Ex Vivo Test of Complement Dysregulation in Atypical Hemolytic Uremic Syndrome Kidney Transplant patients: A Pilot Study.

Introduction: In 2014, a complement assay, which evaluates C5b-9 deposition on endothelial cells, was proposed as a biomarker for atypical hemolytic uremic syndrome (aHUS). Early diagnosis and/or prediction of aHUS (relapse) is pivotal in aHUS kidney transplant recipients who do not receive eculizumab prophylaxis. Methods: In this pilot study, serum samples of transplanted patients with aHUS in remission without eculizumab and patients with other primary kidney diseases (controls) were blinded and evaluated in the complement assay. Results: We included 13 patients with aHUS (4 males, 9 females) of median age of 54 years (range: 35-69) and median of 5.9 years (range: 0.25-14.1) after transplantation; and 13 controls (7 males, 6 females) of median age of 42 years (range: 27-60) and median of 5.8 years (range: 1.6-11.7) after transplantation. There were no significant differences in C5b-9 deposits between patients with aHUS and controls on resting cells (median of 136% [range: 93%-382%] and 121% [range: 75%-200%], respectively) and activated cells (median of 196% [range: 99%-388%] and 170% [range: 113%-260%], respectively). Three patients with aHUS and 4 controls showed elevated C5b-9 deposits on resting cells, which should correspond to active aHUS. None of these patients had laboratory signs of thrombotic microangiopathy (TMA). During follow-up (15.8 months, range: 6-21), estimated glomerular filtration rate remained stable in all. In 5 patients with aHUS with a genetic variant, no increase in C5b-9 deposits was found on activated endothelial cells, which contrasts with the literature suggesting that the test should identify carriers of a genetic variant. Conclusion: Our data question the routine use of the ex vivo complement assay in kidney transplant patients. Future studies should evaluate the test characteristics of assay in kidney transplant patients.

Modeling complement activation on human glomerular microvascular endothelial cells.

Introduction: Atypical hemolytic uremic syndrome (aHUS) is a rare kidney disease caused by dysregulation of the complement alternative pathway. The complement dysregulation specifically leads to damage to the glomerular endothelium. To further understand aHUS pathophysiology, we validated an ex vivo model for measuring complement deposition on both control and patient human glomerular microvascular endothelial cells (GMVECs). Methods: Endothelial cells were incubated with human test sera and stained with an anti-C5b-9 antibody to visualize and quantify complement depositions on the cells with immunofluorescence microscopy. Results: First, we showed that zymosan-activated sera resulted in increased endothelial C5b-9 depositions compared to normal human serum (NHS). The levels of C5b-9 depositions were similar between conditionally immortalized (ci)GMVECs and primary control GMVECs. The protocol with ciGMVECs was further validated and we additionally generated ciGMVECs from an aHUS patient. The increased C5b-9 deposition on control ciGMVECs by zymosan-activated serum could be dose-dependently inhibited by adding the C5 inhibitor eculizumab. Next, sera from five aHUS patients were tested on control ciGMVECs. Sera from acute disease phases of all patients showed increased endothelial C5b-9 deposition levels compared to NHS. The remission samples showed normalized C5b-9 depositions, whether remission was reached with or without complement blockage by eculizumab. We also monitored the glomerular endothelial complement deposition of an aHUS patient with a hybrid complement factor H (CFH)/CFH-related 1 gene during follow-up. This patient had already chronic kidney failure and an ongoing deterioration of kidney function despite absence of markers indicating an aHUS flare. Increased C5b-9 depositions on ciGMVECs were observed in all samples obtained throughout different diseases phases, except for the samples with eculizumab levels above target. We then tested the samples on the patient's own ciGMVECs. The C5b-9 deposition pattern was comparable and these aHUS patient ciGMVECs also responded similar to NHS as control ciGMVECs. Discussion: In conclusion, we demonstrate a robust and reliable model to adequately measure C5b-9-based complement deposition on human control and patient ciGMVECs. This model can be used to study the pathophysiological mechanisms of aHUS or other diseases associated with endothelial complement activation ex vivo.

Clinical and radiological assessment of scoliosis in Koolen-de Vries syndrome.

The Koolen-de Vries syndrome (KdVS) is a multisystem disorder characterized by developmental delay, intellectual disability, characteristic facial features, epilepsy, cardiovascular and urogenital malformations, and various musculoskeletal disorders. Scoliosis is a common feature. The aim of this study is to fill the gap in the current knowledge about scoliosis in individuals with KdVS and to provide recommendations for management and follow-up. In total, 54 individuals with KdVS were included in the study, with a mean age of 13.6 years (range 1.9-38.8 years). Spine radiographs, MR scans, and corresponding radiology reports were analyzed retrospectively for scoliosis and additional anomalies. The presence of scoliosis-related clinical conditions was assessed in participants' medical records and by use of a parent survey. Scoliosis was present in 56% of the participants (30/54) with a mean age of onset of 10.6 years and curve progression during the growth spurt. Prevalence at age 6, 10, and 18 years was, respectively, 9%, 41%, and 65%. Most participants were diagnosed with a single curve (13/24, 54%), of which five participants had a long C-curve type scoliosis. No significant risk factors for development of scoliosis could be identified. Severity was mostly classified as mild, although 29% (7/24) of the curves were larger than 30° at last follow-up. Bracing therapy was received in 13% (7/54), and surgical spinal fusion was warranted in 6% (3/54). Remarkably, participants with scoliosis received less often physical therapy compared to participants without scoliosis (P = 0.002). Scoliosis in individuals with KdVS should be closely monitored and radiologic screening for scoliosis and vertebrae abnormalities is recommended at diagnosis of KdVS, and the age of 10 and 18 years.

Eculizumab Rescue Therapy in Patients With Recurrent Atypical Hemolytic Uremic Syndrome After Kidney Transplantation.

Introduction: Since 2016, kidney transplantation in patients with atypical hemolytic uremic syndrome (aHUS) in the Netherlands is performed without eculizumab prophylaxis. Eculizumab is given in case of posttransplant aHUS recurrence. Eculizumab therapy is monitored in the CUREiHUS study. Methods: All participating kidney transplant patients who received eculizumab therapy for a suspected posttransplant aHUS recurrence were evaluated. Overall recurrence rate was monitored prospectively at Radboud University Medical Center. Results: In the period from January 2016 until October 2020, we included 15 (12 females, 3 males; median age 42 years, range 24-66 years) patients with suspected aHUS recurrence after kidney transplantation in this study. The time interval to recurrence showed a bimodal distribution. Seven patients presented early after transplantation (median 3 months, range 0.3-8.8 months), with typical aHUS features: rapid loss of estimated glomerular filtration rate (eGFR) and laboratory signs of thrombotic microangiopathy (TMA). Eight patients presented late (median 46 months, range 18-69 months) after transplantation. Of these, only 3 patients had systemic TMA, whereas 5 patients presented with slowly deteriorating eGFR without systemic TMA. Treatment with eculizumab resulted in improvement or stabilization of eGFR in 14 patients. Eculizumab discontinuation was tried in 7 patients; however, it was successful only in 3. At the end of the follow-up (median 29 months, range 3-54 months after start of eculizumab), 6 patients had eGFR <30 ml/min per 1.73 m2. Graft loss had occurred in 3 of them. Overall, aHUS recurrence rate without eculizumab prophylaxis was 23%. Conclusions: Rescue treatment of posttransplant aHUS recurrence is effective; however, some patients suffer from irreversible loss of kidney function, likely caused by delayed diagnosis and treatment and/or too aggressive discontinuation of eculizumab. Physicians should be aware that recurrence of aHUS can present without evidence of systemic TMA.

Proteinuria and Exposure to Eculizumab in Atypical Hemolytic Uremic Syndrome.

BACKGROUND: Eculizumab is a monoclonal antibody for the treatment of atypical hemolytic uremic syndrome (aHUS). Kidney damage, a common condition in patients with aHUS, may result in proteinuria. Because proteinuria may affect the pharmacokinetics of therapeutic proteins such as eculizumab, the aim of our study was to investigate the effect of proteinuria on eculizumab pharmacokinetics. METHODS: This study was an ancillary study of a previously performed pharmacokinetic-pharmacodynamic study of eculizumab in aHUS. Proteinuria, measured as urinary protein-creatinine ratios (UPCR), was investigated as covariate for eculizumab clearance. Thereafter, we evaluated the effect of proteinuria on the exposure to eculizumab in a simulation study for the initial phase and for a 2-weekly and 3-weekly interval in the maintenance phase. RESULTS: The addition of UPCR as a linear covariate on clearance to our base model resulted in a statistically improved fit ( P < 0.001) and reduction of unexplained variability in clearance. From our data, we predicted that in the initial phase, 16% of the adult patients with severe proteinuria (UPCR >3.1 g/g) will have inadequate complement inhibition (classical pathway activity >10%) on day 7 of treatment, compared with 3% of the adult patients without proteinuria. None of the pediatric patients will have inadequate complement inhibition at day 7 of treatment. For the 2- and 3-weekly dosing intervals, we predicted that, respectively, 18% and 49% of the adult patients and, respectively, 19% and 57% of the pediatric patients with persistent severe proteinuria will have inadequate complement inhibition, compared with, respectively, 2% and 13% of the adult patients and, respectively, 4% and 22% of the pediatric patients without proteinuria. CONCLUSIONS: Severe proteinuria is associated with a higher risk of underexposure to eculizumab. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: CUREiHUS, Dutch Trial Register, NTR5988/NL5833.

Early Eculizumab Withdrawal in Patients With Atypical Hemolytic Uremic Syndrome in Native Kidneys Is Safe and Cost-Effective: Results of the CUREiHUS Study.

Introduction: The introduction of eculizumab has improved the outcome in patients with atypical hemolytic uremic syndrome (aHUS). The optimal treatment strategy is debated. Here, we report the results of the CUREiHUS study, a 4-year prospective, observational study monitoring unbiased eculizumab discontinuation in Dutch patients with aHUS after 3 months of therapy. Methods: All pediatric and adult patients with aHUS in native kidneys and a first-time eculizumab treatment were evaluated. In addition, an extensive cost-consequence analysis was conducted. Results: A total of 21 patients were included in the study from January 2016 to October 2020. In 17 patients (81%), a complement genetic variant or antibodies against factor H were identified. All patients showed full recovery of hematological thrombotic microangiopathy (TMA) parameters after the start of eculizumab. A renal response was noted in 18 patients. After a median treatment duration of 13.6 weeks (range 2.1-43.9), eculizumab was withdrawn in all patients. During follow-up (80.7 weeks [0.0-236.9]), relapses occurred in 4 patients. Median time to first relapse was 19.5 (14.3-53.6) weeks. Eculizumab was reinitiated within 24 hours in all relapsing patients. At last follow-up, there were no chronic sequelae, i.e., no clinically relevant increase in serum creatinine (sCr), proteinuria, and/or hypertension in relapsing patients. The low sample size and event rate did not allow to determine predictors of relapse. However, relapses only occurred in patients with a likely pathogenic variant. The cost-effectiveness analysis revealed that the total medical expenses of our population were only 30% of the fictive expenses that would have been made when patients received eculizumab every fortnight. Conclusion: It is safe and cost-effective to discontinue eculizumab after 3 months of therapy in patients with aHUS in native kidneys. Larger data registries are needed to determine factors associated with suboptimal kidney function recovery during eculizumab treatment, factors to predict relapses, and long-term outcomes of eculizumab discontinuation.

Proposal for individualized dosing of eculizumab in atypical haemolytic uraemic syndrome: patient friendly and cost-effective.

BACKGROUND: Eculizumab is a lifesaving yet expensive drug for atypical haemolytic uraemic syndrome (aHUS). Current guidelines advise a fixed-dosing schedule, which can be suboptimal and inflexible in the individual patient. METHODS: We evaluated the pharmacokinetics (PK) and pharmacodynamics (PD) [classical pathway (CP) activity levels] of eculizumab in 48 patients, consisting of 849 time-concentration data and 569 CP activity levels. PK-PD modelling was performed with non-linear mixed-effects modelling. The final model was used to develop improved dosing strategies. RESULTS: A PK model with parallel linear and non-linear elimination rates best described the data with the parameter estimates clearance 0.163 L/day, volume of distribution 6.42 L, maximal rate 29.6 mg/day and concentration for 50% of maximum rate 37.9 mg/L. The PK-PD relation between eculizumab concentration and CP activity was described using an inhibitory Emax model with the parameter estimates baseline 101%, maximal inhibitory effect 95.9%, concentration for 50% inhibition 22.0 mg/L and  Hill coefficient 5.42. A weight-based loading dose, followed by PK-guided dosing was found to improve treatment. On day 7, we predict 99.95% of the patients to reach the efficacy target (CP activity <10%), compared with 94.75% with standard dosing. Comparable efficacy was predicted during the maintenance phase, while the dosing interval could be prolonged in ∼33% of the population by means of individualized dosing. With a fixed-dose 4-week dosing interval to allow for holidays, treatment costs will increase by 7.1% and we predict 91% of the patients will reach the efficacy target. CONCLUSIONS: A patient-friendly individualized dosing strategy of eculizumab has the potential to improve treatment response at reduced costs.

COVID-19 vaccination and Atypical hemolytic uremic syndrome.

Introduction: COVID-19 vaccination has been associated with rare but severe complications characterized by thrombosis and thrombocytopenia. Methods and Results: Here we present three patients who developed de novo or relapse atypical hemolytic uremic syndrome (aHUS) in native kidneys, a median of 3 days (range 2-15) after mRNA-based (Pfizer/BioNTech's, BNT162b2) or adenoviral (AstraZeneca, ChAdOx1 nCoV-19) COVID-19 vaccination. All three patients presented with evident hematological signs of TMA and AKI, and other aHUS triggering or explanatory events were absent. After eculizumab treatment, kidney function fully recovered in 2/3 patients. In addition, we describe two patients with dubious aHUS relapse after COVID-19 vaccination. To assess the risks of vaccination, we retrospectively evaluated 29 aHUS patients (n=8 with native kidneys) without complement-inhibitory treatment, who received a total of 73 COVID-19 vaccinations. None developed aHUS relapse after vaccination. Conclusion: In conclusion, aHUS should be included in the differential diagnosis of patients with vaccine-induced thrombocytopenia, especially if co-occuring with mechanical hemolytic anemia (MAHA) and acute kidney injury (AKI). Still, the overall risk is limited and we clearly advise continuation of COVID-19 vaccination in patients with a previous episode of aHUS, yet conditional upon clear patient instruction on how to recognize symptoms of recurrence. At last, we suggest monitoring serum creatinine (sCr), proteinuria, MAHA parameters, and blood pressure days after vaccination.

Validation of an Instrument for Real-Time Assessment of Neonatal Intubation Skills: A Randomized Controlled Simulation Study.

OBJECTIVE: This study aimed to evaluate the construct validity and reliability of real-time assessment of a previously developed neonatal intubation scoring instrument (NISI). STUDY DESIGN: We performed a randomized controlled simulation study at a simulation-based research and training facility. Twenty-four clinicians experienced in neonatal intubation ("experts") and 11 medical students ("novices") performed two identical elective intubations on a neonatal patient simulator. Subjects were randomly assigned to either the intervention group, receiving predefined feedback between the two intubations, or the control group, receiving no feedback. Using the previously developed NISI, all intubations were assessed, both in real time and remotely on video. Construct validity was evaluated by (1) comparing the intubation performances, expressed as percentage scores, with and without feedback, and (2) correlating the intubation performances with the subjects' level of experience. The intrarater reliability, expressed as intraclass correlation coefficient (ICC), of real-time assessment compared with video-based assessment was determined. RESULTS: The intervention group contained 18 subjects, the control group 17. Background characteristics and baseline intubation scores were comparable in both groups. The median (IQR) change in percentage scores between the first and second intubation was significantly different between the intervention and control group (11.6% [4.7-22.8%] vs. 1.4% [0.0-5.7%], respectively; p = 0.013). The 95% CI for this 10.2% difference was 2.2 to 21.4%. The subjects' experience level correlated significantly with their percentage scores (Spearman's R = 0.70; p <0.01). ICC's were 0.95 (95% CI: 0.89-0.97) and 0.94 (95% CI: 0.89-0.97) for the first and second intubation, respectively. CONCLUSION: Our NISI has construct validity and is reliable for real-time assessment. KEY POINTS: · Our neonatal intubation scoring instrument has construct validity.. · Our instrument can be reliably employed to assess neonatal intubation skills directly in real time.. · It is suitable for formative assessment, i.e., providing direct feedback during procedural training..

Case Report: Variable Pharmacokinetic Profile of Eculizumab in an aHUS Patient.

Background: With the introduction of eculizumab, a C5-inhibitor, morbidity and mortality improved significantly for patients with atypical hemolytic uremic syndrome (aHUS). In view of the high costs, actual needs of the drug, and increasing evidence in literature, aHUS patients can be treated according to a restrictive eculizumab regimen. We retrospectively analyzed the pharmacokinetic and dynamic parameters of eculizumab in one patient in time, emphasizing various factors which could be taken into account during tapering of treatment. Case Presentation: A nowadays 18-year-old male with a severe, frequently relapsing form of atypical HUS due to a hybrid CFH/CFHR1 gene in combination with the homozygous factor H haplotype, required chronic plasma therapy (PT), including periods with plasma infusion, from the age of onset at 5 months until initiation of eculizumab at the age of 11 years. A mild but stable chronic kidney disease (CKD) and 9 years of disease remission enabled prolongation of eculizumab interval. At the age of 15 years, a sudden yet multifactorial progression of chronic kidney disease (CKD) was observed, without any signs of disease recurrence. However, an acquired glomerulocystic disease, a reduced left kidney function, and abnormal abdominal venous system of unknown etiology were found. In addition, after an aHUS relapse, an unexpected increase in intra-patient variability of eculizumab concentrations was seen. Retrospective pharmacokinetic analysis revealed a change in eculizumab clearance, associated with a simultaneous increase in proteinuria. Conclusion: High intra-patient variability of eculizumab pharmacokinetics were observed over time, emphasizing the necessity for adequate and continuous therapeutic drug monitoring in aHUS patients. Eculizumab serum trough levels together with complement activation markers (CH50) should be frequently assessed, especially during tapering of drug therapy and/or changing clinical conditions in the patient. In addition, an increase in proteinuria could result in urinary eculizumab loss, indicating that urinary monitoring of eculizumab may be important in aHUS patients with an unexplained decline in serum concentrations.

Appraisal of a scoring instrument for training and testing neonatal intubation skills.

OBJECTIVE: To determine the validity, reliability, feasibility and applicability of a neonatal intubation scoring instrument. DESIGN: Prospective observational study. SETTING: Simulation-based research and training centre (Center for Advanced Pediatric and Perinatal Education), California, USA. SUBJECTS: Forty clinicians qualified for neonatal intubation. INTERVENTIONS: Videotaped elective intubations on a neonatal patient simulator were scored by two independent raters. One rater scored the intubations twice. We scored the preparation of equipment and premedication, intubation performance, tube position/fixation, communication, number of attempts, duration and successfulness of the procedure. MAIN OUTCOME MEASURES: Intraclass correlation coefficients (ICC) were calculated for intrarater and inter-rater reliability. Kappa coefficients for individual items and mean kappa coefficients for all items combined were calculated. Construct validity was assessed with one-way analysis of variance using the hypothesis that experienced clinicians score higher than less experienced clinicians. The approximate time to score one intubation and the instrument's applicability in another setting were evaluated. RESULTS: ICCs for intrarater and inter-rater reliability were 0.99 (95% CI 0.98 to 0.99) and 0.89 (95% CI 0.35 to 0.96), and mean kappa coefficients were 0.93 (95% CI 0.85 to 1.01) and 0.71 (95% CI 0.56 to 0.92), respectively. There were no differences between the more and less experienced clinicians regarding preparation, performance, communication and total scores. The experienced group scored higher only on tube position/fixation (p=0.02). Scoring one intubation took approximately 15 min. Our instrument, developed in The Netherlands, could be readily applied in the USA. CONCLUSIONS: Our scoring instrument for simulated neonatal intubations appears to be reliable, feasible and applicable in another centre. Construct validity could not be established.