Publications by authors named "Danko Milosevic"

34 Publications

Contrast-enhanced voiding urosonography in the diagnosis of intrarenal reflux.

J Ultrasound 2021 Feb 26. Epub 2021 Feb 26.

Children's Hospital Zagreb, Ul. Vjekoslava Klaića 16, 10000, Zagreb, Croatia.

Purpose: Although contrast-enhanced urosonography (ceVUS) has shown capable diagnostic accuracy for the diagnosis of vesicoureteral reflux (VUR) in children, the ability of ceVUS to detect intrarenal reflux (IRR) is considered limited. The purpose of our study is to assess the ability of ceVUS to detect IRR as well as its association with age, gender, and the grade of VUR.

Methods: This study included 5153 children who were referred to our clinic for ceVUS. All children underwent sonographic examinations, which were performed on a LOGIQ S8 machine equipped with dedicated software for contrast-enhanced studies with harmonic imaging. Standard ultrasound of the urinary tract was followed by bladder catheterisation and instillation of physiological normal saline and the US contrast medium (SonoVue, Bracco).

Results: VUR was diagnosed by ceVUS in 1959 out of 5153 children (38%), of whom IRR was found in 233 of 1959 children (11.9%). A total of 285 ureteral units showing IRR were found. High grades of VUR (IV + V) with IRR were found in a total of 235 of 285 (82.81%) renal units. Bilateral IRR was found in 53 patients, usually with a high-grade VUR on both sides. Most children had VUR grade IV, predominantly those < 12 months. The younger the child, the higher the likelihood of higher-grade VUR (p = 0.02).

Conclusion: ceVUS, combined with harmonic imaging and second-generation ultrasound contrast media, enabled IRR detection in almost 12% of our patients with VUR. IRR is most commonly found in children under 1 year of age with VUR grades IV and V.
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http://dx.doi.org/10.1007/s40477-021-00568-wDOI Listing
February 2021

Uroflowmetry in Non-Monosymptomatic Nocturnal Enuresis in Children of Coastal Region of Croatia.

Acta Medica (Hradec Kralove) 2020 ;63(3):113-118

Department of Pediatrics, Zagreb University Hospital, University of Zagreb, Zagreb, Croatia.

Purpose: The aim of the study was to describe clinical characteristics and bladder assessment in children with Non-Monosymptomatic Nocturnal Enuresis (NMNE) in coastal region of Croatia.

Materials And Methods: Records on 85 patients with NMNE were retrospectively reviewed. Bladder assessments were performed in all children. In this research we: (i) compare clinical characteristics and features of bladder assessment: uroflowmetry, post void residuals (PVR) and bladder wall thickness between boys and girls with NMNE and we compare (ii) clinical characteristics and bladder assessment between children with primary and secondary NMNE.

Results: There were 46 girls and 39 boys. The total of 59 children had primary NMNE and 26 children had secondary NMNE. Uroflow pattern was abnormal in 42% of all children with NMNE. Abnormal uroflow pattern in children with NMNE was more often in girls than in boys (P < 0.05) and in children with secondary than in children with primary NMNE (P < 0.05). Ultrasound evidence of bladder wall thickness was more frequent in boys than in girls. Girls were more likely to have dysfunctional voiding and larger residual urinary volume than boys.

Conclusions: Abnormal uroflow pattern in children with NMNE was more often in girls than boys and in children with secondary than in children with primary NMNE.
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http://dx.doi.org/10.14712/18059694.2020.28DOI Listing
January 2020

Whole exome sequencing identified ATP6V1C2 as a novel candidate gene for recessive distal renal tubular acidosis.

Kidney Int 2020 03 22;97(3):567-579. Epub 2019 Oct 22.

Division of Nephrology, Department of Pediatrics, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA. Electronic address:

Distal renal tubular acidosis is a rare renal tubular disorder characterized by hyperchloremic metabolic acidosis and impaired urinary acidification. Mutations in three genes (ATP6V0A4, ATP6V1B1 and SLC4A1) constitute a monogenic causation in 58-70% of familial cases of distal renal tubular acidosis. Recently, mutations in FOXI1 have been identified as an additional cause. Therefore, we hypothesized that further monogenic causes of distal renal tubular acidosis remain to be discovered. Panel sequencing and/or whole exome sequencing was performed in a cohort of 17 families with 19 affected individuals with pediatric onset distal renal tubular acidosis. A causative mutation was detected in one of the three "classical" known distal renal tubular acidosis genes in 10 of 17 families. The seven unsolved families were then subjected to candidate whole exome sequencing analysis. Potential disease causing mutations in three genes were detected: ATP6V1C2, which encodes another kidney specific subunit of the V-type proton ATPase (1 family); WDR72 (2 families), previously implicated in V-ATPase trafficking in cells; and SLC4A2 (1 family), a paralog of the known distal renal tubular acidosis gene SLC4A1. Two of these mutations were assessed for deleteriousness through functional studies. Yeast growth assays for ATP6V1C2 revealed loss-of-function for the patient mutation, strongly supporting ATP6V1C2 as a novel distal renal tubular acidosis gene. Thus, we provided a molecular diagnosis in a known distal renal tubular acidosis gene in 10 of 17 families (59%) with this disease, identified mutations in ATP6V1C2 as a novel human candidate gene, and provided further evidence for phenotypic expansion in WDR72 mutations from amelogenesis imperfecta to distal renal tubular acidosis.
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http://dx.doi.org/10.1016/j.kint.2019.09.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039771PMC
March 2020

Treatment and long-term outcome in primary distal renal tubular acidosis.

Nephrol Dial Transplant 2019 06;34(6):981-991

Department of Paediatric Nephrology, Great Ormond Street Hospital for Children, NHS Foundation Trust, London, UK.

Background: Primary distal renal tubular acidosis (dRTA) is a rare disorder, and we aimed to gather data on treatment and long-term outcome.

Methods: We contacted paediatric and adult nephrologists through European professional organizations. Responding clinicians entered demographic, biochemical, genetic and clinical data in an online form.

Results: Adequate data were collected on 340 patients (29 countries, female 52%). Mutation testing had been performed on 206 patients (61%); pathogenic mutations were identified in 170 patients (83%). The median (range) presentation age was 0.5 (0-54) years and age at last follow-up was 11.0 (0-70.0) years. Adult height was slightly below average with a mean (SD score) of -0.57 (±1.16). There was an increased prevalence of chronic kidney disease (CKD) Stage ≥2 in children (35%) and adults (82%). Nephrocalcinosis was reported in 88%. Nephrolithiasis was more common with SLC4A1 mutations (42% versus 21%). Thirty-six percent had hearing loss, particularly in ATP6V1B1 (88%). The median (interquartile range) prescribed dose of alkali (mEq/kg/day) was 1.9 (1.2-3.3). Adequate metabolic control (normal plasma bicarbonate and normocalciuria) was achieved in 158 patients (51%), more commonly in countries with higher gross domestic product (67% versus 23%), and was associated with higher height and estimated glomerular filtration rate.

Conclusion: Long-term follow-up from this large dRTA cohort shows an overall favourable outcome with normal adult height for most and no patient with CKD Stage 5. However, 82% of adult patients have CKD Stages 2-4. Importance of adequate metabolic control was highlighted by better growth and renal function but was achieved in only half of patients.
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http://dx.doi.org/10.1093/ndt/gfy409DOI Listing
June 2019

A case report of a child with sepsis induced multiorgan failure and massive complement consumption treated with a short course of Eculizumab: A case of crosstalk between coagulation and complement?

Medicine (Baltimore) 2019 Jan;98(4):e14105

University Hospital Centre Zagreb, Kispaticeva, Zagreb.

Rationale: This article describes a child with a life-threatening multiorgan failure with disseminated intravascular coagulation (DIC) and massive complement consumption. To our knowledge this therapeutic approach was for the first time effectively applied in a pediatric patient.

Patient Concerns: A 14-month-old boy was presented with a severe, rapidly progressing, life-threatening disease because of sudden onset of fever, hemathemesis, hematuria, and bloody diarrhoea alongside fast spreading hematomas and general corporeal edema.

Diagnosis: The most plausible diagnosis in our patient is Clostridium difficile sepsis-induced thrombotic microangiopathy alongside with DIC and consumption coagulopathy. The diagnosis was confirmed by positive C difficile bacteria strain in coproculture, clinical, and laboratory tests affirming DIC and global complement activation and consumption.

Interventions: The patient was treated with antibiotics (Metronidazole, Vancomycin), plasmapheresis, dialysis, methylprednisolone, mycophenolate mofetil, and Eculizumab.

Outcomes: The child is in fair overall condition in a 2 year follow-up with no complications save chronic renal failure.

Lessons: In rare cases of sepsis with massive complement consumption, a case-sensitive Eculizumab therapy may be at least considered after the resolution of life-threatening multiorgan failure. The application of this drug can be performed only after sepsis induced disease is put under control. A fast withdrawal of Eculizumab after control of massive complement consumption is recommended to prevent triggering of second sepsis reactivation.
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http://dx.doi.org/10.1097/MD.0000000000014105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358330PMC
January 2019

Whole-Exome Sequencing Identifies Causative Mutations in Families with Congenital Anomalies of the Kidney and Urinary Tract.

J Am Soc Nephrol 2018 09 24;29(9):2348-2361. Epub 2018 Aug 24.

Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts;

Background: Congenital anomalies of the kidney and urinary tract (CAKUT) are the most prevalent cause of kidney disease in the first three decades of life. Previous gene panel studies showed monogenic causation in up to 12% of patients with CAKUT.

Methods: We applied whole-exome sequencing to analyze the genotypes of individuals from 232 families with CAKUT, evaluating for mutations in single genes known to cause human CAKUT and genes known to cause CAKUT in mice. In consanguineous or multiplex families, we additionally performed a search for novel monogenic causes of CAKUT.

Results: In 29 families (13%), we detected a causative mutation in a known gene for isolated or syndromic CAKUT that sufficiently explained the patient's CAKUT phenotype. In three families (1%), we detected a mutation in a gene reported to cause a phenocopy of CAKUT. In 15 of 155 families with isolated CAKUT, we detected deleterious mutations in syndromic CAKUT genes. Our additional search for novel monogenic causes of CAKUT in consanguineous and multiplex families revealed a potential single, novel monogenic CAKUT gene in 19 of 232 families (8%).

Conclusions: We identified monogenic mutations in a known human CAKUT gene or CAKUT phenocopy gene as the cause of disease in 14% of the CAKUT families in this study. Whole-exome sequencing provides an etiologic diagnosis in a high fraction of patients with CAKUT and will provide a new basis for the mechanistic understanding of CAKUT.
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http://dx.doi.org/10.1681/ASN.2017121265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115658PMC
September 2018

Genotype-phenotype Correlation of β-Thalassemia in Croatian Patients: A Specific HBB Gene Mutations.

J Pediatr Hematol Oncol 2018 03;40(2):e77-e82

Department of Pediatrics, University Hospital Centre Zagreb.

An analysis of genotype-phenotype correlation was performed for 14 patients with beta-thalassemia who had been registered in Referral Centre for hematology and oncology of the University Hospital Centre, Zagreb, Croatia. HBB gene mutations were determined using a gene-specific Q5 High-Fidelity PCR analysis with direct DNA sequencing of amplified transcripts. Mahidol score index used for classification of thalassemia severity was found to be low for all the patients enrolled in the study, indicating a mild β-thalassemia phenotype with no signs of disease progression. Most of the patients have already described gene mutations: IVS-II-666 C>T (HBB:c.316-185C>T) and IVS-II-16 G>C (HBB:c.315+16G>C). Each of the aforementioned mutations was found in (11/14; 78,57%) and (10/14; 71,43%) of our patients, respectively. Recently published HBB:c.9T>C mutation was found in 8 of 14 (57,14%) in our study group. IVSII-74 T>G (HBB:c.315+74T>G) is a worldwide mutation found in 6 of 14 (42.86%) of our patients. All these mutations occur among Croatian children with no obvious Indian/Near Eastern/Iranian ancestry. We also identified 7 de novo mutations (c.316-135het_dupT, c.316-133A>G, c.93-54G>A, c.316-68_316-67het_insCGG, c.316-342delA, c.316-312delT, c.316-209delT) of mild severity phenotype according to Mahidol classification score index. We did not find children or adults with thalassemia major severity phenotype.
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http://dx.doi.org/10.1097/MPH.0000000000001039DOI Listing
March 2018

Rational Therapy of Urinary Tract Infections in Children in Croatia

Acta Clin Croat 2016 09;55(3):428-439

Karlovac General Hospital, Karlovac, Croatia

Resistance to chemotherapeutics used in the treatment of urinary tract infection is increasing throughout the world. Taking into account clinical experiences, as well as current bacterial resistance in Croatia and neighboring countries, the selection of antibiotic should be the optimal one. Treatment of urinary tract infection in children is particularly demanding due to their age and inclination to severe systemic reaction and renal scarring. If parenteral antibiotics are administered initially, it should be switched to oral medication as soon as possible. Financial aspects of antimicrobial therapy are also very important with the main goal to seek the optimal cost/benefit ratio. Financial orientation must appreciate the basic primum non nocere as a conditio sine qua non postulate as well.
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http://dx.doi.org/10.20471/acc.2016.55.03.12DOI Listing
September 2016

Whole exome sequencing frequently detects a monogenic cause in early onset nephrolithiasis and nephrocalcinosis.

Kidney Int 2018 01 12;93(1):204-213. Epub 2017 Oct 12.

Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA. Electronic address:

The incidence of nephrolithiasis continues to rise. Previously, we showed that a monogenic cause could be detected in 11.4% of individuals with adult-onset nephrolithiasis or nephrocalcinosis and in 16.7-20.8% of individuals with onset before 18 years of age, using gene panel sequencing of 30 genes known to cause nephrolithiasis/nephrocalcinosis. To overcome the limitations of panel sequencing, we utilized whole exome sequencing in 51 families, who presented before age 25 years with at least one renal stone or with a renal ultrasound finding of nephrocalcinosis to identify the underlying molecular genetic cause of disease. In 15 of 51 families, we detected a monogenic causative mutation by whole exome sequencing. A mutation in seven recessive genes (AGXT, ATP6V1B1, CLDN16, CLDN19, GRHPR, SLC3A1, SLC12A1), in one dominant gene (SLC9A3R1), and in one gene (SLC34A1) with both recessive and dominant inheritance was detected. Seven of the 19 different mutations were not previously described as disease-causing. In one family, a causative mutation in one of 117 genes that may represent phenocopies of nephrolithiasis-causing genes was detected. In nine of 15 families, the genetic diagnosis may have specific implications for stone management and prevention. Several factors that correlated with the higher detection rate in our cohort were younger age at onset of nephrolithiasis/nephrocalcinosis, presence of multiple affected members in a family, and presence of consanguinity. Thus, we established whole exome sequencing as an efficient approach toward a molecular genetic diagnosis in individuals with nephrolithiasis/nephrocalcinosis who manifest before age 25 years.
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http://dx.doi.org/10.1016/j.kint.2017.06.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5750088PMC
January 2018

Association of Generalized Psoriasis and Mixed Glomerulonephritis in a 10-year-old Girl.

Acta Dermatovenerol Croat 2017 Jul;25(2):142-144

Prof. Danko Milošević, MD, PhD, Department of Pediatric Nephrology, Dialysis and Transplantation, University Hospital Center Zagreb, Kišpatićeva 12, 10000 Zagreb, Croatia;

Generalized psoriasis and renal function disorder were previously described in sporadic adult cases, revealing a new entity - psoriatic nephropathy. So far there have been only two cases describing this association in children. We present and discuss a case of 10-year-old girl with the unique biopsy findings of double glomerulonephritis associated with the simultaneous onset of generalized psoriasis.
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July 2017

Neurotoxicity of cyclosporine A in children with steroid-resistant nephrotic syndrome: is cytotoxic edema really an unfavorable predictor of permanent neurological damage?

Wien Klin Wochenschr 2017 Aug 9;129(15-16):579-582. Epub 2017 Jun 9.

School of Medicine, University of Zagreb, Zagreb, Croatia.

Background: Cyclosporine A-associated neurotoxicity has been reported mainly after organ transplantation. Only a small number of children with steroid-resistant nephrotic syndrome and cyclosporine A-associated neurotoxicity have been reported.

Patients: We report three children, aged 4, 11, and 15, with steroid-resistant nephrotic syndrome and cyclosporine A-associated neurotoxicity. In two of the patients, primary diagnosis was idiopathic nephrotic syndrome, and in one it was IgA nephropathy. Magnetic resonance with diffusion-weighted imaging, combined with quantification of apparent diffusion coefficient values, showed lesions caused by cytotoxic edema indicating irreversible brain damage. Nonetheless, the patients fully recovered clinically and radiologically after prompt discontinuation of cyclosporine A.

Conclusions: Neurotoxic effects should be suspected in any child with nephrotic syndrome treated with cyclosporine A in whom sudden neurological symptoms occur. Cytotoxic edema is a rare finding in pediatric patients. However, even in such cases with seemingly irreversible brain damage, full recovery without permanent neurological sequels is possible with prompt cyclosporine A discontinuation and supportive therapy.
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http://dx.doi.org/10.1007/s00508-017-1221-zDOI Listing
August 2017

Analysis of Linear Antibody Epitopes on Factor H and CFHR1 Using Sera of Patients with Autoimmune Atypical Hemolytic Uremic Syndrome.

Front Immunol 2017 30;8:302. Epub 2017 Mar 30.

3rd Department of Internal Medicine, Research Laboratory, Semmelweis University, Budapest, Hungary.

Introduction: In autoimmune atypical hemolytic uremic syndrome (aHUS), the complement regulator factor H (FH) is blocked by FH autoantibodies, while 90% of the patients carry a homozygous deletion of its homolog complement FH-related protein 1 (CFHR1). The functional consequence of FH-blockade is widely established; however, the molecular basis of autoantibody binding and the role of CFHR1 deficiency in disease pathogenesis are still unknown. We performed epitope mapping of FH to provide structural insight in the autoantibody recruitment on FH and potentially CFHR1.

Methods: Eight anti-FH positive aHUS patients were enrolled in this study. With overlapping synthetic FH and CFHR1 peptides, we located the amino acids (aa) involved in binding of acute and convalescence stage autoantibodies. We confirmed the location of the mapped epitopes using recombinant FH domains 19-20 that carried single-aa substitutions at the suspected antibody binding sites in three of our patients. Location of the linear epitopes and the introduced point mutations was visualized using crystal structures of the corresponding domains of FH and CFHR1.

Results: We identified three linear epitopes on FH (aa1157-1171; aa1177-1191; and aa1207-1226) and one on CFHR1 (aa276-290) that are recognized both in the acute and convalescence stages of aHUS. We observed a similar extent of autoantibody binding to the aHUS-specific epitope aa1177-1191 on FH and aa276-290 on CFHR1, despite seven of our patients being deficient for CFHR1. Epitope mapping with the domain constructs validated the location of the linear epitopes on FH with a distinct autoantibody binding motif within aa1183-1198 in line with published observations.

Summary: According to the results, the linear epitopes we identified are located close to each other on the crystal structure of FH domains 19-20. This tertiary configuration contains the amino acids reported to be involved in C3b and sialic acid binding on the regulator, which may explain the functional deficiency of FH in the presence of autoantibodies. The data we provide identify the exact structures involved in autoantibody recruitment on FH and confirm the presence of an autoantibody binding epitope on CFHR1.
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http://dx.doi.org/10.3389/fimmu.2017.00302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5371605PMC
March 2017

Calcium oxalate urolithiasis in children: urinary promoters/inhibitors and role of their ratios.

Eur J Pediatr 2016 Dec 11;175(12):1959-1965. Epub 2016 Oct 11.

Department of Pediatric Nephrology, Dialysis and Transplantation, University of Zagreb School of Medicine, University Hospital Center Zagreb, Kispaticeva 12, 10000, Zagreb, Croatia.

Diagnostic criteria for determination of inclination towards idiopathic calcium oxalate (CaOx) urolithiasis based on biochemical urine parameters are not sufficiently well defined in children. The aim of this study was to determine the risk of CaOx urolithiasis in children from concentrations of calcium, oxalate, citrate, and glycosaminoglycans in urine and their ratios, all standardized in respect to creatinine. We collected and analyzed 24-h urine samples of children with CaOx urolithiasis (n = 61) and compared with urine samples of matched control group of healthy children (n = 25). The study has showed that all stone formers have higher excretion of calcium (mmol/mmol creatinine), calcium/citrate (mol/mmol), and oxalate/(citrate × glycosaminoglycans) ratio (mol Ox × mol cr)/(mol Cit × g GAGs). ROC analysis of these variables gave criteria (>0.28, >1.07, and >0.08, respectively) for distinguishing stone formers from healthy children. Biochemical urine parameters and their ratios (calcium, calcium citrate, and oxalate/(citrate × glycosaminoglycans) enable one to discriminate idiopathic calcium oxalate stone formers from healthy children. Oxalate/(citrate × glycosaminoglycans) ratio per se can serve as an independent risk for stone formation.

Conclusion: Using biochemical urine parameters and their ratios such as calcium, calcium/citrate, and oxalate/(citrate × glycosaminoglycans) enables one to determine diagnostic criteria towards idiopathic calcium oxalate urolithiasis in children. What is known: • The role of urine calcium as a promoter in calcium oxalate urolithiasis is well established. • Seldom used calcium/citrate ratio is acknowledged as a risk factor for calcium/oxalate urolithiasis. What is new: • The values of calcium and citrate in clinically and genetically proven idiopathic calcium oxalate urolithiasis make calcium/citrate ratio useful for diagnostic purposes in such stone formers. • Rarely used calcium independent oxalate/(citrate x glycosaminoglycans) ratio serves as the second best high specificity marker for idiopathic calcium oxalate urolithiasis.
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http://dx.doi.org/10.1007/s00431-016-2792-9DOI Listing
December 2016

Mutations in SLC26A1 Cause Nephrolithiasis.

Am J Hum Genet 2016 06 19;98(6):1228-1234. Epub 2016 May 19.

Division of Nephrology, Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Nephrolithiasis, a condition in which urinary supersaturation leads to stone formation in the urinary system, affects about 5%-10% of individuals worldwide at some point in their lifetime and results in significant medical costs and morbidity. To date, mutations in more than 30 genes have been described as being associated with nephrolithiasis, and these mutations explain about 15% of kidney stone cases, suggesting that additional nephrolithiasis-associated genes remain to be discovered. To identify additional genes whose mutations are linked to nephrolithiasis, we performed targeted next-generation sequencing of 18 hypothesized candidate genes in 348 unrelated individuals with kidney stones. We detected biallelic mutations in SLC26A1 (solute carrier family 26 member 1) in two unrelated individuals with calcium oxalate kidney stones. We show by immunofluorescence, immunoblotting, and glycosylation analysis that the variant protein mimicking p.Thr185Met has defects in protein folding or trafficking. In addition, by measuring anion exchange activity of SLC26A1, we demonstrate that all the identified mutations in SLC26A1 result in decreased transporter activity. Our data identify SLC26A1 mutations as causing a recessive Mendelian form of nephrolithiasis.
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http://dx.doi.org/10.1016/j.ajhg.2016.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908148PMC
June 2016

Prevalence of Monogenic Causes in Pediatric Patients with Nephrolithiasis or Nephrocalcinosis.

Clin J Am Soc Nephrol 2016 Apr 19;11(4):664-72. Epub 2016 Jan 19.

Division of Nephrology, Department of Medicine and Howard Hughes Medical Institute, Chevy Chase, Maryland

Background And Objectives: Nephrolithiasis is a prevalent condition that affects 10%-15% of adults in their lifetime. It is associated with high morbidity due to colicky pain, the necessity for surgical intervention, and sometimes progression to CKD. In recent years, multiple monogenic causes of nephrolithiasis and nephrocalcinosis have been identified. However, the prevalence of each monogenic gene in a pediatric renal stone cohort has not yet been extensively studied.

Design, Setting, Participants, & Measurements: To determine the percentage of cases that can be explained molecularly by mutations in one of 30 known nephrolithiasis/nephrocalcinosis genes, we conducted a high-throughput exon sequencing analysis in an international cohort of 143 individuals <18 years of age, with nephrolithiasis (n=123) or isolated nephrocalcinosis (n=20). Over 7 months, all eligible individuals at three renal stone clinics in the United States and Europe were approached for study participation.

Results: We detected likely causative mutations in 14 of 30 analyzed genes, leading to a molecular diagnosis in 16.8% (24 of 143) of affected individuals; 12 of the 27 detected mutations were not previously described as disease causing (44.4%). We observed that in our cohort all individuals with infantile manifestation of nephrolithiasis or nephrocalcinosis had causative mutations in recessive rather than dominant monogenic genes. In individuals who manifested later in life, causative mutations in dominant genes were more frequent.

Conclusions: We present the first exclusively pediatric cohort examined for monogenic causes of nephrolithiasis/nephrocalcinosis, and suggest that important therapeutic and preventative measures may result from mutational analysis in individuals with early manifestation of nephrolithiasis or nephrocalcinosis.
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http://dx.doi.org/10.2215/CJN.07540715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822665PMC
April 2016

[C1Q NEPHROPATHY: CASE REPORTS AND LITERATURE REVIEW].

Lijec Vjesn 2015 Sep-Oct;137(9-10):283-7

C1q nephropathy is considered a form of glomerulonephritis, defined by histological findings of dominant Clq immune deposits in renal biopsy. It is a rare disease, most often manifested in children and young adults. The most common clinical manifestation of the disease is nephrotic syndrome, but other renal syndromes could also be found. The cause of the disease is not known, but the immune pathogenesis could be assumed. Often, resistance to glucocorticoid or other immunosuppressive therapy is present, potentially leading to chronic renal insufficiency. We present ten patients with renal biopsy and clinical findings of Clq nephropathy. None of the patients had clinical or serological manifestations of systemic lupus. All patients had normal findings of C3 and C4 components of complement, as well as normal ANF, anti-dsD-NA and ANCA antibodies.
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March 2016

Angiotensin-converting enzyme genotype is not a significant genetic risk factor for idiopathic nephrotic syndrome in Croatian children.

Nephron 2015 19;130(1):29-34. Epub 2015 May 19.

Department of Pediatric Nephrology, Clinical Hospital Center Zagreb, University of Zagreb School of Medicine, Zagreb, Croatia.

Aim: The association of the angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism with idiopathic nephrotic syndrome (INS) is controversial. Only scarce information on European populations is available. The aim of the study was to investigate the distribution of the ACE gene I/D polymorphism and its impact on INS in children from Croatia.

Materials And Methods: Ninety-five children with INS were investigated: 30 with minimal change disease (MCD), 35 with mesangial proliferative glomerulonephritis (MesPGN) and 30 with focal segmental glomerulosclerosis (FSGS). The control group consisted of 73 healthy adults. ACE gene was analyzed using the PCR method. The results were correlated with clinical features, renal morphology and response to immunosuppresive therapy.

Results: There was no correlation of ACE genotype with gender, age of the disease onset, level of proteinuria, presence of hematuria or hypertension, and GFR at onset of the disease. No statistically significant differences in ACE genotype or allele frequencies between the controls and whole group of patients, MCD group, MesPGN group, FSGS group, steroid sensitive (SS) patients, steroid resistant (SR) patients, as well as each other, were found, although DD genotype tended to be more frequent in FSGS patients, SR patients, and frequent relapsers. Among 11 children treated with cyclophosphamide the D allele was significantly higher among non-responders (p = 0.003).

Conclusion: DD genotype is not a genetic risk factor for acquiring INS nor significant phenotype modifier regarding to clinical and pathohistological picture and response to steroids in Croatian children. The potential application of ACE genotyping in predicting cyclophosphamide response deserves further investigation.
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http://dx.doi.org/10.1159/000382036DOI Listing
February 2016

Renal thromboembolism during treatment with recombinant activated factor VII (rFVIIa) in a child with hemophilia B with factor IX inhibitors.

BMC Pediatr 2014 Dec 17;14:315. Epub 2014 Dec 17.

Background: Serious thromboembolic events connected with rFVIIa therapy in hemophilia patients are rare. Only three cases are reported in children, all of them with hemophilia A.

Case Presentation: We present unique case of patient with hemophilia B and high titer inhibitors to coagulation FIX, who developed severe renal damage due to thromboembolic event during rFVIIa therapy, associated with unsuspected renovascular anomalies.

Conclusion: Caution is necessary if hematuria B requires administration of rFVIIa. US color doppler renal imaging before and after drug administration should be sufficient as an early warning.
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http://dx.doi.org/10.1186/s12887-014-0315-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4279815PMC
December 2014

Ultrasound distinction between simple recurrent urinary tract infections and a specific bladder wall inflammatory entity called cystitis cystica.

Coll Antropol 2014 Mar;38(1):151-4

A specific representative of recurrent urinary tract infections (UTI) called cystitis cystica (CC) was assessed by ultrasound. The aim of the study was to delineate, by means of ultrasound measurement (US) of bladder wall thickness (BWT), the children with mere repeated UTI from those prone to frequent UTI due to CC. Two groups were compared, the control group of 30 with recurrent UTI without US CC BWT changes, and the group of 30 children with characteristic CC bladder wall thickening in whom cystoscopy was performed for verification the diagnosis of CC. BWT of > 3 mm (> 2.8 mm and > 3.3 mm) was found as cut-of value for distinction of CC versus simple recurrent UTI. US BWT measurement is useful in diagnosing CC and therefore valuable in decision about need of UTI prophylaxis.
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March 2014

Positive feedback loop for cystitis cystica: the effect of recurrent urinary tract infection on the number of bladder wall mucosa nodules.

Acta Clin Croat 2013 Dec;52(4):444-7

The main purpose of this study was to demonstrate positive feedback loop between bladder wall nodules (nodules being one of the key diagnostic factors), bladder wall thickness, and recurrent urinary tract infections. Cystitis cystica was diagnosed in 115 prepubertal girls (mean age 7.79 +/- 3.05 years) by optic examination of bladder mucosal nodules and by ultrasonographic measurement of bladder wall thickness. Bladder wall thickness increased with the frequency of recurrent urinary tract infections as well as with the number of nodules on bladder wall mucosa (3.52 +/- 0.522 mm < or = 5 nodules vs. 4.42 +/- 0.429 mm 6-10 nodules vs. 5.20 +/- 0.610 mm > 10 nodules, respectively). Study results suggested that early control of urinary tract infections by chemoprophylaxis could prevent higher grades of bladder wall mucosal changes and consequently shorten the length of chemoprophylaxis.
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December 2013

Demographic characteristics and metabolic risk factors in Croatian children with urolithiasis.

Eur J Pediatr 2014 Mar 6;173(3):353-9. Epub 2013 Oct 6.

University Children's Hospital, University of Zagreb School of Medicine and Clinical Hospital Center Zagreb, Kišpatićeva 12, 10000, Zagreb, Croatia,

The aim of this study was to assess demographic data, clinical presentation, metabolic features, and treatment in 76 children with urolithiasis presented from 2002 to 2011. Urolithiasis is responsible for 2.5/1,000 pediatric hospitalizations, with new cases diagnosed in 1.1/1,000 admissions. From the observed period, two-fold rise of incidence rate was observed. Compiling the data from other pediatric institutions in our country, we estimated present overall incidence rate in Croatia as 6.5/100,000 children under 18 years. There were 41 boys and 35 girls (ratio 1.17:1). The mean age at diagnosis was 9.7 (range 0.8-16) years and follow-up duration was 5.3 (range 1.8-10) years. Renal colic (75.0 %) and hematuria (57.89 %) were the main symptoms. In 65.78 % of children, stones were unilateral. Stones were located in kidney in 52.63 %, in the ureter in 26.32 %, and in bladder in 6.58 % cases. Stone analysis showed calcium oxalate in 75.0 % of the cases. Associated urinary tract abnormalities were found in 19.73 % children. Most common metabolic disturbances were hypercalciuria (47.37 %) and idiopathic or mild hyperoxaluria (18.42 %). Urine saturation (EQUIL2) was elevated in 61.84 % cases. Spontaneous stone evacuation occurred in 51.21 % children. Extracorporeal shock wave lithotripsy, surgical evacuation, and endoscopic removal of calculi were performed in 21.0, 6.58, and 5.26 % of cases, respectively. Follow-up conservative therapy, consisting of fluid/diet recommendations and additional potassium citrate and/or chlorothiazide in children with increased risk, was sufficient for stone recurrence prevention in 92.1 % of children. In conclusion, the study gave insight in epidemiology and metabolic disturbances of urinary stone disease in Croatian children.
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http://dx.doi.org/10.1007/s00431-013-2165-6DOI Listing
March 2014

Childhood-onset systemic lupus erythematosus in Croatia: demographic, clinical and laboratory features, and factors influencing time to diagnosis.

Clin Exp Rheumatol 2013 Sep-Oct;31(5):803-12. Epub 2013 Jun 14.

Department of Paediatrics, University Hospital Centre Zagreb, University of Zagreb School of Medicine, Zagreb, and Department of Anaesthesiology, Reanimatology and Intensive Care, Varazdin General Hospital, Varazdin, Croatia.

Objectives: Childhood-onset systemic lupus erythematosus (cSLE) presents with diverse clinical features and often with non-classical symptoms that may delay diagnosis and increase risk of morbidity and mortality. This paper aims to analyse incidence, and clinical and laboratory features of cSLE in Croatia between 1991 and 2010, and to identify factors influencing time to diagnosis.

Results: Medical records at three university-based tertiary care centres were analysed retrospectively for 81 children with cSLE (68 girls). Mean age at onset was 13.4±2.8 yr (interquartile range 3), and annual incidence varied from 1-15 per million at risk. The most frequent clinical and laboratory features were musculoskeletal symptoms (80%) and increased erythrocyte sedimentation rate (96%). The most frequent immunological laboratory findings were the presence of antibodies against histones (86%), double-stranded DNA (73%), and Sm protein (64%), as well as low levels of C3 complement (69%). Haematuria was present in 58% of children, proteinuria in 56%, and biopsy-confirmed lupus nephritis in 43%. Median time from symptom onset to diagnosis was 2 months (range 0-96). Time to diagnosis was inversely associated with ECLAM score (p<0.001), but it showed no association with age, gender, clinical features or distance from the nearest paediatric centre.

Conclusions: This is the first large-scale, in-depth study of clinical and laboratory features of cSLE in Croatia. Among all demographic, laboratory and clinical features examined, ECLAM score alone was inversely associated with time to diagnosis. This highlights the need to improve detection of children with fewer symptoms early in the course of the disease, therefore serious consequences for prognosis could be avoided.
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December 2013

Ultrasound bladder wall thickness measurement in diagnosis of recurrent urinary tract infections and cystitis cystica in prepubertal girls.

J Pediatr Urol 2013 Dec 30;9(6 Pt B):1170-7. Epub 2013 May 30.

Department of Pediatrics, Zagreb University Hospital Centre, University of Zagreb, School of Medicine, Kišpatićeva 12, HR-10000 Zagreb, Croatia. Electronic address:

Objective: To evaluate urinary bladder wall thickness (BWT) assessed by ultrasound as a diagnostic tool for cystitis cystica.

Patients And Methods: This was a 9-year prospective study comprising 120 prepubertal girls. Sixty subjects of whom half underwent cystoscopy represented cases while the other 60 (those with a single urinary tract infection and healthy subjects) represented controls.

Results: Based on receiver operating characteristics (ROC) analysis, BWT discriminated very well between cases and controls with area under the ROC curve close to 1.0. At the optimum cut-off defined at 3.9 mm, negative predictive value (NPV) was 100% leaving no probability of cystic cystitis with BWT <3.9 mm. Positive predictive value (PPV) was also very high (95.2%), indicating only around 4.82% probability of no cystic cystitis in patients with BWT values ≥3.9 mm. BWT could also distinguish between healthy subjects and those with a cured single urinary tract infection, although discriminatory properties were moderate (area under ROC 86.7%, PPV 78.8%, NPV 85.2%).

Conclusion: Ultrasound mucosal bladder wall measurement is a non-invasive, simple and quite reliable method in diagnosis of cystitis cystica in prepubertal girls with recurrent urinary tract infections.
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http://dx.doi.org/10.1016/j.jpurol.2013.04.019DOI Listing
December 2013

Vesicoureteral reflux and urodynamic dysfunction.

Urol Int 2013 22;90(4):480-3. Epub 2012 Dec 22.

Nephrology Department, University Children's Hospital, Clinical Hospital Center, Zagreb, Croatia.

Introduction: The concept of vesicoureteral reflux (VUR) as a consequence of congenital anomaly of vesicoureteral junction has undergone changes owing to the finding that such children may have lower urinary tract dysfunction, which produces high intravesical pressure and consequently a predisposition for VUR.

Patients And Methods: The urodynamics was investigated by pressure-flow-EMG study in 132 children with VUR and 162 refluxing units.

Results: Only 33 (25.0%) patients had normal urodynamic finding. The most frequent pathological finding was overactive bladder (OAB), found in 59 (44.7%) children, followed by dysfunctional voiding (DV) in 25 (18.9%) children. Children with VUR grades I and II had a higher percentage of pathological urodynamic findings than children with VUR grades III and IV. OAB was more frequent in children under 5 years of age with unilateral and lower grade VUR. It was found equally in children with and without uroinfections. DV was more frequent in children older than 5 years, with bilateral VUR, higher grade VUR and uroinfections.

Conclusions: Children with VUR have a high incidence of urodynamic disorders. The results of the study indicate the possible role of urodynamic dysfunction in the pathogenesis of VUR, especially mild one.
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http://dx.doi.org/10.1159/000345713DOI Listing
January 2014

Polyarteritis nodosa in Croatian children: a retrospective study over the last 20 years.

Rheumatol Int 2013 Dec 14;33(12):3087-90. Epub 2012 Dec 14.

Division of Paediatric Immunology and Rheumatology, Department of Paediatrics, University Hospital Centre Zagreb, Zagreb University School of Medicine, Kispaticeva 12, 10000, Zagreb, Croatia,

To analyze the disease characteristics, treatment modalities and outcome of polyarteritis nodosa (PAN) in Croatian children. Cross-sectional study included all children with PAN diagnosed according to EULAR/PRES/PRINTO criteria during the last two decades. PAN was diagnosed in 12 patients (6 girls and 6 boys) mean age (±SD) 11.33 ± 3.08 years. The share of PAN among all vasculitides was 3.8 %. Systemic PAN was diagnosed in 7 children (58 %), microscopic polyangiitis in 3 (25 %), cutaneous PAN in 2 (17 %). The most consistent symptoms were skin involvement (90 %) and arthritis/arthralgia (60 %). The CNS was affected in 33 % of patients. Inflammatory markers (C-reactive protein and erythrocyte sedimentation rate [ESR]) were elevated in all patients, and anti-neutrophil cytoplasmatic antibodies were positive in all patients with microscopic polyangiitis. Therapy mode for all patients was corticosteroids. Immunosuppressive drugs were used as additional therapy for patients with severe symptoms. Two patients (17 %), both suffering from microscopic polyangiitis, died due to renal failure during the follow-up. In comparison with available studies, we found a difference in distribution of childhood polyarteritis nodosa as well as some clinical characteristics (e.g., higher prevalence of neurological and pulmonary symptoms), while other researched features, laboratory and treatment were similar.
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http://dx.doi.org/10.1007/s00296-012-2595-xDOI Listing
December 2013

Idiopathic nephrotic syndrome in children: review of 282 Croatian cases.

Clin Nephrol 2012 Aug;78(2):116-21

Department of Pediatrics, University of Zagreb School of Medicine, Croatia.

Recent data suggests increased incidence of focal segmental glomerulosclerosis (FSGS) among children with idiopathic nephrotic syndrome (INS). To determine the causes and possible longitudinal changes in the etiology of INS, 282 Croatian children diagnosed with INS between 1990 and 2009 were evaluated. In total, 122 children were assessed as having minimal change nephrotic syndrome (MCNS) based on their initial presentation, laboratory findings and clinical course. Kidney biopsy was performed in the remaining 160 children. MCNS was present in 18.1% of all biopsies performed. Total incidence of MCNS (assessed + biopsy proven) was only 53.5%. In contrast, FSGS was found in 40.6% of all biopsies and accounted for 23.1% of all cases. Mesangial proliferative glomerulonephritis (MesPGN) was the third most common diagnosis, present in 26.9% of the biopsies, and accounted for 15.2% of all cases. There were no significant longitudinal differences in the incidence of different causes of INS. The overall response to steroids at presentation was 71.6%. A higher proportion of initial steroid responders among children with FSGS (43.1%) and MesPGN (67.4%) than previously reported was noted. A longitudinal tendency of increasing steroid resistance in FSGS and MesPGN groups was observed.
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August 2012

A boy with Dent-2 disease.

Coll Antropol 2011 Sep;35(3):925-8

University of Zagreb, Zagreb University Hospital Centre, Department of Pediatrics, Division of Nephrology, Zagreb, Croatia.

Dent-2 disease is an X-linked renal tubulopathy associated with mutations in OCRL gene. It is characterized by low-molecular weight proteinuria, hypercalciuria, nephrolithiasis/nephrocalcinosis and progressive renal failure. Patients may have some extra-renal symptoms of Lowe syndrome, such as peripheral cataracts, mental impairment, stunted growth or elevation of creatine kinase/lactate dehydrogenase. Our patient was suspected to suffer from Dent disease at 8 months of age because of proteinuria and hypercalciuria. He had no prominent extra-renal symptoms. OCRL mutation in exon 1 (c.217_218 del TT p.L73F, fs X1) was found. He was treated with amiloride+hydroclorthiazide and citrate with good results in reducing calciuria. His renal ultrasound, ophthalmologic and cardiologic examinations, mental development and other laboratory findings are normal till date.
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September 2011

Cystitis cystica and recurrent urinary tract infections in children.

Coll Antropol 2010 Sep;34(3):893-7

Department of Pediatrics, Zagreb University Hospital Center, Zagreb, Croatia.

The pathogenesis of recurrent urinary tract infections (UTIs) in preschool children with anatomically correct urinary tract (UT) is rather obscure. In girls, the bladder wall changes of cystitis cystica (CC) may be per se responsible for UTIs recurrence. During the 20-year period, 127 preschool children (125 girls; median age: 6.1 years) with CC, in whom UT anomalies were excluded, were diagnosed. The mean duration of UTIs symptoms prior to diagnosis was 3.31 +/- 2.51 years. Cystoscopical findings were labelled as mild, moderate and severe in 22.8%, 39.4% and 37.8% of patients, respectively. Following the confirmation of CC, long-term chemoprophylaxis with sulfamethoxazole-trimethoprim/nitrofurantoin was administered. A one year UTI-free period after chemoprophylaxis discontinuation was defined as therapeutic success. With 2.5 years median duration of regular chemoprophylaxis this goal was achieved in 58 children mainly with mild/ moderate CC. Thirty children from "improved/unchanged" group taking regular prophylaxis had significant reduction of UTIs ("improved"). Only 12 children belonging to the same group taking regular prophylaxis and all children with irregular prophylaxis had approximately the same number of UTIs as before treatment ("unchanged"). The "improved/unchanged" outcomes were predominantly found in children with severe form of CC. Although urodynamic disturbances detected in more than 50% of patients in whom urodynamics was performed were not found influential on the disease outcome, they could be responsible for its development. The results of our study suggest that regular and long-lasting chemoprophylaxis remains a basis for successful treatment for majority of patients with CC, even those with severe forms. If not treated properly with chemoprophylactic agents and without fair compliance in taking drugs, the disease is prone to recurrent UTIs.
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September 2010

[Clinical and histopathological characteristics of biopsy-proven renal diseases in Croatia].

Acta Med Croatica 2007 Sep;61(4):361-4

Referentni centar za djecju nefrologiju, Klinika za pedijatriju, Kliniki bolnicki centar Zagreb.

There is little data on the spectrum of renal diseases in children in Croatia. The Croatian Society for Pediatric Nephrology has established the Registry of Biopsy-Proven Renal Diseases in an attempt to address this issue nationwide. Here we report preliminary results of a retrospective analysis of clinical and histopathological data of 565 children aged < or =17 years presenting to 9 hospitals in Croatia from 1991 to 2004, in whom kidney biopsy was performed. The most common indication for renal biopsy was nephrotic syndrome (39.1%), followed by asymptomatic proteinuria/hematuria (22.0%) and acute nephritic syndrome (17.0%). All biopsies were analysed by light-, immunofluorescent and electron microscopy. The majority of children, 552 out of 565 (92.4%), had glomerulonephritis (GN). Tubulointerstitial nephritis was found in 16 (2.8%), congenital renal parenchyma anomalies in 14 (2.5%) and vascular disease in 11 (1.9%) cases. One (0.2%) child had sarcoidosis with nephrocalcinosis. The sample was non-diagnostic in 1 (0.2%) case. Among children with GN, primary GN accounted for 70.9%, secondary GN for 16.1% and hereditary GN for 13.0% cases. The most frequent primary GN forms were focal segmental glomerulosclerosis (FSGS) (24.6%), mesangial proliferative glomerulonephritis (MEPGN) (19.2%) and IgA nephropathy (18.1%). Acute GN in resolution was found in 11.1% and minimal changes GN in 6.8% of cases. Most children with secondary GN had nephritis of Henoch-Schönlein purpura (HSP) (54.7%) and nephritis of systemic lupus erythematosus (SLE) (40.5%), while among hereditary GN Alport syndrome was most common (80.9%). In the group of children with primary GN who presented with nephrotic syndrome, most common forms were FSGS (38.5%) and MEPGN (24.0%). Minimal changes GN accounted for only 10.9% of cases. IgA nephropathy, primary or related to HSP (20.0%), FSGS (16.1%), MEPGN (12.6%) and Alport syndrome (9.7%) were the most common biopsy-proven renal diseases in Croatian children. The analysis provided data on the frequency of histological renal lesions in children in Croatia. The higher frequency of FSGS and MEPGN among Croatian children in comparison with other countries deserves further evaluation.
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September 2007

The significance of ultrasonography in diagnosing and follow-up of cystic cystitis in children.

Coll Antropol 2006 Jun;30(2):355-9

Department of Nephrology of the Pediatrics Clinic, University Hospital Center Zagreb, Croatia.

Cystic cystitis is a separate form of urinary bladder inflammation, detected by cystoscopy in children with recurrent urinary infections. Cystoscopy is an invasive method, so the aim of this investigation was to determine the ultrasonographic characteristics of cystic cystitis and to assess the reliability of ultrasound in relation to cystoscopy in diagnosing cystic cystitis. The study included 115 girls with repeated urinary infections. Cystoscopy and ultrasonography was performed in all. According to the cystoscopic finding the subjects were divided into 4 groups. Lateral and posterior urinary bladder wall thickness was measured during ultrasonography. A statistically significant difference was found between all 4 groups, the method demonstrated a high degree of sensitivity (0.97) and specificity (0.91). Percentile calculations were determined for wall thickness. Ultrasonography can replace endoscopy in diagnosis and follow-up of cystic cystitis in children, with at least 50% fullness of the urinary bladder as a prerequisite.
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June 2006