Pitt-led researchers create quick, simple fluorescent detector for TB

March 20, 2009

Tiny viruses are engineered to inject TB bacteria with green-glowing protein for a fast, on-site diagnosis

PITTSBURGH-Researchers from the University of Pittsburgh and the Albert Einstein College of Medicine have developed an onsite method to quickly diagnose tuberculosis (TB) and expose the deadly drug-resistant strains of Mycobacterium tuberculosis that can mingle undetected with treatable strains. The researchers engineered bacteriophages-tiny viruses that attack bacteria-to inject TB bacteria with a glowing, fluorescent-green protein. They report their findings in the March 19 edition of PLoS ONE, a peer-reviewed online journal from the Public Library of Science.

The method must next undergo clinical trials, but it has potential as a valuable, timesaving tool in rural African areas besieged by TB, explained the paper's senior author Graham Hatfull, chair and Eberly Family Professor of Biological Sciences in Pitt's School of Arts and Sciences. Hatfull conducted the research with Professor William Jacobs Jr. of the Department of Microbiology and Immunology at the Albert Einstein College of Medicine of Yeshiva University in New York and Pitt postdoctoral fellow Mariana Piuri.

"A report from South Africa showed that the extensively drug-resistant TB strains can kill within 16 days, on average," Hatfull said. "In rural Africa, it takes too long to collect samples, send them off, do the test, and have the data sent back. Clinicians need rapid, relatively cheap, and simple methods for detecting TB and drug-resistant strains at the local clinic. This test provides a quick diagnosis so the patient can be isolated and treated."

The group constructed bacteriophages specific to TB that have a green fluorescence protein (GFP) implanted in their genome. Bacteriophages spread by injecting their DNA into bacterial cells-in this case the GFP gene accompanies the DNA into the TB cell, causing the cell to glow. A clinician can detect the GFP's glow with equipment available at many clinics.

Besides quick diagnosis, the test also could be used to distinguish treatable TB strains from those that are drug resistant, a chore that can normally take months, Hatfull said. Hatfull and his colleagues treated M. tuberculosis with antibiotics at the same time the bacteriophages were introduced; the TB strains that were sensitive to antibiotics died, but the drug-resistant cells survived and continued to glow.

The group's research was funded as part of a major new research initiative from Howard Hughes Medical Institute (HHMI). The institute announced March 19 that it will partner with South Africa's University of KwaZulu-Natal to establish an international research center focused on the TB and HIV coepidemics in Africa, called KwaZulu-Natal Research Institute for TB-HIV. Jacobs will direct research into developing rapid and effective TB tests, one of the new institute's primary objectives. His work with Hatfull and Piuri was related to that effort. More information about the HHMI initiative is available on the institute's Web site at www.hhmi.org/news/krith20090319.html

"The development of reporter flurophages," Jacobs said, "allows us to bypass the existing method of diagnosing TB, which requires cultivating slow-growing bacteria in a biosafety level 3 environment, a time-consuming and costly process. By infecting live M. tuberculosis cells with a flurophage, a quick and highly sensitive visual reading can be done. We are optimistic that we can move the diagnostic process from several weeks to several days or even hours, which could have a significant impact on treatment."

University of Pittsburgh

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