Diagnostic Research
A Timely Test: All-in-One Cartridge May Speed TB Detection
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| Image courtesy of Janice Carr and the Centers for Disease Control and Prevention. The rapid detection of multidrug-resistant Mycobacterium tuberculosis bacteria is crucial to curbing the spread of resistant strains. |
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). 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. Individuals infected with MDR TB are significantly more likely to die than individuals with drug-susceptible strains of Mycobacterium tuberculosis (M. tb).
“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 M. tb. 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.”
Colorful Beams Show Where TB Lies
Dr. Alland and his collaborators at Cepheid, Inc., of Sunnyvale, Calif., have had success identifying MDR TB using a test that they say is well suited to conditions in countries where health care 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 M. tb. 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 M. tb 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 M. tb 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 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. 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.
Into the Field
In 2004, the scientists used the molecular beacon assay to correctly distinguish rifampin-resistant and rifampin-susceptible strains of M. tb 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 M. tb 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.
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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