Molecular Epidemiology, Clinical Characteristic And Risk Factors Study Among Children With Human Caliciviruses Diarrhea During Autumn And Winter In Guangzhou

Background, objectives, and significance: Human Calicivirus (HuCV), consisting of norwalk-like virus (NLVs, norovirus) and sapporo-like virus (SLVs, Sappovirus), is a common pathogen causing human acute gastroenteritis, and also is the important pathogen, only less important than rotavirus, causing acute gastroenteritis among children.NLVs and SLVs were found at Norwalk region of Ohio of America in 1968 and at Sapporo region of Japan in 1977 respectively. After this, HuCV was detected in many countries of the world. NLVs were also named picornavirus and small round structured virus. According to the shape, structure, antigen, gene sequence and phylogenetic analysis, the international committee on taxonomy of viruses (ICTV) enrolled NLVs and SLVs in Caliciviridae family in 1998. HuCV strains have great diversity of nucleotide sequence. NLVs consist of three genetic groups, and each genetic group consists of many different clusters. SLVs consist of three genotypes and five clusters. Farkas reported recently SLVs consisted of more genotypes and clusters, and showed the diversity of HuCV genes.The importance of HuCV was commonly underestimated due to the limit of detection method before, and it was recognized gradually with the high sensitivity diagnostic assay of RT-PCR since 1992.HuCV was distributed all over the world, and already was one of the main reasons of nonbacterium acute gastroenteritis. Between July 1997 and June 2000, 93% of the outbreaks of acute gastroenteritis were caused by NLVs in America. In Finland, the detection rate was 29% of HuCV among children between two and twenty-four months age, and it was consistent with rotavirus. The cohort study revealed 17% positive rate of HuCV in diarrhea patients between December 1998 and December 1999 in Netherlands.The studies for HuCV in our country were relatively late, and the earliest report was from Fang Zhaoyin et al. They found two NLVs strains from the stool specimens of diarrhea children in the clinic of a hospital in Henan province between 1990 and 1991. Since then on, HuCV was found in Beijing, Changchun, Heibei, Guangzhou and et al. Its positive rate is from 9.1% to 31.6%. Outbreaks of acute gastroenteritis with NLVs occurred in high school, elementary school and kindergarten of Guangzhou in winter 2003. Those reports showed the infection with HuCV was at large, and it was the important pathogen causing diarrhea among children, and the outbreak caused by HuCV was also commonly occurred.In all, the infection with HuCV prevailed in north China. NLVs was also found in Guangzhou, and caused many outbreaks of acute gastroenteritis. However, the studies in our country were mainly focused on the detection of pathogen and antibody, and the results were short of representative, and were also limited. There was no clinical information and other epidemiological data, also. Hence, here, we want to reveal the incidence, distribution, clinical characteristic, molecule biology characteristic, and risk factors of HuCV infection in Guangzhou. It will be favor of the development ofdetection reagents and effective vaccine. It will also make us clearly understand the danger and the importance of HuCV, in order to determine the pertinent control measure. Therefore, our study has realistic significance, and it is urgently needed.Method: The stool specimens were collected from the young children (less than 5 years) with acute diarrhea who visited the pediatrics of Nanfang hospital in Guangzhou during Oct 2003 to Jan 2004 and Oct 2004 to Jan 2005. Clinical information was collected for these patients with a questionnaire at the same time. The surveyors completed the questionnaire by follow-up. Stool samples were screened for group A rotaviruses by Enzyme-Linked Immunosorbent Assay (ELISA). The rotavirus negative stool specimens were detected for norovirus and sapovirus by RT-PCR. Some positive products of HuCV random selected were purified and sequenced. The sequences were analyzed by using DNAstar and Treeview packages, and phylogenetic analysis was performed. The information of clinical characteristic and risk factors were analyzed with the 1 : 1 pared case (NLVs) - case (RV) comparison study and 1 : 1 pared case - control study. The data between the rotavirus- associated cases and the HuCV-associated cases were tested with McNemar test or paired samples t-test by SPSS package (version 10.0). Using 1 ?' 1 pared one-variable and multivariable logistic regression analyze the risk factors of NLVs infection. Two-tailed tests with a significance level (P value) of <0.05 were used.Results: Six hundred and forty-eight fecal specimens were collected and detected. In the end, 339 RV positive specimens (52.31%), 80 NLVs positive specimens (12.35%) and 2 SLVs positive specimens were found. The four months of our studied all had the high positive rate of NLVs. It was 11.74% (Oct), 14.16% (Dec), 9.09% (Nov) and 13.95% (Jan), respectively, but there was no statistical significance between them. However, the detection rate during Oct 2004 was higher than during Oct 2003 (P<0.05). The ages of NLVs-associated cases were mainly below two yearsold (88.75%). Of these, 6 months to 12 months and 1 year to 2 years groups had the higher positive rate than other groups. The smallest infant was only 25 days old, and the oldest was 4 years and 6 months. There was discrepancy between the number of male and female children, but it caused by the discrepancy of sex of the whole diarrhea children. One of the two SLVs-infected children was 11 months old, and the other was 2 years and 6 months old.Twenty-two strains NLVs random selected were sequenced and analyzed. It revealed 6 strains belong to Gil —3 cluster, 15 strains belong to Gil —4 cluster and 1 strain couldn't determined its belonging at present. In 2003, the GII —3 and Gil — 4 clusters were equal in number, but it was mainly GII — 4 cluster in 2004 (10 GII — 4, and 1 Gil —3). Of the 22 strains, there was no G I genotype. The 2 SLVs both were belong to G I — 1 cluster.The clinical manifestations of NLVs-associated patients were mainly vomiting, diarrhea and fever, and it was different between NLVs cases and RV cases. Among the earliest symptoms, vomiting in NLVs cases (62.82%) was more often occurring than in RV cases (14.10%). The amount of cases with vomiting in NLVs cases was more than in RV cases, also. But the cases with the symptoms such as fever, the highest temperature, the number of days in fever and diarrhea, transfusion or not and the times to see a doctor were more commonly occurring in RV cases than in NLVs cases. It shows that it was more severe of RV cases than NLVs cases in all, but the cost caused by NLVs infection couldn't be neglected. The direct mean cost was 415 Yuan. The comparison of probable risk factors between NLVs and RV cases showed there was no statistical significance.The probable risk factors of NLVs infection were analyzed by one-variable and multivariable logistic regression and four risk factors were determined in the end. They were eating crude or cold foods, touching with diarrhea cases, having the habitof sucking hands and less washing hands. We also analyzed the risk factors of RV infection with the same method and found they both had the same risk factors.Conclusions: Our study confirmed NLVs was the main pathogen causing diarrhea among children in autumn and winter in Guangzhou. It had high incidence, and the epidemic peak of NLVs might occur earlier in north of China than South of China. The ages of the patients cause by HuCV were commonly small, most of them below 2 years old. The clusters of Gil—3 and Gil—4 NLVs both were the prevalent strains in Guangzhou, but it might be different of the prevalent strains existed during different periods. The epidemic of G I genotype NLVs might be minor. Our study confirmed the exiting of SLVs infection, but its epidemic seasons couldn't be determined at present. The two SLVs strains differed from the genotype of the one strain found in north of our country. It showed there existed various genotypes in our country, and the genotypes might be discrepant in different regions.The clinical manifestations of NLVs-associated patients were different from RV-associated patients. It was more severe of RV cases than NLVs cases in all, but the cost caused by NLVs infection couldn't be neglected. NLVs infection and RV infection had the same risk factors. The study of the risk factors about NLVs infection is favor of the establishment of the control measure for NLVs acute gastroenteritis. It was for the fist time of the study about the clinical characteristic and risk factors of NLVs-associated patients.