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Methodist Hospital Develops New Technology to Rapidly Detect Drug-Resistant TB

Technology developed by scientists at The Methodist Hospital Research Institute could halve the time it takes to diagnose tuberculosis infection, and also tell doctors in a day or two whether the bacteria are drug resistant -- a process that currently takes weeks. Initial tests of the device were recently described in Scientific Reports, a Nature online journal.

"Current TB testing techniques take at least two days, and always require the person to be present," said nanomedical engineer Lidong Qin, Ph.D., the report's principal investigator. "And if a person has ever been exposed to TB, even if they don't have an active TB infection, their test will be positive for infection. In as little as one day, we can identify the species of bacteria by isolating single cells. Our method also distinguishes between past and active infections of tuberculosis."

There were about 11,000 new cases of tuberculosis infection in the U.S. in 2010, with a survival rate of about 95 percent. TB causes about 1.5 million deaths around the world each year.

The new technique uses wafers of elastic silicone polymers that are pocked with tens of thousands of square microscopic wells (50 or 100 micrometers wide and 10 micrometers deep) that isolate cells of Mycobacterium tuberculosis or other Mycobacterium species. Antibodies to the TB protein ESAT-6 are affixed to a glass slide, and the slide is placed over the bacteria-containing wells. The sealed plates are then cultured for 24 to 48 hours at 37 degrees C (human body temperature), which gives the bacteria time to produce their own unique bouquets of active proteins and waste. The wafer and slide are separated. The wells, containing bacterial cells, are photographed microscopically so that bacterial cells can be inspected visually, and the slide, to which TB proteins are now stuck, is sent off for fluoroscopic analysis. If TB is present, the squares covering those wells will appear bright green under UV light.

"It can take weeks of culturing bacteria before one can discriminate the species," Qin said. "By switching the culture platform bulky slants to micron-sized wells -- containers whose volume is one millionth of a vitamin pill -- we can make single TB bacteria detectable in less than one day."

Qin is working on the technology with Xin Ma, M.D., Ph.D., and Yen Nguyen, Ph.D., who also coauthored the Scientific Reports paper. The idea for an improved TB test arose from a chance exchange between Ma and Qin.

"A simple conversation between a clinician and an engineer during a coffee break or lunch can stimulate a new idea," Qin said. "Our microwell technology is one such example."

Traditional TB testing, including methods used by The Methodist Hospital and other medical institutions to screen their employees, requires subjects to receive an under-the-skin injection of a harmless protein produced by Mycobacterium tuberculosis (the injection does not contain live bacteria). Two or three days later, the subject is asked to return for a "reading," during which the injection site is evaluated. If a person has ever been infected by TB, or is currently battling a TB infection, the injection site will appear red and irritated -- a positive reading.

Traditional tests also require subjects to be present twice, whereas the technology developed by Qin, Ma, and Nyugen does not require the patient to be present -- cultures could be sent to a lab hundreds of miles away for analysis, if a patient could not present in person.

To determine whether TB bacteria are resistant to antibiotic drugs, current technology requires a separate test that takes 3-6 weeks to complete. Qin, Ma, and Nyugen, however, have found they can affix to the glass slide additional antibodies to defensive proteins the bacteria produce -- meaning the presence of TB bacteria and whether they are drug resistant can be assessed at the same time. Patients could get the right antibiotic sooner.

Qin said each ELIwell test costs about $10 now. With mass production, however, he said the cost should go down to about $1 per test. Current TB testing technology costs about $4-$6 per person tested.

"The technology is not ready for the market quite yet," Qin said. "We need to test it with patient samples to fully evaluate its potential. We may decide to look at other TB proteins as well, such as CFP-10. If multi-antigens are used, the accuracy of the test could be further improved. And it is very easy to do this given our methods. Our current report, however, shows the technology works."

Qin, Ma, and Nguyen examined Mycobacteria species kansasii and smegmatis, which, like tuberculosis, produce ESAT-6. Microscopic photography of the silicon wafer wells would be used to distinguish whether the bacteria present are members of one of these species.

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