Clinical manifestation of norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study

Alpha Fardah Athiyyah; Katsumi Shigemura; Koichi Kitagawa; Nazara Agustina; Andy Darma; Reza Ranuh; Dadik Raharjo; Toshiro Shirakawa; Masato Fujisawa; Subijanto Marto Sudarmo

doi:10.12688/f1000research.21069.3

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Research Article

Revised

Clinical manifestation of norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study

[version 3; peer review: 2 approved]

Previously titled: Norovirus genogroup correlation with acute diarrhea severity in Indonesian pediatric patients aged 1-60 months: a cross-sectional study

Alpha Fardah Athiyyah^1,2, Katsumi Shigemura ^3-5, Koichi Kitagawa^4,6, [...] Nazara Agustina^1,2, Andy Darma^1,2, Reza Ranuh^1,2, Dadik Raharjo^2,7, Toshiro Shirakawa^2-4,6,8, Masato Fujisawa³, Subijanto Marto Sudarmo^1,2

Alpha Fardah Athiyyah^1,2, Katsumi Shigemura ^3-5, [...] Koichi Kitagawa^4,6, Nazara Agustina^1,2, Andy Darma^1,2, Reza Ranuh^1,2, Dadik Raharjo^2,7, Toshiro Shirakawa^2-4,6,8, Masato Fujisawa³, Subijanto Marto Sudarmo^1,2

PUBLISHED 09 Mar 2020

Author details Author details

¹ Indonesia-Japan Collaborative Research Center for Emerging and Re-emerging Infectious Diseases, Institute of Tropical Disease, Institute of Tropical Disease, Airlangga University, Mulyorejo Street, Surabaya, 60115, Indonesia
² Department of Child Health, Faculty of Medicine, Soetomo Hospital, Airlangga University, Moestopo Street 6-8, Surabaya, 60286, Indonesia
³ Department of Infection Control and Prevention, Kobe University Hospital, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan
⁴ Division of Infectious Diseases, Department of International Health, Kobe University Graduate School of Health Science, 7-10-2 Tomogaoka Suma-ku, Kobe, 654-0142, Japan
⁵ Department of Urology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan
⁶ Department of Advanced Medical Science, Kobe University Graduate School of Science, Technology and Innovation, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan
⁷ Institute of Tropical Disease, Institute of Tropical Disease, Airlangga University, Mulyorejo Street, Surabaya, 60115, Indonesia
⁸ Center for Infectious Diseases, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan

Alpha Fardah Athiyyah
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Writing – Original Draft Preparation

Katsumi Shigemura
Roles: Writing – Original Draft Preparation, Writing – Review & Editing

Koichi Kitagawa
Roles: Writing – Original Draft Preparation, Writing – Review & Editing

Nazara Agustina
Roles: Investigation

Andy Darma
Roles: Investigation

Reza Ranuh
Roles: Investigation

Dadik Raharjo
Roles: Investigation, Methodology

Toshiro Shirakawa
Roles: Funding Acquisition, Project Administration, Supervision

Masato Fujisawa
Roles: Supervision

Subijanto Marto Sudarmo
Roles: Project Administration, Resources, Supervision

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background: The objective of this study was to investigate the clinical manifestation of norovirus infection between norovirus genogroup and severity of acute diarrhea in pediatric patients at the Dr. Soetomo Hospital, Surabaya, Indonesia.
Methods: This cross-sectional study involved 31 participants aged 1-60 months admitted to the hospital with acute diarrhea from April 2012 to March 2013. Norovirus genogroups (GI and II) were identified from patient stool using reverse transcription polymerase chain reaction (RT-PCR). Severity was measured using the Ruuska and Vesikari scoring system.
Results: In total, 94 stool samples were obtained, of which 31 (19%) were norovirus positive. Norovirus GI was found in one sample with mild diarrhea. Norovirus GII was found in 30 samples (96.8%); one sample with mild diarrhea (3.3%), 20 samples with moderate diarrhea (66.7%), and nine samples with severe diarrhea (30%).
Conclusion: Norovirus GII was the most prevalent cause of acute diarrhea and 30% of the cases manifested as severe diarrhea.

Keywords

Diarrhea, Infection, Norovirus, Vesikari score

Corresponding author: Katsumi Shigemura

Competing interests: No competing interests were disclosed.

Grant information: The Japan Initiative supported this research for Global Research Network on Infectious Diseases (J-GRID) from Ministry of Education, Culture, Sports, Science & Technology in Japan, and Japan Agency for Medical Research and Development (AMED).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2020 Fardah Athiyyah A et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Fardah Athiyyah A, Shigemura K, Kitagawa K et al. Clinical manifestation of norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study [version 3; peer review: 2 approved]. F1000Research 2020, 8:2130 (https://doi.org/10.12688/f1000research.21069.3) First published: 20 Dec 2019, 8:2130 (https://doi.org/10.12688/f1000research.21069.1) Latest published: 09 Mar 2020, 8:2130 (https://doi.org/10.12688/f1000research.21069.3)

Revised Amendments from Version 2

We have revised the title because our number of patients is so small that we could not evaluate the correlation between G1 positive and severity of diarrheae.

See the authors' detailed response to the review by Mohamad S. Hakim
See the authors' detailed response to the review by Hirokazu Kimura

Introduction

Diarrhea is considered the second-leading cause of death in pediatric patients under the age of five with a worldwide annual mortality of 525,000 children. Diarrhea lasting for even a few days causes dehydration¹. Viruses from the genus Norovirus from the family Caliciviridae are the second-leading cause of acute diarrhea after rotavirus in all age groups of pediatric patients^2–4. Norovirus is responsible for 218,000 pediatric deaths (<15 years old) every year and for 1.1 million pediatric hospitalizations around the world². In Indonesia, previous studies mentioned incidence rate around 17–30% in children^5–8.

Early identification of norovirus strain and genotype is vital for predicting the development of the disease and selecting the most suitable treatment. Genogroup diversity can be checked using reverse transcriptase polymerase chain reaction (RT-PCR). Norovirus is grouped into 40 viral strains, which are further classified into five different genogroups. Among them, GI and GII possess the most diverse genetic components⁹. From the previous study, Norovirus GII.2 genotypes had been the most prevalent norovirus strain in Indonesia (71.4%) followed by norovirus GII.17 (14.3%), one case was GII.4 and one case was GII.1¹⁰.

Norovirus commonly causes mild and short-term diarrheal episodes¹⁰. Nonetheless, this virus can be fatal, particularly in pediatric, geriatric, and immunocompromised patients^11,12. Norovirus patients showed more severe diarrhea compared to those without norovirus infection in pediatric patients. The type of norovirus strain and genome is thought to be related to diarrhea severity¹².

This study aimed to examine the clinical manifestation of norovirus infection in pediatric patients aged 1–60 months in the Dr. Soetomo General Hospital, Surabaya, Indonesia.

Methods

Ethical statement

The study protocol was approved by the Ethical Research Commission of Dr. Soetomo General Hospital, Surabaya, and conducted in line with the 1964 Helsinki declaration and its later amendments or research code of ethic issued by the Ministry of Research, Technology and Higher education. Written informed consent regarding participation in this study, the right to resign, stool and data collection and confidentiality of patient data was obtained and signed from all individuals’ parents. Consent was requested from the patients’ parents because the patients were 1–60 months old.

Study population

This cross-sectional study was conducted between April 2012 and March 2013 of all children aged 1–60 months old with acute diarrhea (described as defecation more than three times per day with change of stool consistency to loose or watery) admitted to the pediatric ward. Patients with a gastrointestinal-anatomical disorder such as Hirschsprung disease, severe systemic disease including sepsis, central nervous system infection or bronchopneumonia, a malabsorption disorder such as cow’s milk allergy, or a compromised immune status were excluded from the study to avoid any bias. On the day of the patients’ admission to the pediatric ward, parents were asked to participate in this study, and they agreed by signing the informed consent form. Stool samples were collected within 24 hours of patient admission with a sterile pot; approximately 3g of stool sample was taken from the middle part of the stool and delivered in no longer than three hours to the laboratory institution. Using a total sampling method, all samples collected until the end of March 2013 were studied.

Patient assessment

All subjects underwent physical examination and the participant’s parents completed a questionnaire.

The patient assessment was carried out by the physicians. The patient’s parents completed questions in the questionnaire regarding characteristic patient data¹³. These data were: patient’s identity (age, gender, body weight, and body height); parent’s identity (maternal education); history of diarrhea, which were divided into diarrhea duration (≤4 days, 5 days, and ≥6 days) and diarrhea frequency within 24 hours (1–3 times/day, 4–5 times/day, and ≥6 times/day); vomiting history, divided into vomiting duration (no vomiting, 1 day, 2 days, and more than 3 days) and vomiting frequency within 24 hours (no vomiting, 1 time/day, 2–4 times/day, and more than 5 times/day); and history of breastfeeding (not received breastfeeding, breastfeeding <6 months, and breastfeeding ≥6 months). Nutritional status was classified to either normal or malnutrition (underweight, stunted, wasted, and overweight) according to the definition by WHO¹⁴.

All patients also underwent physical examination of axillary body temperature (°C), arterial pulse measured with a pulseoxymeter (times/minute), respiratory rate (times/minute) and inspection of the signs of dehydration based on WHO classification¹⁵ and all results were written down in the questionnaire form. The questionnaire was then reviewed by the researchers and entered into the research database.

Norovirus diagnosis

Stool samples were delivered to the laboratory institution and kept in a deep freezer at -80°C until they were thawed at room temperature prior to RT-PCR analysis. To prevent laboratory contamination, our laboratory staff wore complete apparatuses, such as mask, coat, and gloves, throughout the process. RNA extraction was conducted in Bio Safety Cabinet. Before conducting PCR, all containers were disinfected using alcohol. A 10% stool suspension was prepared for each sample prior to RNA extraction by mixing 100µl stool sample with 100µl phosphate buffered saline buffer (Sigma-Aldrich, St. Louis, USA) with a vortex mixer (QL System, MX-2500 Vortex Mixer, UK) for 15 seconds and then centrifuging at 13,000–15,000 rpm for 10 minutes (Microfuge 20, Beckman Coulter, Indiana Polis, USA). The supernatant (1µl) from the stool suspension was transferred into a clean test-tube and the Viral Nucleic Acid Extraction Kit II (Cat # VR100, Geneaid Biotech Ltd., New Taipei, Taiwan) was used to extract viral RNA, carried out according to the kit manufacturer’s instructions. The eluted RNA from the samples was then stored in a deep freezer at -80°C until RT-PCR processing.

Reverse transcription was performed by mixing 75 picomoles of pdN6 random hexamers (Cat # 11034731001, Roche Molecular Biochemicals, Germany), 4U AMV Reverse Transcriptase (Cat # AMS.AMV007-1, AMS Biotechnology, Abingdon, UK) and 5μl of the eluted RNA and incubating at 42°C for 60 minutes. Approximately 10μl of the previous mixture was added to 5μl distilled water, 3μl Ex Taq DNA Polymerase (RR001B, Takara Bio Inc., Kusatsu, Japan) and 2μl of both forward and reverse primer. The primer pair used in this study for G1 were the G1SKF primer with nucleotide chain CTGCCCGAATTYGTAAATGA targeting nucleotide position 5342-5362 and a product size of 329 bp, and the G1SKR primer, with nucleotide chain CCAACCCARCCATTRTACA targeting nucleotide position 5652-5671 and a product size of 329 bp. The primer pair used in this study for G2 were the G2SKF primer, with nucleotide chain CNTGGGAGGGCGATCGCAA targeting nucleotide position 5058-5077 and a product size of 344 bp, and G2SKR, with nucleotide chain CCRCCNGCATRHCCRTTRTACAT targeting nucleotide position 5378-5401 and a product size of 344 bp.

PCR reaction was performed as follows. Initial denaturation was done at 94°C for 7 minutes, followed by 40 amplification cycles with Takara PCR Thermal Cycler Dice (TP600, Takara Bio Inc.). Each cycle consisted of denaturation at 94°C for 30 seconds, primer annealing at 50°C for 30 seconds for G1 or 57°C for 30 seconds for G2, extension reaction at 72°C for 45 seconds, followed by a final extension for 2 hours 24 minutes. The PCR product was then separated via gel electrophoresis in a 2% agarose gel and visualized under the UV light after ethidium bromide staining. The gel patterns were captured with Printgraph Fx Series (AE-6933FXN, Atto Corporation, Tokyo, Japan). The RT-PCR method used in this study was the one used by Rasanen et al. in Finland for identifying norovirus¹⁶, which can reveal the genotype variety via nonstructural proteins within the virus¹⁷. RT-PCR is considered to have the highest sensitivity for diagnosing norovirus infection compared to other methods¹⁸.

Vesikari Scoring System

The severity of diarrhea was measured using the Vesikari Scoring System (see Table 1). This severity scale was originally developed to evaluate the effectiveness of rotavirus vaccines based on 20 points¹⁹. The used parameters have been tested for reliability and validity in a cohort study conducted by Freedman with Cronbach’s α > 0.7.

Table 1. Vesikari Scoring System.

Parameter	Score
Parameter	0	1	2	3
Diarrhea
Maximal no. of diarrhea episodes per 24-hour period	0	1–3	4–5	≥6
Diarrhea duration¹⁷	0	1–4	5	≥6
Vomiting
Maximal no. of vomiting episodes per 24-hour period	0	1	2–4	≥5
Vomiting duration¹⁸	0	1	2	≥3
Temperature (°C)	<37.0	37.1–38.4	38.5–38.9	≥39
Dehydration	<5%	5–10%	>10%
Treatment	None	Oral rehydration solution	Hospitalization

Maximum score = 19; score <7 = mild severity; score 7–10 = moderate severity; score >10 = severe severity.

This table has been reproduced with reference to the study of Ruuska & Vesikari, 1990¹⁹

Diarrhea severity was assessed by evaluating seven clinical symptoms, including the duration of diarrhea, diarrhea frequency within 24 hours, vomiting duration, vomiting frequency within 24 hours, body temperature, dehydration status, and treatment. From those components, we could use the modified Vesikari score²⁰ to assess diarrhea severity. Mild diarrhea is equal to a score of < 7; a score of 7–10 is equivalent to moderate manifestation, and severe manifestation scores > 10.

Statistical analysis

Descriptive analysis was used to determine proportions from patients’ and parents’ identity data (age, gender, nutritional status and maternal education variables) and clinical patient data (diarrhea type, diarrhea duration, diarrhea frequency, vomiting duration and frequency, temperature, dehydration status and causative pathogen). The results of basic and clinical data are presented in tables and divided based on the PCR norovirus result (positive norovirus group and negative norovirus group).

Results

Participant characteristics

Samples were collected in the pediatric wards of the Dr. Soetomo General Hospital Surabaya. A total of 94 stool samples were acquired from eligible subjects within 11 months between April 2012 and March 2013. Of those samples, 31 (33.0%) were positive for norovirus infection using the RT-PCR method (Figure 1)¹³.

Figure 1. Results of norovirus genogroup analysis by polymerase chain reaction.

A. Negative control lines (NC), marker lines (M), DNA stepladder marks (100bp). Second lines (GI, 329bp); arrow shows 300 bp marker. B. Negative control lines (NC), Marker lines (M), DNA stepladder marks (100bp). Sixth, seventh, and eighth lines (GII, 343bp); arrow shows 300 bp marker.

The basic characteristics of all patients participated in this study are presented in Table 2. Most of the participants whose samples were positive for norovirus were male (54.8%), the youngest participant was one month old and the oldest was 24 months old. Twenty-two participants (71%) were between 6–23 months old. As for nutrition status, most of the subjects had adequate nutrition status (67.7%), while 10 subjects (32.3%) were considered malnourished. A total of 26 subjects (83.9%) were breastfed, with 19 subjects (61.3%) breastfed for more than six months and the rest (22.6%) were breastfed for under six months.

Table 2. Basic characteristics data.

Variable	Norovirus positive		Norovirus negative
Variable	n	%	n	%
Age (months)
1–5	9	29.0	10	15.9
6–23	22	71.0	47	74.6
>23	0	0.0	6	9.5
Gender
Male	17	54.8	41	65.1
Female	14	45.2	22	34.9
Nutrition status
Normal	21	67.7	14	22.2
Malnutrition	10	32.3	49	77.8
Breastfeeding status
Never	5	16.1	3	4.8
Breastfeeding ≤ six months	7	22.6	47	74.6
Breastfeeding ≥ six months	19	61.3	13	20.6
Maternal education
Low	5	16.1	7	11.1
Middle	22	71.0	47	74.6
High	4	12.9	9	14.3

Most patients in negative norovirus group were within 6–23 months old (74.6%) and were male (65.1%). Differences were found in the nutritional status of norovirus negative patients, in whom malnutrition was more prevalent (77.8%) than in norovirus positive patients. Breastfeeding for less than six months was also more common in the norovirus negative group (74.6%).

Clinical characteristic data are presented in Table 3. On average, the subjects were brought to the hospital after suffering diarrhea for two days with a frequency of diarrhea of five times within 24 hours. Other symptoms experienced by the subjects included vomiting (71% in positive norovirus group and 63% in negative norovirus group) with the most frequent duration of vomiting being one day (14.9% in positive norovirus group and 40.4% in negative norovirus group) and the most commonly observed frequency of vomiting being 2–4 times (9.6% in positive norovirus group and 21.3% in negative norovirus group) per day. The most frequent body temperature on admission to the hospital was below 37°C for positive norovirus group (48.4%) and sub-febrile (37.1–38.4°C) for the negative norovirus group (50.8%). Dehydration status in this study showed that two of the patients suffered from severe dehydration in both groups, while no dehydration was found only in negative norovirus group (3.2%). Watery stool diarrhea was the most frequent type of diarrhea in both positive norovirus group and negative norovirus group (80.6% and 50.4%, respectively). Bloody or mucoid stool was found only in patients of the negative norovirus group (both 9.5%).

Table 3. Clinical characteristic data.

Variable	Norovirus positive		Norovirus negative
Variable	n	%	n	%
Diarrhea type
Watery	25	80.6	34	54.0
Loose	6	19.4	17	27.0
Bloody	0	0.0	6	9.5
Mucoid	0	0.0	6	9.5
Diarrhea duration
1–4 days	23	24.5	52	55.3
5 days	3	3.2	7	7.4
≥6 days	5	5.3	4	4.3
Diarrhea frequency
1–3 times	13	13.8	18	19.1
4–5 times	7	7.4	25	26.6
≥6 times	11	11.7	20	21.3
Experiencing vomiting
Yes	22	71.0	38	60.3
No	9	29.0	25	39.7
Vomiting duration
No vomiting	9	9.6	26	26.6
1 days	14	14.9	38	40.4
2 days	2	2.1	0	0
≥3 days	6	6.4	0	0.0
Vomiting frequency
No vomiting	9	9.6	26	27.7
1 time	7	7.6	10	10.6
2–4 times	9	9.6	20	21.3
≥5 times	6	6.4	7	7.4
Temperature (°C)
<37.0	15	48.4	27	42.9
37.1–38.4	12	38.7	32	50.8
38.5–38.9	1	3.2	3	4.8
>39	3	9.7	1	1.6
Dehydration status
No dehydration	0	0.0	2	3.2
Some dehydration	29	93.5	59	93.7
Severe dehydration	2	6.5	2	3.2
Causative pathogen
Norovirus GI	1	3.2	0	0
Norovirus GII	30	96.8	0	0

Diarrhea severity

Based on norovirus genogroup identification from gel electrophoresis, GI was found in one sample and GII in 30 samples (96.8%). No products other than norovirus was found.

Distribution between norovirus genogroups and degree of diarrhea severity is presented in Table 4 and shows that norovirus GI was only responsible for one case of diarrhea with moderate severity. From 30 samples that tested positive for norovirus GII, GII was responsible for 20 cases (66.7%) of diarrhea with moderate severity, and nine cases (30%) of diarrhea with severe manifestation.

Table 4. Diarrheal severity distribution by norovirus genogroup.

		Norovirus genogroup		Total
		GI	GII	Total
Severity	Mild (Score <7)	0 (0%)	1 (3.3%)	1 (3.2%)
	Moderate (Score 7–10)	1 (100%)	20 (66.7%)	21 (67.7%)
	Severe (Score ≥11)	0 (0%)	9 (30%)	9 (29.1%)
Total		1	30	31

Discussion

Norovirus has been reported as be the main cause of acute diarrhea worldwide after rotavirus in all age groups of pediatric patients both in developed and developing countries^2,3. Norovirus strain type and genome mutation are thought to correlate with the severity of the diarrhea¹¹. It is important to clarify the pathogenesis of this disease to achieve better treatment for each case.

Norovirus was identified in this study in 31 out of 94 samples (33.0%), with norovirus GII in 30 samples (96.8%) and norovirus GI in one sample (3.2%). This agrees with a previous study mentioning norovirus infection was found in 30% of 102 children aged 0–15 months in Jakarta, Indonesia⁵. However, our study results showed higher norovirus infection incidence than previous studies mentioning incidence of about 17–21%^6–8.

Another study conducted in Rio de Janeiro in Brazil from 2005–2008 showed similar results; 1,087 stool samples obtained from 879 people below 20 years old and 208 people above 20 years old had norovirus in approximately 35% of the samples, and 96% of the norovirus-positive samples were GII positive²¹. A study of 165 participants in Shanghai, China, also showed high prevalence of norovirus GII infection (97.6%), with only 2.42% of those samples positive for norovirus GI²². These worldwide reports suggest that the most prevalent genogroup infecting humans is GII, with GI only seen in a minority of cases.

Norovirus infection, in our study, is most prevalent in 6–23 months population. Similar to other studies, this finding might be due to protection from maternal antibodies during breastfeeding for infant of < 6 months old. After 2 years of age, incidence of norovirus infection will decline due to acquired immunity^23–26.

The degree of diarrheal severity in subjects infected by norovirus GII was mostly moderate and only 30% were classified as severe. This agrees with a study carried out by Japanese group, Nakagomi et al., confirming that norovirus infection could elicit a similar degree of severity to rotavirus infection²⁷. Similarly, a study in Taiwan showed that norovirus caused mild diarrhea in 30.6%, moderate diarrhea in 43.9% and severe diarrhea in 25.5% of cases using the Vesikari Scoring System. Although previous study found that norovirus GII infection could lead to a more severe clinical manifestation diarrhea and vomiting compared to other genogroups, there are also wide range of severity level within the norovirus GII genogroup itself, such as that norovirus GII.4, GII.2, GII.3, GII.6, and GII.7 are associated with higher severity score²⁸. However, it is still a debate whether the genogroup itself or the viral loads that associate with clinical severity²⁹.

In our study, unfortunately, the degree of diarrheal severity in subjects infected by norovirus GI could not be compared to the degree of diarrheal severity in norovirus GII since norovirus GI was only detected in one sample, which is not enough for comparison.

Although this study achieved its aims, there were unavoidable study limitations. First, our sample size was comparatively small compared to previous norovirus studies in other countries. We did not include neonates below 1 month old due to our limitation to reach the neonatal ward. Secondly, we found no recurrent cases in our study, and therefore we did not analyze the relationship between norovirus genogroup classification and recurrence of diarrhea. Thirdly, since all the patients that participated in this study were all being admitted to the hospital, the treatment criteria are relatively more severe. Nevertheless, our findings largely agree with previous studies in Surabaya, Shanghai, and Rio de Janeiro, as explained in our discussion above^10,20,22. Fourth, we did not have data about other pathogens, which might be co-infecting. In addition, we could not classify the norovirus GII into genotype, and then use this genotype to infer the severity of the disease. Finally, this study only categorized norovirus genogroups by RT-PCR. We did not perform gene sequencing for norovirus RNA. We also did not include positive controls for each PCR reaction. Future studies will address these limitations.

Conclusion

This study demonstrated that norovirus was responsible for 33.0% of diarrhea cases in the study group, and norovirus GII was significantly dominant compared to norovirus GI. As many as 30% of norovirus cases had severe diarrheal manifestation, all of which were caused by norovirus GII.

Data availability

Underlying data

Harvard Dataverse: Norovirus PCR data set. https://doi.org/10.7910/DVN/KLBUOE¹³.

This project contains the following underlying data:

- Norovirus PCR data set detaverse.tab (sociodemographic information and clinical findings for norovirus positive patients)
- Master data – Noro negative (1).tab (sociodemographic information and clinical findings for both norovirus negative and positive patients)
- Original PCR gel images in JPEG format

Extended data

Harvard Dataverse: Norovirus PCR data set. https://doi.org/10.7910/DVN/KLBUOE¹³.

This project contains the following extended data:

- Norovirus Study Questionnaire (ENG).pdf (copy of questionnaire in English)
- Ind-Informed for Consent_norovirus.pdf (copy of questionnaire in Indonesian)

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

Faculty Opinions recommended

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21. Ferreira MS, Victoria M, Carvalho-Costa FA, et al.: Surveillance of norovirus infections in the state of Rio De Janeiro, Brazil 2005–2008. J Med Virol. 2010; 82(8): 1442–1448. PubMed Abstract | Publisher Full Text
22. Zeng M, Gong Z, Zhang Y, et al.: Prevalence and genetic diversity of norovirus in outpatient children with acute diarrhea in Shanghai, China. Jpn J Infect Dis. 2011; 64(5): 417–22. PubMed Abstract
23. El Qazoui M, Oumzil H, Baassi L, et al.: Rotavirus and norovirus infections among acute gastroenteritis children in Morocco. BMC Infect Dis. 2014; 14: 300. PubMed Abstract | Publisher Full Text | Free Full Text
24. Oluwatoyin Japhet M, Adeyemi Adesina O, Famurewa O, et al.: Molecular epidemiology of rotavirus and norovirus in Ile-Ife, Nigeria: high prevalence of G12P[8] rotavirus strains and detection of a rare norovirus genotype. J Med Virol. 2012; 84(9): 1489–1496. PubMed Abstract | Publisher Full Text
25. Trang NV, Luan LT, Kim-Anh LT, et al.: Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam. J Med Virol. 2012; 84(2): 290–297. PubMed Abstract | Publisher Full Text
26. Mikounou Louya V, Vouvoungui C, Koukouikila-Koussounda F, et al.: Molecular characterization of norovirus infection responsible for acute diarrhea in Congolese hospitalized children under five years old in Brazzaville, Republic of Congo. Int J Infect Dis. 2019; 88: 41–48. PubMed Abstract | Publisher Full Text
27. Nakagomi T, Correia JB, Nakagomi O, et al.: Norovirus infection among children with acute gastroenteritis in Recife, Brazil: disease severity is comparable to rotavirus gastroenteritis. Arch Virol. 2008; 153(5): 957–960. PubMed Abstract | Publisher Full Text
28. Mathew S, Alansari K, Smatti M, et al.: Epidemiological, Molecular, and Clinical Features of Norovirus Infections among Pediatric Patients in Qatar. Viruses. 2019; 11(5): pii: E400. PubMed Abstract | Publisher Full Text | Free Full Text
29. Chan MC, Leung TF, Chung TW, et al.: Virus Genotype Distribution and Virus Burden in Children and Adults Hospitalized for Norovirus Gastroenteritis, 2012-2014, Hong Kong. Sci Rep. 2015; 5: 11507. PubMed Abstract | Publisher Full Text | Free Full Text

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Version 3

VERSION 3 PUBLISHED 20 Dec 2019

Author details Author details

Alpha Fardah Athiyyah
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Writing – Original Draft Preparation

Katsumi Shigemura
Roles: Writing – Original Draft Preparation, Writing – Review & Editing

Koichi Kitagawa
Roles: Writing – Original Draft Preparation, Writing – Review & Editing

Nazara Agustina
Roles: Investigation

Andy Darma
Roles: Investigation

Reza Ranuh
Roles: Investigation

Dadik Raharjo
Roles: Investigation, Methodology

Toshiro Shirakawa
Roles: Funding Acquisition, Project Administration, Supervision

Masato Fujisawa
Roles: Supervision

Subijanto Marto Sudarmo
Roles: Project Administration, Resources, Supervision

Competing interests

No competing interests were disclosed.

Grant information

The Japan Initiative supported this research for Global Research Network on Infectious Diseases (J-GRID) from Ministry of Education, Culture, Sports, Science & Technology in Japan, and Japan Agency for Medical Research and Development (AMED).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (3)

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Published: 09 Mar 2020, 8:2130

https://doi.org/10.12688/f1000research.21069.3

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Published: 14 Feb 2020, 8:2130

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Published: 20 Dec 2019, 8:2130

https://doi.org/10.12688/f1000research.21069.1

© 2020 Fardah Athiyyah A et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Fardah Athiyyah A, Shigemura K, Kitagawa K et al. Clinical manifestation of norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study [version 3; peer review: 2 approved] F1000Research 2020, 8:2130 (https://doi.org/10.12688/f1000research.21069.3)

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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Version 3

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Reviewer Report 10 Mar 2020

Mohamad S. Hakim, Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Gadjah Mada University (UGM), Yogyakarta, Indonesia

Approved

https://doi.org/10.5256/f1000research.24984.r61038

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Reviewer Report 21 Feb 2020

Mohamad S. Hakim, Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Gadjah Mada University (UGM), Yogyakarta, Indonesia

Approved with Reservations

https://doi.org/10.5256/f1000research.24539.r60048

The authors already made improvements in this revised version of manuscript based on my previous comments. However, I still have some minor questions:

As I have stated earlier in my previous comments, the sample size is

The authors already made improvements in this revised version of manuscript based on my previous comments. However, I still have some minor questions:

As I have stated earlier in my previous comments, the sample size is too small to perform correlation analysis because only one GI positive manifested as moderate diarrhea. The authors should then not use term “correlation” in the objective (last paragraph of introduction section), as well as in the title. How can the authors make correlation analysis if they had only one GI positive samples? Thus, the term "correlation" is not supported by their data.
Therefore, I highly recommend to change to a more general title, such as “Clinical manifestation of norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study” or “Norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study”.
The authors mentioned, “Genogroup diversity can be checked using immunochromatography and reverse transcriptase polymerase chain reaction (RT-PCR)". Previously I was only concerned about electron microscopy, but I just realized that is it true that genogroup diversity can also be determined by immunochromatography? Please support your statement with solid references.
Please change “severer” to “more severe” (in introduction section). I have mentioned this previously, but the authors did not make any changes.
In the Discussion, the authors mentioned, “We did not perform gene sequencing for norovirus DNA.”
Comment: Norovirus is an RNA virus, not DNA virus. Please change.
In the Discussion, the authors mentioned, “One of the possible contributing factors is because we did not use positive controls for our PCR; therefore our result might have high false positive result.”
Comment: high rate of false positive result mostly due to unproper negative control, not positive control or due to high contaminations. In contrast, positive control is employed to reduce false negative results. The best way to confirm the PCR results is by performing sequencing of some or all positive samples. The authors should modify this issue in the next revision.

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Virology, infectious diseases, immunology.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Reviewer Report 17 Feb 2020

Hirokazu Kimura, Department of Health Science, Graduate School of Health Science, Gunma Paz University, Gunma, Japan

Approved

https://doi.org/10.5256/f1000research.24539.r60047

The authors addressed all my comments. Thus, I recommend ... Continue reading

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PUBLISHED 20 Dec 2019

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Reviewer Report 27 Jan 2020

Hirokazu Kimura, Department of Health Science, Graduate School of Health Science, Gunma Paz University, Gunma, Japan

Approved with Reservations

https://doi.org/10.5256/f1000research.23188.r57998

The authors studied the correlation between NoV genogroup and severity of acute diarrhea in the pediatric patients with gastroenteritis at the Hospital, Surabaya, Indonesia. NoV was detected in around 20% of the patients. In many cases, GII virus was detected, while GI was detected in one case. Moreover, 30% of the patients showed severe diarrhea. Overall, the manuscript was well written, while I had some minor comments.

The authors only examined by RT-PCR method. Did you detect nonspecific PCR products in the amplicons? Please add it in the results.
How did you prevent laboratory contamination? Please provide it in the revised manuscript.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Virology and Infectious Diseases.

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Author Response 14 Feb 2020

Katsumi Shigemura, Department of Urology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan

14 Feb 2020

Author Response

1.    The authors only examined by RT-PCR method. Did you detect non-specific PCR products in the amplicons? Please add it in the results.
Response: We suggest that non-specific PCR products refer to ... Continue reading 1.    The authors only examined by RT-PCR method. Did you detect non-specific PCR products in the amplicons? Please add it in the results.
Response: We suggest that non-specific PCR products refer to viruses other than norovirus. If that so, there were no products other than norovirus was found.

2.    How did you prevent laboratory contamination? Please provide it in the revised manuscript.
Response: To prevent laboratory contamination, our laboratory staff wore complete apparatuses, such as mask, coat, and gloves. RNA extraction was conducted in Bio Safety Cabinet. Before conducting PCR, all containers were disinfected using alcohol.
1.    The authors only examined by RT-PCR method. Did you detect non-specific PCR products in the amplicons? Please add it in the results.
Response: We suggest that non-specific PCR products refer to viruses other than norovirus. If that so, there were no products other than norovirus was found.

2.    How did you prevent laboratory contamination? Please provide it in the revised manuscript.
Response: To prevent laboratory contamination, our laboratory staff wore complete apparatuses, such as mask, coat, and gloves. RNA extraction was conducted in Bio Safety Cabinet. Before conducting PCR, all containers were disinfected using alcohol.
Competing Interests: We have no conflict of interest to declare. Close
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Author Response 14 Feb 2020

Katsumi Shigemura, Department of Urology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan

14 Feb 2020

Author Response

1.    The authors only examined by RT-PCR method. Did you detect non-specific PCR products in the amplicons? Please add it in the results.
Response: We suggest that non-specific PCR products refer to ... Continue reading 1.    The authors only examined by RT-PCR method. Did you detect non-specific PCR products in the amplicons? Please add it in the results.
Response: We suggest that non-specific PCR products refer to viruses other than norovirus. If that so, there were no products other than norovirus was found.

2.    How did you prevent laboratory contamination? Please provide it in the revised manuscript.
Response: To prevent laboratory contamination, our laboratory staff wore complete apparatuses, such as mask, coat, and gloves. RNA extraction was conducted in Bio Safety Cabinet. Before conducting PCR, all containers were disinfected using alcohol.
1.    The authors only examined by RT-PCR method. Did you detect non-specific PCR products in the amplicons? Please add it in the results.
Response: We suggest that non-specific PCR products refer to viruses other than norovirus. If that so, there were no products other than norovirus was found.

2.    How did you prevent laboratory contamination? Please provide it in the revised manuscript.
Response: To prevent laboratory contamination, our laboratory staff wore complete apparatuses, such as mask, coat, and gloves. RNA extraction was conducted in Bio Safety Cabinet. Before conducting PCR, all containers were disinfected using alcohol.
Competing Interests: We have no conflict of interest to declare. Close
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Reviewer Report 06 Jan 2020

Mohamad S. Hakim, Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Gadjah Mada University (UGM), Yogyakarta, Indonesia

Not Approved

https://doi.org/10.5256/f1000research.23188.r57997

Summary:
Athiyyah et al. reported the prevalence of norovirus infection during one year study of acute diarrhea patients <5 years old admitted to Dr. Soetomo Hospital, Surabaya, Indonesia. Reports of norovirus prevalence in children <5 years old with acute diarrhea in Indonesia are still limited. Therefore, this paper is highly important to expand the data of norovirus surveillance in Indonesia.

Comments:
However, this paper needs a major revision to improve the way of presenting and discussing the data as I recommend the following:

The sample size is too small to perform correlation analysis. Only one GI positively manifested as moderate diarrhea. The authors should then not use “correlation” in the objective (last paragraph of introduction section), as well as in the title, this does not make any sense. The author can change into a more general title, such as “Clinical manifestation of norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study” or “Norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study.”
The authors mention in the introduction: “Genogroup diversity can be checked using several methods including immunochromatography, reverse transcriptase polymerase chain reaction (RT-PCR), and electron microscopy.”
Comments: any supporting evidence or reference of this statement? Norovirus genogroup is based on genetic analysis, so somehow it is weird that it can be differentiated based on electron microscopy.
The authors did not perform comprehensive literature searching of the previous norovirus study in Indonesia. In fact, there are already some “key” publications in this field, but the authors did not include them in this manuscript.
Please thoroughly check the referenced papers to improve author statement in both introduction and discussion section¹^-⁵.
Please change “severer” to “more severe” (in introduction section).
In the methods: Are there any specific reason to exclude <1 month baby in this study?
Figure 2B, lane 1. Is it considered positive or negative? Because it seems a scanty, positive band there. It looks like Figure 2A, lane 2 which is considered as positive for norovirus GI.
Do the authors include positive controls for each PCR reaction? The prevalence of norovirus in this study (about 32%) is higher than that of global prevalence (about 20%) in countries that did not include Rotavirus vaccination in the NIP. So the authors should ensure that a proper and valid PCR assay has been conducted. Please check: Ahmed SM et al. (2014)⁶.
Figure 1 is not necessary, so please delete it.
The authors mention: “Of those samples, 31 were positive for norovirus infection using the RT-PCR method”. Please provide the percentage of norovirus-positive samples.
The authors mention in discussion: “This agrees with a previous study done in healthy subjects in Surabaya, Indonesia…" This is not a match comparison, because the previous study is in healthy, asymptomatic adult subjects. The authors should check the above papers that I recommended for much better comparison of previous studies in Indonesia.
In the discussion, the authors should also discuss associated risk factors of contracting norovirus based on Table 2. For example, why did in your population is the most prevalent age of norovirus positive 6-23 months?
The authors should add discussion about different severity of norovirus infection based on different genotpe of GII norovirus. Although they did not perform genotype identification in this study, the readers of this paper should still be aware that different GII genotypes can cause different severity of clinical manifestations.
In conclusion, it is mentioned: “This study demonstrated that norovirus was responsible for 48.4% of diarrhea cases in the study group”. Could the authors clarify about the percentage of 48.4%? 31 out of 94 should be 32.9%. It is also not consistent with the abstract (I mention below).
Reference no. 6, what journal that publishes this paper? The authors did not mention this. If this is not published in a peer-reviewed journal, it would be better to change the reference with one I recommended.
The abstract: “This cross-sectional study involved 31 participants” --> please delete “31”. When you design the study, you never know how many patients will participate.
“In total, 91 stool samples were obtained, of which 31 (19%) … “ --> please clarify the percentage. Also, is it 91 or 94? Please thoroughly check all the numbers and percentages you mentioned in this paper.
Table 2, change “Malutrition” to “Malnutrition”. Also, please describe in the methods what the criteria to categorize this nutrition status are.

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

No

References

1. Nirwati H, Donato CM, Mawarti Y, Mulyani NS, et al.: Norovirus and rotavirus infections in children less than five years of age hospitalized with acute gastroenteritis in Indonesia.Arch Virol. 2019; 164 (6): 1515-1525 PubMed Abstract | Publisher Full Text
2. Oyofo BA, Subekti D, Tjaniadi P, Machpud N, et al.: Enteropathogens associated with acute diarrhea in community and hospital patients in Jakarta, Indonesia.FEMS Immunol Med Microbiol. 2002; 34 (2): 139-46 PubMed Abstract | Publisher Full Text
3. Subekti D, Lesmana M, Tjaniadi P, Safari N, et al.: Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.FEMS Immunol Med Microbiol. 2002; 33 (1): 27-33 PubMed Abstract | Publisher Full Text
4. Sudarmo SM, Shigemura K, Athiyyah AF, Osawa K, et al.: Genotyping and clinical factors in pediatric diarrhea caused by rotaviruses: one-year surveillance in Surabaya, Indonesia.Gut Pathog. 2015; 7: 3 PubMed Abstract | Publisher Full Text
5. Hakim MS, Nirwati H, Aman AT, Soenarto Y, et al.: Significance of continuous rotavirus and norovirus surveillance in Indonesia.World J Pediatr. 14 (1): 4-12 PubMed Abstract | Publisher Full Text
6. Ahmed SM, Hall AJ, Robinson AE, Verhoef L, et al.: Global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis.Lancet Infect Dis. 2014; 14 (8): 725-730 PubMed Abstract | Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Virology, infectious diseases, immunology.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Author Response 14 Feb 2020

Katsumi Shigemura, Department of Urology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan

14 Feb 2020

Author Response

RESPONSES

We greatly appreciate your complimentary comments and suggestions.

1. We apologize that our sample size is too small, however, from this limited sample still we could explore many things.

2. We apologize ... Continue reading RESPONSES

We greatly appreciate your complimentary comments and suggestions.

1. We apologize that our sample size is too small, however, from this limited sample still we could explore many things.

2. We apologize that electron microscopy is not a method could be used for genotyping. We will revise the manuscript to: Genogroup diversity can be checked using immunochromatography and reverse transcriptase polymerase chain reaction (RT-PCR).

3. We will check the suggested reference and revise our manuscript accordingly.

4. We will revise the manuscript accordingly.

5. We exclude neonates below 1 month old because of our limitation to involve neonates admitted in the neonatal wing to follow the study.

6. Figure 2B lane 1 is considered positive.

7. It is our limitation that we do not include positive controls for each PCR reaction.

8. We will revise the manuscript accordingly.

9. We involve 94 subjects in this study and 31 subjects (32.9%) were norovirus positive.

10. We will check the suggested reference and revise our manuscript accordingly. Our study result showed higher norovirus infection incidence than previous studies mentioning incidence about 17-21% (Oyofo et al., 2002; Subekti et al., 2002; Nirwati et al., 2019). One of the possible contributing factors is because we do not use positive controls for our PCR.

11. We will revise our manuscript accordingly. Norovirus infection is most prevalent in 6-23 months due to protection from maternal antibodies during breastfeeding for infant < 6 months old. After 2 years of age, cases of norovirus will decline due to acquired immunity (Japhet et al., 2012; Trang et al., 2012; El Qazoui et al., 2014; Mikounou Louya et al., 2019).

12. Previous studies have shown different severity in the clinical manifestation of Norovirus GII. Genogroup of Norovirus GII.4, GII.2, GII.3, GII.6, and GII.7 are associated with higher severity score (Mathew et al., 2019). However, it is also difficult to determine whether genogroups or viral loads that associated with clinical severity (Chan et al., 2015)

13 We involve 94 subjects in this study and 31 subjects (32.9%) were norovirus positive. We will revise our manuscript.

14. We will check the suggested reference and revise our manuscript accordingly.

15. We will revise our manuscript accordingly.

16. We will revise our manuscript accordingly. Nutrition status is based on WHO curve for children aged 1-60 months.

We will check the suggested reference and revise our manuscript accordingly. Thank you very much for reviewing our manuscript.

Reference
Chan, M. C. W. et al. (2015) ‘Virus Genotype Distribution and Virus Burden in Children and Adults Hospitalized for Norovirus Gastroenteritis, 2012-2014, Hong Kong.’, Scientific reports, 5, p. 11507. doi: 10.1038/srep11507.
Japhet, M. O. et al. (2012) ‘Molecular epidemiology of rotavirus and norovirus in Ile-Ife, Nigeria: High prevalence of G12P[8] rotavirus strains and detection of a rare norovirus genotype’, Journal of Medical Virology, 84(9), pp. 1489–1496. doi: 10.1002/jmv.23343.
Mathew, S. et al. (2019) ‘Epidemiological, Molecular, and Clinical Features of Norovirus Infections among Pediatric Patients in Qatar’, Viruses, 11(5), p. 400. doi: 10.3390/v11050400.
Mikounou Louya, V. et al. (2019) ‘Molecular characterization of norovirus infection responsible for acute diarrhea in Congolese hospitalized children under five years old in Brazzaville, Republic of Congo’, International Journal of Infectious Diseases, 88, pp. 41–48. doi: 10.1016/j.ijid.2019.07.034.
Nirwati, H. et al. (2019) ‘Norovirus and rotavirus infections in children less than five years of age hospitalized with acute gastroenteritis in Indonesia’, Archives of Virology, 164(6), pp. 1515–1525. doi: 10.1007/s00705-019-04215-y.
Oyofo, B. A. et al. (2002) ‘Enteropathogens associated with acute diarrhea in community and hospital patients in Jakarta, Indonesia’, FEMS Immunology & Medical Microbiology, 34(2), pp. 139–146. doi: 10.1111/j.1574-695X.2002.tb00615.x.
El Qazoui, M. et al. (2014) ‘Rotavirus and Norovirus infections among acute gastroenteritis children in Morocco’, BMC Infectious Diseases, 14(1), p. 300. doi: 10.1186/1471-2334-14-300.
Subekti, D. et al. (2002) ‘Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.’, FEMS immunology and medical microbiology, 33(1), pp. 27–33. doi: 10.1111/j.1574-695X.2002.tb00568.x.
Trang, N. V. et al. (2012) ‘Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam’, Journal of Medical Virology, 84(2), pp. 290–297. doi: 10.1002/jmv.23185.
Chan, M. C. W. et al. (2015) ‘Virus Genotype Distribution and Virus Burden in Children and Adults Hospitalized for Norovirus Gastroenteritis, 2012-2014, Hong Kong.’, Scientific reports, 5, p. 11507. doi: 10.1038/srep11507.
Japhet, M. O. et al. (2012) ‘Molecular epidemiology of rotavirus and norovirus in Ile-Ife, Nigeria: High prevalence of G12P[8] rotavirus strains and detection of a rare norovirus genotype’, Journal of Medical Virology, 84(9), pp. 1489–1496. doi: 10.1002/jmv.23343.
Mathew, S. et al. (2019) ‘Epidemiological, Molecular, and Clinical Features of Norovirus Infections among Pediatric Patients in Qatar’, Viruses, 11(5), p. 400. doi: 10.3390/v11050400.
Mikounou Louya, V. et al. (2019) ‘Molecular characterization of norovirus infection responsible for acute diarrhea in Congolese hospitalized children under five years old in Brazzaville, Republic of Congo’, International Journal of Infectious Diseases, 88, pp. 41–48. doi: 10.1016/j.ijid.2019.07.034.
Nirwati, H. et al. (2019) ‘Norovirus and rotavirus infections in children less than five years of age hospitalized with acute gastroenteritis in Indonesia’, Archives of Virology, 164(6), pp. 1515–1525. doi: 10.1007/s00705-019-04215-y.
Oyofo, B. A. et al. (2002) ‘Enteropathogens associated with acute diarrhea in community and hospital patients in Jakarta, Indonesia’, FEMS Immunology & Medical Microbiology, 34(2), pp. 139–146. doi: 10.1111/j.1574-695X.2002.tb00615.x.
El Qazoui, M. et al. (2014) ‘Rotavirus and Norovirus infections among acute gastroenteritis children in Morocco’, BMC Infectious Diseases, 14(1), p. 300. doi: 10.1186/1471-2334-14-300.
Subekti, D. et al. (2002) ‘Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.’, FEMS immunology and medical microbiology, 33(1), pp. 27–33. doi: 10.1111/j.1574-695X.2002.tb00568.x.
Trang, N. V. et al. (2012) ‘Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam’, Journal of Medical Virology, 84(2), pp. 290–297. doi: 10.1002/jmv.23185.
RESPONSES

We greatly appreciate your complimentary comments and suggestions.

1. We apologize that our sample size is too small, however, from this limited sample still we could explore many things.

2. We apologize that electron microscopy is not a method could be used for genotyping. We will revise the manuscript to: Genogroup diversity can be checked using immunochromatography and reverse transcriptase polymerase chain reaction (RT-PCR).

3. We will check the suggested reference and revise our manuscript accordingly.

4. We will revise the manuscript accordingly.

5. We exclude neonates below 1 month old because of our limitation to involve neonates admitted in the neonatal wing to follow the study.

6. Figure 2B lane 1 is considered positive.

7. It is our limitation that we do not include positive controls for each PCR reaction.

8. We will revise the manuscript accordingly.

9. We involve 94 subjects in this study and 31 subjects (32.9%) were norovirus positive.

10. We will check the suggested reference and revise our manuscript accordingly. Our study result showed higher norovirus infection incidence than previous studies mentioning incidence about 17-21% (Oyofo et al., 2002; Subekti et al., 2002; Nirwati et al., 2019). One of the possible contributing factors is because we do not use positive controls for our PCR.

11. We will revise our manuscript accordingly. Norovirus infection is most prevalent in 6-23 months due to protection from maternal antibodies during breastfeeding for infant < 6 months old. After 2 years of age, cases of norovirus will decline due to acquired immunity (Japhet et al., 2012; Trang et al., 2012; El Qazoui et al., 2014; Mikounou Louya et al., 2019).

12. Previous studies have shown different severity in the clinical manifestation of Norovirus GII. Genogroup of Norovirus GII.4, GII.2, GII.3, GII.6, and GII.7 are associated with higher severity score (Mathew et al., 2019). However, it is also difficult to determine whether genogroups or viral loads that associated with clinical severity (Chan et al., 2015)

13 We involve 94 subjects in this study and 31 subjects (32.9%) were norovirus positive. We will revise our manuscript.

14. We will check the suggested reference and revise our manuscript accordingly.

15. We will revise our manuscript accordingly.

16. We will revise our manuscript accordingly. Nutrition status is based on WHO curve for children aged 1-60 months.

We will check the suggested reference and revise our manuscript accordingly. Thank you very much for reviewing our manuscript.

Reference
Chan, M. C. W. et al. (2015) ‘Virus Genotype Distribution and Virus Burden in Children and Adults Hospitalized for Norovirus Gastroenteritis, 2012-2014, Hong Kong.’, Scientific reports, 5, p. 11507. doi: 10.1038/srep11507.
Japhet, M. O. et al. (2012) ‘Molecular epidemiology of rotavirus and norovirus in Ile-Ife, Nigeria: High prevalence of G12P[8] rotavirus strains and detection of a rare norovirus genotype’, Journal of Medical Virology, 84(9), pp. 1489–1496. doi: 10.1002/jmv.23343.
Mathew, S. et al. (2019) ‘Epidemiological, Molecular, and Clinical Features of Norovirus Infections among Pediatric Patients in Qatar’, Viruses, 11(5), p. 400. doi: 10.3390/v11050400.
Mikounou Louya, V. et al. (2019) ‘Molecular characterization of norovirus infection responsible for acute diarrhea in Congolese hospitalized children under five years old in Brazzaville, Republic of Congo’, International Journal of Infectious Diseases, 88, pp. 41–48. doi: 10.1016/j.ijid.2019.07.034.
Nirwati, H. et al. (2019) ‘Norovirus and rotavirus infections in children less than five years of age hospitalized with acute gastroenteritis in Indonesia’, Archives of Virology, 164(6), pp. 1515–1525. doi: 10.1007/s00705-019-04215-y.
Oyofo, B. A. et al. (2002) ‘Enteropathogens associated with acute diarrhea in community and hospital patients in Jakarta, Indonesia’, FEMS Immunology & Medical Microbiology, 34(2), pp. 139–146. doi: 10.1111/j.1574-695X.2002.tb00615.x.
El Qazoui, M. et al. (2014) ‘Rotavirus and Norovirus infections among acute gastroenteritis children in Morocco’, BMC Infectious Diseases, 14(1), p. 300. doi: 10.1186/1471-2334-14-300.
Subekti, D. et al. (2002) ‘Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.’, FEMS immunology and medical microbiology, 33(1), pp. 27–33. doi: 10.1111/j.1574-695X.2002.tb00568.x.
Trang, N. V. et al. (2012) ‘Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam’, Journal of Medical Virology, 84(2), pp. 290–297. doi: 10.1002/jmv.23185.
Chan, M. C. W. et al. (2015) ‘Virus Genotype Distribution and Virus Burden in Children and Adults Hospitalized for Norovirus Gastroenteritis, 2012-2014, Hong Kong.’, Scientific reports, 5, p. 11507. doi: 10.1038/srep11507.
Japhet, M. O. et al. (2012) ‘Molecular epidemiology of rotavirus and norovirus in Ile-Ife, Nigeria: High prevalence of G12P[8] rotavirus strains and detection of a rare norovirus genotype’, Journal of Medical Virology, 84(9), pp. 1489–1496. doi: 10.1002/jmv.23343.
Mathew, S. et al. (2019) ‘Epidemiological, Molecular, and Clinical Features of Norovirus Infections among Pediatric Patients in Qatar’, Viruses, 11(5), p. 400. doi: 10.3390/v11050400.
Mikounou Louya, V. et al. (2019) ‘Molecular characterization of norovirus infection responsible for acute diarrhea in Congolese hospitalized children under five years old in Brazzaville, Republic of Congo’, International Journal of Infectious Diseases, 88, pp. 41–48. doi: 10.1016/j.ijid.2019.07.034.
Nirwati, H. et al. (2019) ‘Norovirus and rotavirus infections in children less than five years of age hospitalized with acute gastroenteritis in Indonesia’, Archives of Virology, 164(6), pp. 1515–1525. doi: 10.1007/s00705-019-04215-y.
Oyofo, B. A. et al. (2002) ‘Enteropathogens associated with acute diarrhea in community and hospital patients in Jakarta, Indonesia’, FEMS Immunology & Medical Microbiology, 34(2), pp. 139–146. doi: 10.1111/j.1574-695X.2002.tb00615.x.
El Qazoui, M. et al. (2014) ‘Rotavirus and Norovirus infections among acute gastroenteritis children in Morocco’, BMC Infectious Diseases, 14(1), p. 300. doi: 10.1186/1471-2334-14-300.
Subekti, D. et al. (2002) ‘Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.’, FEMS immunology and medical microbiology, 33(1), pp. 27–33. doi: 10.1111/j.1574-695X.2002.tb00568.x.
Trang, N. V. et al. (2012) ‘Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam’, Journal of Medical Virology, 84(2), pp. 290–297. doi: 10.1002/jmv.23185.
Competing Interests: We have no conflict of interest to declare. Close
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Author Response 14 Feb 2020

Katsumi Shigemura, Department of Urology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan

14 Feb 2020

Author Response

RESPONSES

We greatly appreciate your complimentary comments and suggestions.

1. We apologize that our sample size is too small, however, from this limited sample still we could explore many things.

2. We apologize ... Continue reading RESPONSES

We greatly appreciate your complimentary comments and suggestions.

1. We apologize that our sample size is too small, however, from this limited sample still we could explore many things.

2. We apologize that electron microscopy is not a method could be used for genotyping. We will revise the manuscript to: Genogroup diversity can be checked using immunochromatography and reverse transcriptase polymerase chain reaction (RT-PCR).

3. We will check the suggested reference and revise our manuscript accordingly.

4. We will revise the manuscript accordingly.

5. We exclude neonates below 1 month old because of our limitation to involve neonates admitted in the neonatal wing to follow the study.

6. Figure 2B lane 1 is considered positive.

7. It is our limitation that we do not include positive controls for each PCR reaction.

8. We will revise the manuscript accordingly.

9. We involve 94 subjects in this study and 31 subjects (32.9%) were norovirus positive.

10. We will check the suggested reference and revise our manuscript accordingly. Our study result showed higher norovirus infection incidence than previous studies mentioning incidence about 17-21% (Oyofo et al., 2002; Subekti et al., 2002; Nirwati et al., 2019). One of the possible contributing factors is because we do not use positive controls for our PCR.

11. We will revise our manuscript accordingly. Norovirus infection is most prevalent in 6-23 months due to protection from maternal antibodies during breastfeeding for infant < 6 months old. After 2 years of age, cases of norovirus will decline due to acquired immunity (Japhet et al., 2012; Trang et al., 2012; El Qazoui et al., 2014; Mikounou Louya et al., 2019).

12. Previous studies have shown different severity in the clinical manifestation of Norovirus GII. Genogroup of Norovirus GII.4, GII.2, GII.3, GII.6, and GII.7 are associated with higher severity score (Mathew et al., 2019). However, it is also difficult to determine whether genogroups or viral loads that associated with clinical severity (Chan et al., 2015)

13 We involve 94 subjects in this study and 31 subjects (32.9%) were norovirus positive. We will revise our manuscript.

14. We will check the suggested reference and revise our manuscript accordingly.

15. We will revise our manuscript accordingly.

16. We will revise our manuscript accordingly. Nutrition status is based on WHO curve for children aged 1-60 months.

We will check the suggested reference and revise our manuscript accordingly. Thank you very much for reviewing our manuscript.

Reference
Chan, M. C. W. et al. (2015) ‘Virus Genotype Distribution and Virus Burden in Children and Adults Hospitalized for Norovirus Gastroenteritis, 2012-2014, Hong Kong.’, Scientific reports, 5, p. 11507. doi: 10.1038/srep11507.
Japhet, M. O. et al. (2012) ‘Molecular epidemiology of rotavirus and norovirus in Ile-Ife, Nigeria: High prevalence of G12P[8] rotavirus strains and detection of a rare norovirus genotype’, Journal of Medical Virology, 84(9), pp. 1489–1496. doi: 10.1002/jmv.23343.
Mathew, S. et al. (2019) ‘Epidemiological, Molecular, and Clinical Features of Norovirus Infections among Pediatric Patients in Qatar’, Viruses, 11(5), p. 400. doi: 10.3390/v11050400.
Mikounou Louya, V. et al. (2019) ‘Molecular characterization of norovirus infection responsible for acute diarrhea in Congolese hospitalized children under five years old in Brazzaville, Republic of Congo’, International Journal of Infectious Diseases, 88, pp. 41–48. doi: 10.1016/j.ijid.2019.07.034.
Nirwati, H. et al. (2019) ‘Norovirus and rotavirus infections in children less than five years of age hospitalized with acute gastroenteritis in Indonesia’, Archives of Virology, 164(6), pp. 1515–1525. doi: 10.1007/s00705-019-04215-y.
Oyofo, B. A. et al. (2002) ‘Enteropathogens associated with acute diarrhea in community and hospital patients in Jakarta, Indonesia’, FEMS Immunology & Medical Microbiology, 34(2), pp. 139–146. doi: 10.1111/j.1574-695X.2002.tb00615.x.
El Qazoui, M. et al. (2014) ‘Rotavirus and Norovirus infections among acute gastroenteritis children in Morocco’, BMC Infectious Diseases, 14(1), p. 300. doi: 10.1186/1471-2334-14-300.
Subekti, D. et al. (2002) ‘Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.’, FEMS immunology and medical microbiology, 33(1), pp. 27–33. doi: 10.1111/j.1574-695X.2002.tb00568.x.
Trang, N. V. et al. (2012) ‘Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam’, Journal of Medical Virology, 84(2), pp. 290–297. doi: 10.1002/jmv.23185.
Chan, M. C. W. et al. (2015) ‘Virus Genotype Distribution and Virus Burden in Children and Adults Hospitalized for Norovirus Gastroenteritis, 2012-2014, Hong Kong.’, Scientific reports, 5, p. 11507. doi: 10.1038/srep11507.
Japhet, M. O. et al. (2012) ‘Molecular epidemiology of rotavirus and norovirus in Ile-Ife, Nigeria: High prevalence of G12P[8] rotavirus strains and detection of a rare norovirus genotype’, Journal of Medical Virology, 84(9), pp. 1489–1496. doi: 10.1002/jmv.23343.
Mathew, S. et al. (2019) ‘Epidemiological, Molecular, and Clinical Features of Norovirus Infections among Pediatric Patients in Qatar’, Viruses, 11(5), p. 400. doi: 10.3390/v11050400.
Mikounou Louya, V. et al. (2019) ‘Molecular characterization of norovirus infection responsible for acute diarrhea in Congolese hospitalized children under five years old in Brazzaville, Republic of Congo’, International Journal of Infectious Diseases, 88, pp. 41–48. doi: 10.1016/j.ijid.2019.07.034.
Nirwati, H. et al. (2019) ‘Norovirus and rotavirus infections in children less than five years of age hospitalized with acute gastroenteritis in Indonesia’, Archives of Virology, 164(6), pp. 1515–1525. doi: 10.1007/s00705-019-04215-y.
Oyofo, B. A. et al. (2002) ‘Enteropathogens associated with acute diarrhea in community and hospital patients in Jakarta, Indonesia’, FEMS Immunology & Medical Microbiology, 34(2), pp. 139–146. doi: 10.1111/j.1574-695X.2002.tb00615.x.
El Qazoui, M. et al. (2014) ‘Rotavirus and Norovirus infections among acute gastroenteritis children in Morocco’, BMC Infectious Diseases, 14(1), p. 300. doi: 10.1186/1471-2334-14-300.
Subekti, D. et al. (2002) ‘Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.’, FEMS immunology and medical microbiology, 33(1), pp. 27–33. doi: 10.1111/j.1574-695X.2002.tb00568.x.
Trang, N. V. et al. (2012) ‘Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam’, Journal of Medical Virology, 84(2), pp. 290–297. doi: 10.1002/jmv.23185.
RESPONSES

We greatly appreciate your complimentary comments and suggestions.

1. We apologize that our sample size is too small, however, from this limited sample still we could explore many things.

2. We apologize that electron microscopy is not a method could be used for genotyping. We will revise the manuscript to: Genogroup diversity can be checked using immunochromatography and reverse transcriptase polymerase chain reaction (RT-PCR).

3. We will check the suggested reference and revise our manuscript accordingly.

4. We will revise the manuscript accordingly.

5. We exclude neonates below 1 month old because of our limitation to involve neonates admitted in the neonatal wing to follow the study.

6. Figure 2B lane 1 is considered positive.

7. It is our limitation that we do not include positive controls for each PCR reaction.

8. We will revise the manuscript accordingly.

9. We involve 94 subjects in this study and 31 subjects (32.9%) were norovirus positive.

10. We will check the suggested reference and revise our manuscript accordingly. Our study result showed higher norovirus infection incidence than previous studies mentioning incidence about 17-21% (Oyofo et al., 2002; Subekti et al., 2002; Nirwati et al., 2019). One of the possible contributing factors is because we do not use positive controls for our PCR.

11. We will revise our manuscript accordingly. Norovirus infection is most prevalent in 6-23 months due to protection from maternal antibodies during breastfeeding for infant < 6 months old. After 2 years of age, cases of norovirus will decline due to acquired immunity (Japhet et al., 2012; Trang et al., 2012; El Qazoui et al., 2014; Mikounou Louya et al., 2019).

12. Previous studies have shown different severity in the clinical manifestation of Norovirus GII. Genogroup of Norovirus GII.4, GII.2, GII.3, GII.6, and GII.7 are associated with higher severity score (Mathew et al., 2019). However, it is also difficult to determine whether genogroups or viral loads that associated with clinical severity (Chan et al., 2015)

13 We involve 94 subjects in this study and 31 subjects (32.9%) were norovirus positive. We will revise our manuscript.

14. We will check the suggested reference and revise our manuscript accordingly.

15. We will revise our manuscript accordingly.

16. We will revise our manuscript accordingly. Nutrition status is based on WHO curve for children aged 1-60 months.

We will check the suggested reference and revise our manuscript accordingly. Thank you very much for reviewing our manuscript.

Reference
Chan, M. C. W. et al. (2015) ‘Virus Genotype Distribution and Virus Burden in Children and Adults Hospitalized for Norovirus Gastroenteritis, 2012-2014, Hong Kong.’, Scientific reports, 5, p. 11507. doi: 10.1038/srep11507.
Japhet, M. O. et al. (2012) ‘Molecular epidemiology of rotavirus and norovirus in Ile-Ife, Nigeria: High prevalence of G12P[8] rotavirus strains and detection of a rare norovirus genotype’, Journal of Medical Virology, 84(9), pp. 1489–1496. doi: 10.1002/jmv.23343.
Mathew, S. et al. (2019) ‘Epidemiological, Molecular, and Clinical Features of Norovirus Infections among Pediatric Patients in Qatar’, Viruses, 11(5), p. 400. doi: 10.3390/v11050400.
Mikounou Louya, V. et al. (2019) ‘Molecular characterization of norovirus infection responsible for acute diarrhea in Congolese hospitalized children under five years old in Brazzaville, Republic of Congo’, International Journal of Infectious Diseases, 88, pp. 41–48. doi: 10.1016/j.ijid.2019.07.034.
Nirwati, H. et al. (2019) ‘Norovirus and rotavirus infections in children less than five years of age hospitalized with acute gastroenteritis in Indonesia’, Archives of Virology, 164(6), pp. 1515–1525. doi: 10.1007/s00705-019-04215-y.
Oyofo, B. A. et al. (2002) ‘Enteropathogens associated with acute diarrhea in community and hospital patients in Jakarta, Indonesia’, FEMS Immunology & Medical Microbiology, 34(2), pp. 139–146. doi: 10.1111/j.1574-695X.2002.tb00615.x.
El Qazoui, M. et al. (2014) ‘Rotavirus and Norovirus infections among acute gastroenteritis children in Morocco’, BMC Infectious Diseases, 14(1), p. 300. doi: 10.1186/1471-2334-14-300.
Subekti, D. et al. (2002) ‘Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.’, FEMS immunology and medical microbiology, 33(1), pp. 27–33. doi: 10.1111/j.1574-695X.2002.tb00568.x.
Trang, N. V. et al. (2012) ‘Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam’, Journal of Medical Virology, 84(2), pp. 290–297. doi: 10.1002/jmv.23185.
Chan, M. C. W. et al. (2015) ‘Virus Genotype Distribution and Virus Burden in Children and Adults Hospitalized for Norovirus Gastroenteritis, 2012-2014, Hong Kong.’, Scientific reports, 5, p. 11507. doi: 10.1038/srep11507.
Japhet, M. O. et al. (2012) ‘Molecular epidemiology of rotavirus and norovirus in Ile-Ife, Nigeria: High prevalence of G12P[8] rotavirus strains and detection of a rare norovirus genotype’, Journal of Medical Virology, 84(9), pp. 1489–1496. doi: 10.1002/jmv.23343.
Mathew, S. et al. (2019) ‘Epidemiological, Molecular, and Clinical Features of Norovirus Infections among Pediatric Patients in Qatar’, Viruses, 11(5), p. 400. doi: 10.3390/v11050400.
Mikounou Louya, V. et al. (2019) ‘Molecular characterization of norovirus infection responsible for acute diarrhea in Congolese hospitalized children under five years old in Brazzaville, Republic of Congo’, International Journal of Infectious Diseases, 88, pp. 41–48. doi: 10.1016/j.ijid.2019.07.034.
Nirwati, H. et al. (2019) ‘Norovirus and rotavirus infections in children less than five years of age hospitalized with acute gastroenteritis in Indonesia’, Archives of Virology, 164(6), pp. 1515–1525. doi: 10.1007/s00705-019-04215-y.
Oyofo, B. A. et al. (2002) ‘Enteropathogens associated with acute diarrhea in community and hospital patients in Jakarta, Indonesia’, FEMS Immunology & Medical Microbiology, 34(2), pp. 139–146. doi: 10.1111/j.1574-695X.2002.tb00615.x.
El Qazoui, M. et al. (2014) ‘Rotavirus and Norovirus infections among acute gastroenteritis children in Morocco’, BMC Infectious Diseases, 14(1), p. 300. doi: 10.1186/1471-2334-14-300.
Subekti, D. et al. (2002) ‘Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.’, FEMS immunology and medical microbiology, 33(1), pp. 27–33. doi: 10.1111/j.1574-695X.2002.tb00568.x.
Trang, N. V. et al. (2012) ‘Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam’, Journal of Medical Virology, 84(2), pp. 290–297. doi: 10.1002/jmv.23185.
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Version 3

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Version 3 (revision) 09 Mar 20	read
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Mohamad S. Hakim, Gadjah Mada University (UGM), Yogyakarta, Indonesia
Hirokazu Kimura, Gunma Paz University, Gunma, Japan

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10 Mar 2020 | for Version 3

Mohamad S. Hakim, Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Gadjah Mada University (UGM), Yogyakarta, Indonesia

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Approved

The authors already addressed all my previous comments.

Competing Interests

No competing interests were disclosed.

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Virology, infectious diseases, immunology.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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21 Feb 2020 | for Version 2

Mohamad S. Hakim, Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Gadjah Mada University (UGM), Yogyakarta, Indonesia

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Approved With Reservations

The authors already made improvements in this revised version of manuscript based on my previous comments. However, I still have some minor questions:

As I have stated earlier in my previous comments, the sample size is too small to perform correlation analysis because only one GI positive manifested as moderate diarrhea. The authors should then not use term “correlation” in the objective (last paragraph of introduction section), as well as in the title. How can the authors make correlation analysis if they had only one GI positive samples? Thus, the term "correlation" is not supported by their data.
Therefore, I highly recommend to change to a more general title, such as “Clinical manifestation of norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study” or “Norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study”.
The authors mentioned, “Genogroup diversity can be checked using immunochromatography and reverse transcriptase polymerase chain reaction (RT-PCR)". Previously I was only concerned about electron microscopy, but I just realized that is it true that genogroup diversity can also be determined by immunochromatography? Please support your statement with solid references.
Please change “severer” to “more severe” (in introduction section). I have mentioned this previously, but the authors did not make any changes.
In the Discussion, the authors mentioned, “We did not perform gene sequencing for norovirus DNA.”
Comment: Norovirus is an RNA virus, not DNA virus. Please change.
In the Discussion, the authors mentioned, “One of the possible contributing factors is because we did not use positive controls for our PCR; therefore our result might have high false positive result.”
Comment: high rate of false positive result mostly due to unproper negative control, not positive control or due to high contaminations. In contrast, positive control is employed to reduce false negative results. The best way to confirm the PCR results is by performing sequencing of some or all positive samples. The authors should modify this issue in the next revision.

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17 Feb 2020 | for Version 2

Hirokazu Kimura, Department of Health Science, Graduate School of Health Science, Gunma Paz University, Gunma, Japan

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Approved

The authors addressed all my comments. Thus, I recommend that the revised manuscript is now suitable for indexing.

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27 Jan 2020 | for Version 1

Hirokazu Kimura, Department of Health Science, Graduate School of Health Science, Gunma Paz University, Gunma, Japan

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Approved With Reservations

The authors only examined by RT-PCR method. Did you detect nonspecific PCR products in the amplicons? Please add it in the results.
How did you prevent laboratory contamination? Please provide it in the revised manuscript.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Virology and Infectious Diseases.

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Author Response

14 Feb 2020

Katsumi Shigemura, Department of Urology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan

1. The authors only examined by RT-PCR method. Did you detect non-specific PCR products in the amplicons? Please add it in the results.
Response: We suggest that non-specific PCR products refer to viruses other than norovirus. If that so, there were no products other than norovirus was found.

2. How did you prevent laboratory contamination? Please provide it in the revised manuscript.
Response: To prevent laboratory contamination, our laboratory staff wore complete apparatuses, such as mask, coat, and gloves. RNA extraction was conducted in Bio Safety Cabinet. Before conducting PCR, all containers were disinfected using alcohol.

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06 Jan 2020 | for Version 1

Mohamad S. Hakim, Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Gadjah Mada University (UGM), Yogyakarta, Indonesia

28 Views Cite this report Responses(1)

Not Approved

The sample size is too small to perform correlation analysis. Only one GI positively manifested as moderate diarrhea. The authors should then not use “correlation” in the objective (last paragraph of introduction section), as well as in the title, this does not make any sense. The author can change into a more general title, such as “Clinical manifestation of norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study” or “Norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study.”
The authors mention in the introduction: “Genogroup diversity can be checked using several methods including immunochromatography, reverse transcriptase polymerase chain reaction (RT-PCR), and electron microscopy.”
Comments: any supporting evidence or reference of this statement? Norovirus genogroup is based on genetic analysis, so somehow it is weird that it can be differentiated based on electron microscopy.
The authors did not perform comprehensive literature searching of the previous norovirus study in Indonesia. In fact, there are already some “key” publications in this field, but the authors did not include them in this manuscript.
Please thoroughly check the referenced papers to improve author statement in both introduction and discussion section¹^-⁵.
Please change “severer” to “more severe” (in introduction section).
In the methods: Are there any specific reason to exclude <1 month baby in this study?
Figure 2B, lane 1. Is it considered positive or negative? Because it seems a scanty, positive band there. It looks like Figure 2A, lane 2 which is considered as positive for norovirus GI.
Do the authors include positive controls for each PCR reaction? The prevalence of norovirus in this study (about 32%) is higher than that of global prevalence (about 20%) in countries that did not include Rotavirus vaccination in the NIP. So the authors should ensure that a proper and valid PCR assay has been conducted. Please check: Ahmed SM et al. (2014)⁶.
Figure 1 is not necessary, so please delete it.
The authors mention: “Of those samples, 31 were positive for norovirus infection using the RT-PCR method”. Please provide the percentage of norovirus-positive samples.
The authors mention in discussion: “This agrees with a previous study done in healthy subjects in Surabaya, Indonesia…" This is not a match comparison, because the previous study is in healthy, asymptomatic adult subjects. The authors should check the above papers that I recommended for much better comparison of previous studies in Indonesia.
In the discussion, the authors should also discuss associated risk factors of contracting norovirus based on Table 2. For example, why did in your population is the most prevalent age of norovirus positive 6-23 months?
The authors should add discussion about different severity of norovirus infection based on different genotpe of GII norovirus. Although they did not perform genotype identification in this study, the readers of this paper should still be aware that different GII genotypes can cause different severity of clinical manifestations.
In conclusion, it is mentioned: “This study demonstrated that norovirus was responsible for 48.4% of diarrhea cases in the study group”. Could the authors clarify about the percentage of 48.4%? 31 out of 94 should be 32.9%. It is also not consistent with the abstract (I mention below).
Reference no. 6, what journal that publishes this paper? The authors did not mention this. If this is not published in a peer-reviewed journal, it would be better to change the reference with one I recommended.
The abstract: “This cross-sectional study involved 31 participants” --> please delete “31”. When you design the study, you never know how many patients will participate.
“In total, 91 stool samples were obtained, of which 31 (19%) … “ --> please clarify the percentage. Also, is it 91 or 94? Please thoroughly check all the numbers and percentages you mentioned in this paper.
Table 2, change “Malutrition” to “Malnutrition”. Also, please describe in the methods what the criteria to categorize this nutrition status are.

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

No

References

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Virology, infectious diseases, immunology.

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Author Response

14 Feb 2020

Katsumi Shigemura, Department of Urology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan

RESPONSES

We greatly appreciate your complimentary comments and suggestions.

1. We apologize that our sample size is too small, however, from this limited sample still we could explore many things.

2. We apologize that electron microscopy is not a method could be used for genotyping. We will revise the manuscript to: Genogroup diversity can be checked using immunochromatography and reverse transcriptase polymerase chain reaction (RT-PCR).

3. We will check the suggested reference and revise our manuscript accordingly.

4. We will revise the manuscript accordingly.

5. We exclude neonates below 1 month old because of our limitation to involve neonates admitted in the neonatal wing to follow the study.

6. Figure 2B lane 1 is considered positive.

7. It is our limitation that we do not include positive controls for each PCR reaction.

8. We will revise the manuscript accordingly.

9. We involve 94 subjects in this study and 31 subjects (32.9%) were norovirus positive.

10. We will check the suggested reference and revise our manuscript accordingly. Our study result showed higher norovirus infection incidence than previous studies mentioning incidence about 17-21% (Oyofo et al., 2002; Subekti et al., 2002; Nirwati et al., 2019). One of the possible contributing factors is because we do not use positive controls for our PCR.

11. We will revise our manuscript accordingly. Norovirus infection is most prevalent in 6-23 months due to protection from maternal antibodies during breastfeeding for infant < 6 months old. After 2 years of age, cases of norovirus will decline due to acquired immunity (Japhet et al., 2012; Trang et al., 2012; El Qazoui et al., 2014; Mikounou Louya et al., 2019).

12. Previous studies have shown different severity in the clinical manifestation of Norovirus GII. Genogroup of Norovirus GII.4, GII.2, GII.3, GII.6, and GII.7 are associated with higher severity score (Mathew et al., 2019). However, it is also difficult to determine whether genogroups or viral loads that associated with clinical severity (Chan et al., 2015)

13 We involve 94 subjects in this study and 31 subjects (32.9%) were norovirus positive. We will revise our manuscript.

14. We will check the suggested reference and revise our manuscript accordingly.

15. We will revise our manuscript accordingly.

16. We will revise our manuscript accordingly. Nutrition status is based on WHO curve for children aged 1-60 months.

We will check the suggested reference and revise our manuscript accordingly. Thank you very much for reviewing our manuscript.

Reference
Chan, M. C. W. et al. (2015) ‘Virus Genotype Distribution and Virus Burden in Children and Adults Hospitalized for Norovirus Gastroenteritis, 2012-2014, Hong Kong.’, Scientific reports, 5, p. 11507. doi: 10.1038/srep11507.
Japhet, M. O. et al. (2012) ‘Molecular epidemiology of rotavirus and norovirus in Ile-Ife, Nigeria: High prevalence of G12P[8] rotavirus strains and detection of a rare norovirus genotype’, Journal of Medical Virology, 84(9), pp. 1489–1496. doi: 10.1002/jmv.23343.
Mathew, S. et al. (2019) ‘Epidemiological, Molecular, and Clinical Features of Norovirus Infections among Pediatric Patients in Qatar’, Viruses, 11(5), p. 400. doi: 10.3390/v11050400.
Mikounou Louya, V. et al. (2019) ‘Molecular characterization of norovirus infection responsible for acute diarrhea in Congolese hospitalized children under five years old in Brazzaville, Republic of Congo’, International Journal of Infectious Diseases, 88, pp. 41–48. doi: 10.1016/j.ijid.2019.07.034.
Nirwati, H. et al. (2019) ‘Norovirus and rotavirus infections in children less than five years of age hospitalized with acute gastroenteritis in Indonesia’, Archives of Virology, 164(6), pp. 1515–1525. doi: 10.1007/s00705-019-04215-y.
Oyofo, B. A. et al. (2002) ‘Enteropathogens associated with acute diarrhea in community and hospital patients in Jakarta, Indonesia’, FEMS Immunology & Medical Microbiology, 34(2), pp. 139–146. doi: 10.1111/j.1574-695X.2002.tb00615.x.
El Qazoui, M. et al. (2014) ‘Rotavirus and Norovirus infections among acute gastroenteritis children in Morocco’, BMC Infectious Diseases, 14(1), p. 300. doi: 10.1186/1471-2334-14-300.
Subekti, D. et al. (2002) ‘Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.’, FEMS immunology and medical microbiology, 33(1), pp. 27–33. doi: 10.1111/j.1574-695X.2002.tb00568.x.
Trang, N. V. et al. (2012) ‘Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam’, Journal of Medical Virology, 84(2), pp. 290–297. doi: 10.1002/jmv.23185.
Chan, M. C. W. et al. (2015) ‘Virus Genotype Distribution and Virus Burden in Children and Adults Hospitalized for Norovirus Gastroenteritis, 2012-2014, Hong Kong.’, Scientific reports, 5, p. 11507. doi: 10.1038/srep11507.
Japhet, M. O. et al. (2012) ‘Molecular epidemiology of rotavirus and norovirus in Ile-Ife, Nigeria: High prevalence of G12P[8] rotavirus strains and detection of a rare norovirus genotype’, Journal of Medical Virology, 84(9), pp. 1489–1496. doi: 10.1002/jmv.23343.
Mathew, S. et al. (2019) ‘Epidemiological, Molecular, and Clinical Features of Norovirus Infections among Pediatric Patients in Qatar’, Viruses, 11(5), p. 400. doi: 10.3390/v11050400.
Mikounou Louya, V. et al. (2019) ‘Molecular characterization of norovirus infection responsible for acute diarrhea in Congolese hospitalized children under five years old in Brazzaville, Republic of Congo’, International Journal of Infectious Diseases, 88, pp. 41–48. doi: 10.1016/j.ijid.2019.07.034.
Nirwati, H. et al. (2019) ‘Norovirus and rotavirus infections in children less than five years of age hospitalized with acute gastroenteritis in Indonesia’, Archives of Virology, 164(6), pp. 1515–1525. doi: 10.1007/s00705-019-04215-y.
Oyofo, B. A. et al. (2002) ‘Enteropathogens associated with acute diarrhea in community and hospital patients in Jakarta, Indonesia’, FEMS Immunology & Medical Microbiology, 34(2), pp. 139–146. doi: 10.1111/j.1574-695X.2002.tb00615.x.
El Qazoui, M. et al. (2014) ‘Rotavirus and Norovirus infections among acute gastroenteritis children in Morocco’, BMC Infectious Diseases, 14(1), p. 300. doi: 10.1186/1471-2334-14-300.
Subekti, D. et al. (2002) ‘Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.’, FEMS immunology and medical microbiology, 33(1), pp. 27–33. doi: 10.1111/j.1574-695X.2002.tb00568.x.
Trang, N. V. et al. (2012) ‘Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam’, Journal of Medical Virology, 84(2), pp. 290–297. doi: 10.1002/jmv.23185.

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Competing Interests

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Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

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Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Clinical manifestation of norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study

Abstract

Keywords

Revised Amendments from Version 2

Introduction

Methods

Ethical statement

Study population

Patient assessment

Norovirus diagnosis

Vesikari Scoring System

Table 1. Vesikari Scoring System.

Statistical analysis

Results

Participant characteristics

Figure 1. Results of norovirus genogroup analysis by polymerase chain reaction.

Table 2. Basic characteristics data.

Table 3. Clinical characteristic data.

Diarrhea severity

Table 4. Diarrheal severity distribution by norovirus genogroup.

Discussion

Conclusion

Data availability

Underlying data

Extended data

References

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