Systemic versus free antibiotic delivery in preventing acute exogenous implant related infection in a rat model.

We studied systemic ceftriaxone, and free/local tobramycin and doxycycline in a controlled rat model representing a generic acute exogenous joint infection. We hypothesized that evidence of infection (quantitative colony forming units [CFU], qualitative scanning electron microscopy [SEM], histopathology) (1a) would be reduced with local versus systemic antibiotic, (1b) any antibiotic would be superior to control, (2) there would be a difference among antibiotics, and (3) antibiotic would not be detectable in serum at 4-week euthanasia. Study groups included infected and noninfected (1) control (no treatment), (2) systemic ceftriaxone (daily), (3) local tobramycin, and (4) local doxycycline (10 rats/group; power = 0.8). With IACUC approval, a reliable acute exogenous joint infection was created by slowly injecting 50-µl, 104 CFU Staphylococcus aureus, into the distal femoral medullary canal. The antibiotic formulation was introduced locally to the femoral canal and joint space. After 4 weeks, serum, pin, bone, and synovium were obtained. CFU/ml of bone and synovium were quantified using macrotiter method. SEM imaged biofilm on the surface of the pin, histopathology identified tissue response, liquid chromatography/mass spectrometry quantified plasma antibiotic. (1) Groups receiving any antibiotic reported lower CFU/ml in synovium compared with no treatment. (2) In the synovium, free/local tobramycin reduced CFU/ml to a greater extent than free/local doxycycline (p < 0.05). (3) Antibiotic in plasma after the local application was nondetectable in all groups after 4 weeks. SEM revealed no difference in biofilm on pin among all groups.

Streptococcus agalactiae do not penetrate human chorioamniotic membranes in vitro but alter their biomechanical properties.

INTRODUCTION: Vaginal colonization with Streptococcus agalactiae (group B streptococci) is hypothesized to constitute a risk factor for preterm prelabor rupture of membranes. In vitro studies have shown that S. agalactiae strains isolated from infants with neonatal sepsis adhere to chorion cells of the human chorioamniotic membrane. However, it is still unknown whether S. agalactiae strains penetrate the chorioamniotic membranes and whether S. agalactiae colonization affects the biomechanical properties of the membranes and thus contributes to increased risk of preterm prelabor rupture. The aim of this in vitro study was to explore if different strains of S. agalactiae penetrate and affect the biomechanical properties of human chorioamniotic membranes. MATERIAL AND METHODS: Three different strains of S. agalactiae were obtained, one from an early-onset neonatal infection, one from a case of preterm prelabor rupture of membranes and one from a healthy pregnant carrier. Chorioamniotic membranes from elective cesarean deliveries were either incubated with S. agalactiae or mounted in a two-chamber incubation cell generating a "maternal" and a "fetal" chamber and incubated with S. agalactiae in the maternal chamber. Subsequently the membranes were examined to evaluate S. agalactiae attachment, penetration and the effect on the biomechanical properties. RESULTS: At 5 h after incubation, S. agalactiae adhered to the chorioamniotic membranes with increased number at 20 h. Streptococcus agalactiae did not penetrate the membranes even after 20 h of incubation. Streptococcus agalactiae increased the ultimate tensile stress needed to rupture the membranes and increased the work needed to rupture the membranes as well as the elastic modulus. CONCLUSIONS: Human chorioamniotic membranes constitute a physical barrier against S. agalactiae infections. Moreover, S. agalactiae infection leads to increased strength of the membranes.

Local Vancomycin Concentrations after Intra-articular Injection into the Knee Joint: An Experimental Porcine Study.

Intra-articular injection of vancomycin may be an important antimicrobial prophylactic supplement to systemic administration in the prevention of prosthetic joint infections. In eight female pigs, 500 mg of diluted vancomycin was given by intra-articular injection into the knee joint. Microdialysis was used for dense sampling of vancomycin concentrations over 12 hours in the synovial fluid of the knee joint, and in the adjacent femoral and tibial cancellous bone and subcutaneous tissue. Venous blood samples were obtained as reference. The mean (standard deviation [SD]) peak drug concentration of vancomycin in the synovial fluid of the knee joint was 5,277 (5,668) µg/mL. Only one pig failed to reach a peak drug concentration above 1,000 µg/mL. The concentration remained high throughout the sampling interval with a mean (SD) concentration of 337 (259) µg/mL after 690 minutes. For all extraarticular compartments, the pharmacokinetic parameters (area under the concentration time-curve, peak drug concentration, and time to peak drug concentration) were comparable. The highest extraarticular mean (SD) peak drug concentration of 4.4 (2.3) µg/mL was found in subcutaneous tissue. An intra-articular injection of 500 mg diluted vancomycin was found to provide significant prophylactic mean concentrations for at least 12 hours in the synovial fluid of the knee joint. Correspondingly, the adjacent tissue and plasma concentrations were low but remained stable, signifying low risk of systemic toxic side effects and a slow release or uptake from the synovium to the systemic circulation.

Deleterious mis-splicing of STK11 caused by a novel single-nucleotide substitution in the 3' polypyrimidine tract of intron five.

BACKGROUND: Pathogenic variants in STK11, also designated as LKB1, cause Peutz-Jeghers syndrome, which is a rare autosomal dominant disorder characterized by mucocutaneous pigmentation changes, polyposis, and a high risk of cancer. METHODS: A male meeting the clinical diagnostic criteria for Peutz-Jeghers syndrome underwent next-generation sequencing. To validate the predicted splicing impact of a detected STK11 variant, we performed RNA-Seq on mRNA extracted from patient-derived Epstein-Barr virus-transformed lymphocytes treated with cycloheximide to inhibit nonsense-mediated decay ex vivo. RESULTS: Blood testing identified a novel single-nucleotide substitution, NM_000455.4:c.735-10C>A, at the end of the 3' polypyrimidine tract of intron five in STK11. RNA-Seq confirmed a predicted eight base pair insertion in the mRNA transcript. Following inhibition of nonsense-mediated decay, the out-of-frame insertion was detected in 50% of all RNA-Seq reads. This confirmed a strong, deleterious splicing impact of the variant. CONCLUSION: We characterized a novel likely pathogenic germline variant in intron five of STK11 associated with Peutz-Jeghers syndrome. The study highlights RNA-Seq as a useful supplement in hereditary cancer predisposition testing.

Protein C deficiency; PROC gene variants in a Danish population.

INTRODUCTION: Protein C deficiency is a heritable thrombophilia caused by numerous different genetic alterations in the protein C (PROC) gene. We aimed to identify variants causing protein C deficiency in a Danish population. MATERIAL AND METHODS: Sanger sequencing of the PROC gene was performed in 20 probands and 26 relatives. In total, 30participants were previously diagnosed with protein C deficiency. Protein C activity was measured by a chromogenic substrate method (N = 40) and antigen level by an enzyme-linked immunosorbent assay (N = 26). RESULTS: Ten different single nucleotide variants were detected in 13 probands (65%) and in seven of the relatives previously diagnosed with protein C deficiency. Five variants were novel. The median protein C activity level was lower in participants with an identified variant (50% (range: 38-75%)) than in protein C deficient participants without a variant (65% (range: 36-73%); P = 0.18). A protein C activity of 57% resulted in the highest detection rate (12/13 (92%)). Likewise, the median antigen level was lower in participants with detectable variants than in participants without (49% (range: 35-99%) vs 70% (range: 41-101%); P = 0.09). No difference was found in venous thromboembolism (VTE) prevalence comparing participants with (12/20 (60%)) and without (7/10 (70%)) a variant (P = 0.59). CONCLUSION: In a Danish population, a PROC gene variant was identified in 67% of participants previously diagnosed with protein C deficiency. Five variants were novel. The study confirmed an association between biochemical severity and the presence of a PROC gene variant. The VTE risk did not seem to differ between protein C deficient participants with and without a variant.

Complementary effect of fibrinogen and rFVIIa on clotting ex vivo in Bernard-Soulier syndrome and combined use during three deliveries.

Women with Bernard-Soulier syndrome (BSS) are considered to be at high risk of serious bleeding during childbirth. Due to the frequently occurring platelet transfusion refractoriness, alternative prophylactic therapy is required. Using rotational thromboelastometry, we evaluated the whole blood coagulation profile of a pregnant woman with BSS before and after spiking ex vivo with different concentrations of recombinant activated factor VII (rFVIIa) and fibrinogen. As experiments suggested improved clotting with clinically applicable concentrations of both agents in a complementary manner, the findings were confirmed on blood from a non-pregnant woman and three men suffering from BSS. During delivery, bleeding refractory to platelets occurred and immediately following delivery she received both rFVIIa and fibrinogen intravenously. Immediate cessation of bleeding occurred, and no postpartum hemorrhage was seen. Another woman with BSS later also received the same rFVIIa and fibrinogen treatment prophylactically after delivery without any postpartum bleeding. Eventually, the first woman during her second delivery received the same treatment again prophylactically without any bleeding. No side effects were observed during these three deliveries. Our observations suggest that rFVIIa combined with fibrinogen may provide a beneficial clinical hemostatic effect partly by separate but complementary mechanisms.

Combination of recombinant factor VIIa and fibrinogen corrects clot formation in primary immune thrombocytopenia at very low platelet counts.

Haemostatic treatment modalities alternative to platelet transfusion are desirable to control serious acute bleeds in primary immune thrombocytopenia (ITP). This study challenged the hypothesis that recombinant activated factor VII (rFVIIa) combined with fibrinogen concentrate may correct whole blood (WB) clot formation in ITP. Blood from ITP patients (n = 12) was drawn into tubes containing 3·2% citrate and corn trypsin inhibitor 18·3 µg/ml. WB [mean platelet count 22 × 10(9) /l (range 0-42)] was spiked in vitro with buffer, donor platelets (+40 × 10(9) /l), rFVIIa (1 or 4 µg/ml), fibrinogen (1 or 3 mg/ml), or combinations of rFVIIa and fibrinogen. Coagulation profiles were recorded by tissue factor (0·03 pmol/l) activated thromboelastometry. Coagulation in ITP was characterized by a prolonged clotting time (CT, 1490 s (mean)) and a low maximum velocity (MaxVel, 3·4 mm × 100/s) and maximum clot firmness (MCF, 38·2 mm). Fibrinogen showed no haemostatic effect, whereas rFVIIa reduced the CT and increased the MaxVel. The combination of fibrinogen and rFVIIa revealed a significant synergistic effect, improving all parameters (CT 794 s, MaxVel 7·9 mm × 100/s, MCF 50·7 mm) even at very low platelet counts. These data suggest that rFVIIa combined with fibrinogen corrects the coagulopathy of ITP even at very low platelet counts, and may represent an alternative to platelet transfusion.

The role of fibrinogen: a new paradigm in the treatment of coagulopathic bleeding.

Fibrinogen is involved in both primary and secondary hemostasis, playing an important role in platelet aggregation and the establishment of a fibrin network. Recent evidence suggests that very high levels of fibrinogen act as antithrombin and can reduce endogenous thrombin potential and compromise clot stability, particularly following a low tissue factor stimulus. Several laboratory methods for measuring plasma fibrinogen concentrations are available, but results vary depending on the type of method and the use of artificial colloid plasma expanders. Adopting only the Clauss method can provide erroneously high levels when used in patients who have received colloid plasma expanders. This may contribute to a hazardous delay or complete lack of treatment. Multiple in vitro experiments, animal studies, and proof-of-principle randomized, clinical studies have recently suggested that hemostatic intervention with a fibrinogen concentrate may be efficient and safe in controling perioperative bleeding. In particular, fibrinogen concentrate has a key role in improving clotting function and reducing blood loss in settings such as trauma and cardiothoracic surgery. However, prospective studies are needed to determine the clinical efficacy and safety of fibrinogen concentrate when used as a hemostatic intervention for patients with massive bleeding due to trauma or surgery.

Evaluation of coagulation kinetics using thromboelastometry-methodologic influence of activator and test medium.

Renewed interest has arisen in the use of thromboelastography/thromboelastometry in evaluating coagulation kinetics. The test medium, type of activator, and its concentration may influence the interpretation of coagulation kinetics. This study aimed to investigate methodologic influences of activator and test medium on thromboelastometric parameters of coagulation kinetics. Dynamic clot formation was evaluated by thromboelastometry using whole blood (WB), platelet-rich plasma, or platelet-poor plasma employing different concentrations of extrinsic (tissue factor) and contact activator (synthasil) and with variable concentrations of phospholipids. Plasma samples displayed prolonged clot initiation and enhanced clot propagation compared with WB. Clot firmness was markedly reduced in platelet-poor plasma as compared with platelet-rich plasma and WB. Increasing concentration of activator shortened the clot initiation and increased the velocity of clot propagation, whereas terminal clot firmness remained unaffected. Platelets accelerated clot propagation and raised clot firmness. Phospholipids shortened the time of clot initiation and increased velocity of propagation, while clot firmness remained unchanged. Our results demonstrate that evaluation of coagulation kinetics using thromboelastometry varies according to the composition of the test medium, type, and concentration of activator, as well as the presence and concentration of phospholipids in the test reagent.

Whole blood coagulation in children with thrombocytopenia and the response to platelet replacement, recombinant factor VIIa, and a potent factor VIIa analogue.

The present study evaluated dynamic coagulation profiles, platelet aggregation, and thrombin generation in whole blood (WB) from eight children with thrombocytopenia during chemotherapy, and the haemostatic potential of platelets (+60 x 10(9)/l), recombinant factor VIIa (rFVIIa - NovoSeven), and a potent rFVIIa analogue (NN1731) both at 1 and 4 microg/ml. Dynamic WB coagulation profiles were recorded by thrombelastometry employing activation with tissue factor (TF - Innovin) at low concentrations. The baseline WB coagulation patterns were characterised by a prolonged clotting time (CT) and a pronounced reduction in clot propagation (MaxVel). WB platelet aggregation signal was five times lower in the study group compared with measurements in modelled thrombocytopenic WB from healthy volunteers. In vitro addition of fresh platelets reversed the coagulopathy. Addition of rFVIIa induced no significant changes in the thrombelastographic profile, whereas spiking with NN1731 shortened the CT significantly. The changes in WB thrombin generation reflected the changes in the MaxVel. In modeled thrombocytopenic WB from healthy individuals, both rFVIIa and NN1731 exhibited a pronounced haemostatic effect with NN1731 showing greater potency than rFVIIa. Compromised platelet function in the study group was assumingly responsible for the weakened haemostatic potential of rFVIIa as well as that of NN1731.