Rabies outbreak in Greece during 2012-2014: use of Geographical Information System for analysis, risk assessment and control.

The objectives of this work were (i) geographical analysis of the 2012-2014 outbreak of rabies in Greece using GIS and (ii) comparative analysis of animal cases with data of potential human exposure to rabies together with environmental data, in order to provide information for risk assessment, effective monitoring and control. Most animal cases (40/48) involved red foxes, while domestic animals were also diagnosed with rabies. Overall, 80% of the cases were diagnosed in central northern Greece; 75% of the cases were diagnosed in low altitudes (<343·5 m), within a distance of 1 km from human settlements. Median distance from livestock farms was 201·25 m. Most people potentially exposed to rabies (889/1060) presented with dog bite injuries. Maximum entropy analysis revealed that distance from farms contributed the highest percentage in defining environmental niche profiles for rabid foxes. Oral vaccination programmes were implemented in 24 administrative units of the country during 2013 and 2014, covering a total surface area of ~60 000 km2. Rabies re-occurrence in Greece emphasizes the need for ongoing surveillance in cross-border areas and in areas with intense human activity.

Aspergillus fumigatus chronic colonization and lung function decline in cystic fibrosis may have a two-way relationship.

Aspergillus fumigatus is commonly found in cystic fibrosis (CF) airways. Our aim was to assess the relationship between A. fumigatus chronic colonization and lung function in CF patients. A case-control study of CF patients born from 1989 to 2002 was performed. Medical records were reviewed from the time of initial diagnosis until December 2013. Chronic colonization was defined as two or more positive sputum cultures in a given year. Each patient chronically colonized with A. fumigatus was matched with three control patients (never colonized by A. fumigatus) for age, sex, and year of birth (±3 years). A number of parameters were recorded and analyzed prospectively. The primary outcome measure was the difference in forced expiratory volume in 1 s (FEV1) in percent predicted between groups. Linear mixed models were used for longitudinal analyses to evaluate the relationship between A. fumigatus chronic colonization and lung function during a 7-year period and study the lung function 4 years before the time of enrollment (t0). Twenty patients had chronic colonization and were matched with 60 controls. A significant difference in lung function was detected throughout the 7-year period after adjustment for confounders (est = 8.66, p = 0.020). Four years before t0, FEV1 baseline was the only factor associated with the course of lung function (est = 0.64, p < 0.001) and was significantly different between groups (p = 0.001). In conclusion, a decreased FEV1 baseline appears to be a risk factor for chronic colonization by A. fumigatus, which, in turn, may cause a faster deterioration of lung function.

Re-emergence of animal rabies in northern Greece and subsequent human exposure, October 2012 - March 2013.

Greece has been rabies-free since 1987 with no human cases since 1970. During 2012 to 2013, rabies has re-emerged in wild and domestic animals in northern Greece. By end March 2013, rabies was diagnosed in 17 animals including 14 red foxes, two shepherd dogs and one cat; 104 subsequent human exposures required post-exposure prophylaxis according to the World Health Organization criteria. Human exposures occurred within 50 km radius of a confirmed rabies case in a wild or domestic animal, and most frequently stray dogs were involved.

Contribution of the CFTR gene, the pancreatic secretory trypsin inhibitor gene (SPINK1) and the cationic trypsinogen gene (PRSS1) to the etiology of recurrent pancreatitis.

Acute recurrent/chronic pancreatitis (CP) is a complex multigenic disease. This is a case-control study consisting of 25 Greek patients with CP and a control population of 236 healthy Greek subjects. The whole coding area and neighboring intronic regions of the three genes were screened. Seventeen of 25 patients (68%) had mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene: nine compound heterozygotes with either mild or severe mutations and eight heterozygotes. Four patients (16%) carried CFTR-modulating haplotypes V470-TG11-T5 and V470-TG12-T7. All were negative for PRSS1 gene mutations, while variants c.486C/T and c.738C/T were found in nine patients each, three homozygotes for the minor alleles. Two carried SPINK1 gene mutation p.N34S, one being transheterozygote with CFTR mutation p.F1052V. The promoter variant -253T>C was found in four individuals (one homozygous for the minor allele), all four being transheterozygotes with mutations in the CFTR gene as well. Finally two carried c.272C/T in the 3' untranslated region, one being a p.N34S carrier as well. In total, 80% (20/25) of patients had a molecular defect in one or both of the CFTR and SPINK1 genes, suggesting that mutations/variants in the CFTR plus or minus mutations in the SPINK1, but not the PRSS1 gene, may confer a high risk for recurrent pancreatitis.

Cystic fibrosis in Greece: molecular diagnosis, haplotypes, prenatal diagnosis and carrier identification amongst high-risk individuals.

Cystic fibrosis (CF) mutation analysis on 437 CF patients, characterized 80 different mutations (20 so far specific to our population) accounting for 91% of CF genes and generating 103 different genotypes. Eight mutations were common [F508del (53.4%), 621+1G>T (5.7%), G542X (3.9%), N1303K (2.6%), 2789+5G>A (1.7%), 2183AA>G (1.4%), E822X (1.4%), R1158X (1%)], 12 showed frequencies between 0.5% and 1%, while the remaining (60) were very rare (1 to 3 alleles). Denaturing gradient gel electrophoresis (DGGE) screening of 12 exons (3, 4, 7, 10, 11, 13, 14b, 16, 17b 20 and 21) detected 85.5% of CF alleles. Haplotypes for eight diallelic and three microsatellite markers have been characterized for the common, a few rare and novel Greek mutations. Results of 165 prenatal diagnoses (including 49 due to bowel hyperechogenicity), testing a total of 41 different parental genotypes, are reported. One hundred and sixteen prenatal tests resulted in 22 affected, 59 heterozygous, 34 normal fetuses and one incomplete diagnosis. Of the 49 echogenic bowel fetuses, 3 were heterozygotes. Carrier screening was initiated, with emphasis on individuals and couples in high-risk groups - with a family history of CF, one partner with CF, and couples with male infertility seeking in vitro fertilization (IVF). Mutation analysis on 672 individuals (120 couples, 91 unaffected CF siblings, 283 CF family relatives and 58 general population subjects), identified a total of 176 heterozygotes and 7 couples where both partners were CF heterozygotes. Prenatal diagnosis was performed in 4 cases and 3 were counseled on the availability of a prenatal test.

CFTR gene mutations--including three novel nucleotide substitutions--and haplotype background in patients with asthma, disseminated bronchiectasis and chronic obstructive pulmonary disease.

In order to investigate the incidence of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and unclassified variants in chronic pulmonary disease in children and adults, we studied 20 patients with asthma, 19 with disseminated bronchiectasis (DB) of unknown aetiology, and 12 patients with chronic obstructive pulmonary disease (COPD), and compared the results to 52 subjects from the general Greek population. Analysis of the whole coding region of the CFTR gene and its flanking intronic regions revealed that the proportion of CFTR mutations was 45% in asthma (P<0.05), 26.3% in DB (P>0.05), 16.7% in COPD (P>0.05), compared to 15.4% in the general population. Seventeen different molecular defects involved in disease predisposition were identified in 16 patients. Three potentially disease-causing mutations, T388 M, M1R and V11I, are novel, found so far only in three asthma patients. The hyperactive M470 allele was found more frequently in COPD patients (frequency 70.8%, P<0.01) than in the controls. The study of the TGmTnM470 V polyvariant CFTR allele revealed the presence of CFTR function-modulating haplotypes TG13/T5/M470, TG11/T5/M470, TG12/T5/V470 and TG12/T7, combined with M470 or V470, in six asthma patients, four DB patients (P<0.01), and two COPD patients (P<0.05). These results confirm the involvement of the CFTR gene in asthma, DB and possibly in COPD.

Qualitative and quantitative analysis of mRNA associated with four putative splicing mutations (621+3A-->G, 2751+2T-->A, 296+1G-->C, 1717-9T-->C-D565G) and one nonsense mutation (E822X) in the CFTR gene.

The effects of four splicing mutations and one nonsense mutation on cystic fibrosis transmembrane conductance regulator ( CFTR) gene expression were investigated by reverse transcription-polymerase chain reaction analysis of mRNA extracted from nasal epithelial cells harvested from patients harbouring the mutations. We studied four subjects with 621+3A-->G, two with 2751+2T-->A, one with 296+1G-->C, two with 1717-9T-->C-D565G and seven with E822X and compared the results with CFTR mRNA from normal subjects. Our results showed that mutations 621+3A-->G, 2751+2T-->A, and 296+1G-->C, which disrupt the 5' splice donor sites of introns 4, 14a, and 2, respectively, and 1717-9T-->C-D565G, which possibly disrupts the exonic splicing enhancer sequences of exon 12 (owing to the missense mutation in cis), lead to the production of aberrantly spliced mRNA in nasal epithelial cells. Three of the splicing mutations (621+3A-->G, 2751+2T-->A, and 296+1G-->C) result in severe deficiency of normal CFTR mRNA and severe phenotype in the patients. This information is especially useful for mutation 621+3A-->G, which is found in other populations as well, and was initially reported as a polymorphism. The complex allele 1717-9T-->C-D565G results in aberrant splicing of CFTR mRNA with production of transcripts lacking exon 12 (major product), with minor amounts of transcripts revealing joint exon 11 and 12 skipping. Nonsense mutation E822X results in a severe reduction in mRNA levels to about 6% of wild type. Patients with the mutation have a severe clinical phenotype, with both the pancreatic and the pulmonary function affected.

Plasma immunoreactive endothelin levels in children with cystic fibrosis.

UNLABELLED: Plasma immunoreactive endothelin levels were determined in 31 children and adolescents with cystic fibrosis and it was examined whether these levels correlated with the severity of the disease. The study comprised 16 cystic fibrosis patients (mean (SD) age 13.0 (4.9) y) with impaired lung function (Group A), 15 cystic fibrosis patients (11.2 (5.5) y) with unimpaired lung function (Group B) and 28 healthy controls (10.6 (4.3) y) (Group C). The selection and classification of patients into groups was based on criteria including the grade of finger-clubbing, the Brasfield chest radiograph score and spirometric and arterial blood gas values. In all subjects, plasma immunoreactive endothelin, atrial natriuretic peptide, renin, serum aldosterone levels and serum and urine electrolytes were measured. CONCLUSIONS: Plasma endothelin levels were significantly higher in Group A (range 2.5-8.4 pg/ml, median 3.2 pg/ml) than those in Group B (1.3-3.8 pg/ml, median 2.0 pg/ml, p < 0.001) and Group C (1.5-3.5 pg/ml, median 2.5 pg/ml, p < 0.001), whereas they did not differ between groups B and C. They correlated positively with the severity of finger-clubbing, heart rate, arterial blood PCO2, plasma atrial natriuretic peptide levels and serum aldosterone levels and negatively with the arterial blood PO2, forced vital capacity (FVC), forced expiratory volume in 1 sec (FEV1) and the Brasfield chest radiograph score. In multivariate regression analysis PO2 was the only independent factor found to significantly affect plasma endothelin levels. In conclusion, plasma immunoreactive endothelin levels are increased in cystic fibrosis patients with impaired pulmonary function and are related to the severity of the disease.

Characterization of more than 85% of cystic fibrosis alleles in the Greek population, including five novel mutations.

To completely characterize the spectrum of mutations in the cystic fibrosis transmembrane conductance regulator gene in Greek cystic fibrosis (CF) patients, we screened 500 CF chromosomes by denaturing gradient gel electrophoresis followed by direct sequencing. We identified 48 mutations, accounting for 85.6% of CF chromosomes. They included eight novel mutations, three of which we have described before and five (E822X, Y247X, 2752-26A-->G, 3152delT, and 2751 + T-->A), which are described in this report. The detection of such a high proportion of Greek CF mutations is important for improving prenatal and genetic diagnosis of CF in Greece.

Mutation analysis of ten exons of the CFTR gene in Greek cystic fibrosis patients: characterization of 74.5% of CF alleles including one novel mutation.

To initiate the complete characterization of mutations in the CFTR gene in Greek cystic fibrosis (CF) patients, we screened 184 patients for six relatively common mutations (delta F 508, G542X, G551D, 621 + 1 G-->T, N1303K, W1282X) using allele-specific hybridization and, in addition, analyzed exons 4, 5, 7, 8, 10, 11, 17b, 19, 20 and 21 using the method of denaturing gradient gel electrophoresis (DGGE). Six mutations accounted for 65.9% of the CF alleles in Greek patients, of which the delta F 508 mutation had a frequency of 52.7%. A further 15 previously described mutations accounted for another 8.3% CF alleles and one previously undescribed mutation (3272-4A-->G) was found in one chromosome. The W1282X mutation was not detected at all. Thus, so far, we have identified 21 mutations in the CFTR gene in Greek CF patients, accounting for 74.5% of the CF alleles.

Identification of two novel mutations (296 + 1G-C and A46D) in exon 2 of the CFTR gene in Greek cystic fibrosis patients.

Two novel CFTR mutations were detected in Greek cystic fibrosis patients. One was a missense mutation, A46D, and the other a splice mutation, 296 + 1G-C. Neither was detected on normal chromosomes.