Background: Background. The Xpert MTB/RIF (Xpert) assay, the first near-patient test for tuberculosis (TB), detects Mycobacterium tuberculosis (Mtb) with a limit of detection (LOD) of 133 CFU/ml sputum, and detects mutations in the Mtb rpoB gene which cause Rifampicin resistance (RIF-R). However, despite high sensitivity overall, Xpert sensitivity is only 60 – 80% in smear-negative TB. Also, small numbers of false RIF-R have been reported recently, usually due to abnormal real-time PCR curves or miss-identification of RIF-susceptible (RIF-S) synonymous rpoB mutants as RIF-R. A new molecular TB test with an LOD equivalent to the 10 – 100 CFU/ml LOD of liquid culture and improved RIF-R detection is needed.
Methods: Methods. For the Xpert MTB/RIF Ultra (Ultra) assay, we developed a new sample processing cartridge that doubled the amount of purified DNA delivered to the PCR reaction. Four newly designed probes that detected mutations in rpoB gene replaced the five Xpert real-time probes. Real-time Mtb detecting probes targeting IS6110 and IS1081 were added. Cartridge fluidics and PCR cycling were optimized. Assay LODs were tested by spiking Mtb H37Rv and BCG cells into sputum samples, treating with Sample Reagent, splitting samples, and testing with Xpert and Ultra. RIF-R detection was tested with a panel of 30 different RIF-R Mtb rpoB mutants. LOD was defined as the lowest CFU that could be detected in at least 19/20 (95%) tests.
Results: Results. Ultra was significantly more sensitive than Xpert. In sputum samples spiked with Mtb H37Rv, Ultra had an LOD of 5 CFU/ml compared to an LOD of 50 CFU/ml for Xpert (p=0.001). In sputum samples spiked with BCG, Ultra had an LOD of 25 CFU/ml compared to an LOD of 165 CFU/ml for Xpert (p=0.01). Ultra detected 30 different RIF-R Mtb rpoB mutants as RIF-R (sensitivity 100%). None of the 25 RIF-S rpoB wild type samples and none of the 3 RIF-S synonymous rpoB Q513Q (1) and F514F (2) mutant samples were detected as RIF-R (specificity 100%). Ease of use was identical for Xpert and Ultra.
Conclusions: Conclusions. The new Ultra assay is much more sensitive than Xpert, and is likely to be as sensitive as liquid TB culture. Ultra detects RIF-R as efficiently as Xpert; but the specificity of Ultra RIF-R is likely to be higher due to improvements in assay design. The Ultra assay should significantly increase TB detection in smear-negative patients and provide more reliable RIF-R detection.