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.

The rise in antimicrobial-resistant Neisseria gonorrhoeae (NG) strains poses major challenges to gonorrhea treatment worldwide. Ceftriaxone remains the first-line antibiotic therapy; however, emerging resistance, particularly driven by the mosaic penA 60.001 allele, necessitates vigilant surveillance. This study assesses the antimicrobial susceptibility patterns of NG isolates in the northwestern region of Croatia and evaluates the correlation between phenotypic susceptibility testing for extended-spectrum cephalosporins (ESC) and genotypic detection of the penA 60.001 allele. A total of 39 clinical NG-positive specimens by a multiplex PCR panel for urogenital infections were collected between 1 July 2022, and 30 June 2024. Phenotypic antimicrobial susceptibility testing was performed using the Etest method. Genotypic detection of ceftriaxone resistance determinants was performed using a multiplex nested PCR assay. All NG isolates were susceptible to ceftriaxone and cefixime. High resistance rates were observed for ciprofloxacin (70.6%), tetracycline (44.1%), and azithromycin (20.6%). Mutations in the penA gene associated with decreased susceptibility to ceftriaxone were detected in three samples, although phenotypic resistance was not observed. The high resistance rates to ciprofloxacin, tetracycline, and azithromycin limit their use for empirical therapy in Croatia. While ceftriaxone remains effective, the detection of penA mutations highlights the need for ongoing surveillance.