| BackgroundDiarrheal disease is a group of diseases characterized by increased bowel movements and changes in stool characteristics caused by a variety of pathogens and risk factors.It is an important cause of malnutrition and one of the main causes of death of children in developing or poor countries.Statistics from China’s Disease Control and Prevention in 2014-2015 show that the incidence of infectious diarrhea ranges from 62.4per 100,000 to 74.8 per 100,000.More than half of the affected population are children under 5 years of age.The incidence and number of cases are the highest.Among them,the main cause of diarrhea in children under 5 years old is the infection of various pathogens,mainly viruses,followed by bacteria,parasites and fungi are relatively rare.Viruses include rotavirus,calicivirus(norovirus),star virus,adenovirus,etc.Human Astrovirus(AstV)is one of the important pathogens that cause viral diarrhea in infants,the elderly,and patients with immune insufficiency.The adaptive immunity acquired by its infection does not have a cross-protective effect among various genotypes..HAstV infection is mostly sporadic,but it can also cause an outbreak of infection in nurseries,nursing homes,shelters,hospitals,schools and other places,leading to epidemics.Astrovirus(AstV)is a non-enveloped single-stranded positive-stranded RNA virus with an icosahedral solid shell.It is named after a star shape under an electron microscope.The genome is 6.4-7.3kb in length.Contains 3 open reading flame(ORF).At present,HAstV is classified into classic HAstV according to the serotype of the ORF2region encoding the capsid protein sequence,including 8 genotypes(HAstV-1﹣HAstV-8),of which classic HAstV-1 is the most popular,and new HAstV,including The Melbourne strain of human astrovirus(HAstV-MLB)and the Virginia strain of human astrovirus(HAstV-VA).Research results in many regions of my country show that the detection rate of HAstV in children with acute diarrhea under 5 years old is 2.5%-6.7%.HAstV has obvious seasonality,mostly concentrated in the low temperature season.The main clinical features of acute viral gastroenteritis caused by infection in infants and young children are mild watery diarrhea,fever,loss of appetite,nausea,vomiting and abdominal pain,and less cause dehydration and hospitalization.However,children with stunted growth,weight loss,immune insufficiency,multiple infections,and other basic intestinal diseases are more severely ill.ObjectiveTo understand the molecular epidemiology of HAstV in children with acute diarrhea caused by astrovirus infection in the outpatient clinics of Guangzhou,China from January2018 to December 2019.Methods1.Collect 679 children in the acute diarrhea group and 391 children in the non-diarrhea group from January 2018 to December 2019 in the children’s hospital area of the Guangzhou Women and Children’s Medical Center,the maternity and infant hospital area,and the Zengcheng hospital area.Stool samples and related clinical information.2.Extract viral nucleic acid from collected stool samples,and use primers M269/M270 to perform RT-PCR amplification of the ORF2 region of HAstV in nucleic acid samples to obtain a fragment of 449 bp in size of the RNA extracted.The amplified product is subjected to electrophoresis verification.3.Re-amplification and product purification of the obtained positive samples,purification of specific products,ligation and transfection,cloning bacteria culture,plasmid extraction and identification,to obtain the nucleotide sequence.4.The positive nucleotide sequence was sent for sequencing,and the gene sequence of the reference virus strain used in the control was obtained from the Gen Bank database.Use Geneios10.1.3 software to calculate sequence similarity meter and perform correlation analysis.Use Mega10.0 software for sequence comparison and analysis of sequencing results and reference sequences,and use the Neighbor-joining method to construct and draw a phylogenetic tree map.Result1.Among the 679 cases in the outpatient acute diarrhea group,33 cases were positive for HAstV,and the detection rate was 4.9%.Among the 391 cases in the non-diarrhea group,9 cases were positive for HAstV,the detection rate was 2.3%,there was a statistical difference between the two(X~2=4.306,P=0.038).2.In the acute diarrhea group,there were 19 males with a positive HAstV test,with a positive rate of 5.1%(19/372),and 14 females with a positive rate of 4.6%(14/307).In the non-diarrhea group,the positive rate of males was 1.5%(6/391),and the positive rate of females was 0.8%(3/391),and there was no significant statistical difference.3.The detection rate of astrovirus under the age of 2 in the acute diarrhea group was5.0%(24/481),which was statistically different from that in the non-diarrhea group under the age of 1.2%(2/165)(X~2=4.538,P=0.033).The detection rate of acute diarrhea group over 2 years old was 4.5%(9/198)and 3.1%(7/226)of non-diarrhea group had no significant difference.In the acute diarrhea group,the mean age of HAstV positive months was 19 months,and the 95%confidence interval was(18,20).The mean value of non-diarrhea was 31 months,95%confidence interval(29,33),with significant statistical difference(t=10.608,P<0.0001).In the acute diarrhea group,the detection rates of HAstV in the 0-12 months old,12-24 months old,24-36 months old,36-48 months old,and 48-60 months old in the different month/year groups were 3.5%and 6.6,respectively.%,5.8%,3.6%,3.4%,4.9%,after 2 test,the detection rate of different month age groups has no statistically significant difference.4.In the acute diarrhea group,there was no significant difference in the seasonal detection rate during one year.The detection rate was the highest in spring(6.0%,17/281),and the lowest in summer and autumn(3.2%,5/154).The detection rate was between months.There was no significant statistical difference in the rate,the highest in January(6.7%,9/134),and the lowest in September(1.7%,1/58).5.Children in the acute diarrhea group with positive and negative HAstV tests showed clinical symptoms such as fever,vomiting,dehydration,abdominal pain,convulsions,myocardial damage,acidosis,anemia,and stool morphology,such as the number of red blood cells in the stool and the number of white blood cells in the stool,Watery stool,bloody stool,mucus-like stool,other types of stool,comorbidities such as lactose intolerance,respiratory tract infection,combined with other diseases,systemic lupus erythematosus,allergic purpura,milk protein allergy and other diseases are not significant afterX~2 tests Statistical difference.6.The results of systematic evolution analysis showed that among 33 children in the acute diarrhea group,26 strains(detection rate 3.8%,26/679)HAstV-1 and 5 HAstV-4strains(detection rate 0.7%,5/679)and 2 strains of HAstV-5(detection rate 0.3%,2/679)astrovirus.Among children with non-diarrhea,6 strains(detection rate 1.5%,6/391)HAstV-1,1 strain HAstV-4(detection rate 0.3%,1/391)and 2 strains of HAstV-5astrovirus(Detection rate 0.5%,2/391).Among them,the detection rate of HAstV-1 type in children with acute diarrhea and non-diarrhea is statistically different(X~2=4.503,P=0.0338).7.In the acute diarrhea group,the minimum age for the positive HAstV test was 3months old,the average was 20 months old,0-12 months old accounted for 27.3%(9/33),and 12-24 months old accounted for 45.5%(15/33).The proportion of positive tests was higher in spring,27.3%(9/33)in January,18.2%(6/33)in February,and 21.2%(7/33)in December in winter.8.The result of sequence homology analysis showed that the HAstV-1 type was tested positive.A total of 6 strains were isolates from Hebei,China(Genbank accession number KY311972.1)(2 strains from the non-diarrhea group)and 26 strains from Hungary 2010 isolates(Genbank accession number HQ398856.2)(4 strains from the non-diarrhea group),HAstV-4 type is homologous to the Oxford strain isolated in England(Genbank accession number L38506)and Dresden isolated strain(Genbank accession number AY720891)Similar in sex.The HAstV-5 type is similar to the 2012isolate from Budapest,Hungary(Genbank accession number KF157967).Conclusion1、Astrovirus is one of the most important pathogens of diarrhea in children under 5years old in Guangzhou.HAstV-1 is the dominant strain,which mainly infects children under 2 years old.2、There was no gender difference in the incidence of astrovirus infection,which mainly occurred in cold season(October to February)and rainy season(July)in Guangzhou.3、Diarrhea caused by astrovirus in children is characterized by mild acute gastroenteritis,with mild clinical symptoms and few complications.4、HAstV-1 was mainly isolated from Hebei Province in 2015(Genbank accession number KY311972.1)and Hungary in 2010(Genbank accession number HQ398856.2). |
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