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Researchers improve design of Xpert MTB/RIF assay
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An updated version of the Xpert MTB/RIF assay, which includes new gene target amplifications, improved the detection of tuberculosis and rifampin resistance, according to researchers.
The original Xpert MTB/RIF assay (Cepheid) was endorsed by WHO in 2010 and is currently used in 130 countries. More than 23 million Xpert tests have been distributed, resulting in a three- to eightfold increase in testing for multidrug-resistant TB worldwide, Soumitesh Chakravorty, PhD, adjunct assistant professor in the department of medicine at Rutgers New Jersey Medical School, and colleagues reported in mBio.
Although the Xpert test can detect TB and rifampin resistance with high sensitivity and specificity in pulmonary samples, the researchers said it does not perform as well with smear-negative pulmonary and extrapulmonary samples. In addition, the test has also been associated with false-positive and false-negative results for rifampin resistance in paucibacillary samples.
“Seeking to resolve these limitations associated with Xpert, we developed a next-generation assay for TB and [rifampin resistance] detection that we have termed the Xpert MTB/RIF Ultra assay (Ultra),” the researchers wrote.
Chakravorty and colleagues modified the Xpert assay by refining and adding probes used to detect and differentiate Mycobacterium tuberculosis mutations associated with rifampin resistance. They used the Ultra and Xpert assays to test 25 M. tuberculosis DNA samples, 227 sputum samples spiked with M. tuberculosis H37Rv or Mycobacterium bovis BCG, and clinical samples prospectively obtained from patients with suspected or confirmed TB to compare the efficacy and limits of detection (LOD) of the devices.
For M. tuberculosis H37Rv, the LOD was 15.6 CFU/mL for the Ultra assay vs. 112.6 CFU/mL for the Xpert assay, representing an estimated eightfold improvement in TB detection, according to the researchers. The Ultra assay also substantially improved the detection of M. bovis BCG (143.4 CFU/mL of sputum for Ultra vs. 344 CFU/mL of sputum for Xpert). Although the Ultra assay accurately detected rifampin resistance, the Xpert assay yielded two false-positive results.
“Xpert has been known to occasionally produce false [rifampin resistance] results in paucibacillary samples,” the researchers wrote. “However, we did not observe false [rifampin resistance] in this sample type using Ultra.”
The overall sensitivity of the Ultra assay was 87.5% (95% CI, 82.1-91.7) for clinical sputum samples and 78.9% (95% CI, 10-86.1) for smear-negative samples. In comparison, the Xpert assay was associated with a sensitivity of 81% (95% CI, 74.9-86.2) for clinical sputum samples and 66.1% (95% CI, 56.4-74.9) for smear-negative samples. For rifampin resistance, both tests had a specificity of 98.7% (95% CI, 93-100).
According to the researchers, a prospective multicenter study is currently underway to further investigate the clinical performance of Ultra vs. Xpert in a more representative TB population. They noted that the Ultra test runs on the same GeneXpert software as the original assay, and can therefore be implemented with little additional training at sites already using the Xpert test.
“Our results suggest that Ultra will result in greater TB case detection rates not only in subjects with paucibacillary TB, such as those with HIV coinfection, but also in pediatric patients with TB and those with extrapulmonary TB, which are known to have lower mycobacterial loads,” the researchers concluded. “As TB elimination programs achieve initial successes, rates of paucibacillary TB should increase compared to rates of smear-positive disease. In this setting, assays with increased sensitivity, such as Ultra, may be instrumental in identifying the remaining cases of TB, and these assays are likely to prove valuable in furthering WHO goals to eradicate the disease.” – by Stephanie Viguers
Disclosure: Chakravorty reports filing patents for primers and probes used to detect drug resistance in M. tuberculosis. Please see the study for all other authors’ relevant financial disclosures.
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