Andrej Trampuz

Criteria for the interpretation of synovial fluid are well established for native joint disorders but lacking for the evaluation of prosthetic joint failure. Our aim was to define cutoff values for synovial fluid leukocyte count and neutrophil percentage for differentiating aseptic failure and prosthetic joint infection. We performed a prospective study of 133 patients in whom synovial fluid specimens were collected before total knee arthroplasty revision between January 1998 and December 2003. Patients with underlying inflammatory joint disease were excluded. Aseptic failure was diagnosed in 99 patients and prosthetic joint infection was diagnosed in 34 patients. The synovial fluid leukocyte count was significantly higher in patients with prosthetic joint infection (median, 18.9 x 10(3)/microL; range, 0.3 to 178 x 10(3)/microL) than in those with aseptic failure (median, 0.3 x 10(3)/microL; range, 0.1 to 16 x 10(3)/microL; P <0.0001); the neutrophil percentage was also significa...

We described for the first time the amino acid substitutions conferring rifampicin resistance in eight Propionibacterium acnes strains isolated from patients with biofilm or device-related infections. We identified different mutations in cluster I and one mutation, never reported, in cluster II of the rpoB gene (I480V) associated with the most frequent one in cluster I (S442L). Half of the patients previously received treatment with rifampicin.

Background: We investigated the in-vitro susceptibility, pharmacokinetics and treatment efficacy of daptomycin (DAP), alone and in combination with rifampin (RIF), compared to vancomycin (VAN), in a foreign-body MRSA infection model. Methods: MRSA (ATCC 43300) MIC and MBC was determined in growth media supplemented with 50 mg/l Ca2+. In guinea pigs 4 Teflon cages were s.c. implanted. Pharmacokinetics was determined in sterile cage fluid after a single i.p. dose of DAP using a bioassay. Cages were inoculated with 2.9 x 106 cfu MRSA. After 3 days of infection, i.p. treatment was started for 4 days. Bacteria were determined in cage fluid during and 5 days after treatment (12 cages/regimen). Cages were then explanted and cultured in TSB to detect adherent bacteria. Emergence of RIF resistance was tested by plating cage cultures on agar plates containing 1 µg/ml RIF. Results: MIC, MBClog and MBCstat of DAP (µg/ml) were 0.625, 0.625 and 20, respectively. After single dose of 20, 30 and 40...

Current diagnostic methods in differentiating septic from non-septic arthritis are time-consuming (culture) or have limited sensitivity (Gram stain). Microcalorimetry is a novel method that can rapidly detect microorganisms by their heat production. We investigated the accuracy and time to detection of septic arthritis by using microcalorimetry. Patients older than 18 years of age with acute arthritis of native joints were prospectively included. Synovial fluid was aspirated and investigated by Gram stain, culture and microcalorimetry. The diagnosis of septic arthritis and non-septic arthritis were made by experienced rheumatologists or orthopaedic surgeons. Septic arthritis was diagnosed by considering the finding of acute arthritis together with findings such as positive Gram stain or positive culture of synovial fluid or positive blood culture. The sensitivity and specificity for diagnosing septic arthritis and the time to positivity of microcalorimetry were determined. Of 90 pat...

Propionibacterium acnes colonizes the lipid-rich sebaceous glands of the skin. This preferential anaerobic bacterium is easily identified if cultures are prolonged. It is involved in the inflammation process of acne, but until recently, it was neglected in other clinical presentations. Despite a reported low virulence, the new genomic, transcriptomic, and phylogenetic studies have allowed better understanding of this pathogen's importance that causes many chronic and recurrent infections, including orthopedic and cardiac prosthetic, and breast or eye implant-infections. These infections, facilitated by the ability of P. acnes to produce a biofilm, require using anti-biofilm active antibiotics such as rifampicin. The antibiogram of P. acnes is not systematically performed in microbiology laboratories because of its susceptibility to a wide range of antibiotics. However, in the last 10 years, the rate of antibiotic-resistant bacteria has increased, especially for macrolides and te...

Periprosthetic infections (PPI) represent one of the most complex complications in arthroplasty concerning both, diagnosis and therapy. The incidence of PPI of the hip is approximately 1 % after primary procedures and 4 % after revision surgery. About two thirds of PPIs occur via intraoperative contamination and the remaining PPIs are acquired by hematogenous seeding. This article presents an overview of up to date evidence-based diagnostics and therapy of PPI of the hip with the establishment of a clear algorithm. A selective literature search was carried out with the inclusion of own work. A PPI must be actively excluded in cases of a painful prosthesis or signs of loosening within the first years after implantation. Measurement of C-reactive protein (CRP) can be normal especially in cases of chronic (low grade) PPI and cannot be used as an exclusion criterion. The standard diagnostic procedure includes preoperative joint aspiration with culture and leukocyte counts as well as cul...

Biofilm formation is a multi-step process influenced by surface properties. We investigated early and mature biofilm of Staphylococcus aureus on 4 different biological calcium phosphate (CaP) bone grafts used for filling bone defects. We investigated standardised cylinders of fresh and fresh-frozen human bone grafts were harvested from femoral heads; processed humanand bovine bone grafts were obtained preformed. Biofilm formation was done in tryptic soy broth (TSB) using S. aureus (ATCC 29213) with static conditions. Biofilm density after 3 h (early biofilm) and 24 h (mature biofilm) was investigated by sonication and microcalorimetry. After 3 h, bacterial density was highest on fresh-frozenandfresh bone grafts. After 24 h, biofilm density was lowest on freshbone grafts (p < 0.001) compared to the other 3 materials, which did not differ quantitatively (p > 0.05). The lowest increase in bacterial density was detected on fresh bone grafts (p < 0.001). Despite normal shaped co...

Gamma irradiation is widely used for sterilization; however, its effect on elimination of amplifiable DNA, an issue of relevance to molecular diagnostic approaches, has not been well studied. The effect of gamma irradiation on the viability of Staphylococcus epidermidis and Escherichia coli (using quantitative cultures) and on their DNA (using quantitative 16S rRNA gene PCR) was evaluated. Viability was abrogated at 2.8 and 3.6 kGy for S. epidermidis and E. coli, respectively. The radiation dose required to reduce viable bacteria by one log10 (D10 value) was 0.31 and 0.35 kGy for S. epidermidis and E. coli, respectively. D10 values for amplifiable DNA extracted from bacteria were 2.58 and 3.09 kGy for S. epidermidis and E. coli, respectively, whereas D10 values for amplifiable DNA were significantly higher for DNA extracted from irradiated viable bacterial cells (22.9 and 52.6 kGy for S. epidermidis and E. coli, respectively; P<0.001). This study showed that gamma irradiation of ...

The majority of patients with prosthetic joint replacement (arthroplasty) experience dramatic relief of pain and restoration of satisfactory joint function. In the United States, more than.5 million people have a primary arthroplasty each year. Less than 10% of prosthesis recipients have complications develop during their lifetime, commonly as a result of aseptic biomechanical failure, followed by prosthetic joint infection. The pathogenesis of prosthetic joint infection is related to bacteria in biofilms, in which they are protected from antimicrobial killing and host responses rendering these infections difficult to eradicate. Current microbiology laboratory methods for diagnosis of prosthetic joint infection depend on isolation of a pathogen by culture. However, these methods have neither ideal sensitivity nor ideal specificity. Therefore, culture-independent molecular methods have been used to improve the diagnosis of prosthetic joint infection. In the research setting, detectio...

Capsular contracture is a feared complication following both reconstructive and aesthetic breast surgery. The etiology is uncertain, but bacterial biofilms have been suggested as trigger for chronic peri-implant inflammation, eventually leading to capsular contracture. Data were extracted from patient records included in a prospective cohort between 2008 and 2010. We compared patients who underwent submuscular breast reconstruction using expander implants and those needing implant removal for capsular contracture after aesthetic submuscular breast augmentation. Of 36 included breast implants from 27 patients, 18 implants were inserted for reconstructive reasons and 18 for aesthetic reasons. The median indwelling time was 3 years for aesthetic implants and 3 months for reconstructive expanders. Overall, sonication cultures were positive in 13 implants (36%). In aesthetic implants, sonication cultures were positive in 28% and sonication cultures were positive in expander implants in 44%. Propionibacterium acnes and coagulasenegative staphylococci were predominant. Sonication cultures were positive in approximately 33% of removed breast implants and were comparable for reconstructive expander and aesthetic implants. These findings support the hypothesis that bacterial biofilms play a role in the pathogenesis of capsular contracture, especially after expander reconstruction, as these implants are at the highest risk of contamination during repeated implant-filling procedures.

Emergency treatment of major sub-/total traumatic amputations continue to represent a clinical challenge due to high infection rates and serious handicaps. Effective treatment is based on two columns: surgery and antimicrobial therapy. Detailed identification of pathogen spectrum and epidemiology associated with these injuries is of tremendous importance as it guides the initial empiric antibiotic regimen and prevents adverse septic effents. In this retrospective study 51 patients with major traumatic amputations (n = 16) and subtotal amputations (n = 35) treated from 2001 to 2010 in our trauma center were investigated. All patients received emergency surgery, debridement with microbiological testing within 6 h after admission and empircic antimicrobial therapy. Additionally to baseline patient characteristics, the incidence of positive standardized microbiologic testing combined with clinical signs of infection, pathogen spectrum, administered antimicrobial agents and clinical comp...

No earlier study has investigated the microbiology of negative pressure wound therapy (NPWT) foam using a standardized manner. The purpose of this study is to investigate the bacterial load and microbiological dynamics in NPWT foam removed from chronic wounds (>3 months). To determine the bacterial load, a standardized size of the removed NPWT foam was sonicated. The resulting sonication fluid was cultured, and the colony-forming units (CFU) of each species were enumerated. Sixty-eight foams from 17 patients (mean age 63 years, 71% males) were investigated. In 65 (97%) foams, ≥ 1 and in 37 (54%) ≥ 2 bacterial types were found. The bacterial load remained high during NPWT treatment, ranging from 10(4) to 10(6) CFU/ml. In three patients (27%), additional type of bacteria was found in subsequent foam cultures. The mean bacterial count ± standard deviation was higher in polyvinyl alcohol foam (6.1 ± 0.5 CFU/ml) than in polyurethane (5.5 ± 0.8 CFU/ml) (p = 0.02). The mean of log of sum of CFU/ml in foam from 125 mmHg (5.5 ± 0.8) was lower than in foam from 100 mmHg pressure (5.9 ± 0.5) (p = 0.01). Concluding, bacterial load remains high in NPWT foam, and routine changing does not reduce the load.

The use of biomarkers provides a novel approach to diagnosing infection, its severity and treatment response. Biomarkers, especially procalcitonin and, to a lesser extent, C-reactive protein and interleukin 8, can improve the diagnostic and prognostic assessment of bloodstream infections. Both strengths and weaknesses of biomarkers must be recognized for rational and safe use in clinical settings. Cut-off ranges must be chosen within the specific clinical context. Ultrasensitive assays for procalcitonin, capable of measuring low levels in healthy individuals, may help to identify even ‘subclinical’ inflammatory states before the development of clinically evident sepsis. For immunocompromised patients, the use of biomarkers could lead to an earlier and more targeted antimicrobial therapy for patients at risk of sepsis, whereas those patients with viral illness or a non-infectious aetiology of inflammation who maintain low levels of procalcitonin over time can be withheld from antibio...

The activity of dalbavancin, a representative of the lipoglycopeptide antibiotics, alone and in combination with rifampicin, was investigated against meticillin-resistant Staphylococcus aureus (MRSA) in a foreign-body infection model in guinea pigs. The MIC, MBC and time-kill profile of dalbavancin were determined for MRSA ATCC 43300 in the logarithmic (MBC(log)) and stationary (MBC(stat)) growth phases. The pharmacokinetic profile of dalbavancin was determined in sterile cage fluid in guinea pigs. The activity of intraperitoneal dalbavancin (40, 60 or 80 mg/kg as a single dose), rifampicin (12.5 mg/kg/12 h for 4 days) and their combination was assessed against planktonic and biofilm MRSA. The MIC of dalbavancin was 0.078 mg/L; MBC(log) and MBC(stat) were both >128× MIC. In time-kill studies, bacterial reduction of 3log(10)CFU/mL was achieved after 48 h at ≥32× MIC (logarithmic growth) and at ≥1× MIC (stationary growth). Dalbavancin was neither synergistic nor antagonistic with rifampicin, and prevented the emergence of rifampicin resistance in vitro. The half-life of dalbavancin in cage fluid was 35.8-45.4 h and the concentration remained above the MIC of MRSA during 7 days after a single dose. Dalbavancin reduced planktonic MRSA in cage fluid at high dose (60 mg/kg and 80 mg/kg) but failed to eradicate biofilm MRSA from cages. In combination with rifampicin, dalbavancin at 80 mg/kg cured 36% of infected cages, and emergence of rifampicin resistance was completely prevented. Dalbavancin at 80 mg/kg and in combination with rifampicin eradicated approximately one-third of cage-associated MRSA infections and prevented emergence of rifampicin resistance.

We evaluated the activity of antifungals alone or in combination against Aspergillus fumigatus and Aspergillus terreus by real-time measurement of fungal growth-related heat production. Amphotericin B, voriconazole, caspofungin, and anidulafungin were tested alone or in combination. Heat production was measured in Sabouraud dextrose broth containing 10(5)Aspergillus conidia/mL for 48 h at 37 °C. Antifungal activity was evaluated by measuring the heat detection time relative to the growth control. Against A. fumigatus, the voriconazole-echinocandin combination demonstrated longer heat detection time than each antifungal alone. Against A. terreus, the combination amphotericin B-echinocandin prolonged the heat detection time, compared to each antifungal alone. In contrast, the echinocandin-voriconazole combination did not increase the heat detection time, compared to voriconazole alone. None of the antifungal combinations decreased the heat detection time compared to the antifungals alone (e.g. antagonism was not observed). Microcalorimetry has the potential for real-time evaluation of antifungal combinations against Aspergillus spp.

Enterococcal implant-associated infections are difficult to treat because antibiotics generally lack activity against enterococcal biofilms. We investigated fosfomycin, rifampin, and their combinations against planktonic and adherent Enterococcus faecalis (ATCC 19433) in vitro and in a foreign-body infection model. The MIC/MBClog values were 32/>512 μg/ml for fosfomycin, 4/>64 μg/ml for rifampin, 1/2 μg/ml for ampicillin, 2/>256 μg/ml for linezolid, 16/32 μg/ml for gentamicin, 1/>64 μg/ml for vancomycin, and 1/5 μg/ml for daptomycin. In time-kill studies, fosfomycin was bactericidal at 8× and 16× MIC, but regrowth of resistant strains occurred after 24 h. With the exception of gentamicin, no complete inhibition of growth-related heat production was observed with other antimicrobials on early (3 h) or mature (24 h) biofilms. In the animal model, fosfomycin alone or in combination with daptomycin reduced planktonic counts by ≈4 log10 CFU/ml below the levels before treatment. Fosfomycin cleared planktonic bacteria from 74% of cage fluids (i.e., no growth in aspirated fluid) and eradicated biofilm bacteria from 43% of cages (i.e., no growth from removed cages). In combination with gentamicin, fosfomycin cleared 77% and cured 58% of cages; in combination with vancomycin, fosfomycin cleared 33% and cured 18% of cages; in combination with daptomycin, fosfomycin cleared 75% and cured 17% of cages. Rifampin showed no activity on planktonic or adherent E. faecalis, whereas in combination with daptomycin it cured 17% and with fosfomycin it cured 25% of cages. Emergence of fosfomycin resistance was not observed in vivo. In conclusion, fosfomycin showed activity against planktonic and adherent E. faecalis. Its role against enterococcal biofilms should be further investigated, especially in combination with rifampin and/or daptomycin treatment.