Diagnosis of Helicobacter pylori Infection in a Routine Testing Workflow: Effect of Bacterial Load and Virulence Factors
Round 1
Reviewer 1 Report
In the present article, the authors retrospectively investigated the association with histology findings and H. pylori virulence factors’ effect on the performance of invasive diagnostic testing of H. pylori infection using culture and histology. This report includes an important information about the relation between the result of conventional testing and bacterial load and the virulence factors.
Main comments:
- An ethical statement including the project identification code, date of approval and name of the ethics committee or institutional review board should be cited in the Methods Section of the article.
- The positive result of PCR indicates the presence of bacterial components and does not indicate chronic infection or colonization. In addition, only patients with chronic gastritis are eligible for eradication therapy. Therefore, the author should mention the possibility that this study includes patients who do not have chronic infection or indication for eradication therapy as a limitation.
Author Response
Main comments:
- An ethical statement including the project identification code, date of approval and name of the ethics committee or institutional review board should be cited in the Methods Section of the article. Response: Page 5, lines 94-99: This is a retrospective non-interventional research, carried out in a care structure, which has a correspondent in the National Data Information and Freedom Commission (CNIL) ensuring data compliance. Written informed consent for the endoscopic and diagnostic procedures was obtained from patients or their parents/legal guardians and kept in their medical records. No extra biopsy sampling
- The positive result of PCR indicates the presence of bacterial components and does not indicate chronic infection or colonization. In addition, only patients with chronic gastritis are eligible for eradication therapy. Therefore, the author should mention the possibility that this study includes patients who do not have chronic infection or indication for eradication therapy as a limitation.
Response: We thank the reviewer for the comment. All adults and children underwent endoscopy for gastrointestinal disorders. A sentence was added p 5, lines 84-85.
We agree with the reviewer as mentioned in the discussion (p 15, lines 329-334):”These hypotheses led to provocative questions: What is the clinical significance of the molecular detection of a low density of cagA-negative H. pylori usually missed by histology and conventional culture? Should we consider treating these cases with the same guidelines?”.
Thanks to this comment, we decided to add this sentence to the conclusion:
“H. pylori quantification associated with cagA genotyping in routine practice is essential to determine a sensitive and reliable diagnosis, to distinguish infection from colonization, to identify high-risk patients allowing to manage eradication therapies.”
Reviewer 2 Report
The authors in the work entitled "Diagnosis of Helicobacter pylori infection in a routine testing workflow: effect of bacterial load and virulence factors" present comparison of diagnostic methods of Helicobacter pylori infection.
The European Consensus Group (ECG) during a meeting in Maastricht in 2002 recommended the urea breath testing (UTB) 13C and histological examination of gastric tissue specimens as major diagnostic methods - called the "gold standard"
- To identify Helicobacter -like organisms in tissue, the authors should perform a silvering stainig more sensitive than Giemsa staining or antibody anti-H.pylori to be 100% sure of bacteria identification
- Giemsa staining method, we can not talk about identification of H. pylori but Helicobacter - like organisms - is a serious factual mistake
- The rules for collecting the material for the test are clear, 3-4 pieces of stomach tissue are taken from the antrum part -therefore, it is impossible to explain the own results of the test, defined as false negative, improper sampling of the material and omitting the infection
- In my opinion, it is known that H. pylori CagA + is characterized by greater virulence, better suppression of the immune response on the part of the host organism, which translates into more effective colonization of the gastric epithelium
- I'm not sure what new information the work brings, as data on the sensitivity and specificity of diagnostic tests are generally available, and most of the information is well known
-
TEST
SENSITIVITY
SPECIFICITY
NON-INVASIVE
UBT
90-96%
88-98%
Antigen H. pylori in stool sample
91-98%
94-99%
Antibody antyH. pylori
85-94%
79-95%
INVASIVE
RUT
93-97%
95-100%
Histological examination
95%
100%
Culture
50%
100%
PCR
95%
100%
Author Response
- To identify Helicobacter -like organisms in tissue, the authors should perform a silvering staining more sensitive than Giemsa staining or antibody anti-pylori to be 100% sure of bacteria identification
Response: We thank the reviewer for these comments. As stated in the title, we present the results based on routine testing workflow. Indeed, the pathologists of our university hospital do not perform silvering staining. Giemsa staining was carried out on all biopsies and Immunohistochemical staining was only performed in case of severe gastritis or suspected neoplastic lesions. This histological diagnosis method has recently been published and is discussed lines 270-276
- Giemsa staining method, we cannot talk about identification of pylori but Helicobacter - like organisms - is a serious factual mistake
Response: Thank you for identifying this mistake. We did the correction in the manuscript.
- The rules for collecting the material for the test are clear, 3-4 pieces of stomach tissue are taken from the antrum part -therefore, it is impossible to explain the own results of the test, defined as false negative, improper sampling of the material and omitting the infection
Response: We thank the reviewer to allow specifying. Four biopsies from antrum and four from corpus were taken (p5, lines 89-90). As recommended, one from antrum and one from corpus were used for culture and PCR (line 101) and three from antrum and 3 from fundus were taken for histology (lines 113-114). (2)
- In my opinion, it is known that pylori CagA + is characterized by greater virulence, better suppression of the immune response on the part of the host organism, which translates into more effective colonization of the gastric epithelium
Response: Thank you for the comment; The virulence conferred by CagA is, indeed, perfectly known. One of our objectives was to demonstrate that bacterial density is linked to CagA status. The performance of the various diagnostic tests depends on the bacterial density.
It was added lines 270-276: “We confirmed that a false negative diagnosis by histology staining may be linked to biopsies with few Helicobacter-like organisms (26), in agreement with Benoit et al. findings, stating that H. pylori is present only in case of active gastritis and being always on the standard staining with H-E (in 94% of the cases) (27). The authors concluded, that it is not necessary to systematically perform on all gastric biopsies, a complementary histo- or immuno-histochemical technique (27). In cases of low bacterial load, H. pylori infection can be missed if histology is the only test performed.”
- I'm not sure what new information the work brings, as data on the sensitivity and specificity of diagnostic tests are generally available, and most of the information is well known
Response: Indeed, our results about sensitivity/specificity of diagnostic methods are in agreement with previously published results. One of our objectives was to compare available results of different tests performed in routine practice. We further show that diagnostic tests’ performance can be linked with bacterial density, which is itself linked to the presence / absence of CagA. These data may have an impact on the treatment by differentiating infection from colonization. This last sentence was added (lines 329-334).
[NG1]Pas fotcément d’avis à rajouter cette phrase puisque nous en parlons justement dans le même paragraphe
Round 2
Reviewer 1 Report
I've confirmed sufficient improvements.