General characterization
During the 2-year study period, a total of 44595 diarrhea patients were studied. The mean (±SD) age of the study subjects was 43.51 (±19.06 ) years and 21657 (48.56%) were male. Among the surveillance subjects, a total of 3941 samples (8.84%) were detected (duplicated samples excluded). There were 2114 positive samples detected (positive rate 53.64%) and 903 (detection rate 22.91%) patients were positive for norovirus (referred to as “NoV + ”), consisting of GI (94, 10.41%), GII (769, 85.16%) and co-infections of GI and GII (40, 4.43%). Co-infections of norovirus and other viruses or bacteria were confirmed in 91 cases (2.31%). Excluding co-infection samples, 2947 samples (74.78%) were confirmed as negative for all tested pathogens or positive for other enteric pathogens (referred to as “NoV -”). The positive rates of other enteric pathogens were as follows (excluding co-infections): Shigella 0.51%, Salmonella 3.63%, Vibrio parahemolyticus 3.93%, C. jejuni 0.66%, Yersinia enterocolitica 0.05%, C. coli 0.08%, EPEC 0.74%, ETEC 0.86%, EAggEC 0.10%, EIEC 0.03%, astrovirus 2.54%, rotavirus 10.05%, sapovirus 2.36%, and enteric adenovirus 0.53%.
NoV(+) sample features and comparison with NoV(−)
Epidemiological analysis
Norovirus was detected throughout the year, and the prevailing season lasted as long as half a year (from October to April) (See Figure 1). The seasonal distribution of NoV(+) detection was different from NoV(−) (p < 0.001), but no difference was found between autumn (September-November) and winter (December-February) (p = 0.117). Norovirus spanned all ages, from 0 to 94 years old. The proportion of the child population of NoV(+) patients seemed smaller than that of NoV(−) ones (See Figure 2), but the difference was not statistically significant in a logistic regression model. The sex ratio (male: female) was 1.18:1, with a higher male proportion in the NoV(+) group (p = 0.001, OR = 1.303, 95% CI = 1.110-1.529). The proportion of local citizens infected with norovirus was higher than that of non-norovirus patients (p < 0.001). Norovirus had a higher chance of appearing in: officials/clerks (p = 0.001, OR = 1.348, 95% CI = 1.124-1.618) and a lower chance of appearing in farmers/migrant laborers (p = 0.007, OR = 0.243, 95% CI = 0.087-0.680).
Stratification analysis
In an age stratification analysis, it was discovered that NoV(+) and NoV(−) patients had statistically different seasonal distributions for each age group (0 ~ 4y, p = 0.017; 5 ~ 18y, p = 0.005; 19 ~ 44y, 45 ~ 59y, >60y, all p < 0.001) (See Figure 3), and a significant seasonal difference among NoV(+) patients of different age groups could also be determined (p ≈ 0.027). While a difference in the proportions of male and female NoV(+) patients could be found among different age groups (p = 0.016): in the children and youth groups (<44y), males were dominant, and in the middle-aged and elderly groups (>45y), vice versa (p < 0.001, OR = 1.586, 95% CI = 1.216-2.067), the gender distribution did not differ much from NoV(−) patients, except in the youth (19-44y) group, where male patients had a higher proportion of infections (p = 0.024, OR = 1.302, 95% CI = 1.041-1.629).
Analysis of exposure history
When compared with NoV(−), a higher proportion of NoV(+) patients had a history of consuming suspicious food within 5 days before onset (p = 0.001, OR = 1.319, 95% CI = 1.124-1.550), while a lower proportion had an enteric disease history 6 months prior (p = 0.048, OR = 0.341, 95% CI = 0.117-0.992).Although a large percentage (53.09%) of the children (<18y) group kept or had contact with pets, in a univariate χ2 test, there was no statistically significant difference between NoV(+) and NoV(−) patients (p = 0.451) within this group.
Clinical feature analysis
NoV(+) diarrhea patients featured nausea (p < 0.001, OR = 1.418, 95% CI = 1.176-1.709) and vomiting (p < 0.001, OR = 1.969, 95% CI = 1.618-2.398), while fewer reported fever (p = 0.046, OR = 0.758, 95% CI = 0.577-0.996) and abdominal pain (p = 0.018, OR = 0.815, 95% CI = 0.689-0.965) when compared with NoV(−) patients. In a discriminant analysis, the relationship between symptoms and norovirus infections was also studied. The combination of nausea and vomiting (especially lasting over three days) was typical of norovirus infections, while fever (especially high fever) and abdominal pain were adverse determining factors (p < 0.001). General, epidemiological and clinical comparisons are listed (see Additional file 1).
Comparisons with rotavirus and bacterial infections
A total of 396 samples were confirmed to have rotavirus infections (10.05%), and 432 samples had bacterial infections (10.96%) (co-infections excluded). Comparisons with norovirus regarding their general, epidemiological and clinical features with norovirus are listed (see Additional files 2 and 3).
The seasonal difference between norovirus and rotavirus detection was obvious (p < 0.001): rotavirus mainly occurred in the coldest seasons (from November to February) (See Figure 1). There was also a difference in age distribution between the two viruses (p = 0.002): rotavirus affected children more (p = 0.008, OR = 1.637, 95% CI = 1.136-2.358) (See Figure 2). The proportion of males (p = 0.004, OR = 1.475, 95% CI = 1.133-1.919) and local citizens (p < 0.001) with confirmed norovirus was higher than those with rotavirus. Norovirus and rotavirus were detected from hospitals of different types (p = 0.006). Rotavirus-affected patients had a higher proportion of suspicious food history (p < 0.001, OR = 2.006, 95% = 1.447-2.781). Patients affected with norovirus were more likely to manifest vomiting (p < 0.001, OR = 1.860, 95% CI = 1.373-2.520), but less likely to manifest fever (p = 0.034, OR = 0.626, 95% CI = 0.406-0.964).
The seasonal difference between norovirus and bacteria was more obvious (p < 0.001): bacteria were mostly found in warm seasons (from July to September) (See Figure 1). Age was also an influencing factor p = 0.037): a higher proportion of children were infected with bacteria than norovirus (p = 0.027, OR = 1.511, 95% CI = 1.053-2.169) (See Figure 2). Local citizens had a higher proportion of norovirus infections (p = 0.003). For the following occupations, there was a lower prevalence of norovirus than of bacteria: kindergarten/home-stay children (p = 0.033, OR = 0.090, 95% CI = 0.010-0.822) and farmers/migrant laborers (p = 0.008, OR = 0.180, 95% CI = 0.051-0.643). Norovirus-affected patients had a higher proportion of suspicious food history (p < 0.001, OR = 1.686, 95% CI = 1.266-2.244). Compared with bacteria, norovirus patients less frequently manifested fever (p < 0.001, OR = 0.428, 95% CI = 0.288-0.635) and abdominal pain (p < 0.001, OR = 0.405, 95% CI = 0.299-0.549), but more frequently manifested nausea (p = 0.001, OR = 1.735, 95% CI = 1.247-2.412) and vomiting (p = 0.006, OR = 1.620, 95% CI = 1.149-2.286).
Features of GI and GII genotypes
769 GII strains were detected concomitant with 94 GI ones. The Pearsonχ2 test indicated that the seasonal distribution of two genotypes was different (p < 0.001), with GI highest in spring (March to May) (44.68%) and GII highest in autumn (September to November) (40.44%). Those who work as officials or clerks had a higher possibility of being affected by GI (42.55%) than GII (25.75%) (p = 0.001, OR = 2.136, 95% CI = 1.368-3.289). GI-affected patients seemed to have eaten in a restaurant more often (3.19%) than GII-affected patients (0.65%) (p = 0.047, OR = 5.037, 95% CI = 1.185-21.277). The rate of patients who had consumed contaminated seafood within five days before onset was higher in GI (17.02%) than in GII (10.92%) patients (p = 0.019, OR = 2.294, 95% CI = 1.164-4.525). There were also slight differences in the clinical features of the two genotypes: nausea (54.26% of GI and 42.65% of GII, p = 0.037, OR = 1.595, 95% CI = 1.031-2.439), diarrhea lasting less than three days (87.23% of GI and 81.14% of GII, p = 0.035, OR = 4.008, 95% CI = 0.959-16.667), and hyperactive bowel sounds (37.23% of GI and 23.28% of GII, p = 0.003, OR = 1.955, 95% CI = 1.250-3.077).
In a logistic regression model, officials or clerks were more easily affected with GI than GII (p = 0.006, OR = 1.888, 95% CI = 1.205-2.958). Seasonal differences were statistically significant in both genotypes (p < 0.001). A higher proportion of patients who had eaten in a restaurant was affected with GI than GII (p = 0.048, OR = 4.717, 95% CI = 1.013-21.960).
Co-infection samples
91 norovirus co-infection samples were discovered (excluding co-infections of norovirus GI and GII): 21 with rotavirus, 18 with astrovirus, 15 with sapovirus, 10 with Salmonella, 7 with EPEC, 5 with Vibrio parahemolyticus, 5 with adenovirus, 3 with C. jejuni,2 with C. coli, 1 with Shigella, 1 with EAggEC and 3 triple co-infections.