ABSTRACT
Since 2001 the Istituto Superiore di Sanità established a quality assurance programme for molecular genetic testing that covers four pathologies: Cystic Fibrosis (CF), Beta Thalassemia (BT), Fragile X Syndrome (FX), and Familial Adenomatous Polyposis Coli (APC). Since 2009 this activity is an institutional activity and participation is open to both public and private laboratories. Seven rounds have been performed until now and the eighth is in progress. Laboratories receive 4 DNA samples with mock clinical indications. They analyze the samples using their routine procedures. A panel of assessors review the raw data and the reports; all data are managed through a web utility. In 2010 the number of participants was 43, 17, 15, 5 for CF, BT, FX, APC schemes respectively. Genotyping results were correct in 96%, 98.5%, 100%, and 100% of CF, BT, FX, and APC samples, respectively. Interpretation was correct in 74%, 91%, 88%, and 60% of CF, BT, FX, and APC reports, respectively; however in most of them it was not complete but a referral to genetic counseling was given. Reports were satisfactory in more than 60% of samples in all schemes. This work presents the 2010 results in detail comparing our data with those from other European schemes.
Subject(s)
Genetic Diseases, Inborn/genetics , Genetic Testing/standards , National Health Programs/standards , Quality Assurance, Health Care/standards , Female , Genetic Diseases, Inborn/diagnosis , Genetic Testing/methods , Humans , Italy , Male , National Health Programs/organization & administration , Quality Assurance, Health Care/organization & administrationABSTRACT
BACKGROUND: The Italian external quality assessment scheme in classical cytogenetics was started in 2001 as an activity funded by the National Health System and coordinated by the Italian Public Institute of Health. OBJECTIVES: The aim of our work is to present data from the first 4 years of activity, 2001-2004. METHODS: Italian cytogenetics public laboratories were enrolled on a voluntary basis, and this nationwide program covered prenatal, postnatal and oncological diagnosis. The scheme is annual and retrospective; a panel of experts reviewed the quality of images and reports in order to assess technical, analytical and interpretative performance. RESULTS: Over the 4-year period, the number of participating laboratories increased: from 36 in 2001, 46 in 2002, 49 in 2003 to 51 in 2004. The overall technical performance was satisfactory. Inadequacy or lack of information in reporting was the most frequent analytical inaccuracy identified in all parts of the scheme. However, the percentage of complete reports increased significantly during the period: by 36% in postnatal diagnosis between 2001 and 2004 (p < 0.001) and by 42% in oncological diagnosis between 2002 and 2004 (p = 0.003). CONCLUSIONS: Our experience reveals that participation in external quality assessment programs has significant advantages, helping to standardize and to assure quality in cytogenetic testing.
Subject(s)
Cytogenetic Analysis/methods , Cytogenetic Analysis/standards , Genetic Testing , Molecular Diagnostic Techniques/methods , Molecular Diagnostic Techniques/standards , Neoplasms/diagnosis , Quality Assurance, Health Care , Genotype , Humans , Italy , Neoplasms/genetics , Prenatal Diagnosis , Time FactorsSubject(s)
5' Flanking Region/genetics , Eye Proteins/genetics , Membrane Proteins/genetics , Polymorphism, Genetic , Chromosomes, Human, Pair 17 , CpG Islands , DNA Primers , Gene Frequency , Genotype , Humans , Molecular Epidemiology , Period Circadian Proteins , Polymerase Chain Reaction , R-SNARE Proteins , Sequence DeletionABSTRACT
Biological circadian clocks are endogenous self-sustaining oscillators, where periodically expressed genes control functions at all levels of biological organization. These mechanisms are detectable from prokaryotes to humans, and their basic molecular components are common in most living organisms. This review focuses on the basic properties of biological circadian clocks and their possible involvement in human diseases.
Subject(s)
Biological Clocks/physiology , Circadian Rhythm/physiology , Animals , HumansABSTRACT
We describe the complete sequence, genomic organization, and FISH chromosome mapping of the human VAMP2. We identified a 7-kb clone, pISSHG2b3A, containing the entire structure of VAMP2. Previous studies performed by others identified a 5-kb clone, pVPC5-2, containing the incomplete VAMP2. The pVPC5-2 clone was partially sequenced and mapped to the broad region 17pter-->p12 by somatic cell hybridization. Our clone overlaps the pVPC5-2 clone and extends approximately 2 kb at the 3' end. In this study, we mapped this gene more precisely on 17p12 by FISH and we found a new polymorphic microsatellite, (GT)(7)CC(GT)(5), in exon V. This microsatellite, revealing three alleles with frequencies of 0.778, 0.139, and 0.083, might be useful for future linkage studies. Finally, we localized three previously known markers, stSG12859, TIGR-A002F11, and WIAF-1699 (alias stSG4044), in the 3' untranslated region of the gene.
Subject(s)
Chromosomes, Human, Pair 17/genetics , Genes/genetics , Membrane Proteins/genetics , Base Sequence , Chromosome Mapping , DNA/chemistry , DNA/genetics , Exons , Humans , In Situ Hybridization, Fluorescence , Introns , Microsatellite Repeats , Molecular Sequence Data , Polymorphism, Genetic , R-SNARE Proteins , Sequence Analysis, DNAABSTRACT
Per genes encode components of the circadian clocks controlling metabolic and behavioural rhythms. The human Per1 cDNA, RIGUI, was previously isolated and mapped on chromosome 17p12 (Sun, Z.S., Albrecht, U., Zhuchenko, O., Bailey, J., Eichele, G., Lee, C.C., 1997. RIGUI, a putative mammalian orthologue of the Drosophila period gene. Cell 90, 1003-1011). We have now isolated the entire genomic locus containing the human Per1 gene, in a search for genes associated with CpG-rich sequences. The hPer1 gene spans 15kb of human genomic DNA and is composed of 23 exons, flanked by 5' and 3' regulatory regions. Comparison of the hPer1 genomic clone with the dbEST database revealed homologies with putative alternative transcripts. Functional mapping within the 5' CpG-rich regulatory region enabled us to locate the hPer1 promoter core in a 510bp-long sequence centred around a TATA box, which supports high levels of hPer1 transcription. A second regulatory region was formally identified in intron 1, which appears to exert a negative role in transcriptional control of hPer1. These regions may be differentially involved in tissue-specificity, and/or circadian regulation, of the human hPer1 gene transcription.
Subject(s)
Genes/genetics , Nuclear Proteins/genetics , Promoter Regions, Genetic/genetics , 3T3 Cells , Animals , Base Sequence , Cell Cycle Proteins , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , Chromosome Mapping , Chromosomes, Human, Pair 17/genetics , Cloning, Molecular , DNA/chemistry , DNA/genetics , DNA/isolation & purification , Drosophila Proteins , Exons , Humans , In Situ Hybridization, Fluorescence , Introns , Mice , Molecular Sequence Data , Period Circadian Proteins , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Analysis, DNA , Transcription, GeneticABSTRACT
Multidrug-resistant Vibrio cholerae O1 El Tor strains isolated during the 1994 outbreak of cholera in Albania and Italy were characterized for the molecular basis of antibiotic resistance. All strains were found to be resistant to tetracycline, streptomycin, spectinomycin, trimethoprim, sulfathiazole, and the vibriostatic compound O/129 (2,4-diamino-6,7-diisopropylteridine). Resistance genes were self-transferable by a conjugative plasmid of about 60 MDa, with the exception of spectinomycin resistance, which was conferred by the aadA1 gene cassette located in the bacterial chromosome within a class 1 integron. The resistance to trimethoprim and O/129 was conferred by the dfrA1 gene, which was present on the plasmid. Although the dfrA1 gene is known to be borne on an integron cassette, class 1, 2, or 3 intI genes were not detected as part of the plasmid DNA from the strains studied.
Subject(s)
Conjugation, Genetic , Drug Resistance, Microbial/genetics , Vibrio cholerae/drug effects , Vibrio cholerae/genetics , Albania , Blotting, Southern , Drug Resistance, Multiple , Italy , Microbial Sensitivity Tests , Plasmids , Vibrio cholerae/isolation & purificationABSTRACT
The uropathogenic Escherichia coli strain J96 (04:K6) is able to produce four adherence factors [P-fimbriae (pap and prs), F1C-fimbriae (foc) and Type 1-fimbriae (fim)], two alpha-hemolysins (hlyI and II) and the cytotoxic necrotizing factor type 1 (cnf1). Using phenotypic test systems and genotypic analysis, it has been shown that the mutant strain J96-M1 has lost the hlyII, prs and cnf1 genes. The three virulence associated determinants are linked on one particular region on the chromosome, which is termed 'pathogenicity island II' (Pai II).
Subject(s)
Adhesins, Bacterial/genetics , Bacterial Toxins/genetics , Cytotoxins/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Hemolysin Proteins/genetics , Chromosome Mapping , Multigene Family , MutationABSTRACT
One hundred and sixty-eight isolates of Escherichia coli obtained in Italy from 112 children with diarrhoea and 56 age-matched controls were examined by the HEp-2 cell adhesion assay. Sixteen strains showed localised adherence (LA), 29 showed diffuse adherence (DA) and eight strains showed aggregative adherence (AA). No adhesion pattern was significantly associated with disease. Strains that showed LA or AA were further characterised by serotyping, fluorescent actin staining (FAS) test and hybridisation with the EPEC adherence factor (EAF), E. coli attaching and effacing (eae) and enteroaggregative (EAgg) DNA probes. Strains that showed poor LA were FAS-negative, did not belong to EPEC serotypes and did not hybridise with EPEC probes. Conversely, the two strains that showed a good LA pattern belonged to serotype O128:H2, were FAS positive and hybridised with the eae probe. No isolate hybridised with the EAF probe. Only three of the eight strains with the AA pattern hybridised with the EAgg probe. Probe positivity correlated with the ability to produce clumps at the surface of the liquid culture and to agglutinate rat erythrocytes. In two of these EAgg probe-positive strains, electronmicroscopy revealed the presence of fibrillar bundles which seem to mediate bacterial aggregation.
Subject(s)
Bacterial Adhesion , Diarrhea, Infantile/microbiology , Diarrhea/microbiology , Escherichia coli Infections/microbiology , Escherichia coli/isolation & purification , Cell Line , Child , Child, Preschool , DNA Probes , Escherichia coli/metabolism , Escherichia coli/ultrastructure , Feces/microbiology , Humans , Infant , Italy , Microscopy, Electron , Microscopy, Electron, ScanningSubject(s)
Diarrhea/veterinary , Escherichia coli Infections/veterinary , Escherichia coli Proteins , Escherichia coli/pathogenicity , Horse Diseases/microbiology , Rabbits/microbiology , Animals , Bacterial Toxins/biosynthesis , Cytotoxins/biosynthesis , Diarrhea/epidemiology , Diarrhea/microbiology , Disease Outbreaks/veterinary , Escherichia coli/isolation & purification , Escherichia coli Infections/epidemiology , Escherichia coli Infections/microbiology , Horse Diseases/epidemiology , HorsesABSTRACT
Cytotoxic necrotizing factors (CNFs) are dermonecrotic protein toxins produced by human and animal clinical isolates of Escherichia coli. In this study, the CNF1 determinant was isolated and sequenced, showing that expression of biologically active toxin is governed by a unique open reading frame encoding a protein of 1,014 amino acids with a predicted molecular mass of 113.7 kDa. Nucleotide and protein data base searches showed significant homology between CNF1 and the dermonecrotic toxin of Pasteurella multocida. In particular, the two toxins were found to share a hydrophobic region of about 220 amino acids which is a potential membrane-spanning domain.
Subject(s)
Bacterial Proteins , Bacterial Toxins/genetics , Cytotoxins/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Genes, Bacterial , Amino Acid Sequence , Bacterial Toxins/chemistry , Base Sequence , Cytotoxins/chemistry , Escherichia coli/pathogenicity , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
Cytotoxic necrotizing factors (CNFs) are Escherichia coli protein toxins causing cell multinucleation and enlargement in tissue cultures and necrosis in rabbit skin. In E. coli isolates causing urinary tract infections in humans, the production of CNF1 is closely associated with hemolysin production. In this study, we obtained data suggesting that this phenotypic association is due to the genetic linkage of the determinants of the two toxins on the chromosome of uropathogenic E. coli. The genes encoding hemolysin and CNF1 were shown to be closely linked in a 37-kb cloned DNA fragment from an E. coli urinary tract isolate of serotype O4:K12:H5 (E-B35). A DNA region encoding CNF1 production but not hemolysin production was further subcloned as a 12-kb SalI-EcoRI fragment and used as a CNF1-specific gene probe. DNA hybridization experiments indicated that the CNF1 and hemolysin determinants were closely linked on the chromosomes of isolate E-B35 and six additional extraintestinal isolates belonging to serogroups O2, O4, O6, O22, O75, and O85.
Subject(s)
Bacterial Toxins/genetics , Cytotoxins/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Genes, Bacterial , Hemolysin Proteins/genetics , Bacterial Toxins/immunology , Chromosome Mapping , Cloning, Molecular , Cytotoxins/immunology , Escherichia coli/pathogenicity , Escherichia coli Infections/microbiology , Humans , VirulenceABSTRACT
Escherichia coli strains isolated from 81 fecal samples from red deer (Cervus elaphus), roe deer (Capreoulus capreoulus), chamois (Rupicapra rupicapra) and alpine marmot (Marmota marmota) living in the Stelvio National Park, Italy, were examined for antimicrobial resistance and production of toxic factors. Direct plating of specimens on media containing antimicrobial drugs allowed us to isolate resistant strains of E. coli from 10 of 59 (17%) specimens examined by this technique. Nine of 31 specimens from red deer (29%) contained resistant strains. Different animals were likely colonized by the same resistant strain of E. coli. Conjugative R plasmids were found in four strains isolated from the marmot, roe deer and chamois. A strain from red deer produced heat-stable enterotoxin and another strain produced both hemolysin and cytotoxic necrotizing factor. A marmot isolate produced hemolysin alone. No strains were found to produce heat-labile enterotoxin or verotoxins.
Subject(s)
Animals, Wild/microbiology , Bacterial Toxins/biosynthesis , Escherichia coli/drug effects , Marmota/microbiology , Ruminants/microbiology , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Outer Membrane Proteins/analysis , Deer/microbiology , Drug Resistance, Microbial , Escherichia coli/genetics , Escherichia coli/metabolism , Feces/microbiology , Goats/microbiology , Italy , R FactorsABSTRACT
We examined the relationship between serotype and cytotoxic necrotizing factor (CNF) production in 123 hemolytic strains of Escherichia coli isolated from both stools and extraintestinal infections. Of 76 strains producing both hemolysin (Hly) and CNF, 66 (87%) belonged to one of six serogroups (O2, O4, O6, O22, O75, and O83). In contrast, 47 E. coli strains producing Hly only belonged to 21 different O serogroups, and only 2 of these (O6 and O18ac) were widely represented. Generally, CNF-positive and CNF-negative hemolytic isolates were assigned to different O serogroups, with the exception of O6, often present in both categories of isolates. Serogroups O4 and O18ac were significantly more prevalent among strains from extraintestinal infections than among those from stools. In contrast, the Hly-positive, CNF-negative isolates, belonging to numerous less common serogroups, were hardly ever isolated from extraintestinal infections. Serological typing further confirmed that hemolytic isolates of E. coli may grossly be divided into two main populations on the basis of the ability to produce CNF. Examination of hemolytic E. coli for this property may also be useful in achieving a more detailed characterization of pathogenic clones.
Subject(s)
Bacterial Toxins/biosynthesis , Cytotoxins/biosynthesis , Escherichia coli Proteins , Escherichia coli/pathogenicity , Hemolysin Proteins/biosynthesis , Bacterial Outer Membrane Proteins/analysis , Child , Escherichia coli/classification , Escherichia coli/metabolism , Feces/microbiology , Humans , Serotyping , VirulenceABSTRACT
Nine strains of ETEC isolated in Italy have been compared with 13 isolates from Somalia with respect to toxin production, serotype and antimicrobial resistance pattern. None of the strains isolated from Italy belonged to any serotype or serogroups found among the strains from Somalia. Remarkable differences between the two groups of isolates were also observed with regard to the susceptibility to antimicrobials and the presence of R-plasmids. These findings suggest that ETEC strains isolated in Italy are not related to the strains widespread in Somalia and, generally, in developing countries.
Subject(s)
Bacterial Toxins/biosynthesis , Enterotoxins/biosynthesis , Escherichia coli Proteins , Escherichia coli/classification , Fimbriae Proteins , Antigens, Bacterial/analysis , Drug Resistance, Microbial , Escherichia coli/drug effects , Escherichia coli/immunology , Escherichia coli/metabolism , Hemagglutination Tests , Italy , Serotyping , SomaliaABSTRACT
The effect of the cytotoxic necrotizing factor of Escherichia coli on HEp-2 cells was studied by fluorescence and scanning electron microscopy. This cytotoxin, known for inducing the formation of giant multinucleated cells in several cell lines, caused changes in actin and tubulin organization. The presence of membrane ruffles at the cell border and of numerous thick bundles of actin crossing the cell body, suggests that the factor promotes cell spreading; this probably interferes with cytokinesis, ultimately leading to the formation of very large flattened multinucleated cells. Moreover, the nuclear segmentation observed in treated cells seems to be associated with a rearrangement of actin in the perinuclear region and with the presence of tubulin bundles in proximity to nuclear clefts. Although the primary target is still unknown, these findings suggest that the cytoskeleton is affected accounting for the multinucleation process induced by the factor.
Subject(s)
Bacterial Toxins/toxicity , Cytoskeleton/drug effects , Cytotoxins/toxicity , Escherichia coli Proteins , Escherichia coli/metabolism , Cell Line , Fluorescent Antibody Technique , HumansABSTRACT
A hospital-based systematic sample of 1667 children with severe diarrhoeal disease was studied in Mogadishu, Somalia, throughout 1983 and 1984. One or more enteric pathogens were found in 61% of the patients. Rotavirus (25%), enterotoxigenic Escherichia coli (11%), Shigella spp. (9%), Aeromonas hydrophila (9%), Giardia lamblia trophozoites (8%), Campylobacter jejuni (8%), and Vibrio cholerae non-O1 (6%) were the most frequently identified pathogens. Age-specific detection rates of enteric pathogens and helminths, seasonal patterns, and relationship of some specific infections with feeding status and main clinical features have been defined for all the sample examined.
Subject(s)
Diarrhea/microbiology , Adolescent , Age Factors , Animals , Campylobacter Infections/microbiology , Child , Child, Preschool , Diarrhea/parasitology , Diarrhea, Infantile/microbiology , Diarrhea, Infantile/parasitology , Dysentery, Bacillary/microbiology , Escherichia coli Infections/microbiology , Giardiasis/parasitology , Humans , Infant , Infant, Newborn , Rotavirus Infections/microbiology , Seasons , Somalia , Vibrio Infections/microbiologyABSTRACT
Two hundred and nineteen strains of Escherichia coli from extraintestinal infections and feces of healthy subjects were examined for hemolysin (Hly) and cytotoxic necrotizing factor (CNF) production and for mannose-resistant hemagglutination. Of 105 strains from extraintestinal infections, 42 (40.0%) were positive for production of both Hly and CNF, and 21 (20.0%) were positive for Hly alone; on the contrary, only 1 Hly- and CNF-positive strain and 2 Hly-positive strains were found among 114 strains from normal stools. CNF production was not found to occur among the nonhemolytic strains, confirming the close association existing between these toxic factors. Hemolytic strains positive for CNF showed mannose-resistant hemagglutination less frequently than did Hly-positive, CNF-negative strains (25.6 versus 82.6%), suggesting the existence of two distinct classes among hemolytic strains of E. coli.
Subject(s)
Cytotoxins/biosynthesis , Escherichia coli Infections/microbiology , Escherichia coli/metabolism , Hemolysin Proteins/biosynthesis , Feces/microbiology , Hemagglutination , HumansABSTRACT
A cell-culture assay was used to detect toxins directly in stools from sporadic cases of infantile diarrhoea. Cytotoxins were revealed in 11 out of 58 samples from children with diarrhoea, nine of whom had no common enteric pathogens in their stools. A preliminary characterisation of the cytotoxins was obtained by neutralisation tests with clostridial antitoxins.
Subject(s)
Bacterial Proteins , Bacterial Toxins/analysis , Clostridium , Cytotoxins/analysis , Diarrhea, Infantile/etiology , Feces/analysis , Child, Preschool , Humans , Infant , Infant, NewbornABSTRACT
The dissemination of a gentamicin resistant plasmid, originally found in strains of Klebsiella and termed pk181, into the microbial population of patients of the Orvieto Hospital was studied during 1982. Five hundred and seventy-four strains of Gram-negative bacilli were examined, transferable gentamicin resistance being revealed in five different bacterial species. The resistance was shown to be encoded by 81-megadalton plasmids in Escherichia coli and Enterobacter cloacae, and by 93-megadalton plasmids in Serratia marcescens and Pseudomonas spp. Restriction endonuclease digestion of plasmid DNA showed that the fragment patterns of the 81-megadalton plasmids from E. coli and enterobacter cloacae were identical to one another and to the pattern of plasmid pk181. The fragment patterns of the 93-megadalton plasmids from Serratia and Pseudomonas, on the contrary, differed substantially from those of the 81-megadalton plasmids.