Detection and characterization of human astrovirus and sapovirus in outpatients with acute gastroenteritis in Guangzhou, China

Background Human astrovirus (HAstV) and sapovirus (SaV) are common pathogens that can cause acute gastroenteritis (AGE). However, very few studies have reported the molecular epidemiology and clinical information on HAstV and SaV in China. This study aims to determine the molecular epidemiology and clinical features of HAstV and SaV in patients with AGE in Guangzhou, China. Methods For this study, 656 patients with AGE were enrolled. Their stool samples were screened for 15 enteropathogens using Luminex xTAG® Gastrointestinal Pathogen Panel. HAstV and SaV were detected through an in-house multiplex reverse transcriptase polymerase chain reaction followed by phylogenetic analysis. We described and compared clinical features of AGE in patients with HAstV and SaV. Results Of the 656 stool samples, 63.72% (418/656) were found to be positive, with 550 enteropathogens (296 bacteria and 254 viruses). HAstV and SaV were detected in 20 (3.0%) and 12 (1.8%) samples, respectively. Four genotypes (genotypes 1, 2, 3, and 8) of HAstV and three genotypes (GI.1, GI.2 and GIV) of SaV were identified. Coinfection was observed in ten HAstV-positive and two SaV-positive samples. HAstV was more likely to occur in winter, while SaV in early spring. The median age of the patients with single HAstV infection was higher than that of the patients with other viruses (rotavirus, norovirus, and enteric adenovirus; P = 0.0476) and unknown etiology (P = 0.006). Coinfection with HAstV or SaV were not associated with disease severity (P > 0.05). Conclusion HAstV and SaV are the common causes of AGE in Guangzhou, China.


Background
Acute gastroenteritis (AGE) is a common illness of humans globally. It adversely affects the public health, especially the very young, the elderly, the malnourished, and those with an impaired immune system [1][2][3]. Human AGE is caused by a spectrum of viruses and bacteria. Viruses, including norovirus, rotavirus, enteric adenovirus, astrovirus (HAstV), and sapovirus (SaV), are the major causative agents for AGE [4,5]. Although detailed epidemiological data for both domestic and overseas infectious gastroenteritis caused by rotavirus, norovirus, and enteric adenovirus is available [6,7], little is known about the clinical symptoms, characteristics, and coinfection of HAstV-and SaV-related AGE because of the various limitations of the detection methods and low detection rates. Further epidemiological and molecular information about HAstV and SaV causing AGE may prove useful in the development of HAstV/SaV vaccines and other preventative therapies for AGE in the post rotavirus-vaccination introduction era.
The present study aims to determine the incidence of HAstV and SaV in outpatients with AGE in Guangzhou, China, and characterize HAstV and SaV according to their genotype, age-related distribution, seasonal pattern and clinical symptoms.

Sample collection
From September 2013 to January 2016, 656 stool samples were collected from 656 outpatients who were diagnosed with AGE. The definition of AGE was the sudden onset of > 3 episodes of diarrhea or vomiting in the preceding 24 h and symptom duration < 7 days. All of the enrolled stool samples were routinely collected and stored at − 70 °C prior to investigation. The stool samples were preprocessed using NucliSENS easyMAG Lysis Buffer (BIOMERIEUX, France), followed by Viral RNA and DNA extraction using QIAamp MinElute Virus Spin kit (QIAGEN, Germany), as described previously [8].

Statistical analysis
All statistical analyses were performed using SPSS version 21.0 (SPSS Inc., Chicago, IL, USA). Differences among the proportions were compared by the Chisquare Test, Fisher's Exact Test, or Mann-Whitney U Test. Differences with P < 0.05 were considered statistically significant.

Distribution of enteropathogens in patients with AGE
A total of 656 stool samples were enrolled in the present study. The age distribution of the patients varied from 9 days to 84 years, with the median age of 25 months. Out of the 656 patients, 387 (59.0%) were male and 521 (79.4%) were under 5 years old. We detected 418 (63.72%) positive samples, resulting in 550 enteropathogens (296 bacteria and 254 viruses). HAstV and SaV were detected in 20 (3.0%) and 12 (1.8%) samples, respectively, while rotavirus was detected in 112 (17.1%), norovirus in 103 (15.7%), and enteric adenovirus in 7 (1.1%) samples. Norovirus GI and GII and adenovirus were detected by both the xTAG GPP and the in-house multiplex RT-PCR (Kappa value was 0.884, 0.918, and 0.791, respectively, showing high consistency between xTAG GPP and the in-house multiplex RT-PCR) [13].

Seasonal variation in HAstV and SaV infection is shown in
Age distribution in HAstV and SaV infections is shown in Fig. 2. HAstV showed the highest detection rate in 15-59-year age group (11.9%), follow by 1-5-year age group (5.97%). SaV infection was most common in 1-5-year age group (5.97%). HAstV and SaV were not detected in the 5-14-year-old age group. The median age of people with AGE due to single HAstV infection was higher than that of people with other viruses (rotavirus, norovirus, and enteric adenovirus) (P = 0.0476) and unknown etiology (P = 0.006) ( Table 2).

Clinical features of HAstV and SaV infections in people with AGE
Single SaV infections and infections by other viruses or unknown etiology did not have much different clinical features such as fever, vomit, diarrheal frequency, and stool routine examination (WBC count and OB text). However, AGE with single HAstV infection is more likely to cause fever and has been observed in five of the ten cases (50%) compared to infections caused by unknown etiology (16.81%) (P = 0.0197). No significant difference was observed in the incidence of vomit, diarrheal frequency, and stool routine examination (WBC count and OB text) between single HAstV infection and other virus infections or infections by unknown etiology. The median (IQR) of diarrheal frequency (times/day) in patients with single HAstV,   (Table 2).

Discussion
AGE is one of the most common disease around the world. However, other than rotavirus, norovirus and enteric adenovirus, knowledge about the impact of infections caused by HAstV and SaV is still much needed. In an effort to understand better the role of HAstV and SaV as a cause of AGE, a comprehensive survey on HAstV and SaV was carried out in Guangzhou, China. In-house multiplex RT-PCR and xTAG GPP, which had been validated for enteropathogens detection ability before, were used to detect HAstV, SaV and other enteropathogens associated with AGE [8,14]. The detection rate of HAstV in people with AGE was 3.0% in the present study, which is similar to that previously reported in shanghai (5.22%), Thailand (2.6%), Asian Russia (2.8%) and Germany (5.0%), but was lower  than the mean incidence worldwide of 11.0% [13,[15][16][17][18]. The SaV positive rate was 1.8% in Guangzhou, which is lower than that observed in US (7.0%) and Canada (9.5%) [19,20]. Coinfection was found in 50% of the HAstVpositive samples and 16.7% of the SaV-positive samples in our study. Using real-time RT-PCR, other studies have reported a higher rate of coinfection with HAstV in viral gastroenteritis, ranging between 77 and 80% [21,22]. Zhuo et al. reported a 35% prevalence of coinfection with SaV in Western Canada over a 4-year period [20]. Our slightly lower positivity can be explained by differences in the sample size, geographic location, and detection method. Norovirus and rotavirus were the most frequently detected enteropathogens responsible for coinfection in our study, which is in agreement with earlier reports [21][22][23][24]. It can be suggested that coinfection with HAstV or SaV is not rare. Our data also confirmed HAstV and SaV as the common enteropathogens responsible for AGE in Guangzhou. However, PCR can pick up free nucleic acid or long-term intermittent and asymptomatic shedding of HAstV, SaV or other enteropathogens in patients. A positive detection does not always correspond to active infection. Further investigation of the viral load and pathogenic mechanism is required. SaV was detected most commonly in 1-5-year age group (5.97%), which is in line with those reported previously, ie most of the HAstV, SaV, rotavirus, norovirus, and enteric adenovirus infections occurred predominantly in infants and children < 5 years of age [21][22][23][24]. However, in contrast to the studies above, the prevalence of HAstV was the highest in 15-59-year age group (11.90%) in this study. Our data also showed that the median age of patients with the single HAstV infection was higher than that of the patients with other virus infections (P = 0.0476). 10 of total 20 cases with HAstV infection reported in 15-59-year age group, suggesting that adult may be susceptible to HAstV, not just the very young.
December to February the following year is the winter season in China. In our study, the highest incidence of HAstV occurs in winter (February) and that of SaV in early spring (March). Similar to several other studies of viral gastroenteritis conducted in India, Australia, Italy and Shanghai, HAstV infection was more frequent during the cold weather period in Guangzhou [18,23,25,26]. SaV infection has been found mainly in the cold season [20,27,28]. However, the present study showed that SaV infection was more common during winter to early spring (February to March) in Guangzhou.
HAstV and SaV infections cause low-to-moderate degree of AGE with vomiting, fever, anorexia, abdominal pain, and dehydration [13,29]. In our study, all HAstVand SaV-positive cases were associated with acute watery gastroenteritis. Fever and vomiting were reported in 30% and 35% of HAstV infections and in 8.3% and 16.6% of SaV infections, respectively. In contrast to the study that reported greater diarrhea and fever prevalence, longer duration and greater intensity of diarrhea in HAstV infection compared to that in norovirus infections, our data did not show any significant difference in diarrhea frequency, fever prevalence between single HAstV infection and other virus infections [30]. Some reports suggest that coinfection may lead to more severe diarrhea [31,32]. However, we could not find any significant difference in the specific clinical severity of AGE between single HAstV/SaV infections and mixed HAstV/SaV infections, which is in accordance with the reports from India and other countries [30,33,34]. In one case of Campylobacter, Salmonella, rotavirus, and SaV coinfection, no fever or vomiting was observed; in addition, the test for the presence of white/red blood cell test in the stool was negative, with low degree of gastroenteritis symptoms.

Conclusion
Although this study has established HAstV and SaV as the common causes of AGE in Guangzhou, the true etiologic agent could not be determined with certainty because of the high proportion of coinfection. Hence, further investigation of the viral load or unrecognized agents is required. The results of this study may provide further epidemiological and molecular information about HAstV and SaV strains causing AGE.