David Alland, MD, chief,
Division of Infectious
Disease, New Jersey
MedIcal School
Discoveries:
The UltimateTB Test
by Maryann Brinley
F
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or David Alland, chief of the Division of Infectious Disease at UMDNJ - New Jersey Medical School (NJMS), 2006 was a year filled with good news, capping almost two decades of research effort.
In May, the Foundation for Innovative Diagnostics (FIND), a non-profit organization established by the Bill and Melinda Gates Foundation, announced its intention to fund development of this investigator’s TB test with $3.7 million over the next three years. And in September, the National Institutes of Health (NIH) approved a $4 million grant to provide his laboratory and collaborators with additional support for his other projects involving TB diagnostics research and development.
Designed in a collaborative effort between his lab’s researchers and partners at Cepheid, Inc. in Sunnyvale, California, as well as FIND in Geneva, Switzerland, the new, rapid, affordable tool was created to track and tame a 4,000-year-old germ responsible for killing nearly two million people each year. In an hour and 10 minutes, what might look like a relatively harmless cough or cold can be diagnosed as a clear-cut case of active TB. “This is really a wonderful system now and so sensitive that we can tell whether someone has TB, how much there is in the body, and whether the disease is multi-drug resistant. We want to be able to screen people, and either isolate them or give them a special drug regimen right away.” TB spreads easily through the air in droplets expelled through coughing, and one-third of the world’s population is infected with this bacterium. Removing highly contagious individuals from the “madding crowd” is imperative.
“I’ve been working on rapid tests for tuberculosis since 1988 when I was a research fellow in the lab of Barry R. Bloom, PhD. [Bloom is now the dean of the Harvard School of Public Health.] We were collaborating with researchers Fred Russell Kramer and Sanjay Tyagi at the Public Health Research Institute when it was in New York.”
A 1984 graduate of Columbia College of Physicians and Surgeons, Alland interned at Columbia Presbyterian Hospital before earning a masters in science at the London School of Hygiene and Tropical Medicine. After his infectious disease fellowship, Alland joined the faculty at Montefiore Medical Center. He was recruited to NJMS in 2002. His laboratory has always been committed to addressing Mycobacterium tuberculosis. Active TB is easy to misdiagnose and in this era of regular global travel, multi-drug resistant (MDR) TB strains, and epidemic numbers of HIV immune-compromised individuals who are more susceptible to TB, a fast, inexpensive, portable, diagnostic test has been an urgent public health need. Existing tests to detect latent TB infection, which can remain dormant for years, as well as the disease itself, have relied on skin, blood cultures or sputum smears, but have always fallen short when it comes to speed, ease of use and accuracy. A culture might take from seven days to several months to complete. And, a correct diagnosis of active TB could require a complete physical exam, chest x-ray, sputum testing, and sometimes a closer look at the lung using a bronchoscope.
Alland smiles a little as he recalls one of his first encounters with FIND, a Gates Foundation-funded enterprise dedicated to promoting the health of poor people with new, reasonably priced diagnostics. In a sense, FIND acts as a matchmaker, creating bio-tech marriages for scientists like Alland. “FIND’s goal is to develop diagnostic products that are appropriate for use in the developing world. These products need to address an important health issue. They must be relatively cheap but also commercially viable.” At first, FIND wasn’t sure what was happening, if anything, in Alland’s laboratory in the basement of the Medical Science Building on the Newark campus.
Alland was confident however. After all, he had been working with highly specific, sensitive, molecular PCR (polymerase chain reaction) technology for nearly two decades. PCR is used to uncover hereditary diseases, to identify genetic fingerprints, to test for paternity, to diagnose all sorts of infectious disease, and to do DNA computing. “We had published the first use of molecular beacons [probes that indicate the presence of specific nucleic acids] in bacterial diagnostics,” he says. The molecular beacons invented by Kramer and Tyagi are so extraordinary they can uncover a single cancer cell in a sample of more than 100,000 normal ones. “Essentially we had developed a really good test to detect tuberculosis and some chronic versions of drug resistance. The real stumbling block was with the sample processing.” Getting DNA out of a TB sample had been labor intensive and the processing was not possible in places where equipment was scarce and expensive. “We just needed a way to bring the test to a doctor’s office or clinic and make it so easy that anyone could use it anywhere.”
At first, the Swiss scientists cautioned Alland about being optimistic. “They needed to see our technology but warned in advance that they were not really interested in supporting us,” he says. “By the time they left, they were talking about collaborating on a commercially viable product. We showed them just how good our technology is.”
Cepheid is a company which manufactures sophisticated, small desk-top-size diagnostic instruments for genetic analysis in the clinical, industrial and biotech markets. “We decided to work with them because of their integrated sample processing system but their equipment had only been used to process swabs or liquids.” Tackling thicker sputum from TB patients who have coughed into a little cup proved to be difficult. Finding the right reagent, or buffer, to liquefy, wash and rinse the samples so the system wouldn’t clog took time and collaboration.
“Everything goes into this little cartridge,” Alland explains, holding up a small blue and white box which looks a little like a computer printer cartridge. “You open it here at the top. The sample goes in. Pop it into the machine and press the button. We do what is called nested PCR so we’ll get a first reaction and then the results of that are mixed into a second PCR…all within the cartridge.” What makes the testing go even quicker for a clinician is that each cartridge can be processed individually. “As soon as the sample is obtained, you can run the test and in about an hour, you’ve got your answer.” Depending on the instrument, either four or 16 cartridges can be run staggered or simultaneously. “Even though PCR had always been fast, you were slowed down because you had to wait until you collected 10, 20 or more samples before you could test them. And it required a centrifuge which could be difficult to get.” Meanwhile, these new systems are going to be available at discount rates for public health officials.
“It took two years to seal the deal,” Alland says, including months of negotiations between Cepheid and FIND, trips to California, conference calls and an approval process that was both “easy and difficult. I had to do a budget but this was the first time I didn’t have to write an actual grant.” And, though he isn’t surprised by the successes in 2006, there were times along the way that he “didn’t think it was going to happen at all.” For the two billion human beings worldwide currently infected with M. tuberculosis, the good news is that it did happen.
Credit for this new TB test must go to a long list of collaborators but especially: Danica Helb and Elizabeth Story at UMDNJ; Fred Kramer, Sanjay Tyagi, and Hiyam El-Hajj at PHRI; Bill McMillan, Martin Jones, David Persing, Emily Win-dean at Cepheid; Amy Piatek and Michael Levy at Montefiore Medical Center; and Mark Perkins at FIND.