Molecular Epidemiology Of Viruses Causing Acute Diarrhea In Children In Shanghai Between2006and2011

Objectives This study aimed to survey and analyze molecular epidemiology of four viruses causing diarrhea in inpatients and outpatients in Shanghai. Our research will help to understand comprehensively and to evaluate objectively the role of viruses in children’s diarrhea. It will provide essential data and theoretical guidance for the prophylaxis and treatment of viral diarrhea, and the development and application of vaccine.Methods Fecal samples were collected from children up to age of5years with acute diarrhea treated as inpatients(2006to2011) or outpatients(2010-2011) at Children’s Hospital, Fudan University. RT-PCR assay was applied for detection of rotavirus(RV), human calicivirus(HuCV), human astrovirus(HAstV) and adenovirus (AdV). Rotavirus genotypes were characterized using consensus and type-specific primers. Norovirus(NV), sapovirus(SaV), HAstV and AdV were genotyped directly by sequencing the PCR products and subsequent phylogenetic analysis. All the data were analyzed by SPSS16.0statistical software.Results Molecular epidemiology of viruses causing diarrhea in hospitalized children between2006-2011was as following:1. Overall detection of viruses:At least one virus was found in each of621samples(92.1%). Among the diarrheal viruses detected, group A rotavirus was the most common with a proportion of89.1%(601/674), whereas HuCV, HAstV and AdV were also found respectively in30.6%(206/674),30.1%(203/674) and4.7%(32/674) of patients. There were43.5%(293/674) of cases identified with mono-infection and rotavirus was the predominant single virus, while the detection rate of each other three viruses was less than2%. Mixed virus infection were detected in328(48.6%) of the674children, with RV+HAstV, RV+HuCV and RV+HuCV+HAstV as the most popular co-infections. The detection rate of mixed infection was in a downward trend from2006to2011, and it dropped to11.7%in2011.2. Virus detection in children with nosocomial and non-nosocomial diarrhea:About50.8%(302/626) of hospitalized children with diarrhea were defined as nosocomial infections. The proportion of nosocomial infections was as high as that in2009(60.6%) and2008(60.5%), but it decreased to24.5%in the year of2011. The detection rate of a single viral agent in non-nosocomial diarrhea children(47.8%, 147/308) was significantly higher than that in children with nosocomial infection (38.1%,121/318)(P<0.05). RV was identified as the most prevalent singly infected pathogen. While mixed infections in nosocomial diarrhea children(56.9%,181/318) was remarkably higher than that in non-nosocomial infected children(42.9%,132/308)(P<0.05), with the dual infections of RV+HAstV and RV+HuCV as the most frequent patterns.3. RV genotypes:①G genotypes:The most prevalent type during the study period was G3, but the popularity of G3type showed a downward trend, from64.6%in2006decreasing to12.8%in2011. An obvious increase was observed for G9and G1, with G9identified as the predominant genotype in2011(51.1%) and G1as the secondly prevalent one(19.1%). Mixed G infections with G1、G3or G9were the most prevailing G-mixed types although more different G types were present simultaneously.②P genotypes:The prevalence of P genotypes was in a way fluctuating with year, but P[4], P[8] and Pm genotypes were the predominant strains. The major mixed P infections were genotypes of P[4]+P[8] and P[8]+P[10].③GP combinations:P[8]G3genotype was determined to be the major rotavirus genotype combination(27.5%) in Shanghai during2006-2011and the other prevalent genotypes include PmG3and P[4]G3. P[8]G9strain jumped as the most predominant type in2011(40.5%). Besides, there was no obvious difference of RV genotypes between nosocomial and non-nosocomial diarrhea children.4. HuCV genotypes:All of the206HuCV-positive samples were detected as Norovirus GⅡ. The predominant genotype was GII.4(75.5%,156/206), followed by GII.12(22.3%,46/206), GII.7(1.0%), and GII.b(1.0%). The GII.4-2006b variant was the predominant subtype in our study. The genotypes of NV in children with non-nosocomial infection were similar to that in children with nosocomial children.5. HAstV genotypes:All HAstV identified in hospitalized children were classified as genotype1.6. AdV genotype:Among the32samples positive for adenovirus in this study,50.0%(16/32) belonged to Ad-41, followed by Ad-3(25.0%,8/32). There were some differences of the popularity of various genotypes every year. No difference was found in AdV genotypes between children with nosocomial and non-nosocomial infections.Molecular epidemiology of viruses causing diarrhea in outpatient children between2010-2011was as following:1. General detection of viruses:Among the436specimens,66.7%(291/436) contained at least one viral agent. A total of189samples(43.3%) were identified as group A rotavirus, followed by HuCV(29.4%,128/436), AdV(7.1%,31/436) and HAstV(1.8%,8/436). The mono-infection was most frequently found (51.8%,226/436) in outpatients, with RV(30.0%,131/436) as the most predominant viral agent and HuCV occurred as the second one(16.0%,72/436). The percentage of multiple infections cases was14.9%(65/436). All of these were dual infections while the combination of RV+HuCV was the most prevalent mixed infection.2. Epidemic characteristics of RV:①Age distribution and seasonally:Rotavirus infected diarrhea peaked from September to November and the highest detection rate was found in October(70.0%). The majority of the rotavirus-positive specimens identified (49.7%) were in children0～11months of age and about96.8%of all rotavirus episodes occurred by age3.②Genotypes:Among the G genotypes identified, the most common genotype was G1(31.2%), followed by G3(28.6%) and G9(27.0%). Among the P genotypes, the predominant P genotype was P[8](91.0%,172/189), followed by P[4](5.3%,10/189). The majority of mixed G strain was G1+G9(60.0%,12/20), while mixed P strain was only found in2.6%of patients with P[4]+P[8] as the prevalent one. Genotype combinations of P[8]G3, P[8]G1and P[8]G9were the most popular ones, accounting for27.5%(51/189),26.9%(51/189) and25.4%(48/189) respectively.3. Epidemic characteristics of HuCV:①Cases of HuCV diarrhea occurred throughout the year but peaked in September and October. The highest detection rate of NV infections occurred in September(66.7%), however a high detection rate was also found in June(60.0%). A higher prevalence of HuCV(56.7%,72/128) was observed up to11months of age as compared to the other age groups. Children under3years old comprised97.0%of cases.②Genotypes:A total of128samples(29.4%) were positive for HuCV. Of these,126strains were NV and2belonged to SaV. The predominant NV genotypes were GII.4(71.8%,92/126) and GII.b(15.6%,20/126), followed by GⅡ.12(9.5%), GII.7(0.8%), and GII.2(0.8%). The majority of GII.4clustered as GⅡ4-2006b(65.6%), whereas the other eight belonged to GII.4-2006a subgenotype. Two of SaV cases were identified as GI.2and GⅡ.1, respectively.4. Genotypes of HAstV:All HAstV positive samples belonged to HAstV-1.5. Genotypes of AdV:The predominate type was Ad-41(45.2%,14/31), followed by Ad-3(22.6%,7/31) and Ad-40(16.1%,5/31). Other2cases were Ad-12and the remaining three cases were found as Ad-2,5and9respectively.Conclusion RV and HuCV were the two major viruses both in outpatients and hospitalized children with diarrhea in Shanghai. Compared with data from the year of2001-2005, this study showed new molecular epidemiology characteristics of RV and HuCV during2006-2011in Shanghai, including changes in predominant genotypes and diversification of popular genotypes. Although there were some differences in clinical symptoms and the proportion of multiple infection between diarrhea children with nosocomial and non-nosocomial infection, the major prevalent genotype of viruses causing diarrhea were similar. Besides, the detection rate of AdV in outpatients was significantly higher than that in hospitalized children. Different prevalence of HuCV and AdV genotypes was found between inpatients and outpatients.