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 of Mycobacterium tuberculosis (Mtb) strain H37Rv-mc26230. Swabs processed with 1:1 diluted SR buffer had the lowest Mtb limit 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. Importance Xpert 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 inactivate Mtb and 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. Competing Interest Statement D.A receives financial support from Cepheid in the form of research grants, laboratory equipment, and supplies. Cepheid also pays licensing fees for some Cepheid products and a portion of these fees is paid to Dr. Alland and to his laboratory at Rutgers University. Funding Statement Research reported in this publication was supported by the National Institute of Allergy And Infectious Diseases of the National Institutes of Health under Award Number U01AI152084. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the Rutgers Institutional Review Board (IRB protocol #Pro2021002454). The use of sputum from leftover clinical specimens was approved by the Rutgers Institutional Review Board, IRB protocol numbers 020160000657 and 0120090104. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All data produced in the present work are contained in the manuscript.