Impact of sputum quality on Xpert MTB/RIF Ultra test results for tuberculosis: A multi-country study

ABSTRACT Rationale Sputum-based testing using Xpert MTB/RIF Ultra (Xpert) is the most common molecular testing method for diagnosing tuberculosis (TB). Objectives To evaluate whether sputum quality influences Xpert positivity and diagnostic accuracy. Methods We screened consecutive people for presumptive TB in India, the Philippines, Vietnam, Nigeria, South Africa, Uganda, and Zambia as part of the R2D2 TB Network and ADAPT studies. Participants provided 2-3 sputum samples for Xpert and culture reference testing. The quality of the first sputum sample was graded following standardized procedures by trained research staff and used for Xpert testing. We performed logistic regression to evaluate whether sputum grade was independently associated with Xpert positivity, and calculated sensitivity and specificity of Xpert against a culture-based microbiological reference standard (MRS). Measurements and Main Results Among 1,855 participants, 798 (43%) were female, 348 (19%) were living with HIV (PLHIV), and 1795 (97%) had a cough of ≥2 weeks. Overall, 313 (17%) had a positive Xpert result. Most sputum samples were salivary (83%). Xpert positivity was lowest among salivary samples (16.1%) and highest among purulent samples (31.2%). After adjusting for demographic and clinical variables, there was no significant association between any sputum grade and Xpert positivity. Xpert sensitivity (salivary: 89%, mucoid: 91%, mucopurulent: 87%, purulent: 100%) and specificity (>98%) were high across sputum grades. Conclusions Sputum quality was not independently associated with Xpert positivity and Xpert sensitivity was high across all sputum grades. These findings support molecular testing of all sputum samples for TB diagnosis regardless of macroscopic appearance. Scientific Knowledge on the Subject Xpert MTB/RIF Ultra (Xpert Ultra) is a WHO-recommended initial diagnostic test for pulmonary tuberculosis. Historical laboratory practice, derived from the smear microscopy era, has emphasized sputum macroscopic quality, with many programs recommending rejection of salivary sputum as inadequate. Prior studies evaluating sputum quality and Xpert performance yielded mixed results, and were limited by small sample sizes, single-country designs, heterogeneous sputum grading systems, and predominant use of the first-generation Xpert MTB/RIF assay. What This Study Adds to the Field In this large and geographically diverse evaluation of 1,855 participants across seven high-burden countries, macroscopic sputum quality was not independently associated with Xpert Ultra positivity or diagnostic accuracy. Xpert Ultra demonstrated high sensitivity and specificity across all sputum grades, including salivary specimens, which comprised over 80% of samples. These findings, generated by using a standardized sputum grading protocol and a culture-based microbiological reference standard, provide robust evidence that sputum quality-based rejection criteria are unnecessary for Xpert Ultra testing. Revising laboratory guidelines to accept all expectorated sputum regardless of appearance could improve case detection and reduce diagnostic delays in high-burden settings.