Haematological malignancies as disorders negatively impacting specificities of the direct agglutination and rapid rK39 strip tests as reference diagnostics for visceral leishmanisis
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Introduction. During several years of work in Sudan, we occasionally had been confronted with patients who presented clinical features highly suggestive of visceral leishmaniasis (VL) however direct agglutination test (DAT) readings that were either at the high negative or low positive titre range. Inquiries on the fate of those particular patients revealed mortality, undetermined diagnosis or that in some of them leukaemia was finally diagnosed.
Gap statement. Investigate as to what extent haematological malignancies (HMs) interfere with VL diagnosis.
Aim. Evaluate specificity of DAT version newly developed in this study wherein sodium dodecyle sulphate (SDS) was incorporated as a test sample denaturant in comparison with a standard reference wherein β-mercaptoethanol (β-ME) was used in test execution.
Methodology. Seventy plasma samples from patients with HMs were collected and tested in a primary DAT version (P-DAT). The results obtained were compared with those of the rK39 strip test as VL reference diagnostic. HM samples revealing titres higher than the start dilution (1 : 100) in P-DAT were further tested in a β-ME- and urea-modified DAT versions. The specificity of the newly developed SDS-DAT was assessed against that of β-ME-DAT and rK39 strip tests as current reference diagnostics for VL.
Results. Seven out of 70 patients with HMs scored positive outcomes (titre ≥1 : 3200) in P-DAT and four in the reference rK39 strip test. Of the seven that tested positive in P-DAT or four in the reference rK39, none reacted at titre >1 : 100 in the SDS-DAT. Significant reduction in non-specific agglutination reactions was achieved as a result in respect to the HM plasma samples ( P value <0.05).
Conclusion. To establish desired specificity for VL diagnosis in respect to HMs and subsequently minimize or avoid serious side effects due to unjustified anti-leishmanials prescription the combined application of the SDS-DAT here described and an improved version of the rK39 for confirmation is recommended.
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The work presented is clear and the arguments well formed. The manuscript is well written and contributes to the literature. Thank you for addressing all reviewers comments satisfactorily and in a timely manner.
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Comments to Author
The paper discusses the possibility of cross reactions and thus the effect on the specificity of the well-known direct agglutination test (DAT) and the rapid rK39 strip test for the diagnosis of visceral leishmaniasis (VL). Emerging from the results reported thus far, no plausible justification regarding absolute specificity for any of these two reference tests was given. The availability of non-specific antibodies in non-infectious diseases such as malignancies and auto-immune disorders might provide reasons for cross reactions with serum/plasma samples from these patients. I think this study highlight this possibility and therefore may motivate further confirmation not only with respect to VL diagnosis but also other diseases wherein sero-diagnosis plays an important role. The improvement introduced …
Comments to Author
The paper discusses the possibility of cross reactions and thus the effect on the specificity of the well-known direct agglutination test (DAT) and the rapid rK39 strip test for the diagnosis of visceral leishmaniasis (VL). Emerging from the results reported thus far, no plausible justification regarding absolute specificity for any of these two reference tests was given. The availability of non-specific antibodies in non-infectious diseases such as malignancies and auto-immune disorders might provide reasons for cross reactions with serum/plasma samples from these patients. I think this study highlight this possibility and therefore may motivate further confirmation not only with respect to VL diagnosis but also other diseases wherein sero-diagnosis plays an important role. The improvement introduced to the DAT through incorporating sodium dodecyle sulphate to eliminate these non-specific reactions in DAT present a key finding and added an advantage to this manuscript. 1 - The manuscript also raised an important point concerning use of anti-leishmanials and how risky can unjustified administration of these drugs be. 2 - The results are convincing, well presented and clearly support the conclusions. 3 - The authors have included and adequately discussed several relevant papers. 4 - The manuscript is well organized and well written. 5 - I would like to suggest for the authors to include in their discussion the types of non-specific antibodies or immune-globulins of patients with hematological malignancies that might have resulted in the observed cross-reactions against the Leishmania antigen.
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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Thank you for submitting your manuscript for publication in Access Microbiology. It has been examined by expert reviewers who have concluded that the work is of potential interest to the readership of Access Microbiology. Based on the comments received, a minor amendment of this manuscript will be required. I will be pleased to consider a revised manuscript that addresses all of the reviewers' comments. Your revised manuscript may be returned to one or more of the original reviewers, along with your itemised response to the reviewers’ comments.
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Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data Generally clear, as DAT is an established technique. 2. Presentation of results See specific comments. 3. How the style and organization of the paper communicates and represents key findings Generally clear, as DAT is an established technique, but see specific comments. 4. Literature analysis or discussion Generally OK. 5. Any other relevant comments Specific comments: Line 69. The abbreviation HM should be explained here at its first use. Line76. Add the word 'sulphate' after 'dodecyl'. Line 79/80. Add the section title 'Methods'. Line 83. Replace 'patient's' by 'patient' Line 99. Delete '(L. donovani)' Line 135. The authors should clarify whether titres to 1:1600 are also considered negative. Line 158. Insert a comma …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data Generally clear, as DAT is an established technique. 2. Presentation of results See specific comments. 3. How the style and organization of the paper communicates and represents key findings Generally clear, as DAT is an established technique, but see specific comments. 4. Literature analysis or discussion Generally OK. 5. Any other relevant comments Specific comments: Line 69. The abbreviation HM should be explained here at its first use. Line76. Add the word 'sulphate' after 'dodecyl'. Line 79/80. Add the section title 'Methods'. Line 83. Replace 'patient's' by 'patient' Line 99. Delete '(L. donovani)' Line 135. The authors should clarify whether titres to 1:1600 are also considered negative. Line 158. Insert a comma after 'rK39'. Line158. The authors should clarify that the 4 positive patients by rK39 are different to those positive by P-DAT. Line 161. The authors should check whether '14' here should be '13', to match the data in Table 2. Line 165. Replace 'Agreeable' by 'In agreement'. Line 167. The authors should check whether they mean here 'SDS-DAT' or 'BME-DAT' in this line. Line 191. Replace 'assimilating' with 'similar to'. Line 217. Replace 'equivalent' by 'development'. Lines 316-7 and 324-5. These statistical data should be in the Results section, not as footnotes to the Tables. Discussion. The authors should state more clearly if there are any previous published reports of cross-reactions between HM and VL DAT. Table1. The authors should indicate on the table or in the legend that 1:3200 is the cut-off for positives.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
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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