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UK Researchers Develop New Tuberculosis Testing Method


NEW YORK – A group of researchers largely based out of University College London have developed a new method for testing tuberculosis that would allow lower-level biosafety laboratories to process samples safely without using culture.

The researchers previously developed a molecular bacterial load assay that allowed them to count the number of live Mycobacterium tuberculosis bacteria in a sputum sample by measuring the 16S rRNA, but the original assay was a research tool that wasn't easily applicable to a clinical laboratory, said Isobella Honeyborne, a researcher at the Centre for Clinical Microbiology at University College London and one of the test's developers.  

The new test, which is described in a paper published last month in Clinical Chemistry, was born out of a desire to give the original assay "a new life force" and make it usable in clinical laboratories, as well as in low- and middle-income countries that often don't have the infrastructure to appropriately handle sputum samples.  

Sputum can be a particularly difficult sample type to work with, Honeyborne noted, as it's very different between individuals, there are many inhibitors that can affect the efficacy of an RT-qPCR reaction, and it's hard to take measurements of live bacteria from it. The research team wanted to develop a way to inactivate bacteria in sputum samples so they could be handled in biosafety level 2 laboratories and make tuberculosis testing more accessible.  

In the paper, the researchers noted that six months of treatment are typically required for tuberculosis. "Poor adherence to treatment is one cause of multidrug or rifampicin-resistant ... tuberculosis," and the World Health Organization recommends monthly treatment monitoring with sputum culture. However, the infrastructure for monitoring with culture is often unavailable in low- or middle-income countries, while getting results from a monitoring test can take weeks. 

The new test, called rapid enumeration and diagnostic for tuberculosis (READ-TB), aims to speed up the process of treatment monitoring for tuberculosis without using culture and involves using acetic acid to sterilize the sample while protecting the tuberculosis RNA held within it. First, the researchers tested what was already commercially available for inactivating samples and preserving RNA, but those commercial reagents didn't kill all of the tuberculosis bacteria present, Honeyborne said, meaning they would still need to be handled at a biosafety level 3 laboratory. The team then found a previous paper describing acetic acid as an effective disinfectant against M. tuberculosis, and knowing that it was often used as a component in histological stains, they "wondered whether it might work" for this purpose as well.   

After testing it, they found acetic acid was able to inactivate contaminated samples while still preserving RNA that could then be extracted. The first step of the test involves treating the sample with acetic acid, which could be done at a clinic where the sample is collected. It is then spiked with an internal control based on the phyB gene from a potato that is inhibited at the same rate as the tuberculosis 16S rRNA gene and can be used to standardize readouts from sputum samples, normalize RNA loss during processing, and nullify any inhibitors present in the sample. After that, the sample is centrifuged for 30 minutes, followed by disruption of the bacterial cell wall using silica beads and extraction of the RNA using Qiagen's automated RNA extraction system QIAcube. From there, the RNA is tested using an RT-qPCR instrument. The RT-qPCR process can also be automated, Honeyborne said.   

While there are a few hands-on steps throughout the process, she noted that the RNA extraction process is mostly automated, which may help improve accessibility since less technical knowledge is required.  

The test also requires common instrumentation that could be found easily in most laboratories, even those in low- and middle-income countries, she said. It also doesn't have particularly high energy requirements, which is another constraint found in laboratories in low- and middle-income countries, and it takes only three to four hours to return a result.  

The test is "ready to go now" and is already being used in clinical studies, Honeyborne said. The researchers are hopeful that the Clinical Chemistry paper will help the test gain traction, but Honeyborne noted that there is not a commercial kit available, and it hasn't received regulatory approval. While commercialization may come in the future, it's not the main priority for the researchers right now, she added. 

The test will primarily be used for treatment monitoring in tuberculosis clinical drug trials but also may be useful for patients who are hard to treat or difficult to diagnose – if they still have live tuberculosis in their sputum, the test can inform clinicians whether a patient should stay in the hospital or return home.  

In Europe, where tuberculosis cases are relatively low, patients will likely be tested using standard point-of-care tuberculosis tests like the Cepheid GeneXpert MTB/RIF test. However, according to Honeyborne, that test can't be used in a drug trial because "DNA is not a good measure of live bacteria because you can't see whether the bacterial load is dropping in response to the drug treatment," making READ-TB a good substitute.  Cepheid did not respond to a request for comment on deadline.  

READ-TB is "not a replacement" for standard tuberculosis diagnostics, she added. "Although it could be used as a diagnostic, our main focus was on drug treatment response and clinical trials."