Reflections and proposals to assure quality in molecular diagnostics.

Molecular diagnostic testing has become an important tool in clinical laboratories. Accreditation according to the international quality standard ISO15189:2007 for medical laboratories is required for reimbursement of several molecular diagnostic tests in Belgium. Since the ISO15189:2007 standard applies to medical laboratories in general, the particular requirements for quality and competence are mentioned in general terms, not taking into account the specificities of molecular biology testing. Therefore, the working group "MolecularDiagnostics.be" described a consensus interpretation of chapter 5, Technical requirements, of the ISO standard for application in molecular diagnostic laboratories. The manuscript can be used as an instrument to prepare internal and external audits that meet the 15015189:2007 (chapter 5) criteria.

Contribution of two molecular assays as compared to selective culture for MRSA screening in a low MRSA prevalence population.

BACKGROUND: As the prompt detection of methicillin-resistant Staphylococcus aureus (MRSA) carriers upon admission is fundamental in the MRSA prevention strategy of our hospital, the infection control team is eagerly seeking the most sensitive and rapid screening method. The aim of this study was to compare the performance of two molecular techniques with a conventional MRSA-selective culture test (Bio-Rad chromogenic MRSASelect) in order to elucidate the suitability of the assays specifically in an expected low MRSA prevalence population. PATIENTS AND METHODS: The anterior nares and throat of 500 patients and visitors attending the emergency department of Sint-Jan General Hospital between May and June 2007 were sampled, and MRSA carriage was determined by selective culture after enrichment and the BD GeneOhm StaphSR and the Cepheid Xpert MRSA assays. RESULTS: Eight MRSA carriers were detected by selective culture (1.6% prevalence). The sensitivity, specificity, positive [corrected] predictive value, and negative [corrected] predictive value were 62.5, 99.0, 50.0, and 99.4% for BD GeneOhm StaphSR and 62.5, 97.7, 31.3, and 99.4% for Cepheid Xpert MRSA, respectively. CONCLUSIONS: We conclude that MRSA rapid screening techniques must be interpreted cautiously in a low-prevalence population, as the sensitivity is lower than in selected high-risk populations. MRSA carriers detected with molecular techniques must be confirmed by conventional culture methods for follow-up. The specificity and negative predictive value indicate that molecular rapid methods are worthwhile to be considered in MRSA-preventive strategies.

[Liver transplantation for subcapsular haematoma during HELLP Syndrome]. / Transplantation hépatique pour hématome sous-capsulaire rompu lors d'un HELLP Syndrome.

The hepatic rupture of a subcapsular haematoma during HELLP syndrome is a rare complication carrying a high mortality. There is no clear guideline management in the literature. We report here a case of a subcapsular haematoma which required liver transplantation.

Differentiation and identification of Enterococcus durans, E. hirae and E. villorum.

AIMS: To compare different tests in the identification of Enterococcus durans, E. hirae and E. villorum strains. These bacteria belong to the E. faecium species group and are phylogenetically closely related, as evidenced by 16S rRNA sequence homologies of over 98.8%. METHODS AND RESULTS: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of whole-cell protein, tRNA interpacer polymerase chain reaction (PCR) and arbitrarily-primed (D11344-primed AP) -PCR analysis correctly identified all three species in a collection of strains from very diverse origins. In contrast, biochemical reactions only allowed the unequivocal differentiation of the three species as a group from the other enterococci. Within this group, D-xylose acidification can be used to differentiate E. villorum, but exceptions occur. Strains highly susceptible to clindamycin can be identified as E. durans, but many strains of this species cannot be differentiated from E. hirae and E. villorum due to acquired resistance. CONCLUSIONS: Despite their close relationship, E. durans, E. hirae and E. villorum can be differentiated by genomic methods and by whole-cell protein analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Only a minority of strains of these three enterococcal species can be identified reliably by the currently available and commonly applied phenotypic tests.

Enterococcus villorum sp. nov., an enteroadherent bacterium associated with diarrhoea in piglets.

The taxonomic positions of five enteroadherent bacterial pig isolates, showing phenotypic characteristics most similar to those of Enterococcus durans and Enterococcus hirae, were investigated in a polyphasic study that included 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base-ratio determinations, whole-cell protein fingerprinting, D11344-primed PCR typing and an extensive examination of phenotypic properties. The results demonstrated that the organisms represent a new species in the Enterococcus faecium species group, for which the name Enterococcus villorum sp. nov. is proposed. The type strain is LMG 12287T (= CCM 4887T).

Molecular characterisation of group A streptococci from invasive and non-invasive disease episodes in Belgium during 1993-1994.

Five hundred clinical group A streptococcal (GAS) isolates were collected in Belgium during the period 1 Nov. 1993 to 31 Oct. 1994. Clinical and laboratory data were recorded and isolates were characterised. The presence of the genes encoding streptococcal pyrogenic exotoxin types A (speA), B (speB), C (speC), F (speF) and streptococcal superantigen (ssa) were determined by PCR to target specific sequences. These isolates were also emm-typed and analysed by pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments with the enzyme SmaI. In total, 136 unrelated GAS PFGE types were identified and genetic diversity was clearly demonstrated. Two GAS PFGE types predominated; a first PFGE type comprised 66 (13.2%) emm1 isolates characterised by speA-, speB+, speC-, speF+ and ssa-; the second PFGE type comprised 44 (8.8%) emm12 isolates characterised by speA-, speB+, speC+ (or speC-), speF+ and ssa-. Indistinguishable PFGE types were observed among both invasive and non-invasive isolates. Ten different PFGE types were found among 11 streptococcal toxic shock syndrome (STSS) isolates, and five of these lacked speA. Twenty-five (34.7%) of 72 invasive isolates gave negative results for speA, speC and ssa. This retrospective study confirmed the observation that the dissemination of one specific clone cannot be associated with invasive GAS disease and posed a question regarding the role of SPE A as a major virulence factor. Other streptococcal virulence factors in conjunction with host factors may determine the outcome of invasive GAS infection.

Macrolide resistance and erythromycin resistance determinants among Belgian Streptococcus pyogenes and Streptococcus pneumoniae isolates.

Resistance of streptococci to macrolide antibiotics is caused by target-site modification or drug efflux. The phenotypic expression of target-site modification can be inducible or constitutive. The prevalence of the three phenotypes among Belgian erythromycin-resistant Group A streptococci (GAS) and Streptococcus pneumoniae isolates was surveyed, their MICs for seven antibiotics were determined and the clonality of the isolates was explored. Of the 2014 GAS isolates tested 131(6.5%) were erythromycin resistant (MIC > 1 mg/L): 110 (84.0%) showed the M-resistance phenotype whereas the remaining 21 strains (16.0%) were constitutively resistant. No inducibly resistant strains were detected. Of 100 S. pneumoniae isolates, 33 were erythromycin resistant (MIC > 1 mg/L). In contrast to the GAS isolates, only 9.1% of the 33 erythromycin-resistant S. pneumoniae isolates showed the M-resistance phenotype. The presence of mefA/E and ermB genes in the M-resistant and constitutively and inducibly resistant strains, respectively, was confirmed by PCR analysis. Genomic analysis based on pulsed-field gel electrophoresis (PFGE) using the restriction enzyme SfiI, revealed 54 different PFGE patterns among the 131 erythromycin-resistant GAS isolates, of which an M6 clone represented 16.0% of the strains; all other clones, exhibiting different M-types, represented <7% of the strains. The S. pneumoniae isolates also appeared to be polyclonally based, as determined by arbitrarily primed PCR. The macrolides miocamycin and rovamycin, the lincosamide clindamycin and the ketolide HMR 3647 showed excellent activity against the M-resistant GAS and S. pneumoniae strains.

Prevalence and molecular epidemiology of glycopeptide-resistant enterococci in Belgian renal dialysis units.

The molecular epidemiology of glycopeptide-resistant enterococci (GRE) colonizing the intestinal tracts of Belgian renal dialysis patients was studied among 1318 patients of a population of 1800 dialysis patients from 29 dialysis centers. Of these, 185 patients (14.0%) were colonized with a VANA-positive GRE; GRE harboring the VANB gene were not detected. The majority of the VANA GRE (80.5%) were identified as Enterococcus faecium; 14.8% were identified as E. faecalis; and a limited number were identified as E. avium, E. casseliflavus, E. dispar, E. durans, or E. gallinarum. Genome analysis of 277 VANA-positive GRE by pulsed-field gel electrophoresis revealed a high genetic variability both within the different dialysis centers and within the patients' own GRE flora. No high-level gentamicin-resistant VANA-positive GRE were detected, and most strains remained susceptible to ampicillin. These findings do not support a hospital-driven endemicity of VANA-positive enterococcal isolates in Belgium.

Comparison of glycopeptide-resistant Enterococcus faecium isolates and glycopeptide resistance genes of human and animal origins.

One hundred thirty-two glycopeptide-resistant Enterococcus faecium (GREF) isolates from different hospitals and pig and poultry farms in Belgium were compared on the basis of (i) their antibiotic susceptibilities, (ii) their SmaI pulsed-field gel electrophoresis (PFGE) patterns, and (iii) the organization of their Tn1546 or related elements in order to detect possible phenotypic and genotypic relationships among both groups of isolates. Human and animal vanA-positive GREF isolates were found to have similar susceptibility patterns; they remained susceptible to gentamicin and were, in general, susceptible to ampicillin. PFGE demonstrated a very high degree of genomic heterogeneity in both groups of isolates. However, indistinguishable isolates were found within different farms or hospitals, and in two instances, epidemiologically unrelated pig and human isolates showed indistinguishable PFGE patterns. In total, eight different transposon types were identified, and all were related to the prototype transposon Tn1546. The two predominant types, Tn1546 and type 2 transposons, which differed at three band positions, were present in both human and animal isolates. Type 2 transposons were significantly associated with pig isolates. The other types were seldom detected. These data suggest a possible exchange of glycopeptide resistance markers between animals and humans.

Comparison of direct plating and broth enrichment culture for the detection of intestinal colonization by glycopeptide-resistant enterococci among hospitalized patients.

The results of prevalence studies on glycopeptide-resistant enterococci (GRE) in the intestine may be influenced by the detection methods applied. In most studies different media, different concentrations of antibiotics, and different methods are used, and these differences result in differences in recovery rates. In this cross-sectional study on the carrier state of GRE among patients at the University Hospital Antwerp, Antwerp, Belgium, performed on 21 May 1996, direct plating and broth enrichment were compared by using the same media. Stool samples (n = 213) or rectal swabs (n = 122) were plated directly on Enterococcosel agar (bioMérieux) and after enrichment in Enterococcosel broth. The prevalence of GRE was 12.8%. Direct plating recovered 53.4% of the GRE isolates, and broth enrichment recovered an additional 46.5% of them; in the latter test the isolates were thus present at less than 10(3) CFU per g of feces. The prevalence of GRE among dialysis patients was higher than among the other patients, but the difference was not significant (P = 0.06), possibly as a result of the small numbers of dialysis patients examined. The GRE species isolated included 19 E. gallinarum (44.2%), 13 E. faecium (30.2%), 6 E. faecalis (13.9%), and 5 E. casseliflavus (11.6%) isolates. All E. faecalis and E. faecium strains isolated carried the vanA gene, and E. gallinarum and E. casseliflavus carried the vanC1 and vanC2 gene, respectively. The majority of isolates were polyclonal. Our data indicate that the rate of detection of GRE from both stool samples and rectal swabs is significantly increased with enrichment cultures.

Comparison of screening methods for detection of extended-spectrum beta-lactamases and their prevalence among blood isolates of Escherichia coli and Klebsiella spp. in a Belgian teaching hospital.

Using a set of 33 well-defined extended-spectrum beta-lactamase (ESBL)-producing strains of Escherichia coli and Klebsiella pneumoniae, we compared three screening methods for ESBL detection: (i) a double-disk synergy test, (ii) a three-dimensional test (both the double-disk synergy test and the three-dimensional test were performed with ceftriaxone, ceftazidime, aztreonam, and cefepime), and (iii) the Etest ESBL screen (AB Biodisk, Solna, Sweden), based on the recognition of a reduction in the ceftazidime MIC in the presence of clavulanic acid. In the double-disk test, all four indicator antibiotics scored equally and 31 of the 33 reference strains were recognized. In the three-dimensional test, ceftriaxone was the only satisfactory indicator and 30 ESBL-positive strains were detected by this antibiotic. Both systems produced two false-positive results with cefepime. With the Etest ESBL screen, 15 of 16 TEM-related and 11 of 16 SHV-related ESBL-producing strains scored positive. In 10 cases the clavulanic acid on one end of the strip interfered with the MIC determination for ceftazidime, which was read on the opposite end. This MIC had to be determined with an extra ceftazidime-only strip. No false-positive results were noted. Eighty-six blood isolates of E. coli and Klebsiella species were screened for ESBL expression by the double-disk and three-dimensional tests, both with ceftriaxone. Six strains with suspicious antibiogram phenotypes also gave positive results by the double-disk test. One E. coli strain remained undetected by the three-dimensional test. Identification of the enzymes suspected of being ESBLs by isoelectric focusing (all strains) and DNA sequencing (1 strain) confirmed the screening test results except for one Klebsiella oxytoca strain, which proved to be a hyperproducer of its chromosomal enzyme and which also had a negative Etest score. The five true ESBL producers were all confirmed by the Etest ESBL screen. Pulsed-field gel electrophoresis proved that the E. coli strains were unrelated, but that two of the three K. pneumoniae strains were closely related.

Evaluation of arbitrarily primed PCR analysis and pulsed-field gel electrophoresis of large genomic DNA fragments for identification of enterococci important in human medicine.

The increasing problems encountered with enterococcal nosocomial infections and the intrinsic and acquired resistance of the enterococci to different antimicrobial compounds highlight the need for a rapid identification technique. Enterococcus faecalis is readily identified by biochemical tests, but species differentiation within the Enterococcus faecium and Enterococcus gallinarum species groups is less well established. In the present study, 66 strains representing the most prevalent human enterococci were used to develop a PCR-based species-specific identification protocol. Whole-cell protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used as a reference method for species identification. In addition, the genomic SmaI macro-restriction fragment distribution of all of the strains was examined by pulsed-field gel electrophoresis (PFGE). Oligonucleotide D11344-primed PCR was as discriminative as whole-cell protein analysis and resulted in more easily interpreted band patterns. This PCR-based technique allowed identification of clinical isolates by visual examination of the DNA profiles obtained. The inability of both methods to discriminate between Enterococcus casseliflavus and Enterococcus flavescens brought into question the species status of E. flavescens. PFGE did not result in species-discriminative DNA bands or band patterns, but proved to be superior for interpretation of interstrain relationships.

Identification and classification of Lactobacillus acidophilus, L. gasseri and L. johnsonii strains by SDS-PAGE and rRNA-targeted oligonucleotide probe hybridization.

Thirty-two strains originally identified as Lactobacillus acidophilus and L. gasseri were screened for their taxonomic homogeneity by SDS-PAGE of whole-cell proteins. After numerical comparison of the resulting protein electrophoretic fingerprints, two well-delineated clusters were detected. The majority of the strains grouped in one electrophoretic cluster, which contained the type strain of L. acidophilus and corresponds to DNA group A1 of Johnson, J. L., Phelps, C. F., Cummins, C. S., London, J. & Gasser, F. (1980; International Journal of Systematic Bacteriology 30, 53-68). Another cluster corresponded to DNA group B. It contained two subclusters, which agreed perfectly with DNA subgroups B1 (L. gasseri) and B2 (L. johnsonii), respectively. The 23S rRNA genes were partially sequenced and 23S-rRNA-targeted oligonucleotide probes were designed for identification of DNA groups A1, B1 and B2. Probe Lbg reacted with all strains of electrophoretic cluster B1 (L. gasseri), probe Lbj hybridized with strains of cluster B2 (L. johnsonii) and probe Lba with strains of cluster A1 (authentic L. acidophilus). The probes were successfully used for the identification of strains belonging to the respective species. The phylogenetic relationship of a representative of L. johnsonii was determined by comparative sequence analysis of the 16S rRNA genes. It is very closely related to L. gasseri.