Development of a Novel Method for the Clinical Visualization and Rapid Identification of Multidrug-Resistant Candida auris

ABSTRACT Outbreaks of multidrug-resistant Candida auris infections, associated with a mortality rate of 30% to 60%, are of serious global concern. Candida auris demonstrates high transmission rates in hospital settings; however, its rapid and accurate identification using currently available clinical identification techniques is challenging. In this study, we developed a rapid and effective method for detecting C. auris based on recombinase-aided amplification combined with lateral flow strips (RAA-LFS). We also screened the appropriate reaction conditions. Furthermore, we investigated the specificity and sensitivity of the detection system and its ability to distinguish other fungal strains. Candida auris was accurately identified and differentiated from related species at 37°C within 15 min. The minimum detection limit was 1 CFU (or 10 fg/reaction) and was not affected by high concentrations of related species or host DNA. The simple and cost-efficient detection method established in this study exhibited high specificity and sensitivity and successfully detected C. auris in simulated clinical samples. Compared with other traditional detection methods, this method greatly reduces the time and cost of testing and is thus suitable for hospitals or clinics in remote underfunded areas for screening C. auris infection and colonization. IMPORTANCE Candida auris is a highly lethal, multidrug-resistant, invasive fungus. However, conventional methods of C. auris identification are time-consuming and laborious and have low sensitivity and high error rates. In this study, a new molecular diagnostic method based on recombinase-aided amplification combined with lateral flow strips (RAA-LFS) was developed, and accurate results could be obtained by catalyzing the reaction at body temperature for 15 min. This method can be used for rapid clinical detection of C. auris, consequently saving valuable treatment time for patients.

be sure to reference this table in the text. -Why was the amplification product diluted? -Readers are likely more familiar with picograms and larger quantities than fg. I would consider using these measurements instead. I would also consider reporting concentrations based on the input amounts rather than reaction amounts since you are electing to dilute amplification products. Readers will better understand an input CFU to correlate it with clinical relevance. You can always correlate the input CFU to DNA/reaction as well, but I think it's helpful to have the input amount established.
-For the optimization of temperature and timing, it would be better to use your established limit of detection to be sure these parameters work at lower levels of DNA concentrations -What concentration of organism did you use for the specificity study? It would strengthen the paper if you assess co-culture with C. auris and each of the other organisms, not just C. albicans. -Could you reword section 2.8 to reflect you did 2 separate studies, one with CFU and one with extracted DNA, and not that you did one study where you assessed both.
-What does "treated culture" mean? Diluted? -For the LOD studies, I would like to see lower concentrations since you did not find a concentration it did not detect. Because of this, you cannot conclude a limit of detection. 1 CFU/µL is not that low, clinically. It would be good to go down to 1 CFU/mL instead since this is a concentration that can be seen in blood infections.
-How did you obtain whole blood? Define how was it collected and stored.
-Add section that defines the qPCR method you mention. Be sure to include criteria you used to result tests as detected versus not detected.
Results section: -It is unclear why you chose 37C instead of 39 since the positive line shows up first at that temperature. Just be sure to expand your reasoning.
-Expand on why you chose a 1:10 ratio in section 3.5. Also in this section, I would note there is a slight difference in the strength of positive at 101 between the blood and non-blood samples (blood is slightly lighter). -Expand section 3.6. Specify which samples were positive and negative by either assay. Also include the qPCR data.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
"Development of a Novel Method for the Clinical Visualization and Rapid Identification of Multidrugresistant Candida auris" is a manuscript that describes a novel LFA that can be used to detect C. auris DNA after a chelex 100 extraction. While the assay has a lot of potential for clinical use, I believe a few extra experiments should be done to fully elucidate the diagnostic potential of the LFA. See below for specific comments and requests for modification.
General Comments: Numerous figures were not included in the submission. Tables S1 and S2 and Supplementary Figure 6 were all mentioned but not included. Please submit them for review.
Since this assay requires a boiling step and phenol, I don't believe it would be suitable as a bed-side or POC test, but I do think it has potential for the clinical lab. I would just be careful on how you sell the utility of this test.
Were any of these experiments done in duplicate or triplicate? I believe they should all be replicated to account for variability with extraction or reader bias of the LFA lines.
Is there any way to get true clinical specimens to run on this LFA? It would be good to correlate these to gold standard culture results.
Hospitals are now doing screening tests for C. auris colonization by evaluating swab samples (swabbing nose, axila, and groin). The CDC outlines a protocol for surveillance testing. Though blood infection is important, I would argue surveillance/screening testing for C. auris would have more of a clinical impact. I would consider assessing these types of clinical samples as well on your LFA since utilizing this test as proposed would truthfully just be used as a secondary/confirmatory diagnostic for a very small amount of specimens.
Materials and methods section: -Please elaborate on where the strains were procured from and how they were stored. -Please include a table of all primers and probes used. I assume this was table S2 but am unsure since it was not included. Also be sure to reference this table in the text.
-Why was the amplification product diluted? -Readers are likely more familiar with picograms and larger quantities than fg. I would consider using these measurements instead. I would also consider reporting concentrations based on the input amounts rather than reaction amounts since you are electing to dilute amplification products. Readers will better understand an input CFU to correlate it with clinical relevance. You can always correlate the input CFU to DNA/reaction as well, but I think it's helpful to have the input amount established.
-For the optimization of temperature and timing, it would be better to use your established limit of detection to be sure these parameters work at lower levels of DNA concentrations -What concentration of organism did you use for the specificity study? It would strengthen the paper if you assess co-culture with C. auris and each of the other organisms, not just C. albicans.
-Could you reword section 2.8 to reflect you did 2 separate studies, one with CFU and one with extracted DNA, and not that you did one study where you assessed both.
-What does "treated culture" mean? Diluted? -For the LOD studies, I would like to see lower concentrations since you did not find a concentration it did not detect. Because of this, you cannot conclude a limit of detection. 1 CFU/µL is not that low, clinically. It would be good to go down to 1 CFU/mL instead since this is a concentration that can be seen in blood infections.
-How did you obtain whole blood? Define how was it collected and stored.
-Add section that defines the qPCR method you mention. Be sure to include criteria you used to result tests as detected versus not detected.
Results section: -It is unclear why you chose 37C instead of 39 since the positive line shows up first at that temperature. Just be sure to expand your reasoning.
-Expand on why you chose a 1:10 ratio in section 3.5. Also in this section, I would note there is a slight difference in the strength of positive at 10 1 between the blood and non-blood samples (blood is slightly lighter). -Expand section 3.6. Specify which samples were positive and negative by either assay. Also include the qPCR data. Although the manuscript is interesting, the table and figures are perfect and the methodology is excellent, it requires major revisions with gross English editing and confirmation or proven for my questions.

The English language requires extensive editing.
The language throughout the paper has been carefully checked and improved, and we provide an Editing Certificate as confirmation of this below.

It is not clear what is the clinical significance of the study -how does it help in clinical practice?
The major concern is that the impact of work is not presented enough in the manuscript -authors should at least speculate what could be the importance of their findings in practical application. For clarity and in accordance with the reviewer's concerns, we have added a short description in the Discussion and Conclusions as follows: "Pneumonia has been gradually attracting the attention of researchers due to the COVID-19 pandemic, and Candida is also an important cause of pneumonia. COVID-19 complicated with C. auris infection is a more serious disease, particularly because C. auris readily causes nosocomial infections. In many cases, infections caused by C. auris are not recognized in the first instance, leading to incorrect medication and delayed treatment [32][33][34][35][36]. (Lines 366 to 371.) In addition, "The detection method developed in this study allows patients to self-diagnose early. The clinical test results can then be verified and reasonable antifungal treatment promptly initiated. "(Lines 447 to 449.)

Please add significant data about which method was used recently for diagnosing bloodstream Candida infections.
We have added information about the latest diagnostic methods for Candida bloodstream infections in the Discussion section. (Lines 371 to 392.)

Diagnostic methods have a positive impact on clinical outcomes in patients with Candidiasis.
The original intention of this study was to develop a method for the simple and rapid detection of C. auris. Areas that have sub-optimal health facilities do not have a strong awareness of fungal infections, and the increased numbers of patients in such facilities during the COVID-19 pandemic increased the risk of co-infection The aim of our study was to develop a home testing diagnostic method to enable patients to be diagnosed swiftly and to then promptly seek medical treatment, thereby avoiding delay and further deterioration of the disease due to misdiagnosis or missed diagnosis. Therefore, this method potentially saves lives. In addition, the method enables patients with Candida infection to be regularly monitored, which helps to assess disease progression.

I feel that it is important to mention the efforts to find new combinational methods against these infections
We agree with you, and we have added information about RAA in the discussion section. RAA is a relatively novel detection technique that was originally applied to detect pathogenic bacteria in viruses or foods. Given its quick results, simple operation, low cost, and the ability to visualize the results, we considered its application in difficult-to-detect pathogenic fungi. (Lines 415 to 418.) 6. Line 45 authors mentioned" Since its first report in 2009 from Japan, C.auris has been increasingly reported in cases of invasive candidiasis (IC).WGS analyses of clinical C.auris isolates indicated that the emergence of clonal populations in Asia, South Africa, and South America occurred independently and spread locally within each region. Based on a newly published paper regarding interesting cases of auris from Iran with the literature of review. An isolate representative of a potential fifth clade, distinct from the other clades by >200,000 single-nucleotide polymorphisms, in a patient in Iran who had never traveled outside the country. Please add a paragraph about the prevalence and epidemiological data of C.auris candidiasis. Therefore, those newly published references have been missed; I suggest adding the recent articles both in the introduction and discussion part as follows. Safari We have now included data about the prevalence and epidemiology of C. auris in the Introduction and Discussion sections, and we have also cited the references that you kindly provided. (Lines 52 to 60.)

So, did you check isolates from different clades?
The Supplementary Material was unfortunately not fully uploaded, due to an error. The twelve C. auris strains used in this study were from four different clades, and specific information about the strains is provided in Table S1 in the Supplementary Material. Our assay was based on the conserved sequence of C. auris across all clades, and it is thus reliable and stable for identifying different strains of the same species.
8. The nomenclature of species should be revised. The first time that the name of each species is mentioned the genus should be written in cursive and then it should be abbreviated Check reference format and italic form according to the journal format We apologize for our poor formatting and oversight; the full text has now been revised and re-formatted.

Clarify your isolates (how many clinical auris and non-C. auris yeasts).The specificity and sensitivity test set could be significantly improved by the inclusion of clinically important auris species. All these experiments could be presented preceding the testing of clinical samples.
In this experiment, 12 standard strains of C. auris were used to verify the detection stability of this method among the same species. A total of 24 strains of non-auricular Candida were also used for specific detection (Lines 219 to 220). Although no more clinical strains were used for further verification in this experiment, our detection method was based on the conserved nucleic acid sequence across the clades of C. auris. The experimental results show that our detection method has excellent stability between different strains of the same species and is very specific for the detection of C. auris. Due to the impact of the COVID-19 pandemic, it was difficult to collect clinical strains. However, we hope to apply our detection method to actual clinical samples and compare it with clinical gold standards to evaluate the practical application value of this method in clinical settings. In a follow-up study, we will collect clinical samples to further optimize any problems associated with the detection method and ensure that it is optimally suited to clinical settings. If you have any suitable samples, we would be very pleased if you could share those with us so that we can conduct further tests.
10. The conclusion in the abstract part was unclear if your results suggested that this assay is suitable for the rapid, sensitive, and specific detection of C. auris in clinical samples. The following reference, you probably missed This method had high specificity and high sensitivity, which could successfully detect C. auris strains. We have inserted the following within the conclusion of the Abstract: "The simple and cost-efficient detection method established in this study exhibited high specificity and sensitivity and successfully detected C. auris in simulated clinical blood samples." (Lines 29 to 31.)

Please change strains to isolate. This is the limitation of this study. Why you used only C. auris isolates, how about other species complex
In the subsequent validation and optimization experiments, all the strains used were mixed to evaluate the sensitivity and specificity of the method in the presence of high concentrations of other strains or their DNA. The experimental results showed that the sensitivity and specificity of the detection method were not affected in a complex environment when there were high concentrations of other strains. Considering the limitation of the length of this article, we chose C. albicans, which is more commonly encountered in clinical practice, as the representative for quantification. (Please see Lines 425 to 430 in the main manuscript.) Figure S6 of the Supplementary Material shows the sensitivity and specificity of this detection method in the presence of a high concentration of C. albicans bacterial solution and a high DNA concentration.

What are the limitations of this study?
We believe that the main limitation of this study is that compared to other swab samples that are easier to handle, the processing of simulated clinical blood samples requires an additional phenol purification step. Therefore, it does not meet the requirements of POCT to the point where it can be used at home or in the field as a commercial product. However, in subsequent experiments, we will consider using fungal nucleic acid extraction reagents that do not require boiling or purification, and which only require blood to be collected from the fingertip or the use of nasal and throat swab and other soaks for nucleic acid extraction. By holding the reaction tube in your hand, subsequent reactions can be activated at body temperature, and the results of disposable nucleic acid test strips can be visualized quickly and easily.
Another limitation of this study is that we unfortunately did not use any real clinical samples, but we selected the most complex blood as a representative of simulated samples, and this enabled us to prove that the detection method can directly identify C. auris from blood without affecting the sensitivity. However, we plan to collect actual clinical samples to enable us to further optimize the detection method.

Reviewer #2 (Comments for the Author): "Development of a Novel Method for the Clinical Visualization and Rapid
Identification of Multidrug-resistant C.auris" is a manuscript that describes a novel LFA that can be used to detect C. auris DNA after a chelex 100 extraction. While the assay has a lot of potential for clinical use, I believe a few extra experiments should be done to fully elucidate the diagnostic potential of the LFA. See below for specific comments and requests for modification.
General Comments: 1. Numerous figures were not included in the submission. Tables S1 and S2 and Supplementary Figure 6 were all mentioned but not included. Please submit them for review. We apologize for the error in uploading the material correctly. We have now re-uploaded the relevant material and we would be very grateful if you could now review it.

Since this assay requires a boiling step and phenol, I don't believe it would be suitable as a bed-side or POC test, but I do think it has potential for the clinical lab. I would just be careful on how you sell the utility of this test.
In this study, the boiling method was used to extract fungal nucleic acids simply and efficiently, while phenol was further used to purify complex blood samples. We tried using swabs, urine, and pure serum samples, all of which only needed to be boiled and did not require further phenol purification. Compared to fungal nucleic acid extraction and purification kits that have complex steps, boiling may be the easiest step to implement at home. In the subsequent optimization of this method, we will use a special fungal nucleic acid simple extraction reagent to achieve POCT (similar to the COVID-19 antigen kit), where a nasal swab or fingertip blood is added to the reagent, and it is then shaken to achieve nucleic acid extraction from C. auris without boiling or purification. We anticipate that this will facilitate translation of the test method presented in this study to the field or bedside.
3. Were any of these experiments done in duplicate or triplicate? I believe they should all be replicated to account for variability with extraction or reader bias of the LFA lines. Each of the experiments in this study was repeated at least three times. We set a large number of replicates with the lowest at 1 CFU, and the results showed a result error rate of only 1% due to strip batches and quality control.
4. Is there any way to get true clinical specimens to run on this LFA? It would be good to correlate these to gold standard culture results. Due to the impact of Covid-19, it was difficult to collect clinical samples. However, to enable the practical application of this detection method in clinic, we intend to apply it to actual clinical samples, compare it with clinical gold standards, and conduct a subsequent in-depth evaluation of its performance. In follow-up research, we will collect clinical samples to further optimize problems associated with this detection method. If you could possibly provide us with suitable samples, we would be extremely grateful. (swabbing nose, axila, and groin). The CDC outlines a protocol for surveillance testing. Though blood infection is important, I would argue surveillance/screening testing for C. auris would have more of a clinical impact. I would consider assessing these types of clinical samples as well on your LFA since utilizing this test as proposed would truthfully just be used as a secondary/confirmatory diagnostic for a very small amount of specimens. We agree with your comment. It would be best if there were clinical samples that could be verified using our diagnostic methods. We thank you for your suggestion and look forward to the opportunity of working with you to optimize our approach and meet your clinical needs.

Materials and methods section:
6. Please elaborate on where the strains were procured from and how they were stored. We apologize for our negligence that resulted in the incomplete loading of essential material. All of the supplementary material has now been uploaded and the source information for all strains is provided in Table S1. We have also added the following to Section 2.1, "All strains were stored in the laboratory at −20 °C based on the filter paper preservation method." (Page 6, Lines 111 to 112.)

Please include a table of all primers and probes used. I assume this was table S2 but am unsure since it was not included. Also be sure to reference this table in the text.
We apologize for the problem related to the incomplete uploading of materials. All of the primer probe sequences are listed in Table S2, and we would be extremely grateful if you could take the time to now review this.

Why was the amplification product diluted?
Due to the HOOK effect of the colloidal gold test strip, false negatives or false positives occur when the ratio of antigens and antibodies participating in the reaction is unsuitable. After amplification, the concentration of antibody-labeled products is high, and if the strip is not diluted, false-negative results are likely (i.e., it is possible that the sample has a very high amount of C. auris but the test strip results are negative).
9. Readers are likely more familiar with picograms and larger quantities than fg. I would consider using these measurements instead.

I would also consider reporting concentrations based on the input amounts rather than reaction amounts since you are electing to dilute amplification products. Readers will better understand an input CFU to correlate it with clinical relevance. You can always correlate the input CFU to DNA/reaction as well, but I think it's helpful to have the input amount established.
The common unit of DNA is indeed ng or pg, and if fg was replaced with ng or pg, our detection limit concentration would be expressed as "10 -5 ng" or "10 -2 pg", which would make illustrations complex. We agree that stating the number of bacteria input, as you propose, is very important, and we apologize that we did not mention that the volume of the fungi suspension at different concentrations was actually 1 μL. Similarly, 1 CFU/μL actually contained 1 CFU of fungi, and we have added this information to Section 2.2. In similar references, the unit of DNA is fg, and the bacterial solution is CFU/μL, so we also used this unit. (Lines 127 to 128.) The associated references are as follows:

For the optimization of temperature and timing, it would be better to use your established limit of detection to be sure these parameters work at lower levels of DNA concentrations
The results in Figure S5 show that the detection limit of this method is 10 1 fg/reaction. However, the bands of 10 1 and 10 2 fg/reaction are faint, and if the reaction condition optimization is performed with these two template concentrations, the reaction time needs to be greatly extended and the reaction temperature increased to normalize the brightness of the resulting bands. To increase the convenience and speed of using the detection method at body temperature, which would be more in line with the POCT conditions, we selected 10 3 fg/reaction as the template concentration for optimizing the reaction conditions. 11. What concentration of organism did you use for the specificity study? It would strengthen the paper if you assess co-culture with C. auris and each of the other organisms, not just C. albicans. In the specificity study, we used 10 5 CFU/mL in 1 μL for DNA extraction and detection, and this information has now been provided in Section 2.2. (Lines 127 to 128.) We apologize for our error. In the experiment, we mixed all strains at high concentrations to detect C. auris, and found that the sensitivity remained unchanged; however, considering the word limit of the article, we selected the bacteria most commonly encountered in clinical settings, Candida albicans, as an example. We then quantified the values and presented the results. We have included information about mixing in the Discussion section to support our results. (Lines 425 to 430.) 12. Could you reword section 2.8 to reflect you did 2 separate studies, one with CFU and one with extracted DNA, and not that you did one study where you assessed both. We have rewritten Section 2.8 and have separated the two experiments using DNA and CFU to improve the readability and avoid ambiguity. (Lines 223 to 231.) 13. What does "treated culture" mean? Diluted? "Treated culture" meant that 1 μL of the bacterial solution of C. albicans was taken from different concentrations for DNA extraction, as described in Section 2.2. (Lines 122 to 124.)

For the LOD studies, I would like to see lower concentrations since you did not find a concentration it did not detect. Because of this, you cannot conclude a limit of detection. 1 CFU/μL is not that low, clinically. It would be good to go down to 1 CFU/mL instead since this is a concentration that can be seen in blood infections.
Due to our negligence, the actual amount of C. auris used was not specified, and our detection limit of "1 CFU/μL" meant that the sampling volume of 1 μL at a concentration of 1 CFU/μL could be detected, which implied that 1 C. auris could be detected. To verify this result, we used microdissection to cut individual cells for DNA extraction and detection. The experimental results proved that 1 CFU could indeed be detected. The figure below shows the microdissection and test results. (Lines 425 to 430.)

How did you obtain whole blood? Define how was it collected and stored.
All the blood samples in this study were collected by the researchers using sterile venous blood collection kits to draw a certain amount of their own whole blood. Subsequently, 15 g/L of EDTA-2Na was added, and the contents were mixed and stored at 4℃. This information has now been added to Section 2.9. (Lines 235 to 237.)

Add section that defines the qPCR method you mention. Be sure to include criteria you used to result tests as detected versus not detected.
The qPCR reaction in Section 2.10 was performed using a commercial kit. "A positive result was obtained when the Ct value was less than 35, whereas the result was negative when it was greater than 35. " This information has now been added to Section 2.10. (Lines 246 to 249.) Results section: 17. It is unclear why you chose 37C instead of 39 since the positive line shows up first at that temperature. Just be sure to expand your reasoning. We believe that although 39 ℃ is the first temperature to show a positive result, it is not an easily attainable temperature at home or at the bedside. An appropriate extension of the reaction time to 15 min at 37 ℃ showed stably positive results, and when the time was prolonged and the temperature increased, the positive results were maintained. Considering the ease of implementing the reaction time and temperature, we chose 37 ℃ and 15 min as the reaction conditions. We have added such information to Section 3.2, where we briefly explain the reasons for selecting 37 ℃. (Lines 301 to 303.) 18. Expand on why you chose a 1:10 ratio in section 3.5. Also in this section, I would note there is a slight difference in the strength of positive at 10 1 between the blood and non-blood samples (blood is slightly lighter). We chose a 1:10 dilution based on the amount used in a similar article that employed simulated blood samples to diagnose C. auris. The relevant article is as follows: 1. Sattler, J.; Noster, J.; Brunke, A.; Plum, G.; Wiegel, P.; Kurzai, O.; Meis, J.F.; Hamprecht, A. Comparison of Two Commercially Available qPCR Kits for the Detection of Candida auris. J. Fungi 2021, 7. The light color of the bands in the blood sample occurs because the complex blood background contains a variety of impurities. However, for the test strip results, the positive band shows that our detection method can maintain a sensitivity of 1 CFU in a complex sample, despite the complexity of the test background.
19. Expand section 3.6. Specify which samples were positive and negative by either assay. Also include the qPCR data. In order to better simulate detection in unknown samples, the process of mixing C. auris into blood samples and the process of detecting samples were completely separated and a double-blind experimental process was used. The qPCR curve is shown below, and detailed Ct values of each sample and the results of the two assays are supplemented in Table S3 in the Supplementary Material. We would be extremely grateful if you could please review this information (Lines 354 and 356.) Minor edits: 20. Line 43: should read "C.auris IS a recently discovered...." We apologize for our careless mistake and have now corrected this information. (Line 50.)

Line 73: I think you meant fungi or microbe, not bacteria
We sincerely thank you for reading our manuscript so carefully. Based on your recommendation, we have corrected "bacteria" to "fungi." (Line 95.) Figure  S2A &B We apologize for our careless mistake and have now made associated changes. (Line 185.)

Line 177: Specifically reference Supplementary Figure S2C
We apologize for our careless mistake and have now made associated changes. (Line 201.)

Line 214: Define the 48 samples
We sincerely thank you for reading our manuscript so carefully. Based on your recommendation, "the 48 samples" are defined as follows, "Forty-eight simulated samples containing C. auris ranging from 0-10 5 CFU were prepared by randomly incorporating C. auris into 200 μL of human whole blood." (Lines 244 to 245.) 25. Line 249: "strength" may be a better term than "depth" We sincerely thank you for reading our manuscript so carefully. Based on your recommendation, we have corrected "depth" to "strength."(Line 296.)

Line 266: "observed" instead of "detected"
We sincerely thank you for reading our manuscript so carefully. Based on your recommendation, we have changed "detected" to "observed." (Line 313.)

Figure 4-Specify this is C. auris somewhere in the figure caption
We apologize for our careless mistake and have now revised this. We have changed the legend of Figure 4 to, "Detection of standard reference strains of C. auris." (Line 635.)

Figure S2 -Include label for biotin in the legend
We again apologize for our careless mistake, and have made associated corrections by adding the label of biotin to the legend of Figure S2. Thank you for submitting your manuscript to Microbiology Spectrum. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.

Link Not Available
Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data. If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record. If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. I do not feel previous comments have been thoroughly addressed. Though some have been fully addressed, some were only addressed in comments back to the reviewers and not put in the manuscript, some were partially addressed, and some were not addressed at all. The biggest issue that was not fully addressed is that the authors have still not clarified the clinical impact of this test. As it stands, it cannot be used as a point of care test because it requires a place to set up a nucleic acid amplification which not only requires a tech to use pipettes and a clean space, but it also requires a phenol and boiling step, none of which could be done at home or at the bedside. Further, this is a nosocomial infection (as even pointed out by the authors) where patients could have a pneumonia (where concentrations in the blood may not be that high and therefore not picked up unless grown in blood culture first). Even if a patient were fungaemic, concentrations of yeast in the blood may still be low and require a positive blood culture. You can look at the literature on the T2 Candida panel and see even with their NAAT test, there's only a certain population of patients where this test is most clinically impactful. Because of all these issues, I believe the authors need to reconsider the utility of this test. There is still utility for this in an under-resourced setting, but it still would only have applicability in a lab setting. I think the authors need to completely rework how they present this test and what clinical impact and utility it would have. The other comments that I think need to be further addressed are the following: The English still needs work Any rebuttal the authors gave to the reviewer comments need to be addressed in the manuscript as well. For example, you state that all experiments were done three times, yet this is not stated in the manuscript, nor is there any evidence in the data. There not only as some methodologies that still are not outlined in the methods section (i.e. qPCR), but with the new edits, you mention a microdissection technique that is not mentioned in the manuscript at all. This not only needs to be added, but you must include specifics in the methodology section. You must also be specific with your methods. You cannot say "generally by a factor of 6" [line 434] or "a certain amount of whole blood" [line 235]. The limit of detection of your assay is not consistently stated throughout the manuscript. In some sections it is 1 CFU, in others it is 2 CFU. Similarly in section 3.6 you say there are 48 simulated samples but you only give results for 19.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.

Please see our responses to the reviewers' comments below: both the comments and the replies (in blue) are presented.
We sincerely thank the editor and the reviewers for their valuable feedback that has helped us improve the quality of our manuscript. The editor and reviewer comments are presented in bold, and the specific concerns have been numbered. Our response is presented in normal font, and changes/additions to the manuscript are given in blue.
Editor comments: As you read below, several concerns remained unanswered or unclarified. Please, provide detailed explanations for Reviewer(s) about the raised problems; furthermore, involve the relevant information into the manuscript focusing on the methodological background and clinical applicability of your test.
On behalf of my co-authors, we thank you for giving us a chance to revise and improve the quality of our article. We have read the reviewers'and your comments carefully and have made revision which marked in blue in the paper. We have tried our best to revise our manuscript according to the comments. Attached please find the revised version, which we would like to submit for your kind consideration. Here, we would like to explain the changes briefly as follows: We have found an English native speaker with a research background to review our manuscript during revision. And if you think there is any problem, you can raise it at any time. we will look for professional organizations to improve the language.
Regarding the need to supplement the methodological background and redefine clinical applicability as raised by the reviewers, we have supplemented the material methodology section of the manuscript and made changes to the clinical suitability description of the full text.
We have written a point-by-point response letter for reviewers, you can see the details at the end of this letter. To make the reply more visible, Q represents questions raised by reviewers, and A are our answers for these questions. In all, we found these comments are quite helpful. And special thanks to you and reviewers for your good comments again. I wish this revision will be acceptable for publication in your journal. Thank you for your consideration. I am looking forward to hearing from you.
Reviewer (Comments for the Author): Q1: I do not feel previous comments have been thoroughly addressed. Though some have been fully addressed, some were only addressed in comments back to