Diagnostic Challenges of Non-Sporulating Fungal Isolates Recovered from Traumatic Wounds Following Motor Vehicle Collisions: Subramaniula asteroides and Aspergillus quadrilineatus in the United Arab Emirates

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript reports two uncommon environmental molds recovered from traumatic wounds following motor vehicle collisions and describes the difficulties encountered in identifying initially non-sporulating isolates. The morphological and molecular characterization is of potential interest. However, several issues should be addressed before the manuscript can be considered for publication.

Major comments

1. In neither case is there sufficient evidence to establish that the recovered fungus was responsible for a true fungal infection. In Case 1, histopathologic examination was not performed, no antifungal treatment was documented, and satisfactory wound healing was reported following surgical management and antibacterial therapy. In Case 2, antifungal therapy was discontinued because there were no local or systemic signs of fungal infection. The Discussion itself states that the clinical courses favored environmental colonization rather than invasive infection. The manuscript should therefore clearly distinguish recovery or isolation of a fungus from possible colonization or environmental contamination, and from proven or probable fungal infection. Terms such as “wound infection” should not be used in relation to these fungal isolates unless supported by appropriate clinical, microbiological, or histopathologic evidence. This issue should also be addressed in Table 1, where the present Case 1 is described as “Post-traumatic wound infection.”
2. The clinical significance of the two isolates should be reconsidered accordingly. The main value of these cases appears to be the recovery and accurate identification of rare environmental fungi from traumatic wounds, particularly the difficulty in identifying initially non-sporulating isolates and the limitations of routine identification methods. The available evidence does not demonstrate that either organism caused the patients’ clinical disease. The Abstract, Case Report, Table 1, Discussion, and the concluding statements in the Discussion should therefore be reviewed carefully to avoid implying a causal pathogenic role that has not been established.
3. The timeline in Case 2 appears inconsistent. The isolate was reportedly presumptively identified as Aspergillus versicolor on Day 19, followed by confirmation as quadrilineatus and initiation of voriconazole. However, the following sentence states that voriconazole was discontinued on Day 14. Please verify and correct the chronology of identification and antifungal treatment.
4. The molecular evidence supporting species-level identification of the Case 1 isolate should be presented in greater detail. The manuscript currently reports 98–100% sequence identity with reference strains, including the ex-type strain CBS 123294, without providing locus-specific results. Please report the sequence identities for ITS and D1/D2 separately, particularly their identities to the ex-type strain, together with the corresponding alignment lengths or sequence coverage. The criteria used to assign the isolate to Subramaniula asteroides should also be clarified. If sequence similarity alone does not provide unambiguous species-level resolution, additional phylogenetic support should be considered.
   

   Minor comments

   5. The version of IQ-TREE is inconsistent between the Methods and the Figure 3 legend. The Methods state IQ-TREE v2.4.0, whereas the Figure 3 legend states v3.0.1. Please verify and report the correct version consistently.
   6. The statement that neither case demonstrated histopathologic evidence of invasive fungal disease should be revised. Histopathologic examination was not performed in Case 1, so the absence of histopathologic evidence cannot be concluded for this patient. It would be more accurate to state that histopathologic confirmation was not available for Case 1.
   7. Please check the figures and figure legends carefully for completeness and consistency. In Figure 1, for example, the legend describes panels A–C, D, F, and G–N, but panel E is not clearly described. Minor typographical errors, such as “Thich walled,” should also be corrected.
   8. The manuscript would benefit from careful language editing to improve grammar, clarity, and consistency of terminology. In particular, terms such as “infection,” “colonization,” “contamination,” “isolation,” and “invasive disease” should be used precisely and consistently throughout the manuscript.

Comments on the Quality of English Language

The manuscript is generally understandable, but the English would benefit from careful editing for grammar, sentence structure, and consistency of terminology. In particular, clinical terms such as “infection,” “colonization,” and “isolation” should be used more precisely and consistently.

Author Response

Reviewer 1:

 

The manuscript reports two uncommon environmental molds recovered from traumatic wounds following motor vehicle collisions and describes the difficulties encountered in identifying initially non-sporulating isolates. The morphological and molecular characterization is of potential interest. However, several issues should be addressed before the manuscript can be considered for publication.

Major comments

1. In neither case is there sufficient evidence to establish that the recovered fungus was responsible for a true fungal infection. In Case 1, histopathologic examination was not performed, no antifungal treatment was documented, and satisfactory wound healing was reported following surgical management and antibacterial therapy. In Case 2, antifungal therapy was discontinued because there were no local or systemic signs of fungal infection. The Discussion itself states that the clinical courses favored environmental colonization rather than invasive infection. The manuscript should therefore clearly distinguish recovery or isolation of a fungus from possible colonization or environmental contamination, and from proven or probable fungal infection. Terms such as “wound infection” should not be used in relation to these fungal isolates unless supported by appropriate clinical, microbiological, or histopathologic evidence. This issue should also be addressed in Table 1, where the present Case 1 is described as “Post-traumatic wound infection.”

 

Response: We thank the reviewer for this important comment and agree that recovery of a fungus from a traumatic wound does not, by itself, establish fungal infection. The manuscript has been comprehensively revised to distinguish fungal recovery from possible environmental inoculation, colonization, or contamination and from established fungal infection. Terminology implying a causal fungal wound infection has been removed where this was not supported by the available clinical, microbiological, or histopathologic evidence.

 

For Case 1, we now clarify that histopathologic assessment for fungal tissue invasion was not performed, no antifungal therapy was documented, and the mold was recovered only once, with wound improvement during surgical and antibacterial management. For Case 2, voriconazole was discontinued after 15 days when the isolate was considered more likely to represent colonization or contamination than true wound infection. The Discussion, Limitations, and Conclusion have been revised accordingly to emphasize that species-level identification does not establish pathogenic significance. Table 1 has also been corrected from “post-traumatic wound infection” to “recovery from a post-traumatic left-wrist wound,” with the clinical significance of the fungal recovery stated as uncertain.

 

These revisions are reflected in the Abstract (Lines 39–63), Introduction (Lines 71–73), Case Reports (Case 1: Lines 151–155 and 171–180; Case 2: Lines 211–214), Discussion (Lines 313–318 and 335–343), Limitations (Lines 423–430), Conclusion (Lines 433–439), and Table 1.

 

1. The clinical significance of the two isolates should be reconsidered accordingly. The main value of these cases appears to be the recovery and accurate identification of rare environmental fungi from traumatic wounds, particularly the difficulty in identifying initially non-sporulating isolates and the limitations of routine identification methods. The available evidence does not demonstrate that either organism caused the patients’ clinical disease. The Abstract, Case Report, Table 1, Discussion, and the concluding statements in the Discussion should therefore be reviewed carefully to avoid implying a causal pathogenic role that has not been established.

 

Response: We thank the reviewer for this important comment and agree with this interpretation. The manuscript has been substantially revised to emphasize that the principal value of these cases lies in the recovery and species-level identification of uncommon, initially non-sporulating fungal isolates from traumatic wounds, rather than in establishing a causal pathogenic role.

 

Throughout the revised manuscript, fungal recovery is clearly distinguished from established infection. We clarify that invasive fungal disease was not established in either case and that histopathologic assessment for tissue invasion was not performed. The Discussion and Conclusion have consequently been reframed to emphasize the diagnostic limitations of conventional morphology and routine MALDI-TOF MS, the value of integrated phenotypic and molecular identification, and the distinction between species-level identification and pathogenic significance.

 

These revisions are reflected in the Abstract (Lines 39–63), Case Reports (Lines 174–180 and 203–214), Discussion (Lines 313–318, 335–343, and 414–420), Limitations (Lines 423–430), Conclusion (Lines 433–439), and Table 1.

 

1. The timeline in Case 2 appears inconsistent. The isolate was reportedly presumptively identified as Aspergillus versicolor on Day 19, followed by confirmation as quadrilineatus and initiation of voriconazole. However, the following sentence states that voriconazole was discontinued on Day 14. Please verify and correct the chronology of identification and antifungal treatment.

 

Response: We thank the reviewer for identifying this important chronological inconsistency. We re-reviewed the available clinical, microbiological, referral laboratory, and treatment records and reconstructed the Case 2 chronology using the date of admission following the MVC as Day 0.

 

The revised chronology documents collection of the right-arm wound specimen on Day 7, fungal elements reported on Day 10, referral of the isolate to the UAEU FRL on Day 18, and a designation of “presumptive Aspergillus” on Day 20. Following phenotypic and multilocus molecular characterization, species-level identification as A. quadrilineatus was communicated to the clinical team on Day 30. Voriconazole was initiated on Day 30 with a loading dose of 6 mg/kg every 12 h followed by a maintenance dose of 4 mg/kg every 12 h and was discontinued on Day 45, after the isolate was considered more likely to represent colonization or contamination than true wound infection.

 

The previously reported provisional identification as A. versicolor could not be verified in the reviewed records and has therefore been removed. The revised manuscript now reports only the documented designation of “presumptive Aspergillus” before definitive molecular identification as A. quadrilineatus.

 

The corrected chronology is reflected in the Abstract (Lines 54–59) and Case 2 presentation (Lines 195–214), with the corresponding clinical interpretation updated in the Discussion (Lines 335–343 and 414–420).

 

1. The molecular evidence supporting species-level identification of the Case 1 isolate should be presented in greater detail. The manuscript currently reports 98–100% sequence identity with reference strains, including the ex-type strain CBS 123294, without providing locus-specific results. Please report the sequence identities for ITS and D1/D2 separately, particularly their identities to the ex-type strain, together with the corresponding alignment lengths or sequence coverage. The criteria used to assign the isolate to Subramaniula asteroides should also be clarified. If sequence similarity alone does not provide unambiguous species-level resolution, additional phylogenetic support should be considered.
   
   

Response: We thank the reviewer for this important comment. The Molecular Identification section has been revised to present the ITS and D1/D2 results separately. For the ITS region, UAE40 showed 98.95% sequence identity (100% query coverage) with the ex-type strain of S. asteroides CBS 123294 (GenBank accession no. NR_147625), whereas the D1/D2 region showed 100% sequence identity (100% query coverage) with the same ex-type strain (GenBank accession no. NG_246169).

 

To provide additional support beyond sequence similarity, we performed maximum-likelihood phylogenetic analysis of the ITS region. UAE40 was recovered as sister to the S. asteroides ex-type strain CBS 123294T (UFBoot = 85) within a monophyletic Subramaniula clade. Species-level identification was therefore supported by the locus-specific sequence results together with phylogenetic placement and phenotypic characterization. The corresponding phylogenetic analysis is presented in the new Figure 5.

 

These revisions are reflected in the Molecular Identification section (Lines 289–298), with the phylogenetic methodology described in Lines 258–264 and the corresponding analysis presented in Figure 5.

 

 

Minor comments

1. The version of IQ-TREE is inconsistent between the Methods and the Figure 3 legend. The Methods state IQ-TREE v2.4.0, whereas the Figure 3 legend states v3.0.1. Please verify and report the correct version consistently.

 

Response: We thank the reviewer for identifying this inconsistency. We verified the version of IQ-TREE used for the phylogenetic analyses and corrected the discrepancy throughout the manuscript. The Methods and the legends of the revised phylogenetic figures now consistently report IQ-TREE v3.1.3 (Line 260; Figures 5 and 6).

 

6. The statement that neither case demonstrated histopathologic evidence of invasive fungal disease should be revised. Histopathologic examination was not performed in Case 1, so the absence of histopathologic evidence cannot be concluded for this patient. It would be more accurate to state that histopathologic confirmation was not available for Case 1.

 

Response: We thank the reviewer for this important clarification and agree that absence of histopathologic evidence cannot be inferred when histopathologic examination was not performed. The manuscript has therefore been revised to state that histopathologic assessment for fungal tissue invasion was not performed in either case, rather than implying that histopathologic evidence of invasive fungal disease was absent.

 

This clarification is reflected in the Abstract (Lines 50–53), Case 1 presentation (Lines 177–180), Discussion (Lines 335–343), Limitations (Lines 423–430), and Conclusion (Lines 433–439).

 

1. Please check the figures and legends carefully for completeness and consistency. In Figure 1, for example, the legend describes panels A–C, D, F, and G–N, but panel E is not clearly described. Minor typographical errors, such as “Thich walled,” should also be corrected.

 

Response: We thank the reviewer for identifying these inconsistencies. All figures and corresponding legends have been carefully reviewed and revised for completeness, accuracy, and consistency. In the revised S. asteroides figure (now Figure 3), panel E is explicitly described, and the legend has been updated to correspond to the revised A–P panel layout, including colony morphology and thermotolerance (A–C), prolonged PDA growth with obverse and reverse morphology (D–E), urease activity (F), and microscopic features (G–P). Additional microscopy images with appropriate scale bars have also been incorporated.

 

All panel labels, arrowheads, scale-bar information, figure citations, and typographical errors, including “Thich walled,” have been corrected. The remaining figures and legends were likewise reviewed and revised for consistency with the corresponding descriptions in the manuscript. These revisions are reflected in the Phenotypic and Morphological Identification section (Lines 268–286), Figure 3 legend (Lines 460–469), and Figure 4 legend (Lines 472–480).

 

8. The manuscript would benefit from careful language editing to improve grammar, clarity, and consistency of terminology. In particular, terms such as “infection,” “colonization,” “contamination,” “isolation,” and “invasive disease” should be used precisely and consistently throughout the manuscript.

Response: We thank the reviewer for this important recommendation. The manuscript has been carefully edited throughout to improve grammar, clarity, consistency of terminology, and scientific precision. Particular attention was given to terminology describing the clinical significance of the recovered fungal isolates. We now consistently distinguish fungal recovery or isolation from possible environmental inoculation, colonization, or contamination and avoid using terms such as fungal infection or invasive fungal disease unless supported by the available clinical, microbiological, and, where available, histopathologic evidence.

 

Statements that could imply an unproven causal relationship between the recovered fungi and the patients’ wound processes have been revised accordingly. These changes have been applied throughout the manuscript, with representative revisions in the Introduction (Lines 71–73), Discussion (Lines 313–318 and 335–343), Limitations (Lines 423–430), and Conclusion (Lines 433–439).

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors I have read with great interest the manuscript titled: *Diagnostic Dilemmas of Sterile Clinical Isolates: First UAE Reports of Subramaniula asteroides and Aspergillus quadrilineatus Isolated from Traumatic Wounds Following Motor Vehicle Collisions*.   Strengths: I would like to highlight that this study identified the infectious etiological agents using both culture and gene sequencing (ITS, β-tubulin, and CaM), thereby substantiating the findings presented in the manuscript. In this regard, I offer the following comments:   1. Title   Since the study aims to highlight that alternative methods—such as sequencing—are necessary when sterile mycelia cannot be typed, I consider it appropriate to modify the title. One proposal is:   Sterile mycelia of *Subramaniula asteroides* and *Aspergillus quadrilineatus* obtained from traumatic wounds following motor vehicle collisions: Report of two cases in the United Arab Emirates   Abstract   I strongly recommend modifying this section and enriching it with specific data regarding the two patients. Specifically, the text should highlight the clinical approach and the indicators that led to the suspicion of wound infection, as well as the diagnosis, treatment, and outcome of both cases.   Introduction   I recommend expanding the information regarding cases of invasive aspergillosis; specifically, it is necessary to specify the main *Aspergillus* species associated with cutaneous aspergillosis. The text should link this to the low prevalence of *A. quadrilineatus* and highlight the appropriate diagnostic approach—treating it as a sterile isolate initially—prior to subsequent typing using methods such as MALDI-TOF.   Additionally, please indicate the prevalence of *Subramaniula asteroides* in this type of context: is it frequently isolated? Does it appear at different anatomical sites?   Furthermore, please emphasize the clinical significance of both species and the necessity of identifying them at the species level.   Finally, the objective of the manuscript must be included. Case Reports   Case 1   I strongly recommend including images of the infection on the left hand, as well as the CT scan image.   It is necessary to include the dosages and duration of cefazolin and gentamicin treatment.   It is necessary to state the specific data point and include the reference value.   I recommend specifying how the culture was obtained from the traumatic site with the graft; it is necessary to detail the sampling method used.   Line 76 mentions "completing the course"; please specify the dosage and duration for each medication.   As a recommendation, it would be interesting to see an image of the infected hand and an image taken at the time of discharge.   The acronym MALDI-TOF MS needs to be expanded, particularly upon its first mention in the text; subsequently, it may be used simply as MALDI-TOF MS.   Please cite the images included in each case.   It would be ideal to obtain information regarding the antifungal treatment, as a primary goal of case reports is to provide guidance on managing patients with rare conditions—or, in this instance, regarding the diagnosis of these rare fungi and specifically the typing of *Subramaniula asteroides*. Therefore, it is necessary to detail whether the discharge was against medical advice or to explain why treatment was not initiated before the patient left the hospital.   Case 2   It is necessary to include the patient's chronic kidney disease stage.   I recommend including imaging (X-ray, CT, etc.) that supports the diagnoses regarding the patient's trauma.   It is necessary to include the dosages for the treatments mentioned in lines 95–96.   It is necessary to detail how the wound cultures were obtained (line 99).   Please cite the images within the text.   It is necessary to state the dosage of voriconazole administered.   Phenotypic and Molecular Characterization of Isolates   Regarding the culture media used, it is necessary to add details about the manufacturer, city, and country for all media mentioned in the document. Phenotypic and Morphological Identification   Case 1   Image E is not mentioned in the figure legend; it needs to be included.   I recommend including a scale bar in the images.   Case 2   I recommend including a scale bar in the images.   For greater clarity, I recommend that all information regarding each case be included in its respective section and presented simply as Case 1 and Case 2—that is, integrating the clinical approach, diagnosis, microbiological and sequencing findings, treatment, and outcome as appropriate for each case.

Discussion

The information in lines 206–224 addresses infections in traumatic wounds—similar to the cases presented—yet it is not directly compared to them. Therefore, it is necessary to detail the similarities and differences.

Please update the number of species currently included in the *Aspergillus* genus (10.1016/j.simyco.2020.05.002).

Please standardize the terminology used in Table 1 (e.g., "Not specified" vs. "Not reported").

The final column of that table could simply be labeled "Reference."

I strongly recommend including the limitations of the case reports, such as the lack of information regarding the antifungal treatment for the patient in Case 2.

Given the subject matter, a discussion on how to interpret a sterile mycelium and the additional diagnostic measures to consider is needed.

References
It is necessary to include up-to-date references and format them according to the journal's guidelines.

Regards,

Author Response

Reviewer 2:

I have read with great interest the manuscript titled: *Diagnostic Dilemmas of Sterile Clinical Isolates: First UAE Reports of Subramaniula asteroides and Aspergillus quadrilineatus Isolated from Traumatic Wounds Following Motor Vehicle Collisions*.   Strengths: I would like to highlight that this study identified the infectious etiological agents using both culture and gene sequencing (ITS, β-tubulin, and CaM), thereby substantiating the findings presented in the manuscript. In this regard, I offer the following comments:  

 

1. Title   Since the study aims to highlight that alternative methods—such as sequencing—are necessary when sterile mycelia cannot be typed, I consider it appropriate to modify the title. One proposal is:   Sterile mycelia of *Subramaniula asteroides* and *Aspergillus quadrilineatus* obtained from traumatic wounds following motor vehicle collisions: Report of two cases in the United Arab Emirates

 

Response: We thank the reviewer for this valuable suggestion. The title has been revised to emphasize the diagnostic challenge posed by initially non-sporulating fungal isolates and the role of molecular methods in achieving species-level identification.

 

We chose the term “non-sporulating fungal isolates” rather than “sterile mycelia” because “Mycelia Sterilia” is a historical designation for fungi in which reproductive structures are not observed, whereas “non-sporulating” more precisely describes the initial culture phenotype encountered in our cases. In particular, the A. quadrilineatus isolate initially lacked diagnostic reproductive structures but subsequently developed characteristic Aspergillus structures during prolonged incubation. We also used “recovered from traumatic wounds” to avoid implying a causal role in fungal infection, which was not established in either case.

 

Accordingly, the revised title is: “Diagnostic Challenges of Non-Sporulating Fungal Isolates Recovered from Traumatic Wounds Following Motor Vehicle Collisions: Subramaniula asteroides and Aspergillus quadrilineatus in the United Arab Emirates” (Lines 3–5).

 

 

2. Abstract   I strongly recommend modifying this section and enriching it with specific data regarding the two patients. Specifically, the text should highlight the clinical approach and the indicators that led to the suspicion of wound infection, as well as the diagnosis, treatment, and outcome of both cases.  

 

Response: We thank the reviewer for this recommendation. The Abstract has been revised to provide a concise summary of the clinical presentation, diagnostic approach, treatment, and outcome of both cases. For Case 1, we now describe the severe crush-degloving injury, clinical suspicion of a complicated skin and soft-tissue infection prompting wound culture and antibacterial therapy, molecular identification of the non-sporulating isolate as S. asteroides, absence of histopathologic assessment for fungal tissue invasion, no documented antifungal therapy, and subsequent wound improvement. For Case 2, we summarize the extensive upper-limb trauma, surgical debridement and skin grafting, recovery of an initially non-sporulating mold subsequently identified as A. quadrilineatus by multilocus analysis, and voriconazole treatment followed by discontinuation after 15 days when the isolate was considered more likely to represent colonization or contamination than true wound infection.

 

The concluding statements have also been revised to clarify that invasive fungal disease was not established in either case and to emphasize the diagnostic and interpretive challenges associated with uncommon, initially non-sporulating fungal isolates recovered from traumatic wounds (Lines 37–63).

 

3.Introduction   I recommend expanding the information regarding cases of invasive aspergillosis; specifically, it is necessary to specify the main *Aspergillus* species associated with cutaneous aspergillosis. The text should link this to the low prevalence of *A. quadrilineatus* and highlight the appropriate diagnostic approach—treating it as a sterile isolate initially—prior to subsequent typing using methods such as MALDI-TOF.   Additionally, please indicate the prevalence of *Subramaniula asteroides* in this type of context: is it frequently isolated? Does it appear at different anatomical sites?   Furthermore, please emphasize the clinical significance of both species and the necessity of identifying them at the species level.   Finally, the objective of the manuscript must be included.

 

Response: We thank the reviewer for these constructive recommendations. The Introduction has been substantially revised to provide greater clinical, epidemiological, and diagnostic context for both isolates. We now describe the principal Aspergillus species associated with cutaneous aspergillosis and place the infrequently reported A. quadrilineatus in this context, including its documented clinical presentations and occurrence in the Middle East and Arabian Gulf.

 

The background on S. asteroides has also been expanded to highlight its infrequent clinical recovery, predominant association with ocular disease, additional recovery from skin and sinus-associated specimens, and occurrence in arid environmental sources. We further discuss the diagnostic challenges posed by initially non-sporulating fungal isolates, including the limitations of conventional morphology and MALDI-TOF MS and the complementary role of molecular sequencing for species-level identification.

 

Finally, the revised Introduction emphasizes the clinical relevance of species-level identification while clarifying that fungal recovery does not itself establish infection and must be interpreted in the appropriate clinical, microbiological, and, where available, histopathologic context. The objective of the study is now explicitly stated at the end of the Introduction.

 

These revisions are reflected in Lines 75–117, with the study objective stated in Lines 119–123.

 

4. Case Reports   Case 1   I strongly recommend including images of the infection on the left hand, as well as the CT scan image.   It is necessary to include the dosages and duration of cefazolin and gentamicin treatment.   It is necessary to state the specific data point and include the reference value.   I recommend specifying how the culture was obtained from the traumatic site with the graft; it is necessary to detail the sampling method used.   Line 76 mentions "completing the course"; please specify the dosage and duration for each medication.   As a recommendation, it would be interesting to see an image of the infected hand and an image taken at the time of discharge.  

 

Response: We thank the reviewer for these detailed recommendations. Case 1 has been substantially revised following re-review of the clinical and microbiological records, and the clinical course is now presented chronologically with the additional verified information incorporated.

 

The patient is now specified as having stage 5 chronic kidney disease. Initial antimicrobial management has been clarified, including single intravenous doses of cefazolin 1 g and gentamicin 80 mg, both renally adjusted, followed by amoxicillin-clavulanic acid 1 g every 8 h. Subsequent antibacterial regimens, including doses, frequencies, and treatment durations, have also been incorporated. The clinical and laboratory findings prompting Infectious Diseases assessment, including leukocytosis, C-reactive protein, and procalcitonin values, together with wound findings, antibacterial management, wound evolution, and follow-up, are now described chronologically.

 

The microbiological course has also been clarified, including collection of the left-wrist specimen, subsequent laboratory findings, fungal growth, provisional phenotypic identification, MALDI-TOF MS findings, referral to the UAEU FRL, and definitive molecular identification as S. asteroides. Histopathologic assessment for fungal tissue invasion was not performed, and no antifungal therapy was documented. The available records identify the specimen as originating from the left wrist, but do not further specify the sampling technique; therefore, no unverified collection method has been added.

 

Regarding imaging, relevant CT angiography of the injured left upper extremity has now been included as Figure 1. No suitable clinical photographs of the hand at presentation or near discharge were available for inclusion.

 

These revisions are reflected in the Case 1 presentation (Lines 127–180). Specifically, the CKD stage, trauma and CT findings, and initial antimicrobial doses are detailed in Lines 127–142; the Infectious Diseases assessment, laboratory values, subsequent antibacterial management, and wound evolution in Lines 144–155; and the microbiological course, discharge and follow-up, referral, definitive identification, and antifungal/histopathologic information in Lines 157–180. Figure 1 is cited at Line 135, with the corresponding legend provided at Lines 446–450.

 

5.The acronym MALDI-TOF MS needs to be expanded, particularly upon its first mention in the text; subsequently, it may be used simply as MALDI-TOF MS.

 

Response: Response: We thank the reviewer for this observation. The acronym has been expanded at its first occurrence as “matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)” in both the Abstract and the main text. Thereafter, MALDI-TOF MS is used consistently throughout the manuscript. The platform used for both isolates is also specified as VITEK MS (bioMérieux, Marcy-l'Étoile, France)/subsequent appearance (bioMérieux) in the Case Reports (Abstract, Lines 44–45; Introduction, Lines 107–108; Case 1, Lines 168–169; Case 2, Lines 203–205).

 

6. Please cite the images included in each case.

 

Response: We thank the reviewer for this recommendation. Relevant radiological images have now been included and cited within the respective case presentations. CT angiography of the injured left upper extremity is presented as Figure 1 for Case 1, and contrast-enhanced CT imaging demonstrating the major traumatic findings is presented as Figure 2 for Case 2. The images are cited in the Case 1 and Case 2 presentations at Lines 135 and 191, respectively, with the corresponding legends provided in Figures 1 and 2.

 

7. It would be ideal to obtain information regarding the antifungal treatment, as a primary goal of case reports is to provide guidance on managing patients with rare conditions—or, in this instance, regarding the diagnosis of these rare fungi and specifically the typing of *Subramaniula asteroides*. Therefore, it is necessary to detail whether the discharge was against medical advice or to explain why treatment was not initiated before the patient left the hospital.  

 

Response: We thank the reviewer for raising this important point. Following re-review of the clinical and microbiological records, the Case 1 chronology and antifungal management have been clarified. No antifungal therapy was documented as prescribed or administered. Fungal elements were reported before discharge, while subsequent fungal growth and definitive species-level identification occurred later in the clinical course. No specific clinical intervention in response to the fungal finding was documented.

 

The patient was discharged on Day 32 and subsequently attended outpatient wound-management follow-up on Day 35, with no clinical concerns documented; the available records do not indicate that discharge was against medical advice. The wound had been documented as clean and healing and subsequently clean and dry following surgical and antibacterial management. The fungal isolate was subsequently referred to the UAEU FRL on Day 57 for detailed phenotypic and molecular characterization as part of passive surveillance, where species-level identification as S. asteroides was supported by molecular analysis. Histopathologic assessment for fungal tissue invasion was not performed.

 

The revised manuscript therefore clarifies that S. asteroides was recovered and subsequently identified at the species level, but its etiologic role in the wound could not be established. These revisions are reflected in the Case 1 presentation (Lines 157–180), Discussion (Lines 335–343 and 371–376), Limitations (Lines 423–430), and Conclusion (Lines 433–439).

 

8. Case 2   It is necessary to include the patient's chronic kidney disease stage.   I recommend including imaging (X-ray, CT, etc.) that supports the diagnoses regarding the patient's trauma.   It is necessary to include the dosages for the treatments mentioned in lines 95–96.   It is necessary to detail how the wound cultures were obtained (line 99).   Please cite the images within the text.  

 

Response: We thank the reviewer for these important recommendations. Case 2 has been substantially revised following re-review of the clinical, microbiological, and radiological records, and the clinical course is now presented chronologically.

 

The patient is now specified as having stage 4 chronic kidney disease, with the documented admission serum creatinine and eGFR included. The traumatic findings have also been expanded based on the radiological records, including the right humeral and first-rib fractures, cervical fractures, pulmonary contusions, right-sided pneumothorax, extensive right chest-wall/axillary hematoma, and complete cut-off of the right axillary artery. A representative de-identified contrast-enhanced CT image has been included as Figure 2 and is cited within the Case 2 presentation.

 

Antibacterial treatment has been clarified as intravenous piperacillin-tazobactam 4 g every 8 h and vancomycin 1 g every 12 h during the ICU admission. The microbiological chronology has also been revised: following surgical debridement on Day 7, a right-arm wound specimen was collected and submitted for microbiological culture on the same day; fungal elements were reported on Day 10, followed by a designation of “presumptive Aspergillus” on Day 20. The available records identify the specimen as originating from the right-arm wound but do not further specify the sampling technique; therefore, no unverified collection method has been added.

 

These revisions are reflected in the Case 2 presentation (Lines 182–214), with Figure 2 cited at Line 191 and the corresponding figure legend provided at Lines 453–457.

 

 

9. It is necessary to state the dosage of voriconazole administered.  

 

Response: We thank the reviewer for this important recommendation. The clinical and treatment records were re-reviewed to clarify the voriconazole regimen and treatment chronology in Case 2. The revised manuscript now specifies that voriconazole was initiated on Day 30 with a loading dose of 6 mg/kg every 12 h, followed by a maintenance dose of 4 mg/kg every 12 h. Voriconazole was discontinued on Day 45 after the isolate was considered more likely to represent contamination or colonization than true wound infection. These details are provided in the Case 2 presentation (Lines 211–214).

 

10.Phenotypic and Molecular Characterization of Isolates   Regarding the culture media used, it is necessary to add details about the manufacturer, city, and country for all media mentioned in the document.

 

Response: We thank the reviewer for this suggestion. The Phenotypic and Molecular Characterization of Isolates section has been revised to provide manufacturer, city, and country information for the commercially sourced culture media and reagents used, with catalogue/product numbers included where applicable.

 

The revised Methods now also describe the primary microbiological processing of the clinical specimens at the diagnostic laboratory using routine bacteriological and mycological media supplied by Medysinal FZCO (Dubai, UAE), including the mycological media used for Case 1 and Case 2. For subsequent phenotypic characterization at the UAEU FRL, PDA, MEA, OA, WA, and LPCB are identified as products of HiMedia Laboratories Pvt. Ltd. (Mumbai, India), with their respective product numbers. Commercially prepared urea agar slants are identified with the corresponding catalogue number and supplier information (Medysinal FZCO, Dubai, UAE). The preparation of in-house 10% aqueous lactic acid and banana leaf-PDA has also been clarified.

 

These revisions are reflected in the Materials and Methods section (Lines 218–248).

 

11.Phenotypic and Morphological Identification   Case 1   Image E is not mentioned in the figure legend; it needs to be included.   I recommend including a scale bar in the images.

 

Response: We thank the reviewer for identifying this omission and for the recommendation. Figure 3 (Previously Figure 1) and its corresponding legend have been comprehensively revised. Panel E is now explicitly described as the reverse of the prolonged PDA culture showing dark brown to black pigmentation. The microscopic panels have also been revised and expanded, with scale bars incorporated into the images. Corresponding panel labels, morphological descriptions, and mounting preparations have been updated to accurately reflect the revised figure.

 

The phenotypic and morphological description in the text has been revised for consistency with Figure 3 (Lines 268–274), and the updated Figure 3 legend is provided at Lines 460–469.

 

 

12. Case 2   I recommend including a scale bar in the images.  

 

Response: We thank the reviewer for this helpful recommendation. Scale bars have now been added to all microscopic panels in the revised Case 2 figure (Figure 4) (previously Figure 2), with the corresponding measurement (20 µm) specified in the figure legend. The microscopic panels and legend were also reviewed to ensure consistency of panel labels, morphological descriptions, and mounting preparations. These revisions are reflected in the Phenotypic and Morphological Identification section (Lines 276–286) and the Figure 4 legend (Lines 472–480).

 

13. For greater clarity, I recommend that all information regarding each case be included in its respective section and presented simply as Case 1 and Case 2—that is, integrating the clinical approach, diagnosis, microbiological and sequencing findings, treatment, and outcome as appropriate for each case.

 

Response: We thank the reviewer for this helpful suggestion. To improve clarity, the manuscript has been revised to clearly distinguish Case 1 and Case 2 throughout. The clinical presentation and chronology, diagnostic course, treatment, outcome, and clinical interpretation are now presented separately for each patient, with case-specific information explicitly attributed to the respective case.

 

The isolate-specific phenotypic and molecular findings are likewise presented separately as Case 1 and Case 2 within the corresponding laboratory sections. Laboratory procedures common to both isolates, including culture conditions, microscopy, DNA extraction, sequencing, and phylogenetic analysis, have been retained in shared methodological sections to avoid unnecessary duplication.

 

This revised organization provides a clear case-based presentation while retaining common laboratory methods in a concise and reproducible format. The revised Case 1 and Case 2 presentations are provided at Lines 127–180 and 182–214, respectively; shared laboratory methods at Lines 218–264; and case-specific phenotypic and molecular findings at Lines 268–310.

 

14. Discussion The information in lines 206–224 addresses infections in traumatic wounds—similar to the cases presented—yet it is not directly compared to them. Therefore, it is necessary to detail the similarities and differences.

 

Response: We thank the reviewer for this important suggestion. The Discussion has been revised to directly compare our cases with previously reported post-traumatic invasive fungal infections. We now highlight the shared features, including MVC-related polytrauma, extensive soft-tissue disruption and devitalization, vascular injury, and repeated surgical intervention. We also emphasize the important differences from established post-traumatic invasive fungal disease, particularly the absence of progressive wound necrosis or other local or systemic features attributable to invasive fungal disease and the lack of histopathologic assessment for fungal tissue or vascular invasion in both cases.

 

In Case 1, the wound improved with surgical and antibacterial management without documented antifungal therapy. In Case 2, voriconazole was administered but discontinued after 15 days when the isolate was considered unlikely to represent true wound infection. Thus, despite sharing several recognized risk features of post-traumatic invasive fungal infection, evidence supporting invasive disease was lacking in both cases.

 

These direct comparisons are presented in the revised Discussion (Lines 328–343).

 

 

15. Please update the number of species currently included in the *Aspergillus* genus (10.1016/j.simyco.2020.05.002).

 

Response: We thank the reviewer for this important suggestion. The relevant section has been revised according to the updated taxonomic framework of Houbraken et al. (2020). The previous statement that the genus Aspergillus comprises more than 250 species has been replaced with the updated classification recognizing 446 accepted species distributed across six subgenera, 27 sections, and 75 series. The description of section Nidulantes has also been updated to reflect the 74 accepted species recognized within this section, replacing the previous outdated estimate of more than 25 species.

 

The surrounding Discussion has been revised accordingly to reflect the greater recognized taxonomic diversity of Aspergillus and section Nidulantes and the relevance of uncommon species such as A. quadrilineatus. The reviewer-recommended Houbraken et al. (2020) reference (doi:10.1016/j.simyco.2020.05.002) has been incorporated as Reference 34. These revisions are reflected in the Discussion (Lines 378–389).

 

 

16. Please standardize the terminology used in Table 1 (e.g., "Not specified" vs. "Not reported").

The final column of that table could simply be labeled "Reference."

 

Response: We thank the reviewer for this helpful suggestion. The terminology in Table 1 has been standardized throughout, with “Not reported” used consistently where information was unavailable in the cited reports. In addition, the final column heading has been simplified from “Author (Reference)” to “Reference,” as recommended (Table 1).

 

17. I strongly recommend including the limitations of the case reports, such as the lack of information regarding the antifungal treatment for the patient in Case 2.

 

Response: We thank the reviewer for this important recommendation. A dedicated Limitations paragraph has now been included immediately before the Conclusion to explicitly address the principal limitations of both cases.

 

Most importantly, histopathologic assessment for fungal tissue or vascular invasion was not performed in either case, precluding definitive confirmation or exclusion of invasive fungal disease. Microbiological evidence was also limited to a single fungal recovery in each case without serial cultures to demonstrate persistence, and longer-term clinical follow-up was limited, particularly for Case 1.

 

Regarding the reviewer’s specific concern about antifungal treatment in Case 2, re-review of the treatment records allowed us to clarify this information. The revised Case 2 presentation now documents the voriconazole loading and maintenance doses, initiation on Day 30, discontinuation on Day 45, and the clinical rationale for discontinuation.

 

These revisions are reflected in the Case 2 presentation (Lines 211–214), dedicated Limitations section (Lines 423–430), and Conclusion (Lines 433–439).

 

18. Given the subject matter, a discussion on how to interpret a sterile mycelium and the additional diagnostic measures to consider is needed.

 

Response: We thank the reviewer for this important suggestion. The revised manuscript now includes an expanded discussion on the interpretation and diagnostic work-up of initially non-sporulating (“sterile”) fungal isolates. We clarify that the absence of diagnostic reproductive structures can preclude conventional morphology-based identification and that features such as bulbil- or papulospore-like aggregates may be non-specific and insufficient for reliable species-level identification.

 

This diagnostic challenge was directly reflected in Case 1, in which the isolate was initially non-sporulating and subsequently developed limited, non-diagnostic morphological structures that did not permit reliable species-level identification. MALDI-TOF MS was also non-diagnostic, whereas ITS and D1/D2 sequence analysis together with phylogenetic placement supported identification as S. asteroides. The revised manuscript further emphasizes the complementary role of molecular approaches for morphologically inconclusive isolates and the need to interpret species-level identification in the appropriate clinical and, where available, histopathologic context.

 

These revisions are reflected in the Introduction (Lines 105–117) and Discussion (Lines 352–369).

 

19. References
    
    

It is necessary to include up-to-date references and format them according to the journal's guidelines.

 

Response: We thank the reviewer for this recommendation. The reference list has been comprehensively reviewed and updated to include recent and relevant epidemiological, taxonomic, diagnostic, and clinical literature pertaining to A. quadrilineatus, S. asteroides, and trauma-associated fungal recovery, while retaining appropriate foundational references.

 

All references and in-text citations have been reviewed, reformatted, and renumbered according to the MDPI/Journal of Fungi style. The manuscript has also been checked for consistency in the sequential citation and numbering of references, figures, and tables, together with corresponding revisions to figure legends and table formatting. The updated reference list is provided from Line 519 onward.

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have undoubtedly improved the manuscript; however, some details need to be addressed.

Introduction

Insert the correct acronym for the calmodulin gene (CaM).

Case 1

Remove the repeated sentence: “Tetanus prophylaxis was also provided.”

Case 2

Check the unit of measurement for serum creatinine.

Check the translation of “right axilla” in the Figure 2 description.

Materials and Methods

Include details regarding the manufacturers of the culture media used in both cases. Also, specify the established incubation period.

Ensure that genus and species names are italicized throughout the manuscript.

I would appreciate it if the editorial team could verify that these changes are implemented prior to the manuscript's publication.

Regards,

Author Response

Round 2

 

The authors have undoubtedly improved the manuscript; however, some details need to be addressed.

Revision: We thank the reviewer for their careful reassessment of the revised manuscript and for the additional comments. We have addressed each of the points raised, as detailed below.

Introduction

Insert the correct acronym for the calmodulin gene (CaM).

Revision: We thank the reviewer for this correction. The correct acronym for the calmodulin gene (CaM) has been inserted and used consistently throughout the revised manuscript. The corresponding revisions are reflected at Lines 112, 207, 257, 305, 307, 314, 400, 404–405, and 494.

 

Case 1

Remove the repeated sentence: “Tetanus prophylaxis was also provided.”

Revision: The repeated sentence, “Tetanus prophylaxis was also provided,” has been removed.

Case 2

Check the unit of measurement for serum creatinine.

Revision: Thank you for noting this. The unit of measurement for serum creatinine is µmol/L (micromoles per liter). This has been confirmed/corrected in the manuscript reflected in line 185

Check the translation of “right axilla” in the Figure 2 description.

Revision: Thank you for highlighting this. We reviewed the original CT image and radiology report. Although the complete CT examination documented extension of the hematoma into the right axillary region, the axilla is not directly visualized on the sagittal image presented in Figure 2. We have therefore revised the figure legend to distinguish the findings demonstrated on the selected image from those reported on the complete CT examination.

We have edited the legend under figure 2, and this is now reflected in line 453.

 

 

Materials and Methods

Include details regarding the manufacturers of the culture media used in both cases. Also, specify the established incubation period.

Revision: Response: We thank the reviewer for this suggestion. The Materials and Methods section has been revised to provide the manufacturer and location of the culture media used in both cases, together with the relevant product codes where applicable. Incubation conditions have also been clarified, including the temperatures and established incubation periods used for primary culture and subsequent phenotypic characterization at the UAEU FRL. Primary diagnostic processing is now clearly distinguished from subsequent reference-laboratory characterization. The corresponding revisions are reflected in the Materials and Methods (Lines 226, 229–231, 238–242, and 247–250) and Results (Lines 275–276). The urease incubation condition has also been updated in the corresponding Figure 3 legend (Lines 466–467).

Ensure that genus and species names are italicized throughout the manuscript.

Revision: We thank the reviewer for this observation. Genus and species names were carefully reviewed throughout the manuscript, including the main text, tables, figure legends, and reference list, to ensure consistent italicization. One non-italicized instance of “Aspergillus” at Line 257 was identified and corrected. Scientific names appearing in the reference titles were also reviewed and formatted accordingly. Genus and species names are now consistently italicized throughout the manuscript and references.

 

 

Author Response File: Author Response.docx