There are differences in probe pattern missing in rifampicin resistance determining region of rpoB gene among primary and acquired resistant TB cases. This study aimed to observe the Xpert® MTB RIF Assay presenting probe missing in Hotspot RRDR of rpoB Gene among primary and acquired drug-resistant cases of pulmonary tuberculosis in a tertiary care setting of Lahore. This comparative cross-sectional work was undertaken at The University of Lahore in cooperation with the Department of

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... and HRI-NIH TB Research Centre KEMULahore. Confirmed TB patients being sent for Xpert® MTB RIF Assay were the target population in this study. Based on previous TB treatment history, patients were divided into primary and acquired drug-resistant TB cases. Samples were subjected to Xpert® MTB RIF Assay, and any probes missing, i.e., probes A, B, C, D or E, were observed. Data from a total of 170 cases were finalized for analysis consisting of 84(49.4%) acquired TB cases and 86(50.6%)primary TB cases. A total of 90(52.9%) male and 80(47.1%) females were enrolled. The mean age of patients was 35.45±16.11 years. The proportion of missing probe E (78.2%) remained at the topmost, followed by none of the probes missing at 8.3%, probe A at 5.3%, probe C at 3.5%, probe B at 2.9% and probe D at 1.8%. There are variations in frequencies of probe missing in RDRR regions of rpoB genes among rifampicin-resistant cases of primary andacquired TB. A high frequency of no missing probes among primary resistant was found.