Pathogen Spectrum Analysis With Infectious Diarrhea In Children And Identification Of Sapovirus In Guangzhou,2021-2022

ObjectiveTo describe the characteristics of pathogen spectrum of infectious diarrhea in children in Guangzhou from 2021 to 2022.To establish the method for isolation and identification of Sapovirus(SaV).To explore the evolution and variation of SaV by molecular typing,and provide reference for the establishment of SaV strain’s bank and the improvement of primers and probes for SaV detection.MethodsWe collected stool samples from children with diarrhea in maternal and child health hospital in Guangdong as the sentinel surveillance hospital from December 2021 to December 2022.The diarrhea multi-pathogen detection method was used to detect pathogens in the samples.Meanwhile,the infection characteristics of pathogen spectrum in children with diarrhea combined with basic demographic information were analyzed.At the same time,we attempted to isolate SaV strains from human duodenum carcinoma derived cell line(HuTu-80).Genotyping and mutation sites of SaV positive samples or isolates were analyzed based on Nanopore sequencing platform.The phylogenetic tree was constructed by IQ Tree.ResultsA total of 214 children with diarrhea were included in this study,and 172 cases were detected with diarrhea pathogens.The overall positive rate was 80.37%.The difference between the detection rate of bacteria and virus was statistically significant(χ2=56.939,P<0.001),and the former(70.10%)was higher than the latter(33.64%).The top 4 bacterial species were Salmonella(35.05%),Enteropathogenic Escherichia coli(23.83%),Clostridium difficile toxin A/B(22.43%)and Campylobacter(14.95%).The virus detection rates from high to low were Adenovirus(17.29%),Rotavirus A(13.08%),Norovirus(5.61%),SaV(3.74%)and Astrovirus(1.87%).Co-infection was the main pathogen infection(45.79%,98/214),while single infection was 34.58%(74/214).A total of 20 SaV positive samples(8 sentinel surveillance samples and 12 outbreak samples)were collected in this study.The results showed that:① HuTu-80 was a sensitive cell line for SaV isolation.Before isolation,attention should be paid to the cell coverage degree,with 50%-60%being appropriate.②The virus isolation time was prolonged to 14dpi by fluid exchange.For the separated data using 1%GlyCA,from P1 7 dpi to P3 14 dpi,FC01 Ct decreased from 25.31 to 19.77,FC02 Ct decreased from 27.74 to 18.39,and FC11 Ct decreased from 25.18 to 19.54.Ct value of F187 decreased from 22.34 to 17.41.③ The higher the concentration of GlyCA,the lower the Ct value of the sample,indicating the higher the viral load,but the change range was slightly smaller.At present,FC01,FC02 and FC11 had the highest viral load under 1.75%GlyCA condition,while F187 had the highest viral load under 1.25%GlyCA condition.In this study,the positive rate of SaV from sentinel monitoring was 3.74%(8/214).The results of genome sequencing and phylogenetic tree analysis showed that two genotypes were detected in SaV positive samples(GI.1 and GI.6).1 strain had the highest nucleotide and amino acid homology with Taizhou strain(MN161597.1).The most mutated amino acid domains were VP2 and NS6-7 regions,with 5 mutations in each region.The three strains of GI.6 had the highest nucleotide and amino acid homology with Japanese strain(LC380411.1).The most amino acid mutations were in the VP2 region(33 mutations),followed by the VP1 region(6 mutations).Conclusions1.The positive rate of pathogens in children with diarrhea in Guangzhou was high and the types of pathogens were complex and diverse.The higher positive rate of bacteria were Salmonella,Enteropathogenic Escherichia coli(23.83%),Clostridium difficile toxin A/B,Campylobacter.The detection rate of virus was higher with Adenovirus and Rotavirus A.Multi-pathogen surveillance for infectious diarrhea in children should continue.At the same time,all age groups should strengthen prevention and control throughout the year.2.HuTu-80 was considered to be a sensitive cell line for SaV isolation.The key to isolate SAV was that the optimal cell density before virus inoculation was 50%-60%.Virus isolation time should be extended to 14 days.Meanwhile,the concentration of GlyCA was increased to 1.25%-1.75%.3.The positive rate of SaV from sentinel monitoring was 3.74%in Guangzhou.1 and 3 SaV strains were identified as GI.1 and GI.6 in Guangzhou,respectively.There were only nucleotide substitutions in the four sequences,without nucleotide deletion or insertion.The most amino acid mutations occurred in the VP2 region.