In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model.

BACKGROUND AND AIMS: Pluripotent stem cell-derived hepatocytes differentiated in monolayer culture are known to have more fetal than adult hepatocyte characteristics. If numerous studies tend to show that this immature phenotype might not necessarily be an obstacle to their use in transplantation, other applications such as drug screening, toxicological studies, or bioartificial livers are reliant on hepatocyte functionality and require full differentiation of hepatocytes. New technologies have been used to improve the differentiation process in recent years, usually evaluated by measuring the albumin production and CYP450 activity. Here we used the complex production and most importantly the activity of the coagulation factor IX (FIX) produced by mature hepatocytes to assess the differentiation of hemophilia B (HB) patient's induced pluripotent stem cells (iPSCs) in both monolayer culture and organoids. APPROACH AND RESULTS: Indeed, HB is an X-linked monogenic disease due to an impaired activity of FIX synthesized by hepatocytes in the liver. We have developed an in vitro model of HB hepatocytes using iPSCs generated from fibroblasts of a severe HB patient. We used CRISPR/Cas9 technology to target the genomic insertion of a coagulation factor 9 minigene bearing the Padua mutation to enhance FIX activity. Noncorrected and corrected iPSCs were differentiated into hepatocytes under both two-dimensional and three-dimensional differentiation protocols and deciphered the production of active FIX in vitro. Finally, we assessed the therapeutic efficacy of this approach in vivo using a mouse model of HB. CONCLUSIONS: Functional FIX, whose post-translational modifications only occur in fully mature hepatocytes, was only produced in corrected iPSCs differentiated in organoids. Immunohistochemistry analyses of mouse livers indicated a good cell engraftment, and the FIX activity detected in the plasma of transplanted animals confirmed rescue of the bleeding phenotype.

Long-term immunogenicity, efficacy and tolerability of simoctocog alfa in patients with severe haemophilia A who had completed the NuProtect study in previously untreated patients.

BACKGROUND: The NuProtect study reported data on the immunogenicity, efficacy and tolerability of simoctocog alfa (Nuwiq® ) in 108 previously untreated patients with severe haemophilia A planned to be treated for ≥100 exposure days or up to 5 years. The NuProtect-Extension study collected long-term prophylaxis data in children with severe haemophilia A. METHODS: Patients who completed the NuProtect study according to the protocol were eligible for the NuProtect-Extension study, a prospective, multinational, non-controlled, Phase 3b study. RESULTS: Of 48 patients who entered the extension study, 47 (median age 2.8 years) received prophylaxis with simoctocog alfa for a median of 24 months, with 82%-88% on a twice-weekly or less regimen. No patient developed FVIII inhibitors during the extension study. The median (IQR) annualized bleeding rate (ABR) during prophylaxis was 0 (0-0.5) for spontaneous bleeding episodes (BEs) and 1.00 (0-1.95) for all BEs. ABRs estimated using a negative binomial model were .28 (95% CI: .15, .53) for spontaneous and 1.62 (95% CI: 1.09, 2.42) for all BEs. During the median follow-up of 24 months, 34 (72%) patients had zero spontaneous BEs and 46 (98%) had zero spontaneous joint BEs. Efficacy in treating BEs was excellent or good for 78.2% of rated BEs, and efficacy of surgical prophylaxis was excellent for two rated surgeries. No treatment-related adverse events were reported. CONCLUSION: No FVIII inhibitors developed during long-term prophylaxis in the NuProtect-Extension study. Prophylaxis with simoctocog alfa was efficacious and well-tolerated, and is therefore an attractive long-term option for children with severe haemophilia A.

Revised terminal half-life of nonacog alfa as derived from extended sampling data: A real-world study involving 64 haemophilia B patients on nonacog alfa regular prophylaxis.

BACKGROUND: Nonacog alfa, a standard half-life recombinant factor IX (FIX), is used as a prophylactic treatment in severe haemophilia B (SHB) patients. Its half-life determined in clinical studies involving a limited sampling (72 h) was shown to be rather short. In our clinical practice, we suspected that its half-life could have been underestimated. OBJECTIVES: We aimed to evaluate nonacog alfa pharmacokinetics in real world clinical practice based on FIX levels in patients receiving prophylaxis. METHODS: We retrospectively collected data on patients with SHB receiving prophylaxis from eight centres across France. The terminal half-life (THL), time to reach 5-2 IU/dl and FIX activity at 48, 72 and 96 h were derived by Bayesian estimations using NONMEM analysis. RESULTS AND CONCLUSIONS: Infusion data (n = 455) were collected from 64 patients with SHB. The median THL measured in 92 pharmacokinetic (PK) studies was 43.4 h. In 26 patients ≤12 years of age, 51 PK studies showed a median time to reach 5 IU/dl of FIX of 70.5 h and a median time to reach 2 IU/dl of 121.5 h. In 38 patients 13-75 years of age, 41 PK studies showed a median time to reach 5 IU/dl of FIX of 92.0 h and a median time to reach 2 IU/dl of 167.5 h. Extending the sampling beyond 72 h makes it possible to observe a plateau, with FIX remaining between 2 and 5 IU/dl for several days and shows that the THL of nonacog alfa might be longer than previously described. ESSENTIALS: Nonacog alfa terminal half-life (THL) in patients receiving regular prophylaxis was evaluated in clinical practice. The median THL was estimated to be 36.9 h for patients aged .8-12 years. The median THL was estimated to be 49.9 h for patients aged 13-75 years. For patients aged ≤12 and >12 years, the median times to reach 5 IU/dl were 70.5 and 92 h, respectively; to reach 3 IU/dl, 95.5 and 131.5 h, respectively; to reach 2 IU/dl, 121.5 and 167.5 h, respectively. We suggest that the half-life of nonacog alfa might be longer than previously described in both younger and older patients.

Practical considerations for nonfactor-replacement therapies in the treatment of haemophilia with inhibitors.

New therapeutic agents for haemophilia with inhibitors that are in development or already licensed are expected to provide transformative treatment options. Many of these new therapies are not based on simply replacing the missing factor; new strategies include bispecific antibody technology that mimics factor VIII coagulation function (emicizumab), and inhibition of anticoagulant proteins such as tissue factor pathway inhibitor (eg PF-06741086) and antithrombin (eg fitusiran). These agents are administered subcutaneously and should significantly reduce treatment burden and increase the ability to deliver prophylaxis for patients. Limited real-world data and validated practical guidance on these recently licensed/upcoming treatments resulted in the authors convening to discuss recommendations on their use. Emicizumab is currently the only licenced nonfactor therapy; thus, our recommendations focus on this product. Target candidates for emicizumab prophylaxis are difficult-to-treat patients with haemophilia A and inhibitors and/or venous access issues, frequent bleeds and target joints. In case of breakthrough bleeding while receiving emicizumab, patients still require treatment with bypassing agents; the adjunct treatment of choice is recombinant activated factor VII. This treatment is also recommended to prevent bleeds in patients with inhibitors undergoing surgery. Our recommendations on suitable laboratory assays and monitoring new products, as well as the benefit of patient-reported outcomes (such as pain and physical activity levels), are included. We also briefly discuss future treatment options for patients with haemophilia B and inhibitors. Although these nonfactor treatments offer great promise, further data and real-world evidence are needed.

AsnB is responsible for peptidoglycan precursor amidation in Clostridium difficile in the presence of vancomycin.

Clostridium difficile 630 possesses a cryptic but functional gene cluster vanGCd homologous to the vanG operon of Enterococcus faecalis. Expression of vanGCd in the presence of subinhibitory concentrations of vancomycin is accompanied by peptidoglycan amidation on the meso-DAP residue. In this paper, we report the presence of two potential asparagine synthetase genes named asnB and asnB2 in the C. difficile genome whose products were potentially involved in this peptidoglycan structure modification. We found that asnB expression was only induced when C. difficile was grown in the presence of vancomycin, yet independently from the vanGCd resistance and regulation operons. In addition, peptidoglycan precursors were not amidated when asnB was inactivated. No change in vancomycin MIC was observed in the asnB mutant strain. In contrast, overexpression of asnB resulted in the amidation of most of the C. difficile peptidoglycan precursors and in a weak increase of vancomycin susceptibility. AsnB activity was confirmed in E. coli. In contrast, the expression of the second asparagine synthetase, AsnB2, was not induced in the presence of vancomycin. In summary, our results demonstrate that AsnB is responsible for peptidoglycan amidation of C. difficile in the presence of vancomycin.

Inhibitors in haemophilia A and B: Management of bleeds, inhibitor eradication and strategies for difficult-to-treat patients.

The standard therapy for patients with haemophilia is prophylactic treatment with replacement factor VIII (FVIII) or factor IX (FIX). Patients who develop inhibitors against FVIII/FIX face an increased risk of bleeding, and the likelihood of early development of progressive arthropathy, alongside higher treatment-related costs. Bypassing agents can be used to prevent and control bleeding, as well as the recently licensed prophylaxis, emicizumab, but their efficacy is less predictable than that of factor replacement therapy. Antibody eradication, by way of immune tolerance induction (ITI), is still the preferred management strategy for treating patients with inhibitors. This approach is successful in most patients, but some are difficult to tolerise and/or are unresponsive to ITI, and they represent the most complicated patients to treat. However, there are limited clinical data and guidelines available to help guide physicians in formulating the next treatment steps in these patients. This review summarises currently available treatment options for patients with inhibitors, focussing on ITI regimens and those ITI strategies that may be used in difficult-to-treat patients. Some alternative, non-ITI approaches for inhibitor management, are also proposed.

FranceCoag: a 22-year prospective follow-up of the national French cohort of patients with inherited bleeding disorders.

FranceCoag is an ongoing open prospective multicentre cohort project aimed at improving epidemiological knowledge about inherited bleeding disorders in France. The main objective of this article was to evaluate the project's progress as of the 30th December 2016. Between 1994 and this date, of the 10,047 patients included in the study, 384 (3.8%) were reported by clinicians to have died and 159 (1.6%) to be lost to follow-up. Among the remaining 9504 patients still being followed up, 5748 (60.5%) had haemophilia A, 1300 (13.7%) haemophilia B, 1980 (20.8%) von Willebrand Disease while 476 (5.0%) had another clotting factor deficiency (Factor I, II, V, combined V and VIII, VII, X, XI and XIII). The median age of the population was 32 years (Inter-quartile range (IQR) 18-50 years) at data extraction on December 30th, 2016. The subgroup of children (i.e., < 18 years old) with severe haemophilia and comprehensive information available since the first exposure to treatment was identified as the PUPs (Previously Untreated Patients) cohort. Data for the 643 children included in the PUPs' cohort had been collected since their birth. Follow-up data were collected by the clinicians in haemophilia treatment centres (HTC) every 12.9 months on median (IQR 11.4-21.3). In the PUPS cohort, data were updated every 6.2 months on median (IQR 3.7-11.7). A unique patient number assigned at study inclusion was kept at individual HTC by participating clinicians. The data collected included demographic, clinical, therapeutic and biological items on standard electronic forms. As of December 30th 2016, a plasma and serum samples was available for 2581 patients (27.1%).

Arr-cb Is a Rifampin Resistance Determinant Found Active or Cryptic in Clostridium bolteae Strains.

Clostridiumbolteae, which belongs to the Clostridium clostridioforme complex, is a member of the human gut microbiota. Recent analysis of seven genomes of Cbolteae revealed the presence of an arr-like gene. Among these strains, only 90A7 was found to be resistant to rifampin in the absence of alteration of RpoB. Cloning of arr-cb from 90A7 in Escherichia coli combined with directed mutagenesis demonstrated that Arr-cb was functional but that a Q127→R variant present in 90A9 and 90B3 was inactive. Quantitative reverse transcription-PCR analysis indicated that arr-cb was silent in the four remaining strains because of defective transcription. Thus, two independent mechanisms can make the probably intrinsic arr-cb gene of Cbolteae cryptic.

A national French noninterventional study to assess the long-term safety and efficacy of reformulated nonacog alfa.

BACKGROUND: Nonacog alfa, the recombinant Factor IX (F IX) used for the treatment of hemophilia B, was approved in Europe in 1998. A reformulated version was approved for European use in 2007. STUDY DESIGN AND METHODS: This postmarketing study, as recommended by the risk management plan, was conducted to confirm the safety of reformulated nonacog alfa in a usual care setting in France. This open-label, noninterventional, prospective, longitudinal postmarketing study comprised 19 French hemophilia centers. Patients with hemophilia B receiving reformulated nonacog alfa for prophylaxis or on-demand treatment were followed up on usual care schedule. RESULTS: A total of 58 subjects were enrolled, of whom 29 (50%) were less than 18 years of age. Hemophilia was severe (baseline F IX activity < 1%) in 47 (81%) patients. All subjects except one were already treated with reformulated nonacog alfa before enrollment. One subject was receiving reformulated nonacog alfa as immune tolerance induction at time of enrollment. At enrollment, treatment regimen was mainly prophylactic in subjects less than 18 years and on-demand in subjects 18 years or older. Median duration of follow-up in the survey was 3.3 (2.3-3.8) years. The median annualized bleeding rate was 3.9 (1.5-5.2) for prophylaxis regimen and 12.2 (3.9-22.1) for on-demand regimen. One subject, a previously untreated patient, developed F IX inhibitors during follow-up. No allergic reaction, no blood cell agglutination, no lack of efficacy or recovery, and no thrombotic events were reported. CONCLUSION: Reformulated nonacog alfa was shown to be safe in a usual care setting.

Outcome measures for adult and pediatric hemophilia patients with inhibitors.

Recent advancements in almost all aspects of hemophilia treatment have vastly improved patient care and management, and new and emerging treatments hold the promise of further progress. However, there remains a scarcity of data on long-term outcomes in hemophilia, particularly among those patients with inhibitors, for whom no validated outcome assessment tools are currently available. At the 15th Zürich Haemophilia Forum, an expert panel reviewed the most important outcome measures in inhibitor patients and considered the challenges associated with assessing outcomes in this population. A framework for outcome assessment in inhibitor patients incorporates traditional hemophilia outcome measures, such as bleed frequency and mortality, alongside measures of health, functioning, disability, social participation, quality of life, and economic considerations. It is important to remember that inhibitor patients differ in their clinical needs, perspectives, and priorities according to age, inhibitor status, degree of joint disease, and activity levels; as a result, the relative importance of different outcome measures will change throughout an inhibitor patient's life. Challenges inherent in measuring long-term outcomes in inhibitor patients include the small number of known patients, the subjective nature of many outcome assessment tools, and the risk of overburdening patients with repeated requests to complete questionnaires or participate in studies. Therefore, there is an urgent need to reach consensus on the most important and appropriate assessment tools for measuring outcomes in this population. These tools should ideally be standardized, easily applied, and internationally applicable in order to collect and generate quality outcome data.

Comparative genomics of Clostridium bolteae and Clostridium clostridioforme reveals species-specific genomic properties and numerous putative antibiotic resistance determinants.

BACKGROUND: Clostridium bolteae and Clostridium clostridioforme, previously included in the complex C. clostridioforme in the group Clostridium XIVa, remain difficult to distinguish by phenotypic methods. These bacteria, prevailing in the human intestinal microbiota, are opportunistic pathogens with various drug susceptibility patterns. In order to better characterize the two species and to obtain information on their antibiotic resistance genes, we analyzed the genomes of six strains of C. bolteae and six strains of C. clostridioforme, isolated from human infection. RESULTS: The genome length of C. bolteae varied from 6159 to 6398 kb, and 5719 to 6059 CDSs were detected. The genomes of C. clostridioforme were smaller, between 5467 and 5927 kb, and contained 5231 to 5916 CDSs. The two species display different metabolic pathways. The genomes of C. bolteae contained lactose operons involving PTS system and complex regulation, which contribute to phenotypic differentiation from C. clostridioforme. The Acetyl-CoA pathway, similar to that of Faecalibacterium prausnitzii, a major butyrate producer in the human gut, was only found in C. clostridioforme. The two species have also developed diverse flagella mobility systems contributing to gut colonization. Their genomes harboured many CDSs involved in resistance to beta-lactams, glycopeptides, macrolides, chloramphenicol, lincosamides, rifampin, linezolid, bacitracin, aminoglycosides and tetracyclines. Overall antimicrobial resistance genes were similar within a species, but strain-specific resistance genes were found. We discovered a new group of genes coding for rifampin resistance in C. bolteae. C. bolteae 90B3 was resistant to phenicols and linezolide in producing a 23S rRNA methyltransferase. C. clostridioforme 90A8 contained the VanB-type Tn1549 operon conferring vancomycin resistance. We also detected numerous genes encoding proteins related to efflux pump systems. CONCLUSION: Genomic comparison of C. bolteae and C. clostridiofrome revealed functional differences in butyrate pathways and in flagellar systems, which play a critical role within human microbiota. Most of the resistance genes detected in both species were previously characterized in other bacterial species. A few of them were related to antibiotics inactive against Clostridium spp. Some were part of mobile genetic elements suggesting that these commensals of the human microbiota act as reservoir of antimicrobial resistances.

Origin in Acinetobacter gyllenbergii and dissemination of aminoglycoside-modifying enzyme AAC(6')-Ih.

OBJECTIVES: The aac(6')-Ih gene encoding aminoglycoside 6'-N-acetyltransferase type I subtype h [AAC(6')-Ih] is plasmid-borne in Acinetobacter baumannii where it confers high-level amikacin resistance, but its origin remains unknown. We searched for the gene in the genomes of a collection of 133 Acinetobacter spp. and studied its species specificity, expression and dissemination. METHODS: Gene copy number was determined by quantitative PCR, expression by quantitative RT-PCR, MIC by microdilution and transfer by plasmid mobilization. RESULTS: The aac(6')-Ih gene was present in the chromosome of the two Acinetobacter gyllenbergii of the collection and was detected in all seven A. gyllenbergii clinical isolates. They had indistinguishable flanking regions indicating that the gene was intrinsic to this species. A. baumannii PIS Aba23 promoters were provided by insertion of ISAba23, which disrupted the Pnative promoter in A. gyllenbergii. Both types of promoters were similarly potent in Escherichia coli and A. baumannii. Aminoglycoside MICs for A. baumannii harbouring pIP1858 were higher than for A. gyllenbergii due to gene dosage. The non-self-transferable plasmid could be mobilized to other A. baumannii cells by the broad host range plasmid RP4. CONCLUSIONS: We have found the origin of aac(6')-Ih in A. gyllenbergii, a species isolated, although rarely, in humans, and documented that dissemination of this gene is restricted to the Acinetobacter genus.

High susceptibility of MDR and XDR Gram-negative pathogens to biphenyl-diacetylene-based difluoromethyl-allo-threonyl-hydroxamate LpxC inhibitors.

OBJECTIVES: Inhibitors of uridine diphosphate-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC, which catalyses the first, irreversible step in lipid A biosynthesis) are a promising new class of antibiotics against Gram-negative bacteria. The objectives of the present study were to: (i) compare the antibiotic activities of three LpxC inhibitors (LPC-058, LPC-011 and LPC-087) and the reference inhibitor CHIR-090 against Gram-negative bacilli (including MDR and XDR isolates); and (ii) investigate the effect of combining these inhibitors with conventional antibiotics. METHODS: MICs were determined for 369 clinical isolates (234 Enterobacteriaceae and 135 non-fermentative Gram-negative bacilli). Time-kill assays with LPC-058 were performed on four MDR/XDR strains, including Escherichia coli producing CTX-M-15 ESBL and Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii producing KPC-2, VIM-1 and OXA-23 carbapenemases, respectively. RESULTS: LPC-058 was the most potent antibiotic and displayed the broadest spectrum of antimicrobial activity, with MIC90 values for Enterobacteriaceae, P. aeruginosa, Burkholderia cepacia and A. baumannii of 0.12, 0.5, 1 and 1 mg/L, respectively. LPC-058 was bactericidal at 1× or 2× MIC against CTX-M-15, KPC-2 and VIM-1 carbapenemase-producing strains and bacteriostatic at ≤4× MIC against OXA-23 carbapenemase-producing A. baumannii. Combinations of LPC-058 with ß-lactams, amikacin and ciprofloxacin were synergistic against these strains, albeit in a species-dependent manner. LPC-058's high efficacy was attributed to the presence of the difluoromethyl-allo-threonyl head group and a linear biphenyl-diacetylene tail group. CONCLUSIONS: These in vitro data highlight the therapeutic potential of the new LpxC inhibitor LPC-058 against MDR/XDR strains and set the stage for subsequent in vivo studies.

AAC(3)-XI, a new aminoglycoside 3-N-acetyltransferase from Corynebacterium striatum.

Corynebacterium striatum BM4687 was resistant to gentamicin and tobramycin but susceptible to kanamycin A and amikacin, a phenotype distinct among Gram-positive bacteria. Analysis of the entire genome of this strain did not detect any genes for known aminoglycoside resistance enzymes. Yet, annotation of the coding sequences identified 12 putative acetyltransferases or GCN5-related N-acetyltransferases. A total of 11 of these coding sequences were also present in the genomes of other Corynebacterium spp. The 12th coding sequence had 55 to 60% amino acid identity with acetyltransferases in Actinomycetales. The gene was cloned in Escherichia coli, where it conferred resistance to aminoglycosides by acetylation. The protein was purified to homogeneity, and its steady-state kinetic parameters were determined for dibekacin and kanamycin B. The product of the turnover of dibekacin was purified, and its structure was elucidated by high-field nuclear magnetic resonance (NMR), indicating transfer of the acetyl group to the amine at the C-3 position. Due to the unique profile of the reaction, it was designated aminoglycoside 3-N-acetyltransferase type XI.

Switching treatments in haemophilia: is there a risk of inhibitor development?

Patients with haemophilia A (and their physicians) may be reluctant to switch factor VIII (FVIII) concentrates, often due to concerns about increasing the risk of inhibitors; this reluctance to switch may contribute to patients missing the clinical benefits provided by the arrival of new factor VIII products. This topic was explored at the Eleventh Zürich Haemophilia Forum. Clinical scenarios for which product switching may be cause for concern were discussed; when there is a clinical need, there are no absolute contraindications to switching, but some patients (e.g. previously untreated patients and those undergoing elective surgery) may require more careful consideration. Both patient and physician surveys indicate that the reluctance to switch, and the fear of inhibitor development, does not appear to be evidence based. The evaluation of more recent data did not support previous studies suggesting that particular products (e.g. recombinant vs. plasma-derived and full length vs. B-domain modified) may be associated with increased risk. In addition, data from three national product switches showed that switching was not associated with increased inhibitor risk, but highlighted the need for regular inhibitor testing and for a centralised, unbiased database of inhibitor incidence. To conclude, current evidence does not suggest that switching products significantly influences inhibitor development.

The functional vanGCd cluster of Clostridium difficile does not confer vancomycin resistance.

vanGCd, a cryptic gene cluster highly homologous to the vanG gene cluster of Enterococcus faecalis is largely spread in Clostridium difficile. Since emergence of vancomycin resistance would have dramatic clinical consequences, we have evaluated the capacity of the vanGCd cluster to confer resistance. We showed that expression of vanGCd is inducible by vancomycin and that VanGCd , VanXYCd and VanTCd are functional, exhibiting D-Ala : D-Ser ligase, D,D-dipeptidase and D-Ser racemase activities respectively. In other bacteria, these enzymes are sufficient to promote vancomycin resistance. Trans-complementation of C. difficile with the vanC resistance operon of Enterococcus gallinarum faintly impacted the MIC of vancomycin, but did not promote vancomycin resistance in C. difficile. Sublethal concentration of vancomycin led to production of UDP-MurNAc-pentapeptide[D-Ser], suggesting that the vanGCd gene cluster is able to modify the peptidoglycan precursors. Our results indicated amidation of UDP-MurNAc-tetrapeptide, UDP-MurNAc-pentapeptide[D-Ala] and UDP-MurNAc-pentapeptide[D-Ser]. This modification is passed on the mature peptidoglycan where a muropeptide Tetra-Tetra is amidated on the meso-diaminopimelic acid. Taken together, our results suggest that the vanGCd gene cluster is functional and is prevented from promoting vancomycin resistance in C. difficile.

Inhibitor development according to concentrate in severe hemophilia: reporting on 1392 Previously Untreated Patients from Europe and Canada.

Background: Clotting factor concentrates have been the mainstay of severe hemophilia treatment over the last 50 years. Differences in risk of neutralizing antibody (inhibitor) formation according to concentrate used remain clinically relevant. Objectives: To assess inhibitor development according to type of clotting factor concentrate in previously untreated patients (PUPs) with severe hemophilia A and B. Methods: The European Haemophilia Safety Surveillance (EUHASS) and Canadian Bleeding Disorders Registry (CBDR) have been monitoring adverse events overall and according to concentrate for 11 and 8 years, respectively. Inhibitors were reported quarterly, and PUPs completed 50 exposure days without inhibitor development annually. Cumulative inhibitor incidences and 95% confidence intervals (CIs) were compared without adjustment for other risk factors. Results: Fifty-six European and 23 Canadian centers reported inhibitor development in 312 of 1219 (26%; CI, 23%-28%) PUPs with severe hemophilia A and 14 of 173 (8%; CI, 5%-13%) PUPs with severe hemophilia B. Inhibitor development was lower on plasma-derived factor (F)VIII (pdFVIII, 20%; CI, 14%-26%) than on standard half-life recombinant FVIII (SHL-rFVIII, 27%; CI, 24%-30% and odds ratio, 0.67; CI, 0.45%-0.98%; P = .04). Extended half-life recombinant FVIII (EHL-rFVIII, 22%; CI, 12%-36%) showed an intermediate inhibitor rate, while inhibitor rates for Advate (26%; CI, 22%-31%) and Kogenate/Helixate (30%; CI, 24%-36%) overlapped. For other SHL-rFVIII concentrates, inhibitor rates varied from 3% to 43%. Inhibitor development was similar for pdFIX (11%; CI, 3%-25%), SHL-rFIX (8%; CI, 3%-15%), and EHL-rFIX (7%; CI, 1%-22%). Conclusion: While confirming expected rates of inhibitors in PUPs, inhibitor development was lower in pdFVIII than in SHL-rFVIII. Preliminary data suggest variation in inhibitor development among different SHL-rFVIII and EHL-rFVIII concentrates.

RmtF, a new member of the aminoglycoside resistance 16S rRNA N7 G1405 methyltransferase family.

Multidrug-resistant clinical isolate Klebsiella pneumoniae BM4686 was highly resistant to 4,6-disubstituted 2-deoxystreptamines and to fortimicin. Resistance was due to the presence, on the 40-kb non-self-transferable plasmid pIP849, of the rmtF gene which was cotranscribed with the upstream aac(6')-Ib gene. The deduced RmtF protein had 25 to 46% identity with members of the N7 G1405 family of aminoglycoside resistance 16S rRNA methyltransferases.

Immune tolerance induction in patients with severe hemophilia with inhibitors: expert panel views and recommendations for clinical practice.

For hemophilia patients with inhibitors, immune tolerance induction (ITI) may help to restore clinical response to factor (F) VIII or FIX concentrates. Several ITI regimens and protocols exist; however, despite 30 yr of progressive investigation, the ITI evidence base relies mainly on observational data. Expert opinion, experience, and interpretation of the available evidence are therefore valuable to support clinical decision-making. At the Sixth Zürich Haemophilia Forum, an expert panel considered recent data and consensus to distill key practice points relating to ITI. The panel supported current recommendations that, where feasible, ITI should be offered early to children and adults (ideally ≤ 5 yr of inhibitor detection) when inhibitor titers are <10 Bethesda units (BU) and should be stopped when successful tolerance is achieved. For hemophilia A inhibitor patients, ITI can be founded on recombinant FVIII at high doses. The panel considered that patients with a high bleeding frequency should be offered additional prophylaxis with a bypassing agent. For patients with hemophilia B, there may be a benefit of genetic testing to indicate the risk for inhibitors. ITI is often less effective and associated with a greater risk of side effects in these patients. For high-titer inhibitor (≥ 5 BU) hemophilia B patients, the panel advised that bypassing agents could be offered on demand in addition to ITI. Within future ITI regimens, there may be a role for additional immunosuppressant therapies. Participants agreed that research is needed to find alternatives to ITI therapy that offer durable and sustained effects and reduced rates of complications.