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(Re-)emerging arboviruses, such as tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Usutu virus (USUV), are continuously increasing in incidence. We analyzed the epidemiological characteristics of flavivirus infections in humans, sentinel animals, and mosquitoes detected in the 2022 transmission season in Croatia. From April to November 2022, 110 hospitalized patients with neuroinvasive diseases (NID) were tested for the presence of arboviruses. RT-qPCR was used to detect TBEV, WNV, and USUV RNA. An ELISA and virus neutralization tests were used for the detection of flavivirus antibodies. TBEV infection was confirmed in 22 patients with NID. WNV NID was detected in six patients. TBE showed male predominance (81.8%; male-to-female ratio of 4.5:1). All but one WNV patients were males. TBE occurred from April to August, with the majority of patients (83.3%) being detected during the May–June–July period. WNV infections were recorded in August and September. In addition to human cases, asymptomatic WNV infections (IgM positive) were reported in 10 horses. For the first time in Croatia, WNV NID was observed in one horse that presented with neurological symptoms. Furthermore, USUV was confirmed in one dead blackbird that presented with neurological symptoms. A total of 1984 mosquitoes were collected in the City of Zagreb. Two Ae. albopictus pools tested positive for flavivirus RNA: one collected in July (USUV) and the other collected in August (WNV). A phylogenetic analysis of detected human and avian strains confirmed WNV lineage 2 and the USUV Europe 2 lineage. The presented results confirm the endemic presence of neuroinvasive flaviviruses in continental Croatia. The continuous monitoring of virus circulation in humans, sentinel animals, and mosquitoes is needed to reduce the disease burden.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Background: Toxoplasma gondii (TG), a zoonotic protozoan parasite, belongs to a group of TORCH infectious agents, which can cause severe damage to the fetus if a primary infection occurs during pregnancy. After primary infection, TG rests lifelong in human organisms causing a latent infection. Most studies have analyzed the consequences of acute, but not latent, TG infection. This study analyzed the impact of latent toxoplasmosis on spontaneous abortion history, pregnancy complication rate and neonatal outcome. Methods: IgG and IgM anti-TG antibodies were tested in 806 pregnant women who were consulted at the Timisoara Clinical Emergency Hospital between 2008 and 2010. Demographic data, obstetrical history, and data about the pregnancy complications, birth and neonate were collected for each woman and comparisons between the groups, with and without latent TG infection, were made. Results: This study did not show differences between groups regarding the history of spontaneous abortion (OR = 1.288, p = 0.333), cesarean section (OR = 1.021, p = 0.884), placental abruption (OR 0.995, p = 0.266), pregnancy-induced hypertension rate (OR 1.083, p = 0.846), secondary sex ratio (1.043, p = 0.776), 1′ APGAR score at birth (p = 0.544), gestational age at birth (p = 0.491) or birth weight (p = 0.257). Conclusions: The observed differences between the rate of pregnancy complications in the two groups of pregnant women with and without latent infection with TG, did not reach a statistical significance.

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.

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.

During the four pandemic waves, a total of 560,504 cases and 10,178 deaths due to COVID-19 were reported in Croatia. The Alpha variant, dominant from March 2021 (>50% of positive samples), was rapidly replaced by Delta variants (>90%) by August 2021. Several seroprevalence studies were conducted in different populations (general population, children/adolescents, professional athletes, healthcare workers, veterinarians) and in immunocompromised patients (hemodialysis patients, liver/kidney transplant recipients). After the first pandemic wave, seroprevalence rates of neutralizing (NT) antibodies were reported to be 0.2–5.5%. Significantly higher seropositivity was detected during/after the second wave, 2.6–18.7%. Two studies conducted in pet animals (February-June 2020/July–December 2020) reported SARS-CoV-2 NT antibodies in 0.76% of cats and 0.31–14.69% of dogs, respectively. SARS-CoV-2 NT antibodies were not detected in wildlife. Environmental samples taken in the households of COVID-19 patients showed high-touch personal objects as most frequently contaminated (17.3%), followed by surfaces in patients’ rooms (14.6%), kitchens (13.3%) and bathrooms (8.3%). SARS-CoV-2 RNA was also detected in 96.8% affluent water samples, while all effluent water samples tested negative. Detection of SARS-CoV-2 in humans, animals and the environment suggests that the ‘One Health’ approach is critical to controlling COVID-19 and future pandemics.

Background and Objectives: Toxoplasma gondii, cytomegalovirus (CMV) and rubella virus, besides other agents, belong to a group named the TORCH complex. Research on the epidemiology of these agents in women is of particular interest, as primary infection during pregnancy could cause severe damage to the fetus. Women who had contracted infection before pregnancy develop IgG antibodies, so the fetus is protected in case of contact with the same agent. Our scope was to identify the childbearing women simultaneously protected or susceptible to a primary infection to two or three agents mentioned above. Materials and Methods: A cross-sectional study was performed on 6961 fertile Caucasian women from Western Romania, to analyze the simultaneous seroprevalence to two or three of the pathogens from the TORCH complex: Toxoplasma gondii, CMV, and rubella virus. Sampling was conducted at two time points: 2008–2010 (group 1; 1461 participants) and 2015–2018 (group 2; 5500 participants). Results: The percentage of women simultaneously seropositive to IgG-anti-Toxoplasma gondii/IgG-anti-CMV, IgG-anti-Toxoplasma gondii/IgG-anti-rubella, IgG-anti-CMV/IgG-anti-rubella or IgG-anti-Toxoplasma gondii and IgG-anti-CMV/IgG-anti-rubella antibodies decreased between the two groups (2008–2010 vs. 2015–2018): 41.4% vs. 36.1%, OR = 0.79, p = 0.0002; 41.8% vs. 35.7%, OR = 0.77, p < 0.0001; 88.9% vs. 83.6%, OR = 0.63, p < 0.0001; 39.6% vs. 33.2%, OR = 0.75, p < 0.0001. When comparing women from urban and rural areas, the simultaneous seroprevalence was higher in rural areas. In women tested 2008–2010 (group 1) the simultaneous seroprevalence (urban vs. rural) was: 38.4% vs. 49.1%, OR = 1.54, p = 0.0002; 38.4% vs. 50.6%, OR = 1.64, p < 0.0001; 88.8% vs. 89.2%, OR = 1.04, NS; 36.4% vs. 47.7%, OR = 1.58, p = 0.0001. A similar trend was found in women tested in group 2. Conclusions: The rate of simultaneous seropositivity to Toxoplasma gondii, CMV and rubella virus among Romanian women of reproductive age decreased significantly between 2008–2010 and 2015–2018 and the susceptibility to infections increased. It is necessary to apply increased prevention measures among susceptible pregnant women.

The epidemiology of West Nile (WNV) and Usutu virus (USUV) has changed dramatically over the past two decades. Since 1999, there have been regular reports of WNV outbreaks and the virus has expanded its area of circulation in many Southern European countries. After emerging in Italy in 1996, USUV has spread to other countries causing mortality in several bird species. In 2009, USUV seroconversion in horses was reported in Italy. Co-circulation of both viruses was detected in humans, horses and birds. The main vector of WNV and USUV in Europe is Culex pipiens, however, both viruses were found in native Culex mosquito species (Cx. modestus, Cx. perexiguus). Experimental competence to transmit the WNV was also proven for native and invasive mosquitoes of Aedes and Culex genera (Ae. albopictus, Ae. detritus, Cx. torrentium). Recently, Ae. albopictus and Ae. japonicus naturally-infected with USUV were reported. While neuroinvasive human WNV infections are well-documented, USUV infections are sporadically detected. However, there is increasing evidence of a role of USUV in human disease. Seroepidemiological studies showed that USUV circulation is more common than WNV in some endemic regions. Recent data showed that WNV strains detected in humans, horses, birds, and mosquitoes mainly belong to lineage 2. In addition to European USUV lineages, some reports indicate the presence of African USUV lineages as well. The trends in WNV/USUV range and vector expansion are likely to continue in future years. This mini-review provides an update on the epidemiology of WNV and USUV infections in Southern Europe within a multidisciplinary “One Health” context.

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.

Over a year into the COVID-19 pandemic, there is growing evidence that SARS-CoV-2 infections among dogs are more common than previously thought. In this study, the prevalence of SARS-CoV-2 antibodies was investigated in two dog populations. The first group was comprised of 1069 dogs admitted to the Veterinary Teaching Hospital for any given reason. The second group included dogs that shared households with confirmed COVID-19 cases in humans. This study group numbered 78 dogs. In COVID-19 infected households, 43.9% tested ELISA positive, and neutralising antibodies were detected in 25.64% of dogs. Those data are comparable with the secondary attack rate in the human population. With 14.69% of dogs in the general population testing ELISA positive, there was a surge of SARS-CoV-2 infections within the dog population amid the second wave of the pandemic. Noticeably seroprevalence of SARS-CoV-2 in the dog and the human population did not differ at the end of the study period. Male sex, breed and age were identified as significant risk factors. This study gives strong evidence that while acute dog infections are mostly asymptomatic, they can pose a significant risk to dog health. Due to the retrospective nature of this study, samples for viral isolation and PCR were unavailable. Still, seropositive dogs had a 1.97 times greater risk for developing central nervous symptoms.

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.

Hepatitis E virus (HEV) is the most common cause of viral hepatitis globally. The first human case of autochthonous HEV infection in Croatia was reported in 2012, with the undefined zoonotic transmission of HEV genotype 3. This narrative review comprehensively addresses the current knowledge on the HEV epidemiology in humans and animals in Croatia. Published studies showed the presence of HEV antibodies in different population groups, such as chronic patients, healthcare professionals, voluntary blood donors and professionally exposed and pregnant women. The highest seroprevalence in humans was found in patients on hemodialysis in a study conducted in 2018 (27.9%). Apart from humans, different studies have confirmed the infection in pigs, wild boars and a mouse, indicating the interspecies transmission of HEV due to direct or indirect contact or as a foodborne infection. Continued periodical surveys in humans and animals are needed to identify the possible changes in the epidemiology of HEV infections.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus with a pandemic spread. So far, a total of 349,910 SARS-CoV-2 cases and 7687 deaths were reported in Croatia. We analyzed the seroprevalence and neutralizing (NT) antibody response in the Croatian general population after the first (May–July 2020) and second (December 2020–February 2021) pandemic wave. Initial serological testing was performed using a commercial ELISA, with confirmation of reactive samples by a virus neutralization test (VNT). A significant difference in the overall seroprevalence rate was found after the first (ELISA 2.2%, VNT 0.2%) and second waves (ELISA 25.1%, VNT 18.7%). Seropositive individuals were detected in all age groups, with significant differences according to age. The lowest prevalence of NT antibodies was documented in the youngest (p < 0.001) was observed. SARS-CoV-2 NT antibody titers seem to be age-related, with the highest NT activity in children under 10 years and individuals above 50 years.

Lymphocytic choriomeningitis virus (LCMV) is a neglected rodent-borne zoonotic virus distributed worldwide. Since serologic assays are limited to several laboratories, the disease has been underreported, often making it difficult to determine incidence and seroprevalence rates. Although human clinical cases are rarely recorded, LCMV remains an important cause of meningitis in humans. In addition, a fatal donor-derived LCMV infection in several clusters of solid organ transplant recipients further highlighted a pathogenic potential and clinical significance of this virus. In the transplant populations, abnormalities of the central nervous system were also found, but were overshadowed by the systemic illness resembling the Lassa hemorrhagic fever. LCMV is also an emerging fetal teratogen. Hydrocephalus, periventricular calcifications and chorioretinitis are the predominant characteristics of congenital LCMV infection, occurring in 87.5% of cases. Mortality in congenitally infected children is about 35%, while 70% of them show long-term neurologic sequelae. Clinicians should be aware of the risks posed by LCMV and should consider the virus in the differential diagnosis of aseptic meningitis, especially in patients who reported contact with rodents. Furthermore, LCMV should be considered in infants and children with unexplained hydrocephalus, intracerebral calcifications and chorioretinitis. Despite intensive interdisciplinary research efforts, efficient antiviral therapy for LCMV infection is still not available.

In the period from 2015 to 2020, an entomological survey for the presence of West Nile virus (WNV) and Usutu virus (USUV) in mosquitoes was performed in northwestern Croatia. A total of 20,363 mosquitoes were sampled in the City of Zagreb and Međimurje county, grouped in 899 pools and tested by real-time RT-PCR for WNV and USUV RNA. All pools were negative for WNV while one pool each from 2016 (Aedes albopictus), 2017 (Culex pipiens complex), 2018 (Cx. pipiens complex), and 2019 (Cx. pipiens complex), respectively, was positive for USUV. The 2018 and 2019 positive pools shared 99.31% nucleotide homology within the USUV NS5 gene and both clustered within USUV Europe 2 lineage. The next-generation sequencing of one mosquito pool (Cx. pipiens complex) collected in 2018 in Zagreb confirmed the presence of USUV and revealed several dsDNA and ssRNA viruses of insect, bacterial and mammalian origin.

Lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen associated with aseptic meningitis, severe systemic infections in immunocompromised persons, and congenital anomalies. Data on the prevalence of LCMV infections are scarce. We analyzed the seroprevalence of LCMV in continental Croatian regions. A total of 338 serum samples of professionally exposed (forestry workers, hunters, agriculture workers in contact with rodents) and non-exposed populations (general population, pregnant women) were tested for the presence of LCMV antibodies using indirect immunofluorescence assay. No participants reported recent febrile disease. LCMV IgG antibodies were detected in 23/6.8% of participants: 9.8% exposed persons and 5.1% non-exposed persons (6.1% in the general population and 3.9% in pregnant women). No participants were LCMV IgM positive. Although higher seropositivity was found in males compared to females (8.9% vs. 4.7%), inhabitants of suburban/rural areas compared to inhabitants of urban areas (9.2% vs. 4.6%), and persons who used well as a source of water compared to those who used tap (11.4% vs. 5.6%), these differences did not reach statistical significance. Results of logistic regression showed that the presence of rodents in the house/yard and cleaning rodent nests were associated with an elevated risk for LCMV infection (OR = 2.962, 95% CI = 1.019–8.607).

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.

Several arboviruses have emerged in Croatia in recent years. Tick-borne encephalitis is endemic in continental counties; however, new natural micro-foci have been detected. Two autochthonous dengue cases were reported in 2010. West Nile virus emerged in 2012, followed by emergence of Usutu virus in 2013. Although high seroprevalence rates of Toscana virus have been detected among residents of Croatian littoral, the virus remains neglected, with only a few clinical cases of neuroinvasive infections reported. Lymphocytic choriomeningitis virus is a neglected neuroinvasive rodent-borne virus. So far, there are no reports on human clinical cases; however, the seroprevalence studies indicate the virus presence in the Croatian mainland. Puumala and Dobrava hantaviruses are widely distributing rodent-borne viruses with sporadic and epidemic occurrence. Hepatitis E virus is an emerging food-borne virus in Croatia. After the emergence in 2012, cases were regularly recorded. Seropositivity varies greatly by region and population group. Rotaviruses represent a significant healthcare burden since rotavirus vaccination is not included in the Croatian national immunization program. Additionally, rotaviruses are widely distributed in the Croatian ecosystem. A novel coronavirus, SARS-CoV-2, emerged in February 2020 and spread rapidly throughout the country. This review focuses on emerging and neglected viruses of zoonotic importance detected in Croatia.

Seronegative women are susceptible to primary rubella virus (RV) infection during pregnancy, which can cause fetal damage. Vaccination represents the main strategy in rubella prevention. The aim of this study was to analyze changes in the rubella seroprevalence and identify populations with a high susceptibility to RV. A cross-sectional study was performed on 6914 Caucasian fertile women who had Toxoplasma gondii, other viruses, Rubella, Cytomegalovirus, and the herpes simplex virus (TORCH) screening in two distinct periods—1452 at the Timișoara Municipal Hospital, Romania (Group 1: 2008–2010) and 5462 at the laboratory Bioclinica S.A., Timișoara, Romania (Group 2: 2015–2018). The RV seroprevalence decreased (Group 1 versus Group 2; 94.1% (92.7–95.2) versus 91.4% (90.6–92.1), OR = 0.76 (p = 0.0007)). According to the year of birth and eligibility to vaccination program, RV seroprevalence rates were 82.4% (76.8–86.8)/1997–2004, 85.4% (80.5–89.3)/1995–1996, 90.1% (89.0–91.1)/

Toscana virus (TOSV) is an arthropod-borne virus, transmitted to humans by phlebotomine sandflies. Although the majority of infections are asymptomatic, neuroinvasive disease may occur. We report three cases of neuroinvasive TOSV infection detected in Croatia. Two patients aged 21 and 54 years presented with meningitis, while a 22-year old patient presented with meningoencephalitis and right-sided brachial plexitis. Cerebrospinal fluid (CSF), serum, and urine samples were collected and tested for neuroinvasive arboviruses: tick-borne encephalitis, West Nile, Usutu, TOSV, Tahyna, and Bhanja virus. In addition, CSF and serum samples were tested for the anti-viral cytokine response. High titers of TOSV IgM (1000–3200) and IgG (3200−10,000) antibodies in serum samples confirmed TOSV infection. Antibodies to other phleboviruses (sandfly fever Sicilian/Naples/Cyprus virus) were negative. CSF samples showed high concentrations of interleukin 6 (IL-6; range 162.32−2683.90 pg/mL), interferon gamma (IFN-γ; range 110.12−1568.07 pg/mL), and IL-10 (range 28.08−858.91 pg/mL), while significantly lower cytokine production was observed in serum. Two patients recovered fully. The patient with a brachial plexitis improved significantly at discharge. The presented cases highlight the need of increasing awareness of a TOSV as a possible cause of aseptic meningitis/meningoencephalitis during summer months. Association of TOSV and brachial plexitis with long-term sequelae detected in one patient indicates the possibility of more severe disease, even in young patients.

Usutu virus (USUV) is an emerging arbovirus isolated in 1959 (Usutu River, Swaziland). Previously restricted to sub-Saharan Africa, the virus was introduced in Europe in 1996. While the USUV has received little attention in Africa, the virus emergence has prompted numerous studies with robust epidemiological surveillance programs in Europe. The natural transmission cycle of USUV involves mosquitoes (vectors) and birds (amplifying hosts) with humans and other mammals considered incidental (“dead-end”) hosts. In Africa, the virus was isolated in mosquitoes, rodents and birds and serologically detected in horses and dogs. In Europe, USUV was detected in bats, whereas antibodies were found in different animal species (horses, dogs, squirrels, wild boar, deer and lizards). While bird mortalities were not reported in Africa, in Europe USUV was shown to be highly pathogenic for several bird species, especially blackbirds (Turdus merula) and great gray owls (Strix nebulosa). Furthermore, neurotropism of USUV for humans was reported for the first time in both immunocompromised and immunocompetent patients. Epizootics and genetic diversity of USUV in different bird species as well as detection of the virus in mosquitoes suggest repeated USUV introductions into Europe with endemization in some countries. The zoonotic potential of USUV has been reported in a growing number of human cases. Clinical cases of neuroinvasive disease and USUV fever, as well as seroconversion in blood donors were reported in Europe since 2009. While most USUV strains detected in humans, birds and mosquitoes belong to European USUV lineages, several reports indicate the presence of African lineages as well. Since spreading trends of USUV are likely to continue, continuous multidisciplinary interventions (“One Health” concept) should be conducted for monitoring and prevention of this emerging arboviral infection.

A total of 52 serum samples from patients with symptoms suggestive of tick-borne encephalitis virus (TBEV) infection and positive IgM and/or IgG antibodies were tested for IgG avidity. Acute/recent TBEV infection was confirmed by low/borderline avidity index (AI) in 94.8% IgM positive/IgG positive samples, while in 5.2% high AI was found indicating persisting IgM antibodies. Majority of IgM negative/IgG positive samples (78.6%) showed high AI consistent with past TBEV infection. However, in 21.3% patients without measurable IgM antibodies current/recent infection was confirmed by AI. IgG avidity represents an additional serologic marker that improves diagnosis of TBEV, especially in cases of atypical antibody response.

A 25-years old man from Zagreb, Croatia, was admitted to the University Hospital for Infectious Diseases four days after the onset of symptoms such as fever, intense pain in the calves and anuria. The patient owned a rabbit and, before the onset of the disease, repaired some rubber pipes damaged by rodents. At admission, he had a severe clinical picture with fever, hypotension, jaundice, immobility, and pain in leg muscles. Treatment with ceftriaxone was initiated in combination with volume restitution. Renal failure soon ensued. Consequently continuous venovenous hemodiaphiltration therapy was performed. Due to acute respiratory distress syndrome, the patient was mechanically ventilated. The patient’s condition gradually improved and he recovered fully from multi-organ failure. Diagnosis was confirmed by a microscopic agglutination test (MAT) covering 15 leptospira serovars and real-time polymerase-chain reaction (PCR). The first serum sample taken on day 6 tested negative for leptospira, while PCR showed positive results for leptospiral DNA. The second serum sample taken on day 13 tested positive for serovar Canicola serogroup Canicola, serovar Patoc, serovar Grippotyphosa serogroup Grippotyphosa and serovar Tarassovi serogroup Tarassovi (titre 4000, 4000, 1000 and 2000, respectively), while PCR was negative. This report highlights the benefits of combining MAT and PCR methods in early diagnosis of leptospirosis.

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.