What is tuberculosis?

Tuberculosis is a non-communicable disease, mainly caused by the bacteria Mycobacterium tuberculosis (Mtb). Tuberculosis is contracted by humans through the inhalation of contaminated droplets and aerosols in the air. The air can become contaminated with Mtb as an infected person coughs or sneezes into the air, releasing active Mtb.¹ Tuberculosis (TB)  often causes infections in the lungs, which is known as a pulmonary TB infection. In more serious cases, Mtb can also infect other parts of the body, such as the kidneys, lymph nodes, bones, spine, and the brain.²

Types of tuberculosis

Tuberculosis is distinguished into two types. Active TB involves Mtb multiplying in the body and initiating symptoms. An active TB infection can be transmitted to others through small droplets in the air. On the other hand, a latent TB infection occurs when the infected individual contains Mtb bacteria in their body, but the immune system keeps it contained in an enclosed structure known as a granuloma. A granuloma is produced by the host immune cells in order to prevent Mtb from spreading in the body. ¹

Symptoms

After someone inhales Mtb, the bacteria begin to settle in the lungs. A healthy immune system will usually take control, enclosing the bacteria in a granuloma and causing latent TB. Latent TB shows no symptoms. However, symptoms can appear within a few weeks/months after contraction in cases of immunocompromisation. Individuals with weakened immune systems, such as young children, the elderly, and those suffering from HIV or poorly controlled diabetes, are more susceptible to an active TB infection. This causes symptoms such as:

• Persistent coughing (lasting over 3 weeks)
• Coughing containing mucus and/or blood
• A high temperature
• Unintentional weight loss
• Loss of appetite

What is a biomarker?

A biomarker is a biological molecule in the body that can be used to provide information about the presence of a disease and the severity or stage of a disease. Biomarkers can also measure how the disease responds to treatment therapies, as well as predict if a disease is likely to relapse. Biomarkers can be hormones or proteins found in the tissues or in fluids such as blood, urine, serum, and plasma.

Why are biomarkers important for tuberculosis?

Biomarkers are crucial for the diagnosis and treatment of tuberculosis. Tuberculosis ranks alongside HIV as a leading cause of death worldwide, and it is estimated that one-third of the world’s population latently carries TB, with a 2-10% chance of progression to an active TB infection.¹ With biomarkers, healthcare professionals can track the stage of TB in individuals and predict whether their condition may worsen. Biomarkers can therefore contribute to early detection and prevention of TB infections from worsening. 

Types of biomarkers in tuberculosis

TB biomarkers can be distinguished as they can either be related to the pathogen M. tuberculosis or to the human host.

Host biomarkers

Host biomarkers rely on serum, plasma, urine and blood. A consistent host-derived biomarker is the interferon-inducible gene signature. Interferons are a type of antibody made by the body’s cells in order to respond to bacterial infections such as TB. In TB patients, DNA testing has shown that patients have increased levels of genes which stimulate interferons. Interferons are produced by a type of white blood cell known as a neutrophil. They activate interferons, often causing systemic inflammation during active TB infections. This makes the interferon inducible gene profile a key biomarker as it can help to distinguish patients who are suffering from an active TB infection, and not an asymptomatic latent infection.¹

The interferon-gamma inducible protein (IP10) is a biomarker that is detected in the urine of child and adult patients.¹ IP10 is secreted in response to interferon-gamma during immune activation. This attracts T-cells to the site of infection and inflammation. As M. tuberculosis enters the lungs, IP-10 levels are significantly increased in cases of active TB. This can be used to detect an active TB infection and help to monitor treatment therapies. This is because, with anti-TB treatment, it is expected that the levels of IP10 will decrease as the infection clears. ²

Blood-based studies of biomarkers have shown that there are various immune activation markers which show promise in accurately diagnosing and distinguishing TB from other diseases. These include: CD38 and Ki-67.¹ These biomarkers have previously been tested on mycobacterium-specific T-cells and have been shown to accurately distinguish between active TB infections and latent TB with 100% specificity. Immune activation biomarkers have also classified individuals who have successfully undergone anti-TB therapy, with patients showing a decrease in mycobacterial load following their treatments. ²

Volatile organic compounds can also be TB biomarkers. These compounds originate from Mtb metabolism or oxidative stress within the host. Breath-based tests can detect volatile organic compounds and identify active pulmonary TB with 85% accuracy.1 However, detection of VOCs is often challenging as they are excreted in incredibly small concentrations which current analytical instruments cannot detect.

Pathogen-derived biomarkers

There are limited biomarkers which come from M. tuberculosis itself. This is because the bacterial load is very low in the majority of samples used. M. tuberculosis has a distinct intracellular lifestyle, which also makes pathogen-derived biomarkers scarce. M. tuberculosis typically lives inside macrophages (white blood cells) once it has entered the body, where it is protected from many other host cells. This makes pathogen biomarkers of TB less likely to appear at an identifiable level in bodily fluids.¹

Mycobacterium DNA can be detected in the blood and urine of pulmonary TB patients. This can be achieved by using molecular amplification techniques such as Polymerase Chain Reaction (PCR). PCR can identify Mtb DNA at fast rates, directly from sputum and clinical specimens within hours, and it offers a higher sensitivity and specificity than microscopy. The Xpert MTB/RIF assay (also known as GeneXpert) is a machine which can also identify both Mtb DNA and resistance to certain antibiotics such as rifampicin, aiding diagnosis and monitoring treatment.²

Lipoarabinomannan (LAM) is another pathogen-based biomarker. LAM is a sugar-based component of the cell wall in Mtb. LAM helps the bacterium avoid host immune defences and can aid in the regulation of immune responses e..g, by suppressing T-cell activation and altering immune chemical (cytokine) production. LAM is released from the bacterial cell wall, and it can be detected in blood and urine, acting as a biomarker for active TB. Urine LAM tests have been synthesised, particularly relevant in cases of patients with an HIV-coinfection. However, LAM tests often have low sensitivity in general TB patients.²

Clinical applications of TB biomarkers

Diagnosis of active TB

Current diagnostic methods for an active TB infection include microscopy of sputum (mucus coughed up from the lungs), culture and GeneXpert analysis. Both host and pathogen-derived biomarkers can be detected in various samples (urine, blood, sputum) using these techniques. ²

Monitoring treatment response

Biomarkers can be used to monitor the success of treatment in active TB patients. Sputum culture conversion refers to the removal of Mycobacterium from the lungs with progressive treatment, and different biomarkers can indicate whether this is a success. IP10 and immune biomarkers tend to decrease with effective treatment, and HO-1 and MMPs correlate with the clearance of bacteria from the lungs. ¹

Challenges and limitations

Current diagnostic methods of active TB involve microscopy, culturing, and genetic analysis of sputum coughed up from the patient's lungs. Although sputum culturing is the gold standard for diagnosis, it relies on Mtb-positive sputum, which many TB patients do not have. This makes it difficult to detect TB in sputum, and so current studies are focused on non-sputum-based techniques.³

Biomarkers detecting active TB often have sensitivity and specificity issues, especially in patients without immunocompromising diseases or HIV. Some host biomarkers, such as IP10, can be elevated in lots of other infections and inflammatory conditions, making it harder to detect active TB.⁴

Most biomarkers are identified at a small scale and in an experimental setting; very few have undergone large-scale validation across countries. Additionally, biomarkers that show promise are often expensive and complex as they rely on advanced machinery and techniques, which are not always accessible in regions that are in need of TB research. Many countries facing high cases of TB are in a high-burden setting, such as India, Indonesia and Tanzania.³

Future directions

TB biomarkers have a long journey of progression ahead in terms of future directions in diagnosis and treatment. Many TB biomarkers need larger validation studies (IP10, Interferon-Inducible Gene signature) as they have only been tested in specific groups (e.g. in children or those with HIV). Multicentre testing and trials of these biomarkers need to take place in order to assess their sensitivity, specificity and accuracy in diagnosis and treatment across a wider level. This is crucial as it is the only way to gain insight into applicable results and the possible reproducibility of results, as these biomarkers need to work effectively in different groups of people and countries across the globe.⁵

Biomarkers need to be non-invasive and have to come with a simple procedure for testing in order to effectively be used in low-resource settings and in countries with lower socioeconomic status. The acquisition of these biomarkers needs to be simple, such as using urine samples, finger-pricking to collect blood, and breath tests, so that they can be done quickly and effectively in resource-limited environments.⁵

Incorporating imaging tools such as PET/CT or even AI-based software can help to use biomarker data to provide strong predictive models of TB and potentially aid in treatment monitoring and prognosis.⁵

Summary

Active tuberculosis is a leading cause of death worldwide, spread by coughed aerosols containing the bacterial pathogen Mycobacterium tuberculosis. Diagnosing active TB can be carried out through the microscopy and culturing of sputum samples; however, this is a slow and unreliable process. M. tuberculosis itself can produce biomarkers that help to detect an active TB infection in patients. These include the sugar-based cell wall component lipoarabinomannan, which can be released in patients' urine, and traces of DNA found in the lungs.

There are various biomarkers within the human host that can help to diagnose tuberculosis, such as elevated levels of interferon-gamma inducible protein, interferon-inducible gene signatures, immune markers such as CD38 and volatile organic compounds. IP10, interferon-inducible gene signatures and immune markers are often recruited at the site of infection and are found on cells stimulated by M. tuberculosis. These can help to detect an infection; however, they can also be triggered by other infections and inflammatory conditions, reducing their specificity. 

Finding accurate biomarkers is difficult due to the varying accuracy of current biomarkers and the lack of wide-scale research and validation. There are numerous factors influencing the criteria of the perfect TB biomarker, such as low cost, simplicity in technique, and applicability across different demographics. 

This figure demonstrates the pathogen and host-based biomarkers discussed in this article.⁵

Created in  https://BioRender.com