Title: The vaginal microbiome of South African pregnant women living with Human
Immunodeficiency Virus (HIV) with and without Chlamydia trachomatis infection
Version: 0 Date: 23 Apr 2024
Reviewer's report:
Comparative analysis of the vaginal microbiome of South African pregnant women with and
without Chlamydia trachomatis infection
The aim needs to at least be specific with the issue of the targeted pregnant participants, that they
were living with HIV. Since only them were included. This can be done also looking at the title.
The abstract effectively provides a succinct summary of the key points of the manuscript.
Line 45-46 which control are you referring, wasn’t this part of the investigation / analysis? Or
those that not infected with C. trachomatis, their microbiome is known hence called the control
group.
The introduction and background provide sufficient motivation for the academic importance of
the broad topic under investigation. However, the title needs to be re-looked at.
Line 90: The heading of the section can also include the study design i.e., “study design and
population” to also accommodate all the information provided.
Line 92: the authors mention that “For the larger study, the sample size was calculated based on
the prevalence of HIV and STIs in our current setting”. Which study are you referring to? There's
no mention of weather this study comes from any of the bigger study, please if so, provide that
information or rather rectify.
Line 96-105: Please advise how the confidentiality was maintained in this study, i.e.,
participants’ identity, how the captured data was maintained etc.
Again, advise if there was any consideration regarding the issue of women who were on
antibiotic treatment prior the sample collection. If so, how was this done not to interfere with
the outcomes of this study.
Line 103-105: Was other STIs determined? How was it done and which STIs were considered?
What advise/ measures did the authors give or follow regarding the women who were found
to have C. trachomatis? Please advise on the measures/ procedures followed post detection to
assist the participants.
Line 130: the authors mention that “For the microbiome analysis, there were 20 samples that
only had C. trachomatis in them, and these were selected for sequencing.”  Then what are they
comparing in this study?? Please consider clarifying.
“To ensure that we had matched controls, we selected 20 samples that were free of any STIs.”
For what purpose?? Please consider clarifying this statement.
Line 173: why not all the samples tested positive for C. trachomatis subjected for microbiome
analysis?? Please elaborate and support your decision in this regard.
Line 175: was testing for BV enough to detect other STIs since is not considered a sexually
transmitted infection? Please elaborate.
Line 176: ‘’One sample failed BV screening” how was this determined and why didn’t authors
replace the sample failed the test with the other remaining samples they didn’t choose for this
analysis? Or maybe you may kindly elaborate on how the selection of samples was done.
Line 189-190: Actinobacteria were more abundant in C. trachomatis-infected women (38.18%)
compared to 29.52% in the control group. Which ones were control group? This is now
confusing, what are you comparing if now the other samples are called control group?? Weren’t
this so called “control part of the experiment for comparisons?? What is control??
Line 202: please same as above, rectify.
Line 209-213: wasn’t this supposed to be addressed under methodology section? Please
reconsider rewriting.
Line 227-229: May the authors please refrain from using control group referring to the samples
that were negative for C. trachomatis??
Line 224-226: Taken together, we conclude that the microbiomes of chlamydia patients are
distinct from those of control participants. How they were controls?? Please how did you make
selection for your control participant.
Line 237-238: please rectify the control on your graphs unless there is a valid reason for that.
Line 263: elaborate briefly on its confirmation.
Line 271: Please maintain consistency in your citations i.e. Ceccarani et al. (#number as it
appears on the reference list or whatever the case).
Line 284-285: same as above
Line 288: ‘’L. iners-dominated” write in full. Since it is mentioned for the first time in this
section.
Line 287-296: how is this information related to your findings? Please elaborate.
Line 297-302: this is just the repetition of results. Please consider re-writing to meet the
discussion purpose.
Line 312: was this bacterium found/detected in your study?? In which group was found
dominating? What could be the reason??
Line 318: not only pregnant women but those who are living with HIV.
Line321-322: why was this not included?? Please elaborate or else this needs to be mentioned
as part of your study design. This might have an impact on the C. trachomatis negative group,
thus saying they are negative while they were positive… then how will this be rectified??
Line 324-326; Lastly, a small sample size was used. Future studies with a larger sample size will
provide more clarity on the true association between C. trachomatis infection and the changes
in the vaginal microbiota. Please consider re-writing, in other words you are saying about what
you are presenting in this manuscript? here is not a true reflection, then what is it??
Line 404-407: this is the repeat of what has already been mentioned under the methodology
section. Please make a choice of where you would like to place this.