Christa Noehammer

Identification of human pathogens isolated from blood using microarray hybridisation and signal pattern recognition

BackgroundPathogen identification in clinical routine is based on the cultivation of microbes with subsequent morphological and physiological characterisation lasting at least 24 hours. However, early and accurate identification is a crucial requisite for fast and optimally targeted antimicrobial treatment. Molecular biology based techniques allow fast identification, however discrimination of very closely related species remains still difficult.ResultsA molecular approach is presented for the rapid identification of pathogens combining PCR amplification with microarray detection. The DNA chip comprises oligonucleotide capture probes for 25 different pathogens including Gram positive cocci, the most frequently encountered genera of Enterobacteriaceae, non-fermenter and clinical relevant Candida species. The observed detection limits varied from 10 cells (e.g. E. coli) to 105 cells (S. aureus) per mL artificially spiked blood. Thus the current low sensitivity for some species still represents a barrier for clinical application. Successful discrimination of closely related species was achieved by a signal pattern recognition approach based on the k-nearest-neighbour method. A prototype software providing this statistical evaluation was developed, allowing correct identification in 100 % of the cases at the genus and in 96.7 % at the species level (n = 241).ConclusionThe newly developed molecular assay can be carried out within 6 hours in a research laboratory from pathogen isolation to species identification. From our results we conclude that DNA microarrays can be a useful tool for rapid identification of closely related pathogens particularly when the protocols are adapted to the special clinical scenarios.

Establishment of a semi-automated pathogen DNA isolation from whole blood and comparison with commercially available kits

Molecular methods for bacterial pathogen identification are gaining increased importance in routine clinical diagnostic laboratories. Achieving reliable results using DNA based technologies is strongly dependent on pre-analytical processes including isolation of target cells and their DNA of high quality and purity. In this study a fast and semi-automated method was established for bacterial DNA isolation from whole blood samples and compared to different commercially available kits: Looxster, MolYsis kit, SeptiFast DNA isolation method and standard EasyMAG protocol. The newly established, semi-automated method utilises the EasyMAG device combined with pre-processing steps comprising human cell lysis, centrifugation and bacterial pellet resuspension. Quality of DNA was assessed by a universal PCR targeting the 16S rRNA gene and subsequent microarray hybridisation. The DNA extractions were amplified using two different PCR-mastermixes, to allow comparison of a commercial mastermix with a guaranteed bacterial DNA free PCR mastermix. The modified semi-automated EasyMAG protocol and the Looxster kit gave the most sensitive results. After hybridisation a detection limit of 10(1) to 10(2) bacterial cells per mL whole blood was achieved depending on the isolation method and microbial species lysed. Human DNA present in the isolated DNA suspension did not interfere with PCR and did not lead to non-specific hybridisation events.

Soluble ST2 Is Regulated by p75 Neurotrophin Receptor and Predicts Mortality in Diabetic Patients With Critical Limb Ischemia

Objective—The p75 neurotrophin receptor (p75NTR) contributes to diabetes mellitus−induced defective postischemic neovascularization. The interleukin-33 receptor ST2 is expressed as transmembrane (ST2L) and soluble (sST2) isoforms. Here, we studied the following: (1) the impact of p75NTR in the healing of ischemic and diabetic calf wounds; (2) the link between p75NTR and ST2; and (3) circulating sST2 levels in critical limb ischemia (CLI) patients. Methods and Results—Diabetes mellitus was induced in p75NTR knockout (p75KO) mice and wild-type (WT) littermates by streptozotocin. Diabetic and nondiabetic p75KO and WT mice received left limb ischemia induction and a full-thickness wound on the ipsilateral calf. Diabetes mellitus impaired wound closure and angiogenesis and increased ST2 expression in WT, but not in p75KO wounds. In cultured endothelial cells, p75NTR promoted ST2 (both isoforms) expression through p38MAPK/activating transcription factor 2 pathway activation. Next, sST2 was measured in the serum of patients with CLI undergoing either revascularization or limb amputation and in the 2 nondiabetic groups (with CLI or nonischemic individuals). Serum sST2 increased in diabetic patients with CLI and was directly associated with higher mortality at 1 year from revascularization. Conclusion—p75NTR inhibits the healing of ischemic lower limb wounds in diabetes mellitus and promotes ST2 expression. Circulating sST2 predicts mortality in diabetic CLI patients.

Characterization of the drugged human genome

Human drug targets are a part of our genome of special relevance to human disease. However, the number and nature of drug target genes has not yet been conclusively assessed. We analyzed involvement in biochemical functions, biological processes and pathways, with chromosome, cellular and tissue distribution of the 392 human drug targets collected in DrugBank. Comparison with the whole human genome reveals their scarcely diverse characteristics, largely dominated by rhodopsin-like 7 transmembrane receptors involved in the neuroactive ligand-receptor interaction pathway and located in the plasma membrane. Drug target genes are frequently expressed in multiple tissues, suggesting drug application in distinct disease classes. Intersections with other clinically relevant gene sets, such as the Mendelian disorder-linked genes and various molecular cancer signatures, are discussed.

PCR-Based Analysis of Differentially Methylated Regions of GNAS Enables Convenient Diagnostic Testing of Pseudohypoparathyroidism Type Ib

BACKGROUND Pseudohypoparathyroidism type Ib (PHPIb) is characterized by parathyroid hormone (PTH) resistance, which can lead to hypocalcemia, hyperphosphatemia, and increased serum PTH. The disorder is caused by mutations in regulatory regions of the GNAS gene (GNAS complex locus) that lead to interferences in the methylation status of alternative GNAS promoters, such as exon A/B, NESP55, and XL alpha-s. PHPIb comprises disorders that show distinctive changes in methylation status but share the same clinical phenotype: (a) loss of methylation only at exon A/B of the GNAS gene and involving no other obvious epigenetic abnormalities [e.g., those caused by heterozygous microdeletions in the STX16 (syntaxin 16) region and found in many patients with autosomal dominant (AD) PHPIb]; (b) methylation abnormalities at several differentially methylated regions (DMRs), which are observed in most patients with sporadic PHPIb and some families with AD PHPIb. METHODS To permit early and reliable diagnosis of suspected PHPIb, we designed methylation-sensitive restriction enzyme-based and bisulfite deamination-based PCR tests for exon A/B and NESP55 DMRs. RESULTS Both PCR strategies permit proper methylation testing of GNAS and NESP55 DMRs and elucidate different disease subtypes. We have identified a novel microsatellite repeat polymorphism within GNAS exon A/B, and pedigree analyses have shown its presence to be conclusive evidence for familial disease. CONCLUSIONS We provide a simple diagnostic test for PHPIb, an imprinting disorder caused by different molecular changes within the GNAS complex locus. PHPIb, a complex and diagnostically challenging clinical phenotype, can be treated successfully by taking steps before the manifestation of symptoms to avoid clinical complications in affected patients or asymptomatic members of affected families who show positive results in genetic tests.

Microarray-Based In Vitro Test System for the Discrimination of Contact Allergens and Irritants: Identification of Potential Marker Genes

BACKGROUND Animal tests have been used to characterize the potential of chemicals to produce allergic contact dermatitis, but this approach is increasingly a matter of public and political concern. Our aim was to develop and validate an alternative in vitro test that can identify contact allergens. METHODS We developed a targeted microarray containing oligonucleotide probes for 66 immune-relevant genes and analyzed gene expression in monocyte-derived dendritic cells (Mo-DCs) treated with 1 irritant (SDS) and 2 prominent contact allergens, nickel and Bandrowskis base (BB), which is the oxidation product of the most important hair dye allergen, p-phenylenediamine. RESULTS Comparing RNA amounts in chemical-treated and solvent-treated cells, we identified significant changes in the expression of 21 genes and 10 genes after exposure of immature DCs (iDCs) to nickel and BB, respectively, but not after exposure to SDS. Eight genes were differentially expressed after application of both nickel and BB. Real-time PCR was used to confirm the results for selected genes. CONCLUSION We propose a microarray-based in vitro test that might allow the identification of contact allergens. Independently from donor variability, several immune-relevant genes were up- or downregulated after the application of the tested sensitizers to iDCs, therefore presenting potential marker genes. While reducing the number of laboratory animals used, this test would also enable reliable analysis of chemicals using a human system.

Hepatitis B plasmonic biosensor for the analysis of clinical serum samples

A plasmonic biosensor for rapid detection of protein biomarkers in complex media is reported. Clinical serum samples were analyzed by using a novel biointerface architecture based on poly[(N-(2-hydroxypropyl) methacrylamide)-co-(carboxybetaine methacrylamide)] brushes functionalized with bioreceptors. This biointerface provided an excellent resistance to fouling even after the functionalization and allowed for the first time the direct detection of antibodies against hepatitis B surface antigen (anti-HBs) in clinical serum samples using surface plasmon resonance (SPR). The fabricated SPR biosensor allowed discrimination of anti-HBs positive and negative clinical samples in 10min. Results are validated by enzyme-linked immunoassays of the sera in a certified laboratory. The sensor could be regenerated by simple treatment with glycine buffer.

Strategies for validation and testing of DNA methylation biomarkers

DNA methylation is a stable covalent epigenetic modification of primarily CpG dinucleotides that has recently gained considerable attention for its use as a biomarker in different clinical settings, including disease diagnosis, prognosis and therapeutic response prediction. Although the advent of genome-wide DNA methylation profiling in primary disease tissue has provided a manifold resource for biomarker development, only a tiny fraction of DNA methylation-based assays have reached clinical testing. Here, we provide a critical overview of different analytical methods that are suitable for biomarker validation, including general study design considerations, which might help to streamline epigenetic marker development. Furthermore, we highlight some of the recent marker validation studies and established markers that are currently commercially available for assisting in clinical management of different cancers.

RGG: A general GUI Framework for R scripts

BackgroundR is the leading open source statistics software with a vast number of biostatistical and bioinformatical analysis packages. To exploit the advantages of R, extensive scripting/programming skills are required.ResultsWe have developed a software tool called R GUI Generator (RGG) which enables the easy generation of Graphical User Interfaces (GUIs) for the programming language R by adding a few Extensible Markup Language (XML) – tags. RGG consists of an XML-based GUI definition language and a Java-based GUI engine. GUIs are generated in runtime from defined GUI tags that are embedded into the R script. User-GUI input is returned to the R code and replaces the XML-tags. RGG files can be developed using any text editor. The current version of RGG is available as a stand-alone software (RGGRunner) and as a plug-in for JGR.ConclusionRGG is a general GUI framework for R that has the potential to introduce R statistics (R packages, built-in functions and scripts) to users with limited programming skills and helps to bridge the gap between R developers and GUI-dependent users. RGG aims to abstract the GUI development from individual GUI toolkits by using an XML-based GUI definition language. Thus RGG can be easily integrated in any software. The RGG project further includes the development of a web-based repository for RGG-GUIs. RGG is an open source project licensed under the Lesser General Public License (LGPL) and can be downloaded freely at http://rgg.r-forge.r-project.org

Multiplex detection of antibiotic resistance genes using padlock probes

The elucidation of resistance mechanisms is of central importance to providing and maintaining efficient medical treatment. However, molecular detection methods covering the complete set of resistance genes with a single test are still missing. Here, we present a novel 100-plex assay based on padlock probes in combination with a microarray that allows the simultaneous large-scale identification of highly diverse β-lactamases. The specificity of the assay was performed using 70 clinical bacterial isolates, recovering 98% of the β-lactamase nucleotide sequences present. Additionally, the sensitivity was evaluated with PCR products and genomic bacterial DNA, revealing a detection limit of 10(4) DNA copies per reaction when using PCR products as the template. Pre-amplification of genomic DNA in a 25-multiplex PCR further facilitated the detection of β-lactamase genes in dilutions of 10(7) cells/mL. In summary, we present an efficient, highly specific, and highly sensitive multiplex detection method for any gene.