The New Xpert MTB/RIF Ultra: Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing

Abstract

The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance. Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or Mycobacterium bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For M. tuberculosis H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for M. bovis BCG, it was 143.4 CFU/ml of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert, resulted in an overall sensitivity of 87.5% (95% confidence interval [CI], 82.1, 91.7) versus 81.0% (95% CI, 74.9, 86.2) and a sensitivity on sputum smear-negative samples of 78.9% (95% CI, 70.0, 86.1) versus 66.1% (95% CI, 56.4, 74.9). Both tests had a specificity of 98.7% (95% CI, 93.0, 100), and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection.IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and specificity with pulmonary samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIF-R) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. The present study is the first demonstration of the design features and operational characteristics of an improved Xpert Ultra assay. This study also shows that the Ultra format overcomes many of the known shortcomings of Xpert. The new assay should significantly improve TB detection, especially in patients with paucibacillary disease, and provide more-reliable detection of RIF-R.

Keywords: Xpert MTB/RIF Ultra; diagnosis; sensitive; tuberculosis.

FIG 1

Limit of detection for M. tuberculosis H37Rv. The limit of detection of tuberculosis detection is shown for Xpert (A) versus Ultra (B). The limit of detection for generating a rifampin susceptibility rather than an indeterminate result is shown for Xpert (C) versus Ultra (D).

FIG 2

Detection of M. tuberculosis rpoB gene mutations associated with rifampin resistance. Derivative-transformed curves of four rpoB-specific sloppy molecular beacons (rpo1, rpo2, rpo3, and rpo4) first hybridized and then melted off of their M. tuberculosis rpoB gene amplicon target are shown. Each colored peak indicates the melting temperature of the probe corresponding to the colored line. The peaks for wild-type M. tuberculosis (A) identify a test sample as rifampin susceptible. The shift in one or more of the peaks away from the wild type’s melting temperature (B to G), identify a sample as an rpoB mutant and rifampin resistant. The shift in the melt peak is indicated by arrows.

FIG 3

Detection of hetero-resistance. Samples containing equal quantities of total M. tuberculosis DNA were created using different proportions of wild-type and rifampin-resistant rpoB S531L mutant DNA. Derivative-transformed curves of four rpoB-specific sloppy molecular beacons (rpo1, rpo2, rpo3, and rpo4) first hybridized and then melted off of their M. tuberculosis rpoB gene amplicon target are shown. The presence of rifampin-resistant mutant DNA, as indicated by the peak marked with a red arrow, was consistently detected in samples containing as little as 10% mutant DNA and in a subset of samples containing as little as 5% mutant DNA. WT, wild type; mut., mutant.

FIG 4

Comparative dynamic range of Ultra and Xpert. Log dilutions of M. tuberculosis H37Rv spiked into sputum samples (minimum of 20 samples per dilution) were tested by Ultra and Xpert. The same dilutions of BCG were also tested by Ultra. (A) The C_T values for each CFU dilution are shown. Colored circles show the C_T of each positive result. Assay results that were negative are not shown, as these did not result in any C_T value. Colored shading shows ± 1 standard deviation. (B) The experimental results for the H37Rv dynamic-range study comparing the mean C_T values of each log dilution produced by Ultra versus Xpert are also shown. Error bars show ± 1 standard deviation.