Tuberculosis (TB) : Diagnosing

by National Institute of Health

(Page 8 of 13)

Shining a Molecular Flashlight on the TB Bacterium

William R. Jacobs Jr., Ph.D., has a take-no-prisoners philosophy when it comes to fighting tuberculosis. "Our mission is to eradicate TB and I'll do whatever it takes to get there."

In the campaign against TB, Dr. Jacobs has frequently deployed a miniature army of phages. Phages are viruses that infect only bacteria and are exceedingly common in nature. Dr. Jacobs, who is a Howard Hughes Medical Institute researcher at Albert Einstein College of Medicine in New York City, has isolated phages from Bronx Zoo dirt.

A Miniature Army

In the wild, phages infect their targets by injecting DNA into the bacterial genetic material. This leads to the production of more phages and, eventually, to the death of the bacterium. In the lab, scientists can modify phage DNA with genetic material from other organisms. These genetically engineered phages are like tiny cargo ships that efficiently shuttle the foreign genes into bacteria. So useful is phage technology that Dr. Jacobs says he has used it to surmount every obstacle he has encountered in his research on Mycobacterium tuberculosis (M. tb), the bacterium responsible for TB.

When faced with the problem of developing a rapid, accurate test for determining the antibiotic susceptibility of various strains of M. tb, phages once again proved their mettle. This time, Dr. Jacobs and his colleagues developed a phage that carries the gene for luciferase, the chemical that makes fireflies glow. When luciferase-producing phages infect living cultures of M. tb and begin to reproduce, the cultures glow. If an antibiotic-sensitive strain of M. tb is infected with luciferase phages and subsequently exposed to an antibiotic, the light is extinguished because the phages have no living bacteria to infect. The technique was described in a paper published in 1993.

The Bronx Box

Dr. Jacobs' team built a prototype device, nicknamed the Bronx box, that used inexpensive and sensitive dental X-ray film to detect the glow emitted by phage-infected bacteria.

In 2001, Dr. Jacobs joined with scientists from Stanford University and Mexico's National Institute of Public Health and Nutrition to see if luciferase-reporter phage technology could help clinicians determine to which antibiotics a given strain of TB is susceptible. As Dr. Jacobs notes, drug susceptibility tests are usually not done on clinical samples from TB patients in the developing world; it is simply too expensive and time-consuming. However, incomplete or incorrect drug treatments not only fail to cure the patient, they also lead to ever more prevalent strains of drug-resistant M. tb.

The Bronx box performed admirably in the Mexican study. The technology proved very accurate both in detecting M. tb in samples drawn from Mexican TB patients, and in determining to which antibiotics those isolates were susceptible. The time needed to do these drug susceptibility tests was shortened by more than a week when compared with conventional methods. More studies are underway to evaluate the use of this system in other countries, including India and South Africa.

In 2003, Dr. Jacobs and co-authors from the Centro Internacional de Entrenamiento e Investigaciones Medicas, in Cali, Colombia, published results showing that the low-tech Bronx box and a more sophisticated luciferase-reporter device called a luminometer both performed well in accurately identifying drug resistant strains of TB taken from patients. The luminometer produced a result after only 54 hours, while the Bronx box took 96 hours. Both worked much faster than the "gold standard" test for TB infection, which takes three weeks.

A Timely Test: Real Time PCR

A case of TB that does not respond to two or more of the first-line TB drugs is termed multi-drug-resistant TB (MDR-TB), and it is on the increase in many parts of the world. Patients with MDR-TB are significantly more likely to die of their disease than patients with drug-susceptible strains of Mycobacterium tuberculosis (M. tb) and the cost of treating MDR-TB can run into the thousands of dollars per case.

Fast, accurate diagnosis of MDR-TB, particularly in resource-limited settings, is critical if the rise of this especially deadly form of TB is to be slowed, says David Alland, M.D., of The University of Medicine and Dentistry of New Jersey, in Newark. It is important to distinguish patients who have drug-susceptible disease and can be started on a routine regimen of one or more TB drugs from people whose infections are caused by drug-resistant strains of M. tb. If the latter can be identified as soon as they come to the clinic for treatment, says Dr. Alland, they can be safely separated from the rest of the hospital patients and will not spread MDR-TB to others.

Dr. Alland and his collaborators at Cepheid, Inc., of Sunnyvale, CA, have had success identifying MDR-TB using a test that they say is well suited to conditions in resource-poor countries where TB is rife. Essentially, explains Dr. Alland, the "molecular beacon assay" is a biotech lab in a test tube. The test quickly, automatically, and accurately tells doctors whether a sample of patient sputum (material expelled from the lungs and throat by coughing) contains drug-resistant or drug-susceptible M. tb. The entire test is contained in a sealed cartridge, so the sample cannot become contaminated and give false results.

After the sputum is liquefied and washed, DNA from any M. tb present is first extracted and then rapidly expanded through a technique called real-time PCR. When the sample is large enough, five kinds of lab-made fluorescent molecular beacons are applied simultaneously. Each beacon glows in a different color when it attaches to M. tb DNA. In the presence of drug-susceptible TB, all five colors are visible. If any one of the colors is absent at the end of the test, it means the sample contains drug-resistant M. tb. The initial version of the test detected strains resistant to the TB drug rifampin. In their first publication about this technique, Dr. Alland and his colleagues reported that their molecular beacon assay took less than three hours and correctly identified rifampin-resistant TB in 11 out of 11 sputum samples.

Since then, Dr. Alland and colleagues in the United States, Mexico, and India have improved the assay's accuracy and sensitivity. In 2004, the scientists used the molecular beacon assay to correctly distinguish rifampin-resistant and rifampin-susceptible strains of M. tb in patient sputum samples taken from areas of high TB incidence in north India and Mexico.

Larger trials of the assay and sputum processing techniques are being planned in collaboration with Cepheid, Inc., other investigators from University of Medicine and Dentristry of New Jersey, and the Uganda National TB and Leprosy Control Programme, says Dr. Alland.

Blood Evidence: Antibody Tests that Detect Reactivating TB

Controlling TB hinges on distinguishing latent (symptom-free) TB infection from active disease, notes Maria Laura Gennaro, M.D. Current diagnostic tests, she says, are unsatisfactory because they are too slow, inaccurate, or hard to conduct. Moreover, they do not distinguish between different stages of infection with M. tb. With her colleagues at the Public Health Research Institute in Newark, New Jersey, Dr. Gennaro is examining blood of M. tb-infected people to gain clues to the "evolution of infection."

Simply knowing that a person is infected with M. tb is not very meaningful, notes Dr. Gennaro. In poor countries, where the infection rate can be 70 to 80 percent, it would cost too much to treat people who are infected but who will not become sick. Therefore, clinicians focus treatment efforts mostly on people with active TB and on young children who are in close contact with TB patients. Even in wealthy countries, infected people can be reluctant to start a long course of drug treatment for a condition with no symptoms.

To distinguish among latent, active, and reactivating stages of TB, researchers in Dr. Gennaro's lab are determining "profiles" of blood-borne immune system molecules called antibodies. Antibody assortments, or profiles, change over the course of infection as bacteria themselves shift from one life state to another. If the scientists could discover a correlation between a specific antibody profile and the stage of infection, they could develop powerful tools for distinguishing persons harboring actively growing M. tb from people carrying non-growing M. tb, explains Dr. Gennaro. Also, she says, a shift in antibody profile might provide an early warning that TB is starting to reactivate. Such an early warning signal would allow doctors to treat the awakening TB before symptoms became obvious.

About the Author

NIH is the nation's medical research agency - making important medical discoveries that improve health and save lives. The National Institutes of Health (NIH), a part of the U.S. Department of Health and Human Services, is the primary Federal agency for conducting and supporting medical research.