Blood Aspergillus PCR: The Good, the Bad, and the Ugly
Round 1
Reviewer 1 Report
The present MS entitled " Blood Aspergillus PCR: The Good, the Bad and the Ugly” reviewed the superiorities, applications and limitations of blood Aspergillus PCR in the diagnosis of IA. On the whole, this work analyzed the advantages and problems that the blood Aspergillus PCR facing in clinical application clearly. It’s meaningful for the development of PCR methods in diagnosing invasive fungal infections (IFIs) in the future. But, there are still some aspects in the manuscript that need to be improved.
The section “State of affairs” mainly summarizes the challenges and problems that blood PCR facing now, but the strengths of the method isn’t mentioned. As a summary, it may be better to add this part.
It isn’t the good of blood PCR, it should be a question of standardization of the method in the fifth paragraph of the section “The Good”.
The existing problems are posed, but the existing researches to address these issues are mentioned little in the section “The Bad” and ”The Ugly”. These contents should be supplemented.
The future research directions of blood PCR should be summarized.
The format should be consistent. Some paragraphs are indented, some are not.
Author Response
Response to Reviewers:
We thank the reviewers for their time and for their helpful comments and hope to have adequately addressed their concerns in the revised manuscript.
In the following, we provide answers to the questions raised by the reviewers.
Changes made in the manuscript are highlighted in red color.
Response to Reviewer #1:
Comments:
The section “State of affairs” mainly summarizes the challenges and problems that blood PCR facing now, but the strengths of the method isn’t mentioned. As a summary, it may be better to add this part.
Answer: Thank you the comment. Indeed, the headline “State of affairs” may cause some confusion, as it was supposed to be an introduction. We therefore changed the headline to “Introduction”. The strengths of Aspergillus PCR assays are then stated in the “The Good” as well as in the “Conclusion” section.
It isn’t the good of blood PCR, it should be a question of standardization of the method in the fifth paragraph of the section “The Good”.
Answer: We agree that the lack of standardization of PCR from blood is a problem given the variety of commercially-available assays plus the large number of “in-house” assays that are used. We discuss the lack of standardization in the third paragraph of “The Bad”.
The existing problems are posed, but the existing researches to address these issues are mentioned little in the section “The Bad” and ”The Ugly”. These contents should be supplemented. The future research directions of blood PCR should be summarized.
Answer: We agree that prior to implementation of PCR as a standard diagnostic tool for diagnosis aspergillosis in different cohorts of patients, various issues need to be addressed in future research. We already highlighted some of these current existing drawbacks of PCR in the manuscript (e.g. Line 181 - 187). In addition, we added a statement according to the future direction of PCR based on your comment in the conclusion section.
Modification of the manuscript: “Thus, future research in terms of fungal PCRs is supposed to further address a standardization of PCR assays, to ensure comparability of study results and therefore enable to evaluate PCR performance in a significantly larger amount of patients. This would also increase the number of patients with proven IA, a usually underrepresented classification of IA in most studies, as most patients are classified as having probable IA based on clinical and radiological signs of IA combined with a positive GM test..”, was added at the end of the first paragraph in the conclusion section.
The format should be consistent. Some paragraphs are indented, some are not.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This is a useful discussion re the use of PCR on blood for the diagnosis of aspergillosis. The paper is well organised and generally well-written although there are grammatical errors throughout.
Specific comments
The authors should note and discuss the issue of aitway colonization as a confounding factor in some patients. Also metions problems with sample contamination during processing as a potential problem. The lack of a Gold Standard for infection is also a problem in reviewing the literature and limits test standardization and development. Another problem is that many studies use GM results as the indicator for IA. As a result most cases are only probable with very few proven cases.Author Response
Response to Reviewers:
We thank the reviewers for their time and for their helpful comments and hope to have adequately addressed their concerns in the revised manuscript.
In the following, we provide answers to the questions raised by the reviewers.
Changes made in the manuscript are highlighted in red color.
Response to Reviewer #2:
Comments:
The authors should note and discuss the issue of airway colonization as a confounding factor in some patients. Also mentions problems with sample contamination during processing as a potential problem.
Answer: This is an important comment that is not addressed in the manuscript. Thank you! Indeed, patients may only be colonized with Aspergillus in the LTR, potentially giving a positive PCR result, as they also be positive in culture. We added a paragraph that states these issues.
Modifications of the manuscript: “Positive Aspergillus PCR in BALF samples may also be interpreted with caution and cannot be used as a sole marker for the presence of IA. This is due to the fact, that different cohorts of patients with structural or functional lung disease are likely to be colonized with Aspergillus in the lower respiratory tract but to not necessarily develop invasive disease as long as no additional immunosuppressive disease or treatment is present. In such cases, a positive BALF PCR results may represent either colonization of the patient or actual invasive disease. To discriminate these two entities in a patient with a positive lower respiratory tract Aspergillus PCR, clinical presentation, imaging studies and other biomarkers are helpful. Also contamination of the obtained specimen during sampling or during processing in the laboratory may cause a false positive PCR result..”, was added
The lack of a Gold Standard for infection is also a problem in reviewing the literature and limits test standardization and development. Another problem is that many studies use GM results as the indicator for IA. As a result most cases are only probable with very few proven cases.
Answer: This is correct. We therefore stated “Thus, future research in terms of fungal PCRs is supposed to further address a standardization of PCR assays, to ensure comparability of study results and therefore enable to evaluate PCR performance in a significantly larger amount of patients. This would also increase the number of patients with proven IA, a usually underrepresented classification of IA in most studies, as most patients are classified as having probable IA based on clinical and radiological signs of IA combined with a positive GM test.”, at the end of the first paragraph in the conclusion section.
Author Response File: Author Response.docx
Reviewer 3 Report
The authors in the present review have discussed the various positive and negative aspects of considering the blood PCR for diagnosis of invasive aspergillosis. The article provides an exhaustive literature to understand the current role of PCR in IA diagnosis. The article is very well written. At some instances, the sentences need to shortened or reframed to help readers understand the context better such as 58-63; 71-74 etc.
Author Response
Response to Reviewers:
We thank the reviewers for their time and for their helpful comments and hope to have adequately addressed their concerns in the revised manuscript.
In the following, we provide answers to the questions raised by the reviewers.
Changes made in the manuscript are highlighted in red color.
Response to Reviewer #3:
Comments:
At some instances, the sentences need to shortened or reframed to help readers understand the context better such as 58-63; 71-74 etc.
Answer: Thank you for your comment. We shortened several sentences in order to improve understandability.
Modifications of the manuscript:
Line 58-63:
“A 2019 published systematic review highlighted a mean sensitivity and specificity of 79.2% and 79.6% for a single positive test result and 59.6% and 95.1% for two consecutive positive results, when performing PCR on blood samples (serum or whole blood). The majority of investigated cohorts were hematological malignancy patients at highest risk for IA [i.e., neutropenic cancer patients, hematopoietic stem cell transplant (HSCT) recipients] [18]. In this setting with high prevalence of 16.3%....”
Line 70 -78:
“As a consequence, positive test results, particularly when PCR is combined with other molecular diagnostic tests such as GM (increase of specificity), may be used to initiate further diagnostic work-up. The latter includes bronchoscopy with biopsy or BALF and eventually, initiation of antifungal therapy [21,22]. Although Aspergillus DNA correlates quantitatively to serum GM levels [23], it is important to remember, that at different stages of invasive disease different antigens may be detectable in patient blood [24]. This highlights the importance of combining different biomarkers in the diagnostic process and to repeat available diagnostic tests if they turn out negative, but clinical suspicion of IA is still high..”
Line 125 -128:
“In summary, evidence supports that for the diagnosis of IA in neutropenic patients, Aspergillus PCR in blood is as good as serum GM and superior to 1,3-beta-D-glucan, which cross reacts with Candida spp. and may be elevated in various conditions associated with fungal translocation from the gut [51]. Another benefit of PCR is the potential detection of acquired azole resistance.”
Line 150 – 155:
“The limited utility of PCR from blood specimens in non-neutropenic patients is a very relevant limitation, given that the prevalence of IA continues to increase in non-neutropenic patients with other severe underlying diseases. This includes patients in the ICU where prevalence rates vary between 0.33% – 19% [67-70], solid organ transplant recipients [71], patients receiving systemic glucocorticoids [72], patients with underlying respiratory conditions [67,73,74], patients with solid cancers [67,75] and other patient groups [67,76].”
Author Response File: Author Response.docx
