Background: Due to rapid spread, the Omicron variant has become the dominant SARS-CoV-2 variant responsible for infections worldwide. We present the first detection of the Omicron variant in Croatia which resulted in rapid cross-border spreading. Methods: Whole-genome sequencing was performed using the Illumina MiniSeq sequencing system. SARS-CoV-2 lineages were identified using the PANGOLIN and GISAID databases. Results: The first case of the Omicron variant (BA.1.17) emerged in Croatia after a workshop held in Zagreb in November 2021. The patient reported a history of previous COVID-19 and received two doses of an mRNA vaccine. Three additional cases were detected among Croatian participants of the workshop. At the beginning of December, SARS-CoV-2 infection was confirmed in one participant from Montenegro and her husband. Phylogenetic analysis showed that the detected Omicron variants were closely related to the first Croatian case, confirming the connection with the workshop outbreak and rapid cross-border spreading. Subsequent analyses of SARS-CoV-2 positive samples in Croatia showed the rapid introduction of the Omicron variant and depletion of the Delta variant resulting in the fifth pandemic wave. Conclusions: Genomic monitoring and early detection of novel SARS-CoV-2 variants are essential to implement timely epidemiological interventions and reduce further transmission in the population.

Background: Enterovirus-D68 (EV-D68) was long underreported, with only sporadic cases of respiratory disease worldwide until 2014, when numerous countries experienced significant outbreaks of EV-D68. In Croatia, sporadic detections have primarily resulted from an absence of systematic surveillance. Following the increased incidence of EV-D68 across Europe in 2022, we started to characterize EV-positive respiratory samples in Zagreb to confirm the presence of EV-D68 and identify circulating lineages through phylogenetic analysis. Methods: Respiratory samples from individuals with acute respiratory infection and additional clinical symptoms were tested at the Virology Laboratory of the Croatian Institute of Public Health, and EV-positive samples were further screened using the real-time RT-qPCR method for EV-D68. VP1 sequences were obtained by sequencing and subsequently genotyped. Results: Between March 2022 and December 2024, EV was detected in 2048 respiratory samples. Annual distributions of EV detections were 656 (10.0%) in 2022, 785 (8.1%) in 2023, and 607 (7.4%) in 2024. EV-D68 was identified in 13.1% of EV-positive samples in 2022, 1.4% in 2023, and 19.6% in 2024. The peaks in EV-D68 circulation were observed in July (n = 24) and September (n = 24) in 2022 and in September 2024 (n = 62). Phylogenetic analysis of EV-D68 VP1 sequences revealed the presence of two major clades, A2 and B3. The sequences from 2022 clustered exclusively within clade B3, while in 2024 A2 clade was newly introduced. Conclusions: We confirmed the presence of EV-D68 in Croatia with circulating lineages corresponding to those detected elsewhere in Europe. The absence of routine testing has likely led to an underestimation of EV-D68 prevalence. These findings underscore the urgent need for ongoing surveillance and genomic characterization to clarify EV-D68 epidemiology in Croatia.