## 1 Introduction
Human adenovirus (HAdVs), members of the Adenoviridae family, are non-enveloped, double-stranded DNA viruses known to cause a variety of illnesses, including respiratory infections, conjunctivitis, and gastroenteritis. Among the seven recognized species (A-G), species F, particularly HAdV-F40 and HAdV-F41, is primarily associated with AGE. These enteric adenoviruses are the third leading cause of diarrhea-related mortality in children under five, following rotavirus and _Shigella_. The global prevalence of HAdV-associated gastroenteritis has shown an increasing trend, especially in post-2010 studies, and is most pronounced in regions with poor sanitation and malnutrition, such as Africa. Enteric adenovirus are highly prevalent in children younger than five years due to their underdeveloped immunity. Despite these findings, there are significant gaps in the epidemiology of HAdVs, particularly regarding species and genotype distribution [14]. Human astroviruses (HAstVs) are non-enveloped, positive-sense, single-stranded RNA viruses that belong to the Astroviridae family, which is classified into classic and novel genotypes. Transmission occurs via the fecal-oral route, with infections often presenting as mild, self-limiting diarrhea, though severe cases can occur in immunocompromised individuals. Molecular methods such as RT-PCR and nested RT-PCR are highly sensitive for detecting HAstVs, which are frequently identified in co-infections with other gastrointestinal viruses, like rotavirus and norovirus. Despite their widespread prevalence, research into effective vaccines or targeted therapeutic interventions for HAstVs remains limited [15]. Traditionally, the detection of gastrointestinal (GI) viruses in clinical laboratories has relied on enzyme immunoassay (EIAs) and chromatographic immunoassays (CIAs), which are rapid but often lack sensitivity and specificity, and cannot detect all gastrointestinal viruses, such as sapovirus [16; 17; 1]. More sensitive methods, like cultivation and electron microscopy, are reserved for referral laboratories and are inconvenient for everyday practice. The advent of molecular diagnostic techniques, particularly multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) assays, has revolutionized pathogen detection by enabling simultaneous identification of multiple viruses with superior accuracy [18; 19; 20; 21]. Advances in multiplex RT-PCR panels provide superior accuracy, allowing simultaneous detection of multiple pathogens with high sensitivity and specificity, making them valuable for outbreak management and improving healthcare practices [1; 22]. Emerging diagnostic platforms, like the FilmArray GI Panel and xTAG GPP, demonstrate high accuracy in identifying gastrointestinal pathogens, including mixed infections and underdiagnosed viruses like sapovirus and astrovirus [23; 24]. Molecular methods also facilitate the genotyping and surveillance of evolving strains, as seen with norovirus GII.4 variants, which develop through antigenic drift [25]. However, high costs, infrastructure requirements, and challenges in interpreting co-detections limit their routine use [26; 27]. To optimize clinical utility, a two-step approach combining rapid antigen testing with confirmatory RT-PCR is recommended [27; 28]. Despite these advancements, there remains limited evidence on the clinical utility of these assays compared to traditional methods in routine diagnostic settings. This study aimed to compare the performance of the AccuPower Diarrhea V1&V2 Real-Time RT-PCR, Bioneer (Daejeon, Republic of Korea), with CIA for detecting major GI viruses in stool specimens. Additionally, this study sought to evaluate the diagnostic reliability and clinical applicability of these methods, and to document the first confirmed case of sapovirus in Croatia.