Review Reports - Exploring the Value of Paired Microbiology and Histology in Chronic Osteomyelitis and Fracture-Related Infections

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The topic is relevant, and the manuscript presents an interesting idea: using paired sampling to combine microbiology and histology in the same diagnostic pathway. This approach is particularly appealing for managing culture-negative cases, which remain a major challenge in clinical practice. That said, several methodological and interpretative issues make the conclusions less solid than they appear, and the manuscript would benefit from clearer explanations before being considered for publication.

The cohort is small and limited to a single centre (41 patients, 7 failures), which weakens the strength of the prognostic analyses. The wide confidence intervals of the odds ratios highlight this limitation. Additionally, fracture-related infection and chronic osteomyelitis are grouped together, despite their different biological and clinical behaviour. This may introduce bias, and separate analyses or a more detailed discussion would be helpful.

Author Response

Response to Reviewer 1 Comments

Summary

Thank you very much for taking the time to review our manuscript. We appreciate the thoughtful comments and agree that the combined use of paired microbiology and histology is particularly relevant for managing culture-negative cases, which remain a major clinical challenge and can complicate antimicrobial treatment planning. We have addressed each of the methodological and interpretative concerns in detail below, and all corresponding revisions are highlighted in the resubmitted manuscript and tracked-changes file.

Point-by-point response to Comments and Suggestions for Authors

Comments 1: That said, several methodological and interpretative issues make the conclusions less solid than they appear, and the manuscript would benefit from clearer explanations before being considered for publication.

Response 1: Thank you for this comment. Specifically, we have added a CONSORT flow diagram (Fig. 4) to provide a clearer overview of patient inclusion, sampling, and analysis pathways, and we have expanded the limitations section to address the methodological constraints highlighted. We believe these revisions improve the clarity and interpretability of the manuscript. We have also added a sentence in the conclusion to tie these limitations together.

Conclusion, Lines 279-284.

“Although this is a small study and should be interpreted accordingly, the results present an interesting and novel observation, namely that combining modalities on paired samples may help reveal disease severity and provide prognostic value. However, the observation requires further clinical validation and can currently serve only as a solid foundation for more extensive research on the topic.

Comments 2: The cohort is small and limited to a single centre (41 patients, 7 failures), which weakens the strength of the prognostic analyses.

Comments 3: The wide confidence intervals of the odds ratios highlight this limitation

Comments 4: Additionally, fracture-related infection and chronic osteomyelitis are grouped together, despite their different biological and clinical behaviour. This may introduce bias, and separate analyses or a more detailed discussion would be helpful.

Response 2,3,4: Thank you for these comments. We agree that the small cohort size and single-centre design limit the strength of the prognostic analyses, which is reflected in the wide confidence intervals. We also acknowledge that fracture-related infection and chronic osteomyelitis differ in their biological and clinical behaviour. However, diagnostic use of microbiological and histological tests do not differ between the two entities. We are not aware of any studies advocating different diagnostic criteria or diagnostic modalities for the two conditions. Because of the limited numbers in each subgroup, we combined them to increase statistical power; however, we have now clarified in the manuscript that they remain distinct conditions, despite sharing similar underlying pathophysiological features of bone infection. These methodological constraints have been addressed and expanded upon in the revised limitations section. Larger, multicentre cohorts with adequate subgroup numbers will be necessary to validate the proposed thresholds and allow more robust analyses.

Limitations, Lines 262-275.

“The study lacked a control group with non-paired sampling, as histology is not routinely performed at the current centre. The small cohort size and single-centre design weaken the prognostic values of the proposed thresholds. This is reflected in the wide confidence intervals, and the results should therefore be interpreted with caution. Pooling FRI and cOM patients may be a limitation, as their biological and clinical presentations differ; however, they share similar underlying pathophysiological features of bone infection, which justified combining them in this exploratory cohort. Also, there are no studies which advocate different microbiological or histological diagnostic criteria for these conditions. Larger, multicentre studies with increased sample sizes are needed to validate these thresholds. Furthermore, splitting the samples, for parallel microbiological and histological assessment may introduce error as bacterial load and neutrophil infiltration may vary even within small regions of infected tissue [16]. The proportion of culture-negative cases was high, and molecular diagnostics could have further clarified culture-negative results.”

Response to Comments on the Quality of English Language

Point 1:

None

Additional clarifications

None

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a study investigating the combined diagnostic value of microbiology and histology in assessing chronic osteomyelitis and fracture-related infection. Overall, combining microbiology and histology can improve diagnostic precision, guide treatment decisions, and provide prognostic information, particularly in challenging cases of bone infections. Some revisions are needed to enhance the clarification and comprehension, though.

-Table 1, for consistency, the unit abbreviation of year "y" should be changed to "yr".

-Fig 1: The feature description should be highlighted and indicated in each image for those who are not familiar with the histology.

-Please define the diagnosis or definition of chronic osteomyelitis employed in this study.

-Patient cohort: Name of hospital and other specific details for study location should be provided.

-Methods: Please provide more details for the replicated study, i.e the details of manufacturer, model for the equipments used, and parameters in Microbiological analysis, the name of the stat software and version, the suppliers for the reagent and chemicals, etc.

-IRB detail: the date of COA issued should be given.

-Expand on the limitations, such as the lack of a control group and the potential variability in bacterial load and neutrophil infiltration within small tissue regions. Suggest future research directions to address these gaps

-It may be beneficial to discuss the cost-to-benefit ratio for the combined use of microbiology and histology compared to the typical routine test. Also, the importance of incorporating histology into routine diagnostic protocols for bone infections.

-The consort diagram may be added to aid in the understanding of the volunteered patient status in each step.

Author Response

Response to Reviewer 2 Comments

Summary

Thank you very much for also taking the time to review this manuscript. We greatly appreciate this and agree that this combination can improve diagnostic precision as well as guide treatment, especially in the challenging cases. Please find the detailed responses below, along with the corresponding revisions/corrections highlighted in track changes in the re-submitted files.

Point-by-point response to Comments and Suggestions for Authors

Comments 1: Table 1, for consistency, the unit abbreviation of year “y" should be changed to "yr".

Response 1: Thanks for this.

Table 1, “y” has been changed to “yr” for “Old age (>80 yr)”.

Comments 2: Fig 1: The feature description should be highlighted and indicated in each image for those who are not familiar with the histology.

Response 2: Thank you for pointing this out. As now seen in Fig 1. There are corresponding arrows as well as comments in the figure text.

Results, Lines 103 – 108.

“Figure 1. Localization of Staphylococcus aureus in intraoperative sampled bone biopsies from FRI patients

Immunohistochemical staining of S. aureus. (a–b) Infiltration of S. aureus (red) within the osteocyte lacuna-canalicular network (OLCN) (arrow) and microcracks of viable bone (arrowhead). Surrounding inflammatory cells (dashed arrow) are unable to reach these bacteria. (c) Extracellular aggregation of S. aureus surrounded by immune cells. (d) Intracellular S. aureus within a macrophage (arrow). (e-f) S. aureus observed as single isolated bacteria. Clear cells are erythrocytes. Bar; a-b=100 mm, c=40 mm, d=10 mm, e-f=150 mm. Abbreviations: TB, Trabecular bone; ND, Necrotic debris.

Comments 3: Please define the diagnosis or definition of chronic osteomyelitis employed in this study

Response 3: Thank you for this comment. We have defined chronic osteomyelitis as follows:

Materials and Methods, Lines 293-296.

“cOM was defined as an infection persisting for more than six weeks, with a draining wound or sinus tract communicating with dead bone and with local and/or systemic features of inflammation [38,42].

Comments 4: Patient cohort: Name of hospital and other specific details for study location should be provided.

Response 4: This has now been added to the manuscript to clarify the setup.

Material and Methods, Lines 288-290.

“The study was conducted in a single-centre cohort at a tertiary orthopaedic infection centre, where patients were prospectively enrolled by a specialized multidisciplinary team at Herlev Hospital, Capital Region, Denmark”

Comments 5: Methods: Please provide more details for the replicated study, i.e. the details of manufacturer, model for the equipment used, and parameters in Microbiological analysis, the name of the stat software and version, the suppliers for the reagent and chemicals, etc.

Response 5: We thank the reviewer for this comment. We have added the requested methodological details throughout the Materials and Methods section, including manufacturer and model information, reagent and chemical suppliers, microbiological parameters, and the statistical software and version.

Material and Methods, Lines 368-376.

“Microbiological assessment included inoculation of tissue samples on 5% Columbia blood agar plates and on anaerobe agar plates (Media Preparation Laboratory, Herlev, Denmark) for aerobic (Atmospheric air, 5% CO₂) and anaerobic cultures respectively. Incubation time was 5 days. Additionally, specimens were cultured thioglycolate enrichment broth (Media Preparation Laboratory, Herlev, Denmark) for 14 days at 35°C (Atmospheric air, 5% CO₂). Pathogen identification was performed using MALDI-TOF mass spectrometry (Bruker Daltonics GmbH & Co. KG, Bremen, Germany) using MBT Compass HT software, and MBT Compass library 2023 according to the manufacturer’s protocols. Antimicrobial susceptibility testing followed EUCAST guidelines [43]”

Material and Methods, Lines 446-448.

“Analyses were performed R version 4.2.3 (R Foundation for Statistical Computing, Vienna, Austria); two-sided p < 0.05 was considered statistically significant

Comments 6: IRB detail: the date of COA issued should be given

Response 6: Thank you. We have now included the date of COA into the manuscript. The stat software is now edited as well.

Institutional Review Board Statement, Lines 458-460.

“The study was approved by the Ethics Committee of the Capital Region of Denmark (H-22069974) and was granted on the 15^th of February 2023. All patients gave written and oral consent for inclusion.”

Comments 7: Expand on the limitations, such as the lack of a control group and the potential variability in bacterial load and neutrophil infiltration within small tissue regions. Suggest future research directions to address these gaps

Response 7: We agree that the study has important limitations, particularly the small cohort size and the resulting limited statistical power. We have expanded the limitations section accordingly, and we hope that these additions help address the concerns raised.

Limitations, Lines 262-275.

Comments 8: It may be beneficial to discuss the cost-to-benefit ratio for the combined use of microbiology and histology compared to the typical routine test. Also, the importance of incorporating histology into routine diagnostic protocols for bone infections.

Response 8: Thank you for this comment; it is a valid and interesting point. The cost-benefit of the comparison is not well known, but we have attempted to address this in the discussion:

Discussion, Lines 240-246:

“Although formal cost-effectiveness analyses comparing paired microbiology and histology with microbiology alone are lacking, several studies have demonstrated that the dual approach substantially improves diagnostic accuracy and is therefore considered the diagnostic “gold standard” [12,38,39]. Considering that the cost of histological assessment is low relative to the clinical and economic burden of missed infection, this approach may be particularly valuable in cases managed with antibiotic suppression, where culture yield could be reduced, and the risk of false-negative results is high.”

Comments 9: The consort diagram may be added to aid in the understanding of the volunteered patient status in each step

Response 9: This is a good point and would be helpful and is now included in Materials and Methods as Figure 4.

Materials and Methods, Lines 334-336:

“Figure 4. Consort flow diagram of the study cohort.

Illustrates patient screening, inclusion, allocation into fracture-related infection and chronic osteomyelitis groups, losses to follow-up, and final numbers included in the paired analysis.”

Response to Comments on the Quality of English Language

Point 1:

None

Additional clarifications

None

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have revised the manuscript accordingly. The quality and clarity of the manuscript is substantially improved and could be accepted for publication.