Novel Human Astroviruses: Prevalence and Association with Common Enteric Viruses in Undiagnosed Gastroenteritis Cases in Spain

Abstract

A remarkable percentage of acute gastroenteritis cases remain etiologically undiagnosed. The aim of the study was to determine the prevalence of common and emerging enteric viruses, such as novel human astroviruses, among undiagnosed samples from children with acute gastroenteritis. Epidemiological studies for novel human astroviruses are still scarce. Stool samples collected over two consecutive winter seasons (2016–2017) from children with gastroenteritis in Spain, which were negative for bacteria, rotavirus, and adenovirus by routine diagnostics were screened by real-time RT-PCR assays for the presence of classical and novel astrovirus, rotavirus, norovirus GI and GII, sapovirus, and adenovirus. Overall, 220/384 stool samples (57.3%) were positive for at least one virus. Co-infections were identified in 21% of cases. Among a total of 315 viruses identified, adenovirus was the most prevalent (n = 103), followed by rotavirus (n = 51), sapovirus (n = 50), classical astrovirus (n = 43), novel astroviruses (n = 42), and norovirus (n = 26). Novel astroviruses were present in 13.3% of virus-positive cases. Most novel astroviruses were found in children <2-year-old (30/39 children, 77%, p = 0.01) and were found in co-infection (66%). Only classical astroviruses demonstrated significant differences in the Cq values during mono-infections compared to co-infections. In conclusion, common enteric viruses may be frequently found in children with undiagnosed gastroenteritis, indicating the need to implement more sensitive diagnostic methods. Novel astroviruses circulate in the community and could be the cause of gastroenteritis among young children.

A Spanish case-control study in <5 year-old children reveals the lack of association between MLB and VA astrovirus and diarrhea

“…While Holtz et al did not find any association between MLB1 infection and diarrhea 28 , Meyer et al reported an association between MLB1 and diarrhea and between MLB3 and asymptomatic disease, and no significant association of VA viruses with any of the groups 27 . The results of the present study are also in agreement with the high rate of co-infections observed in our previous study: MLB and VA were identified in co-infection with ≥1 other enteric virus in 78% and 54.8% of cases (all symptomatic), respectively, raising the question of their contribution to the digestive tract symptoms 29 . In this previous study, we could not observe a difference in the novel HAstV mean viral RNA titer during mono-and co-infection.…”

“…We found that 6.3% of case patients were positive for one or several novel HAstV. This is slightly lower than our previous study, which investigated a similar Spanish pediatric population, with a positivity rate of 10% 29 . The difference can be explained by the study period: our first study focused on two consecutive winter seasons from January-April 2016 and January-April 2017, respectively, while the present one extended over a 10-months period (August 2017-May 2018).…”

“…RNA was extracted from 200 µl of a 30% stool suspension in phosphate buffered saline (PBS) using the Viasure RNA-DNA extraction kit (Certest Biotec) following the manufacturer's instructions. Real-time RT-PCR specific assays for HAstV-MLB and HAstV-VA were performed in duplex as previously described 29,45 , using the Kapa Probe Fast Universal One-Step real-time RT-PCR Master Mix (Kapa Biosystems) and following the manufacturer's instructions, on a Stratagene Mx3000P (Thermofischer) and a CFX96 Touch ™ Real-Time PCR Detection System (Bio-Rad). The standard curves for quantification were constructed based on 10-fold serial dilutions of plasmids containing a fragment of the corresponding genome strains (MLB1: nucleotides 4292-4416 from FJ222451; MLB2: nucleotides 3724-3848 from KT224358; VA1: nucleotides 4629-4706 from FJ973620.1; VA2: nucleotides 4480-4558 from GQ502193.2; VA3: nucleotides 3678-3756 from JX857868.1; VA4: nucleotides 4646-4721 from JX857869.1).…”

“…This additional screening was performed in order to complete the information on the asymptomatic carriage of novel astrovirus in healthy children, providing a comparison with prevalence of mostly encountered enteric viruses. We did not performed such complementary screening among the diarrheic group, as this was already done in a previous study 29 .…”

“…Sequences of novel HAstVs have been found in human stools of individuals with diarrhea 15,[24][25][26] , but also in asymptomatic healthy controls 27,28 ; unfortunately, the two latter reports have contradictory results in their case-control study, especially for MLB1, which so far is the most common. We also recently reported a high rate of co-infection with other enteric viruses during episodes of gastroenteritis where a novel astrovirus was identified 29 , raising the question of their causal role in gastroenteritis.…”