Search PubMed⌕ Search

Biomedical subjects

A Deplano

Publications and source records attributed to A Deplano.

At least 19 recordsLinked to original sources
PubMed2006

Validation of pulsed-field gel electrophoresis and spa typing for long-term, nationwide epidemiological surveillance studies of Staphylococcus aureus infections.

Pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments has been used by the Belgian Reference Laboratory for Staphylococci for national hospital surveys of methicillin-resistant Staphylococcus aureus since 1992. The sequencing of the polymorphic X region of the protein A gene (spa typing) offers significant advantages over PFGE in terms of speed, ease of interpretation, and exportability. To validate its potential use for national surveillance, we evaluated the robustness of spa typing compared with that of PFGE based on a collection of 217 S. aureus strains representative of the Belgian S. aureus epidemiology during the last 13 years. spa typing and PFGE both showed high discriminatory power (discriminatory indexes of 0.98 and 0.96, respectively) and achieved high concordance (95.9%) in type classification. Both methods also showed good concordance with multilocus sequence typing (MLST) (95.5%). However, we observed occasional "violations" of MLST clonal complex assignment by spa typing. Our results suggest that both PFGE and spa typing are reliable methods for long-term, nationwide epidemiological surveillance studies. We suggest that spa typing, which is a single-locus-based method, should preferably be used in combination with additional markers, such as staphylococcal cassette chromosome mec typing or resistance or virulence gene detection.

Anti-Bacterial Agents↗
PubMed2006

International dissemination of a high virulence rabbit Staphylococcus aureus clone.

High virulence rabbit Staphylococcus aureus strains cause chronic and spreading problems of mastitis, pododermatitis and subcutaneous abscesses on rabbit flock level, whereas infections with low virulence strains are limited to individual rabbits. In the present report, 13 high virulence rabbit S. aureus strains, selected out of a large collection of strains isolated in five European countries between 1983 and 2004, were genotyped using pulsed-field gel electrophoresis, spa typing, multilocus sequence typing (MLST) and accessory gene regulator (agr) group typing. Two low virulence rabbit S. aureus strains were also included in the study. The results indicate the clonal origin of high virulence rabbit S. aureus strains present in Europe. Furthermore, the results of MLST and spa typing form a basis for international epidemiology of rabbit S. aureus strains, as these DNA sequence-based typing techniques can easily be used for intercentre comparisons.

Animals↗
PubMed2006

New phage type among methicillin-resistant Staphylococcus aureus associated with a local outbreak in Belgium during 2002.

In total, 150 methicillin-resistant Staphylococcus aureus (MRSA) isolates collected during 2002 from a general Belgian hospital were phage-typed at routine test dilution x 100. The majority (45%) belonged to phage group (J)*, while 10% were classified as a new phage type 29/(42E)/54/(D11)*. The isolates belonging to this new type carried the aac(6')-aph(2'') and the aph(3') aminoglycoside resistance genes and showed high-level resistance to oxacillin. Molecular typing revealed that they belonged to the multiresistant clonal pulsed-field gel electrophoresis (PFGE) type D8. PFGE group D, characterised as genotype ST228-MRSA-I, has been present in Belgian hospitals since 1999.

Bacteriophage Typing↗
PubMed2006

External quality assessment of molecular typing of Staphylococcus aureus isolates by a network of laboratories.

A network of laboratories designated Centres for Molecular Diagnosis was funded in 2000 by Belgian National Health Insurance to provide clinically relevant molecular diagnostic tests. These included typing of nosocomial pathogens as a service to local hospital infection control programs. Two external quality assessment (EQA) surveys were performed in 2001 and 2003 to evaluate the proficiencies of the laboratories at Staphylococcus aureus typing. EQA panels included S. aureus isolates with either indistinguishable, clonally related, or unrelated pulsed-field gel electrophoresis (PFGE) patterns. A hypothetical hospital outbreak problem was also submitted for analysis. Typeability, reproducibility, discrimination (D) index, and epidemiological concordance were evaluated. Ten centers participated in each survey. Seven centers performed PFGE analysis, while others used repetitive-element or randomly amplified polymorphic DNA PCR, amplified fragment length polymorphism, or spa typing. Full typeability (100%) was achieved by all centers, and all but one showed 100% reproducibility. Discrimination was appropriate (D index, >or=96%) for centers performing PFGE analysis but not for all those using other methods (D index range, 72% to 97%). Correct answers to the epidemiological questions were provided by 7/10 and 10/10 centers in 2001 and 2003, respectively. Individual feedback of results was provided to each center together with specific technical recommendations for improving performance. Our findings indicate that surveys of lab proficiency are useful for validation and optimization of molecular typing services to local hospital infection control programs.

Bacterial Typing Techniques↗
PubMed2006

High interlaboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study.

Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature.

Bacterial Typing Techniques↗
PubMed2005

Polyclonal emergence and importation of community-acquired methicillin-resistant Staphylococcus aureus strains harbouring Panton-Valentine leucocidin genes in Belgium.

OBJECTIVES: Worldwide spread of a limited number of Panton-Valentine leucocidin (PVL) -producing methicillin-resistant Staphylococcus aureus (MRSA) clones has been reported in various communities. The objective of this study was to describe the molecular characteristics of the first PVL-positive MRSA strains isolated in Belgium. METHODS: Clinical MRSA isolates (n = 41) collected from 2002 to 2004 from Belgian patients were investigated for the PVL gene by PCR. PVL-positive isolates were genotyped by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing, spa sequence typing, accessory gene regulator (agr) polymorphism and multi-locus sequence typing (MLST). Susceptibility to 14 antimicrobials was determined by the disc diffusion method. Genes encoding resistance to tetracyclines, aminoglycosides and macrolide-lincosamide-streptogramin were determined by PCR. RESULTS: Sixteen isolates carried lukS-lukF genes that encode the PVL toxin. All but one isolate were community-acquired. Three patients reported recent travel to North Africa and South America. They were associated with skin or soft tissue infections, bacteraemia and peritonitis. By molecular typing, they belonged to five genotypes: ST80-SCCmec IV, ST8-SCCmec IV, ST30-SCCmec IV, ST153-SCCmec IV and ST88-SCCmec IV. They belonged to the agr type 3 except for ST8 strains, which showed agr type 1. All isolates were susceptible to fluoroquinolones. Approximately, half of them were resistant to tetracycline, fusidic acid and kanamycin. Tetracycline-resistant strains harboured the tet(K) gene and resistance to kanamycin was associated with the aph3'-IIIa gene. The single erythromycin-resistant isolate harboured msr(A/B) genes conferring the M resistance phenotype. CONCLUSIONS: These results indicate the recent emergence and sporadic importation into Belgium of PVL-positive community-associated MRSA strains belonging to five distinct clones.

Adolescent↗
PubMed2005

Staphylococcus aureus carriage among participants at the 13th European Congress of Clinical Microbiology and Infectious Diseases.

The aim of this study was to measure the rate of Staphylococcus aureus nasal colonization among attendees of the 13th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), particularly with regard to methicillin-resistant (MRSA) strains. The 31.4% rate of Staphylococcus aureus colonization detected among the participants was in line with colonization rates reported previously for healthcare workers. A statistical difference was found between the rates of Staphylococcus aureus carriage in physicians (37.4%) and non-physicians (21.7%) but not between males (35.0%) and females (28.9%). Only one participant (a Belgian physician) was found to carry MRSA. Surprisingly, the rate of methicillin-susceptible Staphylococcus aureus carriage was significantly higher among participants from countries with a low prevalence of MRSA.

Carrier State↗
PubMed2005

Molecular characterization of an epidemic clone of panantibiotic-resistant Pseudomonas aeruginosa.

We describe the molecular characterization of a multiresistant Pseudomonas aeruginosa clone causing an outbreak in the intensive care unit (ICU) of a tertiary-care university hospital. Analysis included antimicrobial susceptibility profile, O-serotyping, pulsed-field gel electrophoresis, and amplified fragment length polymorphism. Resistance mechanisms were characterized, including production of naturally occurring and acquired beta-lactamases, porin expression, and efflux pump systems. Eighteen patients were colonized or infected with multiresistant P. aeruginosa. Multiresistant P. aeruginosa was panresistant to penicillins, cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones and remained susceptible only to colistin. Sixteen isolates (89%) belonged to serotype O:11, pulsed-field gel electrophoresis type A1, and amplified fragment length polymorphism type A. Resistance characterization of this epidemic clone showed an overexpression of the chromosomal cephalosporinase AmpC combined with decreased expression of porin OprD and the absence of metallo-beta-lactamase or extended-spectrum beta-lactamase. An upregulation of the MexXY efflux system due to an agrZ mutation in the mexZ repressor was detected. This epidemic clone was restricted to the ICU and was not found elsewhere in hospital. Contamination of the ICU environment and the hands of an ICU nurse with this clone suggests possible hand-borne transmission. Implementation of contact precautions effectively controlled transmission of the epidemic clone. This study illustrates the ability of multiresistant P. aeruginosa to cause an outbreak with significant morbidity and mortality and underscores the need to identify clonal outbreaks, which require targeted infection control measures.

Cross Infection↗
PubMed2004

Management of long-term catheter-related Brevibacterium bacteraemia.

Brevibacterium has been reported as a rare cause of implanted-device infection. In two cases of recurrent Brevibacterium casei bacteraemia associated with infection of surgically implanted intravascular devices, relapse occurred 2 and 5 months, respectively, after completion of therapy with vancomycin via the infected catheter. A second intravenous antibiotic therapy course by the antibiotic-lock technique led to bacteriological cure in one patient. Molecular typing results demonstrated that the recurrent bacteraemia was caused by the same strain. Implanted-device removal may be necessary, in addition to appropriate antibiotics, for successful management of such infections.

Actinomycetales Infections↗
PubMed2001

Epidemiological validation of pulsed-field gel electrophoresis patterns for methicillin-resistant Staphylococcus aureus.

To determine the stability of pulsed-field gel electrophoresis (PFGE) patterns of methicillin-resistant Staphylococcus aureus in the nosocomial setting, we analyzed isolates from long-term carriers (>1 month) and from patients involved in well-defined nosocomial epidemics. The number of fragment differences between the first isolate and subsequent isolates in long-term carriers showed a bimodal distribution, with one group having 0 to 6 fragment differences and the other group having 14 to 24 fragment differences. The PFGE patterns of isolates involved in epidemics also presented a similar bimodal distribution of the number of fragment differences. Typing these isolates with another molecular method (inter-IS256 PCR) showed that isolates of the first group (i.e., with 1 to 6 fragment differences) were clonally related, whereas the second group (with 14 to 24 fragment differences) could be considered genetically different. Among long-term carriers with clonally related isolates, 74 of 84 (88%) of consecutive isolates showed indistinguishable patterns, whereas 10 of 84 (12%) showed related patterns differing by one to six fragments. Moreover, the frequency of apparition of related patterns is higher when the time between the first and the subsequent isolate is longer. During seven nosocomial epidemics lasting from 1 to 15 months, only 2 of 120 isolates (1.7%) showed a pattern which was different, although related, from the predominant one involved in each of these outbreaks.

Bacterial Typing Techniques↗
PubMed2000

Polyclonal Staphylococcal endocarditis caused by genetic variability.

Cultures of blood obtained from a patient with Staphylococcus epidermidis prosthetic valve endocarditis yielded 15 strains of S. epidermidis. Genome macrorestriction and amplified fragment-length polymorphism analyses of these strains showed that they belonged to 4 different, very closely related clones, suggesting that they were the result of genetic variability of an infecting strain during the infectious episode. In vivo experiments in a rat model for foreign body infections using 1 of the S. epidermidis strains from the patient showed genetic variability similar to that of the infecting strain. In the rat model, we also detected the simultaneous presence of different clones that were identical to those isolated from our patient, thus confirming the possibility of genetic variability. It is important to note that the 4 clones isolated from our patient presented with 2 different antibiograms. Therefore, in cases of foreign device-related infections due to coagulase-negative staphylococci, the possibility of polyclonal infection has to be taken into account, particularly as regards differences in antibiotic susceptibility.

Aged↗
PubMed2000

Ommaya-catheter-related Staphylococcus epidermidis cerebritis and recurrent bacteremia documented by molecular typing.

A 26-year-old woman receiving intrathecal chemotherapy for acute leukemia developed Ommaya-catheter-associated cerebritis and bacteremia caused by two clones of Staphylococcus epidermidis. Genomic fingerprinting of 19 staphylococcal isolates from the cerebrospinal fluid, blood, catheter and skull biopsy was necessary to establish the etiologic diagnosis and to guide medical and surgical therapy.

Adult↗
PubMed2000

Clonal dissemination of epidemic methicillin-resistant Staphylococcus aureus in Belgium and neighboring countries.

OBJECTIVES: To determine the diversity of pulsed-field gel electrophoresis (PFGE) types among epidemic strains of methicillin-resistant Staphylococcus aureus (MRSA) recovered in Belgium, France, Germany and The Netherlands over the period 1981-94. METHODS: MRSA strains collected in a multicenter survey in Belgium (n = 171) and from reference laboratories in neighboring countries (n = 102) were characterized by PFGE analysis using the SmaI enzyme. RESULTS: In total, 32 PFGE types were found. Epidemic PFGE type 1, first recognized in 1984, accounted for 82% of Belgian strains (87% of hospitals) and 51% of European MRSA strains. Four other internationally epidemic PFGE types (types 8, 10, 11 and 12) were less widely disseminated and more recently detected (1991-94), each recovered from two or three countries. International spread of two PFGE types was linked to transfer of colonized patients to Dutch hospitals from another country where this type was frequently recovered. CONCLUSIONS: Genotypic analysis indicated widespread distribution of several outbreak-associated MRSA strains over large European regions, which was in some instances related to interhospital patient transfer. These findings underscore the need for standardized international surveillance and control of MRSA transmission between healthcare institutions across Europe.

Bacteriophage Typing↗
PubMed2000

In vivo deletion of the methicillin resistance mec region from the chromosome of Staphylococcus aureus strains.

Two sets of Staphylococcus aureus isolates recovered from two patients exhibited similar susceptibility profiles except for oxacillin susceptibility (MSSA) or resistance (MRSA). SMA:I macrorestriction and inter-IS256 PCR analysis showed patterns closely related to the Belgian epidemic MRSA clone 1 in each pair of MSSA/MRSA strains. Loss of one large SMA:I DNA fragment and concurrent gain of a smaller fragment in the MSSA isolates was observed. The mecA sequence present in the MRSA was absent in the MSSA variant. Therefore, in vivo deletion of the mec region may occur in some lineages of S. aureus more frequently than previously thought.

Bacterial Proteins↗
PubMed2000

Multicenter evaluation of epidemiological typing of methicillin-resistant Staphylococcus aureus strains by repetitive-element PCR analysis. The European Study Group on Epidemiological Markers of the ESCMID.

Rapid and efficient epidemiologic typing systems would be useful to monitor transmission of methicillin-resistant Staphylococcus aureus (MRSA) at both local and interregional levels. To evaluate the intralaboratory performance and interlaboratory reproducibility of three recently developed repeat-element PCR (rep-PCR) methods for the typing of MRSA, 50 MRSA strains characterized by pulsed-field gel electrophoresis (PFGE) (SmaI) analysis and epidemiological data were blindly typed by inter-IS256, 16S-23S ribosomal DNA (rDNA), and MP3 PCR in 12 laboratories in eight countries using standard reagents and protocols. Performance of typing was defined by reproducibility (R), discriminatory power (D), and agreement with PFGE analysis. Interlaboratory reproducibility of pattern and type classification was assessed visually and using gel analysis software. Each typing method showed a different performance level in each center. In the center performing best with each method, inter-IS256 PCR typing achieved R = 100% and D = 100%; 16S-23S rDNA PCR, R = 100% and D = 82%; and MP3 PCR, R = 80% and D = 83%. Concordance between rep-PCR type and PFGE type ranged by center: 70 to 90% for inter-IS256 PCR, 44 to 57% for 16S-23S rDNA PCR, and 53 to 54% for MP3 PCR analysis. In conclusion, the performance of inter-IS256 PCR typing was similar to that of PFGE analysis in some but not all centers, whereas other rep-PCR protocols showed lower discrimination and intralaboratory reproducibility. None of these assays, however, was sufficiently reproducible for interlaboratory exchange of data.

Bacterial Typing Techniques↗
PubMed1999

Molecular characterization of wilson disease in the Sardinian population--evidence of a founder effect.

Wilson disease (WD) in the Sardinian population has an approximate incidence of 1:7,000 live births. Mutation analysis of the WD gene in this population reported in our previous articles led us to the characterization of two common mutations and a group of 13 rare mutations accounting for the molecular defect of 8.5, 7.9, and 15.1% of the WD chromosomes. However, molecular analysis of the WD chromosomes containing the most common haplotype, which accounts for 60.5% of the WD chromosomes, failed to define the disease-causing mutation. In this study, we characterized the promoter and the 5' UTR of the WD gene sequence and carried out a mutation analysis in this DNA region from patients with the most common haplotype. The promoter is contained in a GC-rich island and shows a TATA and a CAAT consensus sequence as well as potential binding sites for transcription factors and metal response elements. In all the analyzed 92 chromosomes with this haplotype, we detected a single mutation consisting of a 15-nt deletion from position -441 to position -427 relative to the translation start site. Expression assays demonstrated a 75% reduction in the transcriptional activity of the mutated sequence compared to the normal control. By adding this mutation to those that have been already characterized, we have now defined the molecular defect in 92% of the WD chromosomes in Sardinians. The high frequency, the expected prevention by preclinical diagnosis and early treatment of the devastating effect of WD on the nervous system and liver tissue, and the feasibility to detect most of molecular defects by DNA analysis indicate that WD in the Sardinian population should be added to the list of diseases currently detected by newborn screening.

5' Untranslated Regions↗