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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.

Najznačajnija primjena seroloških testova u dijagnostici COVID-19 je u svrhu procjene proširenosti bolesti u populaciji. Međutim, imunotestovi mogu poslužiti kao dodatni dijagnostički postupak, posebice kod bolesnika s podatkom o izloženosti COVID-19 i prisutnim kliničkim simptomima, kod kojih je rezultat RT-PCR testa bio negativan. U ovome smo radu analizirali preliminarne rezultate šest seroloških testova za dijagnostiku SARS-CoV-2. Korištena su tri 'point-of care' imunokromatografska testa (POC): ACRO, AMP i ENCODE te tri imunoenzimska testa (DiaPro, Vircell i Euroimmun). Testirano je ukupno 15 uzoraka seruma bolesnika s COVID-19 infekcijom i 15 uzoraka seruma asimptomatskih osoba. Vrijeme uzorkovanja kod bolesnika s COVID-19 iznosilo je 4 – 10 dana (N=4), 11 – 19 dana (N=6) te 20 – 34 dana (N=5) od početka bolesti. Svi su početno reaktivni rezultati potvrđeni testom neutralizacije virusa (VNT). Kod bolesnika s COVID-19 (N=15), učestalost detekcije IgM/IgA protutijela iznosila je 9/60,0% (ACRO), 11/73,3% (AMP, ENCODE, Euroimmun), 12/80,0% (DiaPro) te 13/86,6% (Vircell). Učestalost detekcije IgG protutijela iznosila je 10/66,6% (AMP, Euroimmun) te 11/73,3% (ostali testovi). Ovisno o vremenu uzorkovanja, učestalost detekcije protutijela iznosila je: a) 4-10. dana: 1/25,0% i 2/50,0% (IgM/IgA i IgG); 11-19. dana: 4/66,6%-6/100% (IgM/IgA), 4/66,6% i 5/83,3% (IgG; c) 20-34. dana: 4/80,0% i 5/100% (IgM/IgA), 5/100% (IgG). U jedne asimptomatske osobe s dokazanim IgM/IgA protutijelima testom ACRO, DiaPro i Vircell potvrđena su neutralizacijska protutijela. U skupini seronegativnih asimptomatskih osoba dokazanih VNT testom (N=14), negativan nalaz IgM/IgA je nađen u 12/85,7% (ACRO), 13/92,8% (DiaPro, Vircell) i 14/100% (AMP, ENCODE, Euroimmun) uzoraka, dok je negativan nalaz IgG dokazan kod 13/92,8% (ACRO) te 14/100% (ostali testovi) uzoraka. ELISA testovi pokazali su višu osjetljivost detekcije IgM/IgA protutijela u usporedbi s POC testovima, dok je osjetljivost detekcije IgG protutijela bila podjednaka u POC i ELISA testovima., The most important use of serology in the COVID-19 diagnostics is for determination the extent of disease in the population. However, immunoassays could represent an additional diagnostic method, especially in patients with exposure history and clinical symptoms compatible with COVID-19 who failed to be confirmed by RT-PCR. We analyzed the preliminary results of six serology tests for the diagnosis of SARS-CoV-2. Three point-of-care lateral flow chromatographic immunoassays (POC): ACRO, AMP and ENCODE and three enzyme immunoassays (ELISA): DiaPro, Vircell and Euroimmun were used. A total of 15 serum samples from COVID-19 patients and 15 serum samples from asymptomatic persons were tested. Time of sampling for COVID-19 patients was 4 – 10 days (N=4), 11 – 19 days (N=6) and 20 – 34 days (N=5) after disease onset. Initially reactive results were confirmed using a virus neutralization test (VNT). In COVID-19 patients (N=15), IgM/IgA positive detection rates were 9/60.0% (ACRO), 11/73.3% (AMP, ENCODE, Euroimmun), 12/80.0% (DiaPro) and 13/86.6% (Vircell). Overall IgG detection rates were 10//66.6% (AMP, Euroimmun) and 11/73.3% (other tests). According to the sampling time, positive detection rates were as follows: a) days 4 – 10: 1/25.0% and 2/50.0% (IgM/IgA and IgG); b) days 11 –19: 4/66.6%-6/100% (IgM/IgA), 4/66.6% and 5/83.3% (IgG); c) days 20 – 34: 4/80.0% and 5/100% (IgM/IgA), 5/100% (IgG). One asymptomatic participant tested IgM/IgA positive using ACRO, DiaPro and Vircell was confirmed seropositive using a VNT. In a group of asymptomatic persons detected seronegative using a VNT (N=14), IgM/IgA negative detection rates were 12/85.7% (ACRO), 13/92.8% (DiaPro, Vircell) and 14/100% (AMP, ENCODE, Euroimmun). IgG negative detection rates were 13/92.8% (ACRO) and 14/100% (other tests). ELISA tests showed a higher overall IgM/IgA sensitivity compared to POC tests in patients with COVID-19, while the IgG sensitivity was similar in both POC and ELISA.

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.

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.

In this paper we present a case report of tick-borne encephalitis (TBE) in a professionally exposed person (forestry worker) who acquired this infection three years after he had received a complete primary vaccination. The patient reported multiple tick bites, the last one eighteen days prior to disease onset. The TBE diagnosis was confirmed according to the European Center for Disease Prevention and Control case definition of TBE by clinical criteria (meningitis), epidemiological link (exposure to tick bites in an endemic area) and laboratory criteria (detection of TBEV specific IgM and IgG antibodies in CSF and serum samples). The case presented indicates the need of awareness of possible TBE even in persons who have received complete vaccination, especially in the professionally exposed ones or those exposed to multiple tick bites., U radu prikazujemo slučaj krpeljnog encefalitisa (KE) kod profesionalno izložene osobe (šumar) koja je oboljela tri godine nakon uredno provedenog primarnog cijepljenja. Pacijent je naveo višestruke ugrize krpelja, od kojih je posljednji bio osamnaest dana prije početka bolesti. Dijagnoza KE potvrđena je prema kriterijima Europskog centra za kontrolu bolesti (ECDC) na temelju kliničkih (meningitis), epidemioloških (izloženost krpeljima u endemskom području) te laboratorijskih kriterija (dokaz specifičnih IgM i IgG protutijela za KE u uzorcima seruma i cerebrospinalnog likvora). Prikazani slučaj naglašava potrebu za promišljanjem o mogućoj pojavi KE i kod osoba kod kojih je provedeno kompletno cijepljenje, osobito profesionalno izloženih osoba te onih izloženih čestim ugrizima krpelja.

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.