Keywords
sapovirus
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sapovirus
Sapovirus is a single strand non enveloped RNA virus that belongs to the Caliciviridae family1–4. 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 VPg5,6. The viral genome consists of two to three open reading frames5. There are 15 genotypes of the virus with only four genotypes that 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 sequencing3. The sensitivity and specificity of RT-PCR are good and can be used for routine identification of the virus3. The primers used for the amplification of the virus depend upon the use of a segment from VP1 encoding gene compared to the RdRp region3,9. The classification of sapovirus depends upon complete sequence analysis of VP16,10,11.
Sapovirus infection is associated with viral gastroenteritis, especially in children below five with, around two million deaths around the world, and sapovirus causing viral gastroenteritis represents the second common cause of death12. The common symptoms are vomiting and diarrhoea5. The transmission of sapovirus occurs via ingestion of contaminated food and water and also by direct contact with affected individuals13,14. The infection occurs both sporadically and as an outbreak2,9. Treatment is symptomatic to prevent the aggravation of the disease3,9, and prevention of the infection depends mainly upon access to clean drinking water and food15.
There are data available about sapovirus infection in different countries such as Peru16 Iran17 and Ethiopia18. However, to our best of knowledge, there are no available reports about this in Egypt. Therefore, the present study aimed to evaluate the presence of sapovirus in children with acute gastroenteritis by RT-PCR.
The present study was a cross-sectional study that enrolled 100 children patients (sample size calculated by a statistician) with acute gastroenteritis from outpatient clinics from Mansoura Children’s Hospital, Mansoura, Egypt within the previous 48 hours with excluded bacterial pathogens and parasitic infestation by microbiological culture and microscopic examination to exclude parasites. The study was carried out from January 2019 till February 2020.
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.
Each stool sample was subjected to study by direct microscopic examination, study for rotavirus by ELISA Ridascreen® (R-Biopharm AG- An der Neuen Bergstraße 1764297 Darmstadt, Germany), and the remaining samples was subjected to RNA extraction and RT-PVR for sapovirus.
ELISA for rotavirus. The kit is ELISA for qualitative detection for rotavirus in the stool sample. The kit used monoclonal antibodies in a sandwich-type method. The monoclonal antibody is directed to the protein of the six viral genes (VP6), and it is used for the coating of the wells of the microplate. The VP6 is a group-specific antigen present in all rotaviruses genotypes. In brief, the stool suspension was prepared using ready to use stool diluent (protein-buffered NaCl solution). A total of 1 ml of the diluent was added to 100 microliter of the stool and homogenized the suspension by aspiration into and ejection from a disposable pipette or, alternatively, blending in a Vortex mixer. The suspension was allowed to stand for a short period of time (10 minutes) for the coarse stool particles to settle, and the clarified supernatant of the stool suspension was used directly in the test.
The control supplied with the kit were added to the wells of a microplate with biotinylated monoclonal anti-rotavirus antibodies and incubated at room temperature for one hour. The wells were washed with the supplied wash buffer after incubation and streptavidin poly-peroxidase conjugate was added before a second incubation was performed for a half-hour at room temperature. After a second wash, the substrate of the enzyme was added leading to a colour change of colourless to blue if the test is positive. The samples were then incubated at room temperature for 15 minutes. A stop reagent was added, changing the colour from blue to yellow. The colour intensity was proportional to the concentration of rotavirus present in the stool samples in comparison with a control, and was measured at 450 nm using a microplate ELISA reader (Statfax Chromate 4300).
Extraction of RNA and complementary DNA preparation. The stool samples were subjected to the extraction of RNA of sapovirus using QIAamp Viral RNA kit (Qiagen). The extraction was performed according to the instructions supplied by the manufacturer.
Complementary DNA (cDNA) was generated by adding 100 nanograms of the extracted RNA to 32.5 µl prepared mixture composed of 1 µl hexamers primers (Fermentas, Latvia), 4 µl of 5 × buffer, 0.5 µl of Ribolock RNase inhibitor, 2 µl of dNTP mixture composed of 10 mM each of dNTP, 200 units of reverse transcriptase enzyme and 19 µ1 of RNase free water (Fermentas, Latvia). The RT assay was performed at 42°C for one hour.
PCR for sapovirus. The amplification process was carried out using previously reported primers with nucleotide sequences of primers as follows: SLV5317 forward 5’-CGGRCYTCAAAVSTACCBCCCCA-3’; SLV5317 reverse 5’- CTCGCCACCTACRAWGCBTGGTT-3’19.
The amplification mixture used was ready to use Qiagen Taq PCR Master Mix Kit mixture (Cat No./ID: 201443) with 5 µl of cDNA added to a 20 µl PCR mix. 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 visualised under UV illumination after electrophoresis on a 1% agarose gel stained with ethidium bromide. The estimated amplified fragment size for sapovirus was 434 bp19.
The study included 100 children with acute gastroenteritis manifested by diarrhoea associated predominately with fever (56%), vomiting (47%), abdominal pain (42%). A minority had dehydration (11%). Their mean age± SD was 53.33± 11.71 months. Most cases presented in the spring season (34%) followed by winter (24%). Demographics of the children are shown in Table 1.
The most frequently detected virus was rotavirus by ELISA (25%). RT-PCR detected sapovirus in 7% of the stool samples.
The children with sapovirus were all from rural regions (Belkas, Dekrnes, Aga) and presented mainly during the winter season in Egypt (42.9%). The main presenting symptoms were fever (71.4%) and vomiting (57.1%). None of the children with sapovirus had dehydration. Rotavirus was significantly associated with sapovirus infections in 5 patients (71.4%, P=0.01). There was an insignificant difference between symptoms of gastroenteritis in children with sapovirus and children with gastroenteritis without sapovirus as regards vomiting (P=0.7), fever (P=0.46), abdominal pain (P=0.69) (Table 2).
Viral pathogens represent a significant aetiology for acute gastroenteritis. These infections are usually self-limited in developed countries while it may lead to mortality in underdeveloped countries, especially in children13.
The present study included 100 children with acute gastroenteritis manifested by diarrhoea with the majority experiencing fever (56%), vomiting (47%), and abdominal pain (42%). A minority had dehydration (11%). These are common symptoms of viral gastroenteritis. These patients usually present to outpatient clinics and do not require hospital admission except for dehydration20.
The proper management of children with acute gastroenteritis relies upon appropriate and robust diagnosis of the aetiology. In the present study, the most frequently detected virus was rotavirus by ELISA (25%). Previous meta-analysis study revealed that rotavirus is associated with acute gastroenteritis in 31.5% of children and 25.7% in the general population14. Previous studies in Africa reported that the prevalence of rotavirus infections ranged from 22.73% up to 30%21,22. A previous study from Egypt reported that the prevalence of rotavirus was 31% among children with acute gastroenteritis23.
The study of sapovirus as an emerging pathogen associated with acute gastroenteritis has gained importance in recent years. Research has been facilitated by the emergence of the molecular techniques in laboratory diagnosis16,24,25. In the present study, sapovirus was detected among 7% of children with acute gastroenteritis by RT-PCR. A previous meta-analysis study reported that the prevalence of sapovirus was 6.5% with a remarkable difference in the presence of sapovirus between low income and high-income countries26. Another study reported a lower prevalence of sapovirus 4.6% (10/219)27. Previous studies reported the prevalence of 3% up to 17% of sapovirus in acute gastroenteritis in children with gastroenteritis in high and low-income countries, respectively16,24,25. The variation of the prevalence rates reported may be due to the variation of the climate, environment, socio-economic factors, and cultural practices beside the difference of the used method of diagnoses.
The management of sapovirus depends mainly upon oral rehydration solution and zinc supplementation28. 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 is an argument 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 food29, increasing food and water sanitation 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 mandatory 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 infection30.
In the present study, there was an insignificant difference between the clinical presentation of sapovirus positive and sapovirus negative children. The clinical symptoms associated with acute gastroenteritis 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 the new virus such as sapovirus alongside other pathogens associated with gastroenteritis to improve the control measures30.
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 association with acute gastroenteritis for better management of such infection.
Figshare: Molecular study of sapovirus in acute gastroenteritis in children: a cross-sectional study, https://doi.org/10.6084/m9.figshare.13574933.v131
Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).
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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: Molecular epidemiology of calicivurs
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