Molecular Detection of Sapovirus in Children Under Five Years with Acute Gastroenteritis in Mansoura, Egypt between January 2019 and February 2020

Background: Sapovirus has emerged as a viral cause of acute gastroenteritis. However, there is limited data on sapovirus in Egypt. . The present study aimed to evaluate the presence of sapovirus in children with acute gastroenteritis <5 years in Mansoura, Egypt from January 2019 to February 2020 by reverse transcriptase-polymerase chain reaction (RT-PCR). Methods: The cross-sectional study enrolled a 100 children <5 years who presented with acute gastroenteritis at an outpatient clinic in Mansoura, Egypt between January 2019 and February 2020. Clinical data, demographic data and a stool sample was collected from each child. Stools were screened by microscopy for parasites and culture methods for bacteria and excluded from the study if positive for either. Specimens were also screened for rotavirus by enzyme immune assays (EIA) and sapovirus by reverse transcription PCR. Results: The most frequently detected virus was rotavirus by ELISA 25% (25/100). RT-PCR detected sapovirus in 7% (7/100) of the stool samples. The children with sapovirus were all from rural regions and presented mainly during the winter season in Egypt 42.9% (3/7). The main presenting symptoms were fever 71.4% (5/7) and vomiting 57.1% (4/7). None of the children with sapovirus had dehydration. Rotavirus was significantly associated with sapovirus infections in five samples (5/7) , 71.4%, P=0.01. Conclusion: The present study highlights the emergence of sapovirus as a frequent pathogen associated with acute gastroenteritis in children. There is a need for a national survey program for the study of sapovirus among other pathogens associated with acute gastroenteritis for better management of such infection.


Introduction
Sapovirus is a single strand non enveloped RNA virus that belongs to the Caliciviridae family [1][2][3][4] . The length of its genome is 7.1 to 7.7 kb, and its polyadenylated 3' terminal is responsible for viral replication, while its 5' terminal is associated with viral translation through production of VPg 5,6 . The viral genome consists of two to three open reading frames 5 . There are 15 genogroups of the virus with only four of them can infect humans, namely GI, GII, GIV and GV) 2,5,7,8 .
The laboratory methods for detection of sapovirus include enzyme-linked immunosorbent assay, electron microscopy, reverse transcription-polymerase chain reaction (RT-PCR) and next-generation sequencing 3 . The sensitivity and specificity of RT-PCR are good and can be used for routine identification of the virus 3 . The sensitivity and specificity of most RT-PCR detection assays for sapovirus are above 90% 9,10 . The primers used for the amplification of the virus depend upon the use of a segment from capsid region 3,11 . The classification of sapovirus depends upon complete sequence analysis of VP1 6,12,13 .
Diarrhea represents the second common cause of death in children and is associated with about 525 000 deaths around the world 14 . Sapovirus gastroenteritis occurs via ingestion of contaminated food and water and also by direct contact with affected individuals [13][14][15][16] . The infection occurs both sporadically and as an outbreak 2,11 . Treatment is symptomatic to prevent the aggravation of the disease 3,11 , and prevention of the infection depends mainly upon access to clean drinking water and food and on good hygiene habits (WHO's five keys to food safety) and hand washing 17 .
There are data available about sapovirus infection in different countries such as Peru 18 Iran 19 and Ethiopia 20 . There is limited data on sapovirus in Egypt. Therefore, the present study aimed to evaluate the presence of sapovirus in children with acute gastroenteritis by RT-PCR.

Study design and participants
The cross-sectional study enrolled 100 children <5 years who presented with acute gastroenteritis at an outpatient clinic in Mansoura, Egypt between January 2019 and February 2020. Clinical data, demographic data and a stool sample was collected from each child. Stools were screened by microscopy for parasites and culture methods for bacteria and excluded from the study if positive for either. Specimens were also screened for rotavirus by enzyme immune assays (EIA) and sapovirus by reverse transcription PCR.
The procedures followed were approved by the Mansoura Faculty of Medicine, Egypt ethical committee on human experimentation (R.20.11.1053) and were carried out in accordance with the Helsinki Declaration of 1975, as revised in 1983. Written informed consent was obtained from the parents of the included children.
Each child was subjected to full medical history by asking the parents about the residence area and age of the child, which was then followed by clinical examination. A stool sample was collected from each child for laboratory examination.

Amendments from Version 3
Author Response to the reviewer Thanks Sir for your effort for the revision.
Discussion, paragraph 1: Please rather use low-income, middleincome or high-income as these have been defined by the World Bank rather than underdeveloped which is not specific or well defined. This correction was noted in the previous review and has not been full corrected.

Corrected
Discussion, paragraph 2: Please delete the word hospital. The previous review indicated that either outpatient or hospital should be retained and the other word deleted as it was unclear where the study was conducted.
Deleted Discussion, paragraph 3, line 3 states "A previous systematic review of sapovirus in African countries revealed that rotavirus..." Not sure why the authors are referring to a sapovirus paper for rotavirus prevalence and the reference used for the study is incorrect -should more likely be 4 or 24 and not 14. Please correct the sentence and the reference.

A reference was corrected
Any further responses from the reviewers can be found at the end of the article REVISED PCR for sapovirus. The amplification process was carried out using previously reported primers with nucleotide sequences of primers as follows: SLV5749 forward 5'-CGGRCYTCAAAVSTAC-CBCCCCA-3'; SLV5317 reverse 5'-CTCGCCACCTACRA-WGCBTGGTT-3' 9 .
The cDNA generated from the previous step was used as 2.5 µl and added to ready to use amplification mixture supplied from Qiagen with 0.4 µl of the used primers in total volume 25 µl. The amplification procedures were performed using the following conditions: denaturation at 94°C for 5 minutes, then 35 cycles composed of 94°C for 45 seconds-55°C for 45 seconds and 72°C for 1 minute, then final extension of 7 minutes at 72°C (MiniAmp Thermal Cycler, Applied Biosystem).
PCR products were visualized under UV illumination after electrophoresis on a 1% agarose gel stained with ethidium bromide. The estimated amplified fragment size for sapovirus was 434 bp 9 .

Statistical analysis
Data were analysed with SPSS 22 (SPSS Inc, Chicago, Illinois, USA). Quantitative values were calculated as numbers and percentages. The use of the chi-square test performed comparisons, and the P-value was considered significant if it was <0.05.

Results
The study included 100 children with AGE manifested by diarrhoea associated predominately with fever ( (Table 2).

Discussion
Viral pathogens represent a significant aetiology for acute gastroenteritis. These infections are usually self-limited in high income countries while it may lead to mortality in low income countries, especially in children 15,21 .
The present study including children <5 with diarrhea in an outpatient setting showed that the majority of cases experienced fever, vomiting and abdominal pain; common symptoms of viral gastroenteritis. These patients usually present to outpatient clinics and do not require hospital admission except in rare instance of dehydration. The findings support the results noted in previous study 22 .
The proper management of children with AGE relies upon appropriate and robust diagnosis of the aetiology. In the present study, the most frequently detected virus was rotavirus by ELISA (25% healthy control the prevalence of sapovirus was 7.0% versus 3.0% (P = .07) 29 . The variation of the prevalence rates reported may be due to the variation of the climate, environment, socioeconomic factors, and cultural practices beside the difference of the used method of diagnoses.
The treatment of sapovirus depends mainly upon oral rehydration solution and zinc supplementation 30 . The risk factors for sapovirus infection are not fully understood. The prevention of sapovirus infection depends mainly upon efficient hand hygiene practice, environmental disinfection, proper sewage disposal, and limited contact with ill individuals. There are conflicting data about the role of improvement of water sanitation in the prevention of sapovirus as it is a common pathogen in both high and low-income countries. However, as it is transmitted by contaminated water and food 31 , improving food safety and access to clean water and improved sanitation services will reduce the burden of the infection.
The use of new molecular technologies for sapovirus detection in different samples from patients, food and environment, is important to recognize the mode of sapovirus transmission. Infection at a young age may predispose to durable immunity. Therefore, the development of a vaccine toward this virus may reduce the burden of this infection 32 .
In the present study, there was no significant difference between the clinical presentation of sapovirus positive and sapovirus negative children. The clinical symptoms associated with AGE usually include diarrhoea, vomiting, and fever, making laboratory diagnosis essential for appropriate management. Therefore, there is a need for a national survey program to improve the monitoring of the circulation of enteric viruses including sapovirus alongside other pathogens associated with gastroenteritis to improve the control measures 32 .

Conclusions
The present study highlights the presence of sapovirus as a pathogen associated with AGE in children from Mansoura, Egypt during 2019 and 2020. There is a need for a national survey program for the study of sapovirus among other pathogens association with AGE for better management of such infection.

Nicola A. Page
Center for Enteric Diseases, National Institute for Communicable Diseases (NICD), Johannesburg, South Africa The revised paper has been improved in the current version and describes the detection of sapovirus in children under five presenting to an outpatient clinic for the treatment of diarrhoea between January 2019 and February 2020 in Mansoura, Egypt.
The following minor errors were noted: Discussion, paragraph 1: Please rather use low-income, middle-income or high-income as these have been defined by the World Bank rather than underdeveloped which is not specific or well defined. This correction was noted in the previous review and has not been full corrected.  Introduction, paragraph 2, line 6: The primers used in this study amplify the capsid and not the VP1-RdRp junction. Please modify this sentence to reflect this fact. ○ Introduction, paragraph 3, line 1: This is not an appropriate reference for this statement. Please insert an appropriate and original reference, try looking at the World Health Organization or similar global bodies that conduct these calculations. ○ Introduction, paragraph 3, line 8: Prevention of sapovirus will include access to clean water and uncontaminated food but also on good hygiene habits (WHO's five keys to food safety) and hand washing. Please update sentence to reflect this. ○ Methods, paragraph 5, line 5: This is unclear. Were the children admitted to the hospital or were they enrolled from the outpatient clinic? When was a stool specimen taken to exclude parasites and bacteria? Please also indicate if you mean children under five years of age or if you included slightly older children.
○ Methods, paragraph 5, line 4: "…Mansoura, Egypt. The study was conducted from January 2019 to February 2020. All children provided a stool specimen which was screened for parasites by direct microscopy and bacterial aetiology by culture. Children positive for an enteric parasite or bacteria were excluded from the study." ○ Methods, ELISA for rotavirus: This section is unnecessary. It is sufficient to say that the EIA was completed according to the manufacturer's instructions with EIA plates read at 450nm on a Statfax Chromate 4300 -Please provide manufacturers' details for the Statfax 4300.
○ Methods, Extraction of RNA and cDNA amplification: Please supply enough detail so that someone else could replicate the assay if they wanted to -in the current format no detail was provided for primer concentration, buffer or kit used, manufacturer of the RNase inhibitor or where the dNTPs or RT enzyme were manufactured, concentration used etc. Please rewrite this section.

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Methods, PCR for sapovirus: Primer name incorrect -SLV5749 forward, please correct. Please also provide the concentration that the primers were used at or the reference of the method used in the lab with enough information to be replicated by the person reading it.  Results -Please report prevalence as a percentage followed by the numbers used to calculate the percentage i.e. 10% (10/100). This needs to be corrected throughout the paper. In the current format this paper requires extensive reworking prior to indexing looking at the comments made above.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound?

Marta Diez Valcarce
Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA The article titled 'Molecular study of sapovirus in acute gastroenteritis in children: a cross-sectional study' by El Sayed Zaki et al. includes some very interesting data on the prevalence of sapovirus in Egypt.
Overall, the article is well presented but there are a few minor comments that I would like to make, and also some edits that I consider necessary for clarity and accuracy.
In the abstract section, results paragraph, I think it could be informative which months are considered winter season in Egypt, as this varies in different parts of the world.

○
In the introduction I would suggest to write the family Caliciviridae in italics. Also, and that is a major edit that need to be done, the sapovirus genus contains or can be divided into 15 genogroups, not genotypes. ○ I am not sure what the authors mean when they say that the primers used depend upon the use of a segment from VP1 encoding gene compared to the RdRp region. I am not aware of any comparison made between the capsid and the polymerase for classification of sapovirus. In some cases, both regions are sequenced to give a more accurate classification, but not compared. Maybe the authors could rewrite this sentences so they are clearer.