Review 1: "High-Sensitivity Detection of Mycobacterium Tuberculosis DNA in Tongue Swab Samples"

RR\ID Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: The manuscript demonstrated that tongue swab sampling followed by MTB qPCR may be used as an alternative to sputum samples (reference standard) for pulmonary TB diagnosis. One limitation of tongue swab samples has been the reported decreased sensitivity as compared to sputum for MTB qPCR assays. In order to overcome this limitation, the authors investigated two distinct sample concentration strategies to use with tongue swabs eluent: centrifugation/sedimentation and a sequence specific magnetic capture (SSMaC) method.

Previous literature has reported sensitivity limitations for the detection of TB from oral swabs, especially when not subjected to pre-PCR processing/concentration. This research builds on previous work by assessing two feasible sample concentration strategies for tongue swabs prior to TB qPCR. Additionally, the authors investigated the use of high-capacity open-cell foam swabs for collection to see if this enabled collection of a higher concentration of MTB bacilli, if present on the tongue. The authors described the methods clearly and with appropriate detail, likely for purposes of reproducibility. They also provided essential information about participant eligibility and study design.

Overall, both methods (foam swabs with sedimentation and SSMaC) exhibited higher sensitivity and specificity than FLOQSwabs that did not undergo sedimentation or SSMaC when compared to sputum samples from the same patients - which supports the stated conclusions of the study. However, I think it is necessary to compare these findings with FLOQSwabs that have been concentrated in the same manner as the foam swabs to confirm that foam swabs are superior to FLOQSwabs. Figure 3 helps readers visualize the claim that foam swabs with sedimentation contained higher concentrations of MTB DNA than those without sedimentation. The tables are visually clear and informative. I would recommend adding additional information in the introduction about associations with HIV and co-infections because Table 7, while interesting, seemed a little out of place.

The strengths of this study were the overall study design and presentation of the results, and potential impact on MTB testing (reducing occupational exposures and ease of sample collection).  Some limitations include the use of only frozen, retrospective samples which may bias results (many samples had high levels of MTB) and lack of clinical information for participants. These limit both the ability to generalize the study population and to predict how this process would work in settings that would use fresh (not frozen) specimens. However, the strengths and potential impact of the study are significant and overshadow these limitations.

In summary, this study supports the use of tongue swabs as an alternative sample type for pulmonary TB testing. The authors provide detailed information on two reportedly accurate and feasible methods of tongue swab sample concentration prior to MTB qPCR, which demonstrated increased sensitivity as compared to sputum samples and swabs that did not undergo concentration.