Title: Detection and characterization of human astrovirus and sapovirus in outpatients with acute
gastroenteritis in Guangzhou, China
Version: 2 Date: 02 Oct 2021
Reviewer's report:
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.
Response: We appreciate your comments and suggestions. Through the study, we want to know
if there is a difference in clinical features between HAstV and other virus. We also want to know
whether the co-infection of HAstV with multiple enteropathogens is related to the severity of
the disease. The same to SaV. So Luminex GPP was used to detect the other virus and bacteria
in all patients, not just in HAstV and SaV positive samples. On the other hand, a detailed AGE
etiology assay, including virus and bacteria, has been reported in another manuscript.
Question: Maybe I misread it. Did you mean these data were published in another manuscript
already? I hope not.
Grammar and sentence structure needs to be improved throughout the manuscript. Response:
We carefully reviewed the manuscript and revised the sentences and paragraphs with the kind
assistance from a native English speaker and a scientific writing company.
Comment: Please edit the highlighted sentences in the revised manuscript for clarity.
Line 80, was the use of QIAamp MiniElute Virus Spin kit validated for bacteria and parasites
nucleic acid extraction in the Luminex GPP?
Response: QIAamp MiniElute Virus Spin kit, using to extract virus, bacteria and parasites
nucleic acid in the Luminex GPP, had been validated in our lab. The results were reported in
Reference [15] JianKai D, Xin L, Ruilian W, LingXiao J, Xixia D,
Wei H, Yongzheng P, Yu N, Xiaoyan. C. A Comparison of Luminex XTAG® Gastrointestinal
Pathogen Panel (XTAG GPP) and Routine Tests for the Detection of Enteropathogens
Circulating in Southern China, Diagn. Microbiol. Infect. Dis. 83 (2015) 325–330, https://
doi.org/10.1016/j.diagmicrobio.2015.07.024.
Comment: Please add this citation to line 80 as “…(QIAGEN, Germany), as described
previously [15].”
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?
Response: 1) The in-house multiplex RT-PCR showed high consistency with Luminex GPP on
norovirus GI, norovirus GII and adenovirus detection, in which Kappa value was 0.884, 0.918,
and 0.791 respectively (P =.000). These results were reported in Reference [14] Xin L, Nan Y,
Yonghui G, Jiankai D, Xixia D, Ruilian W, Ning F, Xiaoyan C. Compare a multiplex RT-PCR
method with liquichip technology in the detection of diarrhea-related virus, Chin J Lab Med.
38 (2015) 387–391. https://doi.org/10.3760/cma.j.issn.1009-9158.2015.06.007. 2) The in-house
multiplex RT-PCR designed for adenovirus 40/41. The same to Luminex GPP.
Comment: please report assay concordance and insert citation of reference 14 to Results. Also,
please add the word “enteric” before adenovirus in lines 60, 84 and 86 and please check
throughout the text.
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.
Response: Thanks for your comments and suggestion. The numbers shown on the branches of the
dendrogram were percentages and only those above 50% were shown. We have used neighbor-
joining, Maximum Likelihood and Bayesian method to construct the dendrogram again. The
genotypes obtained from the above methods are consistent. So we still use neighbor-joining
method in our manuscript.
Comment: If so, please change the wording of “the numbers on the branches indicate bootstrap
values” to “the number on the branches indicate percentages and only those above 50% were
shown” in the legend of Figure 3.
C. difficile is commonly found in children under two years of age; those were considered benign
colonization rather than infection.
Response: 2 cases of C. difficile were detected in HAstV and SaV positive samples, including a
28 years old male and a 32 years old female. We think C. difficile was related to AGE in these
two patients.
Comment: AGE in these patients are attributed to C.diff, really? More so than HAstV or SaV?
That’s highly unlikely.
Other comments:
1. Running title should read: Human astrovirus and sapovirus associated with acute
gastroenteritis in southern China
2. Please consider listing the astrovirus and sapovirus coinfections in a table. The text description
was confusing, and the numbers don’t add up for sapovirus co-infections (line 135-136 ).
3. Consider citing: Zhuo, Ran et al. “Molecular Epidemiology of Human Sapovirus among
Children with Acute Gastroenteritis in Western Canada.” Journal of clinical microbiology vol.
59,10 (2021): e0098621. doi:10.1128/JCM.00986-21
to discuss the prevalence of sapovirus and co-infection rates and Tarr, Gillian A M et al. “Clinical
Profiles of Childhood Astrovirus-, Sapovirus-, and Norovirus-Associated Acute Gastroenteritis
in Pediatric Emergency Departments in Alberta, 2014-2018.” The Journal of infectious diseases,
jiab429. 25 Aug. 2021, doi:10.1093/infdis/jiab429 to discuss the comparisons of age group
distribution and clinical features.
4. Please add the geographic locations of the other studies, i.e. ref [16 and 17] to line 188.
5. line 36-37, change the sentence to we described and compared clinical features of acute
gastroenteritis in patients with HAstV and SaV.
6. Please replace “undetected pathogens” with “unknown etiology” throughout the text and in
the header of table 1 because there are other noninfectious etiologies for gastroenteritis.
7. Line 56-57, change to “It is defined as the sudden onset of  # 3 episodes of diarrhea or vomiting
in the preceding 24 hours and symptom duration < 7 days.”
8. line 67, what kind of vaccine?
9. line 68, citation?
10. line 70, change “to” to “and”
11. the descriptions of patient enrollment process and criteria,  patient consent, and ethics
approval are missing in the first paragraph of Methods
12. Line 196-197, the statement that most HAstV infections occurred predominantly in infants
and children <5 years of age contradicts your result that HAstV was most frequently found in
the age group 15-59, line 193-195. Need to revise to clarify.
13. Please discuss the possibility that a positive detection does not always correspond to active
infection in the Discussion. PCR can pick up free nucleic acid or long-term intermittent and
asymptomatic shedding of astrovirus or sapovirus in patients.
14. Line 215-218 “Therefore, rapid detection through multiplex assays of these viral agents in
patients with AGE can help clinicians achieve efficient management of the disease and avoid
unnecessary use of antibiotics.” This sentence is out of context.
15. A limitation paragraph is missing. “Hence, further investigation of the viral load or
unrecognized agents is required.”  is not explained and out of context.
16. please see highlighted text where improvement in clarity is required.
Title: Detection and characterization of human astrovirus and sapovirus in outpatients with acute
gastroenteritis in Guangzhou, China
Version: 2 Date: 02 Oct 2021
Reviewer's report:
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.
Response: We appreciate your comments and suggestions. Through the study, we want to know
if there is a difference in clinical features between HAstV and other virus. We also want to know
whether the co-infection of HAstV with multiple enteropathogens is related to the severity of
the disease. The same to SaV. So Luminex GPP was used to detect the other virus and bacteria
in all patients, not just in HAstV and SaV positive samples. On the other hand, a detailed AGE
etiology assay, including virus and bacteria, has been reported in another manuscript.
Question: Maybe I misread it. Did you mean these data were published in another manuscript
already? I hope not.
Grammar and sentence structure needs to be improved throughout the manuscript. Response:
We carefully reviewed the manuscript and revised the sentences and paragraphs with the kind
assistance from a native English speaker and a scientific writing company.
Comment: Please edit the highlighted sentences in the revised manuscript for clarity.
Line 80, was the use of QIAamp MiniElute Virus Spin kit validated for bacteria and parasites
nucleic acid extraction in the Luminex GPP?
Response: QIAamp MiniElute Virus Spin kit, using to extract virus, bacteria and parasites
nucleic acid in the Luminex GPP, had been validated in our lab. The results were reported in
Reference [15] JianKai D, Xin L, Ruilian W, LingXiao J, Xixia D,
Wei H, Yongzheng P, Yu N, Xiaoyan. C. A Comparison of Luminex XTAG® Gastrointestinal
Pathogen Panel (XTAG GPP) and Routine Tests for the Detection of Enteropathogens
Circulating in Southern China, Diagn. Microbiol. Infect. Dis. 83 (2015) 325–330, https://
doi.org/10.1016/j.diagmicrobio.2015.07.024.
Comment: Please add this citation to line 80 as “…(QIAGEN, Germany), as described
previously [15].”
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?
Response: 1) The in-house multiplex RT-PCR showed high consistency with Luminex GPP on
norovirus GI, norovirus GII and adenovirus detection, in which Kappa value was 0.884, 0.918,
and 0.791 respectively (P =.000). These results were reported in Reference [14] Xin L, Nan Y,
Yonghui G, Jiankai D, Xixia D, Ruilian W, Ning F, Xiaoyan C. Compare a multiplex RT-PCR
method with liquichip technology in the detection of diarrhea-related virus, Chin J Lab Med.
38 (2015) 387–391. https://doi.org/10.3760/cma.j.issn.1009-9158.2015.06.007. 2) The in-house
multiplex RT-PCR designed for adenovirus 40/41. The same to Luminex GPP.
Comment: please report assay concordance and insert citation of reference 14 to Results. Also,
please add the word “enteric” before adenovirus in lines 60, 84 and 86 and please check
throughout the text.
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.
Response: Thanks for your comments and suggestion. The numbers shown on the branches of the
dendrogram were percentages and only those above 50% were shown. We have used neighbor-
joining, Maximum Likelihood and Bayesian method to construct the dendrogram again. The
genotypes obtained from the above methods are consistent. So we still use neighbor-joining
method in our manuscript.
Comment: If so, please change the wording of “the numbers on the branches indicate bootstrap
values” to “the number on the branches indicate percentages and only those above 50% were
shown” in the legend of Figure 3.
C. difficile is commonly found in children under two years of age; those were considered benign
colonization rather than infection.
Response: 2 cases of C. difficile were detected in HAstV and SaV positive samples, including a
28 years old male and a 32 years old female. We think C. difficile was related to AGE in these
two patients.
Comment: AGE in these patients are attributed to C.diff, really? More so than HAstV or SaV?
That’s highly unlikely.
Other comments:
1. Running title should read: Human astrovirus and sapovirus associated with acute
gastroenteritis in southern China
2. Please consider listing the astrovirus and sapovirus coinfections in a table. The text description
was confusing, and the numbers don’t add up for sapovirus co-infections (line 135-136 ).
3. Consider citing: Zhuo, Ran et al. “Molecular Epidemiology of Human Sapovirus among
Children with Acute Gastroenteritis in Western Canada.” Journal of clinical microbiology vol.
59,10 (2021): e0098621. doi:10.1128/JCM.00986-21
to discuss the prevalence of sapovirus and co-infection rates and Tarr, Gillian A M et al. “Clinical
Profiles of Childhood Astrovirus-, Sapovirus-, and Norovirus-Associated Acute Gastroenteritis
in Pediatric Emergency Departments in Alberta, 2014-2018.” The Journal of infectious diseases,
jiab429. 25 Aug. 2021, doi:10.1093/infdis/jiab429 to discuss the comparisons of age group
distribution and clinical features.
4. Please add the geographic locations of the other studies, i.e. ref [16 and 17] to line 188.
5. line 36-37, change the sentence to we described and compared clinical features of acute
gastroenteritis in patients with HAstV and SaV.
6. Please replace “undetected pathogens” with “unknown etiology” throughout the text and in
the header of table 1 because there are other noninfectious etiologies for gastroenteritis.
7. Line 56-57, change to “It is defined as the sudden onset of  # 3 episodes of diarrhea or vomiting
in the preceding 24 hours and symptom duration < 7 days.”
8. line 67, what kind of vaccine?
9. line 68, citation?
10. line 70, change “to” to “and”
11. the descriptions of patient enrollment process and criteria,  patient consent, and ethics
approval are missing in the first paragraph of Methods
12. Line 196-197, the statement that most HAstV infections occurred predominantly in infants
and children <5 years of age contradicts your result that HAstV was most frequently found in
the age group 15-59, line 193-195. Need to revise to clarify.
13. Please discuss the possibility that a positive detection does not always correspond to active
infection in the Discussion. PCR can pick up free nucleic acid or long-term intermittent and
asymptomatic shedding of astrovirus or sapovirus in patients.
14. Line 215-218 “Therefore, rapid detection through multiplex assays of these viral agents in
patients with AGE can help clinicians achieve efficient management of the disease and avoid
unnecessary use of antibiotics.” This sentence is out of context.
15. A limitation paragraph is missing. “Hence, further investigation of the viral load or
unrecognized agents is required.”  is not explained and out of context.
16. please see highlighted text where improvement in clarity is required.
Please review this paper.
user
Title: Detection and characterization of human astrovirus and sapovirus in outpatients with acute
gastroenteritis in Guangzhou, China
Version: 2 Date: 02 Oct 2021
Reviewer's report:
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.
Response: We appreciate your comments and suggestions. Through the study, we want to know
if there is a difference in clinical features between HAstV and other virus. We also want to know
whether the co-infection of HAstV with multiple enteropathogens is related to the severity of
the disease. The same to SaV. So Luminex GPP was used to detect the other virus and bacteria
in all patients, not just in HAstV and SaV positive samples. On the other hand, a detailed AGE
etiology assay, including virus and bacteria, has been reported in another manuscript.
Question: Maybe I misread it. Did you mean these data were published in another manuscript
already? I hope not.
Grammar and sentence structure needs to be improved throughout the manuscript. Response:
We carefully reviewed the manuscript and revised the sentences and paragraphs with the kind
assistance from a native English speaker and a scientific writing company.
Comment: Please edit the highlighted sentences in the revised manuscript for clarity.
Line 80, was the use of QIAamp MiniElute Virus Spin kit validated for bacteria and parasites
nucleic acid extraction in the Luminex GPP?
Response: QIAamp MiniElute Virus Spin kit, using to extract virus, bacteria and parasites
nucleic acid in the Luminex GPP, had been validated in our lab. The results were reported in
Reference [15] JianKai D, Xin L, Ruilian W, LingXiao J, Xixia D,
Wei H, Yongzheng P, Yu N, Xiaoyan. C. A Comparison of Luminex XTAG® Gastrointestinal
Pathogen Panel (XTAG GPP) and Routine Tests for the Detection of Enteropathogens
Circulating in Southern China, Diagn. Microbiol. Infect. Dis. 83 (2015) 325–330, https://
doi.org/10.1016/j.diagmicrobio.2015.07.024.
Comment: Please add this citation to line 80 as “…(QIAGEN, Germany), as described
previously [15].”
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?
Response: 1) The in-house multiplex RT-PCR showed high consistency with Luminex GPP on
norovirus GI, norovirus GII and adenovirus detection, in which Kappa value was 0.884, 0.918,
and 0.791 respectively (P =.000). These results were reported in Reference [14] Xin L, Nan Y,
Yonghui G, Jiankai D, Xixia D, Ruilian W, Ning F, Xiaoyan C. Compare a multiplex RT-PCR
method with liquichip technology in the detection of diarrhea-related virus, Chin J Lab Med.
38 (2015) 387–391. https://doi.org/10.3760/cma.j.issn.1009-9158.2015.06.007. 2) The in-house
multiplex RT-PCR designed for adenovirus 40/41. The same to Luminex GPP.
Comment: please report assay concordance and insert citation of reference 14 to Results. Also,
please add the word “enteric” before adenovirus in lines 60, 84 and 86 and please check
throughout the text.
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.
Response: Thanks for your comments and suggestion. The numbers shown on the branches of the
dendrogram were percentages and only those above 50% were shown. We have used neighbor-
joining, Maximum Likelihood and Bayesian method to construct the dendrogram again. The
genotypes obtained from the above methods are consistent. So we still use neighbor-joining
method in our manuscript.
Comment: If so, please change the wording of “the numbers on the branches indicate bootstrap
values” to “the number on the branches indicate percentages and only those above 50% were
shown” in the legend of Figure 3.
C. difficile is commonly found in children under two years of age; those were considered benign
colonization rather than infection.
Response: 2 cases of C. difficile were detected in HAstV and SaV positive samples, including a
28 years old male and a 32 years old female. We think C. difficile was related to AGE in these
two patients.
Comment: AGE in these patients are attributed to C.diff, really? More so than HAstV or SaV?
That’s highly unlikely.
Other comments:
1. Running title should read: Human astrovirus and sapovirus associated with acute
gastroenteritis in southern China
2. Please consider listing the astrovirus and sapovirus coinfections in a table. The text description
was confusing, and the numbers don’t add up for sapovirus co-infections (line 135-136 ).
3. Consider citing: Zhuo, Ran et al. “Molecular Epidemiology of Human Sapovirus among
Children with Acute Gastroenteritis in Western Canada.” Journal of clinical microbiology vol.
59,10 (2021): e0098621. doi:10.1128/JCM.00986-21
to discuss the prevalence of sapovirus and co-infection rates and Tarr, Gillian A M et al. “Clinical
Profiles of Childhood Astrovirus-, Sapovirus-, and Norovirus-Associated Acute Gastroenteritis
in Pediatric Emergency Departments in Alberta, 2014-2018.” The Journal of infectious diseases,
jiab429. 25 Aug. 2021, doi:10.1093/infdis/jiab429 to discuss the comparisons of age group
distribution and clinical features.
4. Please add the geographic locations of the other studies, i.e. ref [16 and 17] to line 188.
5. line 36-37, change the sentence to we described and compared clinical features of acute
gastroenteritis in patients with HAstV and SaV.
6. Please replace “undetected pathogens” with “unknown etiology” throughout the text and in
the header of table 1 because there are other noninfectious etiologies for gastroenteritis.
7. Line 56-57, change to “It is defined as the sudden onset of  # 3 episodes of diarrhea or vomiting
in the preceding 24 hours and symptom duration < 7 days.”
8. line 67, what kind of vaccine?
9. line 68, citation?
10. line 70, change “to” to “and”
11. the descriptions of patient enrollment process and criteria,  patient consent, and ethics
approval are missing in the first paragraph of Methods
12. Line 196-197, the statement that most HAstV infections occurred predominantly in infants
and children <5 years of age contradicts your result that HAstV was most frequently found in
the age group 15-59, line 193-195. Need to revise to clarify.
13. Please discuss the possibility that a positive detection does not always correspond to active
infection in the Discussion. PCR can pick up free nucleic acid or long-term intermittent and
asymptomatic shedding of astrovirus or sapovirus in patients.
14. Line 215-218 “Therefore, rapid detection through multiplex assays of these viral agents in
patients with AGE can help clinicians achieve efficient management of the disease and avoid
unnecessary use of antibiotics.” This sentence is out of context.
15. A limitation paragraph is missing. “Hence, further investigation of the viral load or
unrecognized agents is required.”  is not explained and out of context.
16. please see highlighted text where improvement in clarity is required.
assistant