Review 3: "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 text is well structured but is partially a bit tricky to read. Please consider making abbreviations that better distinguish GenoType MTBDRplus and FluoroType MTBDR in the text and make supporting tables.

Summary: 

• The objective of the study is not stated

• Line 32: “most in favour of “ What do you mean??

• Lines 40-42: How do you know that Ultra is more correct? What do you mean by “lower load and the Ultra heteroresistance probe pattern were risk factors for Ultra false rifampicin-resistant results”? Rephrase, please

Introduction:

• Line 58: Heteroresistance is not the only possible reason for discordance between direct testing and testing from isolate.

• Line 66: Explain how the detection of mutations works on the GeneXpert and the phenomenon of probe delay

• Lines 75-77: You should consider that the methods have different potentials for detecting specific mutations. With GT-MTBDR, you will be able to find fewer mutations than FT MTBDR, and you will probably detect even more mutations with the targeted sequencing. 

Materials and methods:

Routine diagnostic algorithm

• Was direct microscopy done? Samples taken one hour apart could have different amounts/concentrations of bacteria. If the patient has more than one strain or has heteroresistant bacteria, the composition may differ in the two samples. Some rough estimates of the difference in the number of bacteria can be seen with microscopy. This should be discussed in the Discussion section.

• Line 110: Specify the inactivation reagent

• Line 139: What statistics are evaluated by the prtesti command?

Results:

• The readability would improve if the authors would use one or more tables.

Discussion:

• Discuss the clinical relevance of heteroresistance. When is it essential to detect heteroresistance? At what level of resistance is heteroresistance of clinical significance? In this context, what is the importance of the multi-drug treatment regimens? To me, detecting the resistant part of the heteroresistance is crucial, whereas detecting the susceptible part is not.

• Discuss other potential reasons for false positive or false resistance results with GeneXpert.

• The SMOR method has similarities with the Deeplex method. This should be discussed. 

• Discuss the fact that the potential of detecting mutations is different between methods (see comment for lines 75-77)

• Lines 252-253 I doubt that FT-MTBDR ever will offer a heteroresistance read-out. If you know more, give a reference.

• Line 269, the term sub-phenotypic should not be used. It is a very illogical term, which is supposed to indicate that a strain is phenotypically resistant where a genetic correlate has not been found with the method used. A strain is either phenotypically susceptible or resistant, depending on the breakpoint. There is more than one mechanism that can cause resistance than single-point mutations.