## 3 Results
### Discussion
The detection of sapovirus in a stool sample using RT-PCR in Croatia represents the first documented evidence of this pathogen in human specimens in the region. The infection occurred in a young girl, aged 8, who presented with watery diarrhea. This discovery emphasizes the evolving global importance of sapovirus, which has emerged as a significant cause of acute gastroenteritis, particularly in young children, following the reduction in rotavirus cases due to vaccination. Globally, the pooled prevalence of sapovirus in AGE cases is estimated at 3.4%, rising to 5.6% when more sensitive RT-qPCR assays are employed, showcasing the advantages of molecular diagnostics in pathogen identification. While sapovirus often causes mild, self-limiting gastroenteritis, severe cases requiring hospitalization and complications such as dehydration have been reported, especially in low-resource settings. Unlike other regions with established data, this first report underscores the importance of continued surveillance in underrepresented countries like Croatia, particularly as molecular diagnostic tools facilitate the detection of emerging or previously underreported pathogens [29]. Similarly, no positive cases of astrovirus were identified in this study, which underscores the potential role of multiplex RT-PCR panels in expanding the spectrum of detected viruses and enhancing our understanding of their epidemiology. The manufacturers of the norovirus CIA acknowledge the potential for false positive results in children under 1 year of age, who are often the primary target population for this test. This limitation further underscores the recommendation to opt for the multiplex PCR method instead, which offers greater accuracy [30]. The manufacturer of a rapid antigen test for adenovirus and rotavirus claims an accuracy of over 97.0% overall agreement (OA), based on a comparison with the latex agglutination method. On the contrary, the results of our study have showed a significantly lower percentage of agreement when RT-PCR is used instead of a rapid antigenic test as a comparator. However, we believe that the accuracy of rapid antigen tests should ideally be compared to viral cultures or, when cultures are unavailable, at least to molecular methods. These findings emphasize the limitations of rapid antigen-based tests, particularly their low sensitivity and potential for false positives. Stool samples are inherently challenging for antigen detection due to variability in sample consistency, the presence of interfering substances, and the rapid degradation of viral antigens. RT-PCR, with its superior sensitivity and specificity, offers a more reliable alternative for detecting gastrointestinal viruses. Despite the clear advantages of RT-PCR, its high cost and longer turnaround time compared to EIA/CIA may limit its routine application in all settings. A two-step diagnostic approach, starting with initial CIA screening followed by confirmatory RT-PCR, could balance cost-effectiveness with diagnostic accuracy. In UHS, three gastrointestinal viruses (rotavirus, adenovirus, and norovirus) are routinely tested with CIA, which is cost-effective for our institution. However, if all gastrointestinal viruses were tested with antigenic tests, perhaps not only the diagnostic accuracy but also the cost-effectiveness would favor RT-PCR. This study is limited by the lack of a gold standard reference method, such as viral culture, to establish the true sensitivity and specificity of the assays. Future research should include larger sample sizes and comparisons with other molecular diagnostics to validate the performance of RT-PCR. Additionally, the interpretation of multiplex RT-PCR results in mixed infections warrants further investigation, as distinguishing between true pathogens and incidental findings remains a challenge.