In 2018, Croatia reported the largest outbreak of West Nile virus (WNV) infections as well as the re-occurrence of human Usutu virus (USUV) infections. For the first time, fatal WNV and USUV infections were detected in wild birds. We analysed epidemiological characteristics and molecular epidemiology of WNV and USUV infections detected during 2018 transmission season. From April to November, 178 patients with neuroinvasive disease and 68 patients with febrile disease were tested for WNV and USUV. Viral RNA was detected in cerebrospinal fluid (CSF) and urine samples using a real-time RT-PCR. Positive samples were tested by nested RT-PCR and nucleotide sequencing. IgM/IgG antibodies were detected in serum/CSF samples using ELISA | 1947 VILIBIC-CAVLEK Et AL.

No abstract

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.

The brucellosis and Q-fever coinfection is very rarely reported. To our knowledge, this is the first case report of concomitant brucellosis and Q-fever, most likely imported in Croatia. A 30-year-old male agricultural worker was hospitalized on 22 April 2017 after a ten days fever up to 40°C with chills, shivering, excessive sweating, general weakness, loss of appetite and headache. A month and a half prior to the hospitalization he lost 18 kg of body weight. Three weeks before hospitalization the patient returned from Kupres (Bosnia and Herzegovina) where he was working for the past year on a sheep farm and consumed unpasteurized dairy products of sheep origin. At admission, his condition was moderately severe due to pronounced dehydration. Routine laboratory tests showed slightly elevated erythrocyte sedimentation rate, anemia, thrombocytopenia and elevated liver transaminases. The chest X-ray showed an inhomogeneous infiltrate of the lower right lung. Three sets of blood culture were cultivated. After 48 hours incubation, bacterial growth was detected in aerobic bottles. Gram-stained smear revealed small, gram-negative coccobacilli. Specimens were subcultured on blood and chocolate agar plates. Using a Vitek GN identification card, the isolated organism was identified as Brucella melitensis. 16S rRNA gene sequencing of the isolate confirmed it as a Brucella sp. Rose-Bengal test was positive, while Wright agglutination test showed a significant increase in antibody titer from 80 to 640 in paired sera. Using indirect immunofluorescence assay (IFA), Coxiella burnetii phase II IgM/IgG titers were 50 and 1024, respectively indicating acute Q-fever. The patient was treated with doxycycline and rifampicin. So far, there has been no relapse or signs of chronic infection.

No 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.

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 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: 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.