Title: The vaginal microbiome of South African pregnant women living with Human
Immunodeficiency Virus (HIV) with and without Chlamydia trachomatis infection
Version: 0 Date: 05 Apr 2024
Reviewer's report:
The purpose of this study is to investigate differences in the vaginal microbiome of South
African pregnant women with and without CT infection. The various sections of the paper do
not align well – title, intro, methods, results and discussion should all intersect and feed into
the same axis point. Some of the results that are reported are redundant (e.g., info on Phyla,
Order, etc.) and could be presented as supplementary material. The authors should focus on the
differences in the microbiomes on genus and species level and hone in on these; these results
should then be compared against other studies and unique findings form the current study could
be highlighted in the context of pregnancy, HIV and CT infection. Unfortunately, the manuscript
is not acceptable for publication in its current form and consideration should be given to a
research letter instead as the sample size is small.
The study is hampered by the following factors:
- The findings of the microbiome analysis may well be confounded by gestational age
of participants. No information on gestational age was given. It is a well-known fact that
microbiome diversity is inversely related to gestational age.
- As for HIV infection, no information was given on treatment status or viral load. If participants
had been virally suppressed for a certain (?) period of time it could be assumed that HIV infection
was not a confounder, however, this can’t be excluded as a possibility
- What was the purpose of screening for BV? As the main goal was to look at microbiome
differences between the cohorts. The authors also report on the ‘BV prevalence’ but this is a
flawed statistic as it was in a very narrow, carefully-selected cohort. The authors should rather
have screened the whole cohort of women to give a better prevalence estimate.
- Please comment on the sequencing approach. Were there any technical replicates for the PCR
before amplicons were pooled, to minimise PCR bias? Why was Pacbio sequencing done and
not Illumina short-read sequencing? What was the sequencing depth?
Specific comments:
L35: which microbiomes?
L53: constitutes 9% of – relevance?
L63: competing with what?
L130: do no give results in methods section
L137: normal and positive what? Microflora
L173: refer to parent or study and give brief summary of applicable results
L212: what are ‘the stepwise methods’ ?
L233: It is odd to start a paragraph with ‘therefore’
L235: see ‘conclude’ – never interpret or discuss results in the discussion
L263: the authors did not explicitly lay out the hypothesis under investigation in the beginning
of the paper. Also, a hypothesis is never confirmed; it is rather rejected or not rejected. Please
use the correct terminology
L276: refrain from using non-scientific language such as ‘it is not surprising’
Figure 3 – why would you want to predict CT status? Won’t you rather expect a certain
microbiome given CT infection? You state in the very next paragraph that you want to ‘identify
a microbiome signature’? Again, the aim of this study is to investigate differences in the vaginal
microbiome of South African pregnant women with and without CT infection.
In your results section – focus on genera and species (especially the latter, as that where you
found the most differences). It is not common practice and does not make much sense to compare
Order, Family, etc., especially when bearing in mind that the ‘only’ difference was CT infection
The self-collection of vaginal swabs should also be explained in detail – how was this
standardised; and were these high-vaginal swabs? Collection method also affects results (species
differences in vaginal subsites)