Review Reports - The Prevalence, Risk Factors, and Antimicrobial Resistance Determinants of <i>Helicobacter pylori</i> Detected in Dyspeptic Patients in North–Central Bangladesh

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the manuscript titled "Prevalence, risk factors and antimicrobial resistance determinants of Helicobacter pylori detected in dyspeptic patients in north-central Bangladesh", Syeda Jannatul Ferdaus et al describe the epidemiological features of H. pylori infection in the area outside the capital in Bangladesh, in endoscopic gastrointestinal biopsy samples collected from dyspeptic patients. The study is interesting in terms of showing prevalence of H. pylori in dyspeptic patients, highlighting risk factors, and exploring antimicrobial resistance.

Please see my comments below:

1. The authors mention "H. pylori was most prevalent in an age group of 41-50 year-old, being more common in male" but from the table 2 there is no SD seen in p value between sexes or among difference? Can authors comment how did they conclude this finding?

2. Looking at tables 1 and 2, there is no SD difference for any variable in infected vs non-infected groups which is surprising as it is well established in literature that H.pylori is associated with some of these variables including smoking (PMID: 23853433) Could authors explain why they observed a trend divergent from what is established or presented in the literature?

3. To follow up on previous question, how did authors determine sample size for this study?

4. BMI and family history of peptic ulcer are also associated with H. pylori infections. Did authors measure these parameters for this study?

5. Did any of these patients had history of H.pylori infection before?

6. It is interesting that among PCR-positive specimens (n=71), 36 (51%) were positive for both cagA and 147 vacA, while 8 (11%) was cagA(-)/vacA(+). Since both cagA and vac A genotypes have been shown to be more prevalent in PUD patients.

7. Please write a small paragraph on future directions.

revand The present study revealed the prevalence of H. pylori in dyspeptic patients in north- 237 central Bangladesh, along with the risk factors, AMR and a part of their genetic mecha- 238 nisms. Because H. pylori infection was found to persist as a cause of gastrointestinal dis- 239 eases associated with AMR, further monitoring and prudent antibiotic therapy are neces- 240 sary.

Author Response

Please see my comments below:

The authors mention "H. pylori was most prevalent in an age group of 41-50 year-old, being more common in male" but from the table 2 there is no SD seen in p value between sexes or among difference? Can authors comment how did they conclude this finding?

Response: Thank you for the valuable comments. We agree to the view of the reviewer. Actually, HP was common in the age group 41-50 year-old and male, and urban residents and those of lower socioeconomic status. However, those rates were not significantly different from those of HP-negative group. Though the actual reason was not evident, we presume that it was caused in part by the presence of false-negative samples. As written in “limitation of the study” in Discussion section, there were some reasons for it. In Discussion section, we revised description as follows. “For detection of H. pylori, only one biopsy sample was taken from a patient, which might cause false-negative results because of irregular distribution of bacteria in the gastric mucosa, resulting in a potentially lower positive rate. Thus, it is possible that the significant difference in incidence rates of any risk factors might have been not evident between H. pylori-positive and -negative groups.” (Page 6, 2^nd para.)

Looking at tables 1 and 2, there is no SD difference for any variable in infected vs non-infected groups which is surprising as it is well established in literature that H.pylori is associated with some of these variables including smoking (PMID: 23853433) Could authors explain why they observed a trend divergent from what is established or presented in the literature?

Response. Thank you for the comments. As described above response, presence of false-negative samples may mask the significant difference between HP-positive and -negative groups. In addition, another possible reason is that this study was conducted in rural area of low-income country, where majority of people are living in lower socioeconomic status. In such situation, difference of risk factors might not be so evident. This view was briefly mentioned in revised version in Discussion section, as follows. (Page 5, last para.) “Although significant difference was not found, H. pylori-positive cases showed higher proportion of some risk factors, e.g., residence in rural area, nonfiltered water, and smoking, compared with H. pylori-negative cases. This finding is consistent with the view that H. pylori infection is associated with dietary habits and lifestyle as described previously [5,6]. However, >60% of both H. pylori-positive and -negative cases were categorized into a group of lower socioeconomic status in our study, probably due to local area in low-income country.”

To follow up on previous question, how did authors determine sample size for this study?

Response : Thank you for the question. The minimal sample size was detected by applying a single proportion of the population formula, N = Z^2*(P*(1-P))/E^2. (confidence interval (Z), total error (E), and proportion (P) of the target population). The calculated sample saize was 362. However, in this study, we could collect 221 samples, during the one year period. The actual sample number was lower, because the biopsy samples were taken from individual patients, and the number of patients in Mymensingh Medical College hospital did not reach the aimed number. If more number of samples are obtained, risk factors of H. pylori infections would be more evident.

BMI and family history of peptic ulcer are also associated with H. pylori infections. Did authors measure these parameters for this study?

Response : Thank you for indicating the point. Information of BMI (hight and weight) and family history were obtained from patients, but could not be obtained from all the patients, and thus data collection was incomplete. That was why they were not included to the analysis.

Did any of these patients had history of H.pylori infection before?

Response : Thank you for the question. Yes, history of H. pylori infection was recorded for some part of patients. But not all of them were accurately diagnosed as H. pylori infection. Samples that were analyzed for AMR genes were all those had previously presumptive infection with H. pylori and antimicrobial treatment. Family history of PUD was recorded for all patients and added to Table 2.

It is interesting that among PCR-positive specimens (n=71), 36 (51%) were positive for both cagA and vacA, while 8 (11%) was cagA(-)/vacA(+). Since both cagA and vac A genotypes have been shown to be more prevalent in PUD patients.

Response : Thank you for the comments. Because the number of samples tested was not enough, we need to analyze more samples for understanding relatedness of vacA/cagA and disease type.

Please write a small paragraph on future directions.

Response : Thank you for the suggestion. In the final paragraph of Discussion section, the future directions from our present study was added.

Reviewer 2 Report

Comments and Suggestions for Authors

This is a nice study of H. pylori prevalence and antibiotic resistance in north-central Bangladesh. It is helpful and interesting to see how H. pylori incidence and antibiotic resistance are changing all over the world, especially in places outside the major cities.

The authors describe some of the patients as having lower socioeconomic status, but how this was determined is not clear. Was that assigned by the specific neighborhood of residence? Income? Means of livelihood? Please clarify.

In the abstract, the authors note that H. pylori infection was related to sex, age, and other variables, but one must read the full text to understand that none of these differences were statistically significant. The lack of statistical significance should appear in the abstract. Or those relationships could be omitted from the abstract.

Comments on the Quality of English Language

Detailed comments on the text:

Line 26: Please correct to “determinants”.

Line 27: Please correct to “patients for a one-year”.

L32: Please correct to “more common in males”. Also “41-50 year-olds” as this language refers to the persons in that age range.

L46: Please correct to “of the human stomach”.

L48: Please correct to “Among the H. pylori-positive”.

L50: Please correct to “that H. pylori eradication”.

L51: Word division is non-standard. If the word “although” needs to be divided in the final paper, it should be done as “al-though”.

L53: This text could be improved as “is still a global challenge. The increased risk of”.

L54: This text would be better as “environmental factors, including living in developing”.

L56: Please correct to “dietary habits”. The next sentence is a bit confusing and would be better as “such risk factors may contribute”.

L58: Please correct to “asymptomatic populations was described” for the subject “seroprevalence”.

L59: Please correct to “in India) compared with other Asian regions more than two decades ago”.

L60: Please correct to “In a recent”.

L67: Please correct to “However, high rates of resistance”.

L68: Please correct to “in contrast to a low rate”.

L69: This text would be better as “Furthermore, in Bangladesh, mutations responsible for antimicrobial resistance (AMR) were detected in H. pylori genes including”.

L72: “underscore” is inappropriate here and needs to be replaced with something else, maybe “provide a rationale for”.

L76: To avoid having 2 variants of “study” so close together, this text could be improved as “In the present study, we examined the latest”.

L81: Observational studies are descriptive, so this text could be shortened to “a cross-sectional, observational study”.

L83: Please correct to “attending the gastroenterology”.

L84: Please correct to “Patients who presented with symptoms of dyspepsia for more than one month were included”.

L85: Please correct to “one or more of the following:”

L88: Please correct to “as follows:” (use a colon, not a semicolon).

L92: Please correct to “previously diagnosed stomach cancer”.

L94: Please correct to “were stored at -80”.

L96: Please correct to “in the specimens was performed” because the subject is “Detection”.

L101: Please correct to “size with primers” because there are a pair of primers.

L103: Please correct to “strains”.

L105: Usually if the word “gene” is present in the name, “the” is used. For example, it is more natural to write “sequences of the PBP-1A gene” and in line 107, “Deletion of the rdxA gene”. Also in line 122, “targeting the 16S”.

L106: Please correct to “of PCR products”.

L108: This text would be better as “was examined by PCR”.

L109: Please correct to “PCR products” and “mutations (formerly”.

L110: Please correct to “a common mechanism of CLA resistance, were analyzed”.

L111: H. pylori should be italicized.

L123: Probably the authors mean “positive by both screening methods”.

L124: This text would be better as “cases were defined as those positive in either”.

L139: The 3 age groups should be plural, as “41-50 year-olds”.

L140: Please correct to “more common in males than females”.

L141: Please correct to “lived in rural areas”.

L144: Please correct to “differences in prevalence”.

L145: Please correct to “were not statistically significant”.

L148: Please correct to “were cagA”, because the subject is “8”.

L149: This text would be better as “only one sample showed s2”. Or “only one sample had s2”.

L156: Please correct to “cases that had taken antimicrobial” or “cases that had been given antimicrobial”.

L162: Please correct to “in the quinolone-resistance-determining region”.

L168: Please correct to “a subset of dyspepsias, which represent a wide spectrum”. This change in the verb makes the spectrum refer to the dyspepsias.

L169: Please correct to “25% of the population”.

L181: The text “presumably without regional difference” should be removed, as there is no data on which this statement is based, is there? The logic of this statement is not clear, as the purpose of this paper is to ask the question of whether there are regional differences within Bangladesh.

L188: This text would be better as “indicating that high”.

L189: Please correct to “in some populations. Globally, prevalence”.

L191: Please correct to “hygienic conditions”.

L193: Please correct to “monitoring the same area/populations with”.

L195: Please correct to “in males and in the 41-50 year age group, which was similar to that observed in another study in Bangladesh”.

L199: This text would be better as “This finding is consistent with the view”, because it would usually be people, not a finding, agreeing with a view.

L200: This text could be shortened to “>60% of both H. pylori-positive”.

L204: Please correct to “hygienic conditions”.

L205: This text would be better as “review of AMR in H. pylori”.

L206: Please correct to “the highest resistance rate”.

L207: It is not clear what the authors mean by “latest prevalence with their increasing trend”. Do they mean least? Who corresponds to “their”? The antecedent is unnamed.

L208: What is meant by “more remarkable”? Highest? Please use a more exact word. Please correct to “South Asian”.

L213: Please correct to “those who received CLA”.

L216: Probably the authors mean “a relative decrease in MTZ resistance”. Also “Such a difference”.

L221: Please correct to “of CLA-resistant strains”.

L222: The citation seems to have an error or have something missing.

L223: Please correct to “LVX-resistant”.

L227: Please correct to “and was found in”.

L229: To call attention to physicians means to examine the physicians. Probably the authors mean “which may suggest the need for eradication therapy for H. pylori infection.”

L232: Please correct to “false-negative results”.

L233: Please correct to “a potentially lower positive rate”.

L234: This text would be clearer as “genes and mutations”.

Fig S1: Please correct to “BsaI digestion of PCR products”, because many products are generated.

L14, 16, 256: Department identifiers such as Gastroenterology and Microbiology should be capitalized. Also “College” in L18.

Author Response

This is a nice study of H. pylori prevalence and antibiotic resistance in north-central Bangladesh. It is helpful and interesting to see how H. pylori incidence and antibiotic resistance are changing all over the world, especially in places outside the major cities.

Response : Thank you for the question. Lower socioeconomic status was determined by annual income, based on the standard published by the World Bank (low-income economics, per capita income, Jul.2021). In a data sheet of patient, the socioeconomic status was checked when a patient was interviewed by investigators. This was briefly added to footnote of Table 2 in the revised manuscript.

In the abstract, the authors note that H. pylori infection was related to sex, age, and other variables, but one must read the full text to understand that none of these differences were statistically significant. The lack of statistical significance should appear in the abstract. Or those relationships could be omitted from the abstract.

Response : Thank you for the comment. According to the suggestion, he lack of statistical significance was added, as follows. “H. pylori was most prevalent in an age group of 41-50 year-olds, being more common in males, and rural residents having lower economic status, using nonfiltered water, though the rates of these factors were not significantly different from those of H. pylori-negative group.”

Comments on the Quality of English Language :

Response : Thank you for extensive and careful check for throughout our original manuscript. All the points indicated were corrected accordingly.

Reviewer 3 Report

Comments and Suggestions for Authors

General:

In this study, the authors determined prevalence of H. pylori among dyspeptic patients in north-central Bangladesh, and to analyze risk factors of the infection and antimicrobial resistance determinant in the pathogen. Authors showed that relatively higher frequency was noted for A2147G mutation in 23S rRNA, related to clarithromycin resistance (18%, 7/39).

This study was well written and may have potential.

Major comments:

1. Please revise kinds of diseases (table 1) in normal, atrophic gastritis (mild, moderate, severe), gastric ulcer, duodenal ulcer, and gastric cancer.

2. In general, all strains have vacA. Therefore, 27 samples with vacA-negative may have mismeasurement.

3. In addition, please measure genotype of vacA.

4. How did authors select 30 patients of 80 patients to measure mutations related to antimicrobial resistance in H. pylori?

5. References 12-14 showed that considerably high rate of resistance of H. pylori CAM (10- 30%), MNZ (78-95%), LVFX (66%) have been documented in some reports, in contrast to 68 low rate against AMX (3.6-6.6%). However, in this study, resistance rate of MNZ was 7%. What do authors think this discrepancy?

Author Response

General:

In this study, the authors determined prevalence of H. pylori among dyspeptic patients in north-central Bangladesh, and to analyze risk factors of the infection and antimicrobial resistance determinant in the pathogen. Authors showed that relatively higher frequency was noted for A2147G mutation in 23S rRNA, related to clarithromycin resistance (18%, 7/39). This study was well written and may have potential.

Major comments:

Please revise kinds of diseases (table 1) in normal, atrophic gastritis (mild, moderate, severe), gastric ulcer, duodenal ulcer, and gastric cancer.

Response : Thank you for the valuable suggestion. Accordingly, the order of disease items and some disease names in Table 1 were corrected. We listed “Normal mucosa”, “Gastric ulcer”, “Duodenal ulcer”, “Gastric cancer”, and “GERD”. However, “Antral gastritis”, “Erosive gastritis”, “Nodular gastritis” are kept as they are in the Table, because these are difficult to assign to “Atrophic gastritis (mild, moderate, severe)”, and classification criteria of Atrophic gastritis are different from the former three categories. I am convinced that the reviewer could understand it and approve listing the three names of gastritis.

In general, all strains have vacA. Therefore, 27 samples with vacA-negative may have mismeasurement.

Response : Thank you for your comment. We agree to it. It is possible that some part of vacA-positive samples might be judged as vacA-PCR negative (false-negative) due to any problem of extracted DNA or PCR amplification. However, now we are not able to repeat PCR to confirm due to lack of samples. Therefore, presence of vacA in the 27 samples was uncertain, and only the finding for 44 samples can be described. Thus, in the revised manuscript, the line of “27 vacA(-) samples” in original Table 3 was deleted. In the main text, we revised description as follows. “Among 44 H. pylori-positive samples which were available for virulence factor PCR, 36 were positive for both cagA and vacA, while 8 were /vacA(+)/cagA (-) (Table 3).”

In addition, please measure genotype of vacA.

Response : Thank you for suggestion. The genotype of vacA was determined by size of PCR product. Among the 44 vacA-positive samples, 43 was assigned to genotype s1, while only one to genotype s2. This finding was written in the text.

How did authors select 30 patients of 80 patients to measure mutations related to antimicrobial resistance in H. pylori?

Response : Thank you for the question. In this study, we did not do culture of H. pylori for antimicrobial resistance testing. Thus, we selected patients who might have presumptive resistant H. pylori strains by clinical course. The criteria is as follows : H. pylori-positive patients who received antimicrobials for H. pylori eradica-tion therapy 6-12 months ago. These patients were regarded as those having inappropriate therapy or carrying antimicrobial resistant H. pylori strains. This was written in the revised manuscript. (Page 3, 1^st para.)

References 12-14 showed that considerably high rate of resistance of H. pylori CAM (10- 30%), MNZ (78-95%), LVFX (66%) have been documented in some reports, in contrast to 68 low rate against AMX (3.6-6.6%). However, in this study, resistance rate of MNZ was 7%. What do authors think this discrepancy?

Response : Thank you for your comment. We apologize that the presentation in the manuscript, text and Table 4 might not be understandable, leading to misunderstanding. 7% for MNZ in Table 4 shows the rate of 200-bp deletion in rdxA gene among 30 presumptive MNZ-resistant samples. Actually, there are several other resistance mechanisms for MNZ resistance, but in this study, they were not analyzed. We tried to detect only the mechanism of 200-bp deletion in rdxA. Resistance rate of MNZ (according to clinical course of patients) was 38% (30/80). Still MNZ resistance rate may be low, this may suggest decreasing trend of MNZ resistance due to the less frequency of the use of MNZ in the study site. This view was added to the revised manuscript. In addition, percentage of AMR samples (e.g., 38% for 30/80) was added in Table 4, with revised heading of column, in the revised manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors presented a manuscript entitled "Prevalence, risk factors and antimicrobial resistance determinants of Helicobacter pylori detected in dyspeptic patients in north-central Bangladesh". The conducted a cross sectional study in a specific region. Is a paper with adequate scientific sound and well conducted.

In this paper, the observed a higher prevalence of H. pylori among males ranging 41-50 years old, living in rural areas with smoking habits, non filtered water consumption and family history of peptic ulcer disease.

They observed mutations linked to resistance to the antibiotic used as first line of H. pylori treatment with a moderate frequency. Alternatively, they positively related dyspepsia to other risk factors than H. pylori.

The paper is a shirt communication, but I have some few observations:

- Is more accepted the terms Low, Middle or High Income Countries than "Developing or Developed countries" please consider to change it.

- Line 117, please erase the blank space after and before the open and closing parenthesis sign.

- The authors have access to sequencing data, please verify the entire gene sequence to explore if there're are other mutations not previously reported that might be associated to resistance. It could explain the moderate presence o mutations reported.

Author Response

The authors presented a manuscript entitled "Prevalence, risk factors and antimicrobial resistance determinants of Helicobacter pylori detected in dyspeptic patients in north-central Bangladesh". The conducted a cross sectional study in a specific region. Is a paper with adequate scientific sound and well conducted.

In this paper, the observed a higher prevalence of H. pylori among males ranging 41-50 years old, living in rural areas with smoking habits, non filtered water consumption and family history of peptic ulcer disease.

They observed mutations linked to resistance to the antibiotic used as first line of H. pylori treatment with a moderate frequency. Alternatively, they positively related dyspepsia to other risk factors than H. pylori.

The paper is a shirt communication, but I have some few observations:

Is more accepted the terms Low, Middle or High Income Countries than "Developing or Developed countries" please consider to change it.

Response : Thank you for valuable suggestion. In the revised manuscript, developing countries was replaced by low-income countries.

Line 117, please erase the blank space after and before the open and closing parenthesis sign.

Response : Thank you, this was corrected.

The authors have access to sequencing data, please verify the entire gene sequence to explore if there're are other mutations not previously reported that might be associated to resistance. It could explain the moderate presence o mutations reported.

Response : Thank you for the comment. In this study, we determined sequences of partial 16S rRNA gene, 23srRNA, cagA, ureC, for confirmation of H. pylori and genes, and deposited to GenBank database, as described in text. In addition, PBP-1A and GyrA genes were sequenced. However, among those sequenced in this study, we detected no novel mutation, and the same as those in GenBank database. That is why those sequence data were not deposited to GenBank.