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

Cardiac involvement has rarely been reported in West Nile (WNV) infection. We report a fatal case of WNV encephalitis associated with an acute anteroseptal ST elevation myocardial infarction. The patient was hospitalized with a fever, headache, nausea and vomiting. The physical examination revealed positive meningeal signs and an altered level of consciousness. High levels of cardiac enzymes (creatine phosphokinase/MB fraction, lactate dehydrogenase, myoglobin and cardiac troponin I) and ST elevation on electrocardiogram were found. Both CSF and urine samples were positive for WNV RNA. This case highlights the need of awareness of the possibility of a WNV-related myocardial infection, including myocardial infarction.

Seroprevalence among personnel in the healthcare facilities of Croatia during the first wave of the COVID-19 epidemic.

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.

The West Nile virus (WNV) is a widely distributed emerging mosquito-borne arbovirus. Although the majority of WNV infections are asymptomatic (80%), some infected individuals (20%) present with non- specific febrile disease (WNV fever) and less than 1% develop neuroinvasive diseases (meningitis, encephalitis, and myelitis). Several atypical or rare presentations of WNV infection such as cerebellitis, myocarditis, hepatitis, pancreatitis, and rhabdomyolysis have been described in case reports or small case series1. Although rare, ocular manifestations, including monofocal or multifocal WNV chorioretinitis are the most commonly reported clinical manifestations of WNV infection after fever and neuroinvasive disease.

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 &lt, 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.

Purpose: To present a case of acute anteroseptal ST-elevation myocardial infarction in the West Nile virus (WNV) meningoencephalitis. Methods & Materials: A 77- year-old patient was hospitalized in the late summer of 2017 on the second day of the illness manifested by a fever up to 38.6 °C and diarrhea. The patient did not report recent travel but recalled mosquito bites. Past medical history included hypertension. At admission, routine laboratory tests, electrocardiogram (ECG) and chest x-ray were performed. In addition, cerebrospinal fluid (CSF), urine and blood samples were collected for a virological analysis. Results: At admission, WBC count was 24.6 (reference range 3.4-9.7x10(9)/L) with neutrophilia (92%, range 44-72%) and very high levels of cardiac enzymes: creatinine phosphokinase 1856 (range 17-153 U/I), lactate dehydrogenase 433 (range 2-241 U/L), myoglobin 3116 (range 20-80 ug/L) and troponin I 17.640 (range 0.000-0.056 ug/L). ECG showed ST elevation. In the cardiac intensive care unit, an emergency coronary angiography was performed which confirmed the coronary artery stenosis. The patient's condition complicated on the 4th day of the illness by an altered level of consciousness with progression to coma, accompanied by neck stiffness and positive meningeal signs. Computed tomography of the brain was normal. Cerebrospinal fluid (CSF) showed pleocytosis with 26 cells/mm3, predominantly mononuclears (73%) and elevated protein level (1.151, range 0.170-0.370 g/L). Both CSF and urine were positive for WNV RNA by real- time and nested RT-PCR. Phylogenetic analysis showed WNV lineage 2. The patient was initially treated with acyclovir, ampicillin and cefepime parenterally with supportive therapy (antiedematous, antiaggregation and antihypertensive therapy). On the 8th day of the illness a respiratory insufficiency developed. The patient was intubated and mechanically ventilated, but developed hypotension and low oxygen saturation in spite of an adequate respiratory support. Despite the cardiopulmonary resuscitation, the patient died due to cardiopulmonary arrest. Conclusion: Although cardiac involvement is not frequently reported in the course of a WNV infection, physicians should be aware of the possibility of a WNV-related myocardial infection.

Purpose: To analyze clinical and laboratory characteristics of neuroinvasive viral zoonoses detected in Croatia during 2017-2018. Methods & Materials: From April 2017 to June 2018, a total of 120 patients with neuroinvasive disease from continental Croatian regions were tested for the presence of neuroinvasive zoonotic viruses: tick- borne encephalitis virus (TBEV), West Nile virus (WNV), Usutu virus (USUV), Toscana virus (TOSV), Tahyna virus (TAHV) and lymphocytic choriomeningitis virus (LCMV). Cerebrospinal fluid (CSF) and urine samples were tested for the presence of viral RNA using a real-time RT- PCR and/or nested RT- PCR. Serological tests of serum/CSF samples (IgM/IgG antibodies, IgG avidity) were performed using ELISA (TBEV, WNV, USUV), IFA (TOSV, LCMV) and virus neutralization test (WNV). Results: Etiology was confirmed in 28/23.3% patients: TBEV in 20/16.7% and WNV in 8/6.6% patients by detection of IgM and IgG antibodies of low avidity and/or detection of viral RNA in CSF and urine. Majority of patients with TBEV infection were males (15/75.0%). Although infections were detected in all age groups, 15/75.0% patients were less than 60 years of age. The main clinical symptoms were headache (18/90.0%), weakness (18/90.0%), nausea (12/60.0%) and vomiting (8/40.0%). Fever >39 °C was noted in 16/80.0% patients. CSF leukocyte count ranged from 41- 3520/mm3 with mononuclear cell predominance in 15/75.0% patients. All but one patient fully recovered. WNV infection was reported in 5/62.5% males and 3/37.5% females. All but one patient (7/87.5%) were older than 60 years. Majority of patients reported underlying diseases: hypertension (3/37.5%) and cerebrovascular disease (3/37.5%). The main clinical symptoms were headache (5/62.5%) and weakness (5/62.5%), while fever >39 °C was noted in 4/50.0% patients. CSF leukocyte count ranged from 56-1096/mm3 with mononuclear cell predominance in 4/50.0% patients. One patient died. USUV, TOSV, TAHV and LCMV infections were not detected during the tested period. Conclusion: TBEV infections were more common in patients less than 60 years of age, while WNV infections were most common in elderly. High fever was noted in 80.0% TBEV cases compared to 50.0% WNV cases. CSF pleocytosis was higher in TBEV infection.