When a case of TB does not respond to two or more of the first-line drugs available for treating TB, the infection is considered to be multidrug-resistant TB (MDR TB). In many parts of the world the incidence of MDR TB is on the rise and, at a cost of thousands of dollars per case, of added concern because of the poor outcomes for these patients. People infected with MDR TB are significantly more likely to die than those with drug-susceptible strains of Mycobacterium tuberculosis (Mtb).
“Fast and accurate diagnosis of MDR TB is critical if the increase in 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 those whose infections are caused by drug-resistant strains of Mtb. If the latter can be identified as soon as they come to the clinic for treatment 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, California, have had success identifying MDR TB using a test that they say is well suited to conditions in countries where healthcare resources are limited and where TB is widespread. Dr. Alland explains that the testing method they have developed using a special assay is essentially a biotech lab in a small plastic cartridge. 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 Mtb. The test is contained in a sealed cartridge to protect the patient sample and prevent contamination of the sample. A contaminated sample could result in inaccurate or false test results.
One early hurdle for the researchers was finding a simple way to extract Mtb DNA from the thick, sticky sputum. Ordinarily, processing sputum is labor-intensive and requires expensive equipment, such as centrifuges, which are not always available in resource-limited settings. After some effort, the scientists devised a way to liquefy sputum with chemicals that can be added directly into the sputum collection cup. The sputum can then be easily transferred into the sample processing cartridge using a simple plastic pipette, eliminating the need to spin samples in a centrifuge.
After the Mtb DNA is extracted from the sputum sample, it is amplified for testing, through a technique called real-time polymerase chain reaction. When the sample is large enough, five kinds of lab-made fluorescent molecular tags, called beacons, are applied simultaneously. Each beacon glows in a different color when it attaches to Mtb 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 Mtb. Dr. Alland and his colleagues reported that their molecular beacon assay took less than 2 hours and correctly identified rifampin-resistant TB in almost all of the samples tested.
In 2004, the scientists used the molecular beacon assay to correctly distinguish rifampin-resistant and rifampin-susceptible strains of Mtb in sputum samples taken from patients in areas of high TB incidence in north India and Mexico. Rifampin is one of the first line drugs used to treat TB. It is also one of the drugs to which strains of Mtb have developed resistance, and importantly, resistance to this drug has been shown to be a reliable marker for MDR TB.
Along with Dr. Alland and his collaborators from Cepheid, Inc., the partnership now includes Geneva, Switzerland-based Foundation for Innovative New Diagnostics. The group has initiated field tests of their all-in-one diagnostic cartridge in Azerbaijan, India, Germany, Peru, South Africa, and Uganda.
UPDATE: In September 2010, the research team published the results of a clinical trial of this TB diagnostic test. Known as the Xpert MTB/RIF TB test, it is designed to detect TB and rifampin-resistant Mtb. In the clinical trial, which involved 1,730 patients with suspected drug-sensitive or MDR TB, the test successfully detected 98 percent of confirmed TB cases and 98 percent of patients with rifampin-resistant Mtb. Largely automated, the new test is also more efficient than current diagnostic methods, taking about two hours from start to finish with about two minutes of hands-on technician time. For more information, please see the NIAID Media Availability on this finding at http://www.niaid.nih.gov/news/newsreleases/Archive/2010/Pages/TBdiagnostic.aspx.
El-Hajj HH et al. Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons. J Clin Microbiol. 39(11):4131-7 (2001).
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. 42(12):5512-6 (2004).
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Last Updated September 16, 2010