The ability to provide timely identification of the causative agents of lower respiratory tract infections can promote better patient outcomes and support antimicrobial stewardship efforts. Current ...diagnostic testing options include culture, molecular testing, and antigen detection. These methods may require collection of various specimens, involve extensive sample treatment, and can suffer from low sensitivity and long turnaround times. This study assessed the performance of the BioFire FilmArray Pneumonia Panel (PN panel) and Pneumonia Plus Panel (PNplus panel), an FDA-cleared sample-to-answer assay that enables the detection of viruses, atypical bacteria, bacteria, and antimicrobial resistance marker genes from lower respiratory tract specimens (sputum and bronchoalveolar lavage BAL fluid). Semiquantitative results are also provided for the bacterial targets. This paper describes selected analytical and clinical studies that were conducted to evaluate performance of the panel for regulatory clearance. Prospectively collected respiratory specimens (846 BAL and 836 sputum specimens) evaluated with the PN panel were also tested by quantitative reference culture and molecular methods for comparison. The PN panel showed a sensitivity of 100% for 15/22 etiologic targets using BAL specimens and for 10/24 using sputum specimens. All other targets had sensitivities of ≥75% or were unable to be calculated due to low prevalence in the study population. Specificity for all targets was ≥87.2%, with many false-positive results compared to culture that were confirmed by alternative molecular methods. Appropriate adoption of this test could provide actionable diagnostic information that is anticipated to impact patient care and antimicrobial stewardship decisions.
•BioFire Respiratory Panel 2.1 SARS-CoV-2 assay high sensitivity and specificity.•Comparable performance to gold standard tests for low level viral RNA detection.•Rapid, sample-to-answer, syndromic ...testing for respiratory pathogens with SARS-CoV-2.
We evaluated the performance of the BioFire® Respiratory Panel 2.1 (RP2.1) in the detection of SARS CoV-2 in comparison against three other SARS CoV-2 EUA assays. In these studies, the RP2.1 panel had 98 % positive percent agreement (48/49) and 100 % negative percent agreement (49/49). Since 30 % of nasopharyngeal swab specimens have a SARS CoV-2 Ct >30 and thus detection of virus in low titers is clinically relevant, a sample with a high titer was diluted and each 10 fold dilution was tested in triplicate and compared against 6 other EUA approved SARS CoV-2 assays. These data suggested that the BioFire® RP2.1 panel, along with four other SARS CoV-2 assays (Roche cobas, Cepheid Xpert Xpress, BioFire® Defense COVID19, and NECoV19), consistently detected viral RNA at the 10−7 dilution. Overall, these studies suggest that the BioFire® RP2.1 assay can be used to detect acute cases of SARS CoV2 in addition to patients with low viral titer later in disease presentation.
The genome organizations of eight phylogenetically distinct species from five mammalian orders were compared in order to address fundamental questions relating to mammalian chromosomal evolution. ...Rates of chromosome evolution within mammalian orders were found to increase since the Cretaceous-Tertiary boundary. Nearly 20% of chromosome breakpoint regions were reused during mammalian evolution; these reuse sites are also enriched for centromeres. Analysis of gene content in and around evolutionary breakpoint regions revealed increased gene density relative to the genome-wide average. We found that segmental duplications populate the majority of primate-specific breakpoints and often flank inverted chromosome segments, implicating their role in chromosomal rearrangement.
The domestic pig is being increasingly exploited as a system for modeling human disease. It also has substantial economic importance for meat-based protein production. Physical clone maps have ...underpinned large-scale genomic sequencing and enabled focused cloning efforts for many genomes. Comparative genetic maps indicate that there is more structural similarity between pig and human than, for example, mouse and human, and we have used this close relationship between human and pig as a way of facilitating map construction.
Here we report the construction of the most highly continuous bacterial artificial chromosome (BAC) map of any mammalian genome, for the pig (Sus scrofa domestica) genome. The map provides a template for the generation and assembly of high-quality anchored sequence across the genome. The physical map integrates previous landmark maps with restriction fingerprints and BAC end sequences from over 260,000 BACs derived from 4 BAC libraries and takes advantage of alignments to the human genome to improve the continuity and local ordering of the clone contigs. We estimate that over 98% of the euchromatin of the 18 pig autosomes and the X chromosome along with localized coverage on Y is represented in 172 contigs, with chromosome 13 (218 Mb) represented by a single contig. The map is accessible through pre-Ensembl, where links to marker and sequence data can be found.
The map will enable immediate electronic positional cloning of genes, benefiting the pig research community and further facilitating use of the pig as an alternative animal model for human disease. The clone map and BAC end sequence data can also help to support the assembly of maps and genome sequences of other artiodactyls.
Using the INRA-Minnesota porcine radiation hybrid panel, we have constructed a human-pig comparative map composed of 2274 loci, including 206 ESTs and 2068 BAC-end sequences, assigned to 34 linkage ...groups. The average spacing between comparative anchor loci is 1.15 Mb based on human genome sequence coordinates. A total of 51 conserved synteny groups that include 173 conserved segments were identified. This radiation hybrid map has the highest resolution of any porcine map to date and its integration with the porcine linkage map (reported here) will greatly facilitate the positional cloning of genes influencing complex traits of both agricultural and biomedical interest. Additionally, this map will provide a framework for anchoring contigs generated through BAC fingerprinting efforts and assist in the selection of a BAC minimal tiling path and assembly of the first sequence-ready map of the porcine genome.
The appropriate treatment and control of infectious gastroenteritis depend on the ability to rapidly detect the wide range of etiologic agents associated with the disease. Clinical laboratories ...currently utilize an array of different methodologies to test for bacterial, parasitic, and viral causes of gastroenteritis, a strategy that suffers from poor sensitivity, potentially long turnaround times, and complicated ordering practices and workflows. Additionally, there are limited or no testing methods routinely available for most diarrheagenic Escherichia coli strains, astroviruses, and sapoviruses. This study assessed the performance of the FilmArray Gastrointestinal (GI) Panel for the simultaneous detection of 22 different enteric pathogens directly from stool specimens: Campylobacter spp., Clostridium difficile (toxin A/B), Plesiomonas shigelloides, Salmonella spp., Vibrio spp., Vibrio cholerae, Yersinia enterocolitica, enteroaggregative E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, Shiga-like toxin-producing E. coli (stx1 and stx2) (including specific detection of E. coli O157), Shigella spp./enteroinvasive E. coli, Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, adenovirus F 40/41, astrovirus, norovirus GI/GII, rotavirus A, and sapovirus. Prospectively collected stool specimens (n = 1,556) were evaluated using the BioFire FilmArray GI Panel and tested with conventional stool culture and molecular methods for comparison. The FilmArray GI Panel sensitivity was 100% for 12/22 targets and ≥94.5% for an additional 7/22 targets. For the remaining three targets, sensitivity could not be calculated due to the low prevalences in this study. The FilmArray GI Panel specificity was ≥97.1% for all panel targets. The FilmArray GI Panel provides a comprehensive, rapid, and streamlined alternative to conventional methods for the etiologic diagnosis of infectious gastroenteritis in the laboratory setting. The potential advantages include improved performance parameters, a more extensive menu of pathogens, and a turnaround time of as short as 1 h.
The pregnane X receptor (PXR) plays a crucial role in xenobiotic and drug metabolism, being the major transcriptional regulator of cytochrome P-450 monooxygenase 3A4, which metabolizes more than 50% ...of all clinically used drugs. Recent pharmacodynamic studies have shown that the mouse is not an ideal model for predicting human clinical drug study outcomes. Therefore, we characterized the porcine PXR (pPXR) gene to evaluate the utility of the pig as an alternate preclinical animal model. The complete sequence of pPXR mRNA and 11 kb of genomic sequence were obtained. Similar to the human PXR gene, the pPXR gene revealed multiple splice variants in the ligand-binding domain. All pPXR splice variants (SV) were porcine-specific. The pPXR mRNAs varied in 3'-UTR length due to differential termination and specific deletions. Northern blot analyses identified high levels of pPXR mRNA expression in the liver, small intestine, heart, kidney, and colon. RT-PCR amplification detected lower levels of pPXR expression in multiple tissues. Ninety-three pigs representing eight breeds were analyzed for single nucleotide polymorphisms (SNPs). Only one nonsynonymous SNP (S178L) was found in the pPXR ligand-binding domain. This characterization of the pPXR gene contributes to the development of a porcine model for human drug metabolic studies.
There is a long-standing debate as to how Ireland attained its present fauna; we help to inform this debate with a molecular study of one species. A 1110 base pair fragment of the mitochondrial ...cytochrome b gene was sequenced in 74 specimens of the pygmy shrew, Sorex minutus, collected from throughout its western Palaearctic range. Phylogenetic analysis of these sequences revealed several well-supported lineages. Most of the 65 haplotypes belonged to a northern lineage, which ranged from Britain in the west to Lake Baikal in the east. The other lineages were largely limited to Iberia, Italy and the Balkans. One exception, however, was a lineage found in both Ireland and Andorra. This affinity, and the large difference between the mitochondrial sequences of Irish and British individuals, suggest that pygmy shrews did not colonize Ireland via a land connection from Britain, as has been previously supposed, but instead were introduced by boat from southwest continental Europe. All the Irish pygmy shrews analysed were identical or very similar in cytochrome b sequence, suggesting an extreme founding event.
Ataxia-Telangiectasia (A-T) is a genetic disorder causing cerebellar degeneration, immune deficiency, cancer predisposition, chromosomal instability and radiation sensitivity. Among the mutations ...responsible for A-T, 85% represent truncating mutations that result in the production of shorter, highly unstable forms of ATM (AT-mutated) protein leading to a null ATM phenotype. Several ATM-deficient mice have been created however none reflects the extent of neurological degeneration observed in humans. In an attempt to identify an alternative animal model, we have characterized the porcine ortholog of
ATM (
pATM). When compared to the human
ATM (
hATM), the
pATM showed a high level of homology in the coding region, particularly in the regions coding for functional domains, and had extensive alternative splicing of the 5′UTR, characteristic for the human
ATM mRNA. Six different 5′UTRs resulting from alternative splicing of the first three exons were identified. The porcine 5′UTRs varied in size, had multiple ATG codons and different secondary structures, supporting the possibility of complex transcriptional regulation. Three of the six transcripts demonstrated alternative splicing of exon 3, the first putative coding exon, altering the translation start and giving rise to a putative protein lacking the N-terminus substrate binding domain (82–89 aa) involved in activation of human p53 and BRCA1 pathways. Real time-PCR analysis revealed variable expression levels of total
ATM transcripts in individual tissues. Although each splice variant was ubiquitously expressed among the tissues, differences in the relative abundances of specific 5′UTRs were observed. The extensive alternative splicing of the
pATM gene resembles the complex splicing observed in the
hATM and could provide insights for differences observed between mice and humans with regards to the onset of A-T. Thus, the pig may provide a more relevant clinical model of A-T.
The objective of this study was to compare the aetiologic yield of standard-of-care microbiologic testing ordered by physicians with that of a multiplex PCR platform. Stool specimens obtained from ...children and young adults with gastrointestinal illness were evaluated by standard laboratory methods and a developmental version of the FilmArray Gastrointestinal (GI) Diagnostic System (FilmArray GI Panel), a rapid multiplex PCR platform that detects 23 bacterial, viral and protozoal agents. Results were classified according to the microbiologic tests requested by the treating physician. A median of three (range 1–10) microbiologic tests were performed by the clinical laboratory during 378 unique diarrhoeal episodes. A potential aetiologic agent was identified in 46% of stool specimens by standard laboratory methods and in 65% of specimens tested using the FilmArray GI Panel (p < 0.001). For those patients who only had Clostridium difficile testing requested, an alternative pathogen was identified in 29% of cases with the FilmArray GI Panel. Notably, 11 (12%) cases of norovirus were identified among children who only had testing for Clostridium difficile ordered. Among those who had C. difficile testing ordered in combination with other tests, an additional pathogen was identified in 57% of stool specimens with the FilmArray GI Panel. For patients who had no C. difficile testing performed, the FilmArray GI Panel identified a pathogen in 63% of cases, including C. difficile in 8%. Physician-specified laboratory testing may miss important diarrhoeal pathogens. Additionally, standard laboratory testing is likely to underestimate co-infections with multiple infectious diarrhoeagenic agents.
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