Identification of causative fungus from sterile abscess using metagenomics followed by in situ hybridization
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Introduction. Invasive fungal infections require early diagnosis for treatment. Microscopic observation of biopsy and blood culture is the gold standard for the identification of the causative fungus, but it is difficult to identify the causative pathogen by a sterile abscess biopsy.
Case Presentation. We present a case report of breakthrough invasive trichosporonosis in a 65-year-old Japanese male with acute myeloid leukaemia receiving antifungal prophylaxis. Blood cultures showed no fungal growth, and a liver biopsy and a removed spleen with abscess showed fragmented fungi, but no fungal identification was possible. This report demonstrates that retrospective analyses were able to identify the causative fungus.
Conclusion. We narrowed down the candidate fungi by deep sequencing of the ITS1 region of fungal genome and confirmed that the fungus observed in the specimen was Trichosporon asahii by in situ hybridization using a DNA probe targeting 26S rRNA.
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I am pleased to tell you that your article has now been accepted for publication in Access Microbiology. Thank you for thoroughly addressing your reviewer comments and please accept my apologies for the delay in relaying this accept decision to you following review.
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Authors have clearly made the suggested changes and have addressed all the concerns.
Please rate the quality of the presentation and structure of the manuscript
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To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
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If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
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There was great interest in your paper from our reviewer community and we were lucky to secure 4 reviewer comments. The reviewers were largely positive about your work, and consider it a good fit for Access Microbiology. However, they have raised multiple points which amount to a major revision decision. Please consider all their points and address them in point by point response to reviewers, as well as incorporation these revision into your manuscript. There is mention of plagiarism from one reviewer, and while I can understand the need to strive for originality in newly published work, I have looked through your manuscript and found no evidence of text or content that would pose a problem, so please don't worry about this point. I look forward to receiving your revision, but please let me know if you need more time.
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Comments to Author
The original Identification of causative fungus from sterilized or bacteriostatic abscess using metagenomics following by in situ hybridization is reviewed, and a case in which NGS methods and hybridization are used to make the final etiological diagnosis is correctly described. The work is well structured and provides useful information, therefore, it must be accepted with small clarifications: In the title the term bacteriostatic abscess appears (I understand that because there is no growth by traditional methods of microorganisms), but perhaps only the term sterile abscess would be enough, otherwise a clarification from the authors would be important why they put this term The same thing happens in the abstract when they talk about sterile or static abscess, with one or both of the terms expressed …
Comments to Author
The original Identification of causative fungus from sterilized or bacteriostatic abscess using metagenomics following by in situ hybridization is reviewed, and a case in which NGS methods and hybridization are used to make the final etiological diagnosis is correctly described. The work is well structured and provides useful information, therefore, it must be accepted with small clarifications: In the title the term bacteriostatic abscess appears (I understand that because there is no growth by traditional methods of microorganisms), but perhaps only the term sterile abscess would be enough, otherwise a clarification from the authors would be important why they put this term The same thing happens in the abstract when they talk about sterile or static abscess, with one or both of the terms expressed differently.
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
Congratulations for a good work. Few important points are highlighted which need correction: 1. There is a small amount of plagiarism - may be inadvertantly ..... Authors may please make the changes so that plagiarism does not occur. 2. It seems on reading to me that the patient was on Voriconazole and Liposomal Amphotericin B when the blood culture was taken. If so is this patient being on antifungals not an important reason for failure of blood culture to detect the fungal pathogen. So the authors may include this as the second reason to get a negative blood culture apart from relatively low levels seen in liver and spleen biopsy which they allude to in Line 109-110. Just stating "their reduced biological activity, partly due to antimicrobial agents" does not give the reader especially novices a …
Comments to Author
Congratulations for a good work. Few important points are highlighted which need correction: 1. There is a small amount of plagiarism - may be inadvertantly ..... Authors may please make the changes so that plagiarism does not occur. 2. It seems on reading to me that the patient was on Voriconazole and Liposomal Amphotericin B when the blood culture was taken. If so is this patient being on antifungals not an important reason for failure of blood culture to detect the fungal pathogen. So the authors may include this as the second reason to get a negative blood culture apart from relatively low levels seen in liver and spleen biopsy which they allude to in Line 109-110. Just stating "their reduced biological activity, partly due to antimicrobial agents" does not give the reader especially novices a clear picture. Please categorically mention the role of combination antifungal therapy on the blood culture reports. 3. Further the antimicrobial susceptibility of the finally isolated Trichosoporon (at the time of fatal resurgent infection during transplantation) is required in the results section. 4. Are the units of Mitoxantrone, etoposide and cytarabine dosage (given as mg / body) correct? If not please change. 5. For student readers, please state the dosage of intra aortic infusion of micafungin and itraconazole which the patient received. 6. Please state the full form of IA regimen. Please state the gap in days between the cycles of MEtA regimen. 7. In line 102, you suddenly reveal that "mRNA levels of Wilms' tumor 1 gene increased from below the detection level to 1600 copies/μg of RNA". This will be difficult for student readers to comprehend. Please state the mRNA levels of this same gene before and during the various satges of Induction Therapy also. 8. Could this case reflect that the fungal pathogen was below detection levels when the allogenic hematopoetic transplantation was performed. As such blood cultures and radiology actually showed remission of fungal infection when in fact it was smouldering below and resurfaced when the transplantation was initiated. Could this be suggested in Discussion portion. 9. For benefit of student readers, please provide a picture of the bone marrow biopsy / aspirate of this patient at the time of AML diagnosis. 10. Also for the benefit of student readers mention very briefly why you categorised him as AML with maturation.
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
This manuscript examines the retrospective identification of the causative fungus in sterilized or bacteriostatic abscesses using metagenomics and in situ hybridization. The paper presents a case study of a 65-year-old Japanese male with acute myeloid leukemia (AML) who was receiving antifungal prophylaxis. To prevent the relapse of AML, the patient underwent allogenic hematopoietic transplantation with the administration of prophylactic intravenous micafungin infusion. However, six days after the transplantation, the patient developed a severe fever, ultimately resulted in multiorgan failure and subsequent death. Blood cultures confirmed the presence of fungemia caused by Trichosporon asahii. Major 1. To enhance the introduction, it is recommended that authors dedicate a paragraph to …
Comments to Author
This manuscript examines the retrospective identification of the causative fungus in sterilized or bacteriostatic abscesses using metagenomics and in situ hybridization. The paper presents a case study of a 65-year-old Japanese male with acute myeloid leukemia (AML) who was receiving antifungal prophylaxis. To prevent the relapse of AML, the patient underwent allogenic hematopoietic transplantation with the administration of prophylactic intravenous micafungin infusion. However, six days after the transplantation, the patient developed a severe fever, ultimately resulted in multiorgan failure and subsequent death. Blood cultures confirmed the presence of fungemia caused by Trichosporon asahii. Major 1. To enhance the introduction, it is recommended that authors dedicate a paragraph to trichosporonosis, the organism of interest. This paragraph should address the current challenges associated with identifying this particular organism using existing diagnostic methods and highlight the potential benefits of utilizing Next Generation Sequencing (NGS). However, it is important to acknowledge that not implementing molecular approaches in cases where the fungal agent cannot be isolated by microbiologists may result in delayed diagnosis and treatment and case conundrum, as demonstrated in the presented case. Such modifications will provide the reader with a more direct and informative overview. Introduction should be re-structured. 2. In regards to line 94-95, the authors should clarify whether the laboratory sent out the specimen for molecular testing to identify the organism, instead of stating that "….it as some kind of fungus". Furthermore, it is crucial to explain whether metagenomics was performed at a reference laboratory or in-house, and why this was not conducted earlier in the diagnostic process. This should be addressed.
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
This is an interesting case that fits well in this journal. The presentation and management of the case is appropriate. There are only two concerns about it: 1- What is the antifungal sensitivity profile of the cultivated organism? 2- Please provide any possible explanation for the fact that Candida metapsilosis, Candida parapsilosis, and Candida dubliniesis were detected in the liver and spleen. Why did the authors not conduct hybridization assays for these organisms?
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
…
Comments to Author
This is an interesting case that fits well in this journal. The presentation and management of the case is appropriate. There are only two concerns about it: 1- What is the antifungal sensitivity profile of the cultivated organism? 2- Please provide any possible explanation for the fact that Candida metapsilosis, Candida parapsilosis, and Candida dubliniesis were detected in the liver and spleen. Why did the authors not conduct hybridization assays for these organisms?
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-