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bioRxiv · 10.64898/2026.10.10.758171

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.

Chilambi, G. S.·Modi, K.·Rudra, P.·Chen, Y.·Chakravorty, S.·Alland, D.
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Abstract

A high rate of false rifampicin resistance (RIF-R) was recently reported in paucibacillary sputum samples tested with the Xpert(R) 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 manufacturers 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 370C) to determine the effect on RIF result calls. No false RIF-R results were observed in these samples with 105 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 200C 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.

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Chilambi, G. S., Modi, K., Rudra, P., Chen, Y., Chakravorty, S., Alland, D.. 2026-10-13. 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.. https://doi.org/10.64898/2026.10.10.758171

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