In Croatia, several rodent- and vector-borne agents are endemic and of medical importance. In this study, we investigated hantaviruses and, for the first time, tick-borne encephalitis virus (TBEV) and Rickettsia spp. in small wild rodents from two different sites (mountainous and lowland region) in Croatia. In total, 194 transudate and tissue samples from 170 rodents (A. flavicollis, n=115; A. agrarius, n=2; Myodes glareolus, n=53) were tested for antibodies by indirect immunofluorescence assays (IIFT) and for nucleic acids by conventional (hantaviruses) and real-time RT-/PCRs (TBEV and Rickettsia spp.). A total of 25.5% (24/94) of the rodents from the mountainous area revealed specific antibodies against hantaviruses. In all, 21.3% (20/94) of the samples from the mountainous area and 29.0% (9/31) from the lowland area yielded positive results for either Puumala virus (PUUV) or Dobrava-Belgrade virus (DOBV) using a conventional RT-PCR. All processed samples (n=194) were negative for TBEV by IIFT or real-time RT-PCR. Serological evidence of rickettsial infection was detected in 4.3% (4/94) rodents from the mountainous region. Another 3.2% (3/94) rodents were positive for Rickettsia spp. by real-time PCR. None of the rodents (n=76) from the lowland area were positive for Rickettsia spp. by real-time PCR. Dual infection of PUUV and Rickettsia spp. was found in one M. glareolus from the mountainous area by RT-PCR and real-time PCR, respectively. To our knowledge, this is the first detection of Rickettsia spp. in small rodents from Croatia. Phylogenetic analyses of S- and M-segment sequences obtained from the two study sites revealed well-supported subgroups in Croatian PUUV and DOBV. Although somewhat limited, our data showed occurrence and prevalence of PUUV, DOBV, and rickettsiae in Croatia. Further studies are warranted to confirm these data and to determine the Rickettsia species present in rodents in these areas.

Non-alcoholic fatty liver disease (NAFLD) is identified as a risk factor for developing severe COVID-19. While NAFLD is associated with chronic low-grade inflammation, mechanisms leading to immune system hyperactivation remain unclear. The aim of this prospective observational study is to analyze cytokine profiles in patients with severe COVID-19 and NAFLD. A total of 94 patients with severe COVID-19 were included. Upon admission, clinical and laboratory data were collected, a liver ultrasound was performed to determine the presence of steatosis, and subsequently, 51 were diagnosed with NAFLD according to the current guidelines. There were no differences in age, sex, comorbidities, and baseline disease severity between the groups. Serum cytokine concentrations were analyzed using a multiplex bead-based assay by flow cytometry. Upon admission, the NAFLD group had higher C-reactive protein, procalcitonin, alanine aminotransferase, lactate dehydrogenase, and fibrinogen. Interleukins-6, -8, and -10 and CXCL10 were significantly higher, while IFN-γ was lower in NAFLD patients. Patients with NAFLD who progressed to critical illness had higher concentrations of IL-6, -8, -10, and IFN-β, and IL-8 and IL-10 appear to be effective prognostic biomarkers associated with time to recovery. In conclusion, NAFLD is associated with distinct cytokine profiles in COVID-19, possibly associated with disease severity and adverse outcomes.

Orthohantaviruses are mainly carried and transmitted by wild rodents, although during the last decade, they have also been identified in multiple species of shrews and moles. Orthohantavirus, Orthohantavirus seewisense (Seewis virus, SWSV), first detected in Switzerland in a single Sorex araneus (Eurasian common shrew) specimen, has been further described in several European countries, including Croatia’s neighboring Slovenia and Hungary. Croatia is a well-known endemic region for several zoonotic agents including three different orthohantaviruses: Orthohantavirus puumalaense (PUUV), Orthohantavirus dobravaense (DOBV), and Orthohantavirus tulaense (TULV). In this study, nine shrews were tested and SWSV RNA was detected in liver, lung, and kidney belonging to two shrews (22.22%), one collected on Medvednica mountain in Zagreb County, and the other in the Stara Gradiška area in lowland Croatia. The phylogenetic analysis of the complete S segment’s open reading frame (ORF) and partial L-segment revealed that the Croatian sequences, when compared to sequences from the adjacent geographic regions, form a specific genetic lineage. Two SWSV-positive shrew species—Sorex araneus and Neomys milleri (Mediterranean water shrew)—were identified using barcode-based sequence analysis. Therefore, the SWSV detection in N. milleri throughout the course of this study is seen as a rare find in this shrew species. To our knowledge, this is the first molecular and phylogenetic analysis of SWSV in Croatia.

Coronavirus disease (COVID-19) exhibits a wide spectrum of clinical severity and has been associated with specific biomarkers linked to disease progression and outcomes. This retrospective study analyzed sera from 1222 adult COVID-19 patients hospitalized at the University Hospital for Infectious Diseases in Croatia. We examined the association between several laboratory biomarker levels measured at patient admission and disease severity, fatal outcomes, viral variants and clinical parameters. Deceased patients and surviving patients with severe COVID-19 exhibited significantly elevated levels of several biomarkers on admission, including hs-troponin T, N-terminal pro-brain natriuretic peptide, creatine kinase, C-reactive protein, procalcitonin, interleukin-6, lactate dehydrogenase, lactate, urea and creatinine. Random forest models identified lymphocyte percentage, D-dimers, and hs-troponin T as the most important biomarkers for fatal outcome prediction, achieving 84.1% accuracy. Patients infected with the Delta SARS-CoV-2 variant exhibited significantly higher levels of proinflammatory, cardiac and renal biomarkers. Vaccination correlated with reduced proinflammatory parameters and higher lymphocyte proportions. Hypertension, chronic renal disease and diabetes were associated with increased cardiac, renal and metabolic biomarker levels, respectively. These findings highlight the association of several laboratory biomarkers with COVID-19 severity, viral variants, vaccination status and comorbidities, potentially offering prognostic insights into COVID-19 outcomes.

Hemorrhagic fever with renal syndrome (HFRS) is an acute zoonotic disease caused by viruses of the Orthohantavirus genus. This syndrome is characterized by renal and cardiopulmonary implications detectable with different biomarkers. Here, we explored the role of serum and urine levels of lipocalin-2, endothelin-1 and N-terminal pro-brain natriuretic peptide (NT-proBNP) in HFRS pathology. A total of twenty-eight patients hospitalized due to a Puumala orthohantavirus infection were included, with serum and urine samples collected on patient admission (acute phase) and discharge (convalescent phase). In comparison to healthy individuals, patients exhibited significantly higher acute-phase serum and urine levels of lipocalin-2, serum levels of endothelin-1 and serum and urine levels of NT-proBNP. Patients in the convalescent phase showed a significant decrease in urine lipocalin-2, serum endothelin-1 and serum and urine NT-proBNP levels. We recorded a strong correlation between serum levels of lipocalin-2 and endothelin-1 and urine levels of lipocalin-2 with several kidney injury markers, such as serum creatinine, urea, urine white blood cell count and proteinuria. We also demonstrated an independent correlation of serum and urine lipocalin-2 levels with acute kidney injury in HFRS. All in all, our results show an involvement of NT-proBNP, lipocalin-2 and endothelin-1 in the renal and cardiac pathology of HFRS.

Francisella tularensisis uzročnik je zoonoze tularemije, koja ima široki raspon domaćina, uključujući člankonošce, sisavce i ptice. Dvije podvrste F. tularensis, subsp. tularensis (Tip A) i subsp. holartica (Tip B), opisane su kao najčešći uzročnici bolesti u ljudi (Kingry and Petersen, 2014; Kuehn et al., 2013). Tip A i Tip B razlikuju se po svojim biokemijskim značajkama, patogenosti, ekologiji, epidemiologiji te geografskoj rasprostranjenosti. Rasprostranjenost Tipa A uglavnom je ograničena na sjevernoamerički kontinent, za razliku od Tipa B, koji se javlja duž cijele sjeverne polutke, a ujedno je i najčešći uzročnik tularemije u Europi(Keim et al., 2007; Kuehn et al., 2013). No ekologija i kruženje ove bakterije u prirodi do danas još nisu u potpunosti razjašnjeni. Različiti glodavci (voluharice, miševi, tekunice, bizantski štakori, dabrovi) te dvojezupci (kunić i zec) opisani su kao mogući rezervoari F.tularensis. Zadnja epidemiološka i epizootiološka istraživanja tularemije u Hrvatskoj datiraju iz šezdesetih i sedamdesetih godina prošloga stoljeća(Borcić et al., 1975). Stoga je cilj ovoga rada bio prikupiti nove podatke o prevalenciji tularemije u populaciji sitnih sisavaca u Hrvatskoj. Ukupno 444 uzoraka sitnih glodavaca i kukcojeda sakupljeno je u razdoblju od dvije godine na osam različitih lokaliteta u šumama kontinentalne Hrvatske: Lipovljani, Nova Subocka, Velika Gorica, Stara Gradiška, Županja, Sunja, Koprivnica i Čakovec (Slika 1). Pretraženi su uzorci slezene od: 197 Apodemus agrarius (poljski miš), 78 Apodemus sylvaticus (šumski miš), 92 Apodemus flavicollis (žutogrli miš), 17 Myodes glareolus (šumska voluharica), 27 Mycrotus agrestis (livadna voluharica), 20 Microtus arvalis (poljska voluharica) te 13 Sorex araneus (rovka). Uzorcisu pretraženi na prisutnost DNA Francisella spp. koristeći qRT-PCR metodu. Dobiveni rezultati pokazuju da su od ukupno 444 prikupljene životinje tri uzorka (0.67%), od kojih dva poljska miša te jedan šumski miš, bili pozitivni su na francizelu (Tablica 1). Sva tri pozitivna uzorka prikupljena su na istom području – lokalitetu Lipovljani. Tularemija je bolest koja se primarno povezuje s „nizinskim“ i „vodenim“ ekosustavima (Borcić, 1973). Područje duž toka rijeke Save (Posavina) opisuje se kao prirodno žarište tularemije u Hrvatskoj (Borcic et al., 1976), akao glavni rezervoar tularemije na tom području navodi se poljska voluharica (Borcic et al., 1976). Trimiša pozitivna na F. tularensis upućuju da je ova bakterija i dalje prisutna u populaciji sitnih glodavaca u Hrvatskoj. Nadalje, svi pozitivni uzorci prikupljeni su na lokalitetu Lipovljani, smještenom u srednjem dijelu toka rijeke Save, stoga ova regija zadržava obilježje prirodnog žarišta tularemije u Hrvatskoj.