Review Reports - Synovial Fluid Analysis in Melioidosis: Experiences from the Darwin Prospective Melioidosis Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Campbell et al. detail a straightforward clinical description of B. pseudomallei in synovial fluid. It is an interesting clinical report and I have no major concerns or comments.

Minor. In several places italics are not used appropriately for names of bacteria (line 36, figure 1, etc.). I recommend checking entire manuscript.

Traditionally, letters for stats purposes (i.e. p) are also in italics "p", I would check the entire manuscript.

The new accepted description of the bacterium is "gram-negative" not "Gram negative"

Figure 3 does not have to be that large and should cropped and sized accordingly.

Author Response

Reviewer 1

Comments:

Campbell et al. detail a straightforward clinical description of B. pseudomallei in synovial fluid. It is an interesting clinical report and I have no major concerns or comments.

Minor. In several places italics are not used appropriately for names of bacteria (line 36, figure 1, etc.). I recommend checking entire manuscript.

Traditionally, letters for stats purposes (i.e. p) are also in italics "p", I would check the entire manuscript.

The new accepted description of the bacterium is "gram-negative" not "Gram negative"

Figure 3 does not have to be that large and should cropped and sized accordingly.

Reply:

We thank the reviewer for their comments, and their review of the manuscript. We have reviewed the manuscript and corrected non-italicised species. Unfortunately, we are unable to italicise a specific font in figure 1. Regarding formatting of p values, we have changed these where able (in the texts rather than figure proper). We have altered Gram negative to gram-negative. We agree that figure 3 is quite large (we supplied a high-quality image for possible compression) and are happy for it to be cropped at the editor’s discretion.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript entitled “Synovial fluid analysis in melioidosis: experiences from the Darwin Prospective Melioidosis Study” by Campbell et al. The introdution should provide more background information on the challenges og daignosing melioidosis SA. In the method part, the authors should clarify the criteria which used to define SA in the DPMS. Moreover, the laboratory measurements should clarify the types of methods and anticoagulants in biochemistry tests that the authors used and also add the references. In the result part, the authors should explain how the number of clotted samples may have affected the result of the study. Moreover, the authors ahould explain more detailed information about the timing of antimicrobial exposure between initiation and sampling. For discussion, the authors should give the potential reasons for the differenes between WCC findings in this study and other studies. In additions, the authors should explain about the limitation of this study and the role of PCR and LFA in the diagnosis of melioidosis SA.

Author Response

Comments:

Comments

We thank the reviewer for their review, critical reading, and suggestions for the manuscript.

We thank the reviewer for the suggestions regarding the introduction, and from lines 61-65 added the following: “Accurate diagnosis of this syndrome can be challenging due to the clinical complexity of these cases with often multifocal intra- and extra-articular loci of infection. Further, our clinical experience is notable for late (days post initiation of antimicrobials) manifestations of intraarticular disease after an initial presentation with another syndrome.”

In the DPMS, a label of melioidosis septic arthritis is given based on retrospective review of the case, considering radiographic, biochemical and clinical findings of each specific patient. However, given this is an imprecise method of confirming cases, we focussed our assessment on culture confirmed melioidosis cases. We have altered the wording of the methods to make this more explicit in lines 83-84: “… and then further examined those patients with growth of B. pseudomallei from an intraarticular clinical specimen – IE: culture confirmed melioidosis ….”

Samples are typically only placed in a sterile container and only a single sample was sent to the lab in an anticoagulated blood tube. This sample was not included in the main analysis as it was not sent for culture (and thus not laboratory confirmed melioidosis). We have added the following clarification to lines 99-105 to make the lack of biochemical testing and anticoagulant more explicit:” … without an anticoagulant. Occasionally samples are also placed in ethylenediaminetetraacetic acid (EDTA) or lithium heparin tubes to reduce clotting; this allows accurate cell count but, due to lack of sterility, cannot be used for culture. Some labs, but not ours, use hyaluronidase to dissolve clots. When only small sample volumes are available culture is prioritised”, and removed the following sentence: “Uncommonly samples may be aliquoted into anticoagulated blood tubes or blood culture bottles.’”

Regarding the effect of clotting on the results, we have briefly mentioned this in the limitations section of our discussion (lines 189-192). Specifically we hypothesize that higher inflammatory cell burden in melioidosis infected joints may increase the clotting of specimens.

Regarding further information on the subgroup of pre-antibiotic samples, we have added the following clarification in the methods in lines 96-98: “Samples were considered pre-antibiotics if they were collected before or on the same day as melioidosis-effective intravenous antibiotics (either meropenem or ceftazidime) were initiated.” We have also added to the results section in lines 141-142: “Samples were taken prior to receipt of antibiotic therapy in 32% (22/69), with the median days pre-antibiotics of 1 (IQR: 0-2 days).”

We have updated the discussion to speculate on the possible differences between our WCC findings and others. We have added the following to lines 168-170: “These differences may be due to differences in laboratory reporting as a number (12/77; 15%) of B. pseudomallei infected joints from Teparrukkul et al (2017) had cell counts reported only as ‘frank pus’, thus likely substantially reducing the overall median WCC.”. Further, as we restricted our analysis of joint fluid cell count to those culture positive for B. pseudomallei, our finding of counts usually above 50,000 x10⁶ cells/L are consistent with the literature from septic arthritis studies of other bacterial pathogens.

We have spent lines 178-184 discussing the role of a PCR and LFA, but chose not to go further in-depth in this to retain the focus of the manuscript on the microscopy results. The utility of these diagnostic strategies have been reviewed elsewhere, such as reference 1 (Meumann et al, 2024) and reference 19 (Currie et al, 2022).

Reviewer 3 Report

Comments and Suggestions for Authors

Is it possible to add systemic neutrophil and CRP counts as part of the sub-group analysis?
You mention presence of contiguous osteomyelitis- is this radiologically or macroscopically during surgery. Please can you clarify
Is it possible to either numerically or graphically demonstrate differences in the cell count over time in the post-antibiotic cohort.
For the box plots can you add the datapoints to assist with understanding the numerical breakdowns.

Author Response

Comments:

Is it possible to add systemic neutrophil and CRP counts as part of the sub-group analysis?

You mention presence of contiguous osteomyelitis- is this radiologically or macroscopically during surgery. Please can you clarify

Is it possible to either numerically or graphically demonstrate differences in the cell count over time in the post-antibiotic cohort.

For the box plots can you add the datapoints to assist with understanding the numerical breakdowns.

Comments

We thank the reviewer for their reading and comments on the manuscript.

We have limited data regarding these systemic biochemistry results but have elected not to investigate these due to the marked heterogeneity of collection times, and comorbidities of patients. As such, we focussed on the joint specific findings to help focus the manuscript.

Contiguous osteomyelitis was defined either radiologically, intraoperatively, or microbiologically (if appropriate samples were taken). We have added this clarification to the manuscript (lines 94-95).

We have added an additional figure (new figure 3) which displays the median (IQR) synovial white cell count stratified by days post antibiotic initiation. Given the small numbers for each day we have not performed any specific statistical analysis regarding trend.

We have superimposed the datapoints to the boxplots of figure 2 as requested. The y-axis has also changed slightly to accommodate some outliers.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have already revised the manuscript according to my suggestions. I recommend that this manuscript "Synovial fluid analysis in melioidosis: experiences from the Darwin Prospective Melioidosis Study" is suitable for acceptance.