Review for 10.1186/s12876-023-02838-9 (2023)

Title: Prevalence of Helicobacter pylori Genotypes: cagA, vacA (m1), vacA (s1), babA2, dupA,
iceA1, oipA and Their Association with Gastrointestinal Diseases. A Cross-Sectional Study in
Quito-Ecuador
Version: 2 Date: 05 May 2023
Reviewer's report:
Bustos-Fraga and coworkers aimed to characterize the frequency of the presence of Helicobacter
pylori genes and alleles well known related to pathological characteristics of the stomach in
Quito, Ecuador. They found association of these genetic features of H.pylori with disease. The
results appeal since, to the best of my knowledge, this is the first study reporting those findings
in Ecuator.
Some aspects need to be clarified and addressed.
1. Do authors mean “to carry” verb when they use “expression”? “Expression” sounds good to
refer to the transcription and then translation of a gene in terms of regulation of this process.
Then, is better to use the verb “to carry” (or similar) instead of “expression” throughout the
manuscript.
2. Introduction. Authors generalize the findings of ref. 3 and 4 to Latin America. These
references are based on findings in Brazil.
3. Ref. 12 is not suitable to describe the fact that EPIYAs are associated (not “responsible”) with
gastric cancer. Please use one of the multiple reviews of cagA were this have been consolidated.
4. Introduction. Please consider to rephrase the sentence “ there is no research in Ecuador, which
identifies the bacterium's pathogenic involvement in peptic ulcer disease and specific types of
stomach cancer” to a more effective way to express the content of it.
5. Genotyping. Authors mention that biopsies were taken from antrum, body and incisura
angularis. Did they mix to extract DNA? Specifically, the sentence “ DNA was extracted using
the PureLink Genomic DNA kit” does not mention it.
6. The researchers detected H.pylori using end-point PCR for 16S gene. How they ruled out the
possibility of false negative results? Maybe comparing the rapid urease test result with the PCR
one would solve this aspect.
7. In line with false negative results for end-point PCR, some aspect need to be addressed.
- cagA: A negative result may results from a failure of the PCR or a pour load of bacterium DNA
in the mixture. Did authors replicate those with no band in agarose? A good idea is to run PCR
for empty-site, which primers have been described in literature.
- vacA m1: The literature characterizing vacA alleles always use primers to detect s2 and m2
alleles. If s1 is negative, then s2 should be positive and it rules out the possibility of false
negative. In addition, when one calculate OR for s1/m1 association with disease, s2/m2 is used
as reference category. In summary, I motivate authors to typify s2/m2 vacA alleles.
- iceA1, oipA and babA2: Since authors did not present a table describing the prevalence of
carriers of those genes, I can not evaluate whether there are potential false negative results.
Notwithstanding with it, authors are invited to discuss (at the end of the manuscript as a limitation
of the study) the possibility of false negatives in their results. Do the prevalence of positivity
of those genes be in line with the reported in literature? Is another argument to “validate” the
results in terms of false negative problem.
8. I  motivate authors to present their results of association of genes and alleles with disease in a
table consolidating the prevalence in each group (disease/no disease for example) and the ORs
(do not forget to indicate the reference category -i.e. the absence of the gene, vacA m2 allele,
etc.-). A benchmark for this table is available extensively in literature.
9. Limitations: “larger” instead of “bigger”.
10. Please consider to reduce the number of references to those most important. Seventy nine
references seems to be a large number.