Antiviral treatment significantly reduces the levels of CXCL9, CXCL10 and CXCL11 in chronic hepatitis C.

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.

Steatotic Liver Disease and Sepsis Outcomes-A Prospective Cohort Study (SepsisFAT).

Background: While it has been shown that steatotic liver disease (SLD) is associated with systemic changes in immune response, the impact of SLD on sepsis outcomes has not yet been established. The aim of this study was to investigate the association between SLD and sepsis severity and outcomes. Methods: A prospective observational study included consecutively hospitalized adult patients with community-acquired sepsis during a 16-month period. Results: Of the 378 included patients (49.5% male, median age of 69, IQR 57-78 years), 174 (46%) were diagnosed with SLD. Patients with SLD were older and more frequently fulfilled the criteria for metabolic syndrome. There were no differences in the source and etiology of sepsis between the groups. Patients with SLD exhibited a higher incidence of acute kidney injury (29.3% vs. 17.6%), the need for renal replacement therapy (16.1% vs. 8.8%), and more frequent use of invasive mechanical ventilation (29.3% vs. 18.1%). In-hospital mortality was significantly higher in the SLD group (18.39% vs. 9.8%). The multivariable analysis indicated that SLD was associated with mortality (HR 2.82, 95% CI 1.40-5.71) irrespective of the other elements within metabolic syndrome. Conclusions: SLD might be associated with higher sepsis in-hospital mortality, and more frequent development of acute kidney and respiratory insufficiency requiring more critical care support.

Association of Immune Semaphorins with COVID-19 Severity and Outcomes.

Semaphorins have recently been recognized as crucial modulators of immune responses. In the pathogenesis of COVID-19, the activation of immune responses is the key factor in the development of severe disease. This study aimed to determine the association of serum semaphorin concentrations with COVID-19 severity and outcomes. Serum semaphorin concentrations (SEMA3A, -3C, -3F, -4D, -7A) were measured in 80 hospitalized adult patients with COVID-19 (moderate (n = 24), severe (n = 32), critical, (n = 24)) and 40 healthy controls. While SEMA3C, SEMA3F and SEMA7A serum concentrations were significantly higher in patients with COVID-19, SEMA3A was significantly lower. Furthermore, SEMA3A and SEMA3C decreased with COVID-19 severity, while SEMA3F and SEMA7A increased. SEMA4D showed no correlation with disease severity. Serum semaphorin levels show better predictive values than CRP, IL-6 and LDH for differentiating critical from moderate/severe COVID-19. SEMA3F and SEMA7A serum concentrations were associated with the time to recovery, requirement of invasive mechanical ventilation, development of pulmonary thrombosis and nosocomial infections, as well as with in-hospital mortality. In conclusion, we provide the first evidence that SEMA3A, SEMA3C, SEMA3F and SEMA7A can be considered as new biomarkers of COVID-19 severity.

TGF Beta as a Prognostic Biomarker of COVID-19 Severity in Patients with NAFLD-A Prospective Case-Control Study.

Non-alcoholic fatty liver disease (NAFLD), the leading cause of chronic liver disease in Western countries, has been identified as a possible risk factor for COVID-19 severity. However, the immunological mechanisms by which NAFLD exacerbates COVID-19 remain unknown. Transforming growth factor-beta 1 (TGF-ß1) has an important immunomodulatory and pro-fibrotic role, which has already been described in NAFLD. However, the role of TGF-ß1 in COVID-19 remains unclear, and could also be the pathophysiology link between these two conditions. The aim of this case-control study was to analyze the expression of TGF-ß1 in COVID-19 patients depending on the presence of NAFLD and COVID-19 severity. Serum TGF-ß1 concentrations were measured in 60 hospitalized COVID-19 patients (30 with NAFLD). NAFLD was associated with higher serum TGF-ß1 concentrations that increased with disease severity. Admission TGF-ß1 concentrations showed good discriminative accuracy in predicting the development of critical disease and COVID-19 complications (need for advanced respiratory support, ICU admission, time to recovery, development of nosocomial infections and mortality). In conclusion, TGF-ß1 could be an efficient biomarker for predicting COVID-19 severity and adverse outcomes in patients with NAFLD.

A pilot sentinel surveillance system to monitor treatment and treatment outcomes of chronic hepatitis B and C infections in clinical centres in three European countries, 2019.

BackgroundThe World Health Organization European Action Plan 2020 targets for the elimination of viral hepatitis are that > 75% of eligible individuals with chronic hepatitis B (HBV) or hepatitis C (HCV) are treated, of whom > 90% achieve viral suppression.AimTo report the results from a pilot sentinel surveillance to monitor chronic HBV and HCV treatment uptake and outcomes in 2019.MethodsWe undertook retrospective enhanced data collection on patients with a confirmed chronic HBV or HCV infection presenting at one of seven clinics in three countries (Croatia, Romania and Spain) for the first time between 1 January 2019 and 30 June 2019. Clinical records were reviewed from date of first attendance to 31 December 2019 and data on sociodemographics, clinical history, laboratory results, treatment and treatment outcomes were collected. Treatment eligibility, uptake and case outcome were assessed.ResultsOf 229 individuals with chronic HBV infection, treatment status was reported for 203 (89%). Of the 80 individuals reported as eligible for treatment, 51% (41/80) were treated of whom 89% (33/37) had achieved viral suppression. Of 240 individuals with chronic HCV infection, treatment status was reported for 231 (96%). Of 231 eligible individuals, 77% (179/231) were treated, the majority of whom had received direct acting antivirals (99%, 174/176) and had achieved sustained virological response (98%, 165/169).ConclusionTreatment targets for global elimination were missed for HBV but not for HCV. A wider European implementation of sentinel surveillance with a representative sample of sites could help monitor progress towards achieving hepatitis control targets.

Case Report: Amphotericin B and Mefloquine as a Salvage Treatment of Alveolar Echinococcosis.

Alveolar echinococcosis is an emerging zoonotic disease caused by the parasite Echinococcus multilocularis. Most patients are diagnosed at a late stage, when lifelong treatment with benzimidazoles is required to stop disease progression. However, for patients who do not tolerate benzimidazole therapy, there are no alternatives. Here, we present a patient with advanced alveolar echinococcosis who was successfully treated with amphotericin B deoxycholate and mefloquine as a rescue therapy after he developed albendazole intolerance.

The Role of Non-Alcoholic Fatty Liver Disease in Infections.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease, affecting one third of the Western population. The hallmark of the disease is excessive storage of fat in the liver. Most commonly, it is caused by metabolic syndrome (or one of its components). Even though the development of NAFLD has multiple effects on the human organism resulting in systemic chronic low-grade inflammation, this review is focused on NAFLD as a risk factor for the onset, progression, and outcomes of infectious diseases. The correlation between NAFLD and infections is still unclear. Multiple factors (obesity, chronic inflammation, altered immune system function, insulin resistance, altered intestinal microbiota, etc.) have been proposed to play a role in the development and progression of infections in people with NAFLD, although the exact mechanism and the interplay of mentioned factors is still mostly hypothesized. In this article we review only the selection of well-researched topics on NAFLD and infectious diseases (bacterial pneumonia, COVID, H. pylori, urinary tract infections, C. difficile, bacteremia, hepatitis B, hepatitis C, HIV, and periodontitis).

Distinct Expression Patterns of Genes Coding for Biological Response Modifiers Involved in Inflammatory Responses and Development of Fibrosis in Chronic Hepatitis C: Upregulation of SMAD-6 and MMP-8 and Downregulation of CAV-1, CTGF, CEBPB, PLG, TIMP-3, MMP-1, ITGA-1, ITGA-2 and LOX.

Background and Objectives: The aim of this study was to analyze the expression of genes on transcriptomic levels involved in inflammatory immune responses and the development of fibrosis in patients with chronic hepatitis C. Materials and Methods: Expression patterns of 84 selected genes were analyzed with real-time quantitative RT PCR arrays in the peripheral blood of treatment-naive patients with chronic hepatitis C and healthy controls. The panel included pro- and anti-fibrotic genes, genes coding for extracellular matrix (EMC) structural constituents and remodeling enzymes, cell adhesion molecules, inflammatory cytokines, chemokines and growth factors, signal transduction members of the transforming growth factor- beta (TGF-ß) superfamily, transcription factors, and genes involved in epithelial to mesenchymal transition. Results: The expression of SMAD-6 coding for a signal transduction TGF-beta superfamily member as well as MMP-8 coding for an ECM protein were significantly increased in CHC patients compared with controls. Conclusions: Chronic hepatitis C was also characterized by a significant downregulation of a set of genes including CAV-1, CTGF, TIMP-3, MMP-1, ITGA-1, LOX, ITGA-2, PLG and CEBPB encoding various biological response modifiers and transcription factors. Our results suggest that chronic hepatitis C is associated with distinct patterns of gene expression modulation in pathways associated with the regulation of immune responses and development of fibrosis.

Do Semaphorins Play a Role in Development of Fibrosis in Patients with Nonalcoholic Fatty Liver Disease?

Nonalcoholic fatty liver disease (NAFLD) is associated with systemic changes in immune response linked with chronic low-grade inflammation and disease progression. Semaphorins, a large family of biological response modifiers, were recently recognized as one of the key regulators of immune responses, possibly also associated with chronic liver diseases. The aim of this study was to identify semaphorins associated with NAFLD and their relationship with steatosis and fibrosis stages. In this prospective, case-control study, serum semaphorin concentrations (SEMA3A, -3C, -4A, -4D, -5A and -7A) were measured in 95 NAFLD patients and 35 healthy controls. Significantly higher concentrations of SEMA3A, -3C and -4D and lower concentrations of SEAMA5A and -7A were found in NAFLD. While there was no difference according to steatosis grades, SEMA3C and SEMA4D significantly increased and SEMA3A significantly decreased with fibrosis stages and had better accuracy in predicting fibrosis compared to the FIB-4 score. Immunohistochemistry confirmed higher expression of SEMA4D in hepatocytes, endothelial cells and lymphocytes in NAFLD livers. The SEMA5A rs1319222 TT genotype was more frequent in the NAFLD group and was associated with higher liver stiffness measurements. In conclusion, we provide the first evidence of the association of semaphorins with fibrosis in patients with NAFLD.

COVID-19 and Pulmonary Thrombosis-An Unresolved Clinical Puzzle: A Single-Center Cohort Study.

Pulmonary thrombosis (PT) is a frequent complication of COVID-19. However, the risk factors, predictive scores, and precise diagnostic guidelines on indications for CT pulmonary angiography (CTPA) are still lacking. This study aimed to analyze the clinical and laboratory characteristics associated with PT in patients with COVID-19. We conducted a cohort study of consecutively hospitalized adult patients with COVID-19 who underwent CTPA at the University Hospital for Infectious Diseases in Zagreb, Croatia between 1 April and 31 December 2021. Of 2078 hospitalized patients, 575 (27.6%) underwent CTPA. PT was diagnosed in 178 (30.9%) patients (69.6% males, median age of 61, IQR 50-69 years). The PT group had a higher CRP, LDH, D-dimer, platelets, and CHOD score. PT was more frequent in patients requiring ≥15 L O2/min (25.0% vs. 39.7%). In multivariable analysis, only D-dimer ≥ 1.0 mg/L (OR 1.78, 95%CI 1.12-2.75) and O2 ≥ 15 L (OR 1.89, 95%CI 1.26-2.84) were associated with PT. PT was not associated with in-hospital mortality. In conclusion, our data confirmed a high incidence of PT in hospitalized patients with COVID-19, however, no correlation with traditional risk factors and mortality was found. CTPA should be performed in patients requiring high-flow supplemental oxygen or those with increased D-dimer levels.

The Effect of Treatment-Induced Viral Eradication on Cytokine and Growth Factor Expression in Chronic Hepatitis C.

In this study, we evaluated the effect of hepatitis C virus eradication using direct-acting antivirals (DAA) on the serum cytokine and growth factor profiles of chronic hepatitis C patients (CHC). Serum concentrations of 12 cytokines and 13 growth factors were measured in 56 patients with CHC before, during the DAA treatment and after sustained virological response using bead-based flow cytometry. Cytokine and growth factor levels were also measured in 15 healthy individuals. The majority of the selected cytokines and growth factors exhibited similar concentrations before, during and after successful DAA treatment, the exceptions being IL-10, EGF, HGF and VEGF. Significantly lower concentrations of IL-10, IL-13, IL-4, IL-4, IL-9, TNF- α and higher levels of Ang-2, HGF and SCF were observed in patients with CHC before and after DAA treatment compared with healthy individuals. Patients with severe fibrosis stages exhibited higher levels of Ang-2 and lower levels of EGF, PDGF-AA and VEGF. Furthermore, IL-4, IL-5 and SCF were characterized as potential biomarkers of DAA treatment using random forest. Additionally, logistic regression characterized EGF as a potential biomarker of severe CHC. Our results suggest inhibition of pro-inflammatory processes and promotion of liver regeneration in CHC patients during DAA treatment.

Molecular Epidemiology and Baseline Resistance of Hepatitis C Virus to Direct Acting Antivirals in Croatia.

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.

Distinct Cytokine Profiles in Severe COVID-19 and Non-Alcoholic Fatty Liver Disease.

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-ß, 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.

Association of Nonalcoholic Fatty Liver Disease With COVID-19 Severity and Pulmonary Thrombosis: CovidFAT, a Prospective, Observational Cohort Study.

Background: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease associated with systemic changes in immune response, which might be associated with coronavirus disease 2019 (COVID-19) severity. The aim of this study was to investigate the impact of NAFLD on COVID-19 severity and outcomes. Methods: A prospective observational study included consecutively hospitalized adult patients, hospitalized between March and June 2021, with severe COVID-19. Patients were screened for fatty liver by ultrasound and subsequently diagnosed with NAFLD. Patients were daily followed until discharge, and demographic, clinical, and laboratory data were collected and correlated to clinical outcomes. Results: Of the 216 patients included, 120 (55.5%) had NAFLD. The NAFLD group had higher C-reactive protein (interquartile range [IQR]) (84.7 [38.6-129.8] mg/L vs 66.9 [32.2-97.3] mg/L; P = .0340), interleukin-6 (49.19 [22.66-92.04] ng/L vs 13.22 [5.29-39.75] ng/L; P < .0001), aspartate aminotransferase (58 [40-81] IU/L vs 46 [29-82] IU/L; P = .0123), alanine aminotransferase (51 [32-73] IU/L vs 40 [23-69] IU/L; P = .0345), and lactate dehydrogenase (391 [285-483] IU/L vs 324 [247-411] IU/L; P = .0027). The patients with NAFLD had higher disease severity assessed by 7-category ordinal scale, more frequently required high-flow nasal cannula or noninvasive ventilation (26, 21.66%, vs 10, 10.42%; P = .0289), had longer duration of hospitalization (IQR) (10 [8-15] days vs 9 [6-12] days; P = .0018), and more frequently had pulmonary thromboembolism (26.66% vs 13.54%; P = .0191). On multivariable analyses, NAFLD was negatively associated with time to recovery (hazard ratio, 0.64; 95% CI, 0.48 to 0.86) and was identified as a risk factor for pulmonary thrombosis (odds ratio, 2.15; 95% CI, 1.04 to 4.46). Conclusions: NAFLD is associated with higher COVID-19 severity, more adverse outcomes, and more frequent pulmonary thrombosis.

HCV Elimination in Central Europe with Particular Emphasis on Microelimination in Prisons.

In 2016, the WHO announced a plan to eliminate viral hepatitis as a public health threat by 2030. In this narrative review, experts from Bulgaria, Croatia, the Czech Republic, Hungary, Latvia, Lithuania, Poland and Slovakia assessed the feasibility of achieving the WHO 2030 target for HCV infections in Central Europe. They focused mainly on HCV micro-elimination in prisons, where the highest incidence of HCV infections is usually observed, and the impact of the COVID-19 pandemic on the detection and treatment of HCV infections. According to the presented estimates, almost 400,000 people remain infected with HCV in the analyzed countries. Interferon-free therapies are available ad libitum, but the number of patients treated annually in the last two years has halved compared to 2017-2019, mainly due to the COVID-19 pandemic. None of the countries analyzed had implemented a national HCV screening program or a prison screening program. The main reason is a lack of will at governmental and prison levels. None of the countries analyzed see any chance of meeting the WHO targets for removing viral hepatitis from the public threat list by 2030, unless barriers such as a lack of political will and a lack of screening programs are removed quickly.

Rapid COVID-19 Antigen Testing in Croatia: Risk Perception Plays an Important Role in the Epidemic Control.

Aim: To explore the clinical presentation and epidemiological history of the subjects who underwent SARS-CoV-2 antigen testing. Methods: We included 1,000 consecutive subjects who presented themselves at the diagnostic clinic in Croatia and analyzed their symptoms and epidemiological history. All subjects were classified into three groups, according to their reason of arrival; symptomatic, contacts of confirmed patients, and those who were tested due to administrative reasons. Results: On average, there were 24% of positive antigen results; the positivity rate was 51% among symptomatic, 16% in contacts, and 5% of administrative patients. The commonest symptoms of the disease included febrility and anosmia. We developed a clinical score to predict SARS-CoV-2 positivity, which had an area under the curve of 79.3 [95% confidence intervals (CI) 75.8-82.8]. Contact with the isolated person [odds ratio 0.54 (95% CI 0.31-0.94)] and international travel had a protective effect [0.20 (0.09-0.43)], suggesting that risk perception and mandatory pretravel measures had a key role in the determination of the infection risk. Conclusions: A combination of clinical symptoms can have reasonable predictive power for an antigen-positive test result. Risk perception seems to have a role in the epidemic spread, probably via stricter adherence to personal preventative measures.

Nonalcoholic Fatty Liver Disease-A Novel Risk Factor for Recurrent Clostridioides difficile Infection.

Recurrent Clostridioides difficile infections (rCDI) have a substantial impact on healthcare systems, with limited and often expensive therapeutic options. Nonalcoholic fatty liver disease (NAFLD) affects about 25% of the adult population and is associated with metabolic syndrome, changes in gut microbiome and bile acids biosynthesis, all possibly related with rCDI. The aim of this study was to determine whether NAFLD is a risk factor associated with rCDI. A retrospective cohort study included patients ≥ 60 years hospitalized with CDI. The cohort was divided into two groups: those who were and were not readmitted with CDI within 3 months of discharge. Of the 329 patients included, 107 patients (32.5%) experienced rCDI. Patients with rCDI were older, had higher Charlson Age-Comorbidity Index (CACI) and were more frequently hospitalized within 3 months. Except for chronic kidney disease and NAFLD, which were more frequent in the rCDI group, there were no differences in other comorbidities, antibiotic classes used and duration of antimicrobial therapy. Multivariable Cox regression analysis showed that age >75 years, NAFLD, CACI >6, chronic kidney disease, statins and immobility were associated with rCDI. In conclusion, our study identified NAFLD as a possible new host-related risk factor associated with rCDI.

Is elimination of HCV in 2030 realistic in Central Europe.

According to the recent data presented by Central-European HCV experts, the estimated prevalence of HCV is between 0.2% and 1.7% in certain countries in this region. There are no financial limitations to access to treatment in most countries. Patients in these countries have access to at least one pangenotypic regimen. The most common barriers to the elimination of HCV in Central Europe are a lack of established national screening programmes and limited political commitment to the elimination of HCV. Covid-19 has significantly affected the number of patients who have been diagnosed and treated, thus, delaying the potential elimination of HCV. These data suggest that the elimination of HCV elimination projected by WHO before 2030 will not be possible in the Central Europe.

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.