Comparison of real-time PCR assays for detection, quantification, and differentiation of campylobacter jejuni and campylobacter coli in broiler neck skin samples

We tested the use of multiplex real-time PCR for detection and quantification of Campylobacter jejuni and Campylobacter coli on broiler carcass neck skin samples collected during 2008 from slaughterhouses in Switzerland. Results from an established TaqMan assay based on two different targets (hipO and ceuE for C. jejuni and C. coli, respectively) were corroborated with data from a newly developed assay based on a single-nucleotide polymorphism in the fusA gene, which allows differentiation between C. jejuni and C. coli. Both multiplex real-time PCRs were applied simultaneously for direct detection, differentiation, and quantification of Campylobacter from 351 neck skin samples and compared with culture methods. There was good correlation in detection and enumeration between real-time PCR results and quantitative culture, with real-time PCR being more sensitive. Overall, 251 (71.5%) of the samples were PCR positive for Campylobacter, with 211 (60.1%) in the hipO-ceuE assays, 244 (69.5%) in the fusA assay, and 204 (58.1%) of them being positive in both PCR assays. Thus, the fusA assay was similarly sensitive to the enrichment culture (72.4% positive); however, it is faster and allows for quantification. In addition, real-time PCR allowed for species differentiation; roughly 60% of positive samples contained C. jejuni, less than 10% C. coli, and more than 30% contained both species. Real-time PCR proved to be a suitable method for direct detection, quantification, and differentiation of Campylobacter from carcasses, and could permit time-efficient surveillance of these zoonotic agents.

Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates

Typing of Clostridium perfringens strains by PCR-based determination of toxin genes proved to be a reliable method for diagnosis of enterotoxaemia in various animal species. We report the establishment and validation of three real-time fluorogenic (TaqMan) multiplex PCRs for the detection of C. perfringens alpha-, beta-, beta2-, epsilon-, entero- and iota-toxin genes. The composition of the PCRs was chosen with regard to robustness of the assays and in order to increase sensitivity compared to the conventional simplex PCRs. The combination of probe dyes selected for the real-time assays (FAM/TAMRA, Cy-5/BHQ-2 and VIC/TAMRA) as well as the designation of the chromosome-borne alpha-toxin as internal positive control allowed the creation of highly specific and sensitive, as well as time and cost effective PCRs. One hundred and three strains of C. perfringens isolated in Switzerland derived from clinical or suspected cases of enterotoxaemia in 10 different animal species were tested. The toxin genotypes were in agreement in both the conventional PCRs and the newly designed multiplex PCRs. Furthermore, the real-time PCR carried out as simplex allows to quantitate the copy numbers of plasmid-borne toxin genes in relation to the chromosomally located alpha-toxin gene.

Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples

In order to improve the diagnosis of enzootic pneumonia (EP) in pigs two real-time polymerase chain reaction (rtPCR) assays for the detection of Mycoplasma hyopneumoniae in bronchial swabs from lung necropsies were established and validated in parallel. As a gold standard, the current "mosaic diagnosis" was taken, including epidemiological tracing, clinical signs, macro- and histopathological lesions of the lungs and immunofluorescence. One rtPCR is targeting a repeated DNA element of the M. hyopneumoniae genome (REP assay), the other a putative ABC transporter gene (ABC assay). Both assays were shown to be specific for M. hyopneumoniae and did not cross react with other bacteria and mollicutes from pig. With material from pigs of defined EP-negative farms the two assays showed to be 100% specific. When testing lungs from pig farms with EP, the REP assay detected 50% and the ABC assay 90% of the farms as positive. Both tests together detected all positive farms. Within a positive herd the two assays tested similarly with on average over 90% of the lung samples analysed from a single farm showing positive scores. A series of samples with suspicion of EP and samples from pigs with diseases other than respiratory taken from current routine diagnostic was assayed. None of the assays showed false positive results. The sensitivities in this sample group were 50% for the REP and 70% for the ABC assays and for both assays together 85%. The two assays run in parallel are therefore a valuable tool for the improvement of the current diagnosis of EP.

Finite element based estimation of contact areas and pressures of the human scaphoid in various functional positions of the hand

The scaphoid is the most frequently fractured carpal bone. When investigating fixation stability, which may influence healing, knowledge of forces and moments acting on the scaphoid is essential. The aim of this study was to evaluate cartilage contact forces acting on the intact scaphoid in various functional wrist positions using finite element modeling. A novel methodology was utilized as an attempt to overcome some limitations of earlier studies, namely, relatively coarse imaging resolution to assess geometry, assumption of idealized cartilage thicknesses and neglected cartilage pre-stresses in the unloaded joint. Carpal bone positions and articular cartilage geometry were obtained independently by means of high resolution CT imaging and incorporated into finite element (FE) models of the human wrist in eight functional positions. Displacement driven FE analyses were used to resolve inter-penetration of cartilage layers, and provided contact areas, forces and pressure distribution for the scaphoid bone. The results were in the range reported by previous studies. Novel findings of this study were: (i) cartilage thickness was found to be heterogeneous for each bone and vary considerably between carpal bones; (ii) this heterogeneity largely influenced the FE results and (iii) the forces acting on the scaphoid in the unloaded wrist were found to be significant. As major limitations, accuracy of the method was found to be relatively low, and the results could not be compared to independent experiments. The obtained results will be used in a following study to evaluate existing and recently developed screws used to fix scaphoid fractures.

Finite Element Based Nonlinear Normalization of Human Lumbar Intervertebral Disc Stiffness to Account for Its Morphology

Disc degeneration, usually associated with low back pain and changes of intervertebral stiffness, represents a major health issue. As the intervertebral disc (IVD) morphology influences its stiffness, the link between mechanical properties and degenerative grade is partially lost without an efficient normalization of the stiffness with respect to the morphology. Moreover, although the behavior of soft tissues is highly nonlinear, only linear normalization protocols have been defined so far for the disc stiffness. Thus, the aim of this work is to propose a nonlinear normalization based on finite elements (FE) simulations and evaluate its impact on the stiffness of human anatomical specimens of lumbar IVD. First, a parameter study involving simulations of biomechanical tests (compression, flexion/extension, bilateral torsion and bending) on 20 FE models of IVDs with various dimensions was carried out to evaluate the effect of the disc's geometry on its compliance and establish stiffness/morphology relations necessary to the nonlinear normalization. The computed stiffness was then normalized by height (H), cross-sectional area (CSA), polar moment of inertia (J) or moments of inertia (Ixx, Iyy) to quantify the effect of both linear and nonlinear normalizations. In the second part of the study, T1-weighted MRI images were acquired to determine H, CSA, J, Ixx and Iyy of 14 human lumbar IVDs. Based on the measured morphology and pre-established relation with stiffness, linear and nonlinear normalization routines were then applied to the compliance of the specimens for each quasi-static biomechanical test. The variability of the stiffness prior to and after normalization was assessed via coefficient of variation (CV). The FE study confirmed that larger and thinner IVDs were stiffer while the normalization strongly attenuated the effect of the disc geometry on its stiffness. Yet, notwithstanding the results of the FE study, the experimental stiffness showed consistently higher CV after normalization. Assuming that geometry and material properties affect the mechanical response, they can also compensate for one another. Therefore, the larger CV after normalization can be interpreted as a strong variability of the material properties, previously hidden by the geometry's own influence. In conclusion, a new normalization protocol for the intervertebral disc stiffness in compression, flexion, extension, bilateral torsion and bending was proposed, with the possible use of MRI and FE to acquire the discs' anatomy and determine the nonlinear relations between stiffness and morphology. Such protocol may be useful to relate the disc's mechanical properties to its degree of degeneration.

Speciating Campylobacter jejuni and Campylobacter coli isolates from poultry and humans using six PCR-based assays

Six previously published polymerase chain reaction (PCR) assays each targeting different genes were used to speciate 116 isolates previously identified as Campylobacter jejuni using routine microbiological techniques. Of the 116 isolates, 84 were of poultry origin and 32 of human origin. The six PCR assays confirmed the species identities of 31 of 32 (97%) human isolates and 56 of 84 (67%) poultry isolates as C. jejuni. Twenty eight of 84 (33%) poultry isolates were identified as Campylobacter coli and the remaining human isolate was tentatively identified as Campylobacter upsaliensis based on the degree of similarity of 16S rRNA gene sequences. Four of six published PCR assays showed 100% concordance in their ability to speciate 113 of the 116 (97.4%) isolates; two assays failed to generate a PCR product with four to 10 isolates. A C. coli-specific PCR identified all 28 hippuricase gene (hipO)-negative poultry isolates as C. coli although three isolates confirmed to be C. jejuni by the remaining five assays were also positive in this assay. A PCR-restriction fragment length polymorphism assay based on the 16S rRNA gene was developed, which contrary to the results of the six PCR-based assays, identified 28 of 29 hipO-negative isolates as C. jejuni. DNA sequence analysis of 16S rRNA genes from four hipO-negative poultry isolates showed they were almost identical to the C. jejuni type strain 16S rRNA sequences ATCC43431 and ATCC33560 indicating that assays reliant on 16S rRNA sequence may not be suitable for the differentiation of these two species.

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the "Beijing" sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive.

PCR-based diagnosis of Naegleria sp. infection in formalin-fixed and paraffin-embedded brain sections

We developed a real-time PCR which allowed the highly sensitive detection of Naegleria fowleri in histological brain tissue sections from experimentally infected mice. This genus-specific small-subunit (18S) rRNA gene-based PCR can complement conventional (immuno-) histology for the diagnosis of primary amoebic meningoencephalitis in paraffin-embedded brain necropsy specimens that had been fixed in formalin buffered with phosphate-buffered saline.

New molecular detection tools adapted to emerging rhinoviruses and enteroviruses

Human rhinoviruses (HRV), and to a lesser extent human enteroviruses (HEV), are important respiratory pathogens. Like other RNA viruses, these picornaviruses have an intrinsic propensity to variability. This results in a large number of different serotypes as well as the incessant discovery of new genotypes. This large and growing diversity not only complicates the design of real-time PCR assays but also renders immunofluorescence unfeasible for broad HRV and HEV detection or quantification in cells. In this study, we used the 5' untranslated region, the most conserved part of the genome, as a target for the development of both a real-time PCR assay (Panenterhino/Ge/08) and a peptide nucleic acid-based hybridization oligoprobe (Panenterhino/Ge/08 PNA probe) designed to detect all HRV and HEV species members according to publicly available sequences. The reverse transcription-PCR assay has been validated, using not only plasmid and viral stocks but also quantified RNA transcripts and around 1,000 clinical specimens. These new generic detection PCR assays overcame the variability of circulating strains and lowered the risk of missing emerging and divergent HRV and HEV. An additional real-time PCR assay (Entero/Ge/08) was also designed specifically to provide sensitive and targeted detection of HEV in cerebrospinal fluid. In addition to the generic probe, we developed specific probes for the detection of HRV-A and HRV-B in cells. This investigation provides a comprehensive toolbox for accurate molecular identification of the different HEV and HRV circulating in humans.

Deletion of exon 8 from the EXT1 gene causes multiple osteochondromas (MO) in a family with three affected members.

Multiple osteochondromas (also called hereditary multiple exostoses) is an autosomal dominant disorder characterized by multiple cartilaginous tumors, which are caused by mutations in the genes for exostosin-1 (EXT1) and exostosin-2 (EXT2). The goal of this study was to elucidate the genetic alterations in a family with three affected members. Isolation of RNA from the patients' blood followed by reverse transcription and PCR amplification of selected fragments showed that the three patients lack a specific region of 90 bp from their EXT1 mRNA. This region corresponds to the sequence of exon 8 from the EXT1 gene. No splice site mutation was found around exon 8. However, long-range PCR amplification of the region from intron 7 to intron 8 indicated that the three patients contain a deletion of 4318 bp, which includes exon 8 and part of the flanking introns. There is evidence that the deletion was caused by non-homologous end joining because the breakpoints are not located within a repetitive element, but contain multiple copies of the deletion hotspot sequence TGRRKM. Exon 8 encodes part of the active site of the EXT1 enzyme, including the DXD signature of all UDP-sugar glycosyltransferases. It is conceivable that the mutant protein exerts a dominant negative effect on the activity of the EXT glycosyltransferase since it might interact with normal copies of the enzyme to form an inactive hetero-oligomeric complex. We suggest that sequencing of RNA might be superior to exome sequencing to detect short deletions of a single exon.

Optimized CUDA-Based PDE Solver for Reaction Diffusion Systems on Arbitrary Surfaces

Partial differential equation (PDE) solvers are commonly employed to study and characterize the parameter space for reaction-diffusion (RD) systems while investigating biological pattern formation. Increasingly, biologists wish to perform such studies with arbitrary surfaces representing ‘real’ 3D geometries for better insights. In this paper, we present a highly optimized CUDA-based solver for RD equations on triangulated meshes in 3D. We demonstrate our solver using a chemotactic model that can be used to study snakeskin pigmentation, for example. We employ a finite element based approach to perform explicit Euler time integrations. We compare our approach to a naive GPU implementation and provide an in-depth performance analysis, demonstrating the significant speedup afforded by our optimizations. The optimization strategies that we exploit could be generalized to other mesh based processing applications with PDE simulations.

The FGFRL1 receptor is shed from cell membranes, binds fibroblast growth factors (FGFs), and antagonizes FGF signaling in Xenopus embryos

FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs.

Cost and mortality prediction using polymerase chain reaction pathogen detection in sepsis: evidence from three observational trials

Delays in adequate antimicrobial treatment contribute to high cost and mortality in sepsis. Polymerase chain reaction (PCR) assays are used alongside conventional cultures to accelerate the identification of microorganisms. We analyze the impact on medical outcomes and healthcare costs if improved adequacy of antimicrobial therapy is achieved by providing immediate coverage after positive PCR reports.

Rapid fusion and syncytium formation of heterologous cells upon expression of the FGFRL1 receptor

The fusion of mammalian cells into syncytia is a developmental process that is tightly restricted to a limited subset of cells. Besides gamete and placental trophoblast fusion, only macrophages and myogenic stem cells fuse into multinucleated syncytia. In contrast to viral cell fusion, which is mediated by fusogenic glycoproteins that actively merge membranes, mammalian cell fusion is poorly understood at the molecular level. A variety of mammalian transmembrane proteins, among them many of the immunoglobulin superfamily, have been implicated in cell-cell fusion, but none has been shown to actively fuse cells in vitro. Here we report that the FGFRL1 receptor, which is up-regulated during the differentiation of myoblasts into myotubes, fuses cultured cells into large, multinucleated syncytia. We used luciferase and GFP-based reporter assays to confirm cytoplasmic mixing and to identify the fusion inducing domain of FGFRL1. These assays revealed that Ig-like domain III and the transmembrane domain are both necessary and sufficient to rapidly fuse CHO cells into multinucleated syncytia comprising several hundred nuclei. Moreover, FGFRL1 also fused HEK293 and HeLa cells with untransfected CHO cells. Our data show that FGFRL1 is the first mammalian protein that is capable of inducing syncytium formation of heterologous cells in vitro.