ELISA for leptospiral 3-hydroxyacyl-CoA dehydrogenase in urine is a promising screening tool for acute leptospirosis
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Introduction. Leptospirosis is a zoonotic disease that is prevalent worldwide. Leptospiral 3-hydroxyacyl-CoA dehydrogenase (3-HADH) is excreted in the urine of infected individuals. However, the potential use of 3-HADH as a biomarker for the diagnosis of leptospirosis using enzyme-linked immunosorbent assay (ELISA) has not been investigated. A technique that identifies Leptospira in a patient in urine sample will be valuable in regular diagnostics and epidemic scenarios, as opposed to existing serological approaches. This study aimed to develop and evaluate an ELISA that can detect 3-HADH in the urine of patients with confirmed acute leptospirosis and to assess its potential as a screening test for leptospirosis.
Methods. Laboratory confirmation of acute leptospirosis was done by flaB -nested polymerase chain reaction (PCR) of plasma samples from suspected patients. ELISA-based determination of the presence of 3-HADH in the urine of PCR-positive patients versus PCR-negative patients matched for fever date was performed by coating ELISA plates with urine supernatants and using rabbit anti-3-HADH as the primary antibody. Receiver operating characteristic curve analysis was used to determine the cutoff values for the ELISA. The diagnostic measures between the PCR-positive and PCR-negative patients were compared using the Mann–Whitney U test.
Results. In total, 158 febrile patients were assessed, of whom 121 (76.6 %) were male. Of the 15 flaB- nested PCR-positive patients, 12 were in the acute phase of the febrile illness. The best cutoff was an average optical density (OD av ) value of 0.2200 for febrile patients. Sensitivity and specificity were 83.33% [95 % confidence interval (CI), 51.59–97.91 %) and 83.33 % (95 % CI, 76.05–89.13 %), respectively. The OD av values for PCR-positive patients in the acute phase of the disease (≤7 days of fever) were significantly higher than those for PCR-negative patients ( P <0.001, U =114.0, z =−4.946).
Conclusion. Detection of 3-HADH in urine by ELISA appears to be promising for the screening of acute leptospirosis in suspected patients.
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Thank you for addressing the comments and submitting a revision.
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I think you can emphasize in your introduction that the diagnostic via culture is a very lengthy process and that better and faster diagnostic tests would really help. Unusual reference format in superscript. Might be easier for the reader to just have the numbers in square brackets. Ll 49-50 are these numbers concerning humans? L 70 different font size for partial text Conclusion: I think this can be expanded a bit further. perhaps how will this change the routine in the clinic? Will this lead to faster treatment of the affected patients? Methods: What does convenience sampling mean? Can you explain this in more detail? Are you referring with this term to the fact that patients who were going to be tested anyway on a blood sample to also give a urine sample? How can one access MedCalc statistical package (is there a publication or …
I think you can emphasize in your introduction that the diagnostic via culture is a very lengthy process and that better and faster diagnostic tests would really help. Unusual reference format in superscript. Might be easier for the reader to just have the numbers in square brackets. Ll 49-50 are these numbers concerning humans? L 70 different font size for partial text Conclusion: I think this can be expanded a bit further. perhaps how will this change the routine in the clinic? Will this lead to faster treatment of the affected patients? Methods: What does convenience sampling mean? Can you explain this in more detail? Are you referring with this term to the fact that patients who were going to be tested anyway on a blood sample to also give a urine sample? How can one access MedCalc statistical package (is there a publication or is it a commercial software)? Which version of the software was used? Results: Ll225-227, 290, 297, Table 7, 319 flab instead of flaB, check this throughout Figure 2: why are there no error bars? L 357 genus not in italics It would be better to give the actual concentration of recombinant enzyme that was used for validation of the ELISA compared to dilution of the enzyme which really is meaningless without knowing the initial concentration of the enzyme stock solution. Lines 113-114 This sentence needs to be modified/rewritten.
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This study would be a valuable contribution to the existing literature. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. The language used is poor, which can cause ambiguity at times. Please carefully rewrite it. We offer a discounted translation service, Editage (https://www.editage.com/; see https://www.microbiologyresearch.org/prepare-an-article#13 for more information). Many thanks for your work! Your paper is indeed interesting and will be a welcome addition to the field of diagnostics for this important infectious disease. However, please find below some concerns raised by our reviewers, and I would appreciate they be addressed in a re-submission for further consideration. If we can assist in any further capacity, please do not hesitate to reach out.
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Comments to Author
This paper is a similar work that was published by one of the author (Toma et. al, 2018) with almost the same title; however, the present work describes the development and evaluation of ELISA to detect 3-HADH enzyme in urine which is a worthy and interesting diagnostic approach. In order to demonstrate a meaningful scientific contribution, the authors need to show that their assay is an improvement over standard diagnostic approaches. The English usage in the manuscript overall is quite good; however, there are some issues in that regard throughout the manuscript, and it should be examined carefully by someone whose first language is English. The information presented in some sentences is confusing and this should be revised. Major comments: Title: Since the aim of the study is to develop and …
Comments to Author
This paper is a similar work that was published by one of the author (Toma et. al, 2018) with almost the same title; however, the present work describes the development and evaluation of ELISA to detect 3-HADH enzyme in urine which is a worthy and interesting diagnostic approach. In order to demonstrate a meaningful scientific contribution, the authors need to show that their assay is an improvement over standard diagnostic approaches. The English usage in the manuscript overall is quite good; however, there are some issues in that regard throughout the manuscript, and it should be examined carefully by someone whose first language is English. The information presented in some sentences is confusing and this should be revised. Major comments: Title: Since the aim of the study is to develop and evaluate ELISA (it is clearly written in the abstract) the authors have to change the title. Furthermore, the conclusion of the manuscript is about ELISA. Introduction: - The introduction could be shortened. The authors report a large volume of work, but it quite confusing to read and excessively wordy, leading to repetition in places. It really needs a thorough trimming. The key points could be made in half the length. Several sentences are not necessary. For instance, paragraphs 2 and 5. Method: Lines 136 - delete "once transported to the lab" Line 160- nested PCR is not used to "identify" the presence of Leptospira spp. Line 187 - delete "only if" Results and discussion Authors emphasized the number of males and females but did not discuss their findings. Only the introduction has the information (line 74) General - Insert number of references: line 89 reference 11, lines 99, 105/106, 107 and other lines - Lines 241-243; 391,401- delete "species"
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Poor
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
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Comments to Author
This paper is presenting the results for an innovative biomarker for the early diagnosis an important infectious disease. This is important as it will help improve the case management in this setting. Overall, the paper is a bit well written. However, I will recommend the authors to seek help from English native speaker to improve the fluency. Below are some comments/suggestions that need to be taking into account to improve the article Line 11: please add the corresponding authors sign to make the link with the sign put in the authors list Abstract: Please review the different sections and use the standard subdivision unless require by the journal, as it is uncommon to use: "Gap statement" "Aim" Line 41 Data summary: what is the rational of adding it there? Abbreviations: please use ":" instead of ";"…
Comments to Author
This paper is presenting the results for an innovative biomarker for the early diagnosis an important infectious disease. This is important as it will help improve the case management in this setting. Overall, the paper is a bit well written. However, I will recommend the authors to seek help from English native speaker to improve the fluency. Below are some comments/suggestions that need to be taking into account to improve the article Line 11: please add the corresponding authors sign to make the link with the sign put in the authors list Abstract: Please review the different sections and use the standard subdivision unless require by the journal, as it is uncommon to use: "Gap statement" "Aim" Line 41 Data summary: what is the rational of adding it there? Abbreviations: please use ":" instead of ";" after each word. Line 48: please correct PCR "Polymerace" Introduction: Line 57: what is subclades? Please review the orthograph Line 59: Leptospires : no "s" at the end Lines 72-74: the total DALYs is 2.9 million. However, for male the estimated DALYs is 233 million. Please correct of clarify Line 80: please correct "ofleptospirois". At the same line, please be more specific: "..a positive culture of what??? Line 81: please write the entire PCR as at line 83 as if I am correct, it is the first time at line 81 and not the opposite. The same hold true for MAT Method: Line 120: Study population This section needs to be re-structured as it is a mixture of study population and design. I will suggest to write "Study population and setting" where you will describe the population of interest of the study as well as the study area where you are conducting this study and highlight the importance of the problem in this setting. For example, there is no statement of the "age". Are you recruiting baby of less than one year, adolescent, adult only? In your study, you will need to add a part for the study design in addition to the figure 2. Indeed, you are enrolling patients and healthy participants but it is not well explained in the text. You can add in this part the inclusion and exclusion criteria for the patients group for example. Readers need to go to the figure and try to understand how was the design of the study. Which is a bit hard. In addition, how did you get to the sample size? Line 131: I do not know how a lab request form can be used to collect information on basic clinical and demographic data. Please review as I think it should be a structured study questionnaire. I will suggest to add a section on "Biological samples collection and laboratory procedures" where you will put the different samples collected for each group of patient and how they are stored and processed in the lab as well as all related to the culturing and DNA extraction parts. Statistical analyse: this part needs to be entirely rewritten. Please ask help from a statistician to help explain which variables did you use to describe your population and what kind of test did you use for the comparison. All the software you used need to be put there and not later on in this results section Ethic statement: there is a repetition regarding the consent and Line 430 Results: At the first paragraph you described your population. I will suggest to add a sub-title "demographic characteristic of the study population" and after that a sub-title "results of the PCR and ELISA test". Just an example for your guidance. Line 239: please consider moving the last sentence to the method section Table 1: the title is long. Please review it. I think it is because you add a caption in the title and repeated "All febrile patients described here had a fever
Please rate the manuscript for methodological rigour
Satisfactory
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
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