Comparison of DNA damage in fresh and frozen blood samples: implications for the comet assay in human biomonitoring studies.

The use of the comet assay in large biomonitoring studies may present logistical and technical challenges because of the processing of numerous samples. Proper sample preservation becomes imperative to prevent spurious DNA breakage. Previous research has shown the feasibility of conducting the comet assay on frozen blood samples, highlighting the potential of freezing at - 80 °C in preserving DNA integrity. Nonetheless, this approach presents challenges, including potential DNA damage during freezing and thawing, variability in processing, and the need for standardized protocols. Our objective was to evaluate whether there are comparable results in DNA migration assessed by the comet assay between fresh and frozen blood samples on a larger scale (N = 373). In our findings, elevated DNA migration was evident in frozen samples relative to fresh ones. Additionally, smoking, alcohol consumption, and season were linked to increased DNA damage levels in whole blood cells. Based on our results and available literature, conducting the comet assay on frozen blood samples emerges as a practical and efficient approach for biomonitoring and epidemiological research. This method enables the assessment of DNA damage in large populations over time, with samples, if properly cryopreserved, that may be used for years, possibly even decades. These observations hold significant implications for large-scale human biomonitoring and long-term epidemiological studies, particularly when samples are collected during fieldwork or obtained from biobanks. Continued method optimization and validation efforts are essential to enhance the utility of this approach in environmental and occupational health studies, emphasizing caution when comparing data obtained between fresh and frozen blood samples.

Associating Air Pollution with Cytokinesis-Block Micronucleus Assay Parameters in Lymphocytes of the General Population in Zagreb (Croatia).

Air pollution is recognized as one of the most serious public health issues worldwide and was declared to be a leading environmental cause of cancer deaths. At the same time, the cytokinesis-block micronucleus (CBMN) assay serves as a cancer predictive method that is extensively used in human biomonitoring for populations exposed to environmental contamination. The objective of this cross-sectional study is two-fold: to evaluate genomic instability in a sample (N = 130) of healthy, general population residents from Zagreb (Croatia), chronically exposed to different levels of air pollution, and to relate them to air pollution levels in the period from 2011 to 2015. Measured frequencies of CBMN assay parameters were in agreement with the baseline data for the general population of Croatia. Air pollution exposure was based on four factors obtained from a factor analysis of all exposure data obtained for the examined period. Based on the statistical results, we did not observe a significant positive association between any of the CBMN assay parameters tested and measured air pollution parameters for designated time windows, except for benzo(a)pyrene (B[a]P) that showed significant negative association. Our results show that measured air pollution parameters are largely below the regulatory limits, except for B[a]P, and as such, they do not affect CBMN assay parameters' frequency. Nevertheless, as air pollution is identified as a major health threat, it is necessary to conduct prospective studies investigating the effect of air pollution on genome integrity and human health.

Geographic distribution and risk of upper urothelial carcinomas in Croatia, 2001-2011.

BACKGROUND: Strong associations exist between Balkan endemic nephropathy (BEN) and upper urothelial carcinomas (UUCs). However, the common etiology between the two remains unclear and there are no studies to date that visualize UUC risks in Croatia. In Croatia, 14 villages in the southwestern part of Brod-Posavina County are considered endemic for BEN. The aim of this ecological study is to map cancer risks and describe the case distribution of UUCs in Croatia at the county level during 2001-2011. METHODS: A total of 608 incident cases from the Croatian National Cancer Registry were identified. Indirect standardization was employed to compute standardized incidence ratios (SIRs). RESULTS: Counties with SIRs greater than 1 were concentrated around the agricultural region of Slavonia and the coastal region of Dalmatia. However, only Brod-Posavina County and Vukovar-Srijem County had a statistically significant risk of UUC development, where there were 390 and 210% more UUC cases observed than expected, respectively. Only unique to Brod-Posavina County, females were at higher risk (SIR 4.96; 95% CI 3.59-6.34) of developing UUCs than males (SIR 3.03; 95% CI 2.04-4.01) when compared to their Croatian counterparts. Although Brod-Posavina County only made up 3.7% of the total Croatian population (as of 2011), it had the highest frequency of incident UUC cases after the capital City of Zagreb. No elevated cancer risks were noted in the City of Zagreb, even after stratifying by sex. CONCLUSION: Our findings suggest that Brod-Posavina County had the highest cancer risk for UUCs, especially among females, when compared to Croatia as a whole during 2001-2011. Given that a majority of BEN patients develop associated UUCs, concurrent screening programs for UUCs and BEN should be considered not only in endemic areas of BEN but also the surrounding rural areas and amongst at-risk groups such as those undergoing hemodialysis, who frequently develop UUCs, to help clarify BEN-UUC associations by identifying common risk factors while standardizing disease estimates across endemic regions for BEN.

HEADACHES IN ADOLESCENTS - FREQUENCY, RISK FACTORS AND OTHER HEALTH COMPLAINTS: A CROSS-SECTIONAL STUDY IN CROATIA.

- Headaches are very common in school population. The aim of this study was to determine epidemiological data on headaches among Croatian schoolchildren and their connection to risk factors and other health complaints. In the study, we used the Croatian database of the international research entitled Health Behavior in School-aged Children for 2013/2014. It is a cross-sectional study conducted every four years in 44 countries among children aged 11-16. The students were interviewed using an anonymous standardized questionnaire. The Croatian sample consisted of 5741 students, of which 2857(49.8%) girls and 2884 (50.2%) boys. The response rate was 85.9%. Headaches were reported to be often (once a week to daily) by 23.5% of students and were more often in girls and in higher school grades. They were positively linked to cigarette smoking and health complaints such as back pain and stomachache, bad mood, nervousness, irritability, difficulties with sleeping and dizziness. Headaches are associated to many health problems and can cause difficulties in student functioning. This could be a serious problem. This study pointed to connection of headaches with cigarette smoking and importance to underpin the smoking prevention programs.

Consensus statement on screening, diagnosis, classification and treatment of endemic (Balkan) nephropathy.

Currently used diagnostic criteria in different endemic (Balkan) nephropathy (EN) centers involve different combinations of parameters, various cut-off values and many of them are not in agreement with proposed international guidelines. Leaders of EN centers began to address these problems at scientific meetings, and this paper is the outgrowth of those discussions. The main aim is to provide recommendations for clinical work on current knowledge and expertise. This document is developed for use by general physicians, nephrologists, urologist, public health experts and epidemiologist, and it is hoped that it will be adopted by responsible institutions in countries harboring EN. National medical providers should cover costs of screening and diagnostic procedures and treatment of EN patients with or without upper urothelial cancers.

National program of breast cancer early detection in Brod-Posavina County (East Croatia).

Results of the National Program of Breast Cancer Early Detection in Brod-Posavina County during the 2006-2012 period are presented. Response rate in two National Program cycles, cancers detected according to factors such as first and last menstruation, age at cancer detection, deliveries and mammography findings according to the Breast Imaging Reporting and Data System (BI-RADS) before diagnosis verification were analyzed. Data were obtained from the software connecting Public Health Institutes via Ministry of Health server and questionnaires filled out by the women presenting for screening and processed by the method of descriptive statistics. Mammography findings were classified according to the BI-RADS classification. In two National Program cycles during the 2006-2012 period, women aged 50-69 were called for mammography screening. In the first cycle, the response rate in Brod-Posavina County was 53.2%, with 71 cancers detected at a mean age of 61.3 years. In the second cycle, the response rate was 57.0%, with 44 cancers detected at a mean age of 62.5 years. In the first and second cycles, there were 21.1% and 14.3% of mammography findings requiring additional work-up (BI-RADS 0), respectively. Particular risk factors such as early menarche, late menopause, parity, positive family history and presence of benign breast lesions were not demonstrated in women with verified cancer. There was no increase in the incidence of breast cancer per 100,000 inhabitants in the Brod-Posavina County following implementation of the National Program. In conclusion, efforts should be focused on increasing public health awareness, ensuring appropriate professional staff engaged in screening, and improving medical care in order to reduce the time elapsed from establishing suspicion to confirming the diagnosis of breast cancer.

The risk of developing endemic nephropathy in subjects with proteinuria.

Endemic nephropathy is a chronic tubulointerstitial disease characterized by early damage to the proximal tubule, with low-molecular weight proteinuria being an important hallmark and possible tool for early diagnosis. The aim of this retrospective cohort study was to assess the risk of developing endemic nephropathy in subjects with proteinuria from the endemic region in Croatia. The cohort study included subjects with proteinuria determined by the sulfosalicylic acid method (after 1988 with strip method), involved in the field survey conducted in the Croatian endemic village of Kaniza in 1975 and followed up until 1997. Subjects with endemic nephropathy established at the first visit and patients that failed to present for follow up visits after 1975. were excluded. In the field survey group that consisted of 624 subjects (286 male and 338 female), proteinuria was established in 157 subjects. Upon the application of exclusion and inclusion criteria, the study cohort included 111 of 157 subjects. The mean follow up was 7.26 years (95% confidence interval 4.06-10.46 years). During the follow up period, 19 (17%) subjects with initial proteinuria developed endemic nephropathy. The incidence density of endemic nephropathy among subjects with proteinuria was 1.3 per 100 persons/year. Estimated risk was 0.0137 (confidence interval 0.0087-0.0214) per year of exposure. The presence of proteinuria determined by the sulfosalicylic acid or test strip in subjects from the endemic village indicated that endemic nephropathy would develop in 1.3 of 100 subjects with proteinuria per year.

Air Pollution and Primary DNA Damage among Zagreb (Croatia) Residents: A Cross-Sectional Study.

More than eight million premature deaths annually can be attributed to air pollution, with 99% of the world's population residing in areas below recommended air quality standards. Hence, the present study aimed to examine the association between primary DNA damage and air pollution data among 123 participants enrolled between 2011 and 2015 in Zagreb, Croatia. While most measured air pollutants adhered to regulatory limits, benzo[a]pyrene concentrations bound to PM10 exceeded them. Factorial analysis narrowed down air pollution data to four exposure factors (particulate matter, two metal factors, and other pollutants). Despite the absence of significant positive associations between modeled air pollution exposure factors and comet assay descriptors (tail length, tail intensity, tail moment, and highly damaged nuclei), the critical health implications of air pollution warrant further investigations, particularly with biomarkers of exposure and different biomarkers of effect in populations facing air pollution exposure.

National program of colorectal carcinoma early detection in Brod-Posavina County (east Croatia).

Colorectal carcinoma (CRC) is a major public health problem as the third leading malignant tumor in men and fourth in women in Croatia. Prognosis and treatment greatly depend on tumor stage at the time of detection. Therefore, the National Program of Colorectal Carcinoma Early Detection has been performed since 2007. The aim is to present the response rate, colonoscopy findings and number of newly detected CRC cases in Brod-Posavina County. During five years of the National Program performance, 28 CRC cases were detected in Brod-Posavina County, with the 3.3% rate of carcinoma detection. The majority of CRC cases were found in the 50-64 age group. The response rate in the County was low (20.4%), corresponding to the national rate but far from the recommended one. Such a result could be attributed to the low level of awareness in the population at large, complex testing technique for general population, fear from disease detection and from colonoscopy as a diagnostic procedure. Note should be made of the underestimated role of family physicians; their involvement in the National Program should certainly result in better response rate in our County as well as at the national level.

Could disappearance of endemic (Balkan) nephropathy be expected in forthcoming decades?

BACKGROUND/AIMS: An epidemiological survey of endemic nephropathy (EN) was performed in endemic Croatian areas and the current prevalence was compared to that reported for the same villages several decades ago. METHODS: A total of 2,487 adult farmers from 6 endemic villages and 3 non-endemic villages were enrolled. An extensive epidemiological questionnaire, clinical examination and laboratory analyses of blood and urine were performed. According to the modified WHO criteria, participants were classified into diseased, suspected of having EN, and those at risk of developing EN. RESULTS: The overall prevalence of EN in the Croatian areas was 1.0%, ranging between 0.3 and 2.3% in different villages. Those suspected of having EN amounted to 3.9%. In the endemic villages a decreasing trend in the prevalence of EN was observed comparable to the results obtained in previous surveys. It is interesting to note that no EN patients were recorded in the endemic village of Dubocac. CONCLUSION: The prevalence of EN in the endemic Croatian areas appears to be decreasing. For the first time, we failed to detect any EN patients in a village that was previously considered endemic, which might indicate that EN is diminishing.

Prevalence, treatment and control of hypertension in a Croatian endemic nephropathy area.

BACKGROUND: Hypertension is not considered to be a characteristic of endemic nephropathy (EN). Recent observations suggested that it might be more prevalent than it was reported before. AIM: The aim of our study was to analyze prevalence, treatment and control of hypertension in a Croatian endemic area. METHODS: In the present cross-sectional study, 1,602 farmers were enrolled, 1,246 from EN and 356 from control villages. Epidemiological and medical histories were taken and clinical and laboratory examinations performed for kidney function. Blood pressure was measured following the ESH/ESC guidelines. RESULTS: The prevalence of hypertension in EN villages was higher than in control (50.8 vs. 46.5%, p = 0.005). There was no difference in overall treatment, control of all and treated hypertensives between the villages. In all villages, women were treated more than men (EN 41.6 vs. 28.4%, p < 0.001; control 46.4 vs. 27.3%, p < 0.001), but better control of treated was achieved in men (EN 24.7 vs. 17.4%, p = 0.002; control 29.6 vs. 15.5%, p = 0.002). Women had lower income and level of education than men (p < 0.01). CONCLUSION: Hypertension is highly prevalent in endemic villages. In all villages, men had better blood pressure control despite being treated less. This finding could be explained by poorer education and income in women.

Angiotensin-converting enzyme gene polymorphism and N-Acetyl-ß-D-glucosaminidase excretion in endemic nephropathy.

BACKGROUND: Tubular proteinuria and enzymuria are hallmarks of endemic nephropathy (EN). The role of I/D angiotensin convertase (ACE) gene polymorphism has not yet been elucidated in this peculiar chronic tubulointerstitial nephritis, and our aim was to investigate the role of this polymorphism in EN focusing on the urinary N-acetyl-ß-D-glucosaminidase (NAG) excretion, a biomarker of proximal tubular damage. METHODS: ACE genotype and allele frequencies were determined in 229 farmers (147 women and 82 men) from an endemic Croatian village. The farmers were stratified according to the WHO criteria into the following subgroups: those 'at risk' for EN (n = 37), 'suspected of having EN' (n = 57), and 'others' (n = 135). RESULTS: There were 74 (32.3%) subjects homozygous for the D allele, 99 (43.2%) heterozygous (ID genotype) and 56 (24.4%) homozygous for the I allele. No differences in allele frequency were found between the established WHO subgroups (p > 0.05). In the whole group, DD subjects had significantly higher values of diastolic blood pressure (p = 0.003) and urinary NAG than subjects with ID and II genotype (5.5 ± 1.2 vs. 4.0 ± 3.0 vs. 3.8 ± 4.2, respectively; p = 0.023). The highest values of serum creatinine (p = 0.02), proteinuria (p = 0.03) and urinary NAG (6.0 ± 3.7 vs. 3.7 ± 2.1 vs. 3.0 ± 1.6, respectively; p = 0.008) were observed in those suspected of having EN group with the DD genotype. CONCLUSION: ACE gene polymorphism is not a risk factor for EN. However, it might influence the clinical course of EN, and increased excretion of NAG might be a prognostic marker of this chronic tubulointerstitial nephritis.

[Prevalence of hepatitis B, hepatitis C and HIV among injecting drug users treated outpatiently and in therapeutic community in Brod-Posavina County, Croatia]. / Usporedba prevalencije biljega hepatitisa B, C i HIV-A medu intravenskim ovisnicima lijecenima ambulantno i u terapijskoj zajednici na podrucju Brodsko-posavske zupanije.

Blood transmitted diseases (hepatitis B, hepatitis C, HIV) are major public health problems. Drug users, especially injecting drug users (IDU), are by nature of their illness, a risk population for these diseases. The aim of the study was to determine the prevalence of blood transmitted diseases among IDU in the Brod-Posavina County due to shared use of needle/syringe in outpatients and those treated in a therapeutic community, and to compare the results obtained. First, we analyzed data separately for hepatitis B and C, and then we selected patients with coinfection. The prevalence of HBsAg positive patients in both groups was significantly lower than the prevalence among drug addicts in Croatia (1.16% and 3.28% vs. 13.2%). Significant correlation was found in outpatients with anti-HBs+anti-HBc antibody (p < 0.05) between those who shared needles/syringes and those who did not. Significant correlation was also found among patients treated at therapeutic community (p < 0.01). Comparing the patients treated as outpatients and in therapeutic community, significant correlation was only found between vaccinated patients. HCV positive outpatients had lower and drug addicts in therapeutic communities significantly higher prevalence as compared with the prevalence of HCV among addicts in Croatia (41.86% and 60.66% vs. 44.6%). A significant correlation between those who shared needles/syringes and those who did not was found in both outpatients and patients treated in therapeutic community (P < 0.01). Comparison of HCV positive patients treated as outpatients and those in therapeutic community also yielded significant correlation (p < 0.05). The prevalence of HBV/HCV coinfection was similar in both groups of patients. Significant correlation (P < 0.05) was only found in the group of patients with anti HBC/anti HCV antibodies. There was no HIV-positive patient in any group. We also found a low prevalence of HBsAg/anti-HCV in both groups of patients (1.16% and 2.46%). Upon establishing a network of centers for the treatment of addicts, constant work on prevention and education, systematic testing and vaccination, and implementation of harm reduction programs, we noticed a trend of reducing the number of people with HBV and HCV in the younger population of addicts.

Visual assessment of endemic nephropathy markers relationship.

The aim of this paper was to assess relationship between possible endemic nephropathy (EN) markers visually by the CoPlot methodology, and to illustrate this promising data analysis approach. From 912 screened persons in 3 Croatian endemic villages, 25 persons were diagnosed as confirmed EN patients, 371 as non-EN, and the remainder were classified as suspected of having EN, or at risk. Data on 25 confirmed EN patients were matched with appropriate non-EN examinees. All records with missing data were excluded, resulting in 35 subjects with complete data on the 13 key EN variables for CoPlot mapping. CoPlot solution met the accepted goodness of fit measure thresholds. Result showed relationship between EN markers, identifying some nearly duplicated variables, and possible outliers needing some subsequent analysis.

Reliability of CKD-EPI predictive equation in estimating chronic kidney disease prevalence in the Croatian endemic nephropathy area.

INTRODUCTION: Chronic kidney disease (CKD) is a significant public health problem and it is not possible to precisely predict its progression to terminal renal failure. According to current guidelines, CKD stages are classified based on the estimated glomerular filtration rate (eGFR) and albuminuria. Aims of this study were to determine the reliability of predictive equation in estimation of CKD prevalence in Croatian areas with endemic nephropathy (EN), compare the results with non-endemic areas, and to determine if the prevalence of CKD stages 3-5 was increased in subjects with EN. MATERIALS AND METHODS: A total of 1573 inhabitants of the Croatian Posavina rural area from 6 endemic and 3 non-endemic villages were enrolled. Participants were classified according to the modified criteria of the World Health Organization for EN. Estimated GFR was calculated using Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPI). RESULTS: The results showed a very high CKD prevalence in the Croatian rural area (19%). CKD prevalence was significantly higher in EN then in non EN villages with the lowest eGFR value in diseased subgroup. CONCLUSIONS: eGFR correlated significantly with the diagnosis of EN. Kidney function assessment using CKD-EPI predictive equation proved to be a good marker in differentiating the study subgroups, remained as one of the diagnostic criteria for EN.

[Human leptospirosis in Slavonski Brod, 1995-2005]. / Epidemioloske osobitosti oboljelih od leptospiroze i utjecaj kolicina oborina na broj oboljelih na brodskom podrucju od 1995. do 2005. godine.

Leptospirosis is a worlwide spread zoonosis. During the 1995-2005 period, 54 patients with leptospirosis were recorded at the Institute of Public Health, Brod-Posavina County, yielding an incidence rate of 1.6-8.8 per 100,000 per year. One (1.85 %) patient died. There were 41 (76%) males. The age of leptospirosis patients ranged from 15 to 77 (mean 44) years. Forty-three (79.6 %) patients were from rural areas, and 17%. were farmers. The most frequent serotypes were: australis (20%), pomona (15%), saxkoebing (10%) and grippotyphosa (7%). Thirty-six (68.5%) leptospirosis cases were notified between July and October. A significant correlation (r = 0.77; P < 0.01; r2 = 59.3%) was found betwen the number of leptospirosis cases and average rainfall recorded one month prior to the diagnosis.

[An epidemic of trichinellosis in autumn 2004 in Slavonski Brod]. / Epidemija trihineloze na podrucju Slavonskog Broda u jesen 2004. Godine.

Trichinellosis is a worldwide zoonotic disease caused by a nematode Trichinella sp. Cases of Trichinella spiralis infection in Slavonski Brod were investigated. A total of 64 trichinellosis cases were notified to the Institute of Public Health, Brod-Posavska County between October and November 2004. There were 64 patients, 32 of them female, age range 3-67, mean 35 years. The mean incubation period was 3 (range 1-5) weeks. The epidemic started in October and terminated 39 days later. The patients ate smoked sausages. Trichinella spiralis was identified in sausages by artificial digestion. Among 64 people infected, 65% had facial and/or periorbital edema, 56% diarrhea, 53% myalgia, 48% fever, 26% headache, 23% weakness, 6% conjunctivitis, 6% cough and 3% vomitus, 6% of patients were free from symptoms and 17% were hospitalized. Eosinophils were elevated in 92%, creatinine kinase in 60%, and lactate dehydrogenase in 59% of patients; 97% of patients were positive for Trichinella spiralis on indirect immunofluorescence.

A family outbreak of cryptosporidiosis: Probable nosocomial infection and person-to-person transmission.

Cryptosporidium is an important cause of community gastroenteritis, usually associated with drinking contaminated water, visits to swimming pools or animal exposure. In immunocompetent patients, cryptosporidiosis is usually a self-limiting disease but it can be devastating illness in immunocompromised persons. This report presents a family outbreak of cryptosporidiosis and describes a probable nosocomial infection with Cryptosporidium in one patient and subsequent spread of the parasite among members of his family. The index patient, a 72-year-old man with hemiparesis after his third stroke, died after 28 days of persistent diarrhea. Control measures must be undertaken to prevent nosocomial transmission of cryptosporidial oocysts.

Chronic dietary exposure to aristolochic acid and kidney function in native farmers from a Croatian endemic area and Bosnian immigrants.

BACKGROUND AND OBJECTIVES: Improvements in agricultural practices in Croatia have reduced exposure to consumption of aristolochic acid-contaminated flour and development of endemic (Balkan) nephropathy. Therefore, it was hypothesized that Bosnian immigrants who settled in an endemic area in Croatia 15-30 years ago would be at lower risk of developing endemic nephropathy because of reduced exposure to aristolochic acid. To test this hypothesis, past and present exposure to aristolochic acid, proximal tubule damage as a hallmark of endemic nephropathy, and prevalence of CKD in Bosnian immigrants were analyzed. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In this cross-sectional observational study from 2005 to 2010, 2161 farmers were divided into groups: indigenous inhabitants from endemic nephropathy and nonendemic nephropathy villages and Bosnian immigrants; α-1 microglobulin-to-creatinine ratio >31.5 mg/g and eGFR<60 ml/min per 1.73 m(2) were considered to be abnormal. RESULTS: CKD and proximal tubule damage prevalence was significantly lower in Bosnian immigrants than inhabitants of endemic nephropathy villages (6.9% versus 16.6%; P<0.001; 1.3% versus 7.3%; P=0.003, respectively); 20 years ago, Bosnian immigrants observed fewer Aristolochia clematitis in cultivated fields (41.9% versus 67.8%) and fewer seeds among wheat seeds (6.1% versus 35.6%) and ate more purchased than homemade bread compared with Croatian farmers from endemic nephropathy villages (38.5% versus 14.8%, P<0.001). Both Croatian farmers and Bosnian immigrants observe significantly fewer Aristolochia plants growing in their fields compared with 15-30 years ago. Prior aristolochic acid exposure was associated with proximal tubule damage (odds ratio, 1.64; 95% confidence interval, 1.04 to 2.58; P=0.02), whereas present exposure was not (odds ratio, 1.31; 95% confidence interval, 0.75 to 2.30; P=0.33). Furthermore, immigrant status was an independent negative predictor of proximal tubule damage (odds ratio, 0.40; 95% confidence interval, 0.19 to 0.86; P=0.02). CONCLUSIONS: Bosnian immigrants and autochthonous Croats residing in endemic areas are exposed significantly less to ingestion of aristolochic acid than in the past. The prevalence of endemic nephropathy and its associated urothelial cancers is predicted to decrease over time.