter transit times to minimize exposure to environmental fluctuations.

By implementing these techniques - purification, stabilization, lyophilization – along with proper packaging and temperature control within an acceptable range, it is possible to transport the phages without relying on a cold chain, while maintaining their viability and effectiveness.

Case study: Use of Actiphage TB for detection of viable Mycobacterium tuberculosis

About a quarter of the world’s population is thought to be infected with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), but for most it will remain latent in the body. It is currently difficult to detect those with an infection that will progress to disease – known as incipient disease – and this is one of the greatest challenges for TB biomarkers and diagnostics.

In 2022, an estimated 10.6 million people fell ill with TB worldwide, and 1.3 million people died. But of those who became ill only 7.5 million had been detected and notified, leading to a gap of 3.1 million cases of incipient TB. Ending the TB epidemic by 2030 is among the health targets of the United Nations’ Sustainable Development Goals.

Current diagnostics all have limitations. Mtb is a slow growing bacterium so although culture-based methods are still considered to be the gold standard, they require incubation times ranging from weeks to months and are not always a reliable way to detect low number of cells present in a sample.

The widely used Interferon-Gamma Release Assays are immune-based diagnostics, providing evidence of an immune memory response to TB infection rather than confirming the presence of active pathogen, and are poorly predictive of disease progression.

Traditional PCR-based methods also do not discriminate between viable and non-viable bacteria.

This is why there is growing interest in the phage-based diagnostic Actiphage® TB, developed by PBD Biotech, which can detect low levels of active Mtb in a blood sample.

It is known that Mtb can be contained by the immune system within granulomas in the lungs. If the immune system is compromised, then Mtb can escape into the blood system hidden within the macrophages.

Actiphage can find the Mtb within these peripheral blood mononuclear cells (PBMC) and lyse them, releasing the mycobacterial DNA for analysis with qPCR.

The test requires the collection of a 3 mL blood sample from the patient. The PBMC are isolated, and the sample incubated with Actiphage for 3.5 hours to enable Mtb lysis and DNA release.

The mycobacterial DNA is cleaned up and interrogated using a fluorescent qPCR reaction, giving a qualitative result (positive or negative) for TB.

Actiphage is revolutionary because it enables the rapid detection of TB at the earliest stages of disease before the person becomes infectious to others, thereby enabling clinicians to break the chain of infection.

Advancements and future directions

Mycobacterial infections are one of a number of diseases currently being targeted with a phage-based diagnostic. Other examples include Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae.

In parallel, advances in genetic engineering techniques are enabling the modification of phages for better detection capabilities.

Recent breakthroughs in this field include the development of highly specific and sensitive assays and the integration of phage-based systems with other cutting-edge technologies like microfluidics.

These developments offer the potential to enhance the detection speed and accuracy, marking a significant stride toward more advanced diagnostic approaches.

Phage-based diagnostics are also being explored for their potential in assessing antibiotic resistance in bacterial strains. These tests could provide valuable information about the susceptibility of bacteria to antibiotics, aiding in the selection of effective treatment options.

This is an exciting and dynamic field, and we would welcome contact with other researchers looking to improve the purification and storage of phages.

About PBD Biotech

https://www.pbdbio.com

PBD Biotech Limited specialises in the use of novel bacteriophage-based technology. The company has developed proprietary, patented technology that can be used to detect the presence of mycobacteria that cause tuberculosis in humans and animals in a blood sample.

This includes human TB – Mycobacterium tuberculosis (Mtb) – where the technology has application as a screening tool and for test of cure.