The effect of early broad-spectrum versus delayed narrow-spectrum antibiotic therapy on the primary cure rate of acute infection after osteosynthesis

Purpose: Infection near metal implants is a problem that presents challenging treatment dilemmas for physicians. The aim of this study was to analyse the efficacy of two treatment protocols for acute fracture-related infections. Methods: Seventy-one patients in two level-1 trauma centres in the Netherlands were retrospectively included in this study. These trauma centres had different standardised protocols for acute infection after osteosynthesis: 39 patients were selected from protocol A and 32 from protocol B. Both protocols involve immediate surgical debridement and soft tissue coverage, but differ in antibiotic approach: (A) immediate empirical combination antibiotic therapy with rifampicin, or (B) postponed (1–5 days) targeted antibiotic therapy. The primary outcome of these protocols was success, defined as a fracture healing in the absence of infection. The secondary outcome was antibiotic resistance patterns. Logistic regression was conducted on patients and treatment-related factors in association with primary success. Results: Primary success was achieved in 72% of protocol A patients, in 47% of those in protocol B (P = 0.033), and with prolongation of treatment success was achieved in 90% and 78% of patients, respectively. Protocol A exhibited a better primary success rate (adjusted OR 3.45, CI 1.13–10.52) when adjusted for age and soft tissue injury. There was no significant difference in antibiotic resistance between the two protocols. Conclusion: Both protocols yielded high overall success rates. Immediate empirical antibiotics can be used safely without additional bacterial resistance and may contribute to increased success rates

The dynamic interaction between host and pathogens after trauma

The neutrophil is the first line of defense against invading bacteria. Neutrophils are recognized by its granules and characteristic nuclear segmentation. Neutrophils’ main functions are to phagocytize and, after fusion of the phagolysosome, subsequently degrade the bacteria. After severe trauma, neutrophil dysfunction can occur. A possible consequence is an increased susceptibility to bacterial infections. Part 1 describes heterogeneity within the neutrophil pool in both health and disease. The heterogeneity can be based on receptor expression, cell density, nuclear segmentation, age or their influence on other cells(e.g. T- or cancer cells) (Chapter 2). The term ‘subset’ refers to neutrophils with similar physical characteristics and can mostly be found in inflammatory conditions. In homeostasis ‘subsets’ are much less clear. We found that there is indeed heterogeneity between circulating neutrophils, be it in a more gradual form. A process that was named competitive phagocytosis showed that within the circulating neutrophil pool of healthy adults some neutrophils are more able to phagocytize bacteria compared to others in a non-random way (Chapter 3). Another much studied neutrophil ‘subset’ in disease is the low-density neutrophil, that segregates in the mononuclear layer during density centrifugation. We confirmed the hypothesis that there is a spectrum in neutrophil density in homeostasis which can be related to neutrophil function (bacterial containment, T-cell suppression)(Chapter 4). In Chapter 5 we investigated the accuracy of an automated hematology analyzer in the detection of banded neutrophils after trauma. The automated analyzer was unable to detect banded neutrophils after trauma, were it did accurately detect banded in infection. Suggesting a structural difference between the cells under different conditions. The underlying mechanisms or purpose of this heterogeneity is still not fully understood. In Part 2 the focus is shifted towards infectious complications after trauma, with a special emphasis on fracture-related infections (FRI). Staphylococcus aureus is one of the most important causative pathogens in FRI and is also known for causing metastatic infections. It is hypothesized that this is due to intracellular survival of the bacterium. Chapter 6 reviews the cellular penetration and efficacy against intracellular S. aureus of the most relevant antibiotics. Bacterial identification in FRI is of paramount importance for choosing effective (antibacterial) treatment. In our hospital a standardized tissue sampling protocol for bacterial identification in FRI was introduced. The protocol consisted of obtaining five or more separate, intra-operative deep tissue samples, avoidance of cross-contamination, and a specific culture protocol. Evaluation of the protocol showed increased awareness of the problem at hand in both surgeons and microbiologists, and increased certainty on causative micro-organisms (Chapter 7). In Chapter 8 & 9 the effect of two different protocols for the treatment of FRI were evaluated. The use of early broad-spectrum antibiotics was safe in terms of emergence of antibiotic resistance. Based on our findings, we recommend starting empiric broad-spectrum antibiotic therapy directly after tissue sampling and surgical debridement. Antibiotic therapy should at least cover infections with the most important causative pathogen (S. aureus) and the presence of a potential biofilm should be considered

Neutrophil functional heterogeneity : Identification of competitive phagocytosis

Introduction: Phagocytosis by neutrophils is a key process in the innate immune response against invading microorganisms. Despite reported heterogeneity in other neutrophils functions, little is known regarding differences in phagocytosis by individual cells. Therefore, we tested the hypothesis that heterogeneity is present in the neutrophil compartment in its potency to phagocytize bacteria. Methods: Phagocytosis assays were performed in suspension with isolated neutrophils and Staphylococcus aureus expressing different fluorescent proteins at MOIs between 1 and 10. Repetitive addition of bacteria with different fluorescent proteins and MOIs was used to compare the phagocytic capacity of S. aureus-green fluorescent protein (GFP)-positive and negative neutrophils and exclude randomness. Results: The percentage and mean fluorescence intensity (MFI) of S. aureus-GFP-positive neutrophils increased with higher MOIs. The increase in MFI was due to phagocytosis of multiple bacteria per neutrophil as was confirmed by confocal imaging. Sequential phagocytosis of GFP- and mCherry-expressing S. aureus showed a non-random process, as S. aureus-GFP-positive neutrophils preferentially phagocytized S. aureus-mCherry. Conclusion: All neutrophils were able to phagocytize S. aureus, but some were much more potent than others. Therefore, at physiologically relevant MOIs these potent phagocytizing neutrophils will outcompete the uptake of bacteria by less competent cells in a process we propose to name "competitive phagocytosis.

Effect of a standardized treatment regime for infection after osteosynthesis

BACKGROUND: Infection after osteosynthesis is an important complication with significant morbidity and even mortality. These infections are often caused by biofilm-producing bacteria. Treatment algorithms dictate an aggressive approach with surgical debridement and antibiotic treatment. The aim of this study is to analyze the effect of such an aggressive standardized treatment regime with implant retention for acute, existing <3 weeks, infection after osteosynthesis. METHODS: We conducted a retrospective 2-year cohort in a single, level 1 trauma center on infection occurring within 12 months following any osteosynthesis surgery. The standardized treatment regime consisted of implant retention, thorough surgical debridement, and immediate antibiotic combination therapy with rifampicin. The primary outcome was success. Success was defined as consolidation of the fracture and resolved symptoms of infection. Culture and susceptibility testing were performed to identify bacteria and resistance patterns. Univariate analysis was conducted on patient-related factors in association with primary success and antibiotic resistance. RESULTS: Forty-nine patients were included for analysis. The primary success rate was 63% and overall success rate 88%. Factors negatively associated with primary success were the following: Gustilo classification (P = 0.023), higher number of debridements needed (P = 0.015), inability of primary closure (P = 0.017), and subsequent application of vacuum therapy (P = 0.030). Adherence to the treatment regime was positively related to primary success (P = 0.034). CONCLUSIONS: The described treatment protocol results in high success rates, comparable with success rates achieved in staged exchange in prosthetic joint infection treatment

Effect of a standardized treatment regime for infection after osteosynthesis

BACKGROUND: Infection after osteosynthesis is an important complication with significant morbidity and even mortality. These infections are often caused by biofilm-producing bacteria. Treatment algorithms dictate an aggressive approach with surgical debridement and antibiotic treatment. The aim of this study is to analyze the effect of such an aggressive standardized treatment regime with implant retention for acute, existing <3 weeks, infection after osteosynthesis. METHODS: We conducted a retrospective 2-year cohort in a single, level 1 trauma center on infection occurring within 12 months following any osteosynthesis surgery. The standardized treatment regime consisted of implant retention, thorough surgical debridement, and immediate antibiotic combination therapy with rifampicin. The primary outcome was success. Success was defined as consolidation of the fracture and resolved symptoms of infection. Culture and susceptibility testing were performed to identify bacteria and resistance patterns. Univariate analysis was conducted on patient-related factors in association with primary success and antibiotic resistance. RESULTS: Forty-nine patients were included for analysis. The primary success rate was 63% and overall success rate 88%. Factors negatively associated with primary success were the following: Gustilo classification (P = 0.023), higher number of debridements needed (P = 0.015), inability of primary closure (P = 0.017), and subsequent application of vacuum therapy (P = 0.030). Adherence to the treatment regime was positively related to primary success (P = 0.034). CONCLUSIONS: The described treatment protocol results in high success rates, comparable with success rates achieved in staged exchange in prosthetic joint infection treatment

Intracellular Penetration and Effects of Antibiotics on <i>Staphylococcus aureus</i> Inside Human Neutrophils: A Comprehensive Review

Neutrophils are important assets in defense against invading bacteria like staphylococci. However, (dysfunctioning) neutrophils can also serve as reservoir for pathogens that are able to survive inside the cellular environment. Staphylococcus aureus is a notorious facultative intracellular pathogen. Most vulnerable for neutrophil dysfunction and intracellular infection are immune-deficient patients or, as has recently been described, severely injured patients. These dysfunctional neutrophils can become hide-out spots or &#8220;Trojan horses&#8221; for S. aureus. This location offers protection to bacteria from most antibiotics and allows transportation of bacteria throughout the body inside moving neutrophils. When neutrophils die, these bacteria are released at different locations. In this review, we therefore focus on the capacity of several groups of antibiotics to enter human neutrophils, kill intracellular S. aureus and affect neutrophil function. We provide an overview of intracellular capacity of available antibiotics to aid in clinical decision making. In conclusion, quinolones, rifamycins and sulfamethoxazole-trimethoprim seem very effective against intracellular S. aureus in human neutrophils. Oxazolidinones, macrolides and lincosamides also exert intracellular antibiotic activity. Despite that the reviewed data are predominantly of in vitro origin, these findings should be taken into account when intracellular infection is suspected, as can be the case in severely injured patients

Intracellular Penetration and Effects of Antibiotics on Staphylococcus aureus Inside Human Neutrophils : A Comprehensive Review

Neutrophils are important assets in defense against invading bacteria like staphylococci. However, (dysfunctioning) neutrophils can also serve as reservoir for pathogens that are able to survive inside the cellular environment. Staphylococcus aureus is a notorious facultative intracellular pathogen. Most vulnerable for neutrophil dysfunction and intracellular infection are immune-deficient patients or, as has recently been described, severely injured patients. These dysfunctional neutrophils can become hide-out spots or "Trojan horses" for S. aureus. This location offers protection to bacteria from most antibiotics and allows transportation of bacteria throughout the body inside moving neutrophils. When neutrophils die, these bacteria are released at different locations. In this review, we therefore focus on the capacity of several groups of antibiotics to enter human neutrophils, kill intracellular S. aureus and affect neutrophil function. We provide an overview of intracellular capacity of available antibiotics to aid in clinical decision making. In conclusion, quinolones, rifamycins and sulfamethoxazole-trimethoprim seem very effective against intracellular S. aureus in human neutrophils. Oxazolidinones, macrolides and lincosamides also exert intracellular antibiotic activity. Despite that the reviewed data are predominantly of in vitro origin, these findings should be taken into account when intracellular infection is suspected, as can be the case in severely injured patients

Automated flow cytometry enables high performance point-of-care analysis of leukocyte phenotypes

Introduction: Phagocytes such as granulocytes and monocytes are fundamental players in the innate immune system. Activation of these cells can be quantified by the measurement of activation marker expression using flow cytometry. Analysis of receptor expression on inflammatory cells facilitates the diagnosis of inflammatory diseases and can be used to determine the extent of inflammation. However, several major limitations of this analysis precludes application of inflammation monitoring in clinical practice. Fast and automated analysis would minimalize ex vivo manipulation and allow reproducible processing. The aim of this study was to evaluate a fully automated “load & go” flow cytometer for analyzing activation of granulocytes and monocytes in a clinically applicable setting. Methods: Blood samples were obtained from 10 anonymous and healthy volunteers between the age of 18 and 65 years. Granulocyte and monocyte activation was determined by the use of the markers CD35, CD11b and CD10 measured in the automated AQUIOS CL® “load & go” flow cytometer. This machine is able to pierce the tube caps, add antibodies, lyse and measure the sample within 20 min after vena puncture. Reproducibility tests were performed to test the stability of activation marker expression on phagocytes. The expression of activation markers was measured at different time points after blood drawing to analyze the effect of bench time on granulocyte and monocyte activation. Results: The duplicate experiments demonstrate a high reproducibility of the measurements of the activation state of phagocytes. Healthy controls showed a very homogenous expression of activation markers at T = 0 (immediately after vena puncture). Activation markers on neutrophils were already significantly increased after 1 h (T = 1) depicted as means (95%Cl) CD35: 2.2× (1.5×-2.5×) p =.028, CD11b: 2.5× (1.7×-3.1×) p =.023, CD10: 2.5× (2.1×-2.7×) p =.009) and a further increase in activation markers was observed after 2 and 3 h. Monocytes also showed a increase in activation markers in 1 h (mean (95%Cl) CD35: 1.8× (1.3×–2.2×) p =.058, CD11b: 2.13× (1.6×–2.4×) p =.025) and also a further significant increase in 2 and 3 h was observed. Conclusion: This study showed that bench time of one hour already leads to a significant upregulation and bigger variance in activation markers of granulocytes and monocytes. In addition, it is likely that automated flow cytometry reduces intra-assay variability in the analysis of activation markers on inflammatory cells. Therefore, we found that it is of utmost importance to perform immune activation analysis as fast as possible to prevent drawing wrong conclusions. Automated flow cytometry is able to reduce this analysis from 2 h to only 15–20 min without the need of dedicated personnel and in a point-of-care context. This now allows fast and automated inflammation monitoring in blood samples obtained from a variety of patient groups. Fund: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors

Automated flow cytometry enables high performance point-of-care analysis of leukocyte phenotypes

Introduction: Phagocytes such as granulocytes and monocytes are fundamental players in the innate immune system. Activation of these cells can be quantified by the measurement of activation marker expression using flow cytometry. Analysis of receptor expression on inflammatory cells facilitates the diagnosis of inflammatory diseases and can be used to determine the extent of inflammation. However, several major limitations of this analysis precludes application of inflammation monitoring in clinical practice. Fast and automated analysis would minimalize ex vivo manipulation and allow reproducible processing. The aim of this study was to evaluate a fully automated “load & go” flow cytometer for analyzing activation of granulocytes and monocytes in a clinically applicable setting. Methods: Blood samples were obtained from 10 anonymous and healthy volunteers between the age of 18 and 65 years. Granulocyte and monocyte activation was determined by the use of the markers CD35, CD11b and CD10 measured in the automated AQUIOS CL® “load & go” flow cytometer. This machine is able to pierce the tube caps, add antibodies, lyse and measure the sample within 20 min after vena puncture. Reproducibility tests were performed to test the stability of activation marker expression on phagocytes. The expression of activation markers was measured at different time points after blood drawing to analyze the effect of bench time on granulocyte and monocyte activation. Results: The duplicate experiments demonstrate a high reproducibility of the measurements of the activation state of phagocytes. Healthy controls showed a very homogenous expression of activation markers at T = 0 (immediately after vena puncture). Activation markers on neutrophils were already significantly increased after 1 h (T = 1) depicted as means (95%Cl) CD35: 2.2× (1.5×-2.5×) p =.028, CD11b: 2.5× (1.7×-3.1×) p =.023, CD10: 2.5× (2.1×-2.7×) p =.009) and a further increase in activation markers was observed after 2 and 3 h. Monocytes also showed a increase in activation markers in 1 h (mean (95%Cl) CD35: 1.8× (1.3×–2.2×) p =.058, CD11b: 2.13× (1.6×–2.4×) p =.025) and also a further significant increase in 2 and 3 h was observed. Conclusion: This study showed that bench time of one hour already leads to a significant upregulation and bigger variance in activation markers of granulocytes and monocytes. In addition, it is likely that automated flow cytometry reduces intra-assay variability in the analysis of activation markers on inflammatory cells. Therefore, we found that it is of utmost importance to perform immune activation analysis as fast as possible to prevent drawing wrong conclusions. Automated flow cytometry is able to reduce this analysis from 2 h to only 15–20 min without the need of dedicated personnel and in a point-of-care context. This now allows fast and automated inflammation monitoring in blood samples obtained from a variety of patient groups. Fund: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors

Fragile neutrophils in surgical patients: A phenomenon associated with critical illness.

Leukocyte viability (determined by e.g. propidium iodide [PI] staining) is automatically measured by hematology analyzers to check for delayed bench time. Incidental findings in fresh blood samples revealed the existence of leukocytes with decreased viability in critically ill surgical patients. Not much is known about these cells and their functional and/or clinical implications. Therefore, we investigated the incidence of decreased leukocyte viability, the implications for leukocyte functioning and its relation with clinical outcomes. An automated alarm was set in a routine hematology analyzer (Cell-Dyn Sapphire) for the presence of non-viable leukocytes characterized by increased fluorescence in the PI-channel (FL3:630±30nm). Patients with non-viable leukocytes were prospectively included and blood samples were drawn to investigate leukocyte viability in detail and to investigate leukocyte functioning (phagocytosis and responsiveness to a bacterial stimulus). Then, a retrospective analysis was conducted to investigate the incidence of fragile neutrophils in the circulation and clinical outcomes of surgical patients with fragile neutrophils hospitalized between 2013-2017. A high FL3 signal was either caused by 1) neutrophil autofluorescence which was considered false positive, or by 2) actual non-viable PI-positive neutrophils in the blood sample. These two causes could be distinguished using automatically generated data from the hematology analyzer. The non-viable (PI-positive) neutrophils proved to be viable (PI-negative) in non-lysed blood samples, and were therefore referred to as 'fragile neutrophils'. Overall leukocyte functioning was not impaired in patients with fragile neutrophils. Of the 11 872 retrospectively included surgical patients, 75 (0.63%) were identified to have fragile neutrophils during hospitalization. Of all patients with fragile neutrophils, 75.7% developed an infection, 70.3% were admitted to the ICU and 31.3% died during hospitalization. In conclusion, fragile neutrophils occur in the circulation of critically ill surgical patients. These cells can be automatically detected during routine blood analyses and are an indicator of critical illness