Optimal processing of tongue swab samples for Mycobacterium tuberculosis detection by the Xpert MTB/RIF Ultra assay

Abstract

Tongue swabs represent a potential alternative to sputum as a sample type for detecting pulmonary tuberculosis (TB) using molecular diagnostic tests. The methods used to process tongue swabs for testing in the WHO-recommended Xpert MTB/RIF Ultra (Xpert Ultra) assay vary greatly. We aimed to identify the optimal method for processing diagnostic tongue swabs for subsequent testing by Xpert Ultra. We compared four methods for treating dry tongue swabs with Xpert Ultra sample reagent (SR) mixed with various concentrations of Tris-EDTA-Tween (TET), to treatment with SR alone or to a commonly used SR-free heat-inactivation protocol. In each condition, swabs obtained from volunteers without TB were placed into test buffer spiked with known amounts ofMycobacterium tuberculosis(Mtb) strain H37Rv-mc26230. Swabs processed with 1:1 diluted SR buffer had the lowestMtblimit of detection (LOD) at 22.7 CFU/700 µl (95% CI 14.2-31.2), followed by 2:1 diluted SR buffer at 30.3 CFU/700 µl (95% CI 19.9-40.7), neat SR at 30.9 CFU/700 µl (95% CI 21.5-40.3) and SR prefilled in the Xpert Ultra at 57.1 CFU/700 µl (95% CI 42.4-71.7). Swabs processed using the heat-based protocol had the highest LOD (77.6 CFU/700 µl; 95% CI 51.2–104.0). Similar findings were observed for LOD of RIF-susceptibility. Assay sensitivity using the 2:1 diluted SR buffer did not vary considerably in the presence of sputum matrix or phosphate buffer saline. Further studies are needed to assess the performance of this processing protocol in a clinical setting.ImportanceXpert MTB/RIF Ultra (Xpert Ultra) is approved by the World Health Organization for the diagnosis of tuberculosis (TB). This test is typically performed using sputum specimens obtained from people with presumptive TB. In order to inactivateMtband aid liquefaction, sputum must be mixed with Xpert SR prior to transfer into the Xpert Ultra. However, some people under evaluation for TB are unable to produce sputum. Alternative sample types for TB diagnosis would therefore be of value. Oral-swabs, including tongue-swabs have shown promise, but there are technical challenges associated with sample processing. In this study, several new tongue swab processing conditions were evaluated, utilizing SR, either neat or diluted in buffer. The ability of Xpert Ultra to detect TB was improved under these conditions compared with the previously published heat-processing method (1–3), processing steps were simplified, and technical challenges were overcome.