An Overview of Tick-Borne Encephalitis Epidemiology in Endemic Regions of Continental Croatia, 2017-2023.

Tick-borne encephalitis (TBE) represents an important public health problem in Europe. We analyzed the epidemiology of TBE based on data from humans, animals, and Ixodes ricinus ticks in endemic regions of continental Croatia. In the period from 2017 to 2023, cerebrospinal fluid (CSF) and serum samples of 684 patients with neuroinvasive diseases, 2240 horse serum samples, and 300 sheep serum samples were tested for TBEV. In addition, 8751 I. ricinus ticks were collected. CSF samples were tested using RT-PCR. Serological tests (serum, CSF) were performed using commercial ELISA, with confirmation of cross-reactive samples by a virus neutralization test. Eighty-four autochthonous human TBEV cases were confirmed. The majority of patients were in the age group of 40-69 years (58.3%) with a male predominance (70.2%). TBE showed a bimodal seasonality with a large peak in April-August and a small one in October-November. In addition to humans, TBEV IgG antibodies were found in 12.2% of horses and 9.7% of sheep. Seasonal tick abundance corresponds to the reported number of human infections. Continental Croatia is still an active natural focus of TBE. Continuous monitoring of infections in humans, sentinel animals, and ticks is needed for the implementation of preventive measures.

Detection of Bhanja Bandavirus in Patients with Neuroinvasive Disease of Unknown Etiology in Croatia.

BACKGROUND: Although the Bhanja bandavirus (BHAV) is widely distributed in some European countries, human infections are rarely reported. This study analyzed the prevalence of BHAV antibodies in patients with neuroinvasive diseases of unsolved etiology. METHODS: A total of 254 Croatian patients who developed neurological symptoms during the four consecutive arbovirus transmission seasons (April 2017-October 2021) were tested. Cerebrospinal fluid (CSF) and urine samples were tested using RT-qPCR. In addition, CSF and serum samples were tested using a virus neutralization test. RESULTS: BHAV RNA was not detected in any samples, while neutralizing (NT) antibodies were detected in serum samples of 53/20.8% of patients (95% CI = 16.0-26.3). In two patients, BHAV NT antibodies were detected in the CSF, indicating a recent infection. Both patients were inhabitants of rural areas in continental Croatia, and one reported a tick bite two weeks before symptoms onset. The seropositivity was high in all age groups (15.2-29.1%). The majority of seropositive patients (94.3%) resided at altitudes less than 200 m above sea level. The prevalence rates correlated positively with population density and negatively with certain climate parameters (temperature, number of hot/warm days). CONCLUSIONS: The presented results indicate that BHAV is distributed in Croatia. Further studies are needed to determine the clinical significance of this neglected arbovirus.

Cross-Reactive Antibodies in Tick-Borne Encephalitis: Case Report and Literature Review.

Flaviviruses are a heterogeneous group of viruses that may induce broad antigenic cross-reactivity. We present a patient who was admitted to the infectious disease department with symptoms suggestive of aseptic meningitis. During the clinical workup, the patient reported a tick bite two weeks before the disease onset. High titers of IgM and IgG antibodies to tick-borne encephalitis virus (TBEV) were found in both serum and cerebrospinal fluid (CSF) samples, indicating acute TBEV infection. West Nile virus (WNV) and Usutu virus (USUV) IgM and/or IgG antibodies were also detected, and a virus neutralization test (VNT) was performed. A high titer of TBEV neutralizing (NT) antibodies (640) was detected, which confirmed acute TBE. However, NT antibodies to WNV and USUV were also detected (titer 80 for both viruses). After TBEV and WNV IgG avidity evaluation, previous flavivirus infection was highly suspected (avidity index 82% and 89%, respectively). Blood, CSF, and urine samples were negative for respective viruses' RNA. The presented case highlights the challenges in flavivirus serodiagnosis. In the published literature, different degrees of cross-reactivity or cross-neutralization between TBEV and dengue, louping ill, Omsk hemorrhagic fever, Langat, and Powassan virus were also observed. Therefore, the serology results should be interpreted with caution, including the possibility of cross-reactivity. In areas where several flaviviruses co-circulate VNT is recommended for disease confirmation.

Detection of Tahyna Orthobunyavirus-Neutralizing Antibodies in Patients with Neuroinvasive Disease in Croatia.

BACKGROUND: Tahyna orthobunyavirus (TAHV) is widely distributed in continental Europe. Very few studies have analyzed TAHV seroprevalence in Croatia. We analyzed the prevalence of TAHV RNA and antibodies in Croatian patients with neuroinvasive disease (NID). METHODS: A total of 218 patients with unsolved NID detected during five consecutive arbovirus transmission seasons (April 2017-October 2021) were tested. Cerebrospinal fluid (CSF) and urine samples were tested for TAHV RNA using RT-PCR. In addition, CSF and serum samples were tested for TAHV antibodies using a virus neutralization test (VNT). RESULTS: Clinical presentations in patients with NID were meningitis (141/64.7%), meningoencephalitis (56/25.7%), myelitis (8/3.7%), and 'febrile headache' (13/5.9%). TAHV RNA was not detected in any of the tested CSF or urine samples; however, TAHV-neutralizing (NT) antibodies were detected in 22/10.1% of patients. Detection of NT antibodies in the CSF of two patients presenting with meningitis suggested recent TAHV infection. TAHV seropositivity increased significantly with age, from 1.8% to 24.4%. There was no difference in seroprevalence between genders or areas of residence (urban, suburban/rural). The majority of seropositive patients (90.9%) resided in floodplains along the rivers in continental Croatia. CONCLUSIONS: The presented results confirm that TAHV is present in Croatia. The prevalence and clinical significance of TAHV infection in the Croatian population have yet to be determined.

Severe West Nile Virus Neuroinvasive Disease: Clinical Characteristics, Short- and Long-Term Outcomes.

West Nile Virus Neuroinvasive Disease (WNV NID) requires prolonged intensive care treatment, resulting in high mortality and early disability. Long-term results are lacking. We have conducted an observational retrospective study with a prospective follow-up of WNV NID patients treated at the Intensive Care Unit (ICU), University Hospital for Infectious Diseases, Zagreb, Croatia, 2013-2018. Short-term outcomes were vital status, length of stay (LOS), modified Rankin Scale (mRS), and disposition at discharge. Long-term outcomes were vital status and mRS at follow-up. Twenty-three patients were identified, 78.3% males, median age 72 (range 33-84) years. Two patients (8.7%) died in the ICU, with no lethal outcomes after ICU discharge. The median ICU LOS was 19 days (range 5-73), and the median hospital LOS was 34 days (range 7-97). At discharge, 15 (65.2%) patients had moderate to severe/mRS 3-5, 6 (26.0%) had slight disability/mRS 2-1, no patients were symptom-free/mRS 0. Ten (47.6%) survivors were discharged to rehabilitation facilities. The median time to follow-up was nine months (range 6-69). At follow-up, seven patients died (30.5%), five (21.7%) had moderate to severe/mRS 3-5, one (4.3%) had slight disability/mRS 2-1, six (26.1%) had no symptoms/mRS 0, and four (17.4%) were lost to follow-up. Briefly, ten (43.5%) survivors improved their functional status, one (4.3%) was unaltered, and one (4.3%) aggravated. In patients with severe WNV NID, intensive treatment in the acute phase followed by inpatient rehabilitation resulted in significant recovery of functional status after several months.

Antiviral Cytokine Response in Neuroinvasive and Non-Neuroinvasive West Nile Virus Infection.

Data on the immune response to West Nile virus (WNV) are limited. We analyzed the antiviral cytokine response in serum and cerebrospinal fluid (CSF) samples of patients with WNV fever and WNV neuroinvasive disease using a multiplex bead-based assay for the simultaneous quantification of 13 human cytokines. The panel included cytokines associated with innate and early pro-inflammatory immune responses (TNF-α/IL-6), Th1 (IL-2/IFN-γ), Th2 (IL-4/IL-5/IL-9/IL-13), Th17 immune response (IL-17A/IL-17F/IL-21/IL-22) and the key anti-inflammatory cytokine IL-10. Elevated levels of IFN-γ were detected in 71.7% of CSF and 22.7% of serum samples (p = 0.003). Expression of IL-2/IL-4/TNF-α and Th1 17 cytokines (IL-17A/IL-17F/IL-21) was detected in the serum but not in the CSF (except one positive CSF sample for IL-17F/IL-4). While IL-6 levels were markedly higher in the CSF compared to serum (CSF median 2036.71, IQR 213.82-6190.50; serum median 24.48, IQR 11.93-49.81; p < 0.001), no difference in the IL-13/IL-9/IL-10/IFN-γ/IL-22 levels in serum/CSF was found. In conclusion, increased concentrations of the key cytokines associated with innate and early acute phase responses (IL-6) and Th1 type immune responses (IFN-γ) were found in the CNS of patients with WNV infection. In contrast, expression of the key T-cell growth factor IL-2, Th17 cytokines, a Th2 cytokine IL-4 and the proinflammatory cytokine TNF-α appear to be concentrated mainly in the periphery.

Prevalence and molecular epidemiology of West Nile and Usutu virus infections in Croatia in the 'One health' context, 2018.

In 2018, Croatia reported the largest outbreak of West Nile virus (WNV) infections as well as the re-occurrence of human Usutu virus (USUV) infections. For the first time, fatal WNV and USUV infections were detected in wild birds. We analysed epidemiological characteristics and molecular epidemiology of WNV and USUV infections detected during 2018 transmission season. From April to November, 178 patients with neuroinvasive disease and 68 patients with febrile disease were tested for WNV and USUV. Viral RNA was detected in cerebrospinal fluid (CSF) and urine samples using a real-time RT-PCR. Positive samples were tested by nested RT-PCR and nucleotide sequencing. IgM/IgG antibodies were detected in serum/CSF samples using ELISA with confirmation of cross-reactive samples by virus neutralization test (VNT). WNV neuroinvasive disease was confirmed in 54 and WNV fever in seven patients from 10 continental Croatian counties. Areas affected in 2018 were those in which cases occurred in previous seasons, while in three areas human cases were reported for the first time. Phylogenetic analysis of six strains from patients residing in different geographic areas showed circulation of WNV lineage 2. In three patients, neuroinvasive USUV infection was confirmed by RT-PCR or VNT. Sequence analysis of one detected strain revealed USUV Europe 2 lineage. During the same period, a total of 2,574 horse and 1,069 poultry serum samples were tested for WNV antibodies using ELISA. Acute asymptomatic WNV infection (IgM antibodies) was documented in 20/0.7% horses. WNV IgG antibodies were found in 307/11.9% horses and in 125/12.7% poultry. WNV RNA was detected in two goshawks and USUV RNA was detected in one blackbird from north-western Croatia. In the Zagreb area, 3,670 female mosquitoes were collected. One Culex pipiens pool collected in July tested positive for USUV RNA. Our results highlight the importance of continuous multidisciplinary 'One health' surveillance of these emerging arboviruses.

Diagnostic significance of immunoglobulin G avidity in symptomatic and asymptomatic West Nile virus infection.

INTRODUCTION: West Nile virus (WNV) immunoglobulin M (IgM) antibodies have been shown to persist for up to 500 days in certain patients. To evaluate the usefulness of immunoglobulin G (IgG) avidity assessment in the diagnosis of WNV infection, we analyzed 54 WNV IgM- and/or IgG-positive serum samples from 39 patients with neuroinvasive disease and 15 asymptomatic cases tested during a seroprevalence investigation. METHODS: Serological tests (WNV IgM/IgG antibody detection, IgG avidity) were performed using commercially available enzyme-linked immunosorbent assays. RESULTS: WNV IgM antibodies were detected in 47 (87%) samples. Acute/recent WNV infection was confirmed based on low/borderline avidity index (AI) in 44 IgM-positive samples (93.6%). In three IgM-positive samples (6.4%), high IgG AIs were detected, thus indicating persisting IgM antibodies from previous infections. All IgM-negative samples showed high AIs. Patients with WNV neuroinvasive disease tested within 30 days showed low AIs. In six patients tested 34-50 days after disease onset, AI was borderline (42%-60%), suggesting earlier WNV IgG maturation. Samples with the highest IgM values were associated with the lowest AIs (Spearman's rho coefficient -0.767, p < 0.001). CONCLUSIONS: Our results indicate that IgG avidity differentiates current/recent WNV infection from persistent IgM seropositivity from the previous WNV transmission season both in patients with WNV neuroinvasive disease and in asymptomatic persons. A strong negative correlation between IgM antibody levels and AI indicates that in cases with very high IgM levels, determination of IgG avidity may not be necessary. As many patients showed rapid avidity maturation, low IgG avidity is indicative of WNV infection within the previous month.

Diagnostic significance of immunoglobulin G avidity in symptomatic and asymptomatic West Nile virus infection

Abstract INTRODUCTION West Nile virus (WNV) immunoglobulin M (IgM) antibodies have been shown to persist for up to 500 days in certain patients. To evaluate the usefulness of immunoglobulin G (IgG) avidity assessment in the diagnosis of WNV infection, we analyzed 54 WNV IgM- and/or IgG-positive serum samples from 39 patients with neuroinvasive disease and 15 asymptomatic cases tested during a seroprevalence investigation. METHODS Serological tests (WNV IgM/IgG antibody detection, IgG avidity) were performed using commercially available enzyme-linked immunosorbent assays. RESULTS WNV IgM antibodies were detected in 47 (87%) samples. Acute/recent WNV infection was confirmed based on low/borderline avidity index (AI) in 44 IgM-positive samples (93.6%). In three IgM-positive samples (6.4%), high IgG AIs were detected, thus indicating persisting IgM antibodies from previous infections. All IgM-negative samples showed high AIs. Patients with WNV neuroinvasive disease tested within 30 days showed low AIs. In six patients tested 34-50 days after disease onset, AI was borderline (42%-60%), suggesting earlier WNV IgG maturation. Samples with the highest IgM values were associated with the lowest AIs (Spearman's rho coefficient -0.767, p < 0.001). CONCLUSIONS Our results indicate that IgG avidity differentiates current/recent WNV infection from persistent IgM seropositivity from the previous WNV transmission season both in patients with WNV neuroinvasive disease and in asymptomatic persons. A strong negative correlation between IgM antibody levels and AI indicates that in cases with very high IgM levels, determination of IgG avidity may not be necessary. As many patients showed rapid avidity maturation, low IgG avidity is indicative of WNV infection within the previous month.