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Detection and Comparison of Sapovirus, Rotavirus and Norovirus

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Detection and Comparison of Sapovirus, Rotavirus and Norovirus Abdulazeez et al (2020): Detection and comparison of viruses in acute gastroenteritis Oct 2020 Vol. 23 Issue 14 Detection and comparison of Sapovirus, rotavirus and norovirus among infants and young children with acute gastroenteritis Mariam Sami Abdulazeez1, Arwa Mujahid Al-Shuwaikh2*, Ismail Ibrahim Latif3 1B.Sc, Microbiology Department, College of Medicine, University of Diyala, Iraq 2Ph.D, Assistant Prof, Microbiology Department, College of Medicine, Al-Nahrain University, Iraq, Email: [email protected] 3MBChB, PhD, Microbiology Department, College of Medicine, University of Diyala, Iraq *Corresponding Author: Arwa Mujahid Al-Shuwaikh, Email: [email protected] Abstract Background: Acute gastroenteritis (AGE) remains a global public health problem. Rotavirus (RV), norovirus (NoV) and Sapovirus (SV) are recognized as an important causes of AGE among children worldwide. However, there is no information about prevalence of Sapovirus infection in Iraq. Objective: To assess the frequency of SV, RV and NoV in children with AGE, also to determine the risk factors associated with the disease. Methods: A cross section study was carried out for 93 children under the age of five years old with AGE who attended to the Emergency Department of Pediatrics in Al-Batool Teaching Hospital for Maternity and Children in Baqubah city, Iraq, using real-time Polymerase Chain Reaction (RT-PCR) to determine the presence of human Sapovirus nucleic acid and enzyme-linked immunosorbent assay (ELISA) to evaluate the presence of human rotavirus and human norovirus antigens. Results: The positive samples of SV were (6.5%). And the positive samples of RV and NoV were (79.6%) and (1.1%), respectively. Statistically, RV shows the higher positivity rate among viruses with significant difference (P<0.05). The present study showed that all SV and NoV cases were co- infected with RV. In addition, the present study showed that there was no statistically significant association between viruses positivity and the different sources of water, the level of mother education and clinical features (P>0.05). Conclusion: RV can be consider one of the most causes of diarrhea in children and more research are required to investigate the role of SV as a primary etiology of pediatric diarrhea. Keywords: Viral infection, gastroenteritis, children, Real Time PCR, ELISA, Iraq How to cite this article: Abdulazzez MS, Al-Shuwaikh AM, Latif II (2020): Detection and comparison of Sapovirus, rotavirus among infants and young children with acute gastroenteritis, Ann Trop Med & Public Health; 23(S14): SP231439. DOI: http://doi.org/10.36295/ASRO.2020.231439 List of abbreviation: SV, Sapovirus; RV, rotavirus; NoV, norovirus; AGE, acute gastroenteritis Introduction Diarrhea is the second greatest cause of mortality in children under 5 years of age worldwide (Anderson et al., 2010), with 10% deaths each year. Among the etiologic agents of diarrhea, viruses lead the way with 80% of cases. Among these viruses, rotaviruses (RV) are the most represented (Agbla et al., 2018). Rotaviruses were first identified in humans in 1973 when characteristic particles were observed in the cytoplasm of duodenal epithelial cells obtained from young children admitted to the hospital for treatment of acute diarrhea (Bishop, 1996). Rotaviruses are non-enveloped, icosahedra, double-stranded RNA viruses comprising a genus within the family Reoviridae (Dennehy, 2015; Crawford et al., 2017). In 2009, the World Health Organization (WHO) recommended that all countries introduce rotavirus vaccines into their national immunization programs (World Health Organization, 2009). Enteric Calciviruses in the genera norovirus (NoV) and Sapovirus (SV) are significant causes of gastroenteritis in humans and animals, with noroviruses alone causing approximately 200,000 deaths per annum in children <5 years of age (Patel et al., 2009; Ayukekbong, 2015; Alfajaro et al., 2017). However, in countries where universal rotavirus vaccination has been implemented, noroviruses have become most prevalent in children admitted to hospital with acute gastroenteritis (AGE) (Patel et al., 2009; Ayukekbong et al., 2015). NoV was first recorded in the American town of Norwalk, Ohio after outbreak at a primary school in the town among children and staff. The infection was just distinguished in 1972 after the assessment of feces tests from the incident. It was then named Norwalk infection (Lindesmith et al., 2003). The name norovirus was approved in 2002 (Mayo, 2002). Human noroviruses are positive-strand RNA, non- enveloped viruses (Li et al., 2018). Symptomatic norovirus infections are common within different populations; this renders clinical judgment on its contribution to diarrhoeal disease difficult (Ayukekbong et al., 2015). Sapovirus, formerly known as Sapporo-like viruses, is a significant leading cause of gastroenteritis. Sapovirus particles were first identified in human diarrheic stool samples in 1976 in the United Kingdom by electron microscopy (Yinda et al., 2017). The prototype of SV was first detected in an outbreak of gastroenteritis in an infant’s home in Sapporo, Japan in 1977 (Chiba et al., 1979). SV is a single-stranded positive sense RNA virus. Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.231439 Abdulazeez et al (2020): Detection and comparison of viruses in acute gastroenteritis Oct 2020 Vol. 23 Issue 14 The virions are composed of a single structural capsid protein, with icosahedra symmetry (Romani et al., 2012), the prevalence of Sapovirus in children <5 years of age ranges from 3.3 to 17% (Diez-Valcarce et al., 2018). SV peaks in the winter and is relatively common among infants and toddlers, with almost all children having antibody against Sapovirus by the age of 5 years, while norovirus tends to be more common in older children (Anderson, 2010). Although earlier reports described SV infection as one with less severe clinical symptoms than norovirus and rotavirus, more recent studies have shown that infections with SV can result in hospitalizations and severe dehydration (Diez-Valcarce et al., 2018). The aim of this study is to determine the prevalence of rotaviruses, norovirus and Sapovirus in AGE in Baqubah city, Iraq. Materials and Methods Specimen collection: This cross section study was carried out on children with AGE who admitted to Emergency Department of Pediatrics in Al-Batool Teaching Hospital for Maternity and Children in Baqubah city during the period from 31 October till 22 December, 2019. Stool samples were collected from 93 children with AGE; 57 were males and 36 were females; their ages were ranged from one month to 60 months. For molecular test, stool samples were mixed with 500μl Tris-EDTA buffer (Promega, USA), vortex intensely and centrifuged at 5000 rpm for one minute and then the supernatant was collected and stored frozen at (-80°C) until tested by Real Time PCR (RT-PCR).While for serological tests, samples were collected and divided into two aliquots and stored frozen at (-20) until tested by Enzyme Linked Immunosorbent Assay (ELISA). This study was done after taken the approval of the Ethical Committee at College of Medicine - University of Diyala. Molecular detection of SV by RT-PCR: RNA extraction: The RNA extraction of Sapovirus was done using Maxwell® 16 Viral Total Nucleic Acid Purification Kit (Promega, USA) according to the manufacturer instructions. Internal Control (IC) was used in the extraction procedure which serves as an amplification control for each individually processed specimen and to identify possible reaction inhibition. The extracted RNA was stored frozen at (−70°C) until analysis. Sapovirus RNA amplification: The extracted RNA was transcribed into cDNA by a reverse transcriptase, followed by PCR using direct, qualitative one-step real time PCR. Reactions mix was prepared according to the manufacturer's instructions using RIDA®GENE Sapovirus RT-PCR (R-Biopharm, Germany). RT-PCR was performed with the Mic Qpcr Cycler (Bio Molecular System, Australia) using primers and probe sequences that amplified a specific Sapovirus gene (ORF1).Positive and negative controls were included to verify the validation of the reaction instrument setting according to manufacture instruction for roter type instruments. The real-time thermal condition included reverse transcription step for 10 minutes at 58°C, followed by initial denaturation step for 1 minute at 95°C, then cycling step including 45 cycles of 95°C for 15 second and 60°C for 30 second with fluorescence data collected during the annealing/extension step. Fluorescence was detected in Green and Yellow detection channels for Sapovirus and IC, respectively on Qiagen Rotor-Gene Q. The results were interpreted by the software of Real Time PCR instrument according to manufacturer. Serological detection of RV and NoV by ELISA: Ninety three samples were tested for RV and NoV by using RIDASCREEN® Rotavirus Kit and RIDASCREEN® Norovirus 3rd generation Kit (R-Biopharm, Germany), respectively. Both kits employ monoclonal antibodies (mcAb) in a sandwich type method. In RV detection kit, amcAb to the product of the 6th viral gene (VP6) is coated to the well surface of the microwell plate. This is a group specific antigen that is found in all rotaviruses that cause disease in humans. While in NoV detection kit, the well surface of the microwell plate is coated with specific antibodies to the norovirus antigens of several different genotypes. The procedure was done following manufacturer's instructions. For sample preparation, stool samples were diluted (1:11) before adding into the microwell plate by adding 100μl (100 mg) of stool to 1 ml of sample diluents which supplied with the Kit. The stool suspension was homogenized by vortex mixer and then let the suspension stand for 10 minutes to allow coarse stool particles to settle and clarify the supernatant. After that, supernatant was centrifuged at 2500g for 5 minutes and the supernatant was used directly in the test. Blank, positive and negative control was included when the Kit was run. The optical densities (O.D.) of each well were measured at wave length 450 nm. The cut off value equals the absorbance of negative control plus 0.15.
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