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

Recent studies have shown that the Xpert(R) MTB/RIF Ultra (Ultra) assay can produce a high rate of false rifampicin resistance (RIF-R) results in paucibacillary sputum samples when incubation times exceed the recommended 4-hour limit. To investigate this issue further, a reference laboratory conducted a series of experiments to determine the impact of prolonged SR incubation on RIF result calls.

The researchers tested the Ultra assay using TB-negative sputum spiked with varying levels of Mycobacterium tuberculosis (Mtb) H37Rv, ranging from 300 CFU/mL to 100,000 CFU/mL, under different incubation durations and temperatures. The results showed that no false RIF-R results were observed in the samples with 105 CFU/mL Mtb, even when the incubation conditions significantly deviated from the manufacturer's recommendations.

Furthermore, freezing the 300 CFU/mL SR-treated samples at -20°C for 18 hours also prevented the development of false RIF-R results. However, when the sputum was spiked with 300 CFU/mL Mtb, false RIF-R calls were observed at longer incubation times and higher incubation temperatures than those recommended by the manufacturer. Around half of these results displayed melt patterns consistent with hetero-resistance, which is believed to be due to SR-induced mutagenesis in the wild-type background.

This study highlights the potential risk of false RIF-R results when the recommended SR incubation time is exceeded, particularly in paucibacillary Mycobacterium tuberculosis sputum samples. Freezing the samples in SR may help mitigate this risk, although it is not yet an approved procedure. The findings emphasize the importance of adhering to the manufacturer's recommended incubation times to ensure accurate test results.