Calprest ELISA vs. Liaison^® Chemiluminescence: Evaluating Accuracy, Efficiency, and Clinical Utility in Fecal Calprotectin Testing

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present a comparative study evaluating two commercial fecal calprotectin (FC) assays—Calprest (ELISA) and Liaison (CLIA)—using 135 paired stool samples from 40 patients with Ulcerative Colitis (UC). The study correlates these measurements with endoscopic disease activity (Mayo score). The findings suggest that while the two methods are strongly correlated ($\rho=0.795$), they are not interchangeable due to systematic bias, with Liaison yielding higher values. There are some points to be clarified, as listed below:

1. The authors should perform a sub-analysis of samples collected within a tighter timeframe (e.g., <7 or <10 days from endoscopy) to see if the correlation or AUC improves. If this is not possible, this limitation must be emphasized more strongly.
2. Table 1 and the text indicate that 100% of the patients were on biologics. While the authors acknowledge this in the limitations, it raises questions about the applicability of the derived cut-offs to a general UC population. Please clarify in the Methods why this specific inclusion criterion was used. Was this a study on therapeutic monitoring specifically? If so, the title or abstract should be revised. 
3. The abstract states that Calprest demonstrated slightly better diagnostic accuracy with an AUC of 0.794. Did the authors perform a statistical comparison of the ROC curves to determine if this difference is statistically significant?
4. Grouping a wide range of up to 50 weeks into a single analysis might introduce heterogeneity. Are the follow-up samples mostly maintenance phase?
5. "full conoscopies" should be "colonoscopies".
6. "rectosigmoidoscoipes" should be "rectosigmoidoscopies"
7. Please standardize decimal separators throughout the manuscript

Author Response

Reviewer 1

Comment 1: The authors should perform a sub-analysis of samples collected within a tighter timeframe (e.g., <7 or <10 days from endoscopy) to see if the correlation or AUC improves. If this is not possible, this limitation must be emphasized more strongly.
Response: We agree that a tighter timeframe between sampling and endoscopy would be ideal. Unfortunately, due to the retrospective real-world nature of our sample collection, a robust sub-analysis with a statistically sufficient sample size for the <7-day window is not feasible with the current dataset. However, we strictly followed your suggestion to emphasize this limitation. We have added a dedicated paragraph in the Discussion section explicitly stating that the variable time interval is a limitation that may affect the strength of the observed correlations. However, it should be stressed that ulcerative treatments were not changed between fecal calprotectin sample withdrawal and endoscopy, therefore the impact of different time frame should minimal.

Comment 2: Table 1 and the text indicate that 100% of the patients were on biologics... Please clarify in the Methods why this specific inclusion criterion was used. Was this a study on therapeutic monitoring specifically? If so, the title or abstract should be revised.
Response: Thank you for this observation. The study population consisted of patients undergoing routine therapeutic monitoring for biologic response, which explains the 100% usage rate. We have clarified this in the Materials and Methods section to ensure the context is clear for the reader. To the best of our knowledge the type of treatment does not impact fecal calprotectin levels.

Comment 3: The abstract states that Calprest demonstrated slightly better diagnostic accuracy with an AUC of 0.794. Did the authors perform a statistical comparison of the ROC curves to determine if this difference is statistically significant?
Response: We have clarified in the Results section that while Calprest showed a numerically higher AUC, the difference was not formally tested for statistical significance using methods such as the DeLong test, as the primary aim was to establish method-specific cut-offs rather than prove the superiority of one established method over the other.

Comment 4: Grouping a wide range of up to 50 weeks into a single analysis might introduce heterogeneity. Are the follow-up samples mostly maintenance phase?
Response: Yes, the follow-up samples largely represent patients in the maintenance phase of therapy. We have added a sentence to the Methods section to clarify the phase of treatment for these patients.

Comment 5 & 6: "full conoscopies" should be "colonoscopies" and "rectosigmoidoscoipes" should be "rectosigmoidoscopies".
Response: These typos have been corrected throughout the manuscript.

Comment 7: Please standardize decimal separators throughout the manuscript.
Response: We have reviewed the manuscript and standardized all decimal separators to points (e.g., 0.795) consistent with English scientific convention.

Thank you for your consideration.

Sincerely,

Prof. Joško Osredkar, PhD
(On behalf of all authors)
University Medical Centre Ljubljana
josko.osredkar@kclj.si

Reviewer 2 Report

Comments and Suggestions for Authors

The authors present a study evaluating the accuracy of fecal calprotectin measurement using ELISA and chemiluminescence assays in ulcerative colitis. The topic is clinically relevant, and the dataset appears valuable.

However, I have several questions and suggestions for clarification:

 

Did the authors assess other laboratory or clinical markers (e.g., neutrophil count, hemoglobin, leukocyte count, CRP, or disease activity indices such as CDAI/UCSS)? Including these data could strengthen the interpretation of diagnostic performance.

 

The assays were performed using stool samples, which is standard for fecal calprotectin measurement. Did the authors also evaluate or compare calprotectin levels in serum or blood? If not, please clarify whether such measurements were considered and why they were not included.

 

The study utilized ELISA and chemiluminescence kits from Eurospital and DiaSorin. Since multiple commercial kits are available, how do the authors account for potential variability in assay performance across different manufacturers? Discussion of this point would help contextualize their findings for clinical application.

Author Response

Reviewer 2

Comment 1: Did the authors assess other laboratory or clinical markers (e.g., neutrophil count, hemoglobin, leukocyte count, CRP...)?
Response: While clinical data were available, our study was designed as a direct technical comparison between two fecal calprotectin assays. We have added a sentence to the Discussion acknowledging that integrating these markers would be a valuable direction for future comprehensive studies. Since UC generally does not induce systemic inflammation (with increases in CRP and leucocytes) we used endoscopy to test the reliability of fecal calprotectin, we believe that this is one of the main strengths of our work, as this is rarely reported in the literature.

Comment 2: Did the authors also evaluate or compare calprotectin levels in serum or blood?
Response: We focused exclusively on fecal calprotectin because it is the established gold standard for assessing localized intestinal inflammation. Serum calprotectin is less specific for mucosal healing. We have added a brief explanation in the Discussion regarding why serum levels were not included.

Comment 3: How do the authors account for potential variability in assay performance across different manufacturers?
Response: We have expanded the Discussion to explicitly address inter-kit variability. We emphasize that our findings reinforce the known lack of standardization between manufacturers, highlighting the clinical necessity of establishing method-specific cut-offs rather than relying on universal thresholds.

Thank you for your consideration.

Sincerely,

Prof. Joško Osredkar, PhD
(On behalf of all authors)
University Medical Centre Ljubljana
josko.osredkar@kclj.si

Reviewer 3 Report

Comments and Suggestions for Authors

The paper submitted to me for review “Calprest ELISA vs. Liaison® Chemiluminescence: Evaluating Accuracy, Efficiency, and Clinical Utility in Fecal Calprotectin Testing,” is a research paper. It compares the effectiveness of two tests used to measure calprotectin levels in stool samples. In addition, the authors tested the correlation between calprotectin concentration and endoscopic findings in a group of patients with ulcerative colitis.

Forty patients participated in the study. A total of 138 stool samples were collected from the patients.

In the second chapter, the authors describe the methodology of the scientific research in detail.

In my opinion, the authors used statistical methods appropriate for the research conducted and the results obtained.

The results are presented in descriptive form and in 3 tables and 4 figures.

Chapter 4 discusses the results obtained during the research in a very interesting and substantive manner.

I have no objection to the conclusions drawn by the authors from their research. The paper includes 15 items of current scientific literature.

Author Response

Reviewer 3

Comment: I have no objection to the conclusions drawn by the authors...
Response: We thank the reviewer for your positive assessment of our work.

Thank you for your consideration.

Sincerely,

Prof. Joško Osredkar, PhD
(On behalf of all authors)
University Medical Centre Ljubljana
josko.osredkar@kclj.si