Excessive sample reagent incubation time increases the risk of false rifampin resistance results in Xpert ^® MTB/RIF Ultra tests performed on paucibacillary Mycobacterium tuberculosis sputum

A high rate of false rifampicin resistance (RIF-R) was recently reported in paucibacillary sputum samples tested with the Xpert ^® MTB/RIF Ultra (Ultra) assay. However, 96% of the studied facilities started the test 8 – 12 hours after addition of sample reagent (SR), exceeding the manufacturer’s recommended limit of 4 hours. Assessed whether excessive long SR incubation could lead to similar false RIF-R results in paucibacillary samples in a reference laboratory setting. We evaluated the performance of the Ultra assay following incubation of TB-negative sputum spiked with 300 CFU/mL and 100,000 CFU/mL levels of Mycobacterium tuberculosis (Mtb) H37Rv in SR for different durations (15 min to 24hrs.) at different temperatures (-20 to 37°C) to determine the effect on RIF result calls. No false RIF-R results were observed in these samples with 10 ⁵ CFU/mL Mtb at any of the conditions tested, even when they deviated largely from recommended limits. Similarly, freezing the 300 CFU/mL SR treated samples at minus 20°C for 18 h also prevented the development of false RIF-R results. However, sputum spiked with 300 CFU/ml Mtb, produced false RIF-R calls at longer incubation times and higher incubation temperatures than are recommended by the manufacturer. Approximately half of the results displayed melt patterns consistent with hetero-resistance, indicating SR-induced mutagenesis in a wild-type background. This study demonstrates that extended off-label SR incubation of paucibacillary specimens beyond the recommended 4-hour limit can generate false RIF-R results. Freezing paucibacillary specimens in SR may mitigate this risk although it is not yet an approved procedure.