CLSI-based verification and de novo establishment of reference intervals for common biochemical assays in Croatian newborns.

Introduction: This study aimed to examine whether the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) reference intervals for 19 commonly used biochemical assays (potassium, sodium, chloride, calcium, magnesium, inorganic phosphorous, glucose, urea, creatinine, direct and total bilirubin, C-reactive protein (CRP), total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) and lactate dehydrogenase (LD)) could be applied to the newborn population of one Croatian clinical hospital. Materials and methods: Reference interval verification was performed according to the CLSI EP28-A3c guidelines. Samples of healthy newborns were selected using the direct a posteriori sampling method and analyzed on the Beckman Coulter AU680 biochemical analyzer. If verification wasn't satisfactory, further procedure included de novo determination of own reference intervals by analyzing 120 samples of healthy newborns. Results: After the first set of measurements, 14/19 tested reference intervals were adopted for use: calcium, inorganic phosphorous, glucose, urea, creatinine, total bilirubin, CRP, total protein, albumin, AST, ALT, GGT, ALP and LD. A second set of samples was tested for 5 analytes: potassium, sodium, chloride, magnesium and direct bilirubin. The verification results of the additional samples for sodium and chloride were satisfactory, while the results for potassium, magnesium and direct bilirubin remained unsatisfactory and new reference intervals were determined. Conclusions: The CALIPER reference intervals can be implemented into routine laboratory and clinical practice for the tested newborn population for most of the analyzed assays, while own reference intervals for potassium, magnesium and direct bilirubin have been determined.

Urban heat load assessment in Zagreb, Croatia: a multi-scale analysis using mobile measurement and satellite imagery.

A limited number of meteorological stations and sparse data challenge microclimate assessment in urban areas. Therefore, it is necessary to complement these data with additional measurements to achieve a denser spatial coverage, enabling a detailed representation of the city's microclimatic features. In this study, conducted in Zagreb, Croatia, mobile air temperature measurements were utilized and compared with satellite-derived land surface temperature (LST). Here, air temperature measurements were carried out using bicycles and an instrument with a GPS receiver and temperature probe during a heat wave in June 2021, capturing the spatial pattern of air temperature to highlight the city's microclimate characteristics (i.e. urban heat load; UHL) in extremely hot weather conditions. Simultaneously, remotely sensed LST was retrieved from the Landsat-8 satellite. Air temperature measurements were compared to city-specific street type classification, while neighbourhood heat load characteristics were analysed based on local climate zones (LCZ) and LST. Results indicated significant thermal differences between surface types and urban forms and between street types and LCZs. Air temperatures reached up to 35 °C, while LST exceeded 40 °C. City parks, tree-lined streets and areas near blue infrastructure were 1.5-3 °C cooler than densely built areas. Temperature contrasts between LCZs in terms of median LST were more emphasised and reached 9 °C between some classes. These findings highlight the importance of preserving green areas to reduce UHL and enhance urban resilience. Here, exemplified by the city of Zagreb, it has been demonstrated that the use of multiple datasets allows a comprehensive understanding of temperature patterns and their implications for urban climate research.

Exploring cytokinesis block micronucleus assay in Croatia: A journey through the past, present, and future in biomonitoring of the general population.

In this study, we used the cytokinesis-block micronucleus (CBMN) assay to evaluate the background frequency of cytogenetic damage in peripheral blood lymphocytes of the general population concerning different anthropometric data and lifestyle factors. The background frequency of CBMN assay parameters was analysed in 850 healthy, occupationally non-exposed male and female subjects (average age, 38±11 years) gathered from the general Croatian population from 2000 to 2023. The mean background values for micronuclei (MNi) in the whole population were 5.3±4.3 per 1000 binucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 0.7±1.3 and of nuclear buds (NBUDs) 3.1±3.2. The cut-off value, which corresponds to the 95th percentile of the distribution of 850 individual values, was 14 MNi, 3 NPBs, and 9 NBUDs. Results from our database also showed an association of the tested genomic instability parameters with age and sex but also with other lifestyle factors. These findings underscore the importance of considering several anthropometric and lifestyle factors when conducting biomonitoring studies. Overall, the normal and cut-off values attained here present normal values for the general population that can later serve as baseline values for further human biomonitoring studies either in Croatia or worldwide.

Variability of human Alpha-1-acid glycoprotein N-glycome in a Caucasian population.

AIM: Alpha-1-acid glycoprotein (AGP) is a highly glycosylated protein in human plasma and one of the most abundant acute phase proteins in humans. Glycosylation plays a crucial role in its biological functions, and alterations in AGP N-glycome have been associated with various diseases and inflammatory conditions. However, large-scale studies of AGP N-glycosylation in the general population are lacking. METHODS: Using recently developed high-throughput glycoproteomic workflow for site-specific AGP N-glycosylation analysis, 803 individuals from the Croatian island of Korcula were analyzed and their AGP N-glycome data associated with biochemical and physiological traits, as well as different environmental factors. RESULTS: After regression analysis, we found that AGP N-glycosylation is strongly associated with sex, somewhat less with age, along with multiple biochemical and physiological traits (e.g. BMI, triglycerides, uric acid, glucose, smoking status, fibrinogen). CONCLUSION: For the first time we have extensively explored the inter-individual variability of AGP N-glycome in a general human population, demonstrating its changes with sex, age, biochemical, and physiological status of individuals, providing the baseline for future population and clinical studies.

Cross-cultural adaptation and reproducibility of the EPIC-Norfolk food frequency questionnaire in young people living in Croatia.

AIM: To translate and adapt the European Prospective Investigation of Cancer (EPIC)-Norfolk food frequency questionnaire (FFQ) for use in Croatia, and to assess the reliability and reproducibility of the Croatian version of the EPIC-Norfolk FFQ. METHODS: Translation and cross-cultural adaptation were performed according to published recommendations. Reliability was assessed in 140 respondents (61 men; age range 8-40 years) divided into three groups: young adults, pregnant women, and children and adolescents. Reproducibility was assessed in the group of young adults (32/61 men), who completed the questionnaire on two occasions three months apart. RESULTS: The EPIC-Norfolk FFQ showed good reliability (Cronbach alpha=0.874). Most nutrient intakes showed good reproducibility (intraclass correlation coefficient [ICC] between 0.7 and 0.9). Poor reproducibility was observed for alcohol (ICC=0.337), and moderate reproducibility was observed for beta-carotene (ICC=0.692) and total carbohydrates (ICC=0.698). Nutrient intakes measured by FFQ on two occasions did not significantly differ. CONCLUSION: The Croatian version of the EPIC-Norfolk FFQ can be a useful tool for assessing dietary intakes in young people in Croatia and possibly in neighboring countries with similar languages and dietary habits.

A new subspecies of the subterranean beetle Speoplanes biocovensis Mller, 1934 (Coleoptera: Leiodidae: Cholevinae: Leptodirini) from Bosnia and Herzegovina: the first occurrence of the genus outside Croatia.

A new subterranean leptodirine leiodid beetle, Speoplanes biocovensis bosnicus Vrbica, Vesovi, Raa & uri, ssp. n., is described and diagnosed from a pit in southern Bosnia and Herzegovina. This is the first discovery of the genus Speoplanes Mller, 1911 in this country and the first discovery outside Croatia. Important morphological features of the new taxon are presented, including aspects of the male and female genitalia. The subspecies is endemic to the Dinaric mountain range of Bosnia and Herzegovina. A key to the identification of taxa of the genus Speoplanes is added.

Frequency of regular walking among Croatian adults.

The aim of this study was to determine the share of Croatian adults who walk 210 min or more a week and to explore the relationship between regular walking and demographic factors, health-related behaviours, and chronic non-communicable diseases/conditions. To this end, we used the EHIS-PAQ questionnaire and collected self-reported data on minutes spent walking during a typical week from a total of 3,496 respondents. The data were additionally analysed by gender, age, education, residence (urban/rural), counties and regions, smoking, other types of physical activity, and diseases/chronic conditions. The results show that, overall, 40.9 % of the adult Croatian population walks 210 or more minutes a week, with the largest share found among those from the Lika-Senj County (76.8 %), those who spend 300 min or more weekly on health-enhancing (non-work-related) aerobic physical activity (57.6 %), those who reported having diabetes (49.3 %), and those aged 65-74 years (44.7 %). Despite its limitations, our study gives a valuable insight into the frequency and factors determining healthy walking habits in a representative sample of Croatian adults and provides grounds for further research.

Polychlorinated biphenyls in mussels, small pelagic fish, tuna, turtles, and dolphins from the Croatian Adriatic Sea waters: an overview of the last two decades of monitoring.

This review summarises our two decades of polychlorinated biphenyl (PCB) monitoring in different marine organisms along the eastern Adriatic Sea. The aim was to gain an insight into the trends of PCB distribution in order to evaluate the effectiveness of past and current legislation and suggest further action. Here we mainly focus on PCB levels in wild and farmed Mediterranean mussels, wild and farmed bluefin tuna, loggerhead sea turtles, common bottlenose dolphins, and small pelagic fish. The use of artificial intelligence and advanced statistics enabled an insight into the influence of various variables on the uptake of PCBs in the investigated organisms as well as into their mutual dependence. Our findings suggest that PCBs in small pelagic fish and mussels reflect global pollution and that high levels in dolphins and wild tuna tissues raise particular concern, as they confirm their biomagnification up the food chain. Therefore, the ongoing PCB monitoring should focus on predatory species in particular to help us better understand PCB contamination in marine ecosystems in our efforts to protect the environment and human health.

Hydrogeochemical and environmental isotope study of Topusko thermal waters, Croatia.

Thermal waters in Topusko (Croatia), with temperatures of up to 65 °C, have been used for heating, health, and recreational tourism for the past fifty years. Hydrogeochemical monitoring can provide insights into deeper geological processes and indicate system changes from baseline levels. It helps to identify potential anthropogenic impacts, as well as natural changes. Hydrogeochemical, geothermometrical, and environmental isotope studies of thermal waters in Topusko were conducted to improve the existing conceptual model of the Topusko hydrothermal system (THS), providing a baseline for continuous monitoring of the thermal resource. 2-year thermal springs and precipitation monitoring took place from March 2021 until March 2023. Major anions and cations, stable and radioactive isotopes (i.e. 18O, 2H, SO42-, 3H and 14C) and geothermometers were used to assess the origin of thermal waters in Topusko and their interaction with thermal aquifer. The results indicate the meteoric origin of thermal water, which was recharged in colder climatic conditions around the late Pleistocene-Early Holocene. Thermal water was last in contact with the atmosphere before approximately 9.5 kyr. Ca-HCO3 hydrochemical facies suggests carbonate dissolution as the dominant process driving the solute content. Geothermometrical results indicate an equilibrium temperature in the reservoir of 90 °C.

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.

Gamma knife thalamotomy in treating refractory tremor: initial clinical experience in Croatia.

Tremor refractory to pharmacological therapy significantly reduces the patient´s quality of life, often leading to early retirement and social isolation. Gamma knife (GK) stereotactic radiosurgery of the unilateral thalamic ventral intermediate nucleus is an advanced, minimally invasive surgical procedure for symptomatic tremor suppression. Due to the restricted availability of this type of treatment, literature data on its efficacy and safety are lacking. We present two patients with severe, disabling tremor (one with parkinsonian and one with essential tremor) successfully treated with GK thalamotomy, performed in Croatia for the first time. GK thalamotomy should be considered in patients with refractory tremors and contraindications for deep brain stimulation.

Revealing the Diversity and Complex Relationships of Croatian Olive Germplasm.

Olive growing in Croatia has a long tradition and is of great economic and social impact. The present study includes a set of 108 tree samples (88 samples corresponding to 60 presumed cultivars and 20 trees of unnamed ones) collected from 27 groves in the entire olive growing area, and is the most comprehensive survey to be conducted in Croatia. The genetic diversity, relationships, and structures of olive plants were studied using eight microsatellite loci. All loci were polymorphic and revealed a total of 90 alleles. A total of 74 different genotypes were detected that were subjected to further diversity and genetic relationship studies. The Fitch-Margoliash tree and Bayesian analysis of population structure revealed a complex relationship between the identified olive genotypes, which were clustered into three gene pools, indicating different origins of Croatian olive germplasms. Excluding the redundant germplasms, 44 different genotypes among the sampled trees of well-known cultivars and 16 new local germplasms were identified. In addition, we provide the etymology of 46 vernacular names, which confirms that the vast majority of traditional Croatian cultivars have common and widespread names. The results presented herein underline the importance of safeguarding local cultivars and conducting continuous surveys.

Measuring e-Professional Behavior of Doctors of Medicine and Dental Medicine on Social Networking Sites: Indexes Construction With Formative Indicators.

BACKGROUND: Previous studies have predominantly measured e-professionalism through perceptions or attitudes, yet there exists no validated measure specifically targeting the actual behaviors of health care professionals (HCPs) in this realm. This study addresses this gap by constructing a normative framework, drawing from 3 primary sources to define e-professional behavior across 6 domains. Four domains pertain to the dangers of social networking sites (SNSs), encompassing confidentiality, privacy, patient interaction, and equitable resource allocation. Meanwhile, 2 domains focus on the opportunities of SNSs, namely, the proactive dissemination of public health information and maintaining scientific integrity. OBJECTIVE: This study aims to develop and validate 2 new measures assessing the e-professional behavior of doctors of medicine (MDs) and doctors of dental medicine (DMDs), focusing on both the dangers and opportunities associated with SNSs. METHODS: The study used a purposive sample of MDs and DMDs in Croatia who were users of at least one SNS. Data collection took place in 2021 through an online survey. Validation of both indexes used a formative approach, which involved a 5-step methodology: content specification, indicators definition with instructions for item coding and index construction, indicators collinearity check using the variance inflation factor (VIF), external validity test using multiple indicators multiple causes (MIMIC) model, and external validity test by checking the relationships of the indexes with the scale of attitude toward SNSs using Pearson correlation coefficients. RESULTS: A total of 753 responses were included in the analysis. The first e-professionalism index, assessing the dangers associated with SNSs, comprises 14 items. During the indicators collinearity check, all indicators displayed acceptable VIF values below 2.5. The MIMIC model showed good fit (χ213=9.4, P=.742; χ2/df=0.723; root-mean-square error of approximation<.001; goodness-of-fit index=0.998; comparative fit index=1.000). The external validity of the index is supported by a statistically significant negative correlation with the scale measuring attitudes toward SNSs (r=-0.225, P<.001). Following the removal of 1 item, the second e-professionalism index, focusing on the opportunities associated with SNSs, comprises 5 items. During the indicators collinearity check, all indicators exhibited acceptable VIF values below 2.5. Additionally, the MIMIC model demonstrated a good fit (χ24=2.5, P=.718; χ2/df=0.637; root-mean-square error of approximation<0.001; goodness-of-fit index=0.999; comparative fit index=1.000). The external validity of the index is supported by a statistically significant positive correlation with the scale of attitude toward SNSs (r=0.338; P<.001). CONCLUSIONS: Following the validation process, the instrument designed for gauging the e-professional behavior of MDs and DMDs consists of 19 items, which contribute to the formation of 2 distinct indexes: the e-professionalism index, focusing on the dangers associated with SNSs, comprising 14 items, and the e-professionalism index, highlighting the opportunities offered by SNSs, consisting of 5 items. These indexes serve as valid measures of the e-professional behavior of MDs and DMDs, with the potential for further refinement to encompass emerging forms of unprofessional behavior that may arise over time.

Epidemiology of Bartonella henselae infection in pet and stray cats in Croatia with risk factors analysis.

BACKGROUND: Cats are the primary reservoirs of the bacterium Bartonella henselae, the main cause of cat-scratch disease in humans. The main vector of the bacterium is the cat flea, Ctenocephalides felis. In southeastern Europe, data are lacking on the prevalence of B. henselae infection in cats, the strains of B. henselae involved and the risk factors associated with the infection. METHODS: Blood samples collected in ethylenediaminetetraacetic acid-containing tubes from 189 domestic cats (156 pet cats and 33 stray cats) from Zagreb, the capital city of Croatia, and 10 counties throughout Croatia were cultured for Bartonella spp. Following culture, bacterial isolates were genotyped at eight loci after using PCR to amplify 16S ribosomal RNA (rRNA) and the internal transcribed spacer region between the 16S and 23S rRNA sequences. Univariate and multivariate logistic regression were used to identify risk factors for B. henselae infection in cats. RESULTS: Bartonella spp. was detected in 31 cats (16.4%), and subsequent genotyping at the eight loci revealed B. henselae in all cases. Thirty complete multilocus sequence typing profiles were obtained, and the strains were identified as four sequence types that had been previously reported, namely ST5 (56.7%), ST6 (23.3%), ST1 (13.3%) and ST24 (3.3%), as well as a novel sequence type, ST33 (3.3%). The univariate analysis revealed a significantly higher risk of B. henselae infection in cats residing in coastal areas of Croatia (odds ratio [OR] 2.592, 95% confidence interval [CI] 1.150-5.838; P = 0.0191) and in cats with intestinal parasites (OR 3.207, 95% CI 1.088-9.457; P = 0.0279); a significantly lower risk was identified in cats aged > 1 year (OR 0.356, 95% CI 0.161-0.787; P = 0.0247) and in cats sampled between April and September (OR 0.325, 95% CI 0.147-0.715; P = 0.005). The multivariate analysis that controlled for age showed a positive association with the presence of intestinal parasites (OR 4.241, 95% CI 1.243-14.470; P = 0.0119) and coastal residence (OR 2.567, 95% CI 1.114-5.915; P = 0.0216) implying increased risk of infection, and a negative association with sampling between April and September (OR 0.379, 95% CI 0.169-0.848; P = 0.018) implying a decreased risk of infection. After controlling for the season, an increased risk of infection remained for the coastal region (OR 2.725, 95% CI 1.200-6.186; P = 0.012). CONCLUSIONS: Bartonella henselae is prevalent throughout Croatia and is a public health threat. Environmental and host factors can significantly affect the risk of infection, and these should be explored in more detail. The presence of intestinal parasites highlights the need to eliminate the flea vector, Ctenocephalides felis, as the most effective approach to control infections in cats and humans.

GiOPARK Project: The Genetic Study of Parkinson's Disease in the Croatian Population.

Parkinson's disease is a neurological disorder that affects motor function, autonomic functions, and cognitive abilities. It is likely that both genetic and environmental factors, along with age, contribute to the cause. However, there is no comprehensive guideline for genetic testing for Parkinson's disease, and more research is needed to understand genetic variations in different populations. There has been no research on the genetic background of Parkinson's disease in Croatia so far. Therefore, with the GiOPARK project, we aimed to investigate the genetic variants responsible for Parkinson's disease in 153 Croatian patients with early onset, familial onset, and sporadic late-onset using whole-exome sequencing, along with multiplex ligation-dependent probe amplification and Sanger sequencing in select patients. We found causative variants in 7.84% of the patients, with GBA being the most common gene (4.58%), followed by PRKN (1.96%), ITM2B (0.65%), and MAPT (0.65%). Moreover, variants of uncertain significance were identified in 26.14% of the patients. The causative variants were found in all three subgroups, indicating that genetic factors play a role in all the analyzed Parkinson's disease subtypes. This study emphasizes the need for more inclusive research and improved guidelines to better understand the genetic basis of Parkinson's disease and facilitate more effective clinical management.

Seroepidemiology of Herpes Simplex Viruses Type 1 and 2 in Pregnant Women in Croatia.

Background and Objectives: Herpes simplex viruses (HSV-1 and HSV-2) are one of the most widespread causes of human viral infections. In Croatia, only two published studies have analyzed the seroprevalence of HSV infections in childbearing-aged and pregnant women (2005-2010), while more recent data are lacking. This study aimed to analyze the prevalence and risk factors for HSV-1 and HSV-2 infections among pregnant women in Croatia in the period from 2011 to 2021. Materials and Methods: This study included 667 pregnant women aged 16-45 years submitted for HSV-1 and HSV-2 serology testing. Serum samples were initially screened for HSV-1 and HSV-2 IgM and IgG antibodies using a commercial ELISA test with a confirmation of HSV-2-positive samples using an immunoblot assay. Results: The overall IgG seroprevalence rates were 69.9% for HSV-1 and 3.8% for HSV-2. A significant gradual increase in the HSV-2 seroprevalence with age was observed from 0.5% in participants under 30 years to 8.3% in participants above 40 years. The HSV-1 seroprevalence was stable up to 40 years (70.0 and 68.3%, respectively), with an increase to 86.1%, but this difference did not reach statistical significance. Area of residence (urban or suburban/rural), geographic region (continental or coastal), and obstetric history (normal pregnancy or unfavorable obstetric history) were not associated with HSV-1 and HSV-2 seroprevalence. Older age was found to be a significant risk factor for HSV-2 seropositivity in both univariate and multivariate risk analysis. Conclusions: HSV-1 infection is widely prevalent among pregnant women with a stable trend over time. However, a declining trend in the HSV-2 seroprevalence was observed compared to 2005-2010. Serological screening in pregnant women is important in identifying seronegative women who are susceptible to HSV infection as well as seropositive women who are at risk for genital herpes recurrence during delivery.

Increased number of cryptosporidiosis cases with travel history to Croatia might be related to swimming pools, Germany, 2023.

In August and September 2023, an unusually high number of cryptosporidiosis cases identified by routine German surveillance had travelled to Croatia (n = 23). Nine cases had stayed in the same camping resort and seven further cases had stayed at other camping sites within 15 km. Based on our standardised questionnaires, the most likely source of infection was swimming pools (93%). Further environmental investigations on site might reveal potential common sources of contamination that could be targeted by control measures.

Influence of donor characteristics on the milk donation volume and microbiological quality of donated human milk in first 3 years of work of the Croatian human milk bank-retrospective observational study.

OBJECTIVES: Donated human milk is the best possible alternative when mother's own milk is not available. The aim of this study is to investigate whether there are differences in the milk donation volumes and microbiological quality of donated milk depending on human milk donors (HMDs) characteristics. METHODS: We analyzed data on the HMDs who donated milk in the first three years of work of human milk bank (HMB) - November 2019 to January 2023. The data on the volume of donated milk in L and suitable microbiological quality assessed by the number and isolated species of bacteria were collected from questionnaires filled out by HMDs and documentation administered by HMB employees and are presented using descriptive and comparative statistics. RESULTS: Two hundred HMDs were included in this study. The majority of them are between 26 and 35 years of age, reside in capital city or the surrounding county, have given birth to a full-term child vaginally, and express surplus milk through a breast pump. Donor mothers of preterm born infants (14.5 %) donated greater quantities, there is statistically significant difference in the median of volume of milk donated (9.6 vs. 6.4, p=0.026). Milk expressed manually shows better results in microbiological quality (median percentage 100 vs. 82 vs. 100, p=0.040), while by comparing other characteristics of the donors, no difference was found between the groups. CONCLUSIONS: It is important to be aware of the characteristics of previous HMDs in order to direct the HMB future promotional and educational activities.