Beyond H. pylori: Streptococcal Co-Infections and Their Hidden Impact on Gastric Lesions in Vietnam

Comments 1: Study Design – The cross-sectional nature limits causal inference. The authors could emphasize this limitation and suggest prospective or mechanistic studies to confirm interactions among pathogens.

Response 1: We appreciate the reviewer's insightful comment regarding the limitations of our cross-sectional study design.

To address this, we have revised the limitations paragraph in the Discussion section to explicitly emphasise the inability to infer causality and to recommend future prospective and mechanistic studies.

“[updated text in the manuscript at line 203-217”

Comments 2: Selection of Participants – Additional clarification on inclusion/exclusion criteria (e.g., prior antibiotic or proton pump inhibitor use) would help assess possible biases in bacterial detection.

Response 2: We thank to the reviewer's comment on the need for additional clarification regarding the inclusion and exclusion criteria, particularly concerning prior use of antibiotics or proton pump inhibitors, to better evaluate potential biases in bacterial detection. To address this, we have revised the Study Population subsection (Section 2.1) to provide more explicit details on these criteria and their rationale in minimizing detection biases at the discussion section

Agree. We added into Discussion section, at line 135-145.

“[updated text in the manuscript]”

Comments 3: Mechanistic Discussion – The discussion could be expanded to hypothesize biological mechanisms through which SAG species exacerbate mucosal injury—e.g., local inflammation, biofilm formation, or immune modulation.

Response 3: Thank you for pointing this out. We agree with this comment.

- We added this information to the discussion section, and discussion.

“[updated text in the manuscript at line 185-202]”

Comments 4: The manuscript could benefit from clearer distinction between "infection," "co-infection," and "colonization" when referring to bacterial presence

Response 4: Thank you for your comment. We partly agree with it.

- In gastric microbiology, 'colonization' typically describes the establishment and persistence of bacteria, such as Helicobacter pylori or members of the Streptococcus anginosus group, on the mucosal surface without necessarily causing clinical symptoms or significant pathology, as observed in asymptomatic individuals where these microbes may reside as part of the commensal flora. In contrast, 'infection' refers to scenarios where bacterial presence leads to host immune activation, inflammation, and disease progression, such as chronic gastritis or precancerous lesions, often involving virulence factors and tissue invasion. 'Co-infection' denotes the concurrent detection of multiple pathogenic or opportunistic species, potentially resulting in synergistic interactions that amplify mucosal damage, as evidenced by altered gastric microbiota in H. pylori-positive cases allowing for increased colonization by non-H. pylori bacteria like streptococci. In this study, since all patients exhibited symptomatic gastritis, the PCR-based detection of H. pylori, S. anginosus, and S. constellatus is framed as indicative of infection contributing to pathology, rather than benign colonization; however, we recognize that molecular detection alone may not fully differentiate these states, and functional studies (e.g., assessing bacterial viability or host response) could provide further clarity in future research.

Comments 1: lines 42-45: the references are not recent, find recent studies and works on the topic.

Response 1: Thank you for pointing this out. We agree with this comment.

We have, accordingly, exchanged the recent studies as the references 2-7.

“[updated text in the manuscript at references from 2 to 7]”

Comments 2:  lines 46-47: add note.

Response 2: Agree. We have added recent studies to support this statement.

“[updated text in the manuscript at references 8 and 9]”

Comments 3: lines 47-49: in addition to the reference cited, add other more recent notes.

Response 3: Thank you for bringing this to our attention. We agree with this comment.

- We added 4 recent notes, from 10 to 13

“[updated text in the manuscript at references from 10 to 13, at line 49]”

Comments 4: lines 49-54 in addition to the references cited, add recent note

Response 4: Thank you for your comment. We agree with it.

- We added recent references from line 46-54

“[updated text, and reference in the manuscript at line 46-54]”

Comments 5: ?????? what type of PCR???

Response 5: Thank you for your comment.

- In fact, we used a real-time PCR SYBR Green assay with previously published primer sets to detect H. pylori, S. anginosus, and S. constellatus in gastric tissue. This information has been added to the Methods section.

“[updated text, and reference in the manuscript at line 81,82 ]”

Comments 6: “Previous studies have demonstrated that H. pylori can alter the gastric microbiota, thereby facilitating the colonization and proliferation of opportunistic bacteria such as S. anginosus and S. constellatus [11]”.

(Li, X.X., et al., Bacterial microbiota profiling in gastritis without Helicobacter pylori infection or non-steroidal anti-inflammatory drug use. PLoS One, 2009. 4(11): p. e7985).

““We hypothesized that when H. pylori is not present, other bacterial groups/species may contribute to or be associated with gastritis development. On the microbiota level, we also would like to address a key issue in the field: at what taxon depth(s) does the microbiota appear relatively stable such that perturbations at these levels may be relevant to human health? We used 16S rRNA gene cloning and sequencing to profile the stomach microbiota from normal and NHNN gastritis patients, and taxon-specific real-time quantitative PCR (qPCR) assays to quantify the relative abundance of the Firmicutes phylum and the Streptococcus genus”.

The work explains that the presence of other phyla are found in the stomachs of H. pylori-negative patients with antral gastritis but, it does not seem to me that the Authors are affirming the same thing!!

Response 6: Thank you for your comment. We strongly agree with your point.

This was an error on our part during the process of searching for and citing references. To correct this, I have removed the outdated reference and added two relevant references at line 158.

“[updated text, and reference in the manuscript line 158 ]”

Comments 7: “References 9-10:too old to integrate with newer ones like the other References.

The only References that should not be changed are 15 and 16; the others should absolutely be updated as I recommended.!!

Response 7: Thank you for your comment. We have updated our manuscript by replacing the old references with more recent studies and references throughout the revision process, in accordance with your comments above.

“[updated text, and reference in the manuscript ]”

Comments 8: Reference 11 should be reconsidered if the Authors change the title and the data needs to be revised or explained more clearly.

Response 8: Thank you for your comment. I have already addressed and revised this content above.

“[updated text, and reference in the manuscript ]”

Comments 1: Although the study used PCR analysis of gastric tissue samples to detect H. pylori, which typically indicates current infection, the article did not clearly distinguish between current and past H. pylori infections.

Response 1: Thank you for pointing this out. We partly agree with this comment.

PCR analysis of gastric biopsy samples was employed to detect the DNA of H. pylori, S. anginosus, and S. constellatus, which is indicative of current bacterial presence or active infection within the tissue, as this method amplifies genetic material from viable or recently viable organisms in the mucosal environment. Unlike serological assays, which detect circulating antibodies (e.g., IgG or IgA) and can reflect either current or resolved (past) H. pylori infections due to persistent immune responses post-eradication, PCR on biopsies provides specificity for ongoing colonization or infection at the time of sampling, though it may not differentiate between active replication and dormant states without complementary viability assessments. In this study, no serological testing was performed to evaluate historical exposures, as the focus was on correlating contemporaneous microbial detection with histological lesion patterns; however, this limits insights into prior H. pylori infections that may have contributed to cumulative mucosal damage. Future studies incorporating multimodal diagnostics (e.g., combining PCR with serology or urea breath tests) could better delineate the temporal dynamics of infections in gastritis progression.

Comments 2: The study focused on the infection rates of H. pylori, S. anginosus, and S. constellatus, as well as their effects on gastric lesions; however, it did not address other Helicobacter species. Other Helicobacter bacteria, such as Helicobacter heilmannii, are also known to be associated with gastritis and gastric lesions.

Response 2: We agree with your comments.

Focus of this paper is mainly on the Helicobacter pylori or on certain members of Streptococcus anginosus group, but we should not ignore other Helicobacter species (NHPH). These are transmitted from animals to humans and may cause such human gastric diseases as chronic gastritis, peptic ulcers and MALT lymphoma. H.Heimannii, a spiral bacterial organism, plays a less important role in gastritis than H.pylori. With symptoms, the reported worldwide prevalence ranges from 0.1% to 6% but its discovery among Asian populations like Vietnam is negligible (1-2%). NHPH infections can resemble H.pylori histologically but need special stains or molecular tests for diagnosis, as routine commercial tests often miss them. A limitation resulting from our failure to screen for NHPH according to the study's lack of NHPH identification may have under-estimated their role in lesion severity, but the reason for focusing on H. pylori was because of its higher prevalence in Vietnam. Future metagenomic sequencing could reveal Vietnamese gastritis patients' full Helicobacter diversity and its interplay with SAGs.

Comments 3: The study stated that upper gastrointestinal endoscopy and biopsy were used to diagnose gastritis. Still, no information was provided about the endoscopic equipment used or the experience and technical skill of the performing physicians.

Response 3: Thank you for pointing this out. We agree with this comment.

Upper gastrointestinal endoscopy was performed using high-definition flexible gastroscopes (e.g., Olympus GIF-H190 or equivalent models; Olympus Corporation, Tokyo, Japan) with narrow-band imaging (NBI) functionality to improve visualization of mucosal abnormalities and facilitate targeted biopsies. Procedures were conducted by experienced, board-certified gastroenterologists affiliated with the Institute of Gastroenterology and Hepatology at 108 Military Central Hospital, each possessing at least 10 years of specialized training and clinical practice in diagnostic upper GI endoscopy, thereby minimizing inter-operator variability and ensuring adherence to quality standards. Gastric tissue samples were collected from the antrum and corpus using standard biopsy forceps (e.g., Olympus FB-231K), following the updated Sydney System protocol for histological classification of gastritis. Specimens were initially used for the urease test to determine the presence of H. pylori. Subsequently, total DNA was extracted from the tissue, and PCR was performed to detect the presence of H. pylori, S. anginosus, and S. constellatus. Clinical data, including age, sex, symptoms, and laboratory results, were also recorded

- We edited and added this information to the Method section.

“[updated text in the manuscript at line 70-84]”

Comments 1: The Authors they respected my comments, and I believe the work is sufficient for publication in Gastroenterology Insights.

Remember, the name of the bacteria must always be written in italics!

Check the text..

Response 1: We thank the reviewer for their valuable comments throughout the review process. We also appreciate the positive high assessment provided and the confirmation that our answers appropriately addressed the raised concerns. We are glad to receive a qualitative recommendation to publish our paper in Gastroenterology Insights and support our study in evaluating the role of Helicobacter pylori, Streptococcus anginosus, and Streptococcus constellatus in the development of gastric lesions. Also, the reviewer requested to remind us about proper bacterial nomenclature, so we double-checked and assured that in the revised version of the manuscript, all bacterial names are written in italics following all rules and conventions. Thus, we found no non-italicized names after the repeated revision.

Comments 1: The authors revised their manuscript appropriately; no other comments are required..

Response 1: We thank the reviewer for their positive evaluation of our revisions and for confirming that no additional comments are required. We appreciate the careful review and constructive input, which have strengthened our manuscript on the impact of Helicobacter pylori and streptococcal co-infections in gastric lesions.