An ultrapure plasma-derived monoclonal antibody-purified factor IX concentrate (Nonafact®), results of phase III and IV clinical studies.

Nonafact(®), an ultrapure, monoclonal antibody-purified factor IX concentrate (FIX) was developed to minimize risk of thrombotic complications and viral transmission. To investigate the pharmacokinetics, efficacy and safety, phase III/IV studies were performed in the Netherlands and Poland from 1996 to 2007. The mean half-life, in vivo response and recovery of Nonafact(®) were 18.7 (SD 2.0) h, 1.1 (SD 0.2) IU dL(-1) per IU kg(-1) b.w. of FIX infused and 49% (SD 10%), respectively. Eleven surgical procedures were performed in eight patients. During two surgeries, both high-risk, blood loss was observed. No postoperative bleeding occurred. The in vivo recovery of FIX was higher than expected. In the phase III follow-up study, 26 previously treated patients (PTP) were included with a median follow-up of 1130 days. From the 1617 minor bleedings, 80.5% was stopped after a single infusion. In the phase IV study thirteen patients were treated for a median study period of 737 days. In the two follow-up studies the investigators rated the effect of Nonafact(®) as excellent/good in 95% of major bleedings. Surgeries for which Nonafact(®) was given prophylactically were without bleeding problems. In total more than 10 million units of Nonafact(®) were used during almost 120 person-years. Only one minor adverse event was reported. No inhibitors, viral transmissions and thrombogenic events occurred. In conclusion, Nonafact(®) is safe and provides excellent haemostasis in haemophilia B patients treated for spontaneous bleeding or undergoing surgical procedures. Due to the excellent in vivo recovery characteristic, treatment with Nonafact(®) is cost saving compared to other FIX products.

A randomized, double-blind, placebo-controlled, parallel-group, enriched-design study of nabiximols* (Sativex(®) ), as add-on therapy, in subjects with refractory spasticity caused by multiple sclerosis.

BACKGROUND: Spasticity is a disabling complication of multiple sclerosis, affecting many patients with the condition. We report the first Phase 3 placebo-controlled study of an oral antispasticity agent to use an enriched study design. METHODS: A 19-week follow-up, multicentre, double-blind, randomized, placebo-controlled, parallel-group study in subjects with multiple sclerosis spasticity not fully relieved with current antispasticity therapy. Subjects were treated with nabiximols, as add-on therapy, in a single-blind manner for 4weeks, after which those achieving an improvement in spasticity of ≥20% progressed to a 12-week randomized, placebo-controlled phase. RESULTS: Of the 572 subjects enrolled, 272 achieved a ≥20% improvement after 4weeks of single-blind treatment, and 241 were randomized. The primary end-point was the difference between treatments in the mean spasticity Numeric Rating Scale (NRS) in the randomized, controlled phase of the study. Intention-to-treat (ITT) analysis showed a highly significant difference in favour of nabiximols (P=0.0002). Secondary end-points of responder analysis, Spasm Frequency Score, Sleep Disturbance NRS Patient, Carer and Clinician Global Impression of Change were all significant in favour of nabiximols. CONCLUSIONS: The enriched study design provides a method of determining the efficacy and safety of nabiximols in a way that more closely reflects proposed clinical practice, by limiting exposure to those patients who are likely to benefit from it. Hence, the difference between active and placebo should be a reflection of efficacy and safety in the population intended for treatment.

Diagnosis of factor VIII deficiency.

The correct diagnosis of factor VIII deficiency and the assessment of severity of the disease are essential for a patient-tailored treatment strategy. An optimal diagnostic procedure comprises sensitive and specific screening methods and factor VIII activity assays. Different screening reagents show variable characteristics and receiver operator characteristic curves are presented showing the relation between sensitivity and specificity of eleven activated partial thromboplastin time reagents. The details of the three methods for factor VIII activity assay, one-stage and two-stage assay and chromogenic assays, are discussed. The chromogenic assay seems to be more sensitive than the one-stage assay with regard to the detection of severe haemophilia. Discrepant results obtained with one-stage and two-stage assays are reviewed and discussed.

Factor VIII genotype and inhibitor development in patients with haemophilia A: highest risk in patients with splice site mutations.

The appearance of inhibitory antibodies against factor VIII (FVIII) is the most severe and costly complication of replacement therapy in patients with haemophilia A (HA). To determine the relationship between FVIII genotype and inhibitor development, baseline FVIII activity, genotype and inhibitor development were reviewed in 1104 patients with HA. In patients with severe HA, splicing errors present the highest frequency of inhibitors, ahead of inversion of intron 1 and of intron 22, nonsense mutations and large deletions. The lowest inhibitor frequency in severe HA is found in patients with missense mutations and small deletions/insertions. Subanalyses indicate that nonsense mutations and small deletions/insertions leading to a frameshift in the light chain are associated with a significant higher risk of inhibitor formation than similar mutations occurring in the heavy chain (27% vs. 14%). These mutation types also have a higher frequency of inhibitors when occurring in exons 23-26, where a second FVIII transcript originates, compared with similar mutations in exons 1-22 (28% vs. 17%). These results suggest that complete absence of FVIII because of null mutations, including splice site mutations, or the absence of a second transcript result in an increased risk of inhibitor development.

Prolonged delay for controlling KPC-2-producing Klebsiella pneumoniae outbreak: the role of clinical management.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) are becoming of immediate concern for infection control policies. Prompt detection of CPE on health care setting admission is crucial to halt the spread of an outbreak. We report a cluster of 13 Klebsiella pneumoniae carbapenemase (KPC)-2-producing K pneumoniae cases in a tertiary care hospital.The objective of this study was to identify contributing factors originating the outbreak. METHODS: An outbreak investigation was conducted using descriptive epidemiology, observation of health care practices, and interviews of management staff. A root cause analysis was performed to identify patent and latent failures of infection control measures using the association of litigation and risk management method. RESULTS: The main patent failure was the delay in identifying KPC-2-producing K pneumoniae carriers. Contributing factors were work and environmental factors: understaffing, lack of predefined protocols, staff members' characteristics, and underlying patients' characteristics. Latent failures were as follows: no promotion of the national guidelines for prevention of CPE transmission, no clear procedure for the management of patients hospitalized abroad, no clear initiative for promoting a culture of quality in the hospital, biologic activity recently outsourced to a private laboratory, and poor communication among hospital members. CONCLUSION: Clinical management should be better promoted to control hospital outbreaks and should include team work and safety culture.

Meloxicam, 15 mg/day, spares platelet function in healthy volunteers.

OBJECTIVE: To study the influence of meloxicam, a cyclooxygenase-2 (COX-2) preferential nonsteroidal anti-inflammatory drug, on serum thromboxane and platelet function in healthy volunteers with use of the maximum recommended daily dosage of 15 mg/day. METHODS: This study used an open, randomized crossover design. Indomethacin (INN, indometacin) was given as a positive control for nonsteroidal anti-inflammatory drug-induced inhibition of platelet function. The following variables were recorded: thromboxane B2 serum concentrations by radioimmunoassay, platelet aggregation by whole blood aggregometry in response to collagen 1.1 microg/L and to arachidonic acid 0.35 mmol/L, and closure time with use of the PFA-100. RESULTS: Serum thromboxane B2 at baseline was 535+/-233 nmol/L (mean +/- SD) and was reduced for 95% by indomethacin to 26+/-19 nmol/L (P < .001) and for 66% by meloxicam to 183+/-62 nmol/L (P < .001). Maximal platelet aggregation in response to collagen at baseline was 18.7+/-1.6 ohms (ohms). It was reduced by indomethacin to 7.3+/-4.5 ohms (P < .001), but not by meloxicam (19+/-2.5 ohms). Platelet aggregation in response to arachidonic acid at baseline was 12.2+/-2.0 ohms. It was reduced by indomethacin in all subjects to 0 ohms, but not by meloxicam (11+/-2.4 ohms). Closure time at baseline was 128+/-24 seconds and was prolonged by indomethacin to 286+/-38 seconds (P < .001). Meloxicam caused a minor prolongation of the closure time (141+/-32 seconds; P < .05). CONCLUSION: Meloxicam, 15 mg/day caused a major reduction of maximum thromboxane production but no reduction in collagen- or arachidonic acid-induced platelet aggregation and only minor increase of the closure time.

The type of factor VIII deficient plasma used influences the performance of the Nijmegen modification of the Bethesda assay for factor VIII inhibitors.

We have investigated the influence of the type of factor VIII deficient plasma used on the assay results of the Nijmegen modification of the Bethesda method for factor VIII inhibitors. Immuno depleted factor VIII deficient plasmas, lacking besides factor VIII also von Willebrand factor, gave decreased inhibitor titres compared to assay results with factor VIII deficient plasmas containing von Willebrand factor suggesting the need of the latter in the test system for the stability of factor VIII:C. Moreover the performance of the assay with immuno depleted plasma was contaminated in a certain type of this plasma by the presence of a factor VIII:C inhibitor. Chemically depleted factor VIII deficient plasma appeared to give falsely elevated titres when used in combination with other types of deficient plasmas as substrate plasma in the factor VIII:C assay due to the presence of activated factor Va in the preparation. Suggestions are described with respect to the observed limitations in order to obtain reliable results.

D-dimer determination to assess regression of deep venous thrombosis.

A number of studies evaluating deep venous thrombosis (DVT) have demonstrated that plasma levels of thrombotic and fibrinolytic parameters change during treatment, but the relationship between thrombus regression and evolution of these markers remains unknown. The objective of the present study was to correlate levels of D-Dimer (DD) with thrombus regression as assessed by duplex scanning. From 44 patients treated for acute DVT, DD were determined at diagnosis and at the end of initial heparin therapy of at least 5 days. Thrombus regression was measured by repeated duplex scanning at diagnosis and after 1 and 3 months. DD significantly decreased during heparin treatment as compared with values at presentation. DD levels were significantly higher in the group of patients without normalization of the DVT after 3 months (p = 0.003). A ninefold excess tendency was seen for DD levels > 1200 ng/ml at the end of initial treatment to be associated with poor resolution of the DVT [odds ratio 9.0, 0.95 confidence interval (CI) 2.3-35.4]. When the patients with an established malignancy were excluded, the differences were even more significant (p = 0.0004 for DD levels after initial treatment and an odds ratio of 17.5, 0.95 CI 3.3-92.5). These results suggest that increased DD levels after the initial phase of treatment are related to poor resolution of DVT after 3 months. These findings contribute to further insight into the process of thrombus regression. Furthermore high DD levels might help to identify the patients with a poor prognosis and could be useful to judge the efficacy of anticoagulant treatment.

The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: improved specificity and reliability.

Antibodies against factor VIII coagulant activity can appear in haemophiliacs who are treated with factor VIII preparations but also spontaneously in non-haemophiliacs. The Bethesda assay is the most commonly used method to detect these antibodies, but it lacks specificity especially in the lower range resulting in unreliable data. Two modifications are proposed and tested to resolve the imperfections: 1. Buffering the normal plasma used in the assay- and control mixture with 0.1 M imidazole to pH 7.4. 2. Replacing the imidazole buffer in the control mixture by immunodepleted factor VIII deficient plasma. These modifications allow better discrimination between positive and negative samples and improve reliability.

Fibrinogen Nijmegen: congenital dysfibrinogenemia associated with impaired t-PA mediated plasminogen activation and decreased binding of t-PA.

Congenital dysfibrinogenemia was found in a patient with venous thrombosis. Blood clot lysis was prolonged and suggested an impairment of fibrinolysis. We investigated whether this was related to the fibrinogen abnormality. Fibrinopeptide release was normal but fibrin polymerization was defective in the patient. The stimulating effect of the patient's fibrin on t-PA mediated plasminogen activation was impaired. This could not be attributed to defective binding of plasminogen. However, the binding of t-PA to the patient's fibrin was about 16% less than to normal fibrin. A variant t-PA (G K1 K2 P), which contained only one of the two fibrin binding sites, i.e. the kringle-2 domain, was bound to the abnormal fibrin for only 50% of normal. We conclude that the prolongation of blood clot lysis and the impaired stimulation of t-PA mediated plasminogen activation are related to the defective binding of the kringle-2 domain of t-PA onto the fibrin moiety of the abnormal fibrinogen. The impairment of fibrinolysis might explain the occurrence of thrombosis in the patient.

Reliability of five rapid D-dimer assays compared to ELISA in the exclusion of deep venous thrombosis.

Studies measuring the fibrin degradation product D-Dimer (DD) using enzyme-linked immunosorbent assays (ELISA) in patients with venographically proven deep venous thrombosis (DVT) suggest that it is possible to exclude DVT when DD level is below a certain cut-off level. However, ELISA methods are time-consuming and not available in all laboratories. Different rapid latex-agglutination assays have been investigated, but their sensitivity is considerably lower. In the present study we compared the value of four novel latex DD tests (Tinaquant, Minutex, Ortho and SimpliRed) and one rapid ELISA (VIDAS) to a classical ELISA DD assay (Organon Mab Y18) in 132 patients suspected of DVT. The VIDAS, a new quantitative automated ELISA, had a sensitivity of 100% and a negative predictive value of 100% for both proximal and distal DVT at a cut-off level of 500 ng/ml. The Tinaquant assay, a new quantitative latex method, had a sensitivity of 99% and a negative predictive value of 93% for both proximal and distal DVT at a cut-off level of 500 ng/ml. For proximal DVT only, both assays had a sensitivity and negative predictive value of 100%. VIDAS and Tinaquant correlated well with ELISA (correlation of r = 0.96 and r = 0.98 respectively). Sensitivities of the semi-quantitative latex assays Minutex, Ortho and SimpliRed were considerably lower (77%, 51% and 61% respectively). These results suggest that VIDAS and Tinaquant may be used instead of ELISA DD in the exclusion of DVT. Tinaquant can be performed within 20 min and VIDAS within 35 min. Both assays might be used as a routine screening test and should be evaluated in large clinical management studies.

Fibrinogen binding to ICAM-1 on EA.hy 926 endothelial cells is dependent on an intact cytoskeleton.

Fibrinogen is a ligand for Intercellular Adhesion Molecule-1 (ICAM-1), and enhances monocyte-endothelial cell interaction by coupling Mac-1 on monocytes to ICAM-1 on endothelial cells. We investigated the role of the cytoskeleton in fibrinogen binding to the human endothelial cell line EA.hy 926 using immunofluorescence techniques. In this cell line TNF alpha induced the simultaneous appearance of stress fibers and of ICAM-1, which was clustered predominantly on endothelial cell projections. Incubation of TNF alpha-stimulated endothelial cells with fibrinogen resulted in binding of fibrinogen to ICAM-1 on these cell projections. Disruption of the cytoskeleton by cytocholasin B abolished fibrinogen binding. Activation of protein kinase C with 12-O-tetradecanoyl phorbol-13-acetate resulted in simultaneous loss of both stress fibers and fibrinogen binding. These results suggest that a connection between ICAM-1 and the cytoskeleton results in clustering of ICAM-1 on cell projections, which is required for fibrinogen binding.

Semi-automated detection of the factor V mutation by allele specific amplification and capillary electrophoresis.

Recently a point mutation (G1691A) in the coagulation factor V gene was shown to cause resistance for cleavage by activated protein C. The mutation is associated with an increased thrombotic risk and thus-far the most common genetic cause of thrombophilia. Current techniques to investigate the single base pair mutation at the DNA level use an assay based upon the polymerase chain reaction followed by restriction enzyme digestion or Southern blotting and allele specific probing. The method we describe here consists of a single PCR in which two specially designed allele specific primers and two consensus primers were used in one reaction to distinguish between homozygous normal, heterozygous and homozygous mutant individuals. Amplification products were analysed using Capillary Electrophoresis and on line UV monitoring. The Allele Specific Amplification Protocol and subsequent CE analysis (ASAP-CE) is a convenient, fast, automated and highly reproducible method that can be used in a routine laboratory setting.

Options and limitations of long-term oral ciprofloxacin as antibacterial prophylaxis in allogeneic bone marrow transplant recipients.

The efficacy and safety of ciprofloxacin as long-term antibacterial prophylaxis after allogeneic bone marrow transplantation were assessed prospectively. Eighty-nine recipients of lymphocyte-depleted marrow grafts were each given ciprofloxacin orally, 500 mg twice daily. Fever developed in 71 out of 78 evaluable patients (91%) and was accompanied by positive blood cultures in 42 cases (59%). 'Viridans' streptococci, all but one with reduced in vitro susceptibility to ciprofloxacin, accounted for 35 episodes of bacteraemia. Thirty-three episodes occurred in patients given anthracyclines compared with only two episodes in other patients (chi 2 = 5.58: p less than 0.05). All bacteraemic fevers occurred within 11 days post-transplant. Gram-negative sepsis did not occur in any patient. Sixteen patients died but none due to a bacterial cause. Allergy to ciprofloxacin was registered in three out of 76 assessable cases (4%).

Virus transmission by subcutaneous jet injection.

An animal model was used to establish the risk of transmitting a virus infection by subcutaneous jet injection. Virus transmission was studied with mice chronically infected with LDH virus. The virus infection was transmitted by subcutaneous jet injection in 16 cases out of 49. Other routes of cross-infection were ruled out. Before using the jet injector as a harmless instrument for mass subcutaneous injection, further experiments on the risks of virus transmission should be performed.

Potential sites of infection that develop in febrile neutropenic patients.

Three hundred episodes of neutropenia were reviewed for the occurrence of potential sites of infection. Ninety sites (30 per cent) were identified at the onset of fever independent of initial bacteraemia which was encountered in 104 episodes (35%) and predominantly involved Gram-positive cocci. A further 90 sites were recorded involving mainly the lower respiratory tract (58%) and skin and soft tissue (18%). These changes evolved significantly later (mean of 5.1 and 4.3 days respectively) than did other foci which mainly presented at the onset of fever (p < 0.01). However the infectious aetiology was established in only 54 cases overall with fungi being responsible for 25 of 45 cases of lower respiratory tract infections with a known microbiological aetiology. The mortality associated with initial bacteraemia, focus at onset and unexplained fever was 11-14% while that associated with the development of a subsequent focus was 28% with lung infiltrates carrying the worst prognosis. Therefore rather than being seen as a final solution for possible infectious complications, empiric therapy provides an opportunity for daily review of the patient thereby increasing the likelihood of both explaining initial fever and diagnosing subsequent infection.

Empiric treatment of localized infections in the febrile neutropenic patient with monotherapy.

Empiric therapy is necessary for febrile, neutropenic patients in order to minimise morbidity and mortality. Certain agents are now available for monotherapy which offer comparable success to combinations of either an aminoglycoside with a beta-lactam or two beta-lactams. However, no regimen offers complete treatment under all circumstances in all patients. It is also apparent that febrile, neutropenic patients comprise a more heterogeneous group than just those with bacteraemia, clinically apparent infection and unexplained fever. Localized infections occur in just under a third of cases at the onset of fever and a similar number will develop during the course of fever. Mortality is higher in infections that are accompanied by bacteraemia and also those that develop subsequently, especially when related to the lung. The aetiological agent also differs with each type of infection as does the duration of fever and symptoms. Consequently modifications are required more often. The length of treatment may also differ. Therefore, during the first 3-4 days of empiric therapy, every effort should be made to identify incipient localized infections in addition to detecting bacteraemia. Changes in therapy can then be based on objective grounds rather than continued fever offering more patients individual treatment than is possible when relying only on the temperature chart.

Efficacy of itraconazole in the prevention of fungal infections among neutropenic patients with hematologic malignancies and intensive chemotherapy. A double blind, placebo controlled study.

We studied the efficacy and safety of itraconazole for the prevention of fungal infection in neutropenic patients given cytotoxic chemotherapy for hematologic malignancies. Patients were randomly allocated to receive either itraconazole (200 mg bd) or placebo in addition to oral amphotericin B until the patient either developed fungal infection or had completed antileukemic treatment. Forty six patients (83 neutropenic episodes) treated with itraconazole and 46 placebo treated patients (84 neutropenic episodes) were evaluable. No specific toxicity was noted. Nine fungal infections developed in the itraconazole group, of which four were histologically or microbiologically proven and 15 in the patients given placebo (eight proven) (p < 0.12). All these patients received IV amphotericin B. The incidence of Candida albicans infections tended to be lower in the itraconazole group, but overall, there was no measurable improvement in the prevention of fungal infections and mortality by itraconazole.

Co-trimoxazole in patients with haematological malignancies: a review of 10-years' clinical experience.

Co-trimoxazole has been used in a hospital for over 10 years as a major antibacterial agent in the treatment of malignant haematological diseases. Routine selective gut decontamination with co-trimoxazole combined with colistine and an antifungal agent has led to a reduction in infections in neutropenic patients from 40% to 25% since the strategy was adopted, and this had been accompanied by a change in the most frequent pathogens, from Gram-negative to Gram-positive organisms. Co-trimoxazole has proved to be the drug of choice for Pneumocystis carinii infections. Finally, it is used as first-line therapy in febrile immunosuppressed patients who are not on selective decontamination, with an efficacy of over 90%. Apart from mild abdominal discomfort, an elevated allergy rate of 14% in patients with overt leukaemia is a major disadvantage. On the other hand, substantial prolongation of episodes of bone marrow aplasia has not been observed.