Validation of an improved reference freeze-dried direct agglutination test for detecting leishmaniasis in the canine reservoir
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Introduction Proper identification and management of reservoir post-kala-azar dermal leishmaniasis (PKDL) and canine visceral leishmaniasis (CVL) cases are prerequisites to the effective control of visceral leishmaniasis (VL) worldwide. Unlike PKDL, CVL still awaits effective improvement because of its cryptic nature, absence of Leishmania parasite in lesions or lymph nodes, and insensitivity of tools in current use. Because of the need for certain skills and equipment, both the liquid direct agglutination test (LQ-DAT) and freeze-dried direct agglutination test (FD-DAT) versions, present in comparison with the indirect immunofluorescence (IFAT) or enzyme-linked immunosorbent assay (ELISA), practical and feasible diagnostic alternatives. Aim Validate the performance of an improved FD-DAT to suit routine and large-scale applications in CVL endemic areas. Methodology Introducing of citrate-saline formaldehyde (CSF) as an anti-clumping agent to replace normal saline for antigen reconstitution and drastically however eligibly lowering the concentration of promastigotes (1.4X107) in comparison with the original reference FD-DAT (> 5X107/ml), To ensure optimal safety, β-mercaptoethanol (β-ME) was replaced by urea or sodium dodecyl sulphate (SDS) as a serum reducing agent. Results Through improving the procedure for reconstitution of FD-DAT antigen with CSF a 150% reduction in the test application cost was achieved. Expired test batches (+ 4 years earlier), were successfully revitalized to full validity. As compared to 48-hour shelf-life time for the original, an FD-DAT batch re-constituted here with CSF maintained validity for + 12 months. Conclusions The highly concordant results with IFAT and ELISA (One Way ANOVA Test P = 0.142, Homogeneity of Variances P = 0.009) as routine CVL diagnostics further motivate the application of the improved FD-DAT for detection of the disease in endemic areas.
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Editor's Comments to Author Dear Dr. Hussam Ali Osman, The manuscript "Validation of an improved reference freeze-dried direct agglutination test for detecting leishmaniasis in the canine reservoir" has been positively assessed by two independent reviewers. Yet, both reviewers indicated that minor corrections are necessary, especially regarding the Discussion section. Please notice that even though one reviewer asked for you to contact the kit manufacturer, this is not going to be required for publication. Nevertheless, this is something you may consider doing in order to improve and facilitate the diagnosis of canine leishmaniasis in endemic regions. Once you address the reviewers comments, your paper can be resubmitted and will be considered for publication. Best regards, Gustavo
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Comments to Author
Reliable serological diagnosis of canine VL is a serious problem for One Health. The topic has global epidemiological and epizootiological importance. The paper is very clearly written, and after the first phase of corrections it has been significantly improved. The diagnostic problem is well studied and the solution concept is excellently designed and solved. Have you contacted the manufacturer of the test you modified? Did you inform them of the results obtained after the modification? If there is no problem with that, the paper is very acceptable for publication with minor corrections. 1. In the text, the abbreviation ″ml″ should be corrected to ″mL″. 2. Typos should be corrected throughout the text. For example: line 186 (″1: 10″), line 402 (bold comma), lines 386 and 406 (--), line 455 (double …
Comments to Author
Reliable serological diagnosis of canine VL is a serious problem for One Health. The topic has global epidemiological and epizootiological importance. The paper is very clearly written, and after the first phase of corrections it has been significantly improved. The diagnostic problem is well studied and the solution concept is excellently designed and solved. Have you contacted the manufacturer of the test you modified? Did you inform them of the results obtained after the modification? If there is no problem with that, the paper is very acceptable for publication with minor corrections. 1. In the text, the abbreviation ″ml″ should be corrected to ″mL″. 2. Typos should be corrected throughout the text. For example: line 186 (″1: 10″), line 402 (bold comma), lines 386 and 406 (--), line 455 (double in)… 3. Line 192: How old were the dogs? Were they clinically healthy dogs? Have they been vet checked? 4. What tests were used for the initial diagnosis of VL in 15 dogs from an endemic region? I recommend adding a little to the discussion on the following topics: 5. Why was calf serum replaced with gelatin in 0.2% wt/vol. in normal saline? What components in the composition disqualify it? What are the advantages of gelatin at 0.2% wt/vol. in normal saline? 6. We expect that in dogs with VL from Croatia, the species was Leishmania infantum. I recommend a little more analysis of literature data on the success of applied tests in VL caused by different species of Leishmania.
Please rate the manuscript for methodological rigour
Very good
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
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Comments to Author
This reviewer was invited to review a revised (R1) version of the manuscript. The authors have properly addressed the questions posed by a previous reviewer. Nevertheless, some adaptations are still necessary, including a review of English by a native speaker, to improve to increase the value of this manuscript for the readers. 1. Methodological rigour, reproducibility and availability of underlying data - OK 2. Presentation of results - OK 3. How the style and organization of the paper communicates and represents key findings - different DAT are deemed as equivalent to IFAT and ELISA 4. Literature analysis or discussion - there is a lack of recent reference on CanL and prevention of infection/disease is not addressed Line 25, delete "reservoir" L26, adapt to read as: … are among the prerequisites
Comments to Author
This reviewer was invited to review a revised (R1) version of the manuscript. The authors have properly addressed the questions posed by a previous reviewer. Nevertheless, some adaptations are still necessary, including a review of English by a native speaker, to improve to increase the value of this manuscript for the readers. 1. Methodological rigour, reproducibility and availability of underlying data - OK 2. Presentation of results - OK 3. How the style and organization of the paper communicates and represents key findings - different DAT are deemed as equivalent to IFAT and ELISA 4. Literature analysis or discussion - there is a lack of recent reference on CanL and prevention of infection/disease is not addressed Line 25, delete "reservoir" L26, adapt to read as: … are among the prerequisites L28, Leishmania parasites L29, and not complete sensitivity of some diagnostic tools in use L31, agglutination test (FD-DAT) versions are, in comparison with the indirect immunofluorescence (IFAT) or enzyme-linked immunosorbent assay (ELISA), practical and feasible diagnostic alternatives Line 44, what is the meaning of validity? Keywords, display in alphabetical order L68, Leishmania parasites - replace confirmation with diagnosis L69, do not use Eastern Europe - remove from the manuscript L70, Although lymph node and bone marrow aspiration is L71, delete routinely L72, delete genuine L75, please define the meaning of reliability L76, enzyme-linked immunosorbent assay L77, Leishmania donovani L80, winter stays or summer stays? Transmission of Leishmania is less likely to occur during winter L89, delete high L95, 7 years L97-100, it is not necessary to explain the meaning of abbreviations again L119, 4 years L134, use FCS to abbreviate fetal calf serum, at this point L138, replace efficiency with efficacy, along the manuscript L163, abbreviation has already been presented on L129, it is not necessary to repeat full description again L173, add city (and state), apart from country, to the manufacturers' names L191, why this number of serum samples? L194, The other 64… L200, were results normality distributed? L213, it is not necessary to explain the meaning of abbreviations again In general, titres should be presented as 100, 200, 400, etc. and not 1:100, 1:200, 1:400, etc. L224-255, this should be part of the discussion L233, text is not clear L238, delete as CanL test of choice in Croatia L246, delete Remarkably L248, replace "typical CanL symptoms were observed" with CanL clinical signs were observed L263, replace symptomatic with sick L270, delete very high L277, replace significant with considerable L282, 4 years L312, L314 and L316, replace symptoms with clinical signs L318 and L325, replace symptomatic with sick L323, 3 decades The authors should also address the fact that there are now tolls available for the prevention of CanL, which include insecticides with an anti-feeding effect, vaccines and and immunodulator. Table 1, replace Comparative efficiencies of with Comparison of Table 2, replace Validity with Performance Table, replace Asymptomatic with Clinically healthy Figure 1, provide an explanation for all the abbreviations, including CSF, etc.
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?
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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Dear Dr. Osman, Thank you for submitting your work to Access Microbiology. The paper presented by you and your co-authors is of interest, as it seems to improve the efficacy and the facilitate the use of both valid and expired commercial tests for canine leishmaniasis. Yet, a few points require revision prior to peer-review: 1. As the paper describes a comparison between methodologies, particularly when comparing valid x expired tests, and the different reducing agents, it would be useful to add a figure containing a diagram on how each test was prepared. This is of particular relevance because it is not clear whether the authors tested the expired test using the standard solubilization method as a comparison. It is, of course, impossible to say the expired test vials were "revitalized" with the "improved" protocol if the test …
Dear Dr. Osman, Thank you for submitting your work to Access Microbiology. The paper presented by you and your co-authors is of interest, as it seems to improve the efficacy and the facilitate the use of both valid and expired commercial tests for canine leishmaniasis. Yet, a few points require revision prior to peer-review: 1. As the paper describes a comparison between methodologies, particularly when comparing valid x expired tests, and the different reducing agents, it would be useful to add a figure containing a diagram on how each test was prepared. This is of particular relevance because it is not clear whether the authors tested the expired test using the standard solubilization method as a comparison. It is, of course, impossible to say the expired test vials were "revitalized" with the "improved" protocol if the test perfomance under standard conditions is unknown. 2. Given the paper proposes to reformulate the use of a diagnostic test using a different set of solutions, it would be good to see a table that describes the ammounts and volumes of each reagent used in each solution prepared. 3. The authors have to be clearer on the criteria used to determine a positive versus a negative test. What was the minimum titration required for a sample to be considered positive? Did it change between tests? Addtionally, the authors may take the opportunity to be more thoroughly descriptive in the Methods section. 4. The authors say the tests were read after 18h of incubation in a wide range of temperatures (between 23 and 40 degrees Celsius). Did this affect the performance of the tests? 5. The number of dog samples analysed changed in each table. Also, the authors had 86 samples available, but the maximum number of samples displayed in a table is 73 (Table 2). Why were the other samples not included? Moreover, it would be useful for the authors to discriminate which samples were used as negatives (if any) in the assays. Please address the above questions and resubmit the paper for consideration. Best regards, Gustavo
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