Background: Tick-borne encephalitis (TBE) is a disease with mandatory declaration in the EU since 2012. Information regarding the seroprevalence of the disease across Romania is limited, and only sporadic cases are rarely reported. We aimed to identify new areas of TBEV infection in different counties of Romania. Methods: We conducted a serosurvey assessing the immune response to TBEV infection in adult populations from rural areas in different counties of the country. Seropositivity was defined by a positive TBEV IgM/IgG ELISA test and confirmed by serum neutralization. Results: We collected 1116 samples from 15 different localities in 10 counties (divided into endemic/border/non-endemic counties) across Romania. Overall, 26 (2.3%) of the samples were tested positive using the TBEV ELISA assay in six counties. In those counties, 3.7% of sera were positive, varying from 1.4% to 6.9% per county. After performing the neutralization assay, seven (0.6%) samples were confirmed positive, interestingly all from one site in Sibiu County, where the seroprevalence was 9.7%. Conclusions: The identification of positive serum samples in serosurveys appears to rely on the discovery of TBEV microfoci. Further serological surveys should be conducted in Romania, especially after the identification of positive TBEV patients presenting for medical care.

Background: Mediterranean spotted fever (MSF) is an emerging tick-borne disease caused by Rickettsia conorii subsp. conorii, primarily prevalent in Mediterranean and Southern Europe. We aimed to evaluate MSF seroprevalence and risk factors in non-endemic rural areas of Romania. Methods: We conducted a serosurvey in five counties not under MSF surveillance by testing 459 serum samples from adult volunteers for specific IgG antibodies using ELISA. Participants answered a questionnaire regarding demographics and MSF risk factors. Results: The median age of the participants was 60 years and 329 (71.7%) were female. Overall, 64 (13.9%) samples tested positive for IgG anti-R. conorii, with rates ranging from 7.1% in Sibiu to 22.4% in Hunedoara. The median age of the positive individuals was 68 years, with a significantly higher seropositivity rate of 54.7% among those over 65 years (p = 0.01). Among those positive, 53 (82.8%) owned different household animals; 24 (37.5%) had daily contact with dogs, and 27 (42.2%) with livestock; 17 (26.6%) noted tick infestations of animals, and 23 (35.9%) reported tick bites. Conclusions: This study revealed an important seroprevalence of MSF in Romanian areas considered non-endemic, indicating an expansion of its geographical range, probably due to climate change, and emphasizing the importance of enhanced surveillance and diagnostic capabilities nationwide.

Background: Etiologic diagnosis in suspected viral meningitis is not always achievable, yet it can play a significant role in patient management. Our study aimed to do a comprehensive analysis of current practices regarding etiologic diagnosis in these cases and compare patients with and without known etiologic diagnosis in order to visualize if and how having an etiological diagnosis can impact patient management and/or outcome. Methods and results: We conducted a convergent mixed-methods study. Quantitative data was obtained from 118 patients hospitalized during a one-year period. There were 40.7% (n = 48) cases with unknown etiology. The length of hospitalization was longer in the group with unknown etiology vs. known etiology (12.6 days vs. 9.8 days p = 0.01). Thematic analysis was used for the qualitative approach in order to evaluate physicians’ overall perceptions regarding this subject. Conclusions: Our mixed-methods study shows that while clinicians consider etiologic diagnosis very important, it remains a diagnostic challenge even in modern times. Continued efforts are needed to optimize diagnostic strategies and address existing gaps in the etiologic workup of viral meningitis. There may be overlooked pathogens that have cost-efficient testing methods, like TBEV, that can be introduced in a testing protocol and may enhance patient management and reduce unnecessary hospital stays.

Human granulocytic anaplasmosis is an emerging tick-borne disease. Although Anaplasma phagocytophilum has been identified in vectors and animal reservoirs in Romania, evidence of human exposure has not yet been reported. This study aimed to generate initial evidence of human infection by evaluating A. phagocytophilum antibodies in individuals with recent tick exposure. We conducted a cross-sectional serosurvey between 2023 and 2024 at a tertiary care hospital in Bucharest, enrolling 80 participants 4 to 12 weeks following a tick bite. Serum IgG antibodies against A. phagocytophilum were detected using an indirect immunofluorescence assay, with a titer of ≥1:64 considered indicative of seropositivity. Eight (10%) participants tested positive for A. phagocytophilum IgG antibodies. Seropositivity was not significantly associated with demographics, geographical region, or clinical symptoms. However, fatigue and myalgia were more frequently seen in A. phagocytophilum IgG seropositive individuals. Notably, 43.8% of all participants reported erythema migrans, including five of the eight individuals with positive A. phagocytophilum IgG serology. This study provides the first serological evidence of human exposure to A. phagocytophilum in Romania. A 10% seroprevalence in this high-risk group suggests that anaplasmosis may be underrecognized. Clinicians should consider it in patients with tick exposure and compatible symptoms.

Background/Objectives: Tick-borne encephalitis virus (TBEV) is an emerging vector-borne pathogen in Europe, yet it may be potentially underrecognized in countries reporting few clinical cases. Despite the small number of reported TBE cases, evidence of TBEV circulation in Romania has increased in recent years. Awareness and clinical practices among infectious disease physicians remain insufficiently characterized. This study evaluated changes in knowledge and clinical practices regarding TBEV infection among infectious disease physicians in Romania between 2019 and 2025, following the introduction of TBE vaccination. Methods: We conducted a repeated cross-sectional survey using the same questionnaire in two independent cohorts of infectious disease physicians in Romania (2019, n = 164; 2025 n = 140). Knowledge of tick-borne diseases, TBEV global and local epidemiology, diagnosis, and clinical practice was evaluated and compared. Results: Recognition of TBEV cases in Romania increased significantly from 95/164 (57.9%) in 2019 to 108/140 (77.1%) in 2025 (p = 0.004), while awareness of vaccination as a preventive measure increased from 37.8% to 65.7% (p < 0.001). However, increased awareness did not translate into increased diagnostic testing: TBEV testing remained limited (25.6% in 2019 vs. 20.0% in 2025) and lack of access to serological testing was the most frequently reported diagnostic barrier (65.5% in 2019 vs. 82.1% in 2025). Awareness of alimentary transmission through unpasteurized dairy products also remained limited. Conclusions: Romanian infectious disease physicians’ awareness regarding TBEV improved between 2019 and 2025, but diagnostic practice did not improve accordingly. The persistent gap between awareness and testing capacity may contribute to under recognition of TBE and highlights the need to strengthen diagnostic access alongside physician education and vaccination strategies.

Background: European and Romanian recommendations for Lyme neuroborreliosis (LNB) emphasise cerebrospinal fluid (CSF) pleocytosis and a positive intrathecal antibody index (IAI) for diagnostic confirmation. We assessed whether antibiotic treatment in suspected LNB aligns with diagnostic recommendations. Methods: We conducted a retrospective, single-centre cohort study of patients with suspected LNB between 2008 and 2025, all with positive first-tier ELISA (enzyme-linked immunosorbent assay) Borrelia serology. Patients were classified as confirmed, probable, possible, or ruled-out LNB based on clinical presentation, serology, and CSF findings. We assessed factors associated with antibiotic treatment and temporal changes in diagnostic work-up and treatment patterns. Results: Among 58 patients, LNB was confirmed in 4 (6.9%), probable in 11 (19.0%), possible in 31 (53.4%), and ruled out in 12 (20.7%). Antibiotics were administered to 47 patients (81.0%), of whom 68.1% had possible or ruled out LNB. Antibiotic treatment was more frequent in early-stage than in late-stage disease (38/42, 90.5% vs. 9/16, 56.2%; p = 0.006); no significant association was detected with confirmed (p = 1.000) or ruled-out (p = 0.681) status. Between 2008 and 2019 and 2023–2025, IAI testing increased significantly (35.1% vs. 71.4%, p = 0.013), yet overall antibiotic use remained unchanged (81.1% vs. 81.0%, p = 1.000). Conclusions: Antibiotic treatment was poorly aligned with diagnostic classification, with most treated patients lacking confirmation. Improved diagnostic evaluation over time was not accompanied by a reduction in antibiotic use, highlighting a critical need for stronger diagnostic and antibiotic stewardship.