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UK Researchers Developing Blood-Based TB DNA Test, Eyeing Broader Screening

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NEW YORK – UK researchers said they have used a virus to infiltrate bacteria in white blood cells and boost the sensitivity of a proof-of-concept test used to detect tuberculosis DNA.

The researchers conducted the study at the National Institute for Health Research, Leicester Biomedical Research Centre, and the University of Nottingham's School of Biosciences, and published their results recently in Clinical Infectious Diseases.

During a first human trial, the test demonstrated its potential to diagnose people with active tuberculosis infection with high levels of sensitivity and specificity, said Pranabashis Haldar, the principal investigator of the study who is a clinical senior lecturer at the University of Leicester.

He added that the assay, which is called Actiphage, also identified two people confirmed with latent infection that developed active TB after several months. That finding "suggests that the assay may be capable of working as a test to identify a high-risk subgroup of latent infection," Haldar said. Although important, the finding is preliminary and will require further, more detailed studies to prove it in larger cohorts, he added.

Ruth McNerney, a TB diagnostics researcher and associate professor at the University of Cape Town, said Actiphage is "an intriguing test because people have looked into diagnosing TB in blood and found it difficult." McNerney has evaluated the test but is not involved in its development.

Researchers have encountered challenges with effectively distinguishing TB DNA in a blood sample, she said, "but this looks like a neat way of doing it and it seems to be working quite well."

Since 2016, veterinarians have been using the test to detect tuberculosis in cattle. It's developer, Suffolk, UK-based PBD Biotech, is seeing increasing adoption in that application, said Ben Swift, director of R&D and cofounder of the firm.

In developing the test, he and his colleagues have invented a way to address a challenge associated with current blood tests used to detect TB DNA. Blood tests developed to detect M. tuberculosis DNA within circulating peripheral blood mononuclear cells have trouble accessing DNA to measure it. Actiphage mitigates that problem by using a bacteriophage, or bacterial virus, to infiltrate white blood cells carrying TB bacteria. After the cell walls are broken, mycobacterial DNA are released, and the assay uses PCR to amplify and detect the DNA. 

Swift developed the test at the University of Nottingham in 2015 with fellow researcher and PBD cofounder Catherine Rees. They decided to try it out in humans who are far easier to study than cattle, according to Swift.

In the current trial, Actiphage achieved a sensitivity of 73 percent and a specificity of 100 percent.

The investigators assessed its performance in a study that lasted 21 months and involved testing 66 people separated into four groups. They tested the assay on one group consisting of 15 study participants that had been confirmed as having active pulmonary TB; a second group consisting of 18 study participants had been confirmed with latent TB infection; a third group consisting of patients that presented with suspected TB and were subsequently identified as having community-acquired pneumonia; and a fourth group consisting of healthy people with no TB symptoms or infections.

Development of the assay into a clinical diagnostic test is underway and it may be commercially available within five years if the outcome of further studies validate the current findings, Haldar said.

He and his colleagues are expanding testing to cohorts outside Leicester with an objective to evaluate the test's performance in additional high-income, low-TB-burden settings and in low-income, high-burden settings. "Reproducibility across different settings is critical to the test having global relevance and widespread adoption," Haldar noted.

If successful, the developers anticipate pursuing CE marking, US Food and Drug Administration clearance, and obtaining regulatory approvals in other countries, Swift said. He added that the first test will be done in laboratories, and the firm is looking more long term into developing a version that could be deployed at the point of care.

In the future, Actiphage could be used to extend the reach of screening to children and others who find it difficult to produce enough sputum for existing PCR tests to be effective, Haldar said, adding that clinicians normally use an invasive bronchoscopy to extract fluid for testing in these circumstances.

He said that Actiphage could also be developed for screening patients with extrapulmonary TB, a condition in which the infection manifests in organs other than the lungs. The condition is prevalent in patients co-infected with TB and HIV, Haldar said, adding that the assay still needs to be validated in that subgroup.

TB diagnostics

TB exists in two forms in humans, and a diagnostic test needs to be able to detect both. "Active disease refers to a form of tuberculosis that causes tremendous morbidity across the world," Haldar said. However, in the far more prevalent form of TB, latent infection, people who have been exposed to the bacteria acquire the infection but are able to control it with the immune system. "We estimate that about 90 percent of all TB infection exists in the latent form," Haldar said, adding that it is important to be able to recognize that form of TB because a portion of the population develops an active infection.

Healthcare professionals are tasked with diagnosing active infections early and starting treatments to limit transmission from person to person. They also need to identify people with latent infection and treat them so that they don't run the risk of developing TB in the future.

That is an effective prevention strategy, but it doesn't identify people who are at the greatest risk of developing an active TB infection, Haldar said. In future studies, the researchers will look to validate their preliminary findings and assess how well the assay under development can identify people with latent infection who are at the highest risk of acquiring active TB.

For latent TB diagnostics in developed countries, most clinicians use a skin test or interferon-gamma release assay (IGRA) technology. 

Qiagen and its competitor Oxford Immunotec Global produce blood-based lab tests for latent TB, and their products have achieved strong uptake, especially in developed markets.

Blood-based IGRA tests detect an immune response to infection and are useful when a clinician wants to know whether a patient has been exposed to TB in the past, McNerney said.

"If you're IGRA positive, it means you are at higher risk for acquiring active TB than a person getting a negative IGRA test result," McNerney said. However, clinicians' and patients' responses to a positive IGRA test result vary significantly from region to region, she added. McNerney said that in low-income countries, patients are often reluctant to undergo treatments that are expensive and that are uncomfortable because the drugs produce toxic side effects. Diagnosing and treating people who have an active TB infection is a higher priority in these low-income settings, she said.

The diagnostic researchers noted that Danaher's Cepheid Xpert system is broadly used for rapid TB testing in low-income settings. The World Health Organization has endorsed two versions of the test for use in TB screening.

Swift said he and his colleagues anticipate developing a commercial diagnostic test that is cost competitive with the Xpert TB test.

However, for routine use in a diagnostic lab, the updated version of Actiphage would also need to reduce the number of testing steps and limit the risk of contamination, McNerney said.

If the developers prove in future studies that they can boost the assay's sensitivity over that of existing blood tests, future versions of the assay could advance screening, she said.

Most tests that are used to detect active pulmonary TB use a sputum sample, but some clinicians are also looking to use blood as an alternate type of sample, McNerney said.

Haldar said that PCR tests "are very good provided the patient is able to produce sputum, but we recognize that a significant proportion of people with TB — particularly those early on in the disease — don't produce enough sputum to allow the tests to operate effectively." In that population, the patients require more invasive and expensive testing, or a diagnosis can often be delayed, he added.

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