Performance characteristics and comparison of Abbott and artus real-time systems for hepatitis B virus DNA quantification

Virological monitoring of hepatitis B virus (HBV) DNA is critical to the management of HBV infection. With several HBV DNA quantification assays available, it is important to use the most efficient testing system for virological monitoring. In this study, we evaluated the performance characteristics and comparability of three HBV DNA quantification systems: Abbott HBV real-time PCR (Abbott PCR), artus HBV real-time PCR with QIAamp DNA blood kit purification (artus-DB), and artus HBV real-time PCR with the QIAamp DSP virus kit purification (artus-DSP). The lower limits of detection of these systems were established against the WHO international standards for HBV DNA and were found to be 1.43, 82, and 9 IU/ml, respectively. The intra-assay and interassay coefficients of variation of plasma samples (1 to 6 log(10) IU/ml) ranged between 0.05 to 8.34% and 0.16 to 3.48% for the Abbott PCR, 1.53 to 26.85% and 0.50 to 12.89% for artus-DB, and 0.29 to 7.42% and 0.94 to 3.01% for artus-DSP, respectively. Ninety HBV clinical samples were used for comparison of assays, and paired quantitative results showed strong correlation by linear regression analysis (artus-DB with Abbott PCR, r = 0.95; Abbott PCR with artus-DSP, r = 0.97; and artus-DSP with artus-DB, r = 0.94). Bland-Altman analysis showed a good level of agreement for Abbott PCR and artus-DSP, with a mean difference of 0.10 log(10) IU/ml and limits of agreement of -0.91 to 1.11 log(10) IU/ml. No genotype-specific bias was seen in all three systems for HBV genotypes A, C, and D, which are predominant in this region. This finding illustrates that the Abbott real-time HBV and artus-DSP systems show more comparable performance than the artus-DB system, meeting the current guidelines for assays to be used in the management of hepatitis B.     

Implementation of a methicillin-resistant Staphylococcus aureus (MRSA) prevention bundle results in decreased MRSA surgical site infections

Background

Methicillin-resistant Staphylococcus aureus (MRSA) surgical site infections (SSIs) increase morbidity and mortality. We examined the impact of the MRSA bundle on SSIs.

Methods

Data regarding the implementation of the MRSA bundle from 2007 to 2008 were obtained, including admission and discharge MRSA screenings, overall MRSA infections, and cardiac and orthopedic SSIs. Chi-square was used for all comparisons.

Results

A significant decrease in MRSA transmission from a 5.8 to 3.0 per 1,000 bed-days (P < .05) was found after implementation of the MRSA bundle. Overall MRSA nosocomial infections decreased from 2.0 to 1.0 per 1,000 bed-days (P = .016). There was a statistically significant decrease in overall SSIs (P < .05), with a 65% decrease in orthopaedic MRSA SSIs and 1% decrease in cardiac MRSA SSIs.

Conclusion

Our data demonstrate that successful implementation of the MRSA bundle significantly decreases MRSA transmission between patients, the overall number of nosocomial MRSA infections, and MRSA SSIs.     

Novel SOX2 partner-factor domain mutation in a four-generation family

Anophthalmia (no eye), microphthalmia (small eye) and associated ocular developmental anomalies cause significant visual handicap. In most cases the underlying genetic cause is unknown, but mutations in some genes, such as SOX2, cause ocular developmental defects, particularly anophthalmia, in a subset of patients. Here, we describe a four-generation family with a p.Asp123Gly mutation in the highly conserved partner-factor interaction region of the SOX2 protein, which is important for cell-specific actions of SOX2. The proband in this family has bilateral anophthalmia and several other family members have milder ocular phenotypes, including typical optic fissure coloboma. Expression studies indicate that Sox2 is expressed in the eye at the site of closure of the optic fissure during development. The SOX2 mutation in this family implicates the partner-factor interaction region of SOX2 in contributing to the specificity of SOX2 action in optic fissure closure. Our findings indicate that investigation of SOX2 in a broad range of eye anomaly patients aids in the determination of particular functions of SOX2 in development.     

Rnf19a,a ubiquitin protein ligase,and Psmc3, a component of the 26S proteasome,Tether to the acrosome membranes and the head–tail coupling apparatus during rat spermatid development

We report the cDNA cloning of rat testis Rnf19a, a ubiquitin protein ligase, and show 98% and 93% protein sequence identity of testicular mouse and human Rnf19a, respectively. Rnf19a interacts with Psmc3, a protein component of the 19S regulatory cap of the 26S proteasome. During spermatid development, Rnf19a and Psmc3 are initially found in Golgi‐derived proacrosomal vesicles. Later on, Rnf19a, Psmc3, and ubiquitin are seen along the cytosolic side of the acrosomal membranes and the acroplaxome, a cytoskeletal plate linking the acrosome to the spermatid nuclear envelope. Rnf19a and Psmc3 accumulate at the acroplaxome marginal ring–manchette perinuclear ring region during spermatid head shaping and in the developing sperm head–tail coupling apparatus and tail. Rnf19a and Psmc3 may interact directly or indirectly with each other, presumably pointing to the participation of the ubiquitin–proteasome system in acrosome biogenesis, spermatid head shaping, and development of the head‐tail coupling apparatus and tail. Developmental Dynamics 238:1851–1861, 2009. © 2009 Wiley‐Liss, Inc.     

BAK1 gene variation and abdominal aortic aneurysms

We sought to examine the role of genetics in the multifactorial disease, abdominal aortic aneurysm (AAA), by studying sequence variation in the BAK1 gene (BAK1) that codes for an apoptotic‐promoting protein, as chronic apoptosis activation has been linked to AAA development and progression. BAK1 abdominal aorta cDNA from AAA patients and nondiseased individuals were compared with each other, as well as to the BAK1 genomic sequence obtained from matching blood samples. We found specific BAK1 single nucleotide polymorphism (SNP) containing alleles in both aneurysmic (31 cases) and healthy aortic tissue (5 cases) without seeing them in the matching blood samples. These same BAK1 SNPs have been reported, although rarely (average frequency <0.06%), in reference BAK1 DNA sequences. Based on this and other similar observations, we propose a novel hypothesis postulating that multiple variants of genes may preexist in “minority” forms within specific nondiseased tissues and be selected for, when intra‐ and/or extracellular conditions change. Therefore, the fact that different BAK1 variants can exist in both diseased and nondiseased AA tissues compared to matching blood samples, together with the rare occurrence of these same SNPs in reference sequences, suggests that selection may be a significant factor in AAA ontogeny. Hum Mutat 30:1–5, 2009. © 2009 Wiley‐Liss, Inc.     

Multiplex padlock targeted sequencing reveals human hypermutable CpG variations

Utilizing the full power of next-generation sequencing often requires the ability to perform large-scale multiplex enrichment of many specific genomic loci in multiple samples. Several technologies have been recently developed but await substantial improvements. We report the 10,000-fold improvement of a previously developed padlock-based approach, and apply the assay to identifying genetic variations in hypermutable CpG regions across human chromosome 21. From ∼3 million reads derived from a single Illumina Genome Analyzer lane, ∼94% (∼50,500) target sites can be observed with at least one read. The uniformity of coverage was also greatly improved; up to 93% and 57% of all targets fell within a 100- and 10-fold coverage range, respectively. Alleles at >400,000 target base positions were determined across six subjects and examined for single nucleotide polymorphisms (SNPs), and the concordance with independently obtained genotypes was 98.4%–100%. We detected >500 SNPs not currently in dbSNP, 362 of which were in targeted CpG locations. Transitions in CpG sites were at least 13.7 times more abundant than non-CpG transitions. Fractions of polymorphic CpG sites are lower in CpG-rich regions and show higher correlation with human–chimpanzee divergence within CpG versus non-CpG sites. This is consistent with the hypothesis that methylation rate heterogeneity along chromosomes contributes to mutation rate variation in humans. Our success suggests that targeted CpG resequencing is an efficient way to identify common and rare genetic variations. In addition, the significantly improved padlock capture technology can be readily applied to other projects that require multiplex sample preparation.For decades, DNA sequencing has been pivotal in understanding biology, yielding over 900 whole-genome sequences, and identifying genetic variations and somatic mutations that underlie human diseases (Frazer et al. 2007; Stenson et al. 2008). Recent sequencing-based studies suggest that a large panel of genes is mutated in various cancers (Sjoblom et al. 2006; Jones et al. 2008; Parsons et al. 2008). Individually rare but cumulatively frequent variations contribute to the inheritance of common multifactorial diseases (Cohen et al. 2004, 2006; Bodmer and Bonilla 2008; Ji et al. 2008). Recently, “deep sequencing” has been enabled by “next-generation” technologies that reduce sequencing costs by several orders of magnitude (Shendure and Ji 2008). However, it is still prohibitively expensive to sequence whole human genomes, particularly when sample sizes are large. Thus, multiplexed targeted amplification of many genomic regions of interest is crucial for rapid and cost-effective sequencing-based research projects.Parallel targeted amplification of selected genome regions is a challenging task (Garber 2008). Two different categories of methods have been developed to enrich or capture desired genomic regions, such as exons. One category employs hybridization of sheared genomic DNA to probes complementary to targeted regions. The probes can be oligonucleotides on a microarray surface (Albert et al. 2007; Hodges et al. 2007; Okou et al. 2007) or in solution (Gnirke et al. 2009). Although most of the desired regions are captured, the specificity of enriched genomic DNA tends to be limited due to “off target” and “near target” capture. In addition, due to the low efficiency of hybridization on the surface of a microarray, large amounts of genomic DNA are needed. The other category of methods requires hybridization in regions flanking both sides of the target and subsequent circularization of the targets. One way is to use “selector” oligonucleotides to guide the directed circularization of the target sequences digested with restriction enzymes (Dahl et al. 2005, 2007). Another method, which is independent of the presence of flanking restriction enzyme sites and thus is more flexible, applies padlock (molecular inversion) probes that anchor targeted regions and are circularized after polymerization and ligation (Hardenbol et al. 2003; Porreca et al. 2007). In our initial study, we targeted 55,000 exons but observed only ∼10,000 unique sites in over 2 million end-sequencing reads, and most heterozygous loci were called incorrectly (Porreca et al. 2007), indicating the need for substantial improvement in capturing efficiency.In the human genome, CpG dinucleotides are about fivefold less abundant than expected by chance (Sved and Bird 1990). This is due to the widespread methylation of cytosine in CpG and the deamination of 5-methylcytosine to thymidine (Wang et al. 1982); CpG is thus frequently mutated to TpG (or CpA on the complementary DNA strand). Overall, CpG elevates the mutation rate for transitions by 14- to 15-fold and for transversions by three- to fourfold (Kondrashov 2003; Hwang and Green 2004; Schmidt et al. 2008).Mutations within the CpG context are a predominant cause of human diseases. At least one-third of mutations implicated in Mendelian diseases originated within CpG contexts (Cooper and Youssoufian 1988; Cooper and Krawczak 1993). Although CpG sites are depleted in the bulk of noncoding human DNA, they are selectively maintained in protein-coding genes and other functional genomic regions despite the elevated mutation rate (Subramanian and Kumar 2003; Kondrashov et al. 2006). Thus, the prevalence of CpG-induced mutations among disease mutations is much greater than among all mutations.CpG context plays an important role in somatic mutations involved in human cancer. In the TP53 gene, which is mutated in >50% of all human tumors, ∼30% of all mutations occur at CpG dinucleotides, and all five major mutation hotspots are found at CpGs (Olivier et al. 2002). Recently, sequencing of nearly all protein-coding regions in cancer genomes revealed that 17%, 38%, 43%, and 48% of point mutations occur at CpGs in breast, pancreatic, brain, and colorectal cancers, respectively (Sjoblom et al. 2006; Jones et al. 2008; Parsons et al. 2008).In this work, we describe improvements to our padlock capturing technology that yield an estimated 10,000-fold increase in capture efficiency over previous reports and significant improvements in sensitivity and uniformity. We designed 53,777 padlock probes to cover ∼24% of all CpGs on human chromosome 21, where each probe captured a 40-bp region containing at least one CpG, and applied them to discover genetic variants in the genomic DNA from one HapMap CEPH individual (NA10835) and five volunteers from the Personal Genome Project (PGP; http://www.personalgenomes.org). We report the improved performance and high reproducibility of our optimized methods, and demonstrate the utility of the data for identification of known and novel SNPs and unbiased analysis of CpG variation rates.     

Challenges to effective crisis management: using information and communication technologies to coordinate emergency medical services and emergency department teams

OBJECTIVE: The purpose of this study is to identify the major challenges to coordination between emergency department (ED) teams and emergency medical services (EMS) teams. DESIGN: We conducted a series of focus groups involving both ED and EMS team members using a crisis scenario as the basis of the focus group discussion. We also collected organizational workflow data. RESULTS: We identified three major challenges to coordination between ED and EMS teams including ineffectiveness of current information and communication technologies, lack of common ground, and breakdowns in information flow. DISCUSSION: The three challenges highlight the importance of designing systems from socio-technical perspective. In particular, these inter-team coordination systems must support socio-technical issues such as awareness, context, and workflow between the two teams.     

Predictors of delivery of hospital-based heart failure patient education: a report from OPTIMIZE-HF

BackgroundAlthough recent heart failure (HF) management guidelines recommend delivery of patient education and discharge instructions, little is known about predictors of delivery of these materials or how such materials relate to outpatient disposition postdischarge. This report assesses the degree to which the full set of HF discharge instructions and education comprising the Joint Commission on Accreditation of Healthcare Organizations process-of-care measure (HF-1) was provided, identifies factors predictive of use of HF-1, and determines if HF-1 predicts postdischarge outcome disposition in a registry and performance improvement (PI) program for patients hospitalized for HF.Methods and ResultsIn the Organized Program to Initiate Lifesaving Treatment in Hospitalized Patients with Heart Failure (ie, OPTIMIZE-HF), of 33,681 patients from 259 US hospitals, 54% received HF-1. Some patient and site characteristics, such as symptoms on admission and performance of coronary angiography, were positively associated with delivery of the full set of HF-1 components, and others, such as African-American or Hispanic race and Midwest site location, were negatively associated with HF-1 delivery. However, delivery of the full set of HF-1 components was significantly more likely in the 46% of patients receiving PI tools (OR 2.23, 95% CI 2.12–2.35; P < .0001). Delivery of the full set of HF-1 components was significantly associated with use of specialty referral programs after discharge (P < .0001).ConclusionsDespite recommendations that complete instructions be given to patients with HF before hospital discharge, both PI tools to facilitate HF-1 and HF-1 itself are underused. Efforts should focus on strengthening processes and structures that will improve consistent delivery of HF-1 to all patients.