Diagnostic Research
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.
References
El-Hajj, H. H. et al. Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons. J Clin Microbiol. 2001 Nov;39(11):4131-7.
Varma-Basil, M. et al. Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates from India and Mexico by a molecular beacon assay. J Clin Microbiol. 2004 Dec;42(12):5512-6.
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