When a case of tuberculosis (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). Patients who have MDR TB may require a different type of treatment than those with drug-susceptible disease. In many parts of the world, the incidence of MDR TB is on the rise. People with MDR TB are significantly more likely to die than those infected with drug-susceptible strains of Mycobacterium tuberculosis (Mtb), the bacteria that cause this disease.
"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."
To address the need for rapid diagnosis, Dr. Alland and his collaborators at Cepheid, Inc., of Sunnyvale, California, developed a test to identify MDR TB. Dr. Alland explains that the testing method 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, which 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 DNA is extracted from the sputum sample, it is amplified for testing through a technique called real-time polymerase chain reaction (PCR). After the DNA has been amplified to a high enough level, 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 Mtb that is resistant to the drug rifampin. Rifampin is one of the first line drugs used to treat TB. As an indicator that Mtb may also be resistant to other commonly used drugs, resistance to rifampin has been shown to be a reliable marker for MDR TB. 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.
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. Because the test is easy to use and does not require special equipment, it is well-suited to countries where healthcare resources are limited and where TB is widespread. In 2013, the Food and Drug Administration (FDA) cleared the Xpert MTB/RIF TB test to detect Mtb and resistance to the antibiotic rifampin. In 2015, the FDA cleared the assay to help physicians determine if patients with suspected TB can be removed from isolation. The World Health Organization has endorsed the MTB/RIF assay, which is being rolled out in countries across the world.
NIAID is supporting efforts to test whether the Xpert technology can detect extensively drug-resistant TB (TB that is resistant to all or nearly all first- and second-line TB drugs) and diagnose TB using non-sputum samples, such as stool and blood samples. Using blood or stool would be particularly useful for diagnosing TB in children, as it can be difficult to obtain sputum from this age group. A new version of the Xpert cartridge, known as the Xpert MTB/RIF Ultra, has also been developed. Xpert MTB/RIF Ultra is more sensitive than the original cartridge and may help detect TB in patients who test negative using other diagnostic tests. In addition, the multi-assay GeneXpert platform technology has been used to develop diagnostic assays for other infectious diseases such as sepsis, Ebola, and HIV.
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).
Boehme CC et al. Rapid Molecular Detection of Tuberculosis and Rifampin Resistance. NEJM 363(11):1005-15 (2010).
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Last Updated March 02, 2016