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Background/Objectives: Various biological response modifiers play important roles in the immunopathogenesis of chronic hepatitis C (CHC). While serum levels of cytokines and growth factors change with the disease severity and treatment responses, the impact of concomitant liver steatosis on systemic inflammatory response is largely unknown. The aim of this study was to analyze the characteristics and kinetics of serum profiles of interleukins and growth factors in CHC patients with steatotic liver disease (SLD). Methods: Serum concentrations of 12 cytokines (IL-5, IL-13, IL-2, IL-6, IL-9, IL-10, IFN-γ, TNF-α, IL-17A, IL-17F, IL-4 and IL-22) and 6 growth factors (Angiopoietin-2, EGF, EPO, HGF, SCF, VEGF) were analyzed in 56 CHC patients at four time points (baseline, week 4, week 8 and SVR12) with bead-based flow cytometry assay. Results: At baseline, patients with SLD had significantly lower IL-9, IL-10, IL-13 and IL-22 and higher serum concentrations of EGF, VEGF and ANG. In a subgroup of patients with advanced liver fibrosis, SLD was linked with lower serum concentrations of IL-4, IL-5, IL-9, IL-10, IL-13 and IL-22 and higher concentrations of HGH and VEGF. Distinct cytokine kinetics during DAA treatment was observed, and SLD was identified as the main source of variation for IL-5, IL-9, IL-10, IL-13, IL-17A, IL-22, EGF, VEGF and ANG. Patients with SLD at SVR12 had significantly higher VEGF and HGF serum concentrations. Conclusions: SLD is associated with distinct cytokine and growth factor profiles and kinetics during CHC treatment, which might be associated with disease severity and the capacity for liver regeneration and contribute to fibrosis persistence.

In this study, we aimed to elucidate the changes in the immune response during antiviral treatment of patients with chronic hepatitis C, with an emphasis on the chemokine dynamics and their association with liver fibrosis. Serum concentrations of 12 chemokines. (CCL2, CCL3, CCL4, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10 and CXCL11) were measured in 32 patients with chronic hepatitis C before direct-acting antiviral treatment and after sustained virological response using bead-based flow cytometry. Chemokine levels were also measured in 14 sex- and age-matched healthy individuals. Concentrations of CXCL9, CXCL10, CXCL11 and CCL20 were significantly higher in chronic hepatitis C patients before direct-acting antiviral treatment compared to healthy individuals. We also observed a significant reduction in CXCL9, CXCL10 and CXCL11 levels after sustained virological response. Furthermore, we demonstrated a strong positive correlation between CXCL9, CXCL10 and CXCL11 levels before antiviral treatment. When considering liver fibrosis, we found significantly higher levels of CXCL10 and lower levels of CCL17 and CXCL5 in pre-treatment patients with severe fibrosis. None of the analysed chemokines were able to predict METAVIR fibrosis score reduction after sustained virological response. The results of this study emphasize the importance of proinflammatory pathways in liver fibrosis immunopathology during chronic hepatitis C. Finally, our results also characterized CXCL10 as the chemokine which most accurately distinguished pre-treatment CHC patients and healthy individuals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) are the most important neuroinvasive arboviruses detected in Europe. In this study, we analyzed cerebrospinal fluid (CSF) concentrations of 12 proinflammatory chemokines (CCL2, CCL3, CCL4, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, and CXCL11) in 77 patients with neuroinvasive diseases (NIDs). Flavivirus infection was confirmed in 62 patients (TBEV and WNV in 31 patients each), while in 15 patients the etiology of NID was not determined (NDE). Similar patterns of high-level expression of chemokines regulating monocyte/macrophage responses (CCL2), neutrophil recruitment (CXCL1 and CXCL8), and interferon-inducible chemoattractants for leukocytes (CXCL10 and CXCL11) have been observed in WNV and TBEV groups. None of the tested chemokines significantly differed between patients with TBEV or WNV. Concentrations of CCL17, CCL20, CXCL5, CXCL10, and CXCL11 were significantly lower in both WNV and TBEV groups compared to NID NDE patients. The logistic regression model showed that CSF concentrations of CXCL11, CXCL5, and CXCL10 could potentially be used for the classification of patients into the WNV or TBEV group versus groups with other NIDs. This study identified, for the first time, similar patterns of CSF chemokine expression in WNV and TBEV infections, suggesting common immunopathogenic mechanisms in neuroinvasive flavivirus infections that should be further evaluated.

Background and Objectives : John Cunningham polyomavirus (JCPyV) is a highly prevalent virus in the human population. The prevalence of JCPyV in patients with central nervous system disorders has not been examined extensively. The aim of this study was to analyze the prevalence of JCPyV DNA/antibodies in patients with neuroinvasive diseases (NID) of unknown etiology. Materials and Methods : The study included 132 patients with NID (febrile headache, meningitis, encephalitis) tested from January 2021 to December 2022. The control group consisted of 47 asymptomatic individuals. In patients with NID, serum and cerebrospinal fluid (CSF) samples were collected in the acute phase of the disease. CSF samples were tested for JCPyV DNA (PCR), while serum samples were tested for JCPyV IgG antibodies (ELISA). In controls, serum samples were tested for JCPyV IgG antibodies (ELISA). Results : JCPyV DNA was not detected in any of the CSF samples from patients with NID. JCPyV IgG antibodies were detected in 88.6% of patients and 74.5% of controls ( p < 0.001). In the patients' group, a significant difference in the IgG prevalence was observed between males (94.6%) and females (81.0%). In addition, significant differences in the seropositivity between age groups were found. The lowest seroprevalence (28.6%) was in patients less than 20 years, followed by a sharp increase in the 20-29-year group (69.2%), after which the seroprevalence remained stable (90.0-94.1%) in patients up to 69 years. All patients older than 70 years were JCPyV IgG-seropositive. No significant difference in the seroprevalence was found in patients presenting with febrile headache (81.6%), meningitis (93.3%), or meningoencephalitis (91.3%). No difference in the seropositivity between genders was found in controls. Although the seropositivity steadily increased in older participants, these differences were not significant. Analyzing the JCPyV antibody levels in patients with NID, the median antibody titers differed significantly between groups, ranging from 248 AU/mL (younger age groups) to 400 AU/mL (older age groups). Conclusions : Higher seroprevalence in the patients' group highlights the need to further investigate the possible association of JCPyV and NID.

Molecular epidemiology of HIV-1 infection is challenging due to the highly diverse HIV-genome. We investigated the genetic diversity and prevalence of transmitted drug resistance (TDR) followed by phylogenetic analysis in 270 HIV-1 infected, treatment-naïve individuals from Croatia in the period 2019-2022. The results of this research confirmed a high overall prevalence of TDR of 16.7%. Resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside RTIs (NNRTIs), and protease inhibitors (PIs) was found in 9.6%, 7.4%, and 1.5% of persons, respectively. No resistance to integrase strand-transfer inhibitors (INSTIs) was found. Phylogenetic analysis revealed that 173/229 sequences (75.5%) were part of transmission clusters, and the largest identified was T215S, consisting of 45 sequences. Forward transmission was confirmed in several clusters. We compared deep sequencing (DS) with Sanger sequencing (SS) on 60 randomly selected samples and identified additional surveillance drug resistance mutations (SDRMs) in 49 of them. Our data highlight the need for baseline resistance testing in treatment-naïve persons. Although no major INSTIs were found, monitoring of SDRMs to INSTIs should be continued due to the extensive use of first- and second-generation INSTIs.

The molecular diversity of Epstein-Barr virus (EBV) is exceptionally complex and based on the characterization of sequences coding for several viral genes. The aim of this study was to analyze the distribution of EBV types 1 and 2 and to characterize LMP1 variants in a cohort of 73 patients with infectious mononucleosis (IM), as well as to investigate a possible association between viral diversity and relevant clinical parameters. Population-based sequencing of EBNA-2 gene showed the presence of EBV type 1 in all IM patients. Analysis of LMP1 gene found a restricted repertoire of LMP1 variants with the predominance of wild-type B95-8, China1, Mediterranean and North Carolina variants with the presence of more than one LMP1 variant in 16.4% of patients. Co-infections with different LMP1 variants were associated with significantly higher levels of C-reactive protein and lower levels of maximal neutrophil counts and minimal platelet count. The results of this study have shown a narrow repertoire of LMP1 variants and an exclusive presence of EBV type 1 in a cohort of IM from Croatia, suggesting a characteristic local molecular pattern of this virus. The clinical importance of distinct immunobiological features of IM patients with LMP1 variant co-infections needs to be investigated further.

Viral hepatitis is a significant cause of morbidity and mortality worldwide. In Croatia, hepatitis B virus (HBV) and hepatitis C virus (HCV) are widely distributed, especially in some high-risk groups such as people who inject drugs (PWID), prisoners, and highly promiscuous groups. The seroprevalence of HBV ranges from 7.0% in the general population to 38.8% in PWID, depending on the region. The seroprevalence of HCV is highest among PWID (29-75.5%) as compared to 0.9% in the general population. Analyzing the distribution of HCV genotypes, no substantial changes in the molecular epidemiology of the two most frequent HCV genotypes (1 and 3) in the past 20 years were observed. However, the predominance of subtype 1b compared to subtype 1a as detected in 1996-2005 was not confirmed in 2008-2015. Hepatitis A virus (HAV) incidence was high in the past with a decreasing trend since the 2000s, except for an outbreak in 2017-2018 as part of the large European outbreak, which was mainly among men who have sex with men. Hepatitis E virus (HEV) is an emerging virus detected for the first time in Croatia in 2012. The seroprevalence of HEV is high among hemodialysis patients (27.9%) and liver transplant recipients (19.3-24.4%). In addition, higher seroprevalence rates were observed in animal-related professions (e.g., veterinarians, 15.2%; hunters, 14.9%). All detected HEV strains belonged to genotype 3.

Toll-like receptors (TLRs), as a family of pattern recognition receptors, play an important role in the recognition of HIV-1 molecular structures by various cells of the innate immune system, but also provide a functional association with subsequent mechanisms of adaptive immunity. TLR7 and TLR8 play a particularly important role in the innate immune response to RNA viruses due to their ability to recognise GU-rich single-stranded RNA molecules and subsequently activate intracellular signalling pathways resulting in expression of genes coding for various biological response modifiers (interferons, proinflammatory cytokines, chemokines). The aim of this review is to summarise the most recent knowledge on the role of TLRs in the innate immune response to HIV-1 and the role of TLR gene polymorphisms in the biology and in the clinical aspects of HIV infections. In addition, the role of TLR agonists as latency reversing agents in research to treat HIV infections and as immunomodulators in HIV vaccine research will be discussed.

Objectives: To analyze phylogenetic relations and assess the role of cross-border clusters in the spread of HIV-1 subtype B across the Balkans, given the general trends of new HIV diagnoses in seven Balkan countries., Design: Retrospective phylogenetic and trend analysis., Methods: In-depth phylogenetic, phylodynamic and phylogeographic analysis performed on 2415 HIV-1 subtype B sequences from 1999 to 2019 using maximal likelihood and Bayesian methods. The joinpoint regression analysis of new HIV diagnoses by country and modes of transmission using 2004-2019 ECDC data., Results: Ninety-three HIV-1 Subtype B transmission clusters (68% of studied sequences) were detected of which four cross-border clusters (11% of studied sequences). Phylodynamic analysis showed activity of cross-border clusters up until the mid-2000s, with a subsequent stationary growth phase. Phylogeography analyses revealed reciprocal spread patterns between Serbia, Slovenia and Montenegro and several introductions to Romania from these countries and Croatia. The joinpoint analysis revealed a reduction in new HIV diagnoses in Romania, Greece and Slovenia, whereas an increase in Serbia, Bulgaria, Croatia and Montenegro, predominantly among MSM., Conclusion: Differing trends of new HIV diagnoses in the Balkans mirror differences in preventive policies implemented in participating countries. Regional spread of HIV within the countries of former Yugoslavia has continued to play an important role even after country break-up, whereas the spread of subtype B through multiple introductions to Romania suggested the changing pattern of travel and migration linked to European integration of Balkan countries in the early 2000s., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Growth factors play a significant role in the immunopathogenesis of liver diseases, especially in liver fibrosis and cirrhosis. They can also play a role in liver regeneration and tissue repair. The regenerative capacity of the liver has been well established. Molecular mechanisms leading to regeneration involve a complex network of diverse molecules. Chronic liver injury leads to the dysregulation of regenerative mechanisms in the liver that, in addition to molecular oncogenesis, lead to uncontrolled cell proliferation and development of hepatocellular carcinoma (HCC). Stem cell factor (SCF), epidermal growth factor (EGF) and Angiopietin-2 (Ang-2) have been shown to be extremely important in the pathogenesis of liver diseases, and given their role in hepatitis B (HBV) or C virus (HCV), HCC and nonalcoholic fatty liver disease (NAFLD), they seem to be potential targets for future research into antifibrotic drugs. The role of SCF receptor c-kit in the liver is debatable, as it has impact on both liver regeneration and liver disease. EGF is a potential indicator of the survival of patients with HCC and can be a biomarker and therapeutic target structure in HCC. Further research is needed to investigate the potential role of Ang-2 for NAFLD associated with liver damage as a non-invasive circulating biomarker.

Hodgkin lymphomas (HLs) are a heterogeneous group of lymphoid neoplasia associated with Epstein-Barr virus (EBV) infection. EBV, considered to be an important etiological co-factor in approximately 1% of human malignancies, can be classified into two genotypes based on EBNA-2, EBNA-3A and EBNA-3C sequences, and into genetic variants based on the sequence variation of the gene coding for the LMP1 protein. Here, we present the results on the distribution of EBV genotypes 1 and 2 as well as LMP1 gene variants in 50 patients with EBV-positive classical HL selected from a cohort of 289 histologically verified cases collected over a 9-year period in a tertiary clinical center in the Southeast of Europe. The population-based sequencing of the EBNA-3C gene showed the exclusive presence of EBV genotype 1 in all cHL samples. The analysis of EBV LMP1 variant distribution showed a predominance of the wild-type strain B95-8 and the Mediterranean subtype with 30 bp deletion. These findings could contribute to the understanding of EBV immunobiology in cHL as well as to the development of a prophylactic and therapeutic vaccine.

Background: Parvovirus B19 (B19V) is associated with a wide range of clinical manifestations. The major presentation is erythema infectiosum. However, a persistent infection may cause pure red cell aplasia and chronic anemia in immunocompromized patients. The B19V seroprevalence varies with age and geographical location., Aim: To determine the B19V serological status and DNAemia in kidney, liver, and pancreas transplant candidates., Methods: Patients who underwent kidney, liver, or simultaneous kidney and pancreas/liver transplantation between January 2021 and May 2022 were included in the study. The serum samples were collected before transplantation. For detection of B19V DNA, a LightMix Kit B19V EC (TIB MOLBIOL, Berlin, Germany) was used. B19V IgM and IgG antibodies were detected using a commercial ELISA test (Euroimmun, Lübeck, Germany)., Results: One hundred and thirty-one transplant candidates were included in the study, 71.0% male, with an average age of 53.27 years ± 12.71 years. There were 68.7% liver, 27.5% kidney, 3.0% simul taneous pancreas/kidney transplant (SPKT), and 0.8% simultaneous liver/kidney transplant recipients. No patients had detectable B19V DNA. B19V IgG seroprevalence was 77.1%. No acute or recent infections were detected (IgM antibodies). There was no difference in the mean age of seronegative and seropositive patients (51.8 years ± 12.9 years vs 53.7 years ± 12.7 years, t = -0.603; P = 0.548). Although seropositivity was lower in patients aged less than 30 years (66.6%) compared to the patients aged 30-59 years and > 60 years (80.4% and 78.1%, respectively), this difference was not significant. In addition, there was no difference in seropositivity between male and female transplant candidates, 76.3% and 78.9% ( χ 2 = 0.104; P = 0.748). The seroprevalence did not differ among organ recipients, with 77.8%, 80.6%, and 50.0% for liver, kidney, and SPKT, respectively, ( χ 2 = 5.297; P = 0.151). No significant difference was found in the seroprevalence in kidney transplant patients according to dialysis modality. Seroprevalence was 71.1% in hemodialysis patients, and 100% in peritoneal dialysis patients ( χ 2 = 0.799; P = 0.372)., Conclusion: The B19V seroprevalence is expectedly high among kidney, liver, and pancreas transplant candidates, but there are still 22.9% of seronegative individuals who remain at risk for primary disease and severe manifestations. Further research should elucidate the necessity of B19V screening in peri-transplant management., Competing Interests: Conflict-of-interest statement: The authors have no conflicts of interest to declare., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

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.

Molecular epidemiology of hepatitis C virus (HCV) is exceptionally complex due to the highly diverse HCV genome. Genetic diversity, transmission dynamics, and epidemic history of the most common HCV genotypes were inferred by population sequencing of the HCV NS3, NS5A, and NS5B region followed by phylogenetic and phylodynamic analysis. The results of this research suggest high overall prevalence of baseline NS3 resistance associate substitutions (RAS) (33.0%), moderate prevalence of NS5A RAS (13.7%), and low prevalence of nucleoside inhibitor NS5B RAS (8.3%). Prevalence of RAS significantly differed according to HCV genotype, with the highest prevalence of baseline resistance to NS3 inhibitors and NS5A inhibitors observed in HCV subtype 1a (68.8%) and subtype 1b (21.3%), respectively. Phylogenetic tree reconstructions showed two distinct clades within the subtype 1a, clade I (62.4%) and clade II (37.6%). NS3 RAS were preferentially associated with clade I. Phylogenetic analysis demonstrated that 27 (9.0%) HCV sequences had a presumed epidemiological link with another sequence and classified into 13 transmission pairs or clusters which were predominantly comprised of subtype 3a viruses and commonly detected among intravenous drug users (IDU). Phylodynamic analyses highlighted an exponential increase in subtype 1a and 3a effective population size in the late 20th century, which is a period associated with an explosive increase in the number of IDU in Croatia.

Lymphocytic choriomeningitis virus (LCMV) is a neglected rodent-borne zoonotic virus distributed worldwide. Since serologic assays are limited to several laboratories, the disease has been underreported, often making it difficult to determine incidence and seroprevalence rates. Although human clinical cases are rarely recorded, LCMV remains an important cause of meningitis in humans. In addition, a fatal donor-derived LCMV infection in several clusters of solid organ transplant recipients further highlighted a pathogenic potential and clinical significance of this virus. In the transplant populations, abnormalities of the central nervous system were also found, but were overshadowed by the systemic illness resembling the Lassa hemorrhagic fever. LCMV is also an emerging fetal teratogen. Hydrocephalus, periventricular calcifications and chorioretinitis are the predominant characteristics of congenital LCMV infection, occurring in 87.5% of cases. Mortality in congenitally infected children is about 35%, while 70% of them show long-term neurologic sequelae. Clinicians should be aware of the risks posed by LCMV and should consider the virus in the differential diagnosis of aseptic meningitis, especially in patients who reported contact with rodents. Furthermore, LCMV should be considered in infants and children with unexplained hydrocephalus, intracerebral calcifications and chorioretinitis. Despite intensive interdisciplinary research efforts, efficient antiviral therapy for LCMV infection is still not available.

Lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen associated with aseptic meningitis, severe systemic infections in immunocompromised persons, and congenital anomalies. Data on the prevalence of LCMV infections are scarce. We analyzed the seroprevalence of LCMV in continental Croatian regions. A total of 338 serum samples of professionally exposed (forestry workers, hunters, agriculture workers in contact with rodents) and non-exposed populations (general population, pregnant women) were tested for the presence of LCMV antibodies using indirect immunofluorescence assay. No participants reported recent febrile disease. LCMV IgG antibodies were detected in 23/6.8% of participants: 9.8% exposed persons and 5.1% non-exposed persons (6.1% in the general population and 3.9% in pregnant women). No participants were LCMV IgM positive. Although higher seropositivity was found in males compared to females (8.9% vs. 4.7%), inhabitants of suburban/rural areas compared to inhabitants of urban areas (9.2% vs. 4.6%), and persons who used well as a source of water compared to those who used tap (11.4% vs. 5.6%), these differences did not reach statistical significance. Results of logistic regression showed that the presence of rodents in the house/yard and cleaning rodent nests were associated with an elevated risk for LCMV infection (OR = 2.962, 95% CI = 1.019-8.607).

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.

Late presentation to care is the major obstacle to receiving treatment for chronic hepatitis C (CHC). Our aim was to analyze the prevalence and trends of late presenters (LP) at first consultations in Croatia during a 10-year period. This retrospective cross-sectional study included all adult CHC patients ( n = 854) entering specialist medical care at the University Hospital for Infectious Diseases Zagreb between 2009 and 2018. LP was defined as liver stiffness measurement ≥ 9.5 kPa or biopsy METAVIR F ≥ 3. During the study period, mean patients' age increased from 37 to 52 years while HCV genotype distribution changed leading to the replacement of genotype 1b with 1a (g1b 32% to 21%; g1a 19% to 38%). A total of 320 (37.4%) were LP; they were older (47.5, IQR 40.5-57.6), and more commonly infected with g1b (34.1%) and g3 (42.5%). The prevalence of LP significantly increased from 31.9% in 2009 to 46.5% in 2018. Late presentation for care of CHC is increasing in Croatia suggesting a gap of diagnosing strategies in patients over 50 years.

Objectives: Understanding the public health impact of lymphogranuloma venereum (LGV) in Europe is hampered by inadequate diagnostics and surveillance systems in many European countries. We developed and piloted LGV surveillance in three European countries without existing systems and performed a preliminary investigation of LGV epidemiology, where little evidence currently exists., Methods: We recruited STI or dermatovenereology clinics and associated laboratories serving men who have sex with men (MSM) in Austria, Croatia and Slovenia, using the UK for comparison. We undertook centralised LGV testing of Chlamydia trachomatis (CT)-positive rectal swabs collected between October 2016 and May 2017 from MSM attending these clinics. Stored specimens from Austria (2015-2016) and Croatia (2014) were also tested. Clinical and sociodemographic data were collected using a standardised proforma. The ompA gene of LGV-positive specimens was sequenced., Results: In total, 500 specimens from CT-positive MSM were tested, and LGV positivity was 25.6% (128/500; 95% CI 22.0% to 29.6%) overall, and 47.6% (79/166; 40.1% to 55.2%) in Austria, 20.0% (3/15; 7.1% to 45.2%) in Croatia, 16.7% (1/6; 3.0% to 56.4%) in Slovenia and 14.4% (45/313; 10.9% to 18.7 %) in the UK. Proformas were completed for cases in Croatia, Slovenia and in the UK; proformas could not be completed for Austrian cases, but limited data were available from line listings. Where recorded, 83.9% (78/93) of LGV-CT cases were HIV-positive compared with 65.4% (149/228) of non-LGV-CT cases; MSM with LGV-CT were more likely to have proctitis (Austria, 91.8% vs 40.5%, p<0.001; Croatia, 100% vs 25%, p=0.04; UK, 52.4% vs 11.7%, p<0.001) than those with non-LGV-CT. Six different ompA sequences were identified, including three new variants; the L2 ompA sequence predominated (58.6%, 51/87)., Conclusions: LGV is substantially underdiagnosed in MSM across Europe. Unified efforts are needed to overcome barriers to testing, establish effective surveillance, and optimise diagnosis, treatment and prevention., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Semaphorins are a diverse family of immunoregulators recently recognized to play a major role in various phases of immune responses. Their role in chronic viral hepatitis C (CHC) and contribution to the progression of liver disease is unknown. The aim of this study was to analyse the association of secreted semaphorins with the severity of liver disease in patients with CHC. Serum concentrations of semaphorins were measured in 114 treatment-naive CHC patients and 36 healthy controls. Serum concentrations of SEMA3A, SEMA3C, SEMA5A, SEMA6B and SEMA6D were significantly increased in patients with CHC compared to controls. While serum concentrations of SEMA3C and SEMA6D significantly increased with fibrosis stage in both HCV-g1 and HCV-g3 infections, the concentration of SEMA5A inversely correlated with fibrosis stage in both HCV genotypes. ROC analysis showed that serum concentrations of SEMA3C (>4.0ng/mL, AUC 0.88) and SEMA6D (>4.5, AUC 0.82) had higher AUC than widely used APRI (AUC 0.71) and FIB-4 (AUC 0.74) scores. Serum concentrations of SEMA3C and SEMA6D significantly decreased after DAA and PEG IFN-α/ribavirin therapy, while the serum concentration of SEMA5A significantly increased after DAAs therapy. Immunohistochemistry confirmed the expression of SEMA3C and SEMA5A in hepatocytes, endothelial cells and lymphocytes of cirrhotic livers from CHC patients but not in controls. In conclusion, we provide the first evidence that SEMA3C, SEMA5A and SEMA6D can be considered as markers of liver injury in CHC., Competing Interests: The authors have declared that no competing interests exist.

The aim of this study was to compare cytokine expression on both gene and protein levels in acute and chronic phase of HIV type 1 (HIV-1) infection. Thirty four patients were enrolled for cytokine expression analysis on protein level in acute and chronic stage of HIV-1 infection. Using PCR array technology, expression of 84 cytokine genes was measured in 3 patients in acute and 3 patients in chronic stage of HIV-1 infection. Bead-based cytometry was used to quantify levels of Th1/Th2/Th9/Th17/Th22 cytokines. The results showed statistically significant increase of 13 cytokine gene expression (cd40lg, csf2, ifna5, il12b, il1b, il20, lta, osm, spp1, tgfa, tnfsf 11, 14 and 8) and downregulation of the il12a expression in chronic HIV type 1 infection. Concentrations of IL-10, IL-4 and TNF-α were increased in the acute HIV type 1 infection when compared to control group. During chronic HIV type 1 infection there was an increase of IL-10, TNF-α, IL-2, IL-6, IL-13 and IL-22 levels when compared to control group. Comparison of cytokine expression between two stages of infection showed a significant decrease in IL-9 concentration. This study showed changes in cytokine profiles on both gene and protein levels in different stages of HIV-infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Numerous studies have shown that baseline drug resistance patterns may influence the outcome of antiretroviral therapy. Therefore, guidelines recommend drug resistance testing to guide the choice of initial regimen. In addition to optimizing individual patient management, these baseline resistance data enable transmitted drug resistance (TDR) to be surveyed for public health purposes. The SPREAD program systematically collects data to gain insight into TDR occurring in Europe since 2001., Methods: Demographic, clinical, and virological data from 4140 antiretroviral-naive human immunodeficiency virus (HIV)-infected individuals from 26 countries who were newly diagnosed between 2008 and 2010 were analyzed. Evidence of TDR was defined using the WHO list for surveillance of drug resistance mutations. Prevalence of TDR was assessed over time by comparing the results to SPREAD data from 2002 to 2007. Baseline susceptibility to antiretroviral drugs was predicted using the Stanford HIVdb program version 7.0., Results: The overall prevalence of TDR did not change significantly over time and was 8.3% (95% confidence interval, 7.2%-9.5%) in 2008-2010. The most frequent indicators of TDR were nucleoside reverse transcriptase inhibitor (NRTI) mutations (4.5%), followed by nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations (2.9%) and protease inhibitor mutations (2.0%). Baseline mutations were most predictive of reduced susceptibility to initial NNRTI-based regimens: 4.5% and 6.5% of patient isolates were predicted to have resistance to regimens containing efavirenz or rilpivirine, respectively, independent of current NRTI backbones., Conclusions: Although TDR was highest for NRTIs, the impact of baseline drug resistance patterns on susceptibility was largest for NNRTIs. The prevalence of TDR assessed by epidemiological surveys does not clearly indicate to what degree susceptibility to different drug classes is affected., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America.)

Objectives: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance., Methods: This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score ≥ 10 to at least one InSTI. To rule out circulation of minority InSTI-resistant HIV, 65 samples were selected for 454 integrase sequencing., Results: For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIs were detected. Eleven (4%) subjects had mutations at resistance-associated positions with an HIVdb score ≥ 10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutations were detected, whereas integrase substitutions with an HIVdb score ≥ 10 were found in 8 (14.3%) individuals., Conclusions: No signature InSTI-resistant variants were circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistance were not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Successfully treated HIV-infected patients may still have an increased risk for cardiovascular morbidity and mortality, which might be related not only to traditional risks, but also to inflammation and dyslipidemia induced by HIV and/or antiretroviral therapy [1, 2]. We examined the relationship of serum lipid levels with plasma biomarkers of inflammation using a composite inflammatory burden score (IBS) from the following seven markers of inflammation: CD40L, tPA, MCP-1, IL-8, IL-6, hCRP and P-selectin., Materials and Methods: Subjects were selected among consecutive HIV-infected males ≥18 years of age with an undetectable viral load (<50 copies/mL of HIV1-RNA), seen at the University Hospital for Infectious Diseases, Zagreb, Croatia, in the period from January 2012 to March 2013. Plasma inflammatory biomarkers (CD40L, tPA, MCP-1, IL-8, IL-6, hCRP and P-selectin, quantified by bead-based cytometry) >75th percentile were considered elevated and an IBS was constructed as the presence of zero, one, two, or three or more elevated biomarkers. Correlations between the IBS and lipid parameters were examined using Spearman's Rho and by ordered logistic regression proportional odds model to estimate the odds of more elevated (>75th percentile) biomarkers., Results: 181 male patients were included into the study, the median age was 46.7 (Q1-Q3, 39.9-55.0) years and the median current CD4 cell count was 553.0 (Q1-Q3, 389-729) per microliter. The patients were mainly treated with two nucleoside reverse transcriptase inhibitor (NRTI) plus one non-NRTI (NNRTI) (N=100, 60.8%) or two NRTI plus lopinavir (N=50, 27.6%). There was a significant correlation between the IBS and serum cholesterol (Rho=0.23, 95% CI, 0.09-0.37), triglycerides (Rho=0.30, 95% CI, 0.16-0.42) and cholesterol/HDL-cholesterol ratio (Rho=0.25, 95% CI 0.11-0.38). In the multivariable model a one unit increase in cholesterol/HDL-cholesterol ratio was associated with a 1.72-fold (95% CI, 1.27-2.33) increased odds of having a greater IBS. One unit increase (mmol/L) of cholesterol and triglycerides was associated with a 1.41-fold (95% CI, 1.13-1.76) and 1.37-fold (95% CI, 1.18-1.60) increased odds of having a greater IBS, respectively., Conclusions: Our study suggests that in virologically suppressed patients there is a significant association between markers of inflammation and serum levels of cholesterol and triglycerides as well as the cholesterol/HDL-cholesterol ratio.

Unlabelled: Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts and laryngeal papillomas and has been included in both the quadrivalent and nonavalent prophylactic HPV vaccines. This study investigated the global genomic diversity of HPV6, using 724 isolates and 190 complete genomes from six continents, and the association of HPV6 genomic variants with geographical location, anatomical site of infection/disease, and gender. Initially, a 2,800-bp E5a-E5b-L1-LCR fragment was sequenced from 492/530 (92.8%) HPV6-positive samples collected for this study. Among them, 130 exhibited at least one single nucleotide polymorphism (SNP), indel, or amino acid change in the E5a-E5b-L1-LCR fragment and were sequenced in full. A global alignment and maximum likelihood tree of 190 complete HPV6 genomes (130 fully sequenced in this study and 60 obtained from sequence repositories) revealed two variant lineages, A and B, and five B sublineages: B1, B2, B3, B4, and B5. HPV6 (sub)lineage-specific SNPs and a 960-bp representative region for whole-genome-based phylogenetic clustering within the L2 open reading frame were identified. Multivariate logistic regression analysis revealed that lineage B predominated globally. Sublineage B3 was more common in Africa and North and South America, and lineage A was more common in Asia. Sublineages B1 and B3 were associated with anogenital infections, indicating a potential lesion-specific predilection of some HPV6 sublineages. Females had higher odds for infection with sublineage B3 than males. In conclusion, a global HPV6 phylogenetic analysis revealed the existence of two variant lineages and five sublineages, showing some degree of ethnogeographic, gender, and/or disease predilection in their distribution., Importance: This study established the largest database of globally circulating HPV6 genomic variants and contributed a total of 130 new, complete HPV6 genome sequences to available sequence repositories. Two HPV6 variant lineages and five sublineages were identified and showed some degree of association with geographical location, anatomical site of infection/disease, and/or gender. We additionally identified several HPV6 lineage- and sublineage-specific SNPs to facilitate the identification of HPV6 variants and determined a representative region within the L2 gene that is suitable for HPV6 whole-genome-based phylogenetic analysis. This study complements and significantly expands the current knowledge of HPV6 genetic diversity and forms a comprehensive basis for future epidemiological, evolutionary, functional, pathogenicity, vaccination, and molecular assay development studies., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Low-level viremia during antiretroviral therapy and its accurate measurement are increasingly relevant. Here, we present an international collaboration of 4,221 paired blood plasma viral load (pVL) results from four commercial assays, emphasizing the data with low pVL. The assays compared were the Abbott RealTime assay, the Roche Amplicor assay, and the Roche TaqMan version 1 and version 2 assays. The correlation between the assays was 0.90 to 0.97. However, at a low pVL, the correlation fell to 0.45 to 0.85. The observed interassay concordance was higher when detectability was defined as 200 copies/ml than when it was defined as 50 copies/ml. A pVL of ∼100 to 125 copies/ml by the TaqMan version 1 and version 2 assays corresponded best to a 50-copies/ml threshold with the Amplicor assay. Correlation and concordance between the viral load assays were lower at a low pVL. Clear guidelines are needed on the clinical significance of low-level viremia.

Post-transplant lymphoproliferative disorder (PTLD) is an increasingly recognized condition as the number of solid organ and bone marrow transplant recipients increases. It can be a life threatening fulminant disorder and affects approximately 8% of solid organ transplant recipients. Epstein-Barr virus (EBV) is closely involved in the pathogenesis of PTLD and the majority of PTLD cases arise in response to primary infection with EBV or to re-activation of previously acquired EBV. The principal risk factors underlying the development of PTLD are the degree of overall immunosuppression and EBV serostatus of the recipient. The most commonly used pathologic classification of PTLD is the World Health Organization classification, which divides PTLD into three categories: early lesions, polymorphic PTLD, and monomorphic PTLD. Early lesions are characterized by reactive plasmacytic hyperplasia. Polymorphic PTLD may be either polyclonal or monoclonal and is characterized by destruction of the underlying lymphoid architecture, necrosis, and nuclear atypia. In monomorphic PTLD, the majority of cases (>80%) arise from B cells, similar to non-Hodgkin's lymphoma in immunocompetent hosts. The most common subtype is diffuse large B-cell lymphoma, but Burkitt's/Burkitt's-like lymphoma and plasma cell myeloma are also seen. Rarely T-cell variants occur, which include peripheral T-cell lymphomas and, rarely, other uncommon types, including gamma/delta T-cell lymphoma and T-natural killer (NK) cell varieties. Hodgkin's disease-like lymphoma is very unusual. An accurate diagnosis of PTLD requires a high index of suspicion, since the disorder may present subtly and/or extranodally. Radiologic evidence of a mass or the presence of elevated serum markers (such as increased LDH levels) are suggestive of PTLD, with positive finding on ultrasonography, computed tomography, magnetic resonance and/or positron emission tomography scanning (possibly indicating metabolically active areas) also favoring the diagnosis. The management of PTLD poses a major therapeutic challenge and although there is reasonable agreement about the overall principles of treatment, there is still considerable controversy about the optimal treatment of individual patients. EBV-related PTLDs are a significant cause of mortality in patients undergoing orthotopic liver transplantation with the observed mortality rate of up to 50%. This paper presents the experience acquired at Merkur University Hospital in the diagnosis and treatment of patients with liver transplantation and PTLD.

Background: Although considered an essential tool for monitoring the effect of combination antiretroviral treatment (CART), HIV-1 RNA (viral load, VL) testing is greatly influenced by cost and availability of resources., Objectives: To examine whether HIV infected patients who were initially successfully treated with CART have less frequent monitoring of VL over time and whether CART failure and other HIV-disease and sociodemographic characteristics are associated with less frequent VL testing., Methods: The study included patients who started CART in the period 1999-2004, were older than 18 years, CART naive, had two consecutive viral load measurements of <400 copies/ml after 5 months of treatment and had continuous CART during the first 15 months. The time between two consecutive visits (days) was the outcome and associated factors were assessed using linear mixed models., Results: We analyzed a total of 128 patients with 1683 visits through December 2009. CART failure was observed in 31 (24%) patients. When adjusted for the follow-up time, the mean interval between two consecutive VL tests taken in patients before CART failure (155.2 days) was almost identical to the interval taken in patients who did not fail CART (155.3 days). On multivariable analysis, we found that the adjusted estimated time between visits was 150.9 days before 2003 and 177.6 in 2008/2009. A longer time between visits was observed in seafarers compared to non-seafarers; the mean difference was 30.7 days (95% CI, 14.0 to 47.4; p<0.001); and in individuals who lived more than 160 kilometers from the HIV treatment center (mean difference, 16 days, p = 0.010)., Conclusions: Less frequent monitoring of VL became common in recent years and was not associated with failure. We identified seafarers as a population with special needs for CART monitoring and delivery.

Molecular methods are a well-established part of routine diagnostic work-up in patients infected with hepatitis B virus (HBV) and hepatitis C virus (HCV). Confirmation of active viral replication in infected patients is based on detection and/or quantification of viral genome in serum by molecular assays. Diagnostic algorithm for hepatitis C includes detection and/or quantification of HCV RNA in serum of infected patients and HCV genotyping. Diagnostic work-up in patients with hepatitis B includes quantification of HBV DNA in serum, HBV genotyping, and determination of resistance to nucleoside and nucleotide analogues. Real-time polymerase chain reaction (PCR) is the standard recommended molecular method for quantification of HCV RNA and HBV DNA in clinical samples. Due to superior sensitivity, real-time PCR assays can provide both qualitative detection of viral genome and quantification. Molecular diagnosis of HCV and HBV infections in clinical laboratories should be limited to certified standardized assays.

We examined a total of 194 patients over 18 years of age with chronic prostatitis syndrome and no evidence of structural or functional lower genitourinary tract abnormalities. The following data were obtained for each patient: clinical history--the severity of chronic prostatitis symptoms scored by a Croatian translation of the NiH CPSI questionnaire, clinical status including digitorectal examination, urethral swab specimens, and selective samples of urine and expressed prostatic secretion, according to the 4-glass localization test (meares and Stamey localization technique). Patients were treated orally with antimicrobial agents in doses and duration according to clinical practice in Croatia. An infectious etiology was determined in 169 (87%) patients. Chlamydia trachomatis was the causative pathogen in 38 (20%), Trichomonas vaginalis in 35 (18%), Enterococcus in 36 (19%) and Escherichia coli in 35 (18%) patients. In the remaining 25 patients the following causative pathogens were found: Ureaplasma urealyticum, Proteus mirabilis, Klebsiella pneumoniae, Streptococcus agalactiae and Pseudomonas aeruginosa. Comparison of symptoms scores and effect on quality of life has shown that the most severe clinical presentation of disease was recorded in patients with chronic bacterial prostatitis caused by E. coli and Enterococcus (p<0.001). Clinical success was paralleled by bacteriological eradication in chronic bacterial prostatitis caused by C. trachomatis, Enterococcus and E. coli (kappa >0.2<0.5), but not in inflammatory chronic pelvic pain syndrome caused by T. vaginalis.

Prometryne is a methylthio-s-triazine herbicide used to control annual broadleaf and grass weeds in many cultivated plants. Significant traces are documented in environment, mainly water, soil and plants used for human and domestic animal nutrition. Data on the toxic effects of prometryne and other methylthio-s-triazine have scorcely been published. The goal of this study was to investigate if prometryne, applied orally, could induce DNA damage in mouse leukocytes, in subchronical in vivo experimental design. Three different doses of prometryne were applied per os repeatedly every 48 hours. After the 7th dose (day 14) and the 14th dose (day 28) blood leucocytes were analyzed by alkaline Single Cell Gel Electrophoresis (Comet) assay. The results of three different comet parameters showed general increase in Olive tail moment, tail length and tail intensity values in treated groups of animals. The increase in measured values was almost proportional to the dose received and the time of exposure. We conclude that prometryne or its metabolic residues have the potential to induce processes that cause genotoxic effects on leukocytes on mice in in vivo repeated exposure.

Prometryne is a methylthio-s-triazine herbicide. Significant traces are documented in environment, mainly waters, soil and plants used for nutrition. The aim of this study was to estimate prometryne immunotoxic properties through induction of apoptotic and/or necrotic changes in thymocytes, splenocytes and lymph node cells after repeated subchronical exposure. Three different doses of prometryne (185, 375, 555mgkg(-1)) were applied per os every 48h, over 28 days. Flow cytometry assay (annexinV-FITC and PI) was conducted to record apoptotic and necrotic damage. In the spleen significant changes in the percentage of apoptotic cells were not detected between treated and control groups respectively. In thymus and lymph node, within the lowest dose group (185mgkg(-)1), an increase in percentage of early apoptosis without any significant increase in necrosis was detected. Medium (375mgkg(-1)) as well as high dose triggered increase in late apoptosis in lymph node while in thymus; late apoptosis was increased only in animals exposed to the highest dose (555mgkg(-1)). The highest applied dose, in thymus and lymph node respectively, caused a general decrease in percentage of vital cells in favour of marked increase of percentages of all types of dying cells (apoptotic, late apoptotic/early necrotic and necrotic). Prometryne caused disbalance in major organs of immune system, markedly lymph nodes and thymus, by induction of early apoptotic changes in dose/time specific manner., (Copyright © 2008 Elsevier B.V. All rights reserved.)