Intrapartum group B Streptococcus screening in the labor ward by Xpert® GBS real-time PCR.

Group B Streptococcus (GBS) is the leading cause of neonatal infections in industrialized countries. Intrapartum antibiotic prophylaxis (IAP) given to colonized parturients is a key step for the prevention of neonatal early-onset infection. We compared the performances of Xpert® GBS polymerase chain reaction (PCR) (Cepheid, Sunnyvale, CA, USA) as a point-of-care system in labor wards to standard culture for intrapartum GBS detection. Pregnant women with a GBS-positive antenatal screening were prospectively included. A vaginal double swab was collected at the time of delivery for point-of-care Xpert® GBS PCR and GBS culture. A total of 565 pregnant women were included. Valid Xpert® GBS results were obtained for 488 (86.4%) women on the first attempt. Repeat testing improved the PCR success to 516 (91.3%) women. Among the 305 women positive for GBS by culture at delivery, only 238 (78.0%) were positive by Xpert® GBS PCR, cycle thresholds being correlated to culture quantification. Among 260 women negative for GBS culture, 56 (21.5%) were positive by Xpert® GBS PCR, including 50 where IAP was initiated before vaginal sampling. Overall, among the 565 women with GBS antenatal positive culture, only 335 (59.3%) were still positive at delivery whatever the technique used, resulting in unnecessary IAP for 40% of them. This large cohort study comparing intrapartum to antepartum GBS detection provides evidence that (i) Xpert® GBS PCR might be a valuable solution for intrapartum GBS detection compared to culture-based strategies and (ii) laboratory training of non-specialized staff is mandatory to reach the performances required for point-of-care tests.

Highly selective recovery of Ni(II) in neutral and acidic media using a novel Ni(II)-ion imprinted polymer.

In this work, an original ion-imprinted polymer (IIP) was synthetized for the highly selective removal of Ni(II) ions in neutral and acidic media. First a novel functional monomer (AMP-MMA) was synthetized through the amidation of 2-(aminomethyl)pyridine (AMP) with methacryloylchloride. Following Ni(II)/AMP-MMA complex formation study, the Ni(II)-IIP was produced via inverse suspension polymerization (DMSO in mineral oil) and characterized with solid state 13C CPMAS NMR, FT-IR, SEM and nitrogen adsorption/desorption experiments. The Ni(II)-IIP was then used in solid-phase extraction of Ni(II) exploring a wide range of pH (from neutral to strongly acidic solution), several initial concentrations of Ni(II) (from 0.02 to 1 g/L), and the presence of competitive ions (Co(II), Cu(II), Cd(II), Mn(II), and Mg(II)). The maximum Ni(II) adsorption capacity at pH 2 and pH 7 reached values of 138.9 mg/g and 169.5 mg/g, that are among the best reported in literature. The selectivity coefficients toward Cd(II), Mn(II), Co(II), Mg(II) and Cu(II) are also very high, with values up to 38.6, 32.9, 25.2, 23.1 and 15.0, respectively. The Ni(II)-IIP showed good reusability of up to 5 cycles both with acidic and basic Ni(II) eluents.

[Evaluation of the impact of different lengths of pre-enrichment in a nutritive broth and prolonged incubation of MRSA-ID, a chromogenic agar medium, on its performances for identifying methicillin-resistant Staphylococcus aureus in screening samples]. / Evaluation de l'influence des conditions de culture sur les performances du milieu gélosé sélectif MRSA-ID pour l'identification de Staphylococcus aureus résistant à la méticilline dans les prélèvements de dépistage.

MRSA-carrier screening is recommended to prevent MRSA dissemination in hospitals. Rapid and specific detection of MRSA in the laboratory is a key element in enabling control measures. Our objective was to evaluate the impact of different lengths of pre-incubation in a nutritive broth and prolonged incubation of MRSA-ID, a chromogenic agar medium, on its performances for identifying MRSA in screening samples. According to our results, short-length pre-enrichments only provided a weak increase of sensitivity as compared to the absence of pre-enrichment. On the contrary, the sensitivity increase provided by an overnight pre-enrichment was significant. The prolongation of incubation in the chromogenic agar medium (48 hours instead of 24 hours) did not provide any significant increase of sensitivity but was associated with a strong and significant loss of specificity. Therefore, it seems relevant to reject prolonged incubation of selective agar media and to make a choice between the absence of pre-enrichment (faster results) and an overnight pre-enrichment (higher sensitivity), according to local epidemiology and local practices implemented for prevention.

PlaScope: a targeted approach to assess the plasmidome from genome assemblies at the species level.

Plasmid prediction may be of great interest when studying bacteria of medical importance such as Enterobacteriaceae as well as Staphylococcus aureus or Enterococcus. Indeed, many resistance and virulence genes are located on such replicons with major impact in terms of pathogenicity and spreading capacities. Beyond strain outbreak, plasmid outbreaks have been reported in particular for some extended-spectrum beta-lactamase- or carbapenemase-producing Enterobacteriaceae. Several tools are now available to explore the 'plasmidome' from whole-genome sequences with various approaches, but none of them are able to combine high sensitivity and specificity. With this in mind, we developed PlaScope, a targeted approach to recover plasmidic sequences in genome assemblies at the species or genus level. Based on Centrifuge, a metagenomic classifier, and a custom database containing complete sequences of chromosomes and plasmids from various curated databases, PlaScope classifies contigs from an assembly according to their predicted location. Compared to other plasmid classifiers, PlasFlow and cBar, it achieves better recall (0.87), specificity (0.99), precision (0.96) and accuracy (0.98) on a dataset of 70 genomes of Escherichia coli containing plasmids. In a second part, we identified 20 of the 21 chromosomal integrations of the extended-spectrum beta-lactamase coding gene in a clinical dataset of E. coli strains. In addition, we predicted virulence gene and operon locations in agreement with the literature. We also built a database for Klebsiella and correctly assigned the location for the majority of resistance genes from a collection of 12 Klebsiella pneumoniae strains. Similar approaches could also be developed for other well-characterized bacteria.

Impact of selective screening in the emergency department on meticillin-resistant Staphylococcus aureus control programmes.

Identification of the meticillin-resistant Staphylococcus aureus (MRSA) reservoir by active screening followed by the implementation of contact precautions is one of the major components of MRSA control programmes. The objective of this study was to evaluate the results of a programme of selective screening in an emergency department (ED) and the appropriateness of the contact precautions implemented. This was estimated by distinguishing necessary and unnecessary days of contact precautions. This estimation was performed for all days of contact precautions and, more specifically, for days of preventive contact precautions implemented before the availability of screening results. During a three-year period, screening of MRSA carriers was performed on 0.95% (N=605) of patients visiting the emergency ward. Among the 193 (31.9%) MRSA carriers identified, 159 were hospitalized in the short-length-hospitalization area (SLHA) of the ED and/or in other wards. Among the 140 patients admitted to the SLHA, 44 were hospitalized for at least 48 h, with a mean length of hospitalization of 5.9 days. The cumulative duration of hospitalization of carriers identified by screening was 1897 days. In total, 2370 days of contact precautions (including 924 days of preventive precautions) were implemented for patients screened in the ED. Considering the whole hospital, the appropriateness of this entire programme of contact precautions for patients screened in the ED was 80.0% (52.1% for the SLHA), whereas the specific appropriateness of preventive isolation days was 48.6% (43.6% for the SLHA). This study underscores the risk of MRSA cross-transmission in the SLHA, and the usefulness of implementing a control programme of screening carriers in the ED.

[Consequences of replacing abacavir for indinavir in successful antiretroviral treatment of a patient with HIV infection]. / Conséquences de la substitution de l'indinavir par l'abacavir chez un patient infecté par le VIH en succès thérapeutique.

INTRODUCTION: Simplification of combined antiretroviral therapy in HIV-infected patients is possible, but virological success can be compromised by the development or emergence of resistant viruses. CASE: Worsening renal functioning in a patient under successful combination antiretroviral therapy resulted led to the replacement of indinavir by abacavir. Eight weeks later, his viral load rose and he developed a mutant virus resistant to all the nucleoside analogs. DISCUSSION: Our case report illustrates the danger of streamlining combined antiretroviral therapy composed only of nucleoside analogs in patients already successfully treated with nucleoside analogs, by exposing them to the risk of the emergence of a mutant virus.

Isolation and properties of carboxylesterase P4 from Yersinia pseudotuberculosis.

The carboxylesterase P4 produced by Yersinia pseudotuberculosis was purified 330-fold by gel permeation and DEAE-trisacryl chromatography with a final yield of 21%. The apparent molecular weight, as determined by fast-protein liquid chromatography, was 45 kDa. The hydrolytic activity of esterase P4 was higher with the 1-naphthyl esters than with the 2-naphthyl esters of acetic, propionic and butyric acids. The apparent Km values were identical for 1-naphthyl acetate and 1-naphthyl propionate (0.15 mM). The enzyme was unstable at pH values below 5, but retained 80% of its initial activity after 30 min at 65 degrees C. It was unaffected by EDTA, eserine, tosyl-L-lysine chloromethylketone, iodoacetamide or 4-hydroxymercuribenzoate, but was strongly inhibited by low concentrations of diisopropyl fluorophosphate, suggesting the presence of serine in its active site. The purified enzyme gave a single precipitin line on Ouchterlony double immunodiffusion with homologous antiserum. This antiserum cross-reacted with the esterase bands E3 and E5 of Y. enterocolitica biotype 1, whereas there was no cross-reaction with the esterase bands produced by Y. enterocolitica biotypes 2 to 5, Y. intermedia, Y. frederiksenii, Y. kristensenii or Y. aldovae. The carboxylesterase P4 produced by Y. pestis was physicochemically, biochemically and immunologically indistinguishable from Y. pseudotuberculosis carboxylesterase P4. The latter enzyme and carboxylesterase B of Escherichia coli showed some biochemical similarities, but were antigenically unrelated. Our data confirm the relevance of esterases to phylogenetic and taxonomic studies of Enterobacteria.

Differentiation of human and animal strains of Streptococcus dysgalactiae by pulsed-field gel electrophoresis.

The genetic diversity among 54 human isolates and 33 animal isolates belonging to the species Streptococcus dysgalactiae (20 alpha-haemolytic Streptococcus dysgalactiae, 23 Streptococcus equisimilis, 43 group G streptococci and one group L streptococcus) was evaluated by macrorestriction analysis of chromosomal DNA with SmaI and resolution by pulsed-field gel electrophoresis. This technique revealed a high degree of intraspecies polymorphism, leading to the differentiation of 80 distinct banding patterns, and identified the presence of two major clusters, one containing isolates of human origin and the other isolates of animal origin. These results suggest than human and animal isolates of S.dysgalactiae are genetically distinct, and support the recent proposal of the subspecies S. dysgalactiae subsp. equisimilis for human isolates. The heterogeneity revealed within isolates from the same host type indicates that pulsed-field gel electrophoresis is a powerful epidemiological tool for studying S. dysgalactiae infections.

Esterase electrophoretic polymorphism of methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus.

Methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus from diverse geographic origins were analysed by polyacrylamide-agarose gel electrophoresis for esterase polymorphism. Three kinds of esterase bands, designated A, B and C, were defined by their ranges of activity toward five synthetic substrates and their resistance to di-isopropyl fluorophosphate. There were five allozymes of esterase A, four of esterase B and four of esterase C. Eighteen distinct combinations of allozymes (zymotypes) were distinguished amongst 105 strains analysed. Two major zymotypes were represented by 35 and 19 strains respectively, whereas other zymotypes were represented by one or, at most, seven strains. The coefficient of genetic diversity was lower for methicillin-resistant strains than for methicillin-sensitive strains. Most of the methicillin-resistant strains are represented by the two major zymotypes which differed from each other by the electrophoretic behaviour of the three esterases. These results indicate that, on the basis of esterase electrophoretic polymorphism, methicillin resistance is expressed in genetically different strains.

Persistence of Staphylococcus aureus strains among cystic fibrosis patients over extended periods of time.

Pulsed-field gel electrophoresis (PFGE) of SmaI macrorestriction fragments of chromosomal DNA was used to confirm the persistence of methicillin-sensitive Staphylococcus aureus isolates in the sputum of 25 cystic fibrosis patients in five French hospitals. Three-to-eight consecutive isolates, with the same esterase electrophoretic type isolated from each patient over a period of 12-28 months, were analysed. Consecutive isolates with indistinguishable PFGE profiles were found in 12 patients (48%) and consecutive isolates with similar PFGE profiles showing minor differences of one-to-four fragments (similarity coefficient >/=84%) were found in 11 patients. Consecutive isolates with different PFGE profiles were obtained from only two patients, but the profiles found in each patient were more closely related to each other than to other profiles. The results were in agreement with esterase electrophoretic typing for 23 patients, and we considered that those patients were infected with a single persistent strain. For any given patient, variations in antibiotypes and phage types of consecutive isolates were not associated with major genotypic variations. PFGE is useful in confirming the persistence of S. aureus strains in cystic fibrosis patients over long periods.

Analysis of genetic relationships among strains of groups A, C and G streptococci by random amplified polymorphic DNA analysis.

Random amplified polymorphic DNA (RAPD) analysis was evaluated for its capacity to distinguish species and strains within species of groups A, C and G streptococci. The 99 strains tested, previously typed by multilocus enzyme electrophoresis (MLEE), included 41 group A streptococci (Streptococcus pyogenes), 25 group G Streptococcus spp. (GGS), seven S. dysgalactiae, 11 S. equisimilis, four S. canis, three S. equi and eight S. zooepidemicus. The combined data obtained with three single primers distinguished 82 types. RAPD analysis provided taxonomic results that were in general agreement with previous species classification based on DNA-DNA homology and MLEE. The intraspecies typing efficiency of the technique was significantly improved by the parallel use of several primers. RAPD analysis had greater discriminatory power than MLEE for GAS and GGS. There was not total agreement between the two techniques as RAPD distinguished strains with identical electrophoretic types, whereas MLEE differentiated strains with identical PCR types. RAPD analysis did not distinguish all GAS strains with different biotypes and its already high discriminatory power was further enhanced by concomitant biotyping.

Long-term investigation of the clonal dissemination of Klebsiella pneumoniae isolates producing extended-spectrum beta-lactamases in a university hospital.

Seventy isolates of Klebsiella pneumoniae with extended-spectrum beta-lactamases (ESBLs) were compared. These were isolated from 51 patients on 10 separate wards in one hospital over an 18-month period between 1992 and 1994. Antibiograms were determined and the isolates were typed by pulsed-field gel electrophoresis of their DNA digestion with XbaI. The isolates were compared to three genotypically different epidemic strains responsible for previous outbreaks at the hospital between 1988 and 1991. Isolates from 84% of the present patients had closely related XbaI patterns, and most (74%) produced an ESBL with an iso-electric point (pI) of 7.0. A similar pattern was found for one of the previous epidemic strains, but it produced an ESBL with a pI of 7.8; isolates with this latter enzyme variant were found only in six of the present patients. The two other previous epidemic strains had ESBLs with a pI of 6.3 and organisms related to them were found in one and two of the present patients, respectively.

TEM-24 extended-spectrum beta-lactamase-producing Enterobacter aerogenes: long-term clonal dissemination in French hospitals.

OBJECTIVE: To investigate interstrain relatedness of TEM-24-producing Enterobacter aerogenes clinical strains isolated between 1993 and 1998 in 10 French hospitals from nine areas by pulsed-field gel electrophoresis (PFGE) and plasmid patterns. METHODS: Fifteen TEM-24-producing strains and a set of 16 control strains having various other antibiotic resistance phenotypes were genotyped by PFGE. Plasmid DNA from TEM-24-producing strains and transconjugants was analyzed. RESULTS: Analysis of XbaI macrorestriction patterns revealed only minor variations, and showed that all 15 TEM-24-producing strains were closely related. Some isolates originating from distant areas had indistinguishable patterns. According to their clustering correlation coefficients, they were also genomically distant from the control strains. Two plasmid patterns were observed in TEM-24-producing strains, one of them in 13 of the strains. Large plasmids of 85 kb encoding TEM-24 beta-lactamase were present in all isolates and, in all except one strain, could be transferred with high frequency by conjugation. CONCLUSIONS: These results confirm that the spread of the TEM-24 extended-spectrum beta-lactamase in France was essentially due to the dissemination of a single clone.

Genetic heterogeneity in methicillin-resistant strains of Staphylococcus aureus revealed by esterase electrophoretic polymorphism.

136 methicillin-resistant strains of Staphylococcus aureus recovered from hospitalized patients in 18 countries were characterized by electrophoretic mobilities of three types of esterases. These were defined by their ranges of activity toward five synthetic substrates and their resistance to di-isopropyl fluorophosphate. Fourteen zymotypes were distinguished. Two, designated as 6 and 14, were found in 53 and 50 strains, respectively. Genetic diversity coefficients were lower for strains from France and from other European countries (H = 0.47 and 0.53, respectively) than for strains from North America (H = 0.79). On the basis of electrophoretic polymorphism of esterases, our work provides evidence that methicillin-resistance is expressed in genetically different strains. Variations in esterase electrophoretic pattern within methicillin-resistant strains of S. aureus can make a significant contribution to the study of their epidemiology.

Arbitrary primed PCR rules out Clostridium difficile cross-infection among patients in a haematology unit.

Eight out of 20 (40%) patients with haematological malignancies hospitalized in the same unit of our hospital from 24 January to 24 April 1995, suffered from diarrhoea due to Clostridium difficile. The C. difficile isolates were characterized by serotyping and by arbitrary primed polymerase chain reaction (AP-PCR) using three different 10-mer oligonucleotides. It was found by serotyping that five patients had non-typeable isolates and three had serogroup H isolates. The AP-PCR typed all the isolates and yielded various patterns suggesting that there had been no cross-transmission between the patients. Control faecal sample cultures showed that two patients were still carrying the same isolates after specific treatment with vancomycin or metronidazole, and that one patient had acquired an isolate with a new AP-PCR type. AP-PCR was found to be a rapid, effective discriminative method for the immediate epidemiological tracking of hospital-acquired infections due to C difficile.

Epidemiological typing of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates responsible for five outbreaks in a university hospital.

Thirty-seven isolates of extended-spectrum beta-lactamase-producing (ESBL) Klebsiella pneumoniae implicated in five nosocomial outbreaks (I-V) on three distinct wards of our hospital were compared using capsular typing, biotyping, antibiotyping, enzyme electrophoresis typing and DNA macrorestriction analysis with Xba I resolved by pulsed-field gel electrophoresis. The isolates from each outbreak had common phenotypic and genotypic characteristics indicating that they were related epidemiologically. Isolates from outbreaks I (four patients) and V (13 patients), although they occurred in two different wards (neurology and surgery) and three years apart, produced the same ESBL with a pI of 7.8 (SHV-4) and were of serotype K25. The Xba I patterns were closely related. The isolates of outbreaks II (seven patients), III (four patients) and IV (seven patients), which occurred in a single surgical intensive care unit, produced an ESBL with a pI of 6.3 (TEM-3). Isolates from outbreaks III and IV, which occurred six months apart, were of serotype K68 and had similar Xba I patterns suggesting that the two outbreaks were due to a single strain which persisted endemically in the ward. The isolates from outbreak II were of serotype K62, and had distinct characteristics from the two later outbreaks. The Xba I patterns of the isolates from outbreaks "I and V', II and "III and IV' had Dice similarity coefficients under 40% showing that the three groups were genetically distant. DNA macrorestriction analysis was a useful complement to phenotypic methods for identifying K. pneumoniae strains responsible for outbreaks harbouring a common ESBL.

Synthesis of 4'-iodo-5-methoxy-valerophenone O-(2-aminoethyl)oxime as an agent for exploration of serotoninergic transporter.

The serotonin reuptake process is observed in the central nervous system and in cells derived from the neural crest. It would therefore be of great interest to visualize this reuptake for brain exploration and to visualize the tumors derived from these cells (Apudome). Fluvoxamine has been described as a specific uptake inhibitor for serotonin uptake and we therefore supposed that an iodinated derivative of this compound would be a suitable tracer for this purpose. We had shown by computer-assisted investigation that the trifluoromethyl group of fluvoxamine can be replaced by iodine without changing the steric hindrance of the structure. We therefore expected that this result would allow the development of a new iodinated ligand for human exploration by SPECT which would inhibit for the serotoninergic transporter. This new ligand is 4'-iodo-5-methoxyvalerophenone O-(2-aminoethyl)oxime in its E configuration. In vitro binding studies demonstrated that this iodinated ligand has a weaker affinity for the serotonin uptake sites than fluvoxamine. Steric hindrance is not sufficient to predict affinity, other structural factors such as electronic density and dipole moment must be considered to explain the biological difference between fluvoxamine and its iodinated analog.

Comparison of the efficacy of three commercial bacterins in preventing canine leptospirosis.

Twenty-four specific pathogen-free beagles were randomly allocated into four groups (three vaccinated groups and one control group) and inoculated at nine and 12 weeks of age with one of three commercial inactivated Leptospira vaccines: A (Vanguard 7; Pfizer Santé Animale), B (Dohyvac 7L; Fort Dodge), and C (Nobivac DHPPi + Lepto; Intervet International); the control group received Nobivac DHPPi (Intervet International). Seven weeks after the second vaccination all the dogs were challenged with Leptospira interrogans serogroup canicola. All the vaccinated dogs developed a mild serological response (microscopic agglutination titres) after the booster vaccination. A significant serological response after the challenge was observed, particularly in the controls. The challenge induced fever and clinical disorders in the control group, whereas in the vaccinated groups the clinical signs were mild. Blood cultures became positive in all control dogs, and in one of six dogs vaccinated with vaccine A and two of four dogs vaccinated with vaccine B; none of the six dogs vaccinated with vaccine C was leptospiraemic at any stage of the experiment. Urine cultures were positive in all the control dogs two weeks after the challenge. One of six dogs vaccinated with vaccine A and two of four dogs vaccinated with vaccine B shed bacteria in their urine after the challenge, but none of the dogs vaccinated with vaccine C shed bacteria in their urine at any time during the experiment.

[Comparative pharmacokinetics of antibiotics in blood, csf and brain]. / Pharmacocinétique comparée d'antibiotiques dans le sang, le L.C.R. et le cerveau.

The Knowledge of the antibiotic's cerebral diffusion is essential to define a of neurosurgical antibioprophylaxis' strategy. Without references in the medical world-literature, we've decided to compare the pharmacological kinetics in the blood, the cerebrospinal fluid and the sound cerebral tissue of common used molecules in neurosurgery that is to say: amoxicillin, cefamandole, metronidazole and pefloxacin. The results show that the cerebral levels of metronidazole and pefloxacine are rapidly high with an extended duration (> 10 times the M.I.C. of the sensitive bacteriae), but the tissue penetration of amoxicillin is hazardous and shot-duration for cefamandole (undosable after 3 hours).

[pH and the bacteriology of gastroplasties after esogastric resection]. / pH et bactériologie des gastro-plasties après résection oeso-gastrique.

A study of pH of digestive fluid in 24 patients six days after esophagogastric resection (EGR) showed conservation of acidity (pH: 3 or less) in one-thirds of cases. Immediate postoperative course was invariable with respect to incidence of pneumopathy and fistulae. Fungal infection is almost a constant finding in digestive grafts together with microbial pullulation, with a linear relation to pH. Ecology of germs was that of digestive flora of patients with obstruction, selected by the antibiotic cover administered. Group D streptococci and Gram negative bacilli were usually resistant to cephalosporins. Regurgitation pneumopathy therefore requires modification of antibiotic therapy and possible antifungal treatment. When functional disturbance is provoked by EGR, as for example a reflux syndrome; only those patients (1/3) with a pH of 3 or less can obtain relief from treatment with antacids.