Proinflammatory Chemokine Levels in Cerebrospinal Fluid of Patients with Neuroinvasive Flavivirus Infections.

Tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) are the most important neuroinvasive arboviruses detected in Europe. In this study, we analyzed cerebrospinal fluid (CSF) concentrations of 12 proinflammatory chemokines (CCL2, CCL3, CCL4, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, and CXCL11) in 77 patients with neuroinvasive diseases (NIDs). Flavivirus infection was confirmed in 62 patients (TBEV and WNV in 31 patients each), while in 15 patients the etiology of NID was not determined (NDE). Similar patterns of high-level expression of chemokines regulating monocyte/macrophage responses (CCL2), neutrophil recruitment (CXCL1 and CXCL8), and interferon-inducible chemoattractants for leukocytes (CXCL10 and CXCL11) have been observed in WNV and TBEV groups. None of the tested chemokines significantly differed between patients with TBEV or WNV. Concentrations of CCL17, CCL20, CXCL5, CXCL10, and CXCL11 were significantly lower in both WNV and TBEV groups compared to NID NDE patients. The logistic regression model showed that CSF concentrations of CXCL11, CXCL5, and CXCL10 could potentially be used for the classification of patients into the WNV or TBEV group versus groups with other NIDs. This study identified, for the first time, similar patterns of CSF chemokine expression in WNV and TBEV infections, suggesting common immunopathogenic mechanisms in neuroinvasive flavivirus infections that should be further evaluated.

The Impact of Latent Cytomegalovirus Infection on Spontaneous Abortion History and Pregnancy Outcomes in Romanian Pregnant Women.

Cytomegalovirus (CMV), a DNA virus that belongs to the Orthoherpesviridae family, infects 40-100% of people. Primary/non-primary CMV infection during pregnancy could cause fetal disabilities. After primary infection, CMV causes a latent infection and resides in cells of the myeloid compartment (CD34+, monocytes). Few studies have analyzed the impact of latent CMV infections on miscarriage history, pregnancy complications, and neonatal outcomes. METHODS: Serum samples from 806 pregnant women (28.29 ± 4.50 years old) who came for a consultation at the Timisoara Clinical Emergency City Hospital between 2008 and 2010 were tested for anti-CMV IgM/IgG antibodies, and data about demography, obstetrical history, pregnancy complications, birth, and neonate were collected. The data were compared between the groups with and without latent CMV infection, and statistical significance was calculated. RESULTS: We did not find a difference regarding cesarean section (OR = 0.916, p = 0.856), placental abruption (OR = 1.004, p = 1.00), pregnancy-induced hypertension rate (OR = 1.078, p = 1.00), secondary sex ratio (0.882, p = 0.857), APGAR score (p = 0.225), gestational age at birth (p = 0.434), or birth weight (p = 0.365). A borderline significant difference was found regarding the presence of miscarriage history: OR = 8.467, p = 0.051. CONCLUSIONS: The presence of latent CMV infection does not affect the likelihood of complications in healthy women. A borderline significantly higher prevalence of miscarriage history was found in women with latent CMV infection.

Seroepidemiology of Herpes Simplex Viruses Type 1 and 2 in Pregnant Women in Croatia.

Background and Objectives: Herpes simplex viruses (HSV-1 and HSV-2) are one of the most widespread causes of human viral infections. In Croatia, only two published studies have analyzed the seroprevalence of HSV infections in childbearing-aged and pregnant women (2005-2010), while more recent data are lacking. This study aimed to analyze the prevalence and risk factors for HSV-1 and HSV-2 infections among pregnant women in Croatia in the period from 2011 to 2021. Materials and Methods: This study included 667 pregnant women aged 16-45 years submitted for HSV-1 and HSV-2 serology testing. Serum samples were initially screened for HSV-1 and HSV-2 IgM and IgG antibodies using a commercial ELISA test with a confirmation of HSV-2-positive samples using an immunoblot assay. Results: The overall IgG seroprevalence rates were 69.9% for HSV-1 and 3.8% for HSV-2. A significant gradual increase in the HSV-2 seroprevalence with age was observed from 0.5% in participants under 30 years to 8.3% in participants above 40 years. The HSV-1 seroprevalence was stable up to 40 years (70.0 and 68.3%, respectively), with an increase to 86.1%, but this difference did not reach statistical significance. Area of residence (urban or suburban/rural), geographic region (continental or coastal), and obstetric history (normal pregnancy or unfavorable obstetric history) were not associated with HSV-1 and HSV-2 seroprevalence. Older age was found to be a significant risk factor for HSV-2 seropositivity in both univariate and multivariate risk analysis. Conclusions: HSV-1 infection is widely prevalent among pregnant women with a stable trend over time. However, a declining trend in the HSV-2 seroprevalence was observed compared to 2005-2010. Serological screening in pregnant women is important in identifying seronegative women who are susceptible to HSV infection as well as seropositive women who are at risk for genital herpes recurrence during delivery.

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.

Hantavirus Pulmonary Syndrome Caused by Puumala Orthohantavirus-A Case Report and Literature Review.

In this article, we report on a rare case of acute respiratory distress syndrome (ARDS) caused by the Puumala orthohantavirus (PUUV), which is typically associated with hemorrhagic fever with renal syndrome (HFRS). This is the first documented case of PUUV-associated ARDS in Southeast Europe. The diagnosis was confirmed by serum RT-PCR and serology and corroborated by phylogenetic analysis and chemokine profiling. The patient was a 23-year-old male from Zagreb, Croatia, who had recently traveled throughout Europe. He presented with fever, headache, abdominal pain, and sudden onset of ARDS. Treatment involved high-flow nasal cannula oxygen therapy and glucocorticoids, which resulted in a full recovery. A systematic literature review identified 10 cases of hantavirus pulmonary syndrome (HPS) caused by PUUV in various European countries and Turkey between 2002 and 2023. The median age of patients was 53 years (range 24-73), and six of the patients were male. Most patients were treated in intensive care units, but none received antiviral therapy targeting PUUV. Eight patients survived hospitalization. The presented case highlights the importance of considering HPS in the differential diagnosis of ARDS, even in areas where HFRS is the dominant form of hantavirus infection.

Neutralizing Activity of SARS-CoV-2 Antibodies in Patients with COVID-19 and Vaccinated Individuals.

BACKGROUND: Serological diagnosis of COVID-19 is complex due to the emergence of different SARS-CoV-2 variants. METHODS: 164 serum samples from (I) patients who recovered from COVID-19 (n = 62) as well as (II) vaccinated individuals (n = 52) and (III) vaccinated individuals who were infected with different SARS-CoV-2 variants after vaccination (n = 50) were included. All samples were tested using EIA (binding antibodies) and a virus neutralization test (VNT) using the Wuhan strain (NT antibodies). Group III was further tested with a VNT using the Alpha/Delta/Omicron strains. RESULTS: The highest antibody index (AI) was observed in vaccinated individuals infected with COVID-19 (median AI = 50, IQR = 27-71) and the lowest in vaccinated individuals (median AI = 19, IQR = 8-48). Similarly, NT antibody titer was highest in vaccinated individuals infected with COVID-19 (median 128; IQR = 32-256) compared to vaccinated individuals (median 32, IQR = 4-128) and patients with COVID-19 (median 32, IQR = 8-64). The correlation between AI and NT titer was strongly positive in vaccinated individuals and moderately positive in patients with COVID-19. No significant correlation was observed in vaccinated individuals infected with COVID-19. In patients infected with Alpha and Delta, the lowest VNT positivity rate was for the Omicron variant (85.0%/83.3%). Patients infected with the Alpha variant showed the lowest NT titer for the Omicron variant (median titer 32) compared to the Wuhan/Delta variants (64/128). Patients infected with the Delta variant had the lowest NT titer to the Omicron variant (median 32), compared to the Wuhan/Alpha variants (64/128). Patients infected with the Omicron variant showed similar titers to the Delta/Wuhan variants (128) and higher to the Alpha variant (256). CONCLUSIONS: The cross-immunity to SARS-CoV-2 is lowest for the Omicron variant compared to the Alpha/Delta variants.

Current Status of Vector-Borne Diseases in Croatia: Challenges and Future Prospects.

Different vector-borne pathogens are present or have (re-)emerged in Croatia. Flaviviruses tick-borne encephalitis (TBEV), West Nile (WNV), and Usutu (USUV) are widely distributed in continental regions, while Toscana virus (TOSV) and sandfly fever viruses are detected at the Croatian littoral. Recently, sporadic clinical cases of Tahyna orthobunyavirus (TAHV) and Bhanja bandavirus infection and seropositive individuals have been reported in continental Croatia. Acute infections and serologic evidence of WNV, TBEV, USUV, and TAHV were also confirmed in sentinel animals and vectors. Autochthonous dengue was reported in 2010 at the Croatian littoral. Lyme borreliosis is the most widely distributed vector-borne bacterial infection. The incidence is very high in northwestern and eastern regions, which correlates with numerous records of Ixodes ricinus ticks. Acute human Anaplasma phagocytophilum infections are reported sporadically, but there are many records of serologic evidence of anaplasmosis in animals. Mediterranean spotted fever (Rickettsia conorii) and murine typhus (Rickettsia typhi) are the main rickettsial infections in Croatia. Human leishmaniasis is notified sporadically, while serologic evidence of leishmaniasis was found in 11.4% of the Croatian population. After the official eradication of malaria in 1964, only imported cases were reported in Croatia. Since vector-borne diseases show a growing trend, continuous monitoring of vectors is required to protect the population from these infections.

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.

Serological Response to SARS-CoV-2 Vaccine in Hemodialyzed Patients and the Association with Later COVID-19 Positivity.

BACKGROUND: The effectiveness of the COVID-19 vaccine may differ in hemodialysis patients. The aim of this prospective multicenter study was to determine the degree of serological response to the SARS-CoV-2 vaccine in the population of dialysis patients and its association with later SARS-CoV-2 infections. METHODS: A blood sample was taken for the determination of COVID-19 serological status (IgG antibodies) in 706 dialysis patients 16 weeks after vaccination with the second dose (Pfizer-BioNTech). RESULTS: Only 314 (44.5%) hemodialyzed patients had a satisfactory response to the COVID-19 vaccine. Eighty-two patients (11.6%) had a borderline response, while 310 patients (43.9%) had an unsatisfactory (negative) post-vaccinal antibody titer. A longer dialysis vintage had an increased odds ratio (OR) of 1.01 for the occurrence of COVID-19 positivity after vaccination. In the group of subsequently positive patients, 28 patients (13.6%) died from complications of COVID-19. We have found differences in mean survival time between patients with and without appropriate responses to vaccination in favor of patients with a satisfactory serological response. CONCLUSIONS: The results showed that the dialysis population will not have the same serological response to the vaccine as the general population. The majority of dialysis patients did not develop a severe clinical picture or die at the time of positivity for COVID-19.

Detection of SARS-CoV-2 Antibodies: Comparison of Enzyme Immunoassay, Surrogate Neutralization and Virus Neutralization Test.

BACKGROUND: Since sensitivity and specificity vary widely between tests, SARS-CoV-2 serology results should be interpreted with caution. METHODS: The study included serum samples from patients who had recovered from COVID-19 (n = 71), individuals vaccinated against SARS-CoV-2 (n = 84), and asymptomatic individuals (n = 33). All samples were tested for the presence of binding antibodies (enzyme immunoassay; EIA), neutralizing (NT) antibodies (virus neutralization test; VNT), and surrogate NT (sNT) antibodies (surrogate virus neutralization test; sVNT) of SARS-CoV-2. RESULTS: SARS-CoV-2-binding antibodies were detected in 71 (100%) COVID-19 patients, 77 (91.6%) vaccinated individuals, and 4 (12.1%) control subjects. Among EIA-positive samples, VNT was positive (titer ≥ 8) in 100% of COVID-19 patients and 63 (75.0%) of the vaccinated individuals, while sVNT was positive (>30% inhibition) in 62 (87.3%) patients and 59 (70.2%) vaccinated individuals. The analysis of antibody levels showed a significant moderate positive correlation between EIA and VNT, a moderate positive correlation between EIA and sVNT, and a strong positive correlation between VNT and sVNT. The proportion of positive sVNT detection rate was associated with VNT titer. The lowest positivity (72.4%/70.8%) was detected in samples with low NT titers (8/16) and increased progressively from 88.2% in samples with titer 32 to 100% in samples with titer 256. CONCLUSIONS: sVNT appeared to be a reliable method for the assessment COVID-19 serology in patients with high antibody levels, while false-negative results were frequently observed in patients with low NT titers.

Seroprevalence Trends and Molecular Epidemiology of Viral Hepatitis in Croatia.

Viral hepatitis is a significant cause of morbidity and mortality worldwide. In Croatia, hepatitis B virus (HBV) and hepatitis C virus (HCV) are widely distributed, especially in some high-risk groups such as people who inject drugs (PWID), prisoners, and highly promiscuous groups. The seroprevalence of HBV ranges from 7.0% in the general population to 38.8% in PWID, depending on the region. The seroprevalence of HCV is highest among PWID (29-75.5%) as compared to 0.9% in the general population. Analyzing the distribution of HCV genotypes, no substantial changes in the molecular epidemiology of the two most frequent HCV genotypes (1 and 3) in the past 20 years were observed. However, the predominance of subtype 1b compared to subtype 1a as detected in 1996-2005 was not confirmed in 2008-2015. Hepatitis A virus (HAV) incidence was high in the past with a decreasing trend since the 2000s, except for an outbreak in 2017-2018 as part of the large European outbreak, which was mainly among men who have sex with men. Hepatitis E virus (HEV) is an emerging virus detected for the first time in Croatia in 2012. The seroprevalence of HEV is high among hemodialysis patients (27.9%) and liver transplant recipients (19.3-24.4%). In addition, higher seroprevalence rates were observed in animal-related professions (e.g., veterinarians, 15.2%; hunters, 14.9%). All detected HEV strains belonged to genotype 3.

Prevalence of JC Polyomavirus in Patients with Neuroinvasive Disease of Unknown Etiology in Croatia.

Background and Objectives: John Cunningham polyomavirus (JCPyV) is a highly prevalent virus in the human population. The prevalence of JCPyV in patients with central nervous system disorders has not been examined extensively. The aim of this study was to analyze the prevalence of JCPyV DNA/antibodies in patients with neuroinvasive diseases (NID) of unknown etiology. Materials and Methods: The study included 132 patients with NID (febrile headache, meningitis, encephalitis) tested from January 2021 to December 2022. The control group consisted of 47 asymptomatic individuals. In patients with NID, serum and cerebrospinal fluid (CSF) samples were collected in the acute phase of the disease. CSF samples were tested for JCPyV DNA (PCR), while serum samples were tested for JCPyV IgG antibodies (ELISA). In controls, serum samples were tested for JCPyV IgG antibodies (ELISA). Results: JCPyV DNA was not detected in any of the CSF samples from patients with NID. JCPyV IgG antibodies were detected in 88.6% of patients and 74.5% of controls (p < 0.001). In the patients' group, a significant difference in the IgG prevalence was observed between males (94.6%) and females (81.0%). In addition, significant differences in the seropositivity between age groups were found. The lowest seroprevalence (28.6%) was in patients less than 20 years, followed by a sharp increase in the 20-29-year group (69.2%), after which the seroprevalence remained stable (90.0-94.1%) in patients up to 69 years. All patients older than 70 years were JCPyV IgG-seropositive. No significant difference in the seroprevalence was found in patients presenting with febrile headache (81.6%), meningitis (93.3%), or meningoencephalitis (91.3%). No difference in the seropositivity between genders was found in controls. Although the seropositivity steadily increased in older participants, these differences were not significant. Analyzing the JCPyV antibody levels in patients with NID, the median antibody titers differed significantly between groups, ranging from 248 AU/mL (younger age groups) to 400 AU/mL (older age groups). Conclusions: Higher seroprevalence in the patients' group highlights the need to further investigate the possible association of JCPyV and NID.

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.

Quantification of Antiviral Cytokines in Serum, Cerebrospinal Fluid and Urine of Patients with Tick-Borne Encephalitis in Croatia.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is one of the most significant arboviruses affecting the human central nervous system (CNS) in Europe. Data on cytokine response in TBEV infection are limited. METHODS: We analyzed the cytokine response in serum, cerebrospinal fluid (CSF) and urine samples of patients with TBE. The control group consisted of patients with 'febrile headache' who had normal CSF cytology. The panel included 12 cytokines: TNF-α, IL-6, Th1 (IL-2, IFN-γ), Th2 (IL-4, IL-5, IL-13), Th9 (IL-9), Th17 (IL-17A, IL-17F), Th22 (IL-22) cytokines and IL-10. RESULTS: TBE patients were more likely to have increased levels of IL-6 and IFN-γ in CSF compared to controls (85.7% vs. 58.8% and 85.7% vs. 47.1%, respectively). However, concentrations of IL-6 (the most abundant cytokine in the CSF of both groups), IL-10 and IL-9 were lower in TBEV patients compared with controls, but the difference was statistically significant for IL-9 only (p = 0.001). By analyzing the cytokine levels in different clinical samples, all measured cytokines were detected in the serum, with the highest concentrations found for IFN-γ, TNF-α, IL-10, IL-17F and IL-22. Higher concentrations of cytokines in the CSF compared with serum were observed for IL-5, IL-6 and IL-22. All cytokines except IL-13 were detectable in urine but in a small proportion of patients, except for IL-22, which was detectable in 95.8% of patients. CONCLUSIONS: Cytokine composition in different clinical samples of TBE patients reveals a different network of early innate immune response cytokines, Th1, Th2, Th9, Th22, Th17 and anti-inflammatory cytokines.

Surveillance of Tahyna Orthobunyavirus in Urban Areas in Croatia-The "One Health" Approach.

BACKGROUND: Tahyna orthobunyavirus (TAHV) is a neglected mosquito-borne bunyavirus. Although the virus is widespread in continental Europe, TAHV infections are rarely reported. We analyzed the prevalence of TAHV in humans and different animal species as well as mosquitoes collected in urban areas of Zagreb and its surroundings in the period from 2020 to 2022. METHODS: The study included 32 patients with neuroinvasive disease (NID), 218 asymptomatic individuals, 98 horses, 94 pet animals (dogs and cats), and 4456 Aedes vexans mosquitoes. Cerebrospinal fluid (CSF) and urine samples of patients with NID were tested for the TAHV RNA using a real-time reverse transcription-polymerase chain reaction (RT-qPCR). Human and animal serum samples were tested for TAHV-neutralizing (NT) antibodies using a virus-neutralization test (VNT). Mosquito pools were tested for TAHV RNA using an RT-qPCR. RESULTS: TAHV NT antibodies were detected in 3/9.4% of patients with NID, 8/3.7% of asymptomatic individuals, 29/29.6% of horses, and 11/11.7% of pet animals. There was no difference in the seroprevalence according to age, sex, and area of residence in asymptomatic individuals. In addition, TAHV seropositivity did not differ according to age and sex in pet animals. None of the tested mosquito pools was TAHV RNA-positive. CONCLUSIONS: The presented results highlight the importance of interdisciplinary surveillance ("One Health") of this neglected viral zoonosis.

Insights into hepatitis E virus epidemiology in Croatia.

BACKGROUND: Hepatitis E virus (HEV) is an emerging virus of global health concern. The seroprevalence rates differ greatly according to geographic region and population group. AIM: To analyze the seroprevalence of HEV in exposed (animal-related professions) and nonexposed populations, as well as solid organ and hematopoietic stem cell transplant patients. METHODS: Forestry workers (n = 93), hunters (n = 74), and veterinarians (n = 151) represented the exposed population. The general population (n = 126) and pregnant women (n = 118) constituted the control group. Transplant patients included liver transplant recipients (LTRs) (n = 83), kidney transplant recipients (KTRs) (n = 43), and hematopoietic stem cell transplant recipients (HSCRs) (n = 39). HEV immunoglobulin G antibodies were detected using the enzyme-linked immunosorbent assay and confirmed by the immunoblot test. RESULTS: The HEV seroprevalence significantly differed between groups: Veterinarians 15.2%, hunters 14.9%, forestry workers 6.5%, general population 7.1%, and pregnant women 1.7%. In transplant patients, the seropositivity was highest in LTRs (19.3%), while in KTRs and HSCRs, the seroprevalence was similar to the general population (6.9% and 5.1%, respectively). A significant increase in seropositivity with age was observed from 2.9% in individuals less than 30 years to 23.5% in those older than 60 years. Sociodemographic characteristics (sex, educational level, area of residence, and number of household members), eating habits (game meat, offal, and pork products consumption), and environmental and housing conditions (drinking water supply, type of water drainage/sewer, waste disposal, domestic animals) were not associated with HEV seropositivity. However, individuals who reported a pet ownership were more often seropositive compared to those who did not have pet animals (12.5% vs 7.0%). CONCLUSION: The results of this study showed that individuals in professional contact with animals and LTRs are at higher risk for HEV infection. In addition, age is a significant risk factor for HEV seropositivity.

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.

Comparison of indirect immunofluorescence and western blot method in the diagnosis of hantavirus infections.

BACKGROUND: Serologic cross-reactivity between hantaviruses often complicates the interpretation of the results. AIM: To analyze the diagnostic value of indirect immunofluorescence assay (IFA) and western blot (WB) in the diagnosis of hantavirus infections. METHODS: One hundred eighty-eight serum samples from Puumala (PUUV) and Dobrava (DOBV) orthohantavirus infected patients were analyzed. Serology was performed using commercial tests (Euroimmun, Lübeck, Germany). RESULTS: Using IFA, 49.5% of acute-phase samples showed a monotypic response to PUUV, while 50.5% cross-reacted with other hantaviruses. The overall cross-reactivity was higher for immunoglobulin G (IgG) (50.0%) than for immunoglobulin M (IgM) (25.5%). PUUV IgM/IgG antibodies showed low/moderate reactivity with orthohantaviruses Hantaan (12.3%/31.5%), Seoul (7.5%/17.8%), DOBV (5.4%/ 28.1%), and Saaremaa (4.8%/15.7%). Both DOBV IgM and IgG antibodies were broadly reactive with Hantaan (76.2%/95.2%), Saaremaa (80.9%/83.3%), and Seoul (78.6%/85.7%) and moderate with PUUV (28.5%/38.1%). Using a WB, serotyping was successful in most cross-reactive samples (89.5%). CONCLUSION: The presented results indicate that WB is more specific than IFA in the diagnosis of hantavirus infections, confirming serotype in most IFA cross-reactive samples.

Parvovirus B19 in Croatia: A Large-Scale Seroprevalence Study.

Background and Objectives: Seroepidemiological studies indicate that parvovirus B19 circulates in all areas of the world, although with some differences. The aim of this study is to analyze the seroprevalence of parvovirus B19 in the Croatian population. Materials and Methods: From 2010 to 2021, 1538 serum samples from different populations were tested for the presence of parvovirus B19 IgM/IgG antibodies. Serological tests were performed using a commercial enzyme-linked immunosorbent assay. Results: IgG antibodies were detected in 986/64.1% of participants with differences (p < 0.001) among the following population groups: 42.4% of children and adolescents, 67.1% of the adult general population, 66.7% of hemodialysis patients, and 65.6% of liver transplant recipients. Seroprevalence increased with age, from 30.0% in the 6 months-9 years age group to 69.0% in the 40-49 years age group, and remained stable thereafter (68.8-73.3%). There was no difference in the seropositivity among males (66.1%) and females (63.1%), as well as the place of residence (suburban/rural 63.9%, urban 64.1%). IgM antibodies (current/recent infection) were found in 61/4.0% of participants with the highest seropositivity in the youngest age group (11.1%). In pregnant women, seroprevalence was higher in women with an unfavorable obstetric history compared with a normal pregnancy (IgG 71.0% vs. 62.6%; IgM 6.5% vs. 2.4%), but these differences were not significant. Logistic regression showed that the adult population had almost three times higher risk of IgG seropositivity compared to children/adolescents (general population OR = 2.777, 95% CI = 2.023-3.812; hemodialysis patients OR = 2.586, 95% CI = 1.531-4.367; and transplant patients OR = 2.717, 95% CI = 1.604-4.603). A one-year increase in age increased the risk of IgG seroprevalence (OR = 1.017; 95% CI = 1.011-1.022). Conclusions: Older age was the main risk factor for IgG seropositivity. Hemodialysis and organ transplantation seem unrelated to the increased parvovirus B19 seroprevalence. The role of parvovirus B19 in the etiology of TORCH infections needs to be studied further.

Emerging Trends in the West Nile Virus Epidemiology in Croatia in the 'One Health' Context, 2011-2020.

West Nile virus (WNV) is one of the most widely distributed (re-)emerging arboviruses. In Croatia, acute WNV infections as well as seropositivity were detected in humans, horses, birds and poultry. Although serologic evidence of WNV human infections dates back to the 1970s, no clinical cases were reported until 2012. WNV outbreaks, as well as sporadic infections, were continuously recorded in continental Croatian counties from 2012 to 2018. In addition, acute asymptomatic infections (IgM antibodies) in horses have been regularly notified in continental regions since 2012, while seropositive horses (seroprevalence rates 3.7-21.4%) were detected in both continental and coastal regions. Moreover, WNV seropositivity in poultry (1.8-22.9%) was reported from 2013 to 2020. During the largest WNV outbreak in 2018, WNV RNA was detected for the first time in two dead goshawks (Accipiter gentilis) from the same aviary in North-West Croatia, while WNV antibodies were found in one buzzard (Butteo butteo) from the same region. In addition, WNV RNA was detected in a dead blackbird (Turdus merula) at the Croatian littoral. The phylogenetic analysis of 11 strains detected in urine samples of patients with neuroinvasive disease and 1 strain detected in a goshawk showed circulation of WNV lineage 2. Thus far, WNV has not been detected in mosquitoes in Croatia.