Challenges, innovations and considerations for the use of tongue swabs in Mycobacterium tuberculosis complex detection

Abstract

Background Sputum-based testing is most used for diagnosing pulmonary tuberculosis (TB), but non-sputum specimens such as tongue swabs (TS) offer additional approaches for detecting Mycobacterium tuberculosis complex (MTBC). This two-phase study evaluated various storage, transport, and processing conditions to improve MTBC detection using TS tested on quantitative PCR (qPCR) and Xpert MTB/RIF Ultra (Ultra). Methods Adults (≥18 years) with Ultra-positive sputum were enrolled at a healthcare facility in Johannesburg, South Africa. In Phase 1, five serial TS were collected per participant and transported “dry” at 2–8°C, to the testing laboratory. In Phase 2, seven TS were collected: three stored “dry,” two in Tris-EDTA (TE) buffer, and two in PrimeStore® Molecular Transport Medium (MTM). Across both phases, pre-testing methods included heat lysis (HL)-only or HL combined with vortex bead beating (VBB), and storage at varying temperatures before qPCR. Swabs stored in MTM were tested on Ultra. Results A paired t -test revealed no statistically significant differences in Ct values between sequentially collected TS (adjusted p   > 0.05). Similarly, mean IS 6110 /IS 1081 Ct values did not differ between fresh and frozen samples (t (85) = −0.031, p  = 0.98; 95% CI: –0.44 to 0.43). VBB significantly reduced Ct values compared to HL alone (t (85) = −9.67, p  = 2.422 x 10 -15 ; 95% CI: −2.44 to −1.61). While storage conditions influenced Ct value and consistency to some extent, mean Ct values varied slightly from 32.48–34.28. Ultra detection improved when TS were processed with MTM and SR buffer. Conclusions MTBC detection from serially collected TS was variable, but Ct values were consistent across swab order and storage conditions. VBB improved yield on qPCR, and Ultra detection was enhanced with diluted SR buffer, highlighting the value of optimized processing. These findings support the continued development of TS-based TB diagnostics.