Clinical evaluation of the BioFire® Respiratory Panel 2.1 and detection of 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.

Multicenter Evaluation of the BioFire FilmArray Pneumonia/Pneumonia Plus Panel for Detection and Quantification of Agents of Lower Respiratory Tract Infection.

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.

Detection of 23 Gastrointestinal Pathogens Among Children Who Present With Diarrhea.

BACKGROUND: Diarrheal diseases are a major cause of ambulatory care visits and hospitalizations among children. Because of overlapping signs and symptoms and expensive and inefficient testing methods, the etiology of pediatric diarrhea is rarely established. METHODS: We identified children <18 years of age who were evaluated for diarrhea at Primary Children's Hospital in Salt Lake City, Utah, between October 2010 and September 2012. Stool specimens submitted for testing were evaluated by using the FilmArray gastrointestinal diagnostic system, which is a rapid multiplex polymerase chain reaction platform that can simultaneously detect 23 bacterial, viral, and protozoal agents. RESULTS: A pathogen was detected in 561 (52%) of 1089 diarrheal episodes. The most commonly detected pathogens included toxigenic Clostridium difficile (16%), diarrheagenic Escherichia coli (15%), norovirus GI/GII (11%), and adenovirus F 40/41 (7%). Shiga toxin-producing E coli were detected in 43 (4%) specimens. Multiple pathogens were identified in 160 (15%) specimens. Viral pathogens (norovirus, adenovirus, rotavirus, and sapovirus) were more common among children <5 years old than among those 5 to 17 years old (38% vs 16%, respectively; P < .001). Bacterial pathogens were identified most commonly in children 2 to 4 years of age. Children with 1 or more underlying chronic medical conditions were less likely to have a pathogen identified than those without a chronic medical condition (45% vs 60%, respectively; P < .01). Viral pathogens were detected more commonly in the winter, whereas bacterial pathogens were detected more commonly in the summer. CONCLUSIONS: Toxigenic C difficile, diarrheagenic E coli, and norovirus were the leading organisms detected among these children with diarrhea. Viral pathogens are identified frequently among young children with acute gastroenteritis.

Multicenter evaluation of the BioFire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis.

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.

Non-invasive sample collection for respiratory virus testing by multiplex PCR.

BACKGROUND: Identifying respiratory pathogens within populations is difficult because invasive sample collection, such as with nasopharyngeal aspirate (NPA), is generally required. PCR technology could allow for non-invasive sampling methods. OBJECTIVE: Evaluate the utility of non-invasive sample collection using anterior nare swabs and facial tissues for respiratory virus detection by multiplex PCR. STUDY DESIGN: Children aged 1 month-17 years evaluated in a pediatric emergency department for respiratory symptoms had a swab, facial tissue, and NPA sample collected. All samples were tested for respiratory viruses by multiplex PCR. Viral detection rates were calculated for each collection method. Sensitivity and specificity of swabs and facial tissues were calculated using NPA as the gold standard. RESULTS: 285 samples from 95 children were evaluated (92 swab-NPA pairs, 91 facial tissue-NPA pairs). 91% of NPA, 82% of swab, and 77% of tissue samples were positive for ≥1 virus. Respiratory syncytial virus (RSV) and human rhinovirus (HRV) were most common. Overall, swabs were positive for 74% of virus infections, and facial tissues were positive for 58%. Sensitivity ranged from 17 to 94% for swabs and 33 to 84% for tissues. Sensitivity was highest for RSV (94% swabs and 84% tissues). Specificity was ≥95% for all viruses except HRV for both collection methods. CONCLUSIONS: Sensitivity of anterior nare swabs and facial tissues in the detection of respiratory viruses by multiplex PCR varied by virus type. Given its simplicity and specificity, non-invasive sampling for PCR testing may be useful for conducting epidemiologic or surveillance studies in settings where invasive testing is impractical or not feasible.

Piggy-BACing the human genome I: constructing a porcine BAC physical map through comparative genomics.

Availability of the human genome sequence and high similarity between humans and pigs at the molecular level provides an opportunity to use a comparative mapping approach to piggy-BAC the human genome. In order to advance the pig genome sequencing initiative, sequence similarity between large-scale porcine BAC-end sequences (BESs) and human genome sequence was used to construct a comparatively-anchored porcine physical map that is a first step towards sequencing the pig genome. A total of 50,300 porcine BAC clones were end-sequenced, yielding 76,906 BESs after trimming with an average read length of 538 bp. To anchor the porcine BACs on the human genome, these BESs were subjected to BLAST analysis using the human draft sequence, revealing 31.5% significant hits (E < e(-5)). Both genic and non-genic regions of homology contributed to the alignments between the human and porcine genomes. Porcine BESs with unique homology matches within the human genome provided a source of markers spaced approximately 70 to 300 kb along each human chromosome. In order to evaluate the utility of piggy-BACing human genome sequences, and confirm predictions of orthology, 193 evenly spaced BESs with similarity to HSA3 and HSA21 were selected and then utilized for developing a high-resolution (1.22 Mb) comparative radiation hybrid map of SSC13 that represents a fusion of HSA3 and HSA21. Resulting RH mapping of SSC13 covers 99% and 97% of HSA3 and HSA21, respectively. Seven evolutionary conserved blocks were identified including six on HSA3 and a single syntenic block corresponding to HSA21. The strategy of piggy-BACing the human genome described in this study demonstrates that through a directed, targeted comparative genomics approach construction of a high-resolution anchored physical map of the pig genome can be achieved. This map supports the selection of BACs to construct a minimal tiling path for genome sequencing and targeted gap filling. Moreover, this approach is highly relevant to other genome sequencing projects.

Characterization of the porcine ATM gene: towards the generation of a novel non-murine animal model for Ataxia-Telangiectasia.

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.

A high utility integrated map of the pig genome.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Comparative genomics of xenobiotic metabolism: a porcine-human PXR gene comparison.

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.

Piggy-BACing the human genome II. A high-resolution, physically anchored, comparative map of the porcine autosomes.

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.

Dynamics of mammalian chromosome evolution inferred from multispecies comparative maps.

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.

Genomic organization of mouse ZAKI-4 gene that encodes ZAKI-4 alpha and beta isoforms, endogenous calcineurin inhibitors, and changes in the expression of these isoforms by thyroid hormone in adult mouse brain and heart.

OBJECTIVE: ZAKI-4 was identified as a thyroid hormone-responsive gene in cultured human fibroblasts. A single ZAKI-4 gene encodes two isoforms, ZAKI-4 alpha and beta, both inhibiting calcineurin activity. ZAKI-4 alpha and beta differ at their N termini, and show distinct distribution profiles in human tissues. The aim of this study was to elucidate the organization of the mouse ZAKI-4 gene and to determine the effect of thyroid hormone on the expression of ZAKI-4 isoforms in vivo. DESIGN: We cloned mouse homologues of human ZAKI-4 alpha and beta cDNA. Fluorescence in situ hybridization and bioinformatics analysis were employed to determine the gene organization. The effect of thyroid hormone on the expression of ZAKI-4 isoforms in mouse brain and heart was also studied. METHODS: Total RNA extracted from mouse cerebellum was used to clone ZAKI-4 alpha and beta cDNAs by RT-PCR followed by rapid amplification of cDNA ends. Mice were rendered hypothyroid by feeding a low iodine diet supplemented with propylthiouracil for 2 weeks. In one group (hyperthyroid) L-T(3) was injected i.p. for the last 4 days whereas another group (hypothyroid) received vehicle only. Non-treated mice were controls. RESULTS AND CONCLUSION: Mouse ZAKI-4 alpha and beta cDNAs were highly homologous to the human isoforms. The gene was mapped on chromosome 17qC, syntenic to human chromosome 6 where the human ZAKI-4 gene is located. As observed in human, ZAKI-4 alpha mRNA was expressed only in brain whereas beta mRNA was distributed in other tissues as well, such as heart and skeletal muscle. ZAKI-4 alpha mRNA was lower in the cerebral cortex of hypothyroid mice. Injection of L-T(3) caused an increase in ZAKI-4 beta mRNA in heart; however, expression of neither ZAKI-4 alpha nor beta mRNA was influenced by thyroid status in other tissues. These results indicate that expression of ZAKI-4 alpha and beta isoforms is regulated by thyroid hormone in vivo, and the regulation is isoform- and tissue-specific.

Creating porcine biomedical models through recombineering.

Recent advances in genomics provide genetic information from humans and other mammals (mouse, rat, dog and primates) traditionally used as models as well as new candidates (pigs and cattle). In addition, linked enabling technologies, such as transgenesis and animal cloning, provide innovative ways to design and perform experiments to dissect complex biological systems. Exploitation of genomic information overcomes the traditional need to choose naturally occurring models. Thus, investigators can utilize emerging genomic knowledge and tools to create relevant animal models. This approach is referred to as reverse genetics. In contrast to 'forward genetics', in which gene(s) responsible for a particular phenotype are identified by positional cloning (phenotype to genotype), the 'reverse genetics' approach determines the function of a gene and predicts the phenotype of a cell, tissue, or organism (genotype to phenotype). The convergence of classical and reverse genetics, along with genomics, provides a working definition of a 'genetic model' organism (3). The recent construction of phenotypic maps defining quantitative trait loci (QTL) in various domesticated species provides insights into how allelic variations contribute to phenotypic diversity. Targeted chromosomal regions are characterized by the construction of bacterial artificial chromosome (BAC) contigs to isolate and characterize genes contributing towards phenotypic variation. Recombineering provides a powerful methodology to harvest genetic information responsible for phenotype. Linking recombineering with gene-targeted homologous recombination, coupled with nuclear transfer (NT) technology can provide 'clones' of genetically modified animals.

How did pygmy shrews colonize Ireland? Clues from a phylogenetic analysis of mitochondrial cytochrome b sequences.

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.

Harvesting the genomic promise: recombineering sequences for phenotypes.

The past decade has witnessed the construction of linkage and physical maps defining quantitative trait loci (QTL) in various domesticated species. Targeted chromosomal regions are being further characterized through the construction of bacterial artificial chromosome (BAC) contigs in order to isolate and characterize genes contributing towards phenotypic variation. Whole-genome BAC contigs are also being constructed that will serve as the tiling path for genomic sequencing. Harvesting this genetic information for biological gain requires either genetic selection or the production of genetically modified animals. This later approach when coupled with nuclear transfer technology (NT) provides "clones" of genetically modified animals. However, to date, the production of genetically modified animals has been limited to either microinjection of small gene constructs into embryos with random insertion or complex gene constructs designed to knock-out targeted gene expression. Neither of these approaches provides for introducing directed genetic manipulation allowing for allelic substitution [knock-in], subsequent analyses of gene expression, and cloning. An alternative approach utilizing genomic sequence information and recombineering to direct gene targeting of specific porcine BACs is presented here.

Type 1 iodothyronine deiodinase in the house musk shrew (Suncus murinus, Insectivora: Soricidae): cloning and characterization of complementary DNA, unique tissue distribution and regulation by T(3).

The house musk shrew Suncus murinus (Insectivora: Soricidae) has been reported as having low thyroxine to 3,3'5-triiodothyronine (T(3)) converting activity in liver and kidney homogenates and was assumed to be type 1 iodothyronine deiodinase (D1)-deficient. To study whether this is due to structural abnormality of shrew D1, we cloned the cDNA and characterized the enzyme. The deduced amino acid sequence of shrew D1 was found to be highly homologous to other known D1s and the enzyme itself to have similar catalytic activity. However, unlike in other species, the D1 activity was detected only in liver. Moreover, the D1 activity in liver of the shrew was less than half of that in rat liver and its expression was not up-regulated by T(3). In contrast, a very high activity of D2 was demonstrated in brain and brown adipose tissue. The present study also revealed that the serum level of T(3) in the shrew was in the same range as these in other mammals. These results suggest that D2 contributes to the production and maintenance of T(3) levels in the house musk shrew.