The results of a proof-of-concept study in humans suggest that a new blood test used to detect tuberculosis (TB) and paratuberculosis in livestock could be adapted as a rapid mainstream diagnostic for Mycobacterium tuberculosis (Mtb) infection in humans, and also as a screening tool to identify people at risk of developing TB.

The test, called ActiphageTM, has been developed by researchers at the University of Nottingham. Reporting on their pilot evaluation of Actiphage to diagnose TB in humans, the researchers, and collaborators at the National Institute for Health Research (NIHR) Leicester Biomedical Research Centre (BRC), suggest that their results support the existence of TB infection in a symptomless transitional state that can subsequently develop into active disease.

The team describes its findings in Clinical Infectious Diseases. “Our observations provide new insights into how human TB develops and support recent evidence of the existence of a transitional state of TB infection called incipient TB that does not produce symptoms but carries a high risk of progressing to active TB,” stated Pranabashis Haldar, PhD, clinical senior lecturer at the University of Leicester and consultant in respiratory medicine at Leicester’s Hospitals. “As a blood test, it is particularly suitable for patients unable to produce sputum, including children, and may help support diagnosis in underserved groups that struggle to access freely available healthcare resources.”

The team’s published paper is titled, “A novel high sensitivity bacteriophage-based assay identifies low level M. tuberculosis bacteremia in immunocompetent patients with active and incipient TB.”

About a quarter of the world’s population carries M. tuberculosis infection, which represents the leading cause of death from an infectious disease, the authors explained. “It most commonly affects the lungs and from this site is transmitted to others by coughing and sneezing,” Haldar commented. “As there is a lack of diagnostic tools for people unable to bring up sputum, diagnosis is delayed, increasing the likelihood that the disease is spread.”

In most cases, the Mtb bacterium establishes what is known as a latent tuberculosis infection (LTBI) that causes no symptoms, but about 5–10% of individuals with LTBI will go on to develop TB, usually within two years. The mechanisms that underlie this conversion to active TB aren’t well understood, the researchers wrote.

Detecting Mtb can be problematic because the organism is very slow growing, which makes traditional culture methods inefficient. Molecular tests that identify Mtb DNA are also limited because the bacterium’s tough cell wall makes it hard to extract DNA. “TB is the leading cause of death from an infectious disease. It most commonly affects the lungs and from this site is transmitted to others by coughing and sneezing,” Haldar noted. “As there is a lack of diagnostic tools for people unable to bring up sputum, diagnosis is delayed, increasing the likelihood that the disease is spread.”

The Actiphage test developed by the Nottingham researchers exploits a bacteriophage that specifically infects Mtb by penetrates the bacterial cell wall, and releasing Mtb DNA. The DNA can then be amplified and detected. The whole testing process can be completed in about six hours. Actiphage is commercialized by PBD Biotech for diagnosing Mtb diseases in livestock, and primarily as a blood or milk test for bovine TB and Johne’s disease. “Actiphage is a novel blood test for mycobacteria that was developed to identify mycobacterial infections in farmed animals, helping farmers to control these difficult diseases,” explained study co-author Catherine Rees, PhD, associate professor in microbiology at the University of Nottingham, and CSO at PBD Biotech.

For their first evaluation of the test in humans, the researchers recruited 66 individuals in four cohorts, including 15 patients with active pulmonary TB (PTB), 18 individuals with latent TB infection (LTBI), a control group of 5 patients with non-TB respiratory illness, and a control group of healthy individuals. Actiphage was used to test all patients twice, 12 months apart.

The test was found to demonstrate 73.3% sensitivity, and 100% specificity. Importantly, none of the control patients tested positive, and none of the LTBI patients who tested negative with Actiphage went on to develop active TB. Interestingly, two of three participants with LTBI who did test positive with Actiphage did go on to develop active TB more than six months later. While these patients had no clinical or radiological evidence of active TB at the three-month follow-up stage, they did have detectable Mtb in their blood.

“Our data suggests that incipient disease associates with, and may be identified by, detecting Mtb in blood during early infection,” the authors wrote. In fact, the tests detected circulating Mtb in the majority of participants with active PTB and clinical features of single-organ disease, which indicated low-grade bacteremia that was below the sensitivity threshold of existing techniques. Bacteremia was also associated with greater disease extent, the authors commented. “In contrast, failure to detect Mtb in the blood of four PTB participants with clinically milder disease expression suggests a distinct phenotype of earlier disease characterized by effective immune-mediated retention of Mtb in the lung, with any circulating bacteria present at levels below the sensitivity threshold of the assay.”

“The data from our initial study in humans suggest that following infection, Mtb is circulating in the blood at levels that were previously undetectable, and that the immune system may be failing to effectively contain the bacteria within the lungs,” Rees noted.

The researchers concluded that while their results may be preliminary, the  study demonstrates “early promise with Actiphage as a blood-based diagnostic tool for infectious PTB to improve earlier diagnosis in patients unable to expectorate sputum.” While the test will require some modification to simplify sample preparation and molecular detection for use in low resource settings. “While we are cautious about generalizing from a small sample size, we are optimistic that these initial findings indicate that Actiphage can be used as a tool to help us better understand the dynamics of the infection in humans,” Rees stated. “The new Actiphage blood test offers the potential to target those at risk of TB and allow treatment to start early. This is a very exciting development that invites further study.”

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