Prosthetic infection: improvement of diagnostic procedures using 16S ribosomal deoxyribonucleic acid polymerase chain reaction.

PURPOSE: Prosthetic infection is the worst complication in joint arthroplasty. The diagnostic procedure is time consuming and in many cases unrewarding. The aim of this investigation was to raise the sensitivity of the diagnostic procedure. METHODS: Altogether, 229 implants were removed from 229 patients. Complete data from 157 patients could be analysed. On explantation of the respective arthroplasty, tissue was removed, puncture fluid aspirated and biofilm scratched from the implant surface with a surgical knife. Specimens were investigated with conventional culture methods and with 16S ribosomal DNA (rDNA) polymerase chain reaction (PCR) and sequencing. RESULTS: In 123 cases, no pathogen could be identified by routine culture methods. In three of these culture-negative cases, bacteria could be identified with 16S rDNA sequencing of the removed biofilm. In 34 cases, bacteria could be identified with culture methods. In two of these cases, sequencing detected additional pathogens. CONCLUSIONS: The process of 16S ribosomal deoxyribonucleic acid polymerase chain reaction (rDNA PCR) and sequencing of biofilm removed from the explanted prosthesis is an important addition to conventional culture methods in prosthetic joint infection. Polymerase chain reaction detects additional pathogens and improves diagnostic sensitivity. The examination of tissue, puncture fluid and biofilm should be performed in cases of prosthesis loosening and explantation.

Implant stability in the treatment of MRSA bone implant infections with linezolid versus vancomycin in a rabbit model.

The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infections represents a significant healthcare burden. Vancomycin and linezolid exhibit potent clinical and microbiological activity in MRSA infections. Our purpose was to investigate the efficacy of linezolid versus vancomycin in experimental implant infections and the influence on implant stability in a rabbit model. Thirty-six female New Zealand White rabbits received surgical insertion of titanium implants into their distal femurs and were randomly assigned to six groups (A: infected, no treatment; B: infected, vancomycin; C: infected, linezolid; D: no infection, no treatment; E/F: no infection, vancomycin or linezolid, respectively). Antibiotics were administered, and plasma levels determined. Bone-implant specimens were tested for mechanical stability of fixation. Quantitative histomorphometry of bone and soft tissue was performed using computerized image analysis. Plasma levels of linezolid and vancomycin were within the respective therapeutic ranges. Microbiological analysis of specimens from infected rabbits showed MRSA tissue colonization in all untreated animals, in two of six vancomycin-treated animals, and in none of the linezolid-treated animals. Antibiotic treatment improved mechanical stability significantly (p = 0.004) with both vancomycin and linezolid. Mechanical testing correlated with histomorphometry results. A significant negative correlation was found between displacement of the implant and the percentage of calcified tissue around the implant, and a significant positive correlation was found between displacement of the implant and the amount of noncalcified tissue. Our data indicate that both treatment regimens improved implant stability.

A simple isocratic HPLC assay to determine linezolid concentrations in different biomatrices for in vivo and in vitro studies.

BACKGROUND: Linezolid is an important therapeutic option for the treatment of infections caused by multiresistant Gram-positive bacteria such as vancomycin-resistant Enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). However, the clinical benefit of linezolid is threatened by the emergence of resistant strains of MRSA and VRE reported in North America and Europe. For effective antimicrobial treatment, it is extremely important to have exact knowledge of drug concentrations at the site of action. METHODS: A simple HPLC method for the rapid and precise determination of linezolid in different biomatrices (e.g., plasma, soft tissue, bone, dialysis fluid and used microbiological broth) was developed and validated. Proteins were precipitated with acetonitrile and separation was performed on a reversed-phase C8 column with a mobile phase consisting of water/acetonitrile (80:20, v/v). UV detection was performed at 251 nm. RESULTS: This method has a lower limit of quantification of 0.3 microg/mL and a linear calibration range of 0.5-40 microg/mL. The method showed excellent reproducibility, with an inter- and intra-day assay precision of <5% (% relative standard deviation), as well as excellent accuracy, with inter- and intra-day assay accuracy ranging from 100.6% to 103.2%. Stability up to 6 months in water and plasma was proven. Quantitative recovery was possible after up to three freeze thaw cycles. CONCLUSIONS: The method is useful in the acquisition of in vivo and in vitro data. It is simple, flexible, specific, precise and reproducible, as well as of adequate sensitivity for clinical use.

Detection of extended-spectrum beta-lactamases among Enterobacteriaceae by use of semiautomated microbiology systems and manual detection procedures.

Three commercially available microbiology identification and susceptibility testing systems were compared with regard to their ability to detect extended-spectrum beta-lactamase (ESBL) production in Enterobacteriaceae, i.e., the Phoenix Automated Microbiology System (BD Diagnostic Systems, Sparks, MD), the VITEK 2 System (bioMérieux, Marcy l'Etoile, France), and the MicroScan WalkAway-96 System (Dade Behring, Inc., West Sacramento, CA), using routine testing panels. One hundred fifty putative ESBL producers were distributed blindly to three participating laboratories. Conventional phenotypic confirmatory tests such as the disk approximation method, the CLSI double-disk synergy test, and the Etest ESBL were also evaluated. Biochemical and molecular characterization of beta-lactamases performed at an independent laboratory was used as the reference method. One hundred forty-seven isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Serratia marcescens, Proteus mirabilis, Proteus vulgaris, and Morganella morganii were investigated. Of these isolates, 85 were identified as ESBL producers by the reference method. The remaining isolates were identified as non-ESBL producers; they were either hyperproducers of their chromosomal AmpC, Koxy, or SHV enzymes or lacked any detectable beta-lactamase activity. The system with the highest sensitivity for the detection of ESBLs was the Phoenix (99%), followed by the VITEK 2 (86%) and the MicroScan (84%); however, specificity was more variable, ranging from 52% (Phoenix) to 78% (VITEK 2). The performance of the semiautomated systems differed widely with the species investigated. The sensitivities of the conventional test methods ranged from 93 to 94%. The double-disk synergy test showed the highest specificity and positive predictive value among all test methods, i.e., 97% and 98%, respectively.

Chlorhexidine and silver-sulfadiazine coated central venous catheters in haematological patients--a double-blind, randomised, prospective, controlled trial.

BACKGROUND: Central venous catheters (CVCs) are essential for the intensive care of patients with haematological illness. Catheter-related infections (CRI) are an important problem in modern medicine, which may lead to life-threatening situations, to prolonged hospitalisation and increased cost. In immunocompromised patients suffering from haemato-oncological diseases, CRI is a significant factor for adverse outcome. Several clinical studies have shown that CVCs coated with antiseptics such as chlorhexidine and silver-sulfadiazine (CHSS) reduce the risk of catheter-related bacteraemia. Most studies, however, were performed on intensive care patients not suffering from chemotherapy-induced immunosuppression. PATIENTS AND METHODS: A prospective double-blind, randomised, controlled trial was performed to investigate the effectiveness of CHSS-coated catheters in haemato-oncological patients. A total number of 184 catheters (median duration of placement, 11 days) were inserted into 184 patients (male 115, female 69), of which 90 were antiseptically coated. After removal, all catheters were investigated for bacterial growth. MAIN RESULTS: Catheters coated with CHSS were effective in reducing the rate of significant bacterial growth on either the tip or subcutaneous segment (26%) compared to control catheters (49%). The incidence of catheter colonisation was also significantly reduced (12% coated vs 33% uncoated). Data obtained show a significant reduction of catheter colonisation in CHSS catheters. There was no significant difference in the incidence of catheter-related bacteraemia (3% coated vs 7% uncoated). However, due to the overall low rate of CRI, we could not observe a significant reduction in the incidence of catheter-related bacteraemia. CONCLUSION: Our data show that the use of CHSS catheters in patients with haematological malignancy reduces the overall risk of catheter colonisation and CRI, although the incidence of catheter-related bacteremia was similar in both groups.

Varying linezolid susceptibility of vancomycin-resistant Enterococcus faecium isolates during therapy: a case report.

OBJECTIVES: Linezolid is an oxazolidinone antibiotic used in the treatment of infections caused by vancomycin-resistant enterococci. Resistance to linezolid has been associated with a G2576U mutation in domain V of the 23S rRNA. PATIENT AND METHODS: We present clinical details and susceptibility data from multiple Enterococcus faecium strains isolated from a liver transplant patient over 13 months. MICs of linezolid, vancomycin and quinupristin/dalfopristin were determined using Etest. Molecular typing was performed by pulsed-field gel electrophoresis. Domain V of the 23S rRNA gene in the vancomycin-resistant Enterococcus faecium was amplified. Linezolid concentrations were analysed by HPLC. RESULTS: We report the emergence of resistance to linezolid in a vancomycin-resistant Enterococcus faecium during linezolid treatment. After discontinuation of the linezolid therapy, the isolate reverted to susceptibility. However, after re-administration of linezolid the vancomycin-resistant Enterococcus faecium became resistant to linezolid again. The isolates that were resistant to linezolid had a G2576T mutation in their 23S rDNA. CONCLUSION: We describe a clinical case that shows the shift of a vancomycin-resistant Enterococcus faecium from linezolid resistance to susceptibility and then back to resistance again related to linezolid therapy.

Surgical site infection - a European perspective of incidence and economic burden.

This retrospective review of reported surgical site infection (SSI) rates in Europe was undertaken to obtain an estimated scale of the problem and the associated economic burden. Preliminary literature searches revealed incomplete datasets when applying the National Nosocomial Infection Surveillance System criteria. Following an expanded literature search, studies were selected according to the number of parameters reported, from those identified as critical for accurate determination of SSI rates. Forty-eight studies were analysed. None of the reviewed studies recorded all the data necessary to enable a comparative assessment of the SSI rate to be undertaken. The estimated range from selected studies analysed varied widely from 1.5-20% - a consequence of inconsistencies in data collection methods, surveillance criteria and wide variations in the surgical procedures investigated - often unspecified. SSIs contribute greatly to the economic costs of surgical procedures - estimated range: 1.47-19.1 billion Euro dollars. The analysis suggests that the true rate of SSIs, currently unknown, is likely to have been previously under-reported. Consequently, the associated economic burden is also likely to be underestimated. A significant improvement in study design, data collection, analysis and reporting will be necessary to ensure that SSI baseline rates are more accurately assessed to enable the evaluation of future cost-effective measures.