Proinflammatory Chemokine Levels in Cerebrospinal Fluid of Patients with Neuroinvasive Flavivirus Infections.

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.

Human Cytomegalovirus (HCMV) Genetic Diversity, Drug Resistance Testing and Prevalence of the Resistance Mutations: A Literature Review.

Human cytomegalovirus (HCMV) is a pathogen with high prevalence in the general population that is responsible for high morbidity and mortality in immunocompromised individuals and newborns, while remaining mainly asymptomatic in healthy individuals. The HCMV genome is 236,000 nucleotides long and encodes approximately 200 genes in more than 170 open reading frames, with the highest rate of genetic polymorphisms occurring in the envelope glycoproteins. HCMV infection is treated with antiviral drugs such as ganciclovir, valganciclovir, cidofovir, foscarnet, letermovir and maribavir targeting viral enzymes, DNA polymerase, kinase and the terminase complex. One of the obstacles to successful therapy is the emergence of drug resistance, which can be tested phenotypically or by genotyping using Sanger sequencing, which is a widely available but less sensitive method, or next-generation sequencing performed in samples with a lower viral load to detect minority variants, those representing approximately 1% of the population. The prevalence of drug resistance depends on the population tested, as well as the drug, and ranges from no mutations detected to up to almost 50%. A high prevalence of resistance emphasizes the importance of testing the patient whenever resistance is suspected, which requires the development of more sensitive and rapid tests while also highlighting the need for alternative therapeutic targets, strategies and the development of an effective vaccine.

Infection with human cytomegalovirus, Epstein-Barr virus, and high-risk types 16 and 18 of human papillomavirus in EGFR-mutated lung adenocarcinoma.

AIM: To assess the frequency of human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and high-risk types of human papillomavirus (HPV16 and HPV18) infections in lung adenocarcinoma samples. METHODS: Lung adenocarcinoma cytological smears and their DNA isolates were obtained from patients hospitalized at the Department for Lung Diseases Jordanovac, Zagreb, in 2016 and 2017. Overall, 67 lung adenocarcinoma samples were examined: 34 with epidermal growth factor receptor gene (EGFR) mutations and 33 without EGFR mutations. The EGFR mutation status and virus presence were assessed with a polymerase chain reaction, and random samples were additionally tested for EBV with Sanger sequencing. HCMV, EBV, HPV16, and HPV18 infections were evaluated in relation to EGFR mutation, smoking status, and sex. A meta-analysis of available data about HPV infection in non-small cell lung cancer was performed. RESULTS: More frequent HCMV, EBV, HPV16, and HPV18 infections were observed in lung adenocarcinoma samples with EGFR mutations than in samples without these mutations. Coinfection of the investigated viruses was observed only in lung adenocarcinoma samples with mutated EGFR. In the group with EGFR mutations, smoking was significantly associated with HPV16 infection. The meta-analysis showed that non-small cell lung cancer patients with EGFR mutations had a higher odds of HPV infection. CONCLUSION: HCMV, EBV, and high-risk HPV infections are more frequent in EGFR-mutated lung adenocarcinomas, which indicates a possible viral impact on the etiology of this lung cancer subtype.

Effects of combining flow intermittency and exposure to emerging contaminants on the composition and metabolic response of streambed biofilm bacterial communities.

Freshwater ecosystems are characterised by the co-occurrence of stressors that simultaneously affect the biota. Among these, flow intermittency and chemical pollution severely impair the diversity and functioning of streambed bacterial communities. Using an artificial streams mesocosm facility, this study examined how desiccation and pollution caused by emerging contaminants affect the composition of stream biofilm bacterial communities, their metabolic profiles, and interactions with their environment. Through an integrative analysis of the composition of biofilm communities, characterization of their metabolome and composition of the dissolved organic matter, we found strong genotype-to-phenotype interconnections. The strongest correlation was found between the composition and metabolism of the bacterial community, both of which were influenced by incubation time and desiccation. Unexpectedly, no effect of the emerging contaminants was observed, which was due to the low concentration of the emerging contaminants and the dominant impact of desiccation. However, biofilm bacterial communities modified the chemical composition of their environment under the effect of pollution. Considering the tentatively identified classes of metabolites, we hypothesised that the biofilm response to desiccation was mainly intracellular while the response to chemical pollution was extracellular. The present study demonstrates that metabolite and dissolved organic matter profiling may be effectively integrated with compositional analysis of stream biofilm communities to yield a more complete picture of changes in response to stressors.

Fate and effects of microplastics in combination with pharmaceuticals and endocrine disruptors in freshwaters: Insights from a microcosm experiment.

Microplastic contamination of freshwater ecosystems has become an increasing environmental concern. To advance the hazard assessment of microplastics, we conducted a microcosm experiment in which we exposed a simplified aquatic ecosystem consisting of moss and caddisflies to microplastics (polyethylene, polystyrene and polypropylene) and pharmaceuticals and personal care products (1H-benzotriazole, bisphenol A, caffeine, gemfibrozil, ketoprofen, methylparaben, estriol, diphenhydramine, tris (1-chloro-2-propyl) phosphate) over the course of 60 days. We monitored the flux of microplastics within the microcosm, as well as the metabolic and total protein variation of organisms. This study offers evidence highlighting the capacity of moss to act as a sink for free-floating microplastics in freshwater environments. Moss is also shown to serve as a source and pathway for microplastic particles to enter aquatic food webs via caddisflies feeding off of the moss. Although most ingested microparticles were eliminated between caddisflies life stages, a small fraction of microplastics was transferred from aquatic to terrestrial ecosystem by emergence. While moss exhibited a mild response to microplastic stress, caddisflies ingesting microplastics showed stress comparable to that caused by exposure to pharmaceuticals. The molecular responses that the stressors triggered were tentatively identified and related to phenotypic responses, such as the delayed development manifested through the delayed emergence of caddisflies exposed to stress. Overall, our study provides valuable insights into the adverse effects of microplastics on aquatic species, compares the impacts of microplastics on freshwater biota to those of pharmaceuticals and endocrine disrupting compounds, and demonstrates the role aquatic organisms have in redistributing microplastics between aquatic and terrestrial ecosystems.

Quantification of Antiviral Cytokines in Serum, Cerebrospinal Fluid and Urine of Patients with Tick-Borne Encephalitis in Croatia.

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.

The curious case of methylparaben: Anthropogenic contaminant or natural origin?

The widespread use of methylparaben as a preservative has caused increased exposure to natural aquatic systems in recent decades. However, current studies have suggested that exposure to this compound can result in endocrine disrupting effects, raising much concern regarding its environmental impact. In contast, methylparaben has also been found to be part of the metabolome of some organisms, prompting the question as to whether this compound may be more natural than previously assumed. Through a combination of field studies investigating the natural presence of methylparaben across different taxa, and a 54-day microcosm experiment examining the bioaccumulation and movement of methylparaben across different life stages of aquatic insects (order Trichoptera), our results offer evidence suggesting the natural origin of methylparaben in aquatic and terrestrial biota. This study improves our understanding of the role and impact this compound has on biota and challenges the current paradigm that methylparaben is exclusively a harmful anthropogenic contaminant. Our findings highlight the need for further research on this topic to fully understand the origin and role of parabens in the environment which will allow for a comprehensive understanding of the extent of environmental contamination and result in a representative assessment of the environmental risk that may pose.

Severe West Nile Virus Neuroinvasive Disease: Clinical Characteristics, Short- and Long-Term Outcomes.

West Nile Virus Neuroinvasive Disease (WNV NID) requires prolonged intensive care treatment, resulting in high mortality and early disability. Long-term results are lacking. We have conducted an observational retrospective study with a prospective follow-up of WNV NID patients treated at the Intensive Care Unit (ICU), University Hospital for Infectious Diseases, Zagreb, Croatia, 2013-2018. Short-term outcomes were vital status, length of stay (LOS), modified Rankin Scale (mRS), and disposition at discharge. Long-term outcomes were vital status and mRS at follow-up. Twenty-three patients were identified, 78.3% males, median age 72 (range 33-84) years. Two patients (8.7%) died in the ICU, with no lethal outcomes after ICU discharge. The median ICU LOS was 19 days (range 5-73), and the median hospital LOS was 34 days (range 7-97). At discharge, 15 (65.2%) patients had moderate to severe/mRS 3-5, 6 (26.0%) had slight disability/mRS 2-1, no patients were symptom-free/mRS 0. Ten (47.6%) survivors were discharged to rehabilitation facilities. The median time to follow-up was nine months (range 6-69). At follow-up, seven patients died (30.5%), five (21.7%) had moderate to severe/mRS 3-5, one (4.3%) had slight disability/mRS 2-1, six (26.1%) had no symptoms/mRS 0, and four (17.4%) were lost to follow-up. Briefly, ten (43.5%) survivors improved their functional status, one (4.3%) was unaltered, and one (4.3%) aggravated. In patients with severe WNV NID, intensive treatment in the acute phase followed by inpatient rehabilitation resulted in significant recovery of functional status after several months.

Hepatitis A Outbreak in Men Who Have Sex with Men Using Pre-Exposure Prophylaxis and People Living with HIV in Croatia, January to October 2022.

The hepatitis A virus (HAV) is a highly hepatotropic virus transmitted mainly via the fecal-oral route. The purpose of this study is to describe a prolonged HAV outbreak in HIV-infected men who have sex with men (MSM) and pre-exposure prophylaxis (PrEP) users in Croatia in 2022. Croatia has a centralized system of HIV care and the PrEP service is only available at the University Hospital for Infectious Diseases (UHID), Zagreb. We reviewed all MSM living with HIV and MSM PrEP users at UHID and identified those diagnosed with HAV between January and October 2022. During this period, a total of 1036 MSM living with HIV and 361 PrEP users were followed, and 45 (4.4%) and 32 (8.9%) were diagnosed with HAV, respectively. Most cases were diagnosed in mid-February. A total of 70.1% (726/1036) MSM living with HIV and 82.3% (297/361) PrEP users were susceptible to HAV. Sequencing information was available for 34 persons; in all cases the HAV subtype IA was found. Our findings indicate that both MSM living with HIV and HIV-uninfected PrEP users are vulnerable to HAV infection and might be a potential source for a more widespread HAV epidemic.

Effects of sodium bicarbonate supplementation on exercise performance: an umbrella review.

BACKGROUND: We aimed to perform an umbrella review of meta-analyses examining the effects of sodium bicarbonate supplementation on exercise performance. METHODS: We systematically searched for meta-analyses that examined the effects of sodium bicarbonate supplementation on exercise performance. The methodological quality of the included reviews was evaluated using the Assessing the Methodological Quality of Systematic Reviews 2 (AMSTAR 2) checklist. Grading of Recommendations Assessment, Development, and Evaluation (GRADE) framework for downgrading the certainty in evidence was used, which included assessments of risk of bias, inconsistency, indirectness, imprecision, and publication bias. RESULTS: Eight reviews of moderate and high methodological quality met inclusion criteria. Using the GRADE framework, evidence for the ergogenic effects of sodium bicarbonate supplementation on peak and mean power in the Wingate test and Yo-Yo test performance was classified as being of moderate quality. The evidence for these outcomes did not receive a point on the indirectness GRADE item, as "serious indirectness" was detected. Low-quality evidence was found for the ergogenic effect of sodium bicarbonate supplementation on endurance events lasting ∼45 s to 8 min, muscle endurance, and 2000-m rowing performance. Evidence for these outcomes was classified as low quality, given that risk of bias, indirectness, and publication bias were assessed as "unclear", "serious", and "strongly suspected", respectively. The ergogenic effects ranged from trivial (pooled effect size: 0.09) to large (pooled effect size: 1.26). Still, for most outcomes, sodium bicarbonate elicited comparable ergogenic effects. For example, sodium bicarbonate produced similar effects on performance in endurance events lasting ∼45 s to 8 min, muscle endurance tests, and Yo-Yo test (pooled effect size range: 0.36 to 0.40). No significant differences between the effects of sodium bicarbonate and placebo were found for general mean power, muscle strength, and repeated-sprint ability. CONCLUSION: Based on meta-analyses of moderate to high quality, it can be concluded that sodium bicarbonate supplementation acutely enhances peak anaerobic power, anaerobic capacity, performance in endurance events lasting ∼45 s to 8 min, muscle endurance, 2000-m rowing performance, and high-intensity intermittent running. More research is needed among women to improve the generalizability of findings.

UV filters as a driver of the antibiotic pollution in different water matrices.

Antibiotic pollution is frequently detected in fresh waters and wastewaters where they represent an environmental risk for the development of global antibiotic resistance. Due to their excessive use in personal care products, UV filters have also been found to be pseudo-persistent in the aquatic environment. In contrast to antibiotics, which can undergo photodegradation, UV filters are compounds designed to stably absorb UV radiation. This study explored the light based remediation of representative antibiotics from seven classes of antibiotics (fluoroquinolones, tetracyclines, penicillins, macrolide, glycolpeptide, sulphonamides and trimethoprim) in order to assess whether antibiotic pollution persists longer in the presence of organic UV filters. We show that the presence of UV filters either completely supresses or significantly alters the photodegradation of antibiotics in water. Advanced technologies in wastewater treatment, such as the use of UV C radiation, both effectively minimise the effect of UV filters and degrade most of the tested antibiotics proving to be effective management strategy. However, the half-life of erythromycin and amoxicillin, widely used antibiotics from macrolide- and penicillin-like classes, is extended in the presence of UV filters, even during UV C irradiation. Overall, the UV filters present within environmental mixtures are identified as important drivers of mixture toxicity, as they prolong antibiotic contamination of aquatic and engineered environments. The ramification of such finding is that inadequate consideration of UV filters may result in an imperfect prediction of the solar and UV light-based remediation of antibiotics, lead to improper classification of antibiotics persistence in the environment and cause non-optimal chemical fate and transport model performance. Use of the more benign compounds and assessment of the UV filters were identified as feasible management options in minimizing the influence of UV filters onto the remediation of antibiotics in aquatic environments.

Antiviral Cytokine Response in Neuroinvasive and Non-Neuroinvasive West Nile Virus Infection.

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.

Distribution of human papillomavirus genotypes in women with high-grade cervical intraepithelial lesions and cervical carcinoma and analysis of human papillomavirus-16 genomic variants.

AIM: To analyze the distribution of high-risk human papillomavirus (HR-HPV) genotypes and the diversity of HPV-16 genomic variants in Croatian women with high-grade squamous intraepithelial lesions (HSIL) and cervical carcinoma. METHODS: Tissue biopsy specimens were obtained from 324 women with histopathologically confirmed HSIL or cervical carcinoma, 5 women with low-grade SIL, and 49 women with negative histopathology. HR-HPV DNA was detected with Ampliquality HPV-type nucleic-acid hybridization assay, which identifies 29 different HPV genotypes. HPV-16 genomic variants were analyzed by an in-house sequencing. RESULTS: The most common HPV type in women with HSIL was HPV-16, detected in 127/219 (57.9%) specimens. HPV-16 was also the dominant type in squamous cell cervical carcinoma (46/69 or 66.7%) and in adenocarcinoma (18/36 or 50.0%). Out of 378 patients, 360 had HR-HPV (282 single infections and 79 multiple infections), 3 (0.8%) patients had low-risk HPV, and 15 (4%) tested negative. HPV-16 variants were determined in 130 HPV-16 positive specimens, including 74 HSIL and 46 carcinoma specimens. In HSIL specimens, 41 distinct variants were found, 98.6% belonging to the European branch and 1.4% belonging to the African branch. In cervical carcinoma specimens, 95% isolates grouped in 41 variants belonging to the European branch, one isolate (2.5%) belonged to the North American, and one (2.5%) to the Asian-American branch. CONCLUSION: HPV-16, mainly belonging to the European branch, was the most frequent HPV genotype in women from Croatia with histologically confirmed HSIL and cervical cancer.

Clinical, Virological, and Immunological Findings in Patients with Toscana Neuroinvasive Disease in Croatia: Report of Three Cases.

Toscana virus (TOSV) is an arthropod-borne virus, transmitted to humans by phlebotomine sandflies. Although the majority of infections are asymptomatic, neuroinvasive disease may occur. We report three cases of neuroinvasive TOSV infection detected in Croatia. Two patients aged 21 and 54 years presented with meningitis, while a 22-year old patient presented with meningoencephalitis and right-sided brachial plexitis. Cerebrospinal fluid (CSF), serum, and urine samples were collected and tested for neuroinvasive arboviruses: tick-borne encephalitis, West Nile, Usutu, TOSV, Tahyna, and Bhanja virus. In addition, CSF and serum samples were tested for the anti-viral cytokine response. High titers of TOSV IgM (1000-3200) and IgG (3200-10,000) antibodies in serum samples confirmed TOSV infection. Antibodies to other phleboviruses (sandfly fever Sicilian/Naples/Cyprus virus) were negative. CSF samples showed high concentrations of interleukin 6 (IL-6; range 162.32-2683.90 pg/mL), interferon gamma (IFN-γ; range 110.12-1568.07 pg/mL), and IL-10 (range 28.08-858.91 pg/mL), while significantly lower cytokine production was observed in serum. Two patients recovered fully. The patient with a brachial plexitis improved significantly at discharge. The presented cases highlight the need of increasing awareness of a TOSV as a possible cause of aseptic meningitis/meningoencephalitis during summer months. Association of TOSV and brachial plexitis with long-term sequelae detected in one patient indicates the possibility of more severe disease, even in young patients.

Wake up and smell the coffee: caffeine supplementation and exercise performance-an umbrella review of 21 published meta-analyses.

OBJECTIVE: To systematically review, summarise and appraise findings of published meta-analyses that examined the effects of caffeine on exercise performance. DESIGN: Umbrella review. DATA SOURCES: Twelve databases. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: Meta-analyses that examined the effects of caffeine ingestion on exercise performance. RESULTS: Eleven reviews (with a total of 21 meta-analyses) were included, all being of moderate or high methodological quality (assessed using the Assessing the Methodological Quality of Systematic Reviews 2 checklist). In the meta-analyses, caffeine was ergogenic for aerobic endurance, muscle strength, muscle endurance, power, jumping performance and exercise speed. However, not all analyses provided a definite direction for the effect of caffeine when considering the 95% prediction interval. Using the Grading of Recommendations Assessment, Development and Evaluation criteria the quality of evidence was generally categorised as moderate (with some low to very low quality of evidence). Most individual studies included in the published meta-analyses were conducted among young men. SUMMARY/CONCLUSION: Synthesis of the currently available meta-analyses suggest that caffeine ingestion improves exercise performance in a broad range of exercise tasks. Ergogenic effects of caffeine on muscle endurance, muscle strength, anaerobic power and aerobic endurance were substantiated by moderate quality of evidence coming from moderate-to-high quality systematic reviews. For other outcomes, we found moderate quality reviews that presented evidence of very low or low quality. It seems that the magnitude of the effect of caffeine is generally greater for aerobic as compared with anaerobic exercise. More primary studies should be conducted among women, middle-aged and older adults to improve the generalisability of these findings.

Frequency of baseline NS5A resistance-associated substitutions in patients infected with genotype 1 of hepatitis C virus in Croatia.

The backbone of current treatment for chronic Hepatitis C virus (HCV) infection are direct-acting antivirals targeting viral nonstructural proteins (NS3, NS4A, NS5A, NS5B). To date, there are six NS5A inhibitors approved for treatment of chronic HCV infection. The presence of drug-associated resistance substitutions is mainly due to fast error-prone replication, showing differential frequency between genotypes and subtypes. The aim of this study was to determine the frequency of baseline resistance to NS5A protein inhibitors in patients with genotype 1 HCV in Croatia. Resistance-associated substitutions (RAS) were detected by Sanger sequencing of HCV NS5A region amplified from 84 patients followed by phylogenetic analysis and analysis with Geno2Pheno algorithm. The frequency of NS5A RAS was 14.3% and highly dependent on viral subtype. The overall frequency of NS5A RAS was higher in patients infected with HCV subtype 1b (24.2%) than in those infected with HCV subtype 1a (7.8%). Overall, three resistance-conferring mutations were detected (Q30R, M28T and Y93H) along with two mutations (M28V and L31I) that cause reduced susceptibility to NS5A inhibitors. Analysis of the sequences showed two distinct subtype 1a clades with RAS detected in 4.3% (1/23) clade I and 10.7% (3/28) clade II sequences. Only a few distinct NS5A RAS were detected suggesting a high degree of homogeneity of the viral population. High frequency of clinically relevant NS5A RAS in Croatia suggest that the analysis of frequency and patterns of resistance mutations in local populations and evaluation of their possible clinical impact could be beneficial.

The association of semaphorins 3C, 5A and 6D with liver fibrosis stage in chronic hepatitis C.

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.

Cardiovascular markers of inflammation and serum lipid levels in HIV-infected patients with undetectable viremia.

Patients successfully treated for HIV infection 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. We examined the relationship of serum lipid levels with plasma biomarkers of inflammation using a composite inflammatory burden score (IBS) based on individual (>75th percentile) measurements from the following seven markers: CD40L, tPA, MCP-1, IL-8, IL-6, hCRP and P-selectin. IBS was categorized as 0 (none of the biomarkers >75th percentile), 1, 2 and 3 or more scores. Correlations between the IBS and lipid parameters were examined by ordered logistic regression proportional odds models to estimate the odds of more elevated biomarkers. 181 male patients with undetectable HIV-viremia were included into the study. In the multivariate model, a one-unit increase (mmol/L) of total 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. Those with an IBS score ≥1 compared to none had 2.14 (95% CI, 1.43-3.20) higher odds of having a one-unit increased total cholesterol/HDL-cholesterol ratio. In successfully treated HIV-infected persons dyslipidemia was associated with inflammation.

Total and cytosolic concentrations of twenty metals/metalloids in the liver of brown trout Salmo trutta (Linnaeus, 1758) from the karstic Croatian river Krka.

Total and cytosolic concentrations of twenty metals/metalloids in the liver of brown trout Salmo trutta (Linnaeus, 1758) were studied in the period from April 2015 to May 2016 at two sampling sites on Croatian river Krka, to establish if river water contamination with metals/metalloids downstream of Knin town has influenced metal bioaccumulation in S. trutta liver. Differences were observed between two sites, with higher concentrations of several elements (Ag, As, Ca, Co, Na, Se, Sr, V) found downstream of Knin town, whereas few others (Cd, Cs, Mo, Tl) were, unexpectedly, increased at the Krka River spring. However, total metal/metalloid concentrations in the liver of S. trutta from both sites of the Krka River were still mainly below previously reported levels for pristine freshwaters worldwide. The analysis of seasonal changes of metal/metalloid concentrations in S. trutta liver and their association with fish sex and size mostly indicated their independence of fish physiology, making them good indicators of water contamination and exposure level. Metal/metalloid concentrations in the metabolically available hepatic cytosolic fractions reported in this study are the first data of that kind for S. trutta liver, and the majority of analyzed elements were present in the cytosol in the quantity higher than 50% of their total concentrations, thus indicating their possible availability for toxic effects. However, the special attention should be directed to As, Cd, Cs, and Tl, which under the conditions of increased exposure tended to accumulate more within the cytosol. Although metal/metalloid concentrations in S. trutta liver were still rather low, monitoring of the Krka River water quality and of the health status of its biota is essential due to a trend of higher metal/metalloid bioaccumulation downstream of Knin town, especially taking into consideration the proximity of National Park Krka and the need for its conservation.