Molecular epidemiology and mechanisms of carbapenem and colistin resistance in Klebsiella and other Enterobacterales from treated wastewater in Croatia.

Among the most problematic bacteria with clinical relevance are the carbapenem-resistant Enterobacterales (CRE), as there are very limited options for their treatment. Treated wastewater can be a route for the release of these bacteria into the environment and the population. The aim of this study was to isolate CRE from treated wastewater from the Zagreb wastewater treatment plant and to determine their phenotypic and genomic characteristics. A total of 200 suspected CRE were isolated, 148 of which were confirmed as Enterobacterales by MALDI-TOF MS. The predominant species was Klebsiella spp. (n = 47), followed by Citrobacter spp. (n = 40) and Enterobacter cloacae complex (cplx.) (n = 35). All 148 isolates were carbapenemase producers with a multidrug-resistant phenotype. Using multi-locus sequence typing and whole-genome sequencing (WGS), 18 different sequence types were identified among these isolates, 14 of which were associated with human-associated clones. The virulence gene analysis of the sequenced Klebsiella isolates (n = 7) revealed their potential pathogenicity. PCR and WGS showed that the most frequent carbapenemase genes in K. pneumoniae were blaOXA-48 and blaNDM-1, which frequently occurred together, while blaKPC-2 together with blaNDM-1 was mainly detected in K. oxytoca, E. cloacae cplx. and Citrobacter spp. Colistin resistance was observed in 40% of Klebsiella and 57% of Enterobacter isolates. Underlying mechanisms identified by WGS include known and potentially novel intrinsic mechanisms (point mutations in the pmrA/B, phoP/Q, mgrB and crrB genes) and acquired mechanisms (mcr-4.3 gene). The mcr-4.3 gene was identified for the first time in K. pneumoniae and is probably located on the conjugative IncHI1B plasmid. In addition, WGS analysis of 13 isolates revealed various virulence genes and resistance genes to other clinically relevant antibiotics as well as different plasmids possibly associated with carbapenemase genes. Our study demonstrates the important role that treated municipal wastewater plays in harboring and spreading enterobacterial pathogens that are resistant to last-resort antibiotics.

Comparison of MALDI-TOF mass spectrometry and 16S rDNA sequencing for identification of environmental bacteria: a case study of cave mussel-associated culturable microorganisms.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is routinely used as a rapid and cost-effective method for pathogen identification in clinical settings. In comparison, its performance in other microbiological fields, such as environmental microbiology, is still being tested, although isolates of environmental microbes are essential for in-depth in vivo studies of their biology, including biotechnological applications. We investigated the applicability of MALDI-TOF MS for the identification of bacterial isolates from a highly oligotrophic environment - Dinaric Karst caves, which likely harbor specific microorganisms. We cultured bacteria from the shell surface of the endemic mussel Congeria jalzici, one of the three known cave mussels in the world that lives in the Dinaric karst underground. The bacterial isolates were obtained by swabbing the shell surface of mussels living in microhabitats with different amounts of water: 10 air-exposed mussels, 10 submerged mussels, and 10 mussels in the hygropetric zone. A collection of 87 pure culture isolates was obtained, mostly belonging to the phylum Bacillota (72%), followed by Pseudomonadota (16%), Actinomycetota (11%), and Bacteroidota (1%). We compared the results of MALDI-TOF MS identification (Bruker databases DB-5989 and version 11, v11) with the results of 16S rDNA-based phylogenetic analysis, a standard procedure for bacterial identification. Identification to the genus level based on 16S rDNA was possible for all isolates and clearly outperformed the results from MALDI-TOF MS, although the updated MALDI-TOF MS database v11 gave better results than the DB-5989 version (85% versus 62%). However, identification to the species-level by 16S rDNA sequencing was achieved for only 17% of isolates, compared with 14% and 40% for the MALDI-TOF MS databases DB-5989 and v11 database, respectively. In conclusion, our results suggest that continued enrichment of MALDI-TOF MS libraries will result with this method soon becoming a rapid, accurate, and efficient tool for assessing the diversity of culturable bacteria from different environmental niches.

Treated municipal wastewater as a source of high-risk and emerging multidrug-resistant clones of E. coli and other Enterobacterales producing extended-spectrum ß-lactamases.

Extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales are a major public health problem, and wastewater from municipal wastewater treatment plants (WWTPs) is a potential means of spreading them into the environment and community. Our objective was to isolate ESBL-producing E. coli and other Enterobacterales from wastewater after treatment at Croatia's largest WWTP and to characterize these isolates by phenotypic and genotypic testing. Of the 200 bacterial isolates, 140 were confirmed as Enterobacterales by MALDI-TOF MS, with Escherichia coli and Klebsiella spp. predominating (69% and 7%, respectively). All 140 enterobacterial isolates were multidrug-resistant (MDR) and produced ESBLs. The most prevalent ESBL genes among the isolates tested were blaCTX-M-15 (60%), blaTEM-116 (44%), and blaCTX-M-3 (13%). Most isolates (94%) carried more than one ESBL gene in addition to blaCTX-M. Genes encoding plasmid-mediated AmpC, most notably blaEBC, were detected in 22% of isolates, whereas genes encoding carbapenemases (blaOXA-48, blaNDM-1, blaVIM-1) were less represented (10%). In E. coli, 9 different sequence types (ST) were found, with the emerging high-risk clones ST361 (serotype A-O9:H30) and pandemic ST131 (serotype B2-O25:H4) predominating (32% and 15%, respectively). Other high-risk E. coli clones included ST405 (3%), ST410 (3%), CC10 (3%), ST10 (3%), and ST38 (2%), and emerging clones included ST1193 (2%) and ST635 (2%). Whole-genome sequencing of three representative E. coli from two dominant clone groups (ST361 and ST131) and one extensively drug-resistant K. oxytoca revealed the presence of multiple plasmids and resistance genes to several other antibiotic classes, as well as association of the blaCTX-M-15 gene with transposons and insertion sequences. Our findings indicate that treated municipal wastewater contributes to the spread of emerging and pandemic MDR E. coli clones and other enterobacterial strains of clinical importance into the aquatic environment, with the risk of reintroduction into humans.

Metabolic Associated Fatty Liver Disease (MAFLD) and COVID-19 Infection: An Independent Predictor of Poor Disease Outcome?

Background and Objectives: Early reports on COVID-19 infection suggested that the SARS-CoV-2 virus solely attacks respiratory tract cells. As the pandemic spread, it became clear that the infection is multiorganic. Metabolic associated fatty liver disease (MAFLD) is a chronic liver disease strongly associated with insulin resistance and diabetes. The aim of this study was to assess a possible interplay between MAFLD and COVID-19 infection and its implication in COVID-19 outcome. Materials and Methods: A retrospective observational study, including 130 COVID-19 positive patients was conducted. MAFLD diagnosis was made based on the International Consensus criteria. Patients were divided into two groups, group A (MAFLD) and group B (nonMAFLD). Anthropometric and laboratory analysis were obtained. COVID-19 severity was assessed using the NEWS2 score. Disease outcome was threefold and regarded as discharged, patients who required mechanical ventilation (MV), and deceased patients. Results: MAFLD prevalence was 42%, 67% of patients were discharged, and 19% needed MV. Mortality rate was 14%. MAFLD patients were significantly younger (p < 0.001), and had higher body mass index (p < 0.05), respiratory rate (p < 0.05) and systolic blood pressure (p < 0.05) than nonMAFLD patients. Regarding metabolic syndrome and inflammatory markers: group A had significantly higher glycemia at admission (p = 0.008), lower HDL-c (p < 0.01), higher triglycerides (p < 0.01), CRP (p < 0.001), IL-6 (p < 0.05) and ferritin (p < 0.05) than group B. MAFLD was associated with more prevalent type 2 diabetes (p = 0.035) and hypertension (p < 0.05). MAFLD patients had a more severe disease course (NEWS2 score, 6.5 ± 0.5 vs. 3 ± 1.0, p < 0.05). MAFLD presence was associated with lower patient discharge (p < 0.01) and increased need for MV (p = 0.024). Multiple regression analysis showed that BMI (p = 0.045), IL-6 (p = 0.03), and MAFLD (p < 0.05) are significant independent risk factors for a poor COVID-19 outcome. Conclusions: The prevalence of MAFLD is relatively high. MAFLD patients had a more severe COVID-19 clinical course and worse disease outcome. Our results imply that early patient stratification and risk assessment are mandatory in order to avoid poor outcomes.

Current Trends in the Management of Helicobacter pylori Infection in Serbia: Preliminary Results from the European Registry on H. pylori Management.

BACKGROUND: Helicobacter pylori (H. pylori) is the most common chronic bacterial infection. Treatment effectiveness remains a subject of debate considering bacterial antimicrobial resistance. Our aim was to analyze the diagnostic methods and eradication treatments for H. pylori infection in Serbia. METHODS: An observational multicenter prospective study was conducted in Serbia, as part of the European Registry on H. pylori Management (Hp-EuReg). Demographics, treatment indication, diagnostic methods, previous eradication attempts, and treatment were collected at AEG-REDCap e-CRF. Modified intention-to-treat (mITT) and per-protocol (PP) effectiveness analyses were performed. Safety, compliance, and bacterial antimicrobial resistance rates were reported. Data were quality checked. RESULTS: Overall, 283 patients were included, with a mean age of 55 ± 15 years. Dyspepsia (n = 214, 77%) was the most frequent treatment indication, and histology (n = 144, 51%) was the most used diagnostic method. Overall eradication rate was 95% (PP) and 94% (mITT). Most prevalent first-line therapy was quadruple PPI + clarithromycin + amoxicillin + metronidazole, with a 96% effectiveness (p < 0.001). Second-line main treatment choice was triple amoxicillin + levofloxacin, with a 95% effectiveness (p < 0.05). Single-capsule Pylera® was the most prescribed third-line therapy, with 100% effectiveness (p < 0.05). Longer treatment duration was associated with a higher eradication rate in first-line therapy (p < 0.05). Clarithromycin and quinolone resistance rates in first-line were 24% and 8.3%, respectively. The overall adverse events' incidence rate was 13.4%, and therapy compliance was 97%. CONCLUSIONS: Considering the high eradication rate, 14-day non-bismuth quadruple concomitant therapy is a reasonable first-line choice, while quinolone-based therapy and single-capsule Pylera® should be considered as rescue therapy options.

Resistance to critically important antibiotics in hospital wastewater from the largest Croatian city.

The emergence of extended-spectrum ß-lactamase (ESBL)- and especially carbapenemases in Enterobacterales has led to limited therapeutic options. Therefore, it is critical to fully understand all potential routes of transmission, especially in high-risk sources such as hospital wastewater. This study aimed to quantify four enteric opportunistic pathogens (EOPs), total, ESBL- and carbapenem-resistant coliforms and their corresponding resistance genes (two ESBL and five carbapenemase genes) and to characterize enterobacterial isolates from hospital wastewater from two large hospitals in Zagreb over two seasons. Culturing revealed similar average levels of total and carbapenem-resistant coliforms (3.4 × 104 CFU/mL), and 10-fold lower levels of presumptive ESBL coliforms (3 × 103 CFU/mL). Real-time PCR revealed the highest E. coli levels among EOPs (105 cell equivalents/mL) and the highest levels of the blaKPC gene (up to 10-1 gene copies/16S copies) among all resistance genes examined. Of the 69 ESBL- and 90 carbapenemase-producing Enterobacterales (CPE) isolates from hospital wastewater, all were multidrug-resistant and most were identified as Escherichia coli, Citrobacter, Enterobacter, and Klebsiella. Among ESBL isolates, blaCTX-M-15 was the most prevalent ESBL gene, whereas in CPE isolates, blaKPC-2 and blaNDM-1 were the most frequently detected CP genes, followed by blaOXA-48. Molecular epidemiology using PFGE, MLST and whole-genome sequencing (WGS) revealed that clinically relevant variants such as E. coli ST131 (blaCTX-M-15/blaTEM-116) and ST541 (blaKPC-2), K. pneumoniae ST101 (blaOXA-48/blaNDM-1), and Enterobacter cloacae complex ST277 (blaKPC-2/blaNDM-1) were among the most frequently detected clone types. WGS also revealed a diverse range of resistance genes and plasmids in these and other isolates, as well as transposons and insertion sequences in the flanking regions of the blaCTX-M, blaOXA-48, and blaKPC-2 genes, suggesting the potential for mobilization. We conclude that hospital wastewater is a potential secondary reservoir of clinically important pathogens and resistance genes and therefore requires effective pretreatment before discharge to the municipal sewer system.

Prevalence of enteric opportunistic pathogens and extended-spectrum cephalosporin- and carbapenem-resistant coliforms and genes in wastewater from municipal wastewater treatment plants in Croatia.

Extended-spectrum ß-lactamase (ESBL)- and carbapenemase-producing Enterobacterales are a critical global health problem and wastewater treatment plants (WWTPs) can promote their spread into the environment; yet their efficacy is not well characterized. Here, we have used conventional culturing to monitor coliform bacteria and quantitative PCR to monitor 2 ESBL and 5 carbapenemase (CP) genes and 4 enteric opportunistic pathogens (EOPs) in the influent and effluent of 7 Croatian WWTPs in two seasons. In general, levels of total, cefotaxime- and carbapenem-resistant coliforms were significantly reduced but not eliminated by conventional treatment in most WWTPs. Most WWTPs efficiently removed EOPs such as K. pneumoniae and A. baumannii, while E. coli and Enterococcus spp. were reduced but still present in relatively high concentrations in the effluent. ESBL genes (blaTEM and blaCTX-M-32) were only slightly reduced or enriched after treatment. CP genes, blaKPC-3, blaNDM and blaOXA-48-like, were sporadically detected, while blaIMP and blaVIM were frequently enriched during treatment and correlated with plant size, number or size of hospitals in the catchment area, and COD effluent concentration. Our results suggest that improvements in wastewater treatment technologies are needed to minimize the risk of environmental contamination with top priority EOPs and ARGs and the resulting public health.

D variants in the population of D-negative blood donors in the north-eastern region of Croatia.

OBJECTIVES: The aim of this study was to determine RHESUS D GENE (RHD) allelic variants among Croatian D-negative blood donors and compare our results with respective data from other European countries. BACKGROUND: Altered or reduced D antigen expression can result in D variants, which can be mistyped and can lead to the alloimmunisation of the blood recipient. RHD genotyping can distinguish D variants: weak D, partial D and DEL, thus preventing alloimmunisation. MATERIAL/METHODS: A total of 6523 samples obtained from D-negative Croatian donors were screened for the presence of RHD using the real-time polymerase chain reaction (PCR) method. PCR-SSP was performed for D variant genotyping by using commercial genotyping kits (Inno-Train, Kronberg, Germany). Genomic DNA sequencing for all 10 exons of the RHD was performed when the genotyping kits failed to assign a D variant. RESULTS: RHD molecular screening revealed 23 (0.35%) RHD-PCR positive samples, all C/E positive, in decreasing frequency: 11 hybrid RHD-CE (2-9) D-CE variants, 4 weak partial D type 11 and 2 weak D type 2. Six samples remained unresolved and were sequenced. For 12 of 23 samples (excluding large hybrids), an adsorption/elution of anti-D serum was performed, confirming that all 12 were RhD+. The calculated frequency of clinically significant D alleles in RhD-negative blood donors was 1:543 (0.18%) or 1:53 (1.89%) in C/E blood donors. CONCLUSION: Data on the significant frequency of D variants among serologically D-negative blood donors in the north-eastern region of Croatia could help in introducing RHD molecular screening of blood donors in a routine workflow.

Title does matter: a cross-sectional study of 30 journals in the Medical Laboratory Technology category.

INTRODUCTION: First impression on potential readers is created by the title; therefore, authors should give importance to the title structure. The aim of this study was to establish whether articles created by a smaller number of authors and with shorter, descriptive or declarative titles gain more citations and whether article title length and number of authors correlate to the number of citations. MATERIAL AND METHODS: A cross-sectional study on article citation data for 30 scientific journals published in 2016 in Medical Laboratory Technology field according to Web of Science database was conducted. The type of article, type of title, as well as number of words in the title and number of authors was recorded. RESULTS: In the group of original articles (N = 2623), articles with declarative titles (N = 336, 13%) showed statistically higher number of citations in multiple comparison analysis when compared to descriptive titles (P < 0.001). No correlation was found between number of citations and title word count (r = 0.07, P < 0.001) nor between number of citations and number of authors in group of original articles (r = 0.09, P < 0.001). Original articles with descriptive titles longer than 15 words or with more than six authors are cited more (P = 0.005 and P < 0.001, respectively). CONCLUSION: Based on results of our study, titles do matter. Therefore, authors of original articles might want to consider including their findings in the title and having longer titles.

Antithrombotic activity of flavonoids and polyphenols rich plant species.

Cardiovascular diseases represent one of the most notable health problems of the modern civilization. Stroke and heart attack often lead to lethal outcome; essential problem underneath being thrombus formation. Prophylactic approaches include acetylsalicylic acid and clopidogrel therapy on the level of primary hemostasis, i.e., primary clot formation. In the last five years, in the USA, health care expenses related to cardiovascular diseases have increased 50 %, to over 350 billion dollars. Thus, application of plant species and medicinal plants rich in polyphenols in prevention of thrombus formation are of interest. This is supported by the fact that the number of publications on antiaggregatory effect of polyphenols has doubled in the last decade. In this review we focus on antiaggregatory effect of most abundant polyphenols - flavonoids, the effect of plant extracts rich in polyphenols (propolis, species Salvia sp., Calamintha nepeta L., Lavandula angustifolia Mill., Melissa officinalis L, Mentha x piperita L., Ocimum basilicum L., Origanum vulgare L., Rosmarinus officinalis L.) on platelet aggregation, association of chemical composition and antioxidant properties with the observed biological effect, and possible clinical significance of the published results.

Influence of flavonoids' lipophilicity on platelet aggregation.

Flavonoids are natural polyphenolic compounds present in a wide spectrum of plants that have a beneficial effect on human health. In the context of cardiovascular diseases related to plaque and thrombus formation, flavonoids exhibit an anti-aggregatory effect. Previously, it has been reported that all tested flavonoids exhibit an antiaggregatory effect on platelet aggregation when measured by impedance aggregometry on whole blood, in the test of aggregation induced by adenosine diphosphate (ADP). As not all flavonoids have the same targets within signaling pathways, an assumption of a common non-specific mechanism related to lipophilicity is to be considered. To test this hypothesis, reverse-phase thin layer chromatography was used to assess the lipophilicity of flavonoids; impedance aggregometry was used for testing of platelet aggregation and flow cytometry to monitor the influence of flavonoids on platelet activation. Lipophilicity analysis showed a highly negative correlation of logP and MINaAC for groups of flavones and flavanones. As determined by flow cytometry, the exposition of receptors necessary for the promotion of platelet activation and primary clot formation was diminished, i.e., lowered expression of the activated form of integrin αIIbß3 was observed in the presence of flavanone. Platelet membrane stabilization by flavonoids as a mechanism of antiaggregatory effect has been supported by impedance aggregometry experiments when specific inhibitors of platelet aggregation signaling pathways (U73122, indomethacin, verapamil) were used in the presence of a weak (ADP) and a strong (TRAP-6) agonist of aggregation. While individual flavonoids can have specific targets within aggregation signaling pathways, all flavonoids share a common non-specific mechanism of platelet aggregation inhibition related to their lipophilicity and membrane stabilization that, to some extent, contributes to their antiaggregatory effect.

Anti-HBc prevalence among Croatian blood donors in a 14-year period (2004-2017): Assessment of trends, risks and need for implementing routine testing.

OBJECTIVES: The anti-HBc prevalence over a 14-years period (2004-2017), trends, infectivity, residual risk, and need for testing in blood donors (BD) of the Croatian Institute of Transfusion Medicine were assessed. MATERIAL AND METHODS: Anti-HBc was tested in 19,969 BD serum samples collected in 2004 (N=7561), 2013 (N=7318) and 2017 (N=5090). All serums were initially screened for HBsAg, anti-HCV, HIV Ag/Ab, and anti-TP. 2013 and 2017 samples were also tested by ID-NAT. RESULTS: Over a 14-years period, the anti-HBc prevalence significantly decreased among Croatian BD (5.24% in 2004, 2.56% in 2013, and 1.32% in 2017). Similarly, the prevalence of anti-HBc-only profiles decreased from 0.62% in 2004, 0.25% in 2013, and 0.21% in 2017. The 4-time decreasing trend was observed in all age groups of BD from 2017 but mostly among repeat donors (5.90% to 1.38%). First-time donors showed no significant difference in anti-HBc prevalence probably due to their younger age (<29 years) and HBV vaccine status. However, similar anti-HBs carriage rates (80.56%, 87.57%, and 82.09%) were reported in anti-HBc positive donors over the study period. HBsAg and HBV DNA were not detected. No OBI infection was found in the study despite an OBI frequency of 1:10,900 donations previously reported in Croatia. A HBV decreasing residual risks of 68, 88, and 12 per million donations were estimated for years 2004, 2013, and 2017, respectively. CONCLUSION: Anti-HBc testing is an additional measure of preventing HBV infection by transfusion. Implementation of anti-HBc testing will result in the deferral of 1.3% BD and should be supported by cost-benefit analyses.

Efficiency of Target Larvicides Is Conditioned by ABC-Mediated Transport in the Zoonotic Nematode Anisakis pegreffii.

Anisakiasis is among the most significant emerging foodborne parasitoses contracted through consumption of thermally unprocessed seafood harboring infective Anisakis species larvae. The efficacy of the currently applied anthelminthic therapy in humans and in model organisms has not proven sufficient, so alternative solutions employing natural compounds combined with chemical inhibitors should be explored. By testing toxicity of the natural monoterpenes nerolidol and farnesol and the conventional anthelminthics abamectin and levamisole in the presence/absence of MK-571 and Valspodar, which inhibit the ABC transporter proteins multidrug resistance protein (MRP-like) and P-glycoprotein (P-gp), we determined the preliminary traits of Anisakis detoxifying mechanisms. We found that Anisakis P-gp and MRP-like transporters have a role in the efflux of the tested compounds, which could be useful in the design of novel anthelminthic strategies. As expected, transporter activation and efflux fluctuated over time; they were synchronously active very early postexposure, whereas the activity of one transporter dominated over the other in a time-dependent manner. MRP-like transporters dominated in the efflux of farnesol, and P-gp dominated in efflux of nerolidol, while both were active in effluxing levamisole. The highest toxicity was exerted by abamectin, a P-gp inhibitor per se, which also elicited the highest oxidative stress in treated Anisakis larvae. We suggest that ß-tubulin, observed for the first time as a core element in Anisakis cuticle, might represent an important target for the tested compounds.

Collaborative global health E-learning: A Massive Open Online Course experience of young family doctors.

Massive Open Online Courses (MOOCs) are unlimited web-based courses accessed through computers, smartphones, or other digital devices. Although they have multiple advantages, a common challenge is the low course completion rates. Young family doctors of the World Organization of Family Doctors (WONCA) from more than 20 countries recently completed an initiative that combined social media platforms with a MOOC offered by Harvard University. This resulted in a completion rate five times greater than the baseline rate. We propose perfecting and expanding this method to augment continued medical education, collaboration, and best practice exchange among WONCA's members and participating organizations worldwide.

Distribution and diversity of marine picocyanobacteria community: Targeting of Prochlorococcus ecotypes in winter conditions (southern Adriatic Sea).

Adriatic, the northernmost part of the Mediterranean Sea, due its oligotrophy, topography, and hydrology dynamics, and complex circulation patterns, was suggested as an important study site for rapid climatology impacts. Its southern part is mainly oligotrophic and dominated by picophytoplankton, with cyanobacteria as main representatives. Diversity and distribution patterns of different Prochlorococcus ecotypes were investigated by molecular tools and flow cytometry during the winter convection event in the southern Adriatic (BIOTA winter cruise; February/March 2015). Phylogenetic diversity based on clone libraries of the 16S-23S ribosomal DNA ITS region, as well as flow cytometry (histograms of red fluorescence), indicated presence of 2 different Prochlorococcus in the Adriatic. HLI, as a typical clade for Mediterranean Sea, was likewise found to be dominant Prochlorococcus in the Adriatic, followed by less abundant LLI clade. In addition, Prochlorococcus were found to co-occur with diverse Synechococcus population (53% and 47% of obtained ITS sequences, respectively). Different Prochlorococcus ecotypes had similar patterns of vertical distribution, predominantly occupying upper 100m depth layer, but their distribution was clearly affected by the heterogeneity of hydrological conditions, nitrogen concentration and temperature along vertical and horizontal sampling points. Different studies pointed out that, as a consequence of climate changes, serious alteration of biological and ecological patterns are already taking place Therefore, understanding of the distribution and abundance of picophytoplankton in Adriatic, being still limited, is much needed baseline for predicting possible biogeochemical impact of future environmental changes.

Three-Year Experience in NAT Screening of Blood Donors for Transfusion Transmitted Viruses in Croatia.

BACKGROUND: Croatia implemented individual donation (ID)-NAT testing of blood donors in 2013 for three viruses HBV, HCV, and HIV-1 as a mandatory test for all blood donors. This study assessed the impact of NAT screening 3 years after its implementation. METHODS: A total of 545,463 donations were collected and screened for HBV, HCV, and HIV-1 using the Procleix Ultrio Plus Assay. All initially reactive (IR) NAT samples were retested in triplicate and, if repeatedly reactive (RR), NAT discriminatory assay (dNAT) was performed. ID-NAT positive donations were confirmed by RT-PCR on the COBAS AmpliPrep/TaqMan platform. RESULTS: Out of 545,463 samples tested, 108 (0.02%) were RR in NAT. There were 82 (75,9%) HBV reactive, 16 (14.8%) HCV reactive, and 10 (9.3%) HIV-1 reactive samples. 51 (47.2%) samples were ID-NAT positive only. Out of these 51 NAT yield cases, 1 window period HIV-1 and 50 occult HBV infections (OBI) were determined. There were only two potential HBV DNA transmissions from OBI donors. CONCLUSION: The implementation of NAT screening for three viruses has improved blood safety in Croatia. During the 3-year period, 1 window period HIV-1 and a number of occult HBV donations were identified.

Quantitative Analysis of Phenolic Acids and Antiplatelet Activity of Melissa officinalis Leaf Extracts.

The contents of total phenolic acids and rosmarinic acid were determined and the antiplatelet potential assessed of extracts of lemon balm (Melissa officinalis L., Lamiaceae) leaf samples collected during 2014 and 2015. The quantity of total phenolic acids ranged from 4.8 to 8.5%, while the rosmarinic acid content ranged from 1.78 to 4.35%. Results of statistical analysis showed that the undiluted extracts have antiplatelet activity in the ADP test (final concentration 31.25 mg/L). While there are reports on antiplatelet activity of lemon balm essential oil, to the best of our knowledge, this is the first report on antiplatelet activity of lemon balm leaf extracts.

Trends in preventive activities for the adult population in family medicine in Croatia: 1995-2012.

A few preventive activities were recorded in Croatian family medicine (FM) from 1995 until 2003, and then in 2004, additional fee-for-service reimbursement for general check-ups for people aged 45 to 65 years was introduced. The aim of this study was to investigate the trends in preventive activities before and after the introduction of those measures by using the Croatian Health Service Yearbooks for 1995-2012 as the main database. Data on the number of preventive, general, and total number of check-ups were collected. The results showed that the total number of check-ups registered in FM was low, suggesting that the additional reimbursement did not bring any improvements. In fact, the trend in the number decreased after 2004. These results are not unexpected because of the ineffectiveness of general check-ups as indicated in the literature. General check-ups should be replaced by targeted preventive interventions with evidence-based effectiveness.

Preventive health care for women in Croatia: ongoing trends from 1995 to 2012.

The privatization of gynecological services and the introduction of additional reimbursements to capitation fees are ongoing mini reforms in Croatia. In order to evaluate the outcomes of this, study was performed with the main aim of determining trends in preventive activities carried out in public and private gynecological practices from 1995 to 2012. The Croatian Health Service Yearbooks served as the basis for data collection. Data were collected on the number of general check-ups, the number of targeted check-ups, and the number of follow-up check-ups. The results indicate a trend of continuous decline in the number of general and follow-up check-ups, as well as breast examinations and Pap smears, in public gynecological practices even after the introduction of contractual obligations and additional reimbursements and fee-for-service payments. One important note is that many resources were invested in general checks-up interventions, which proved to be ineffective, while fewer resources were invested in the more effective Pap smear interventions.