Heidi Misteli

The timing of surgical antimicrobial prophylaxis.

Objective:To obtain precise information on the optimal time window for surgical antimicrobial prophylaxis. Summary Background Data:Although perioperative antimicrobial prophylaxis is a well-established strategy for reducing the risk of surgical site infections (SSI), the optimal timing for this procedure has yet to be precisely determined. Under todays recommendations, antibiotics may be administered within the final 2 hours before skin incision, ideally as close to incision time as possible. Methods:In this prospective observational cohort study at Basel University Hospital we analyzed the incidence of SSI by the timing of antimicrobial prophylaxis in a consecutive series of 3836 surgical procedures. Surgical wounds and resulting infections were assessed to Centers for Disease Control and Prevention standards. Antimicrobial prophylaxis consisted in single-shot administration of 1.5 g of cefuroxime (plus 500 mg of metronidazole in colorectal surgery). Results:The overall SSI rate was 4.7% (180 of 3836). In 49% of all procedures antimicrobial prophylaxis was administered within the final half hour. Multivariable logistic regression analyses showed a significant increase in the odds of SSI when antimicrobial prophylaxis was administered less than 30 minutes (crude odds ratio = 2.01; adjusted odds ratio = 1.95; 95% confidence interval, 1.4–2.8; P < 0.001) and 120 to 60 minutes (crude odds ratio = 1.75; adjusted odds ratio = 1.74; 95% confidence interval, 1.0–2.9; P = 0.035) as compared with the reference interval of 59 to 30 minutes before incision. Conclusions:When cefuroxime is used as a prophylactic antibiotic, administration 59 to 30 minutes before incision is more effective than administration during the last half hour.

Surgical Glove Perforation and the Risk of Surgical Site Infection

HYPOTHESIS Clinically apparent surgical glove perforation increases the risk of surgical site infection (SSI). DESIGN Prospective observational cohort study. SETTING University Hospital Basel, with an average of 28,000 surgical interventions per year. PARTICIPANTS Consecutive series of 4147 surgical procedures performed in the Visceral Surgery, Vascular Surgery, and Traumatology divisions of the Department of General Surgery. MAIN OUTCOME MEASURES The outcome of interest was SSI occurrence as assessed pursuant to the Centers of Disease Control and Prevention standards. The primary predictor variable was compromised asepsis due to glove perforation. RESULTS The overall SSI rate was 4.5% (188 of 4147 procedures). Univariate logistic regression analysis showed a higher likelihood of SSI in procedures in which gloves were perforated compared with interventions with maintained asepsis (odds ratio [OR], 2.0; 95% confidence interval [CI], 1.4-2.8; P < .001). However, multivariate logistic regression analyses showed that the increase in SSI risk with perforated gloves was different for procedures with vs those without surgical antimicrobial prophylaxis (test for effect modification, P = .005). Without antimicrobial prophylaxis, glove perforation entailed significantly higher odds of SSI compared with the reference group with no breach of asepsis (adjusted OR, 4.2; 95% CI, 1.7-10.8; P = .003). On the contrary, when surgical antimicrobial prophylaxis was applied, the likelihood of SSI was not significantly higher for operations in which gloves were punctured (adjusted OR, 1.3; 95% CI, 0.9-1.9; P = .26). CONCLUSION Without surgical antimicrobial prophylaxis, glove perforation increases the risk of SSI.

Differential effects of the tryptophan metabolite 3-hydroxyanthranilic acid on the proliferation of human CD8 + T cells induced by TCR triggering or homeostatic cytokines

Production of indoleamine 2,3‐dioxygenase (IDO) by tumor cells, leading to tryptophan depletion and production of immunosuppressive metabolites, may facilitate immune tolerance of cancer. IDO gene is also expressed in dendritic cells (DC) upon maturation induced by lipopolysaccarides or IFN. We investigated IDO gene expression in melanoma cell lines and clinical specimens as compared to mature DC (mDC). Furthermore, we explored effects of L‐kynurenine (L‐kyn) and 3‐hydroxyanthranilic acid (3‐HAA) on survival and antigen‐dependent and independent proliferation of CD8+ cells. We observed that IDO gene expression in cultured tumor cells and freshly excised samples is orders of magnitude lower than in mDC, providing highly efficient antigen presentation to CD8+ T cells. Non toxic concentrations of L‐kyn or 3‐HAA did not significantly inhibit antigen‐specific CTL responses. However, 3‐HAA, but not L‐kyn markedly inhibited antigen‐independent proliferation of CD8+ T cells induced by common receptor γ‐chain cytokines IL‐2, ‐7 and ‐15. Our data suggest that CD8+ T cell activation induced by antigenic stimulation, a function exquisitely fulfilled by mDC, is unaffected by tryptophan metabolites. Instead, in the absence of effective T cell receptor triggering, 3‐HAA profoundly affects homeostatic proliferation of CD8+ T cells.

The association of preoperative anemia and perioperative allogeneic blood transfusion with the risk of surgical site infection

BACKGROUND: The purpose of the study was to investigate allogeneic blood transfusion (ABT) and preoperative anemia as risk factors for surgical site infection (SSI).

High-throughput flow cytometry purification of transduced progenitors expressing defined levels of vascular endothelial growth factor induces controlled angiogenesis in vivo

Delivery of therapeutic genes by genetically modified progenitors is a powerful tool for regenerative medicine. However, many proteins remain localized within or around the expressing cell, and heterogeneous expression levels can lead to reduced efficacy or increased toxicity. For example, the matrix‐binding vascular endothelial growth factor (VEGF) can induce normal, stable, and functional angiogenesis or aberrant angioma growth depending on its level of expression in the microenvironment around each producing cell, and not on its total dose. To overcome this limitation, we developed a flow cytometry–based method to rapidly purify transduced cells expressing desired levels of a therapeutic transgene. Primary mouse myoblasts were transduced with a bicistronic retrovirus expressing VEGF linked to a nonfunctional, truncated form of the syngenic molecule CD8a. By using a clonal population uniformly expressing a known VEGF level as a reference, cells producing similar VEGF amounts were rapidly sorted from the primary population on the basis of their CD8a fluorescence intensity. A single round of sorting with a suitably designed gate yielded a purified population that induced robust, normal, and stable angiogenesis, and completely avoided angioma growth, which was instead always caused by the heterogeneous parent population. This clinically applicable high‐throughput technique allowed the delivery of highly controlled VEGF levels in vivo, leading to significantly improved safety without compromising efficacy. Furthermore, when applied to other suitable progenitor populations, this technique could help overcome a significant obstacle in the development of safe and efficacious vascularization strategies in the fields of regenerative medicine and tissue engineering. STEM CELLS 2010;28:611–619

Prevention and control of surgical site infections: review of the Basel Cohort Study.

INTRODUCTION Surgical site infections (SSI) are the most common hospital-acquired infections among surgical patients, with significant impact on patient morbidity and health care costs. The Basel SSI Cohort Study was performed to evaluate risk factors and validate current preventive measures for SSI. The objective of the present article was to review the main results of this study and its implications for clinical practice and future research. SUMMARY OF METHODS OF THE BASEL SSI COHORT STUDY The prospective observational cohort study included 6,283 consecutive general surgery procedures closely monitored for evidence of SSI up to 1 year after surgery. The dataset was analysed for the influence of various potential SSI risk factors, including timing of surgical antimicrobial prophylaxis (SAP), glove perforation, anaemia, transfusion and tutorial assistance, using multiple logistic regression analyses. In addition, post hoc analyses were performed to assess the economic burden of SSI, the efficiency of the clinical SSI surveillance system, and the spectrum of SSI-causing pathogens. REVIEW OF MAIN RESULTS OF THE BASEL SSI COHORT STUDY The overall SSI rate was 4.7% (293/6,283). While SAP was administered in most patients between 44 and 0 minutes before surgical incision, the lowest risk of SSI was recorded when the antibiotics were administered between 74 and 30 minutes before surgery. Glove perforation in the absence of SAP increased the risk of SSI (OR 2.0; CI 1.4-2.8; p <0.001). No significant association was found for anaemia, transfusion and tutorial assistance with the risk of SSI. The mean additional hospital cost in the event of SSI was CHF 19,638 (95% CI, 8,492-30,784). The surgical staff documented only 49% of in-hospital SSI; the infection control team registered the remaining 51%. Staphylococcus aureus was the most common SSI-causing pathogen (29% of all SSI with documented microbiology). No case of an antimicrobial-resistant pathogen was identified in this series. CONCLUSIONS The Basel SSI Cohort Study suggested that SAP should be administered between 74 and 30 minutes before surgery. Due to the observational nature of these data, corroboration is planned in a randomized controlled trial, which is supported by the Swiss National Science Foundation. Routine change of gloves or double gloving is recommended in the absence of SAP. Anaemia, transfusion and tutorial assistance do not increase the risk of SSI. The substantial economic burden of in-hospital SSI has been confirmed. SSI surveillance by the surgical staff detected only half of all in-hospital SSI, which prompted the introduction of an electronic SSI surveillance system at the University Hospital of Basel and the Cantonal Hospital of Aarau. Due to the absence of multiresistant SSI-causing pathogens, the continuous use of single-shot single-drug SAP with cefuroxime (plus metronidazole in colorectal surgery) has been validated.

FACS‐purified myoblasts producing controlled VEGF levels induce safe and stable angiogenesis in chronic hind limb ischemia

We recently developed a method to control the in vivo distribution of vascular endothelial growth factor (VEGF) by high throughput Fluorescence‐Activated Cell Sorting (FACS) purification of transduced progenitors such that they homogeneously express specific VEGF levels. Here we investigated the long‐term safety of this method in chronic hind limb ischemia in nude rats. Primary myoblasts were transduced to co‐express rat VEGF‐A164 (rVEGF) and truncated ratCD8a, the latter serving as a FACS‐quantifiable surface marker. Based on the CD8 fluorescence of a reference clonal population, which expressed the desired VEGF level, cells producing similar VEGF levels were sorted from the primary population, which contained cells with very heterogeneous VEGF levels. One week after ischemia induction, 12 × 106 cells were implanted in the thigh muscles. Unsorted myoblasts caused angioma‐like structures, whereas purified cells only induced normal capillaries that were stable after 3 months. Vessel density was doubled in engrafted areas, but only approximately 0.1% of muscle volume showed cell engraftment, explaining why no increase in total blood flow was observed. In conclusion, the use of FACS‐purified myoblasts granted the cell‐by‐cell control of VEGF expression levels, which ensured long‐term safety in a model of chronic ischemia. Based on these results, the total number of implanted cells required to achieve efficacy will need to be determined before a clinical application.

Spectrum of pathogens in surgical site infections at a Swiss university hospital

BACKGROUND The type of surgical antimicrobial prophylaxis (SAP) is determined by the spectrum and antimicrobial resistance of pathogens causing surgical site infections (SSI). The aim of this study was to define the microbiological features of SSI in general surgery patients at Basel University Hospital in order to validate our current strategy of single-shot SAP with 1.5 g cefuroxime (plus 500 mg metronidazole in colorectal surgery). METHODS A prospective observational cohort of consecutive vascular, visceral and trauma procedures was analysed to evaluate the incidence of SSI. Surgical wounds and resulting infections were assessed to centres for disease control standards. Microbiological evaluation was performed by microscopic direct preparation, cultures and testing for antibiotic resistance. RESULTS A total of 293 instances of SSI were detected in this cohort of 6283 surgical procedures (4.7%). Microbiological species were identified in 129 of 293 SSI (44%). Staphylococcus aureus (29.5%) was the most common pathogen causing SSI in trauma and vascular surgery, whereas Escherichia coli (20.9%) was more frequently responsible for SSI in visceral surgery. Importantly, not a single case of SSI was caused by antimicrobial-resistant pathogens in this series. CONCLUSIONS The spectrum of pathogens causing SSI identified and the very low incidence of antimicrobial resistance at Basel University Hospital validate the continuous use of single-shot single-drug SAP with cefuroxime (plus metronidazole in colorectal surgery).

Timing of surgical antimicrobial prophylaxis: a phase 3 randomised controlled trial

BACKGROUND Based on observational studies, administration of surgical antimicrobial prophylaxis (SAP) for the prevention of surgical site infection (SSI) is recommended within 60 min before incision. However, the precise optimum timing is unknown. This trial compared early versus late administration of SAP before surgery. METHODS In this phase 3 randomised controlled superiority trial, we included general surgery adult inpatients (age ≥18 years) at two Swiss hospitals in Basel and Aarau. Patients were randomised centrally and stratified by hospital according to a pre-existing computer-generated list in a 1:1 ratio to receive SAP early in the anaesthesia room or late in the operating room. Patients and the outcome assessment team were blinded to group assignment. SAP consisted of single-shot, intravenous infusion of 1·5 g of cefuroxime, a commonly used cephalosporin with a short half-life, over 2-5 min (combined with 500 mg metronidazole in colorectal surgery). The primary endpoint was the occurrence of SSI within 30 days of surgery. The main analyses were by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT01790529. FINDINGS Between Feb 21, 2013, and Aug 3, 2015, 5580 patients were randomly assigned to receive SAP early (2798 patients) or late (2782 patients). 5175 patients (2589 in the early group and 2586 in the late group) were analysed. Median administration time was 42 min before incision in the early group (IQR 30-55) and 16 min before incision in the late group (IQR 10-25). Inpatient follow-up rate was 100% (5175 of 5175 patients); outpatient 30-day follow-up rate was 88·8% (4596 of 5175), with an overall SSI rate of 5·1% (234 of 4596). Early administration of SAP did not significantly reduce the risk of SSI compared with late administration (odds ratio 0·93, 95% CI 0·72-1·21, p=0·601). INTERPRETATION Our findings do not support any narrowing of the 60-min window for the administration of a cephalosporin with a short half-life, thereby obviating the need for increasingly challenging SAP timing recommendations. FUNDING Swiss National Science Foundation, Hospital of Aarau, University of Basel, Gottfried und Julia Bangerter-Rhyner Foundation, Hippocrate Foundation, and Nora van Meeuwen-Häfliger Foundation.

Therapeutische Angiogenese mittels FACS-sortierter transduzierter Myoblasten

Background: Therapeutic angiogenesis aims at generating new blood vessels by delivering growth factors such as Vascular Endothelial Growth Factor (VEGF), and thus is a promising treatment for patients with peripheral arterial disease. It has previously been shown that a discrete threshold in VEGF dose exists, below which normal stable capillaries are induced and above which angioma growth occurs. However, VEGF dose must be homogeneously distributed in the microenvironment, as VEGF remains localized around each producing cell. By using retrovirally-transduced myoblasts, normal angiogenesis can be induced by implanting clonal populations, in which every cell expresses the same VEGF level. While providing proof-of-concept, this approach is not directly applicable to clinical practice. Therefore, we developed a Fluorescence Activated Cell Sorting (FACS)-based method to allow the rapid isolation of engineered myoblasts homogeneously expressing predictable levels of VEGF from a heterogeneous primary population in vitro, in order to achieve controlled microen-vironmental VEGF doses in vivo. Methods: Primary mouse myoblasts were transduced with a retroviral construct in which the VEGF164 gene is linked through an Internal Ribosomal Entry Site (IRES) to a truncated version of CD8a, acting as a reporter gene. Therefore, changes in the level of VEGF expression are reflected by the cell-surface expression of CD8a, which can be detected and quantified on live cells by FACS. Results: To determine the relationship between expression of VEGF (by ELISA) and that of CD8 (by FACS) at the single cell level, a standard curve was produced by isolating clones from all regions of the expression spectrum of the heterogeneous polyclonal population. VEGF and CD8 expression were found to be stable during expansion over 49 cell doublings in 5 sample clones producing levels of VEGF in a range of 2–142 ng/106 cells/day. We determined the VEGF dose at which aberrant angiogenesis is induced in vivo, representing the upper threshold above which safety is lost. The clone which produced the highest VEGF amount (∼40 ng/106 cells/day), while at the same time completely avoiding aberrant angiogenesis (highest safe dose) was selected as a reference to identify and sort the cells expressing similar levels of CD8a, and therefore of VEGF, out of the heterogeneous polyclonal population. We tested up to 3 successive rounds of sorting, with 2 different gates (large and narrow), in order to optimize the purity of the selection. VEGF and CD8a expression of the sorted cells was stable during in vitro expansion over 23 doublings. When implanted in vivo, the heterogeneous population always caused angioma growth by four weeks. In contrast, the sorted populations caused exclusively normal angiogenesis in 100 % of the animals implanted with narrowgate cells and 65 % with large-gate cells after four weeks. Therefore, selective sorting with a narrow gate appears to reliably avoid expression of excessive VEGF levels. Conclusion: The ability to isolate homogeneous populations expressing predictable levels of the transgene will allow the optimization of both efficacy and safety in cell-based strategies for VEGF gene delivery.