Spread of Puumala Hantavirus to New Areas in a Large Croatian Outbreak of Hemorrhagic Fever with Renal Syndrome, 2021.

Background: The largest documented outbreak of hemorrhagic fever with renal syndrome occurred in Primorje-Gorski Kotar County, Croatia, in 2021, marking the first-time cases of hantavirus infection recorded outside of the known endemic region in the north of the county. Aim: To identify the factors contributing to the spread of the outbreak and to compare risk factors for acquiring hantavirus infection in the endemic and newly affected regions. Methods and Results: A total of 189 cases were confirmed by positive Puumala IgM/IgG antibodies (93.6%), and 13 probable cases were identified by clinical and epidemiological data (6.4%) using a structured questionnaire. Of the 179 cases with available clinical data, 59 (33.0%) were hospitalized. Three cases received hemodialysis, and no deaths were reported. Among 170 cases with information on exposures, 66 (38.8%) reported occupational risk. Cases in the northern part of county were more likely to have been infected in early spring (OR 27.1, 95% CI 2.93-250.7), to report seeing a rodent (OR 6.5; 95%CI 2.3-18.4), and to know someone with hemorrhagic fever with renal syndrome (HFRS) (OR 3.0; 95%CI 1.2-8.0) than cases from the southern part of the county. Data from Croatian Forests Ltd. suggested that an unusually good production of beech seeds in 2020 may have contributed to an increased rodent population in 2021. However, average temperature, rainfall, and humidity data from 2021 did not illustrate a significant difference from previous years (Kruskal-Wallis p = 0.837, p = 0.999, p = 0.108). Conclusion: The 2021 HFRS outbreak was likely fueled by an abundant rodent population and virus transmission in rodent hosts. Human activity, environmental factors, and the ensuing animal-human interactions have spread hantavirus infection from Croatia's mountainous region to a previously nonendemic coastal area with a Mediterranean climate.

Screening for TORCH Antibodies in Croatian Childbearing-Aged Women, 2014-2023.

TORCH infections usually result in mild maternal morbidity, but may cause severe congenital abnormalities. Therefore, it is important to detect maternal infections, monitor the fetus after the disease has been recognized, and define the seronegative women who are at risk of primary infection during pregnancy. From 2014 to 2023, serum samples from 1032 childbearing-aged and pregnant women (16-45 years) were tested for IgM/IgG antibodies to the most common TORCH pathogens: Toxoplasma gondii, rubella virus (RUBV), cytomegalovirus (CMV), and herpes simplex viruses (HSV-1 and HSV-2). The overall IgG seroprevalence rates were 20.1% for T. gondii, 91.3% for RUBV, 70.5% for CMV, 66.8% for HSV-1, and 3.5% for HSV-2. Only HSV-2 seroprevalence was age-related, with a significant progressive increase in seropositivity from 0% in those aged less than 26 years to 9.3% in those older than 40 years. The seroprevalence of T. gondii was higher in residents of suburban/rural areas than in residents of urban areas (27.4% vs. 17.1%). In addition, participants from continental regions were more often toxoplasma-seropositive than those from coastal regions (22.2% vs. 15.3%). HSV-1 seroprevalence was also higher in suburban/rural areas (71.7% vs. 64.7%). Obstetric history was not associated with TORCH seropositivity. Univariate and multivariate risk analysis showed that suburban/rural areas of residence and continental geographic regions were significant risk factors for T. gondii seroprevalence. Furthermore, suburban/rural area of residence was a significant risk factor for HSV-1 seroprevalence, while older age was a significant risk factor for HSV-2 seroprevalence. A declining trend in the seroprevalence of all TORCH pathogens was observed compared to previous Croatian studies (2005-2011). Similarly, the proportion of women simultaneously IgG-seropositive to two or three pathogens decreased over time. The maternal serology before pregnancy could potentially reduce the burden of congenital TORCH infections.

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.

Large Anthrax Outbreak in a New Locality in Croatia, 2022.

Bacillus anthracis is a well-known zoonotic pathogen that can cause disease in both animals and humans. Moreover, it has a high bioterrorism potential as its lethal spores are resistant to inactivation, are easy to produce in large quantities, and are easily spread over large areas. Anthrax cases occur in different parts of the world, including most European countries. Specific areas of Croatia are long known as anthrax districts, but with sporadically reported cases over the years. Here, we present a major outbreak of animal and human anthrax in the region of Lonjsko Polje in Croatia, a region not known to have anthrax cases in the past. The outbreak started in July 2022 and lasted several months, but most human and animal cases were reported in the first month. During the outbreak, there were 17 reported human cases of cutaneous anthrax and 29 laboratory-confirmed animal cases. However, due to issues in reporting in animals and the late finding of the carcasses, which made laboratory diagnostics challenging, the actual number of animal cases was probably significantly higher.

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.

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.

Hepatitis E seroprevalence and associated risk factors in Croatian liver transplant recipients

Abstract INTRODUCTION Solid-organ transplant recipients are at risk of hepatitis E virus (HEV) infection. We analyzed the seroprevalence/risk factors of HEV in Croatian liver transplant recipients. METHODS Two hundred forty-two serum samples were tested for HEV immunoglobuline IgG/IgM and HEV RNA. Sociodemographic data and risk factors were collected using a questionnaire. RESULTS HEV IgG seroprevalence rate was 24.4%. Positive/equivocal HEV IgM were found in two patients. HEV RNA was not detected. Logistic regression showed that older age, female gender, rural area/farm, water well, and septic tank were associated with HEV seropositivity. CONCLUSIONS This study revealed a high exposure rate to HEV in Croatian liver recipients.

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.