Same-day identification and antibiotic susceptibility testing on positive blood cultures: a simple and inexpensive procedure.

Fast diagnostic tools are becoming a hot topic in microbiology, especially in the case of septic patients. Therefore, we attempted to develop a fast, inexpensive, accurate and easy method to identify bacteria and perform an antibiotic susceptibility test directly on positive blood cultures that could be used in a routine laboratory. A procedure based on centrifugation and washing steps was performed on 110 non-duplicated (including nine seeded) positive blood culture bottles. Direct identification (DID) and antimicrobial susceptibility testing (AST) was conducted on the pellet with the MALDI Biotyper and Phoenix, respectively. Identification (ID) to the species level was correct in 44/45 (97%) cases for Gram-negative bacteria and 44/56 (79%) cases for Gram-positive bacteria. In total, 98.9% of the AST results were identical to the routine laboratory result. No very major errors, four major errors and eight minor errors were detected. A reliable identification and a high AST agreement were obtained from blood cultures seeded with multi-resistant bacteria. We simulated the timeline of DID and demonstrated an identification and AST result within 24 h using Escherichia coli- and Staphylococcus aureus-positive blood cultures as examples. We developed an easy, fast and cheap method to generate reliable ID and AST results. Moreover, this method may be used to obtain results within 24 h after incubating the blood culture bottles in the microbiology lab.

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.

Genetic diversity of methicillin-resistant Staphylococcus aureus in a tertiary hospital in The Netherlands between 2002 and 2006.

The aim of this study was to investigate the methicillin-resistant Staphylococcus aureus (MRSA) clones isolated in a Dutch university hospital, situated near the borders of Belgium and Germany, between 2002 and 2006. MRSA strains (n = 175) were characterized using spa and SCCmec typing. The presence of Panton Valentine leukocidin (PVL) was determined. Between 2002 and 2005, ST5-MRSA-IV was predominant, and the spa type of ST5-MRSA-IV changed from t002 to t447. ST5-MRSA-I, ST5-MRSA-II, ST228-MRSA-I, and ST247-MRSA-I were also observed in this period. From 2004, the MRSA genetic background became more diverse, and in 2006, ST5-MRSA-IV was only sporadically observed. From 2005, ST5-MRSA-II, ST8-MRSA-IV, ST22-MRSA-IV, and ST45-MRSA-IV were increasingly observed. Several other MRSA clones, such as ST239-MRSA-III, were found sporadically. Four PVL-positive MRSA isolates were observed, associated with ST80-MRSA-IV and ST8-MRSA-IV. ST5-MRSA-I, ST5-MRSA-II, ST5-MRSA-IV, and ST228-MRSA-I have not been described previously in The Netherlands.

Cost of the meticillin-resistant Staphylococcus aureus search and destroy policy in a Dutch university hospital.

Costs related to a search and destroy policy and treatment for Staphylococcus aureus bacteraemia in the University Hospital Maastricht were calculated for the period 2000 and 2004. The financial cost-benefit break-even point of the search and destroy policy was determined by modelling. On average 22,412 patients were admitted per year for an average of 8.7 days. Each year 246 patients were screened for meticillin-resistant Staphylococcus aureus (MRSA) and 74 patients were decolonised and nursed in preventive isolation. The prevalence of MRSA in the University Hospital Maastricht was 0.7%, as calculated from positive blood cultures, and mean length of stay for all patients with S. aureus bloodstream infections was 39.9 days. The annual cost of pro-active searching for MRSA in the University Hospital Maastricht was euro 1,383,200, and euro 2,736,762 for MRSA prevention and treatment of S. aureus bloodstream infections. Simulation of a variety MRSA/meticillin-susceptible S. aureus (MSSA) ratios showed that even if the MRSA prevalence reaches 8%, prevention costs are still lower than the cost of treating S. aureus infections. In conclusion, the total cost of a search and destroy policy is lower than the cost of treating S. aureus bloodstream infections in the University Hospital Maastricht. At an MRSA prevalence of

Comparison of five different immunoassays for the detection of Borrelia burgdorferi IgM and IgG antibodies.

The performances of five commercially available enzyme immunoassays were compared for the detection of Borrelia burgdorferi IgM and IgG antibodies. Sensitivity was assessed with European serum samples collected from 45 patients with clinically defined Lyme disease in conjunction with a positive immunoblot (n = 44) or other serological test (n = 1). Sensitivities for the detection of IgM and IgG with each test were: Dako IgM 64%; Dako IgG 53%; Serion IgM 89%; and Serion IgG 88%. The Immunetics assay makes no distinction between IgM and IgG antibodies and had a sensitivity of 91%. Specificity was calculated by testing a control group comprising 40 patients with acute Epstein-Barr virus infection, cytomegalovirus infection, syphilis or rheumatoid factor positivity. The specificities achieved for each test were: Dako IgM 78%; Dako IgG 100%; Serion IgM 52%; Serion IgG 92%; and Immunetics 92%. The discriminatory power between control and patient samples appeared highest for the Immunetics assay. Between-run variation was comparable for the five tests and did not exceed 13%. When the Immunetics assay was used as an initial screening test, with low-titre positive results confirmed by an immunoblot, a sensitivity of 91% and a specificity of 100% were achieved. To attain maximal sensitivity, the Serion IgM and IgG tests were also performed on samples with negative Immunetics results. All positive Serion IgM and IgG results were also confirmed by immunoblot. In conclusion, the Immunetics assay, based on a synthetic C6 peptide, can be used reliably as an initial screening test for the serodiagnosis of Lyme disease.

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.

Laboratory diagnosis and biosafety issues of biological warfare agents.

Bioterrorism events have been rare until recently. Many clinical laboratories may not be familiar with handling specimens from a possible bioterrorism attack. Therefore, they should be aware of their own responsibilities and limitations in the handling and treatment of such specimens, and what to do if they are requested to process clinical samples. The Centers for Disease Control and Prevention has developed the Laboratory Response Network to provide an organized response system for the detection and diagnosis of biological warfare agents based on laboratory testing abilities and facilities. There are potentially many biological warfare agents, but probably a limited number of agents would be encountered in case of an attack, and their identification and laboratory safety will be discussed.

Recurrent bacteremia by Chryseobacterium indologenes in an oncology patient with a totally implanted intravascular device.

Chryseobacterium indologenes was isolated from the blood cultures of an oncological patient with a totally implantable device. Because a catheter-related infection was suspected, the Port-A-Cath was removed after a 10-day course of piperacillin-tazobactam. Differences in susceptibility may exist if either the criteria for either Pseudomonas or Enterobacteriaceae are used.

Reactivity to p52 and CM2 recombinant proteins in primary human cytomegalovirus infection with a microparticle agglutination assay.

We evaluated the reactivities of sera against p52 and CM2 recombinant antigens of human cytomegalovirus (HCMV), coated on microparticles, for the differentiation of primary HCMV infection from an established infection. Two different test formats of the CMV Multiplex Copalis assay were evaluated. The 214 serum samples tested were immunoglobulin M (IgM) positive or equivocal by our reference assay. Reactivities against p52 and CM2 antigens were tested for sera from 37 patients with a well-documented seroconversion within the preceding 3 months (119 serum specimens), 31 patients known to have had a seroconversion at least 8 months earlier (31 serum specimens), and 57 patients without a documented seroconversion (64 serum specimens). The assay had a sensitivity for the detection of a primary infection of 70 or 86% by the first test format and a sensitivity of 88 or 94% by the second test format, according to the criteria used to indicate a primary infection by this test. A good correlation of the results of the assay with our in-house avidity index was found. The specificity of the assay warrants further evaluation. With IgM-positive sera, the assay was not sufficiently specific to make a distinction between a primary infection and an established infection.

Use of the polymerase chain reaction (PCR) for the detection of aacA genes encoding aminoglycoside-6'-N-acetyltransferases in reference strains and gram-negative clinical isolates from two Belgium hospitals.

Genes encoding aminoglycoside 6'-N-acetyltransferases, were identified using the polymerase chain reaction (PCR). Four sets of primers delineating DNA fragments of 209 bp, 250 bp, 260 bp and 347 bp, specific for the four known aacA genes, and probes within these fragments, were constructed based on the nucleotide sequences of the aacA genes. The specificity of the primers was evaluated using reference strains encoding various aminoglycoside-modifying enzymes. The primers reacted with their corresponding aacA genes and did not cross-react with genes coding for other aminoglycoside-modifying enzymes. One hundred and sixty-one aminoglycoside resistant clinical isolates showing AAC(6')I activity were tested using the PCR assays. The gene described by Tran Van Nhieu & Collatz (1987) was the most frequently identified aacA gene. One strain of Citrobacter freundii contained two distinct aacA genes. However, in 46% of the strains, the majority being Serratia spp. and Acinetobacter spp. none of the specific amplified DNA fragments for any of the known aacA genes could be detected.

Serotypes and extended spectrum beta-lactam resistance in aminoglycoside resistant Pseudomonas aeruginosa isolates from two Belgian general hospitals: a seven year study.

A total of 1896 isolates of Pseudomonas aeruginosa resistant to aminoglycosides and isolated during the period 1983-1989 in two Belgian general hospitals were included in this study. The most frequently encountered O serotypes were O4, O11, O12 and non-typable isolates. The majority of the isolates showed resistance to extended spectrum beta-lactam antibiotics (cefotaxime, ceftriaxone and cefepime). However, a low degree of resistance was found for ceftazidime. By contrast, amikacin and isepamicin, remained active on a significant number of aminoglycoside resistant isolates. In both hospitals, impermeability and AAC(3)II enzyme production were the most prevalent aminoglycoside resistance mechanisms. There were marked differences between the two hospitals with regard to the distribution of the O-serotypes and resistance profiles.

Comparative in vitro activity of cefepime and four extended-spectrum beta-lactams on 1,251 aminoglycoside-resistant gram-negative hospital strains.

Cefepime was the most active compound on the Enterobacteriaceae with a MIC90 of 0.26 microgram/ml and a resistance rate of 0.1%. Ceftazidime was the most active drug on the non-fermenting bacilli (MIC90 9.65 micrograms/ml; resistance rate 3%). Amikacin- and gentamicin-resistant strains showed a decreased susceptibility to the beta-lactams, though the Enterobacteriaceae and the non-fermenters remained fairly sensitive to cefepime and ceftazidime, respectively. Aminoglycoside-3-N-acetyltransferase was the most prevalent enzyme and was often associated with intermediate resistance or resistance to beta-lactams. Non-fermenters showing aminoglycoside impermeability were very often intermediately resistant or resistant to beta-lactams.

Activity of cefotiam in combination with beta-lactam antibiotics on enterobacterial hospital strains.

By using checkerboard titrations the effect of cefotiam combined with different beta-lactam antibiotics on fifty strains of Enterobacteriaceae moderately susceptible (minimal inhibiting concentration greater than or equal to 8 mg/l) or resistant (minimal inhibiting concentration greater than or equal to 64 mg/l) to cefotiam was evaluated. The following compounds were tested: cefamandole, cefazolin, cefmenoxime, cefotaxime, cefotiam, ceftazidime, cefuroxime, mecillinam and piperacillin. The synergistic effect varied markedly. The combination cefotiam-mecillinam showed the highest rate of synergistic activity. Antagonism was found in 1% of the combinations.

[Antibacterial activity of carumonam and cefpirome on hospital strains resistant to gentamicin and cephalothin: comparison with other beta-lactam antibiotics, new fluoroquinolones, aminoglycosides and other antibiotics]. / Activité antibactérienne du carumonam et du cefpirome sur des souches hospitalières résistantes à la gentamicine et la céfalotine:comparaison avec d'autres bêta-lactamines, de nouvelles fluoroquinolones, aminoglycosides et d'antibiotiques divers.

The antibacterial in vitro activity of carumonam, a new monobactam, and cefpirome, a new cephalosporin, was studied on 483 hospital strains resistant to gentamicin and cephalothin, in comparison with amikacin, azlocillin, aztreonam, cefmenoxim, cefoperazone, cefotaxim, cefsulodin (for Pseudomonas), ceftazidime, ceftriaxone, cefuroxim, chloramphenicol, ciprofloxacin, doxycycline, enoxacin, netilmicin, norfloxacin, pefloxacin, piperacillin, rifampicin, tobramycin and trimethoprim. In general the two compounds have a very good in vito activity on Enterobacteriaceae but are less active on non-fermenting microorganisms. For the Enterobacteriaceae the minimal inhibitory concentrations 90% for carumonam was less than or equal to 1.1 mg/l excepted for Enterobacter spp. (43,6 mg/l) and M. morganii (56.8 mg/l) . All the Enterobacteriaceae are susceptible to cefpirome (minimal inhibitory concentrations 90% less than or equal to 5.3 mg/l). The activity of carumonam and cefpirome on Enterobacteriaceae is comparable with that of the third generation cephalosporins. Carumonam is more active than cefpirome and other beta-lactams, ceftazidime excepted, on Pseudomonas aeruginosa and Pseudomonas spp. On the other hand, both compounds reveal to have only a low activity on the other non-fermenters which minimal inhibitory concentrations 90% values of 115.4 mg/l for carumonam and 32.0 mg/l for cefpirome.

The comparative activity of pefloxacin, enoxacin, ciprofloxacin and 13 other antimicrobial agents against enteropathogenic microorganisms.

In this study, we compared the activity of pefloxacin, enoxacin and ciprofloxacin against 269 enteropathogenic strains (Campylobacter jejuni, enteropathogenic Escherichia coli, Salmonella typhi, Shigella spp., Vibrio cholerae and Yersinia enterocolitica) with that of rosoxacin, flumequin, nifuroxazide, erythromycin, chloramphenicol, ampicillin, cefotaxime, tetracycline, amikacin, netilmicin, sulfamethoxazole, trimethoprim and co-trimoxazole. Pefloxacin, enoxacin and ciprofloxacin were always among the most active compounds. Furthermore, resistant strains or strains with elevated MIC values were not found. The MIC90 value for these three compounds was less than or equal to 0.25 mg/l, except for C. jejuni where it was 0.3 mg/l and 1.4 mg/l for pefloxacin and enoxacin, respectively.