Thelazia callipaeda--first human case of thelaziosis in Croatia.

A case of the first ocular thelaziosis in a human male patient in Croatia is presented. Thelaziosis is a zoonosis caused by the nematodes of the genus Thelazia. In all, two types of the genus have been described as causes of infection in humans, Thelazia callipaeda and Thelazia californiensis. This zoonosis, called "oriental eye worm" is very rare in humans in European countries, with high incidence in Asian countries, especially in China, Thailand, and Japan.

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.

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.

Malondialdehyde and 8-oxo-7.8-dihydro-2'deoxyguanosine in the urine of residents from Balkan endemic nephropathy area in Croatia--a pilot study.

Balkan endemic nephropathy (BEN) is a human chronic tubulointerstitial renal disease that occurs in rural areas of some Balkan countries. The disease is insidious and fatal, and mostly affects persons in their sixties or seventies. BEN areas have unusually high rates of otherwise rare upper urinary tract tumors (UTT). Since extensive production of reactive oxygen species leading to oxidative stress has been implicated in tumor development, the aim of this study was to see whether oxidative stress is involved in the development of BEN and UTT. Urine samples were collected from a BEN village (N = 22) and a control village (N = 16) residents and analyzed for malondialdehyde (MDA) and 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxodG). The levels of both oxidative stress parameters were significantly higher in the BEN village residents than controls. However, there was no correlation between MDA and 8-oxodG results. Our results confirm that oxidative stress could be implicated in development of both, BEN and UTT.

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.

Genetic evidence for the presence of two distinct hantaviruses associated with Apodemus mice in Croatia and analysis of local strains.

In Europe, Dobrava-Belgrade (DOBV), Saaremaa (SAAV), and Puumala (PUUV) viruses are known to cause hemorrhagic fever with renal syndrome (HFRS). All three hantaviruses are now found in Croatia. Lung tissue samples of 315 Apodemus mice trapped in 2003-2004 were screened for the presence of hantaviral N-Ag and 20 mice (6.3%) were found either strongly positive or weak/suspected-positive. Partial sequences of hantavirus M and S segments were recovered by RT-PCR from six mice and subjected to (phylo)genetic analysis that revealed the presence of four novel strains of DOBV and one of SAAV. Curiously, one of the newly described DOBV strains was found in Apodemus agrarius mouse, that is, not in the traditional host, A. flavicollis mice, suggesting a spillover event. S segment sequences recovered previously from HFRS cases [Markotic et al., 2002] were confirmed as DOBV sequences; one of which appeared particularly close to the prototype Slovenian DOBV isolate. Taken together with earlier data on PUUV in Croatia, these results show a co-circulation of three European hantavirus pathogens in this country. So far, not a single SAAV sequence has been recovered from HFRS patients either in Croatia or neighboring Slovenia and Hungary nor in Slovakia suggesting a somewhat lower fequency of acute SAAV infection in humans in this part of Europe than for example in the Baltics.

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.

Zoonoses and vector-borne diseases in Croatia - a multidisciplinary approach.

Emerging and re-emerging infectious diseases create constant and serious concerns for public health. The majority of emerging infectious diseases (EID) are wildlife zoonotic diseases and vector-borne diseases. Croatia has a long tradition in the control, management and research of EID zoonotic diseases and vector-borne diseases. There has also been a long and advantageous tradition in the collaboration of different experts and professionals in EID research in Croatia involving physician clinicians in infectious diseases, microbiologists, pathologists, veterinarians and animal scientists, ecologists, forestry experts, wildlife scientists, public health specialists and epidemiologists and laboratory scientists. The University Hospital for Infectious Diseases in Zagreb established the Centre for Emerging and Re-emerging Infectious Diseases in liaison with national and international partners from Europe and the United States. This Centre is working in line with the 'One Health initiative' which recognises the inter-relationships between human, animal and environmental health.

The involvement of mycotoxins in the development of endemic nephropathy.

Endemic nephropathy is a human kidney disease that still escapes scientific explanation. It is accompanied by a high incidence of urothelial tumors in rural populations in endemic areas, which suggests that a natural nephrotoxic and carcinogenic compound may be involved in the etiology. The most imputed causative agent of endemic nephropathy is the mycotoxin ochratoxin A (OTA), because of its confirmed nephrotoxic and carcinogenic action. This paper presents a review of studies of OTA in food collected in the endemic areas and in blood and urine of their residents. Data on the co-occurrence of OTA and other nephrotoxic and carcinogenic mycotoxins such as citrinin and fumonisin B(1) in food are also presented. Unfortunately, there is no study on the co-occurrence of OTA and other mycotoxins in humans and there is only one study on fumonisin B(1) exposure in endemic areas. The paper also presents experimental data on cultured cells and laboratory animals treated with combinations of OTA and other nephrotoxic mycotoxins, because most such combinations show a synergistic effect. The occurrence of OTA- and aristolochic acid-DNA adducts is also presented.

[Epidemiologic characteristics of endemic nephropathy in Croatia in 2005]. / Epidemioloske osobitosti endemske nefropatije u Hrvatskoj 2005. Godine.

INTRODUCTION: Endemic nephropathy (EN) is chronic renal disease of unknown etiology, characterized with ominous onset and progressive clinical course. It occurs in rural areas in values of tributaries of the river Danube. According to the census from 2001, 10865 farmers live in Croatian endemic focus, i. e. 14 villages in Brod Posavina County. Apart from kidney impairment, high incidence of urothelial cancers is a hallmark of EN. Due to this, most agents that have been investigated were nephrotoxic and carcinogen. PARTICIPANTS AND METHODS: In the last 25 years approximately 70% of 13 Croatian endemic villages' inhabitants were examined following WHO criteria. In spring 2005, 1081 inhabitants of 3 endemic villages (Kaniza, Bebrina, Banovci) and control village of Klakar were examined using the same criteria, and persons were divided as follows: diseased of EN, suspect of having EN, at risk for EN and others. RESULTS: In the group of 1081 persons, we found 25 diseased of EN (12 men and 13 women): Bebrina 11, Banovci 9, Kaniza 5 (chi2 = 8.06; p < 0.05). Prevalence of EN in Kaniza was 0.6%, Bebrina 2.1% and Banovci 2.3%, while prevalence of suspect of EN was 1.7%, 6%, 6%, respectively. One fifth of inhabitants were at risk of having EN. CONCLUSION: This epidemiologic survey that was conducted in spring 2005 found out that prevalence of EN in Croatia is approximately the same as it was in the last decades. Apart from renal impairment, high incidence of urothelial cancers is characteristic of EN in this region. In addition to medical and scientific, we will need social and political help in elucidating the etiology of EN as well as in improving treatment and cure of this poor rural population.

[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.

Short report: dual infections with Puumala virus and Leptospira interrogans serovar lora in a bank vole (Clethrionomys glareolus).

Leptospirosis and hemorrhagic fever with renal syndrome are public health problems in Croatia. Diagnosis and epidemiology of these diseases are complicated because these two diseases are sympatric in certain areas. We describe a natural dual infection of Puumala virus and a leptospire in a bank vole (Clethrionomys glareolus).

Puumala virus in Croatia in the 2002 HFRS outbreak.

HFRS is an endemic disease throughout Croatia. The incidence of HFRS varies in a cyclic fashion, with peaks occurring every couple of years, coinciding with peaks in vole populations. PUUV was shown to be dominant pathogen during the last HFRS outbreak in Croatia in 2002. We focused our research on two newly discovered localities (Okucani and Nova Gradiska) with a high number of reported HFRS cases and a significant increase in rodent population. PUUV infection was verified in 84.2% of patients at this region during the 2002 outbreak. Genetic analysis of wild-type (wt) PUUV strains was performed. Fifty seven bank voles Clethrionomys glareolus originating from PUUV-associated HFRS areas were screened for the presence of PUUV N antigen and 15 (26%) were found positive. Total RNA isolated from rodent lung tissues was reverse transcribed followed by PCR amplification with primers specific for PUUV medium (M) or small (S) genome segments. Partial PUUV M segment sequences (approximately 450 bp long) were recovered from five bank voles and partial S segment sequences (app. 250 nt long)-from two bank voles. Genetic analysis of Croatian wt-PUUV strains revealed their close relatedness suggesting that the two localities belong to the same natural focus of infection. On phylogenetic trees Croatian PUUV strains clustered together with the strains from Slovenia and Austria forming distinct Alpe-Adrian genetic lineage.

Recent data on endemic nephropathy and related urothelial tumors in Croatia.

Endemic nephropathy (EN) is a renal disease of unknown etiology. In Croatia it occurs in the rural population in 14 villages located in the western part of Brodsko-Posavska county. This region also has an unusually high incidence of otherwise rare upper urothelial cancers. Between 1991 and 2002 the average general mortality for both sexes in the endemic region was 10.3 per thousand and the specific mortality for patients with EN was 0.65 per thousand (M 0.58/10((3)), F 0.72/10(3)). The average age of death of patients with EN was 69.2 years (M 67.7, F 70.3), which is similar to the life expectancy for the rest of the population in the county (67.8, M 64.4, F 71.8). This life expectancy is significantly higher than in the period 1957-1960 when the average age of EN-related death was 45.1 years. Between 1995 and 2002, in contrast to both Croatia as a whole and the respective county, the specific mortality with tumors of the pyelon and ureter in the endemic region was much higher in women than in men (9.020 and 4.697 per 100,000, respectively). The specific mortality of all patients with urothelial tumors was 14 times higher in the endemic region than in Brodsko-Posavska county and 55 times higher than in Croatia overall. The much higher specific urothelial-tumor mortality in women than in men (in contrast to the rest of the country) and the higher specific EN mortality indicate that the causative agent of the two nosological entities is the same.

Two different clean-up procedures for liquid chromatographic determination of ochratoxin A in urine.

This paper describes two different procedures for extraction of ochratoxin A (OTA) from urine samples: one using acidic chloroform-methanol mixture, followed by solid-phase extraction (SPE) clean-up and the other using commercial Chem Elut columns and a chloroform-formic acid mixture. The recovery of OTA using the procedure with silica gel columns was 82% with a R.S.D. < 8.4% and the detection and quantitation limits were 0.5 and 1.5 ng OTA/ml, respectively. The recovery of OTA in the second procedure with urine samples purified only on commercial Chem Elut columns was 95% with R.S.D. < 4.0%, and detection and quantitation limits 0.3 and 0.9 ng/ml, respectively. Both procedures of OTA extraction effectively eliminate interfering substances and give reliable and repeatable results. However, the procedure with Chem Elut columns gave higher recovery and lower detection and quantitation limits. It was successfully applied in determining OTA in human urine samples.

[An epidemic of hemorrhagic fever with renal syndrome at the therapeutic community for treatment of psychoactive drug dependence in Ivanovac]. / Epidemija hemoragijske vrucice s bubreznim sindromom u Zajednici lijecenih ovisnika o psihoaktivnim drogama u Ivanovcu.

GOAL: The outbreak of hemorrhagic fever with renal syndrome (HFRS) at the Ivanovac Drug Therapeutic Community near Nova Gradiska was analyzed. The major epidemiologic, clinical and serologic findings are presented. PATIENTS AND METHODS: During the HFRS outbreak, serum samples were obtained from 31 men from the Community and analyzed by ELISA IgM and IgG tests for Puumala (PUUV) and Dobrava (DOBV) viruses. The main epidemiologic and clinical data were analyzed. RESULTS: At the beginning of March 2002, a-30-year-old male from the Community developed severe clinical picture of HFRS with fulminant systemic hemorrhage, acute renal failure and consecutive death. IgM antibodies to DOBV were detected. HFRS was serologically confirmed in 15 subjects in total. All others were infected with PUUV. During the mild and dry winter, the Community members cut bushes, collected trash, branches and dry leaves near the beech-wood forest border to prepare that part of the field as a garden. They observed a number of rodents, their nests and holes. Most of the patients developed mild to moderate clinical picture with fever, headache, backache, myalgia, abdominal pain, melena, oliguria, polyuria, epistaxis, and conjunctival injection. Hepatitis B (HBV) and/or C (HCV) infection was recorded in 58.1% of Community members. Interestingly, 86.7% of the HFRS patients were HBV and/or HCV positive. CONCLUSIONS: This is the first HFRS outbreak described among drug addicts in Europe, mostly caused by PUUV. Further studies are needed to identify additional risk factors that may contribute to hantavirus infection among drug addicts in HFRS endemic regions in Croatia as well as in Europe.