Metagenomic Analysis of Enteropathogenic Diversity Using Next-Generation Sequencing in Norovirus-Positive Stool Samples from Children Under 3 Years of Age Hospitalized With Acute Gastroenteritis at Dr. Luis Calvo Mackenna Hospital, Santiago, Chile

Carlos Garrido, University of Antofagasta

Carlos Garrido-Soto ¹ ³; Richard A. Olivares¹ ² ³; Mariella Rivas¹ ³; Mauricio J. Farfán⁴; Jan Vinje⁵; Margarita K. Lay¹ ² ³¹ Department of Biotechnology, University of Antofagasta, Antofagasta, Chile² Millennium Institute on Immunology and Immunotherapy, University of Antofagasta, Chile³ Research Center for Immunology and Biomedical Biotechnology of Antofagasta (CIIBBA), University of Antofagasta, Antofagasta, Chile⁴ Molecular Biology Laboratory, Hospital Dr. Luis Calvo Mackenna, Santiago, Chile⁵ National Center for Immunization and Respiratory Diseases, U.S. Centers for Disease Control and Prevention, Atlanta, Georgia

Molecular Epidemiology & Evolution

Human noroviruses (HuNoV) are the leading cause of acute gastroenteritis (AGE), with high morbidity worldwide, especially in children under 5 years of age, immunocompromised individuals and the elderly. Often, AGE cases involve co-infection by multiple enteric pathogens. In this study, 11 norovirus-positive stool samples from pediatric patients (<3 years old) hospitalized for AGE at the Hospital Dr. Luis Calvo Mackenna, Santiago, Chile, were first screened by FilmArray GI®, revealing 5 norovirus-rotavirus A co-infections, 1 norovirus-adenovirus co-infection and 5 norovirus single infections. Conventional RT-PCR analysis yielded: 4 GI, 4 GI/GII co-infections and 3 GII norovirus positive samples. All samples were subjected to RNA-seq to assemble viral genomes and transcripts. Across 9 of 11 samples, we detected a wide variety of enteric pathogens that were not detected in the initial screening like Clostridium difficile, Shigella flexneri, Escherichia albertii, Salmonella enterica, Campylobacter jejuni and Parechovirus, thereby uncovering the polymicrobial etiology of AGE that missed by targeted pathogen assays. In addition, it was recovered two complete norovirus genomes (GII.6[P7] and GII.4 Sydney[P16]) and two partial fragments (GII.4 Sydney[P16] and GII.3), enabling high-resolution capsid–polymerase genotyping and identification of ORF1–ORF2 recombination events, key drivers of norovirus evolution. These constitute the first complete pediatric norovirus genomes reported from Chile, supporting surveillance of emerging lineages. Furthermore, our results demonstrate that Next-Generation Sequencing outperforms conventional PCR panels by detecting both expected and unexpected pathogens and enabling recovery of transcript of non-target enteric pathogens, thereby expanding AGE diagnostic and surveillance capabilities.