Transnasal Brain Sampling for Human Rabies Diagnosis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Major Comments

1. I am not sure if it is good to write someone's name on the methodology section of the paper; if I were you, I wouldn't do that. 

2. In 2.2, I strongly suggest showing an anatomical figure of the human head and nose, showing the direct way of taking the brain tissue sample from the nasal cavity; this will help authors to understand more clearly and allow other medical professionals to replicate this procedure.

3. From lines 111-117. While going inside the nasal cavity, we encounter the ethmoid bone. Can we simply break it using forceps and enter the brain? Please describe it more anatomically and in detail. 

4. Please add an ethical statement at the bottom of the manuscript. I saw it mentioned in the acknowledgment, but add a separate ethical statement and write the whole story. 

5. WHO suggests using the FAT test for rabies diagnosis, yet you used the RT-PCR way. Please describe why and how in one more additional paragraph. 

6. You mentioned "One Health" five times in this manuscript; yes, I understand its importance and personally advocate it, but is it necessary in your paper to show alternative techniques to obtain a tissue sample from the brain? 

Minor Comments 

1. Intext citation:  this is the correct way [1], not this one (1) for this journal.

2. "Otolorin and colleagues" I suggest writing Otolorin et. al. (2015) and (10) at the end of the sentence. 

3. De Benedictis and colleagues, please change this also similarly as above. 

Comments on the Quality of English Language

Minor editing of English language required. 

Author Response

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Reviewer 2 Report

Comments and Suggestions for Authors

Djimatey et al describe a relatively easy and non-invasive method for collecting brain tissue from human suspect cases. The manuscript is well written and the approach very well described. 

i. Can the authors confirm the rabies variant associated with this human case?

ii. Can the authors also compare this method to the one used in animals were the samples are extracted through the occipital foramen?

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

Material and Methods

Lines 74-86. I don´t think this paragraph is part of material and methods, maybe at the Introduction or Discussion will be better placed.

Line 96. I think it will be better to described as hydrophobia, not fear of water.

Line 116. How can you be sure that you are taken the more accurate brain structures for rabies diagnosis? How many different structures did you take in this way? I recommend a figure and or video showing how do you performed this technique.

Line 131. This section needs to be improved, since is all about diagnosis, please add the targets, the primers sequences, concentrations, the size of the expected products, RT-PCR reactions and cycling conditions. How many reactions you performed at each lab, what kind of controls do you included…

Also you need to explain and justify, why you did not use the fluorescent antigen test, that is the gold standard for rabies diagnosis for WHO and WOHA.

You also have to give the Gen Bank accession number of the sequences of the virus obtained and the BLAST analysis. What was the origin of the virus that you obtained?

Line 167. I do not consider that with just one case you can be sure of this, I will rather say that it is promising method but more cases need to be confirmed with this technique, also you have to compare with the gold standard the FAT in order to propose this as the recommended and optimal method for post-mortem brain tissue collection in human rabies diagnosis

Author Response

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Reviewer 4 Report

Comments and Suggestions for Authors

1. It is indeed important to confirm rabies infection in man post-mortem to discover the true prevalence of the disease. Ignorance of the number of deaths is inhibiting efforts to prevent rabies. This also applies to diagnosing dog rabies, but that is not the subject of this paper. The Introduction should concentrate more on the human aspect. 

2. The method of brain biopsy to confirm rabies infection post-mortem has been used before but  the biopsy has usually been taken with a needle via the inner canthus of the eye. This also leaves no mark on the body.

 3. Reference 11 [should be Emerging Infectious Diseases Vol. 13, No. 1, 2007]  States:

‘For fatal cases, permission was sought for autopsy or supraorbital needle biopsy. Samples of human brain tissue were analyzed for rabies virus by using the fluorescent antibody test’ 

The method has been described in more detail in Trop. Med Infect Dis 2017: 2(4), 52. The Imperative of Palliation in the Management of Rabies Encephalomyelitis.

‘Brain necropsies are taken with a long biopsy needle via the medial canthus of the eye through the superior orbital fissure; via the nose through the ethmoid bone; through the foramen magnum or open fontanelles in children.’

 The method of sample taking in rabies is described in: Tong TR, Leung KM, Lee KC, Lam AWS. Trucut needle biopsy through superior orbital fissure for diagnosis of rabies. Lancet 1999; 354: 2137–38.

 The technique has most often been used to diagnose malaria, but also other infections.

 4. This patients’ samples were tested by PCR, however the immunofluorescence technique, which has long been used to diagnose rabies in fresh dog brains, is simpler, quicker, cheaper and less demanding. This should also be mentioned in the discussion.

Author Response

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Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made significant improvements to the manuscript. I would recommend accepting the paper in its current form.

Author Response

Thank you for your contributions to our work

Reviewer 2 Report

Comments and Suggestions for Authors

This is a much improved version of the manuscript. The issues I raised in the first review have been addressed. However, I have these minor issues for the authors to attend to:

i. Line 121 - replace the word dropped with suspended,

ii. Line 122 - sterile saline, provide the concentration of this preservative,

iii. Line 148 - replace rabies with either rabies virus or Rabies lyssavirus. 

Author Response

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Thank you

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Reviewer 3 Report

Comments and Suggestions for Authors

Trans-nasal Brain Sampling for Human Rabies Confirmation

Although this new version has been sufficiently improved, they are some changes that should be done in order to warrant publication in Anatomia.

Title:

I will recommend to modify the title in order to adequate it: Trans-nasal Brain Sampling for Human Rabies Diagnosis, since confirmation depends not only in a good sample taking, but also a good lab performance.

Introduction:

Line 49.  Please give the full name here and later you can use only the acronym WHO, move from line 52.

Material and Methods

Lines 87-95. This paragraph should be placed as part of the Case report section.

Line 110. This sentence is not correct, you shall mention that the personal involved in the sample taking (cause it was not an autopsy) was wearing the recommended PEP as refereed in WHO expert consultation on rabies: third report, 2018 [4].

Lines 111-125.This seems to be a description in a book, please clarify if you did it as described: The orbitofrontal cortex was sampled (not can be…) since you are proposing this technique, please give the more accurate description of what you done.

Line 120. Nutral, please clarify is natural o neutral.

Figure 1. Please refer it in the text.

Line 135. This is not necessary to be clarified since it raises more doubts:  it was a video call or just messages?  Do you consider that this method can be reproduced if the author receive more calls from other physicians???? Please delete this sentence.

Lines 145-148. You can simplify this sentence by mentioning that both labs performed the RT-PCR as described by…..

Line 152. Why you used warm buffer? Do you mean room temperature? Otherwise you have not followed manufacturer’s instructions.

Lines 154 and 156.  RT-PCR or you used only for the PCR? If you done that please describe how do you made the Retro transcription as well.

Line 159. In vitro transcripts kindly donated by the laboratory of Prof Christian Drosten, Institute of Virology, Charite, Germany were used as positive control (in singular).

Line 161. Negative control (singular) or you ran more than one reactions without RNA?

Line 164. I suggest: retro transcription.

Line 166. This is the first time that you mentioned like RT-qPCR, please standardize the term throughout the document.

Table 1. Please refer this table in the text. Add the sense of the DNA chain of the primers and probe sequences.

Discussion

Line 177. trans-nasal

I suggest you mention that a negative result does not exclude rabies, this technique helps to confirm but not to rule out. Although in countries with a high rate of non-confirmed human rabies cases, this will be helpful to have a better idea of the epidemiology of the disease.

References:

4 and 5.  These are the same one with a different style of citation.

Author Response

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Reviewer 4 Report

Comments and Suggestions for Authors

1. The highlighted sections of the revised version do not indicate the changes made.

Please highlight only the new text and show deleted text as strike through, like this. 

2. Your assumption that orbital route demands expertise is unfounded, especially when compared with your description of the nasal method. Inserting a needle vertically downwards into the brain, it may well be easier to enter brain tissue, see this in your diagram.

 3. As described, the PCR testing is complicated, technically demanding and prone to false positives. It is rarely available in African countries.

The FAT test is simpler, quicker, cheaper and is more reliable. A veterinary laboratory is most likely to rely on this simpler test. All testing can be done in one laboratory in the locality. Has your laboratory had any experience of using the FAT?

 Conclusion: More precise editing is essential.

Author Response

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Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

Why do you deleted the RT-PCR description? You confirmed the diagnosis by this method, his information is important in order to reproduce your findings.

Author Response

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Thank you

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Reviewer 4 Report

Comments and Suggestions for Authors

The authors have commented on but not responded to comments on v2. It is still impossible to detect how the manuscript has been changed especially the differences in each version.

 1. 210  ‘approach provides a promising method for optimal post-mortem  brain tissue collection in human rabies’

‘promising method for optimal’ is not a helpful statement. The method is not new but the paper describes in detail a means of diagnosis without a biopsy needle.

2. Discussion

‘This is the first reported case of human rabies..’. insert ‘reported’ 

3. 146 ‘This novel method circumvents disfigurement..’ This is not a unique to the trans-nasal method. It also applies to the two other sampling methods.

Suggest change to: This  method, as with needle biopsies, leaves no mark on the body.

Author Response

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Thank you

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