Review 2: "Xpert MTB/RIF Ultra Resistant and MTBDRplus Susceptible Rifampicin Results in People with Tuberculosis: Utility of FluoroType MTBDR and Deep Sequencing"

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 provides a detailed and compelling analysis of the discordance between Xpert MTB/RIF Ultra and MTBDRplus in detecting rifampicin-resistant tuberculosis (TB), with significant clinical implications. Discrepancies between two widely used WHO approved molecular approaches Xpert Ultra and MTBDRplus complicate clinical management potentially due to heteroresistance and potential culture bias. The study is commendable for its rigorous methodology, including the use of FluoroType MTBDR and targeted deep sequencing as comparators, which enhance the robustness of its findings. However, several aspects could be improved for clarity, relevance, and impact. 

1. Strengths:

   • The study addresses a critical gap in understanding discrepancies in TB diagnostic tools, particularly in high-burden settings like South Africa.

   • Likely one of the first few studies to describe rifampicin susceptibility discordance between two rapid molecular tests Xpert Ultra and MTBDRplus.

   • The use of multiple advanced diagnostic methods, including single-molecule overlapping reads (SMOR), provides a strong reference standard and lends credibility to the findings.

   • The discussion highlights practical implications for laboratory algorithms and the need for isoniazid susceptibility testing, which are crucial for improving TB management.

2. Areas for Improvement:

   • Introduction:

     • While the background is well-articulated, the introduction could benefit from a clearer justification for selecting FluoroType MTBDR as a comparator over other potential diagnostics. 

     • What have been some of the main challenges in clinical management of patients that have been because of drug susceptibility and discordant results?

   • Methods:

     • More details are needed on the selection process for discordant isolates and the statistical rationale for sample size. This would help contextualize the findings and their generalizability. This is also needed to describe how the patient specimens included were selected.

     • The definition of heteroresistance and its operational implications could be better linked to clinical decision-making.

     • Was there any data collected/available on clinical outcomes, and some data on treatment initiation/regimen?

   • Results:

     • Although comprehensive, the results section could be streamlined to improve readability. The first paragraph describing the frequency of discordant rifampicin results sets a good pace. Grouping findings under thematic subheadings would enhance clarity. The other sections where the more technical aspects are presented could be restructured to improve readability and also more relevant to programmatic aspects. Suggested groupings include i) heteroresistance, ii) performance characteristics (sensitivity/specificity, positive predictive value etc) and iii) parameters associated with discordance/heteroresistance and rifampicin mono-resistance, could be presented as such to improve readability.

     • The study mentions the association of low bacterial load with false-positive Ultra results, but additional data or discussion on how this can be mitigated in practice would be valuable.

   • Discussion:

     • While the findings are contextualized effectively, the authors could delve deeper into the broader policy implications of scaling up FluoroType MTBDR or similar methods in resource-limited settings.

     • It would be beneficial to address how these findings compare with other studies investigating Ultra’s performance in different populations.

     • What are the implications in routine management of patients in South Africa?

     • Comment on the practicality of resolving discordant results in routine laboratory settings and in the context of the new testing algorithm implemented in South Africa which now excludes MTBDRplus.

     • What are some of the key considerations and implications for introducing FT MTBDR, sequencing and other molecular approaches in high burden settings with limited resources?

     • What are the implications of heteroresistance in clinical management of patients?

   • Figures and Tables:

     • The figures and tables are informative but could benefit from improved labeling and captions that summarize key takeaways. For example, highlighting the clinical significance of specific discordant categories would make the data more accessible.

3. Conclusion:

   • The conclusion effectively summarizes the findings but should explicitly call out actionable recommendations for implementing the study insights into TB diagnostic programs.

4. Minor Suggestions:

   • Ensure consistent terminology, particularly for abbreviations like "Ultra," "MTBDRplus," and "FT-MTBDR."

   • Proofreading for minor typographical errors and grammatical improvements would enhance readability.

Overall, this manuscript significantly contributes to the field by identifying limitations in existing TB diagnostics and proposing a path forward with alternative tools. Addressing the suggested improvements will enhance the manuscript's clarity, accessibility, and practical relevance to global TB control efforts.