Etiological Analysis of Viral Encephalitis in Children in Zhejiang Province from 2018 to 2019

Round 1

Reviewer 1 Report

The article by Liu et al. is potentially interesting.

My comments are as below.  

1.       Abstract (conclusions):

The authors state, “In children, VE is more prevalent in the summer than in other seasons”. However, I think the present data show the seasonal epidemiology in Zhejiang. 

2.       Primer sequences and product sizes are shown in Table 1. Did the authors design these primers? No reference was found.

3.       In results 3.3, “the 10 obtained PCR products were verified using Sanger sequencing. “

Only 10 PCR products were sequenced?

4.       In the discussion (lines 187-189), the authors describe as below.

In this study, we 187 used multiplex PCR to simultaneously detect multiple infectious agents in a small 188 amount of CSF [10]. 

Reference 10 seems to use FilmArray. I was confused.

Author Response

Response to Reviewer 1 Comments

 

Point 1: Abstract (conclusions): The authors state, “In children, VE is more prevalent in the summer than in other seasons”. However, I think the present data show the seasonal epidemiology in Zhejiang.

Response 1: The advice makes the conclusion more accurate. We have revised the conclusions as suggested. (Page 1, line 35)

 

Point 2: Primer sequences and product sizes are shown in Table 1. Did the authors design these primers? No reference was found.

 

Response 2: The primer sequences in Table 1 were designed by our team.

 

Point 3: In results 3.3, “the 10 obtained PCR products were verified using Sanger sequencing. “Only 10 PCR products were sequenced?

 

Response 3: Thank you for your comments.  Indeed, only 10 PCR products were sequenced in this study.  To further confirm the reliability of multiplex and singleplex RT-PCR results, PCR products were sequenced.  The 10 PCR products included all virus species detected in our study (EB/EV/HSV-1/CMV/VZV/MuV/HHV-6). However, due to the high cost of sequencing, we were unable to sequence all PCR products.  We finally decided to publish the sequencing results of the 10 PCR products in order to show readers that the technical methods we adopted were credible.  Thanks again to the expert for this question.

 

Point 4: In the discussion (lines 187-189), the authors describe as below.In this study, we 187 used multiplex PCR to simultaneously detect multiple infectious agents in a small 188 amount of CSF [10]. Reference 10 seems to use FilmArray.

 

Response 4: Many thanks to the expert for timely finding the labeling errors of the references in this paper.  It has been corrected in the article.  At the same time, all quotations in this article have been checked again.  (Page 6, line 178-181) ( Page 7, line 220-224)

Reviewer 2 Report

Language:

Line 23: “the samples were detected”, samples were analyzed or tested.

Line 33: “was confirmed”, should be plural.

Line 213: “The encephalitis caused by viral pathogens” should be ‘viral encephalitis’

Line 214: “the highest number… were”, should be singular

Line 263: “In conclusioon”

 

Introduction:

The introduction clearly describes the background and objectives of the study. I would suggest to remove “treatment” from line 60, as this manuscript only discusses the diagnosis.

 

Patients and Methods:

-          The inclusion and exclusion criteria are overall well considered and described. It is not fully clear whether any child between 0 and up to 18 years old was eligible. The white blood cell count cut-off values for inclusion should be provided in more detail than ‘normale or moderate’.

-          Was the quantity of available CSF an inclusion criterion?

-          The laboratory methods are clearly described by the authors

 

Results

-          I believe the results section would benefit from a more complete description of the study population including the clinical features (e.g. frequency of symptoms such as seizures, fever). This will be helpful to allow for comparison with other cohorts. Furthermore, it is important to mention on what day from symptom onset the CSF was collected, as this will affect the PCR positivity rate. Furthermore, it would be interesting to see an analysis of how these variables related to the virus detection rate in this study. Currently the authors only explore differences in detection rate according to age and gender.

-          Figure 2 could be improved by showing the distribution of pathogens per age group, perhaps as a table. The current graph is not very insightful because total numbers are not presented, furthermore it is unclear why the authors only report a statistical test of a comparison between the age distribution of 2 viruses.

-          In section 3.2 the authors should report the actual p-value, rather than the significance level of 0.05.

 

Discussion

-          The objectives as defined in the introduction “simplify and expand the scope of multi-pathogen detection, and will provide a reference for the empirical diagnosis and treatment of patients with VE” should be reflected in the discussion. In its current state, the discussion mostly focuses on clinical characteristics of the different causes of viral encephalitis. Some questions that would be interesting to address: How did the virus detection rate in this study compare to other studies? What were positive and negative predictors of positivity? How does multiplex PCR improve the time to diagnosis?

 

Overall the authors present interesting data and the manuscript is well written. The manuscript would benefit from some adaptations as suggested above and I would suggest to have the language checked by a native English speaker.

Author Response

Response to Reviewer 2 Comments

 

Point 1: Language:

Line 23: “the samples were detected”, samples were analyzed or tested.

Line 33: “was confirmed”, should be plural.

Line 213: “The encephalitis caused by viral pathogens” should be ‘viral encephalitis’

Line 214: “the highest number… were”, should be singular

Line 263: “In conclusioon”

 

Response 1: Thanks for the suggestions given by experts in the text, the above errors have been revised in the article.  In addition, we have checked the grammar and text expression of the article again, thanks for the guidance of experts. The revised part has been marked in red in this paper.

 

Point 2: Introduction:

The introduction clearly describes the background and objectives of the study. I would           suggest to remove “treatment” from line 60, as this manuscript only discusses the diagnosis .

 

Response 2: This study is mainly related to the etiological diagnosis of viral encephalitis in children. Therefore, the expression in this section has been modified according to the advice of experts and the treatment has been deleted. (Page 2, line 57)

 

Point 3: Patients and Methods:

-        The inclusion and exclusion criteria are overall well considered and described. It is not fully clear whether any child between 0 and up to 18 years old was eligible. The white blood cell count cut-off values for inclusion should be provided in more detail than ‘normale or moderate’.

-         Was the quantity of available CSF an inclusion criterion?

 

Response 3:

-  Thanks to the guidance of the experts, we did not make a clear description of "age". Now the paper has been revised.  We studied hospitalized children with an age range of 0-16 years.  In addition, the number of cerebrospinal fluid cells cut-off value is described in this paper. (Page 2, line 62; Page 2, line 68)

-    The enrolled children were required to collect 1.5-2ml of cerebrospinal fluid for virus-related pathogen detection and retesting.  Included children who did not have sufficient cerebrospinal fluid for etiological analysis would be excluded. Therefore, we added insufficient cerebrospinal fluid retention (< 1.5ml) to the exclusion criteria. ( Page 2, line 69)

 

Point 4: Results

-          I believe the results section would benefit from a more complete description of the study population including the clinical features (e.g. frequency of symptoms such as seizures, fever). This will be helpful to allow for comparison with other cohorts. Furthermore, it is important to mention on what day from symptom onset the CSF was collected, as this will affect the PCR positivity rate. Furthermore, it would be interesting to see an analysis of how these variables related to the virus detection rate in this study. Currently the authors only explore differences in detection rate according to age and gender.

-          Figure 2 could be improved by showing the distribution of pathogens per age group, perhaps as a table. The current graph is not very insightful because total numbers are not presented, furthermore it is unclear why the authors only report a statistical test of a comparison between the age distribution of 2 viruses.

-          In section 3.2 the authors should report the actual p-value, rather than the significance level of 0.05.

 

Response 4:

-   Thank you for your guidance and for informing us of the importance of detailed description of clinical features in this type of study.  The clinical data in this paper, such as fever, convulsions, lethargy and other clinical manifestations, were used to evaluate whether they met the inclusion criteria. Meanwhile, we mainly focused on the demographic characteristics of viral encephalitis pathogen distribution and the seasonal characteristics of virus distribution, so we did not analyze the clinical manifestations.  For cerebrospinal fluid collection, we collected it within 48 hours of hospitalization (basically 2-5 days after onset).  The association between CSF collection time and pathogen detection rate mentioned by experts is a significant study, which can be added to our future research work.  (Page 2, line 73)

-   Figure 2 changed to a table as suggested by the expert. Since only 1-2 cases of HCMV/MuV/HSV-1/VZV were detected, no statistical comparison of age at onset was conducted. We compared the mean age of onset of the three viruses with the highest number of cases between and within groups.  ANOVA was used for comparison between the three groups(EV,EBV, and HHV-6), F=3.972, P=0.021. Pair-wise comparison between the two groups suggested that there was a statistically significant difference in onset age between EB and HHV-6 groups, P < 0.017 (P=0.006, LSD method).  In Figure 2, only the comparison between EB and HHV-6 groups was marked, which caused confusion to readers. Now, it has been corrected into a table. (Page 5, line 146-147)

- According to the advice of the expert, we have reported the P value. (Page 6, line 170)

 

Point 5: Discussion

-          The objectives as defined in the introduction “simplify and expand the scope of multi-pathogen detection, and will provide a reference for the empirical diagnosis and treatment of patients with VE” should be reflected in the discussion. In its current state, the discussion mostly focuses on clinical characteristics of the different causes of viral encephalitis. Some questions that would be interesting to address: How did the virus detection rate in this study compare to other studies? What were positive and negative predictors of positivity? How does multiplex PCR improve the time to diagnosis?

 

Overall the authors present interesting data and the manuscript is well written. The manuscript would benefit from some adaptations as suggested above and I would suggest to have the language checked by a native English speaker.

 

Response 5:

-   In our discussion, we added statements about simplifying the experimental process and expanding the scope of pathogen detection. (Page 6, line 187-196)

-   At present, the etiological diagnosis rate of viral encephalitis is 27.5%-79% [1], and the detection rate of PCR virus in this study is 39.65%, which is similar to that of PCR virus in other studies (38%) [2].

[1] Boucher A, Herrmann JL, Morand P, Buzelé R, Crabol Y, Stahl JP, Mailles A. Epidemiology of infectious encephalitis causes in 2016. Med Mal Infect. 2017 May;47(3):221-235

[2] Ben Abid F, Abukhattab M, Ghazouani H, Khalil O, Gohar A, Al Soub H, Al Maslamani M, Al Khal A, Al Masalamani E, Al Dhahry S, Hashim S, Howadi F, Butt AA. Epidemiology and clinical outcomes of viral central nervous system infections. Int J Infect Dis. 2018 Aug;73:85-90.

-   Multiple convulsions and cranial imaging abnormalities may be the predictors of a high positive rate of pathogen detection. However, there was a lack of analysis on the influencing factors of pathogen detection rate in our study, which is meaningful and can be used as our future research direction.

-   In this study, multiple PCR can detect multiple pathogens at one time, and the detection results of multiple pathogens can be fed back to clinicians at one time, reducing the time cost of communication between the laboratory and clinicians, including HHV-6, HSV-1, and MuV, etc., which cannot be detected by traditional single pathogen PCR in our hospital.  Rapid diagnosis of these pathogens will benefit from multiple PCR applications.  Therefore, we believe that the application of multiplex PCR can shorten the diagnosis time. 

-     This article has been proofread again by native Speakers with expert advice.  Thanks again. 

Round 2

Reviewer 2 Report

Manuscript has improved and is acceptable in its current form. 

Additional comments:

-        Line 50: “such as ganciclovir”. This is not the drug of choice for treatment of HSV encephalitis

-          Line 282: “detection of EBV in the CSF has clinical significance”. The interpretation of EBV DNA detection in CSF is not straightforward and does not prove EBV encephalitis. EBV DNA can also be found in CSF in patients with EBV-associated malignancies and is also found together with other microbial agents (coinfection, reactivation secondary to infection with another agent or latent virus carried in inflammatory cells).

Author Response

Please see the attachment.

Author Response File: Author Response.pdf