Title: Detection and characterization of human astrovirus and sapovirus in outpatients with acute
gastroenteritis in Guangzhou, China
Version: 1 Date: 25 Aug 2021
Reviewer's report:
Major comments:
Luo et al. determined the incidence of human astrovirus and sapovirus in outpatients with acute
gastroenteritis from a hospital in Guangzhou, China, between September 2013 and January
2016. They characterized the seasonality, demographic and clinical characteristics of HAstV
and SaV infections. This manuscript provides data on the molecular epidemiology of two
important enteric viral pathogens, viz. HAstV and SaV, which are considered emerging in the
post rotavirus-vaccination introduction era. These results are of interest to the field, however the
overall study design needs improvement. HAstV and SaV are not included as targets of Luminex
GPP. I suggest that if the authors want to have an astrovirus and sapovirus centric manuscript,
they should focus on describing the use of GPP on the HAstV and SaV positive samples as
a secondary assay to show co-detection of other enteropathogens and include the majority of
GPP results in a separate manuscript. Otherwise, this manuscript can be modified to become an
AGE etiology paper and describe the same clinical picture presented here of the major pathogens
detected: rotavirus, norovirus, enteric bacteria, HAstV and SaV, etc.
Another major comment is that the data presented in Figures 1 and 2 should be normalized to
percent of the total number of specimens tested each month/age group. Plotting raw numbers is
misleading as a low total number in a specific month or age group would automatically lead to
a low number of HAstV and SaV positives.
Table 1 is redundant; its data were repeated in the texts (co-infections) or Figure 1-3, table 2 or
the Methods (accession numbers). There was no mention of table 1 in the text either. Consider
deleting table 1. Multivariate logistic regression analysis would strengthen the results in table 2
The conclusion that HAstV and SaV are major causative agents of AGE in Guangzhou, China
is not supported by the results as rotavirus, norovirus and enteric bacteria are more prevalent in
your study. The importance of studying Hast V and SaV should be set up in the introduction
better and the conclusion can be HAstV and SaV are common causes of AGE, especially in
children in south China.
Grammar and sentence structure need to be improved throughout the manuscript.
Minor comments:
Enteric virus seasons are usually shown from July the previous year to June the following year
to have the seasonal peaks (in the winter months) in the middle of the graph instead of splitting
it as shown in figure 1. Please add “in #C” after Mean Temperature in the figure legend.
Although the total number of positive cases would be very low, it would be interesting to separate
the data by year and see whether genotype prevalence changes in different years.
Line 76-78, the sentence “The age distribution of …were under 5 years old” should be placed
in Methods
Line 80, was the use of QIAamp MiniElute Virus Spin kit validated for bacteria and parasites
nucleic acid extraction in the Luminex GPP?
The Multiplex RT-PCR paragraph in Methods is missing the description of gel electrophoresis
and sequencing.
What was the concordance of the in-house multiplex RT-PCR and Luminex GPP on norovirus
and adenovirus detection? Luminex GPP was shown to have challenges in detecting certain NoV
GII.2 strains. Was the in-house RT-PCR designed for pan-serotype detection of adenovirus or
specific enteric adenoviruses (40/41) as Luminex GPP was?
Because in line 110, “1000 bootstrap(s)” was mentioned, I assume that the numbers shown
on the branches of the dendrogram were percentages and only those above 50% were shown.
Also, consider constructing the dendrogram with the Maximum Likelihood or even the Bayesian
method in MEGA. They have superior statistical properties, especially when you have only a
few sequences to incorporate.
Please move the description of the seasons to Methods. Only define the winter season if you are
going to use that term later on (line 140).
C. difficile is commonly found in children under two years of age; those were considered benign
colonization rather than infection.
Sentences in line 173-176 should be placed in Methods.
Line 177, GI and GIV are genogroups, not genotypes of sapovirus. Please report the genotypes
as well.
Line 180-182, the discussion of assay characteristics is irrelevant to this manuscript unless put
into context. I suggest that the authors summarize the study design and findings in the first
paragraph of the Discussion.
Title: Detection and characterization of human astrovirus and sapovirus in outpatients with acute
gastroenteritis in Guangzhou, China
Version: 1 Date: 25 Aug 2021
Reviewer's report:
Major comments:
Luo et al. determined the incidence of human astrovirus and sapovirus in outpatients with acute
gastroenteritis from a hospital in Guangzhou, China, between September 2013 and January
2016. They characterized the seasonality, demographic and clinical characteristics of HAstV
and SaV infections. This manuscript provides data on the molecular epidemiology of two
important enteric viral pathogens, viz. HAstV and SaV, which are considered emerging in the
post rotavirus-vaccination introduction era. These results are of interest to the field, however the
overall study design needs improvement. HAstV and SaV are not included as targets of Luminex
GPP. I suggest that if the authors want to have an astrovirus and sapovirus centric manuscript,
they should focus on describing the use of GPP on the HAstV and SaV positive samples as
a secondary assay to show co-detection of other enteropathogens and include the majority of
GPP results in a separate manuscript. Otherwise, this manuscript can be modified to become an
AGE etiology paper and describe the same clinical picture presented here of the major pathogens
detected: rotavirus, norovirus, enteric bacteria, HAstV and SaV, etc.
Another major comment is that the data presented in Figures 1 and 2 should be normalized to
percent of the total number of specimens tested each month/age group. Plotting raw numbers is
misleading as a low total number in a specific month or age group would automatically lead to
a low number of HAstV and SaV positives.
Table 1 is redundant; its data were repeated in the texts (co-infections) or Figure 1-3, table 2 or
the Methods (accession numbers). There was no mention of table 1 in the text either. Consider
deleting table 1. Multivariate logistic regression analysis would strengthen the results in table 2
The conclusion that HAstV and SaV are major causative agents of AGE in Guangzhou, China
is not supported by the results as rotavirus, norovirus and enteric bacteria are more prevalent in
your study. The importance of studying Hast V and SaV should be set up in the introduction
better and the conclusion can be HAstV and SaV are common causes of AGE, especially in
children in south China.
Grammar and sentence structure need to be improved throughout the manuscript.
Minor comments:
Enteric virus seasons are usually shown from July the previous year to June the following year
to have the seasonal peaks (in the winter months) in the middle of the graph instead of splitting
it as shown in figure 1. Please add “in #C” after Mean Temperature in the figure legend.
Although the total number of positive cases would be very low, it would be interesting to separate
the data by year and see whether genotype prevalence changes in different years.
Line 76-78, the sentence “The age distribution of …were under 5 years old” should be placed
in Methods
Line 80, was the use of QIAamp MiniElute Virus Spin kit validated for bacteria and parasites
nucleic acid extraction in the Luminex GPP?
The Multiplex RT-PCR paragraph in Methods is missing the description of gel electrophoresis
and sequencing.
What was the concordance of the in-house multiplex RT-PCR and Luminex GPP on norovirus
and adenovirus detection? Luminex GPP was shown to have challenges in detecting certain NoV
GII.2 strains. Was the in-house RT-PCR designed for pan-serotype detection of adenovirus or
specific enteric adenoviruses (40/41) as Luminex GPP was?
Because in line 110, “1000 bootstrap(s)” was mentioned, I assume that the numbers shown
on the branches of the dendrogram were percentages and only those above 50% were shown.
Also, consider constructing the dendrogram with the Maximum Likelihood or even the Bayesian
method in MEGA. They have superior statistical properties, especially when you have only a
few sequences to incorporate.
Please move the description of the seasons to Methods. Only define the winter season if you are
going to use that term later on (line 140).
C. difficile is commonly found in children under two years of age; those were considered benign
colonization rather than infection.
Sentences in line 173-176 should be placed in Methods.
Line 177, GI and GIV are genogroups, not genotypes of sapovirus. Please report the genotypes
as well.
Line 180-182, the discussion of assay characteristics is irrelevant to this manuscript unless put
into context. I suggest that the authors summarize the study design and findings in the first
paragraph of the Discussion.
Please review this paper.
user
Title: Detection and characterization of human astrovirus and sapovirus in outpatients with acute
gastroenteritis in Guangzhou, China
Version: 1 Date: 25 Aug 2021
Reviewer's report:
Major comments:
Luo et al. determined the incidence of human astrovirus and sapovirus in outpatients with acute
gastroenteritis from a hospital in Guangzhou, China, between September 2013 and January
2016. They characterized the seasonality, demographic and clinical characteristics of HAstV
and SaV infections. This manuscript provides data on the molecular epidemiology of two
important enteric viral pathogens, viz. HAstV and SaV, which are considered emerging in the
post rotavirus-vaccination introduction era. These results are of interest to the field, however the
overall study design needs improvement. HAstV and SaV are not included as targets of Luminex
GPP. I suggest that if the authors want to have an astrovirus and sapovirus centric manuscript,
they should focus on describing the use of GPP on the HAstV and SaV positive samples as
a secondary assay to show co-detection of other enteropathogens and include the majority of
GPP results in a separate manuscript. Otherwise, this manuscript can be modified to become an
AGE etiology paper and describe the same clinical picture presented here of the major pathogens
detected: rotavirus, norovirus, enteric bacteria, HAstV and SaV, etc.
Another major comment is that the data presented in Figures 1 and 2 should be normalized to
percent of the total number of specimens tested each month/age group. Plotting raw numbers is
misleading as a low total number in a specific month or age group would automatically lead to
a low number of HAstV and SaV positives.
Table 1 is redundant; its data were repeated in the texts (co-infections) or Figure 1-3, table 2 or
the Methods (accession numbers). There was no mention of table 1 in the text either. Consider
deleting table 1. Multivariate logistic regression analysis would strengthen the results in table 2
The conclusion that HAstV and SaV are major causative agents of AGE in Guangzhou, China
is not supported by the results as rotavirus, norovirus and enteric bacteria are more prevalent in
your study. The importance of studying Hast V and SaV should be set up in the introduction
better and the conclusion can be HAstV and SaV are common causes of AGE, especially in
children in south China.
Grammar and sentence structure need to be improved throughout the manuscript.
Minor comments:
Enteric virus seasons are usually shown from July the previous year to June the following year
to have the seasonal peaks (in the winter months) in the middle of the graph instead of splitting
it as shown in figure 1. Please add “in #C” after Mean Temperature in the figure legend.
Although the total number of positive cases would be very low, it would be interesting to separate
the data by year and see whether genotype prevalence changes in different years.
Line 76-78, the sentence “The age distribution of …were under 5 years old” should be placed
in Methods
Line 80, was the use of QIAamp MiniElute Virus Spin kit validated for bacteria and parasites
nucleic acid extraction in the Luminex GPP?
The Multiplex RT-PCR paragraph in Methods is missing the description of gel electrophoresis
and sequencing.
What was the concordance of the in-house multiplex RT-PCR and Luminex GPP on norovirus
and adenovirus detection? Luminex GPP was shown to have challenges in detecting certain NoV
GII.2 strains. Was the in-house RT-PCR designed for pan-serotype detection of adenovirus or
specific enteric adenoviruses (40/41) as Luminex GPP was?
Because in line 110, “1000 bootstrap(s)” was mentioned, I assume that the numbers shown
on the branches of the dendrogram were percentages and only those above 50% were shown.
Also, consider constructing the dendrogram with the Maximum Likelihood or even the Bayesian
method in MEGA. They have superior statistical properties, especially when you have only a
few sequences to incorporate.
Please move the description of the seasons to Methods. Only define the winter season if you are
going to use that term later on (line 140).
C. difficile is commonly found in children under two years of age; those were considered benign
colonization rather than infection.
Sentences in line 173-176 should be placed in Methods.
Line 177, GI and GIV are genogroups, not genotypes of sapovirus. Please report the genotypes
as well.
Line 180-182, the discussion of assay characteristics is irrelevant to this manuscript unless put
into context. I suggest that the authors summarize the study design and findings in the first
paragraph of the Discussion.
assistant