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Glow-in-the-Dark Assay Illuminates TB Diagnosis
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Accession Number
A00024
Author
National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID)
Source
NIAID Press Release
Release Date
May 6, 1993
Major Descriptors
Diagnosis
Drug resistance
Tuberculosis (TB)
Topic
HIV Testing
Opportunistic Infection and Other AIDS-related Conditions
Text
With help from the firefly, investigators supported by the National Institute of Allergy and Infectious Diseases (NIAID) and the World Health Organization (WHO) have developed an experimental technique that holds promise for speeding the diagnosis of tuberculosis (TB) and rapidly determining which drugs can be used to kill the TB strain a patient is carrying. The technique may also offer researchers an efficient method to screen large numbers of potential anti-TB compounds, a crucial concern as scientists work to develop effective new drugs against strains of the TB bacterium resistant to the current drugs.
The new approach uses luciferase, an enzyme that is part of the "glow-in-the-dark" system in the tail of fireflies, to produce light in living TB bacteria. Using the technique, the investigators have detected the presence of TB bacteria in one to two hours and determined within 48 hours which drugs can kill a particular strain of the TB bacteria.
With the recent rise in drug-resistant tuberculosis, it is especially critical that physicians confirm a patient's TB diagnosis at the earliest possible moment and determine which drugs are effective," says Anthony S. Fauci, M.D., NIAID director. "This new laboratory technique for rapidly identifying TB and the drug susceptibility of TB isolates holds particular promise for use in clinical settings."
As reported in the May 7 Science and at the second annual meeting of the NIAID International Centers for Tropical Disease Research in April, the study investigators, led by William R. Jacobs, M.D., of the Howard Hughes Medical Institute at the Albert Einstein College of Medicine, used genetic engineering to insert the firefly luciferase gene into the genetic material of a virus specific to the TB organism.
When this genetically altered virus infects the TB bacterium, the viral DNA with the luciferase gene is integrated into the bacterium's genes. The TB organism now can produce luciferase. Luciferase reacts with another substance, luciferin, to change chemical energy in cells into light. Current TB diagnostic tests rely on cell cultures and can take weeks because the TB bacterium, Mycobacterium tuberculosis, grows slowly, multiplying only once every 24 hours. By comparison, other bacteria such as those that cause strep throat multiply every 20 minutes, forming colonies that are detected easily.
While waiting for the results of TB cultures, physicians are forced to make treatment decisions with limited information. Patients may receive drugs that are not effective, while possibly transmitting the disease to others.
With the new procedure, known as the luciferase phage assay, the genetically engineered virus and luciferin may be added to specimens suspected of containing TB bacteria. The luciferin diffuses easily into the cells and if TB bacteria are present, they are infected and make luciferase. Light is produced and can be measured by a light-sensitive instrument known as a luminometer.
To determine the drug susceptibility of the TB strain a patient is carrying, various anti-TB drugs are added to TB cultures along with the altered virus and luciferin. If light is seen, the investigators know that the antibiotic is ineffective, because the living bacterium continues to produce luciferase and, consequently, light. However, if the TB bacteria are treated with an effective antibiotic, they will be killed and no light will be emitted.
This is a quick and sensitive method for detecting drug-resistant TB strains, and it could potentially be automated to process large numbers of samples in a short time," says Dr. Jacobs. "Refinement of the luciferase phage diagnostic system for use in the clinical setting is currently under way. It is anticipated that this assay also will be used in the near future to efficiently screen compounds for anti-TB activity."
TB, an airborne disease, is a significant and growing problem worldwide. In 1991, the Centers for Disease Control and Prevention received reports of 26,283 active cases in the United States, an increase of 18 percent since 1985. Each year, eight million people worldwide develop active TB, and three million die, according to the WHO. Drug-resistant cases of TB also have increased rapidly in the last few years. In 1991 in New York City, one-third of all TB patients had infections resistant to one or more antibiotic drugs.
Dr. Jacobs' co-authors include Rupa Udani, Gabriel Sosne and Barry R. Bloom, Ph.D., all of the Howard Hughes Medical Institute/Albert Einstein College of Medicine; John Chan, M.D. and Gary Kalkut, M.D., of Albert Einstein; Raul Barletta, Ph.D., of the University of Nebraska; Tobias Kieser, Ph.D., of the John Innes Institute and AFRC Institute of Plant Science Research (U.K.); and Gary Sarkis and Graham F. Hatfull, Ph.D., of the University of Pittsburgh.
NIAID, the lead institute for TB research at the National Institutes of Health, supports more than 50 research projects related to TB. NIH is one of eight agencies of the U.S. Public Health Service within the Department of Health and Human Services.
FOR PRESS ONLY: A Backgrounder on TB and the NIAID TB research agenda is available from the NIAID Office of Communications. Please call (301) 402-1663.
Dr. Jacobs can be reached through Art Oshins at the Albert Einstein College of Medicine Public Affairs Office at (718) 430-3101.
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