Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Line 60: “particularly Klebsiella pneumoniae producing”. Please italicize the species name.
2. Lines 96-107: Please be more concise, in order to short the Introduction section.
3. Lines 108-109: “…located in a high-endemic European setting…”. Please be more specific.
4. Line 131: “Overall, 1,084 patients met these criteria and underwent microbiological screening.”. This should be placed on the Results section.
5. Lines 134-155: This is related to the information provided in the previous section (2.2) and on Figure 1. It should be integrated in that previous section and/or in the Figure 1 itself.
6. There is a lack of information regarding the sample collection methodology. Please provide it.
7. Lines 158-162: “…on selective chromogenic media for carbapenem-resistant Enterobacterales. Bacterial identification was performed using routine automated laboratory identification systems. Suspected isolates underwent confirmatory testing using molecular methods based on polymerase chain reaction (PCR) for the detection of major carbapenemase genes.”. Please provide details (or a reference) on the chromogenic media, automated laboratory identification systems and the PCR reactions.
8. Lines 172-173: Ethics details should appear in its own section.
9. Line 186: Please indicate the percentage for recent hospitalization, as for the other criterions it was presented further.
10. Lines 191-192: “Overall, 180 validated patients were found to carry carbapenemase-producing Enterobacterales, corresponding to a positivity rate of 16.6% among screened individuals.”. This should be the first statement of the paragraph. Details should follow that point of situation.
11. Line 190: “The molecular epidemiology of microbiologically positive patients is reported in Table 3”. Determination of the bla_{carbapenemase} gene is not a “molecular epidemiology” information. It is a molecular characterization of the carbapenemase enzyme. Please correct this on along the manuscript text and Table(s). The Table 3 title and respective footnote should be improved and concise.
12. Line 197: “..high-endemic molecular setting.”. What is a molecular setting??
13. Lines 208-209: “Univariable analyses suggested strong associations between several predefined 208 screening criteria and CPE positivity.”. Which ones? Please improve.
14. Lines 218: Include de percentage again.

 

 

FIGURES

15. Figure 1: In the second statement of the checklist appears “…admiiting…”. Please correct.

Moreover, in the sixth statement of the checklist, the acronym PICC should be preceded by its description in extension.

Please improve a little the presentation of the image.

 

TABLES

16. Table 1: The line presenting the total of patients is a repetition of the first line of the Table. Please improve.
17. Table 2: The total of the “n” column is not 1084. Please clarify.

 

References

18. The references section should be improved, as the most recent references are 1 from 2022, and 1 from 2026 (among a total of 26). All others are from or before 2019.
19. Reference 24: please add the date of the publication.

Author Response

We sincerely thank the Reviewer for the careful reading of our manuscript and for the constructive comments. We have revised the manuscript accordingly, improving the Introduction, Methods, Results, figures, tables, terminology, and references. The main changes include a more specific description of the study setting, reorganisation of the Methods and Results sections, addition of details on sample collection and microbiological methods, clarification of non-mutually exclusive screening indications, correction of molecular terminology, and update of the reference list.

Comment 1.
Line 60: “in particular Klebsiella pneumoniae producing…”. Please italicize the species name.

Response:
We thank the Reviewer for this comment. The species name Klebsiella pneumoniae has been italicized throughout the manuscript, in accordance with taxonomic nomenclature.

Changes made:
The manuscript was revised to ensure that Klebsiella pneumoniae appears in italics wherever mentioned.

Comment 2.
Lines 96–107: Please be more concise in order to shorten the Introduction section.

Response:
We agree with the Reviewer. The Introduction has been revised and shortened to improve clarity and avoid repetition. The revised Introduction now focuses more directly on antimicrobial resistance, CRE/CPE relevance, the Italian high-endemic setting, the rationale for risk-based screening, and the aim of the study.

Changes made:
The Introduction was substantially condensed. Some broader organisational considerations were removed from the Introduction and, where appropriate, discussed later in the Discussion section.

Comment 3.
Lines 108–109: “…located in a high-endemic European setting…”. Please be more specific.

Response:
We thank the Reviewer for this useful suggestion. The study setting has now been specified more precisely. The revised manuscript states that the study was conducted at the Azienda Ospedaliero-Universitaria Consorziale Policlinico di Bari, Bari, Apulia Region, Southern Italy, a large tertiary-care referral hospital located in an Italian region where CRE/CPE circulation is considered endemic according to national and European surveillance data.

Changes made:
The setting was clarified in both the Introduction and the Materials and Methods section.

Comment 4.
Line 131: “Overall, 1,084 patients met these criteria and underwent microbiological screening.” This line should be included in the Results section.

Response:
We agree with the Reviewer. This information has been removed from the Study population subsection and moved to the Results section.

Changes made:
The Results section now reports that, during the study period, 2,694 patients were evaluated for hospital admission and that 1,084 met predefined screening criteria and underwent rectal swab testing, corresponding to 40.2% of the overall cohort evaluated for admission.

Comment 5.
Lines 134–155: These are related to the information provided in the previous section and Figure 1. They should be integrated into the previous section and/or into Figure 1 itself.

Response:
We thank the Reviewer for this suggestion. The information on screening criteria has been reorganised and integrated into the revised subsection “Screening criteria and data management”. This section now explains how checklist items were grouped into predefined risk domains and clarifies the operational definitions used for analysis.

Changes made:
The former separate subsection on screening criteria was merged and reorganised within the Methods section. The revised Methods now clarify that patients could meet more than one screening criterion and that patient-level deduplication was applied when multiple records referred to the same patient.

Comment 6.
Information on the sample collection methodology is missing. Please provide it.

Response:
We agree with the Reviewer. The Methods section has been expanded to include details on sample collection.

Changes made:
A new subsection entitled “Sample collection and microbiological methods” has been added. The revised text specifies that rectal swabs were collected during the pre-admission infectious risk assessment by trained healthcare personnel and processed by the hospital microbiology laboratory according to routine diagnostic procedures.

Comment 7.
Lines 158–162: “…on selective chromogenic media for carbapenem-resistant Enterobacterales. Bacterial identification was performed using routine automated laboratory identification systems. Suspected isolates underwent confirmatory testing using molecular methods based on polymerase chain reaction (PCR) for the detection of major carbapenemase genes.” Please provide details or a reference on chromogenic media, automated identification systems, and PCR reactions.

Response:
We thank the Reviewer for highlighting this important methodological point. The microbiological methods section has been expanded. We clarified that resistant strains were identified using conventional microbiological techniques followed by molecular confirmation when indicated, and that PCR was used for the rapid detection of the main carbapenemase resistance genes, including bla_KPC, bla_NDM, bla_OXA-48-like, and bla_VIM.

As this was a retrospective observational study based on routinely generated diagnostic reports, the research dataset contained validated laboratory results rather than raw molecular run files. Therefore, primer sequences, amplification conditions, and commercial assay specifications were not available in the research dataset and could not be reported in full. This methodological limitation has now been explicitly stated.

Changes made:
The Methods section now specifies that molecular results were extracted from validated laboratory reports generated within the routine diagnostic workflow, which included standard internal quality-control procedures and positive and negative controls according to laboratory practice. The absence of primer sequences, amplification conditions, and full commercial assay specifications has also been acknowledged.

Comment 8.
Lines 172–173: Ethical details should appear in a separate section.

Response:
We agree with the Reviewer. Ethical information has been moved to a dedicated subsection.

Changes made:
A separate subsection entitled “Ethical considerations” has been added to the Materials and Methods section. It specifies that the study was retrospective and observational, used anonymised routinely collected clinical, administrative, and microbiological data, and did not involve any intervention or deviation from routine practice.

Comment 9.
Line 186: Please indicate the percentage related to recent hospitalization, as for the other criteria presented afterwards.

Response:
We thank the Reviewer for this comment. The percentage has now been explicitly reported.

Changes made:
The Results section now states that recent hospitalization or recent admission from another healthcare/residential facility was the most frequent indication, accounting for 510/1,084 screened patients, corresponding to 47.0%.

Comment 10.
Lines 191–192: “Overall, 180 validated patients were found to carry carbapenemase-producing Enterobacterales, corresponding to a positivity rate of 16.6% among screened individuals.” This should be the first statement of the paragraph. The details should follow.

Response:
We agree with the Reviewer. The paragraph has been restructured so that the main result is presented first, followed by the details on molecular characterization.

Changes made:
The revised Results section now begins this paragraph by reporting that 191 unique patients were found to carry carbapenemase-producing Enterobacterales after patient-level deduplication, corresponding to a positivity rate of 17.6% among screened individuals and 7.1% of the overall cohort evaluated for admission.

Comment 11.
Line 190: “The molecular epidemiology of microbiologically positive patients is reported in Table 3.” The determination of the bla carbapenemase gene is not molecular epidemiology. It is a molecular characterization of the carbapenemase enzyme. Please correct this error in the manuscript and tables. The title of Table 3 and the footnote should be improved and made more concise.

Response:
We thank the Reviewer for this important clarification. We agree that the term “molecular epidemiology” was not appropriate in this context. The terminology has been revised throughout the manuscript. The text now refers to “molecular characterization of carbapenemase genes” rather than “molecular epidemiology”.

Changes made:
The title of Table 3 has been revised to “Carbapenemase genes detected among deduplicated CPE-positive patients (n = 191)”. The footnote has also been shortened and now states that percentages are calculated over all deduplicated CPE-positive patients.

Comment 12.
Line 197: “…high-endemic molecular setting”. What is a molecular setting?

Response:
We agree with the Reviewer that this wording was unclear. The expression “high-endemic molecular setting” has been removed and replaced with clearer terminology referring to the local CPE pattern and carbapenemase gene distribution.

Changes made:
The revised text now states that KPC and NDM accounted for 83.2% of detected carbapenemase genes, indicating a local CPE pattern dominated by KPC, with a substantial contribution of NDM.

Comment 13.
Lines 208–209: “Univariable analyses suggested strong associations between several predefined screening criteria and CPE positivity.” Which ones? Please improve.

Response:
We thank the Reviewer for this observation. During revision, we reconsidered the statistical presentation of the data. Because screening indications were non-mutually exclusive and microbiologically positive records were consolidated through patient-level deduplication, inferential estimates for individual criteria were not retained in the final revised analysis. The revised manuscript now presents the screening pathway descriptively, focusing on programme yield and carbapenemase gene distribution, thereby avoiding overinterpretation of unstable associations.

Changes made:
The former univariable association table was removed from the revised analysis. The Statistical analysis section now explains that the final analysis was descriptive and that no multivariable model was presented because the available retrospective dataset did not allow stable and fully reproducible patient-level modelling of independent associations between individual screening criteria and CPE positivity.

Comment 14.
Line 218: Include the percentage again.

Response:
We thank the Reviewer. The relevant percentages have been added and repeated where needed for clarity.

Changes made:
The revised manuscript now reports that 191 CPE carriers were identified among 1,084 screened patients, corresponding to 17.6% of screened individuals and 7.1% of the overall cohort evaluated for admission.

Figures

Comment 15.
Figure 1: In the second checklist statement, “…admitting…” appears incorrectly. Please correct it.

Response:
We thank the Reviewer for noticing this. The wording in Figure 1 has been corrected.

Changes made:
The checklist wording was revised to remove the typographical/wording error.

Comment 16.
In the sixth checklist item, the acronym PICC should be preceded by its full description.

Response:
We agree with the Reviewer. The acronym PICC has been defined in full.

Changes made:
The revised text now reports “peripherally inserted central catheter (PICC)” when referring to invasive devices.

Comment 17.
Please improve the presentation of the image.

Response:
We thank the Reviewer for this suggestion. Figure 1 has been revised to improve clarity, readability, and presentation quality.

Changes made:
The checklist figure was reformatted, and the caption was revised to better describe its function within the pre-admission risk-assessment pathway.

Tables

Comment 18.
Table 1: The row presenting the total number of patients repeats the first row of the table. Please improve it.

Response:
We agree with the Reviewer. The redundant row has been removed.

Changes made:
Table 1 now reports sex distribution and median age only, while the total cohort size is indicated in the table heading as “Overall cohort (n = 2,694)”.

Comment 19.
Table 2: The total of the “n” column is not 1,084. Please clarify.

Response:
We thank the Reviewer for this important comment. We clarified that screening indications were not mutually exclusive and that some patients could present more than one screening indication. Therefore, the sum of the “n” column exceeds the number of screened patients.

Changes made:
The Results section now explicitly states that 1,128 screening indications were recorded among 1,084 screened patients. Most patients had one screening indication, while 38 had two indications and 3 had three indications. Percentages in Table 2 are calculated using the 1,084 screened patients as the denominator.

References

Comment 20.
The reference section should be improved, since the most recent references are one from 2022 and one from 2026, while all others are from 2019 or earlier.

Response:
We agree with the Reviewer. The reference list has been updated to include more recent and relevant sources, including recent WHO, ECDC, ISS, and contemporary literature on CRE/CPE epidemiology, infection prevention, and screening strategies.

Changes made:
Recent references were added, including updated WHO, ECDC, and ISS documents and recent literature relevant to carbapenem-resistant Enterobacterales and infection prevention.

Comment 21.
Reference 24: Please add the publication date.

Response:
We thank the Reviewer. The publication date has been added to the reference.

Changes made:
Reference 24 was revised accordingly.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Risk-based pre-admission screening for carbapenem-resistant Enterobacterales (CRE): a patient-level observational study in a high-endemic European setting” addresses an epidemiologically important topic. The focus on risk-based screening and the patient-level analytical approach in a high-endemic European setting is valuable and relevant to infection control practices.

However, several major concerns must be addressed before the manuscript can be considered for publication.

Major Comments

1. Lack of methodological detail for molecular detection

The Materials and Methods section does not provide sufficient detail regarding the PCR protocols used for the detection of carbapenemase genes. Critical information, such as primer sequences, amplification conditions, controls, and validation procedures, is missing. This lack of methodological transparency significantly limits reproducibility and raises concerns about the reliability of the molecular findings.

2. Inadequate and superficial Discussion section

The Discussion section is notably weak and does not meet the standards expected for a scientific manuscript. The authors fail to adequately contextualize their findings within the existing body of literature. There is insufficient comparison with studies conducted in similar high-endemic regions or other international settings. A robust discussion should critically evaluate similarities and differences with previously published data and provide possible explanations for observed discrepancies.

3. Insufficient discussion of carbapenemase diversity

Although major carbapenemases such as KPC and NDM are mentioned, the manuscript does not provide a comprehensive discussion of other relevant carbapenemase types identified in the study. In particular, findings related to KPC/NDM co-producers, OXA-like enzymes, and VIM are not sufficiently elaborated. Given the epidemiological importance of these resistance mechanisms, a more detailed and critical interpretation is required.

4. Discussion structured as a conclusion rather than a critical analysis

The current Discussion section largely summarizes the findings without offering in-depth interpretation. As such, it resembles a conclusion rather than a critical scientific discussion. The authors should substantially revise this section to include interpretation, comparison with the literature, implications for clinical practice, and limitations of the study.

Author Response

We sincerely thank the Reviewer for the positive evaluation of the relevance of our study and for the constructive critical comments. We agree that the previous version of the manuscript required more methodological transparency and a more developed Discussion section. In response, we substantially revised the manuscript by expanding the microbiological Methods, adding a dedicated methodological subsection on molecular detection, restructuring and deepening the Discussion, improving the interpretation of carbapenemase diversity, and strengthening the limitations of the study. The revised manuscript now better contextualizes the findings within the existing literature and more clearly distinguishes the operational value of the screening pathway from the methodological constraints of the retrospective dataset.

Response 1.
We thank the Reviewer for this important comment. We agree that molecular methods should be reported as transparently as possible. In response, we expanded the Methods section to better describe the sample collection and microbiological workflow. The revised manuscript now specifies that rectal swabs were collected during the pre-admission infectious risk assessment by trained healthcare personnel and processed by the hospital microbiology laboratory according to routine diagnostic procedures. We also clarified that resistant strains were identified using conventional microbiological techniques, followed by molecular confirmation when indicated, and that PCR was used for the rapid detection of the major carbapenemase resistance genes, including blaKPC, blaNDM, blaOXA-48-like, and blaVIM.

As this was a retrospective observational study based on routinely generated diagnostic reports, the research dataset contained validated laboratory results rather than raw molecular run files. Therefore, primer sequences, amplification conditions, and full assay-validation parameters were not available for independent reporting. To avoid overclaiming reproducibility, we explicitly added this point both in the Methods and in a dedicated Discussion subsection entitled “Methodological considerations regarding molecular detection”.

The Methods section was expanded to describe the routine diagnostic workflow, the use of rectal swabs, the molecular targets investigated, and the fact that molecular results were extracted from validated laboratory reports. In addition, a new Discussion subsection was added to clarify that the study was not designed to develop, compare, or analytically validate PCR assays, and that future prospective studies should collect and report complete molecular platform information, including commercial assay name, manufacturer, target genes, internal controls, positive and negative controls, amplification conditions, interpretation criteria, and external quality-assessment participation.

Response 2.
We agree with the Reviewer. The Discussion section has been substantially rewritten and expanded. The revised Discussion now includes a dedicated subsection entitled “Principal findings and comparison with published evidence”. In this section, we contextualize the observed screening yield within the epidemiological background of Southern Italy and the broader European CRE/CPE context. We now clarify that the positivity rate observed in our cohort should not be interpreted as the prevalence of CRE/CPE colonisation among all hospital admissions, but rather as the operational yield of a risk-enriched screening pathway applied in a high-endemic tertiary-care hospital.

We also added a more critical discussion of the implications of targeted screening. In particular, we emphasize that a direct estimate of colonisation among non-screened patients would require a different study design, because patients without screening indications did not routinely undergo rectal swab testing. Therefore, the overall cohort positivity rate should be considered a conservative measure of detected burden rather than a true prevalence estimate.

The Discussion was restructured into thematic subsections. The revised version now discusses the principal findings and comparison with published evidence, the interpretation of the risk-based screening strategy, the carbapenemase gene distribution and epidemiological interpretation, the clinical, microbiological and organisational implications, the methodological considerations regarding molecular detection, and the limitations of the study. This revision transforms the Discussion from a descriptive summary into a more critical interpretation of the study findings.

Response 3.
We thank the Reviewer for this valuable suggestion. We agree that the diversity of carbapenemase genes deserves a more detailed interpretation. The revised manuscript now includes a dedicated subsection entitled “Carbapenemase gene distribution and epidemiological interpretation”.

In the Results section, we now report the corrected and deduplicated carbapenemase gene distribution among 191 CPE-positive patients: KPC was detected in 102/191 patients, NDM in 57/191, KPC/NDM co-production in 14/191, VIM in 8/191, OXA-48 in 3/191, KPC/OXA-48 in 3/191, NDM/OXA-48 in 2/191, KPC/NDM/OXA-48 in 1/191, and KPC/NDM/VIM in 1/191.

The Discussion now emphasizes that the predominance of KPC is consistent with the Italian epidemiological background, but that the substantial proportion of NDM-positive patients and the presence of KPC/NDM co-producers indicate a more heterogeneous resistance landscape. We also discuss the epidemiological relevance of OXA-48 and VIM, noting that OXA-48-like carbapenemases may be difficult to detect phenotypically in some contexts, whereas VIM and NDM belong to the metallo-beta-lactamase group and may limit therapeutic options.

A new, expanded interpretation of carbapenemase diversity was added. The revised Discussion now addresses KPC, NDM, KPC/NDM co-production, OXA-48, VIM, and combined carbapenemase profiles. The manuscript also highlights that local screening programmes should not be designed around a single resistance mechanism, but should be able to detect multiple carbapenemase families.

Response 4.
We fully agree with the Reviewer. The Discussion section has been substantially revised to provide a more analytical and critical interpretation of the findings. Rather than simply summarizing the Results, the revised Discussion now explains the operational meaning of the screening yield, the selective nature of the risk-based screening pathway, the uncertainty regarding non-screened patients, the implications of carbapenemase diversity, and the clinical, microbiological, and organisational relevance of pre-admission CRE/CPE screening.

We added a specific discussion of the clinical implications of early identification of CPE carriers, particularly for patients with severe illness, invasive devices, immunosuppression, or recent healthcare exposure. The revised text also highlights that colonisation does not necessarily imply infection, but may inform empirical therapy in selected high-risk scenarios and support antimicrobial stewardship.

From a microbiological perspective, the revised Discussion now explains the importance of combining phenotypic detection with molecular confirmation, since CRE is a phenotypic category while CPE indicates carbapenemase production confirmed by molecular testing. From an organisational perspective, we added that screening results are actionable only if they trigger timely isolation, clear communication, appropriate documentation in the electronic record, and coordination between clinical teams and infection prevention personnel.

The Limitations section was also expanded. It now explicitly states that the study cannot estimate CRE colonisation prevalence among all hospital admissions, cannot evaluate the sensitivity of the screening checklist among patients without risk factors, and has limited molecular-method reproducibility because detailed laboratory platform information was not available in the retrospective research dataset.

We thank the Reviewer again for these constructive and important comments. In response, we made substantial revisions to the manuscript. We expanded the microbiological Methods, clarified the limits of PCR reporting in a retrospective routine-diagnostics dataset, added a dedicated methodological subsection on molecular detection, substantially rewrote the Discussion, improved the contextualization of the findings within high-endemic settings, expanded the interpretation of carbapenemase diversity, and strengthened the clinical, microbiological, organisational, and methodological implications of the study. We believe these revisions have significantly improved the transparency, balance, and scientific quality of the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Thanks for the opportunity to review this paper on CRE screening criteria. This is a well structured and written manuscript showing which screening criteria were strong risk factors for being colonized. 

It would be nice to know the % colonization among the non-screened individuals (even an estimate). Do you have a sense of what proportion of colonizations you are capturing with your criteria?

It would be interesting to speak to the resources required for this screening. 

I'm a bit confused by the presented #s; with additional clarification/confirmation, this will read nicely. 

Some comments below: 

Results – why stopping at univariate analyses? Would be helpful to understanding the high ORs to present a multivariable or speak to the fact that so many of the cases had multiple criteria that you can’t separate effects (if that’s the case). 

Table 2 & table 4 – The sum of the tables is 1021 (510+182+102+133+91+3) – why is this less than the 1084 patients (particularly if patients can have >1 screening criterion)?

Figure 1 - typo in the word “admitted”

Not all criteria in Figure 1 are included in the screening criteria in Table 2 / 4. We’re some criterion dropped from the analyses? Should they be included or were they merged? Are they in the denominator groups? 

Table 4 – I think something strange might be happening with your math. I know I don’t have all the duplicates response #s of course, but for a gut check….

OR = odds of being a case among Recent hospitalization / odds of being a case in other categories

= (177/333) / (445/56)

= 0.53 / 8.125

= 0.07

We need more information on the denominator categories to know what is going on (do all cases basically have 3+  multiple risk factors? If this is the case how is the n=1084? each patient needs at least one criteria so # should be higher number in the table?)  

 

  

Author Response

We sincerely thank the Reviewer for the positive assessment of the manuscript and for the careful comments regarding the statistical presentation and interpretation of the screening criteria. We agree that the previous version required clearer explanation of the denominators, screening indications, non-screened patients, and the limitations of the inferential analysis. During the revision process, we also identified discrepancies in the dataset exported from the SPSS-based data collection and management workflow. For this reason, we rechecked the original records, reconciled the microbiological and screening datasets, applied patient-level deduplication, and recalculated the descriptive results. Consequently, the revised manuscript now presents a corrected descriptive analysis rather than unstable univariable odds ratios.

Response 1.
We thank the Reviewer for this important comment. We agree that estimating colonisation among non-screened individuals would be valuable for understanding the proportion of CPE carriers captured by the risk-based strategy. However, in the present retrospective pathway, patients without screening indications did not routinely undergo rectal swab testing. Therefore, the colonisation rate among the 1,610 non-screened individuals cannot be directly estimated from the available data.

To avoid overinterpretation, we clarified in the revised Results and Discussion that the observed positivity rate in the overall admission cohort should be interpreted as a minimum detected burden rather than as the true prevalence of CRE/CPE colonisation among all patients evaluated for admission. We also added that a prospective validation phase, including systematic screening of a representative sample of patients without risk criteria, would be required to estimate missed colonisation, sensitivity of the screening algorithm, and negative predictive value.

Response 2.
We agree with the Reviewer that the resource implications of expanding screening or estimating missed colonisation should be discussed. We have therefore added a specific paragraph in the Discussion addressing the resources required for universal screening or for a hybrid validation strategy.

The revised Discussion now explains that systematic screening of non-screened patients would increase the number of rectal swabs, microbiology laboratory workload, consumable use, personnel time, reporting activity, and infection-prevention follow-up. We also added that additional positive findings would increase demand for single rooms, cohort areas, contact precautions, environmental cleaning, and bed-management coordination. This addition allows the screening strategy to be interpreted not only in terms of microbiological yield, but also in terms of operational feasibility and resource allocation.

Response 3.
We thank the Reviewer for pointing out the confusion in the numbers. During revision, we performed a complete recheck of the dataset and identified discrepancies related to the SPSS-based data collection and export process. Specifically, some records had been omitted or inconsistently retained during the initial SPSS data extraction and matching procedure. We therefore returned to the original source files, reconciled the screening and microbiological datasets, removed duplicate patient-level records, and recalculated the descriptive results.

After this correction, the revised manuscript now reports 2,694 patients evaluated for admission, 1,084 patients undergoing CRE rectal swab screening, and 191 unique CPE-positive patients after patient-level deduplication. The previous value of 180 positive patients has therefore been corrected to 191. Because of these data reconciliation issues and because screening indications were non-mutually exclusive, we removed the previous inferential Table 4 and no longer present the unstable odds ratios.

Response 4.
We agree with the Reviewer that the previous univariable analysis was potentially misleading. The very high odds ratios were strongly influenced by the selective nature of the screening pathway, the non-mutually exclusive screening indications, and the structure of the retrospectively collected dataset. In addition, after identifying discrepancies in the SPSS-based data management process and after performing patient-level reconciliation, we concluded that a stable and fully reproducible multivariable model could not be reliably presented.

For this reason, the revised manuscript no longer reports the previous univariable odds ratios. The Statistical analysis section now states that, because screening indications were non-mutually exclusive and microbiologically positive records were analysed after patient-level deduplication, the final analysis was descriptive. We also clarified that no multivariable model was presented because the available retrospective dataset did not allow stable and fully reproducible patient-level modelling of independent associations between individual screening criteria and CPE positivity.

This change was made to avoid overinterpretation of unstable estimates and to present the study more accurately as an evaluation of the operational yield of a risk-based screening programme.

Response 5.
We thank the Reviewer for highlighting the apparent inconsistency between the total number of screened patients and the sum of the criteria in the previous version of Table 2 and Table 4. We rechecked this issue carefully during revision.

The discrepancy was due to two factors. First, some screening triggers present in Figure 1 had not been clearly represented in the original analytical grouping. Second, inconsistencies were identified in the SPSS-based data extraction and export process. After reconciling the source files, we corrected Table 2 and included all screening indications used in the institutional checklist or local screening pathway.

The revised Table 2 now reports 1,128 screening indications among 1,084 screened patients. Most patients had one screening indication, whereas 38 patients had two indications and 3 patients had three indications. Percentages are calculated using the 1,084 screened patients as the denominator. This clarification explains why the total number of indications may exceed the number of screened patients.

Response 6.
We thank the Reviewer for noticing the typographical error in Figure 1. The wording has been corrected in the revised version of the figure.

In addition, Figure 1 has been reformatted to improve readability and presentation quality. The acronym PICC has also been defined as “peripherally inserted central catheter (PICC)” in the revised manuscript.

Response 7.
We agree with the Reviewer that the relationship between Figure 1 and Tables 2/4 required clarification. In the revised manuscript, we reorganised the screening criteria and clarified that checklist items were grouped into predefined operational risk domains for analysis.

Some items from Figure 1 were not excluded, but were grouped into broader screening indication categories. For example, dialysis and recent antineoplastic chemotherapy were grouped with immunosuppression; ileostomy and colostomy were grouped with fecal or urinary incontinence/diarrhoea/ostomy; and several invasive devices were grouped under the broader category of invasive or indwelling medical devices. We also added previously documented CRE/CPE colonisation or infection, planned admission to selected high-risk hospital units, and origin from a country endemic for CPE infections where applicable.

The revised Table 2 now better reflects the institutional checklist and clarifies the categories included in the denominator.

Response 8.
We thank the Reviewer for carefully checking the odds ratio calculation. We agree that the previous Table 4 generated confusion and could not be adequately interpreted without clearer denominator information. During revision, we re-evaluated the statistical dataset and identified discrepancies in the SPSS-based data collection/export process. We therefore corrected the dataset, applied patient-level deduplication, and decided not to retain the previous inferential analysis.

The previous odds ratios have been removed from the revised manuscript. The study is now presented as a descriptive patient-level evaluation of a risk-based pre-admission screening programme. This approach is more appropriate because each patient required at least one screening indication to be tested, screening criteria were not mutually exclusive, and the available retrospective data did not allow stable modelling of the independent effect of each individual criterion.

Response 9.
We agree that additional information on denominators was necessary. The revised manuscript now clearly distinguishes between:
the overall cohort evaluated for admission, consisting of 2,694 patients;
the screened population, consisting of 1,084 patients;
the non-screened population, consisting of 1,610 patients;
the number of screening indications, equal to 1,128;
and the number of unique CPE-positive patients after deduplication, equal to 191.

This structure clarifies that Table 2 reports screening indications rather than mutually exclusive patient groups. It also clarifies that the non-screened population cannot be treated as microbiologically negative, because these patients did not routinely undergo rectal swab testing.

We thank the Reviewer again for these valuable comments. They prompted a complete re-evaluation of the statistical presentation and data structure. Following this revision, we corrected discrepancies related to the SPSS-based data collection and export process, recalculated the descriptive results, updated the number of CPE-positive patients from 180 to 191, clarified all denominators, removed the unstable odds-ratio analysis, expanded the discussion of non-screened patients and resource implications, and revised the manuscript to present the study as a descriptive evaluation of the operational yield of a risk-based pre-admission CRE/CPE screening pathway.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Regarding Comment 7 of Report 1: "Lines 158-162......Please provide details (or a reference) on the chromogenic media, automated laboratory identification systems and the PCR reactions."

Your answer was: "As this was a retrospective observational study based on routinely generated diagnostic reports, the research dataset contained validated laboratory results rather than raw molecular run files. Therefore, primer sequences, amplification conditions, and commercial assay specifications were not available in the research dataset and could not be reported in full. This methodological limitation has now been explicitly stated."

 

However, if you contact the microbiological laboratory(ies) involved, they should be able to give you some details about the routinely used methods. If they used validated methods, they should inform you which ones were and/or provide references to include in the manuscript text. This is important in order to ensure reproducibility. This will also guarantee quality of the publication.

Author Response

We thank the reviewer for this important suggestion. We agree that
additional details on the microbiological procedures are necessary to
improve transparency and reproducibility. Following the reviewer’s
comment, we revised the Methods section by providing further information
on the routine microbiological workflow used during the study period,
including culture media, chromogenic media, automated identification
systems, antimicrobial susceptibility testing, and molecular assays.
We add in methods section the following paragraph.

Reviewer 2 Report

Comments and Suggestions for Authors

The concerns raised during my initial evaluation have been addressed to a large extent in the revised manuscript. In addition, the authors have made the necessary revisions and provided appropriate clarifications in response to the comments and suggestions raised by the other reviewers.

Therefore, the revised manuscript is acceptable for publication.

Author Response

Thank you very much for your positive evaluation of our revised manuscript. We are grateful for your careful review and constructive comments, which helped us improve the quality and clarity of the work. We sincerely appreciate your acknowledgment that the concerns raised during the initial evaluation have been addressed and that the manuscript is now acceptable for publication.

Reviewer 3 Report

Comments and Suggestions for Authors

Thanks for the opportunity to re-review. The removal of the odds ratios does remove the concerns regarding the odds ratios and the numbers are presented more clearly. 

It doesn't appear that the information is available, but more detailed breakdown of estimated costs / resources at your facility would be a valuable addition. 

 

 

Author Response

Thank you very much for your careful re-review of our manuscript and for your positive feedback. We are pleased that the removal of the odds ratios and the revised presentation of the data have improved the clarity of the results. We also appreciate your valuable suggestion regarding a more detailed breakdown of estimated costs and resources at our facility. Unfortunately, these data are not currently available in sufficient detail to allow a robust facility-level analysis. However, we agree that this would represent an important addition and a relevant direction for future research, particularly for assessing the organizational and economic sustainability of the proposed screening strategy.