Simultaneous determination of five systemic azoles in plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and automatable HPLC assay was developed for a simultaneous determination of systemic azoles (fluconazole, posaconazole, voriconazole, itraconazole and its metabolite hydroxyl-itraconazole, and ketoconazole) in plasma. The major advantage of this assay was sample preparation by a fully automatable solid phase extraction with Varian Plexa cartridges. C6-phenyl column was used for chromatographic separation, and UV detection was set at a wavelength of 260 nm. Linezolid was used as an internal standard. The assay was specific and linear over the concentration range of 0.05 to 40 μg/ml excepted for fluconazole which was between 0.05 and 100 μg/ml, and itraconazole between 0.1 and 40 μg/ml. Validation data for accuracy and precision for intra- and inter-day were good and satisfied FDA's guidance: CV between 0.24% and 11.66% and accuracy between 93.8% and 108.7% for all molecules. This assay was applied to therapeutic drug monitoring on patients hospitalized in intensive care and onco-hematologic units.     

Asthma phenotypes based on health services use for allergic diseases in a province-wide birth cohort

Background

Many previous studies on asthma phenotypes were conducted in selected clinical populations and overlooked changes throughout the life course.

Management of human resources of a pharmacy department during the COVID-19 pandemic: Take-aways from the first wave

The coronavirus disease 2019 (COVID-19) is the biggest public health threat the world has seen in many years and poses new challenges and opportunities to healthcare systems. The new reality imposed by the pandemic requires a modification of practices to ensure the health and safety of patients and medical teams. The purpose of this article is to share the experiences of the pharmacy department of the Centre hospitalier de l’Université de Montréal (CHUM) in response to the COVID-19 pandemic. Seven of the most important issues will be addressed: crisis management, internal communications, employee stress, reorganisation of workspaces, reorganisation of pharmacist workforce, telework and schedule management. Some of the changes made in human resources deployment will likely remain even post-pandemic.     

Helper functions required for wild type and recombinant adeno-associated virus growth

The human parvovirus Adeno-Associated virus (AAV-2) has been classified as a Dependovirus because it requires the presence of a helper virus to achieve a productive replication cycle. Several viruses such as Adenovirus (Ad), Herpes Simplex Virus (HSV), Vaccinia virus, and human papillomaviruses (HPV) can provide the helper activities required for AAV growth. The studies on the helper activities provided by adenovirus have provided useful information not only to understand the AAV-2 biology but also to develop tools for the production of recombinant AAV particles (rAAV). This review will focus on the current knowledge about the helper activities provided by the most extensively studied helper viruses, Ad and HSV-1, and also illustrate the methods used to supply the helper functions rAAV assembly.     

Influence of the Duplication of CFTR Exon 9 and Its Flanking Sequences on Diagnosis of Cystic Fibrosis Mutations

The DNA sequences of seven regions in the human genome were examined for sequence identity with exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is mutated in cystic fibrosis, and its intronic boundaries. These sequences were 95% to 96% homologous. Based on this nucleotide sequence similarity, PCR primers for CFTR exon 9 can potentially anneal with other homologous sequences in the human genome. Sequence alignment analysis of the CFTR exon 9 homologous sequences revealed that five registered mutations in the Cystic Fibrosis Mutation Database may be due to the undesired annealing of primers to a homologous sequence, resulting in inappropriate PCR amplification. For this reason, we propose that certain pseudomutations may result from the similarity between CFTR exon 9 (and its flanking introns) and related sequences in the human genome. Here we show that two mutations previously described in the CFTR database (c.1392 + 6insC; c.1392 + 12G>A) were inappropriately attributed to two individuals who sought carrier testing. A more detailed study by either direct sequencing or subcloning and sequencing of PCR products using specially designed primers revealed that these apparent mutations were not, in fact, present in CFTR. In addition, we present new PCR conditions that permit specific amplification of CFTR exon 9 and its flanking regions.The cystic fibrosis (CF) gene, encoding the cystic fibrosis transmembrane conductance regulator (CFTR), is located on the long arm of chromosome 7, at position 7p31. CFTR is involved in the active transport of ions through the apical membrane of epithelial cells.¹ The 250-kb gene, containing 27 exons, appears highly susceptible to mutations due to its large size.² More than 1500 genetic alterations have been described to date. Most are disease-causing mutations; about half lead to amino acid substitutions (missense mutations), 20% lead to splicing errors, and 30% appear to be nonsense and frame shift (including small deletions and insertions) or promoter mutations (http://www.genet.sickkids.on.ca/cftr/, last accessed November 29, 2006). Moreover, the type and distribution of mutations vary substantially between populations.^3,4Previous studies on the CFTR gene have reported that exon 9 and its flanking introns are present in multiple copies in the human genome. Indeed, this region is part of the large duplicated sequence unit LCR7-20 (low-copy repeats 7 to 20), which is dispersed on different chromosomes in human genome.^5,6Screening for CFTR exon 9 mutations is difficult due to the polymorphism of the (TG)m (T)n repeats located at the end of intron 8. This variation eludes common PCR-based techniques for mutation detection in this region, including direct sequencing, as well as denaturing high-performance liquid chromatography. Thus, if denaturing high-performance liquid chromatography analysis is used, it is necessary to use primers that have been documented⁷ to prevent the variability that T/TG repeats can cause. Using this method, however, only the beginning of exon 9 is amplified. Because this region of CFTR has been duplicated in several regions of the genome, we suggest that using these classical primers could lead to misidentification of a CFTR pseudogene mutation as a CF-causative mutation.Here, we analyzed two cases in which there were molecular diagnosis difficulties in the exon 9 region of CFTR. We demonstrate that technical anomalies leading to incorrect molecular diagnosis are due to the primers used in the PCR protocol. The consequence of these primer mismatches could be that multiple mutations already registered in the Cystic Fibrosis Mutation Database (http://www.genet.sickkids.on.ca/cftr/; last accessed December 24, 2008; Hospital for Sick Children, Toronto, Canada) could be irrelevant.To our knowledge, there has been no previous report demonstrating the effect of sequence similarity between CFTR exon 9 (encoding a part of the first nucleotide-binding domain⁸) and related sequences on CFTR mutation screening. Therefore, our primary aim in this paper is to provide evidence of mutations in the database that are in fact pseudomutations in duplicated regions of the genome, with normal sequences in exon 9 of the CFTR gene. Since traditional methods for amplification of exon 9 and its flanking sequences will amplify several ectopic regions on chromosomes other than chromosome 7, we define here conditions that can be used to study this region exclusively. We suggest that each patient who presents such mutations should be re-examined by our proposed method.     

Transradial approach for coronary angioplasty in the setting of acute myocardial infarction: a dual-center registry.

Although transradial angioplasty has been shown to have no major entry site-related complications, its clinical applicability for balloon angioplasty and stenting in acute myocardial infarction (AMI) is unclear. In order to assess the feasibility, safety, and clinical outcome of transradial access for coronary angioplasty (PTCA) and stenting during AMI, transradial angioplasty for AMI was registered on a prospective database at two European sites (A and B) with experience in the radial approach (RA); 6 Fr catheters with an inner lumen of at least 0.064" and low-profile rapid-exchange balloons were used. Primary success rates and procedural complications of 6 Fr RA were determined and compared to 6 Fr femoral approach (FA) procedures. A total of 1,224 AMI patients entered the registry. Study site A enrolled 185 RA patients (13.6% AMI) and study site B 92 RA patients (63.4%). Patient baseline demographics were similar in both study centers and showed no differences between RA and FA patients, except a more frequent use of abciximab in study site B compared to A. PTCA was successful in > 95% of both RA and FA patients. Total procedural time did not differ between RA and FA patients. Severe access site-related bleeding complications, however, were observed in FA patients only: study site A used closure devices routinely and found 2% severe bleedings; study site B used no closure device for FA patients and observed 7% severe bleedings. In selected patients and in experienced hands, transradial PTCA in AMI has a high success rate, is clinically safe, and could become an attractive alternative access site for patients being at high or even low risk for bleeding complications.