Improving the sensitivity of the Xpert MTB/RIF assay on sputum pellets by decreasing the amount of added sample reagent: a laboratory and clinical evaluation.

ABSTRACT

The Xpert MTB/RIF (Xpert) assay permits rapid near-patient detection of Mycobacterium tuberculosis in sputum; however, the test sensitivity remains suboptimal in paucibacillary specimens that are negative for acid-fast bacilli using smear microscopy. Xpert testing includes dilution with sample reagent, and when processed sputum pellets are tested, the recommended sample reagent/pellet ratio is 31. We evaluated whether a decreased sample reagent/pellet ratio of 21 increased Xpert sensitivity compared to the recommended 31. The limit of detection was determined by inoculating serial dilutions of M. tuberculosis into sputum samples, preparing sputum pellets, and testing each pellet by Xpert at both sample reagent ratios. Processed sputum pellets obtained from M. tuberculosis culture-positive clinical specimens were also tested by Xpert at both ratios. Among spiked sputum pellets, the limit of detection was 1,478 CFU/ml (95% confidence interval [CI], 1,211 to 1,943) at a 31 ratio and decreased to 832 CFU/ml (95% CI, 671 to 1,134) at 21. The proportion of specimens in which M. tuberculosis was detected was greater at 21 than at 31 for almost all numbers of CFU/ml; this difference was most prominent at lower numbers of CFU/ml. Among 134 concentrated sputum pellets from the clinical study, the sensitivity of Xpert at 21 was greater than at 31 overall (80% versus 72%; P=0.03) and for smear-negative specimens (67% versus 58%; P=0.12). For Xpert testing of sputum pellets, using a lower sample reagent/pellet ratio increased M. tuberculosis detection, especially for paucibacillary specimens. Our study supports use of a 21 sample reagent/pellet dilution for Xpert testing of sputum pellets.