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International Team Developing Reagent Test That Could Broaden Access to TB Screening

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NEW YORK (360Dx) – By creating a reagent that's consumed by tuberculosis bacteria and causes infected cells to emit light, international researchers believe they have a new method for diagnosing tuberculosis that could broaden access to screening for people with TB symptoms.

The process, which is undergoing clinical trials to investigate its clinical utility, uses a reagent developed at Stanford University that combines a sugar molecule, called trehalose, and a chemical, called DMN, or 4-N,N-dimethylamino-1,8-naphthalimide.

"The reagent can be metabolized by metabolically active, or viable, TB," Carolyn Bertozzi, one of the lead investigators, who is a professor of chemistry at Stanford and a researcher at the Howard Hughes Medical Institute, said in an interview.

In practice, TB bacteria eat the reagent, modify it, and insert it within a lipid membrane, she said. The TB cells emit light, but only when the modified reagent is part of the lipid membrane. Because of that property the investigators are better able to detect active TB, she added.

"We think that the reagent might be useful in helping people diagnose tuberculosis, especially in low-resource, point-of-care settings where TB is endemic, such as parts of Africa, South East Asia, and India," said Bertozzi.

With colleagues, Bertozzi recently published a study in the journal Science Translational Medicine in which they conducted clinical testing on the reagent.

In it, scientists at the University of the Witwatersrand, Johannesburg, added DMN-trehalose to sputum smears from TB patients in Soweto. In tests on sputum samples from 16 people with TB, DMN-Tre identified Mycobacterium tuberculosis cells in all of the samples. The new technique performed similarly to a standard labeling method, which is based on the Auramine O stain.

Other tests showed that DMN-Tre is selective to Actinobacteria, the bacterial phylum that includes M. tuberculosis. Human cells and other types of bacteria, both of which are plentiful in sputum samples, don't incorporate the sugar molecule, the researchers said.

The new reagent creates a simpler process that "provides the opportunity to stain TB bacteria in a smear more quickly and with high specificity," Bavesh Kana, head of the Center of Excellence in Biomedical TB Research at Wits, said in a statement.

Early clinical results have shown that by tracking the glowing molecules, it's possible to identify living bacteria with levels of performance that could make the reagent valuable for clinicians looking to diagnose and treat TB and monitor the progress of treatment, Bertozzi said.

"We can see the effects of tuberculosis drugs on bacterial cells within a matter of hours," she said.

Eric Rubin, a professor of immunology and infectious diseases at the Harvard TH Chan School of Public Health, said in an interview that he is familiar with the research of Bertozzi and her colleagues. "It sounds like this is very easy to do and has the huge advantage of detecting only living bacteria," he said.

Rubin recently wrote a paper about TB diagnostics for the journal Nature Microbiology, noting that effective treatment and eradication of TB "requires highly sensitive and specific, easy-to-use detection methods."

He said that he sees many potential research applications for the new reagent and that he would "undoubtedly use it" in his lab, which has an objective of obtaining a better understanding of the basic biology of M. tuberculosis and developing new therapeutic strategies.

"If [the new reagent] works reproducibly on patient samples, it might become the way to determine if patients are responding to therapy," he added.

M. tuberculosis can be difficult to identify using an old-school technique that consists of using a microscope, which allows viewing of bacteria on a glass slide with a colored stain. According to Bertozzi, the method has not changed in about 100 years, and though such tests have clearly shown clinical utility, their levels of sensitivity, ranging from 32 percent to 94 percent, need improvement.

The existing staining reagent identifies live and dead TB bacteria, but it does not differentiate among them, and clinicians can find it difficult to clearly see the stained TB cells and separate them from other debris on glass slides, Bertozzi added.

In clinical use, the new reagent could become part of a TB diagnostic workflow that would also involve using the existing testing method and include follow-up lab testing with PCR or other assays to confirm initial results, she noted. Although the existing method identifies live and dead cells without differentiating among them, there are clinical applications when you need to identify all cells, dead and alive, she said.

Among the companies producing lab-based blood tests for TB are Qiagen, Oxford Immunotec, and Cepheid. PCR and other diagnostic tests are extremely accurate and work very well in the lab, "but you normally can't deploy them in the rural clinics," which usually lack refrigeration storage, funding, and technical competencies needed to run the tests, Bertozzi said.

Further, growing TB bacterial cultures in the lab, a process used by clinicians to test tuberculosis cells for their response to drugs, can take more than four weeks to provide results, she added. That can prove particularly impractical in rural settings, where tracking down patients a few weeks after testing is sometimes impossible.

Patients can wait for weeks only to find out that they have been treated with the wrong drug, she said, "so it is much better to be able to make an accurate decision about treating them when they are in the clinic and doing testing," she added.

Bertozzi and her team are producing reagents that are being used for clinical testing and for test development by diagnostic companies, and her hope is that a company, whether it is a startup or more established entity, will take on commercializing a test by licensing the reagent from Stanford. The main investment needed to conduct the test is for a battery-powered microscope. Manu Prakash, an assistant professor, physicist, and bioengineer at Stanford University, is developing microscopes for low-resource settings that could cost less than $150, Bertozzi added.  

A few additional studies are underway involving the use of the new reagent, and the results "are encouraging," Valerie Mizrahi, director of the Institute of Infectious Disease and Molecular Medicine at the University of Cape Town, said in an interview.

However, it’s important to bear in mind that the reagent "can detect bacteria other than M. tuberculosis, which may affect its clinical utility," she added. Only time and clinical testing will tell whether the new reagent shows the requisite clinical utility, but in the meantime, the reagent is "a very exciting new tool for both basic and clinical TB research,” she noted.

Bertozzi noted that more work remains before the molecule is ready for use in the field, but she is optimistic it could prove useful in the global fight against TB. From her perspective, she said, the reagent is ready for integration into test platforms for clinical testing.

A group of investigators led by Digby Warner, a researcher in the Institute of Infectious Disease and Molecular Medicine at the University of Cape Town, has already tested the use of DMN-Tre in detecting M. tuberculosis in bioaerosols captured from TB patients. That research is part of a study on TB transmission led by Robin Wood, a researcher in the University of Cape Town, Department of Medicine. The early results from this study look very promising, Mizrahi said.

In parallel, David Barr, a researcher of microbiology and immunology at the University of Liverpool, has used DMN-Tre to develop a test for M. tuberculosis bloodstream infection (MTB-BSI), which is "a major presentation" of HIV-associated TB in adult inpatients and is associated with high mortality, according to Mizrahi.

"MTB-BSI is also under-recognized, in part due to limitations of blood culture as a diagnostic assay," Mizrahi said. Barr's "results suggest that DMN-Tre-based microscopy of blood will offer a promising method for rapid detection and quantification of MTB BSI in this patient population."