Distribution of rotavirus genotypes in three Croatian regions among children ≤5 years of age (2012-2014).

OBJECTIVES: Rotavirus is the major cause of severe diarrhea in young children worldwide. In countries like Croatia, where rotavirus vaccine has not been introduced in the national immunization program, prospective surveillance is necessary to establish the diversity of rotavirus strains. The aim of this study was to describe the prevalence and geographical distribution of rotavirus strains in Croatia and to detect the possible emergence of novel strains. METHODS: The study was conducted among children ≤5 years of age with acute gastroenteritis at three hospitals located in different geographical regions of Croatia, during the years 2012 to 2014. Rotavirus was detected in stools using an immunochromatographic assay and then sent for further molecular analysis. RESULTS: Genotyping of 822 rotaviruses showed that the predominant circulating strain was G1P[8] (61.9%), followed by G2P[4] (19.5%), G1P[4] (3.9%), and G3P[8] (2.9%). A high prevalence of reassortants among common human rotavirus genotypes was detected (7.7%). Possible zoonotic reassortants were found, including G8 and G6 strains. The latter is described for the first time in Croatia. CONCLUSIONS: This study represents pre-vaccination data that are important for decisions regarding immunization strategies in Croatia. The high prevalence of 'common' rotavirus strains circulating in Croatia may advocate for rotavirus vaccine introduction, but further surveillance is necessary to monitor the possible emergence of novel genotypes.

Late-Onset Hypertrophic Pyloric Stenosis in a 14-Weeks-Old Full Term Male Infant.

BACKGROUND: Hypertrophic pyloric stenosis is the most common cause of gastric outlet obstruction in infants, and classically presents at 2 to 6 weeks of age. Delayed presentation is an extremely rare occurrence after early infancy. CASE REPORT: A 14-weeks-old full term male infant presented with non-bilious vomiting, dehydration and hypocloremic metabolic alkalosis. Abdominal ultrasonography revealed tubular mass 20 mm in lenght. Because of unusual age, diagnosis was confirmed with upper gastrointestinal contrast study. Laparoscopic pyloromyotomy was performed. After surgery the child was free of symptoms, had gained weight, and was tolerating a regular diet. Message: Despite the age hypertrophic pyloric stenosis should be kept in mind in any child who presents with non-bilious vomiting.

PHACES Syndrome with Intestinal Hemangiomatosis.

We present a rare case of a neonate with PHACES syndrome (posterior fossa malformations, large facial hemangiomas, cerebral arterial anomalies, cardiovascular anomalies, eye anomalies and sternal clefting or supraumbilical raphe) and diffuse hemangiomatosis of the ileum, presenting with multiple intestinal perforations and peritonitis. The infant was successfully treated with propranolol and methylprednisolone as well as octreotide, tranexamic acid, and supportive therapy for massive intestinal bleeding.

Molecular basis and clinical presentation of classic galactosemia in a Croatian population.

BACKGROUND: Classic galactosemia is an autosomal recessive disorder of galactose metabolism caused by severely decreased activity of galactose-1-phosphate uridylyltransferase (GALT) due to pathogenic mutations in the GALT gene. To date more than 330 mutations have been described, with p.Q188R and p.K285N being the most common in Caucasian populations. Although acute manifestations can be fully avoided by a galactose-restricted diet, chronic complications, such as neurological ones, cannot be prevented in a significant number of patients despite compliance with the dietary treatment. METHODS: A cohort of 16 galactosemic Croatian patients, including one pair of siblings, was studied. Molecular characterization was performed by direct sequence analysis of the GALT gene. RESULTS: Sixteen patients were analyzed and only four different mutations were detected. As expected, p.Q188R and p.K285N were common, accounting for 40% and 37% of unrelated alleles, respectively. The third mutation accounting for 20% of mutant alleles was p.R123X causing a premature stop codon, is thus considered to be severe, which is in accordance with the phenotype presented by the homozygous patient described here. The fourth mutation p.E271D was found in a single allele. More than half of our patients manifested some chronic neurological complications. CONCLUSIONS: This is the first report on mutational and phenotypic spectra of classic galactosemia in Croatia that expands the knowledge on the mutational map of the GALT gene across Europe and reveals the genetic homogeneity of the Croatian population.

Molecular Characterization of Glucose-6-phosphate Dehydrogenase Deficiency in Families from the Republic of Macedonia and Genotype-phenotype Correlation.

INTRODUCTION: Glucose-6-phospahte dehydrogenase deficiency (G6PD) is one of the most common inherited disorders affecting around 400 million people worldwide. Molecular analysis of the G6PD gene identified more than 140 distinct mutations, the majority being single base missense mutations. G6PD Mediterranean is the most common variant found in populations of the Mediterranean area. AIM: The aim of our study was to perform molecular characterization of G6PD deficiency in families from the Republic of Macedonia and correlate the findings to disease phenotype. PATIENTS AND METHODS: Six patients and seven other family members were selected for genetic characterization, the selection procedure involved clinical evaluation and G6PD quantitative testing. All patients were first screened for the Mediterranean mutation, and subsequently for the Seattle mutation. Mutations were detected using PCR amplification and appropriate restriction endonuclease cleavage. RESULTS: Four hemizygote and 3 heterozygous carriers for G6PD Mediterranean were detected. All G6PD deficient patients from this group showed clinical picture of hemolysis, and in 66.6% neonatal jaundice was confirmed based on history data. To our knowledge, this is the first study concerned with molecular aspects of the G6PD deficiency in R. Macedonia. CONCLUSION: This study represents a step towards a more comprehensive genetic evaluation in our population and better understanding of the health issues involved.

Influence of the Inherited Glucose-6-phosphate Dehydrogenase Deficiency on the Appearance of Neonatal Hyperbilirubinemia in Southern Croatia.

BACKGROUND: Neonatal hyperbilirubinemia is a common clinical manifestation of the inherited glucose-6-phosphate dehydrogenase (G6PD) deficiency. AIM OF THE STUDY: The aim of this study was to investigate the influence of the inherited G6PD deficiency on the appearance of neonatal hyperbilirubinemia in southern Croatia. METHODS: The fluorescent spot test (FST) was used in a retrospective study to screen blood samples of 513 male children who had neonatal hyperbilirubinemia, of unknown cause, higher than 240 µmol/L. Fluorescence readings were performed at the beginning and at the fifth and tenth minute of incubation and were classified into three groups bright fluorescence (BF), weak fluorescence (WF) and no fluorescence (NF). Normal samples show bright fluorescence. All NF and WF samples at the fifth minute were quantitatively measured using the spectrophotometric method. RESULTS: Bright fluorescence was present in 461 patients (89.9%) at the fifth minute. The remaining 52 (10.1%) were quantitatively estimated using the spectrophotometric method. G6PD deficiency was observed in 38 patients (7.4%). CONCLUSIONS: Prevalence rate of G6PD deficiency among male newborns with hyperbilirubinemia in southern Croatia is significantly higher (p < 0.01) compared with the previously reported prevalence rate among male in general population of southern Croatia (0.75%). We recommend FST to be performed in hyperbilirubinemic newborns in southern Croatia.

Lab-score is a valuable predictor of serious bacterial infection in infants admitted to hospital.

Parents frequently bring their children to the Emergency Department (ED) because of the fever without apparent source (FWAS). To avoid possible complications, it is important to recognize serious bacterial infection (SBI) as early as possible. Various tests, including different clinical scores and scales, are used in the laboratory evaluation of patients. However, it is still impossible to predict the presence of SBI with complete certainty. Galetto-Lacour et al. developed and validated a risk index score, named Lab-score. Lab-score is based on the three predictive variables independently associated with SBI: procalcitonin (PCT), C-reactive protein (CRP), and urinary dipstick. The objective of this study was to assess the performance of the Lab-score in predicting SBI in well-appearing infants ≤ 180 days of age with FWAS, who presented to ED and were hospitalized with suspicion of having SBI. Based on this study findings, white blood cells count (WBC), CRP, PCT, and lab-score ≥ 3 were confirmed as useful biomarkers for differentiation between SBI and non-SBI. Also, receiver operating characteristic curve (ROC) analysis confirmed that all of them were useful for differentiation between SBI and non-SBI patients with the highest area under curve (AUC) calculated for the Lab-score. The results of this research confirmed its value, with calculated sensitivity of 67.7% and specificity of 98.6% in prediction of SBI in infants aged ≤ 180 days. Its value was even better in infants aged ≤ 90 days with sensitivity of 75% and specificity of 97.7%. In conclusion, we demonstrated the high value of lab-score in detecting SBI in infants under 6 months of age with FWAS.

Evaluation of Neonatal Hemolytic Jaundice: Clinical and Laboratory Parameters.

BACKGROUND: Neonatal jaundice that occurs in ABO or Rhesus issoimunisation has been recognized as one of the major risk factors for development of severe hyperbilirubinemia and bilirubin neurotoxicity. AIM: Aim of our study was to investigate clinical and laboratory parameters associated with hemolytic jaundice due to Rh and ABO incompatibility and compare results with the group of unspecific jaundice. MATERIAL AND METHODS: One hundred sixty seven (167) neonatal hyperbilirubinemia cases were included in the study, 24.6% of which presented with ABO/Rhesus type hemolytic jaundice, and the rest with unspecific jaundice. Evaluation included: blood count, reticulocites, serum bilirubin, aminotransferases, blood grouping, and Coombs test, also the day of bilirubin peak, duration of the hyperbilirubinemia, and additional bilirubin measurements. RESULTS: We showed significantly lower mean values of hemoglobin, erythrocytes and hematocrit and significantly higher values of reticulocytes in the group of ABO/Rh incompatibility compared to the group of jaundice of unspecific etiology; also an earlier presentation and a higher-grade jaundice in this group. CONCLUSIONS: The laboratory profile in ABO/Rh isoimmunisation cases depicts hemolytic mechanism of jaundice. These cases carry a significant risk for early and severe hyperbilirubinemia and are eligible for neurodevelopmental follow-up. Hematological parameters and blood grouping are simple diagnostic methods that assist the etiological diagnosis of neonatal hyperbilirubinemia.

The OSR1 rs12329305 polymorphism contributes to the development of congenital malformations in cases of stillborn/neonatal death.

BACKGROUND: Involvement of development-related gene polymorphisms in multifactorial/polygenic etiology of stillborn/neonatal deaths due to malformations has been insufficiently tested. Since these genes showed evolutional stability and their mutations are very rare, we can assume that their polymorphic variants may be a risk factor associated with the occurrence of developmental disorders of unknown etiology or can enhance the phenotypic variability of known genetic disorders. MATERIAL AND METHODS: To determine the association of 3 polymorphisms involved in the regulation of the early embryonic development of different organs, we conducted an association study of their relation to the particular malformation. We selected 140 samples of archived paraffin tissue samples from deceased patients in which fetal/neonatal autopsy examination had shown congenital abnormalities as the most likely cause of death. The polymorphisms of OSR1 rs12329305, rs9936833 near FOXF1, and HOXA1 rs10951154 were genotyped using the TaqMan allelic discrimination assay. RESULTS: After Bonferroni correction for multiple testing, significant allelic association with stillborn/neonatal deaths was observed for rs12329305 (p=7×10-4). In addition, association analysis for the same polymorphism was shown in the subgroup with isolated anomalies (1.25×10^-5), particularly in the subgroup of cases with kidney and heart anomalies (p=4.18×10^-5, p=5.12×10^-8, respectively). CONCLUSIONS: The findings of the present study showed, for the first time, the role of the OSR1 rs12329305 polymorphism in the development of congenital malformations in cases of stillborn/neonatal death, particularly in those with congenital kidney and heart developmental defects.

Mechanism of cystogenesis in nephrotic kidneys: a histopathological study.

BACKGROUND: Nephrotic syndrome (NS) is pathological condition characterized by heavy proteinuria. Our study investigates hypothesis that change in cell proliferation of proximal tubules influences primary cilia structure and function and promotes cystogenesis in congenital nephrotic syndrome of the Finnish type (CNF) and focal segmental glomerulosclerosis (FSGS). METHODS: CNF kidneys were analyzed genetically. Proliferation (Ki-67), apoptosis (caspase-3), and primary cilia (α-tubulin) length and structure were analyzed immunohistochemically and ultrastructurally in healthy, CNF and FSGS kidneys. Cyst diameters were measured and correlated with proliferation index. RESULTS: Proximal tubules cells of healthy kidneys did not proliferate. In nephrotic kidneys, tubules with apparently normal diameter covered by cuboidal/columnar epithelium (PTNC) contained 81.54% of proliferating cells in CNF and 36.18% in FSGS, while cysts covered with columnar epithelium (CC) contained 37.52% of proliferating cells in CNF and 45.23% in FSGS. The largest cysts, covered with squamous epithelium (CS) had 11.54% of proliferating cells in CNF and 13.76% in FSGS. Increase in cysts diameter correlated with changes in proliferation index, tubular cells shape, primary cilia formation and appearance of apoptotic cells. CONCLUSIONS: We present a novel histopathological data on the structure and possible changes in function of tubular cell in NS kidneys during cystogenesis. We suggest existence of common principles of cystogenesis in CNF and FSGS kidneys, including serious disturbances of tubular cells proliferation and apoptosis, and faulty primary cilia signaling leading to deterioration of proteinuria in NS kidneys.

Association of NOS3 tag polymorphisms with hypoxic-ischemic encephalopathy.

AIM: To test the association of NOS3 gene with hypoxic-ischemic encephalopathy (HIE). METHODS: The study included 110 unrelated term or preterm born children (69 boys and 41 girls) with HIE and 128 term and preterm born children (60 boys and 68 girls) without any neurological problems after the second year of life. Children with perinatal HIE fulfilled the diagnostic criteria for perinatal asphyxia. All children were admitted to the Clinical Hospital Split between 1992 and 2008. We analyzed 6 tagging single nucleotide polymorphisms (SNP) within NOS3 gene (rs3918186, rs3918188, rs1800783, rs1808593, rs3918227, rs1799983), in addition to previously confirmed NOS3-associated SNP rs1800779. Genotyping was conducted using real-time polymerase chain reaction (PCR). Association analyses were performed according to allelic and genotypic distribution. RESULTS: Allelic test did not show any SNP association with HIE. SNP rs1808593 showed genotype association (P=0.008) and rs1800783-rs1800779 TG haplotype showed an association with HIE (P<0.001). The study had 80% statistical power to detect (α=0.05) an effect with odds ratio (OR)=2.07 for rs3918186, OR=1.69 for rs3918188, OR=1.70 for rs1800783, OR=1.80 for rs1808593, OR=2.10 for rs3918227, OR=1.68 for rs1800779, and OR=1.76 for rs1799983, assuming an additive model. CONCLUSION: Despite the limited number of HIE patients, we observed genotypic and haplotype associations of NOS3 polymorphisms with HIE.

Evaluation of cardiovascular risk in school children.

Atherosclerosis is a pathological condition that begins in early childhood, but clinically the disease manifests in older age. The aim of work was to determine frequency of atherosclerosis risk factors in healthy school children. Cross-sectional study included 214 children in mean age 10,99+/-2,52 years, within range 7 to 15 years. Patients body mass index, blood pressure, lipid status, dietary habits, physical activity and sedentary habits have been evaluated. Cardiovascular risk factors are significantly present in children (P<0,05) i.e. one cardiovascular risk factor is present in 47/214 (21,96%) children, two risk factors had 25/214 (11,68%) children, while 17/214 (7,94%) children had three or more cardiovascular risk factors. Obesity was present in 20/214 (9,34%) children, while overweight was present in 23/214 (10,74%) children. Hypertension was present in 10/214 (4,67%) children, and it was significantly present (p<0,05) in obese and overweight children. Total cholesterol was increased in 17/214 (7,94%) children, LDL-cholesterol was increased in 11/214 (5,14%) [corrected], increased triglycerides had 4/214 (1,86%) children, while decreased HDL-cholesterol was found in (3/214, 1,40%) children. Unhealthy dietary habits were present in 45/214 (21,02%) children, 42/214 (19,62%) children is physically inactive, while sedentary habits were shown in 39/214 (18,22%) children. Research shows that a large number within study group has one or more cardiovascular risk factors that can lead to premature atherosclerosis. Using massive screening of cardiovascular risk factors, along with adequate physical activity, healthy dietary habits, reduced sedentary habits, doctors and teacher's education, parents and children can reduce premature clinical sequels in atherosclerotic process.

Genome-wide association study of anthropometric traits in Korcula Island, Croatia.

AIM: To identify genetic variants underlying six anthropometric traits: body height, body weight, body mass index, brachial circumference, waist circumference, and hip circumference, using a genome-wide association study. METHODS: The study was carried out in the isolated population of the island of Korcula, Croatia, with 898 adult examinees who participated in the larger DNA-based genetic epidemiological study in 2007. Anthropometric measurements followed standard internationally accepted procedures. Examinees were genotyped using HumanHap 370CNV chip by Illumina, with a genome-wide scan containing 316730 single nucleotide polymorphisms (SNP). RESULTS: A total of 11 SNPs were associated with the investigated traits at the level of P<10(-5), with one SNP (rs7792939 in gene zinc finger protein 498, ZNF498) associated with body weight, hip circumference, and brachial circumference (P=3.59-5.73 x 10(-6)), and another one (rs157350 in gene delta-sarcoglycan, SGCD) with both brachial and hip circumference (P=3.70-6.08 x 10(-6). Variants in CRIM1, a gene regulating delivery of bone morphogenetic proteins to the cell surface, and ITGA1, involved in the regulation of mesenchymal stem cell proliferation and cartilage production, were also associated with brachial circumference (P=7.82 and 9.68 x 10(-6), respectively) and represent interesting functional candidates. Other associations involved those between genes SEZ6L2 and MAX and waist circumference, XTP6 and brachial circumference, and AMPA1/GRIA1 and height. CONCLUSION: Although the study was underpowered for the reported associations to reach formal threshold of genome-wide significance under the assumption of independent multiple testing, the consistency of association between the 2 variants and a set of anthropometric traits makes CRIM1 and ITGA1 highly interesting for further replication and functional follow-up. Increased linkage disequilibrium between the used markers in an isolated population makes the formal significance threshold overly stringent, and changed allele frequencies in isolate population may contribute to identifying variants that would not be easily identified in large outbred populations.

Glucose-6-phosphate dehydrogenase deficiency and idiopathic presenile cataract in Dalmatia, Croatia.

BACKGROUND: Impaired activity of the enzyme glucose-6-phosphate dehydrogenase (G6PD) has been suggested as a risk factor in cataractogenesis. The aim of this study was to determine the G6PD activity level in 89 male subjects of Dalmatian origin with idiopathic presenile cataracts. METHODS: G6PD activity was determined by a quantitative spectrophotometric method. RESULTS: Of 89 males with presenile cataracts only one (1.1%) had G6PD deficiency. The G6PD deficiency prevalence rate among males with presenile cataracts is not significantly different (p > 0.05) from the prevalence of G6PD deficiency in the general population of Dalmatia (0.75% among men). INTERPRETATION: The results of this study suggest that G6PD deficiency does not represent a pathogenetic factor in presenile cataract, at least not in the population of the southern part of Croatia.

High incidence of glucose-6-phosphate dehydrogenase deficiency in Croatian island isolate: example from Vis island, Croatia.

AIM: To determine the prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency in the population of the town of Komiza on the island of Vis, which has previously been reported as a place with several cases of favism. METHODS: We screened 302 randomly selected men, using the fluorescent spot test. Fluorescence readings were performed at the beginning and 5, 10, and 20 minutes after incubation, and were classified into three groups: bright fluorescence, weak fluorescence, and no fluorescence. All men found to be G6PD deficient were tested with a quantitative spectrophotometric UV method. RESULTS: Of the 302 tested blood samples, 36 (11.9%) samples showed weak fluorescence or no fluorescence spots. Spectrophotometric UV test showed that 18 (5.96%) men were G6PD deficient. The prevalence of G6PD deficiency in the population of Komiza is significantly higher (P<0.001) than the prevalence in the whole population of Dalmatia in the south of Croatia (0.75% in men). CONCLUSION: On the basis of these findings, we recommend including the newborns from the island of Vis into a screening program for G6PD deficiency. Our results indicate that G6PD deficiency should be determined for all the island isolates in the Mediterranean basin and they warrant further studies.

The influence of chronic health conditions on susceptibility to severe acute illness of children treated in PICU.

OBJECTIVES: Our study aimed to assess differences in the susceptibility to severe acute illness in children with and without chronic health conditions treated in a pediatric intensive care unit (PICU). PATIENTS AND METHODS: Data on age, gender, need for ventilator support, length of stay, as well as other parameters for the Paediatric Index of Mortality (PIM2) score were collected. Data were analyzed and compared across three patient groups: those with a neurodevelopmental disability, those with a chronic condition other than a neurodevelopmental disability, and those with no chronic condition. Reasons for admission of patients were classified according to the Australia and New Zealand Paediatric Intensive Care Registry (ANZPIC Registry) diagnostic codes. In the multidisciplinary, seven-bed, level I PICU of the Split University Hospital, the admission data were collected prospectively for 591 consecutively admitted patients aged

Characterization of G6PD deficiency in southern Croatia: description of a new variant, G6PD Split.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency protects from severe forms of malaria. It is interesting therefore to analyze the molecular basis underlying G6PD deficiency in regions such as the Mediterranean basin where malaria was present for a long time in history. Here we report on the genetic characterization of G6PD deficiency among inhabitants of one Mediterranean region-the Dalmatian region of south Croatia. We analyzed 24 unrelated G6PD-deficient male subjects. Molecular testing revealed several different mutations: G6PD Cosenza 9, G6PD Mediterranean 4, G6PD Seattle 3, G6PD Union 3, and G6PD Cassano 1. Furthermore, we have identified one novel G6PD variant that we named G6PD Split. This variant is caused by a nucleotide change 1442 C-->G leading to the amino acid substitution 481 Pro-->Arg and is characterized by moderate enzyme deficiency (class III variant). This study reveals a higher prevalence (37.5%) of the Cosenza mutation in the Dalmatian region than anywhere else previously investigated and overall shows the considerable molecular heterogeneity underlining G6PD deficiency that can be observed in Mediterranean populations.

Prevalence of factor V Leiden and G6PD 1311 silent mutations in Dalmatian population.

BACKGROUND: Factor V Leiden has been described as a common genetic risk factor for venous thromboembolism. The geographic distribution of this abnormality varies greatly, being high in Europe and almost absent in Asia and Africa. Particularly high prevalence is observed in some Mediterranean countries, which suggests the Mediterranean origin of this mutation. Similarly, prevalence of silent mutation 1311 of the G6PD gene seems to be higher among Mediterranean populations. Since the Dalmatian population (of south Croatia) geographically belongs to the Mediterranean populations we analyzed the prevalence of FV-Leiden and silent mutation 1311 in this region. Furthermore, because the coincidence of G6PD deficiency and venous thromboembolism was described earlier, we tested a possible association of FV-Leiden and G6PD deficiency. METHODS: One hundred sixty-eight healthy blood donors and 55 G6PD deficient individuals originating from the Dalmatian region were tested for the presence of FV-Leiden mutation and silent mutation 1311. RESULTS: Prevalence of FV-Leiden among blood donors was 2.4%, while among G6PD deficient individuals it was significantly higher, 11% (p=0.011). Prevalence of silent mutation 1311 among blood donors and G6PD deficient individuals was 21 and 15%, respectively. CONCLUSIONS: Observed allele frequencies among individuals originating from the Dalmatian region is similar to the neighboring European and Mediterranean populations. Interestingly, our results indicate the association of the FV-Leiden and G6PD deficiency and warrant further studies.