Prevalence and molecular epidemiology of West Nile and Usutu virus infections in Croatia in the 'One health' context, 2018.

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.

[The biggest epidemic of hemorrhagic fever with renal syndrome in Croatia]. / Najveca epidemija hemoragijske vrucice s bubreznim sindromom u Hrvatskoj.

INTRODUCTION: The largest outbreak of hemorrhagic fever with renal syndrome (HFRS) to date occurred in Croatia in the year 2002. The epidemic started in winter, lasted throughout spring to summer months, ending not earlier than November. AIM: The aim of this study was to investigate and analyze the basic epidemiologic and clinical features of HFRS in Croatia by uniform and standardized prospective-retrospective analysis of all patients affected by the epidemic. PATIENTS AND METHODS: When the epidemic started, a patient questionnaire with questions on the basic demographic data, site of infection and other epidemiologic characteristics, clinical symptoms, disease severity and laboratory results was designed. Data on 401 patient with a clinical diagnosis of HFRS were collected. The etiologic diagnosis of the disease was confirmed by ELISA, and in some patients by indirect immunofluorescence test (IFT). The results were analyzed using a descriptive statistical method. RESULTS: HFRS was clinically diagnosed in 401 patients from all over Croatia. A total of 320 (79.8%) cases were reported to the Epidemiology Service of the National Institute of Public Health. The majority of patients (n = 128) were registered in June. Males were three times more affected than females. Apart from its long duration, this epidemic was characteristic for the involvement of general population, with only a small number of the affected from the potential risk groups (forestry workers 28, soldiers 14, farmers 18). The epidemic spread almost throughout inland Coratia. At least 44 patients were infected in the Plitvice Lakes area, 32 in Slunj, 27 on Sljeme, 24 in Velika, and at least 19 in the area of Kutjevo. The youngest patient was aged 4 and the oldest 80 years. The majority of patients were treated in Zagreb (University Hospital for Infectious Diseases--110, Zagreb University Hospital Center--3), followed by Karlovac (71 inpatients and 39 outpatients), Pozega (n = 79), and Rijeka (n = 37). Serologic analysis (ELISA method) detected Puumala virus in 161 and Dobrava virus in only 17 patients. The disease was confirmed by immunofluorescence method in 53 patients (mostly in Kariovac). During the 2002 outbreak, HFRS clinically manifested mostly in a milder form with general symptoms and transitory renal insufficiency, while hemorrhages were rarely recorded. According to our disease severity score, a mild form of the disease was recorded in 65%, moderately severe in 28%, severe in 5% and extremely severe form in 2% of the patients. One patient died. Two thirds of the patients were hospitalized during the febrile stage of the disease. All patients had fever, whereas headache and pain in the lumbar region were recorded in more than 90% of cases, polyuria in 75%, oliguira and vomiting in approximately 50%, respiratory symptoms in 35%, and hemorrhages (mostly on the skin and mucous membranes), vision disturbances, conjunctivitis and diarrhea in approximately 25% of patients. ESR was elevated in 64% and CRP in 93% of patients. Leukocytosis was recorded in 25% and thrombocytopenia in 70% of patients. Increased values of urea and creatinine and signs of liver damage were recorded in approximately 50% of the patients. CONCLUSION: The largest outbreak of HFRS occurred in Croatia in 2002, with more than 400 diseased throughout Croatia. This epidemic confirmed our previous assumption that the whole Croatia, apart from its narrow coastline area and islands, is a natural focus of HFRS with different causative types of hantaviruses. Efforts should be made to conduct a comprehensive ecologic and mammologic study on hantaviruses and their biologic characteristics in these areas.