Development and application of molecular tools for the detection of the human pathogenic protozoan Giardia, Cryptosporidium and Toxoplasma

Es besteht ein enormer Bedarf an der Entwicklung effektiver molekularer Methoden, die die Nachteile bisheriger Nachweisverfahren für parasitäre Protozoen wie Giardia, Cryptosporidium und Toxoplasma überwinden. Die vorliegende Arbeit beschreibt die Entwicklung und Anwendung der Loop mediated-isothermal-amplification (LAMP) Technik zum Nachweis von Cryptosporidium und Toxoplasma. Darüber hinaus wurde eine phylogenetische Analyse von aus Haustieren isolierten Giardia und Cryptosporidium durchgeführt. SNP basierende SAM-LAMP-Methode für Cryptosporidium Das auf der Amplifizierung des SAM-Gens basierende SNP- (single nucleotid polymorphism-) LAMP-Verfahren wurde anhand der entsprechenden Nukleotid-sequenzen für den spezifischen Nachweis der wichtigsten humanpathogenen Cryprosporidium-Arten (C. parvum, C. hominis und C. meleagridis) entwickelt, anschließend in Wasser- sowie klinischen und Tierproben angewendet. Für die Speziescharakterisierung der Proben wurde die Methode mit zwei nPCR-Methoden verglichen, die auf dem Nachweis des SSU rRNA-Gens mit der Amplifizierung von 435 bp und einer 850 bp langen polymorphen Regionen des Gens beruhen. Das auf SNP basierende SAM-LAMP-Verfahren wurde mit den Ergebnissen des Immunofluoreszenz-Tests (IFT) für Cryptosporidium und mit dem zuvor entwickelten GP60-LAMP-Verfahren zum ausschließlichen Nachweis von C. parvum verglichen. Die ermittelte Nachweisgrenze von 800 fg/µl entspricht 0,1 Cryptosporidium- Oozysten. Das entwickelte Verfahren wurde zunächst an 53 Wasserproben, die mit einer bekannten Anzahl an Cryptosporidium-Oozysten kontaminiert wurden, getestet. Die Sensitivität bei dem LAMP-Verfahren betrug 100% für die aufgestockten Proben, während die Sensitivität beider PCRs bei nur 3% und 7% lag. Bei den gleichen ursprünglichen (natürlichen) Proben wurden mittels auf SNP basierendem SAM-LAMP-Verfahren 50% und mittels GP60-Verfahren 24% Cryptosporidium DNA-Positive ermittelt; mit den nPCR Verfahren wurden hingegen nur 5% bzw. 7% Positivergebnisse generiert. Von 15 Wasserproben aus Thailand waren 12 (80%) positiv; ebenso positiv waren 4 von 8 (50%) Muschelproben, 11 von 44 (25%) Stuhlproben von HIV/AIDS Patienten und 12 von 22 (55%) tierischen Fäkalproben. Mit dem GP60-LAMP-Test und den beiden nPCR-Verfahren konnte vergleichsweise eine geringere Anzahl an positiven Proben ermittelt werden als durch SNP-basierende LAMP-Verfahren. Unter den durch Sequenzanalyse identifizierten Arten aus den untersuchten Proben war C. parvum am häufigsten, gefolgt von C. hominis in einer Muschelprobe, C. andersoni in einer tierischen Fäkalprobe und C. fragile in einer Wasserprobe. B1 und TgOWP LAMP-Methode für den Nachweis von Toxoplasma in Wasserproben Das auf der Amplifizierung der B1 und TgOWP-Gene basierende LAMP-Verfahren wurde für den schnellen Nachweis von Toxoplasma gondii in Wasserproben entwickelt und evaluiert. Die Nachweisgrenze der LAMP lag für beide Gene bei 0,1 Tachyzoiten-DNA. Das LAMP-Verfahren wurde vorher evaluiert und an 26 mit Toxoplasma-Oozysten aufgestockten Wasserproben mit der nested PCR verglichen. Die LAMP-Sensitivität betrug 100%, hingegen erreichte die nPCR der gleichen Proben nur 53.8% bei den gleichen aufgestockten ursprünglichen Wasserproben. Der Vergleich von IFT, LAMP und nPCR anhand von 52 ursprünglichen Wasserproben zeigte, dass 25 (48%) der mittels LAMP untersuchten Proben und keine mit IFT–Verfahren untersuchten Proben positiv waren, während nPCR- untersuchte in 7 (13,5%) der Proben generiert wurden. Haustierinfektionen und phylogenetische Analyse von Giardia und Cryptosporidium Die molekulare Diagnose von Giardia-Zysten und Cryptosporidium-Oozysten von Haustieren mit Ursprung aus Deutschland mittels nPCR und Sequenzanalyse war ein Teilgegenstand dieser Arbeit. Die Tierkotproben wurden im Anschluss an die Amplifizierung des Glutamat-Dehydrogenase-Locus (GDH Locus) auf das Vorhandensein von Giardia und die SSU-rRNA auf das Vorhandensein von Cryptosporidium untersucht. Von den 81 untersuchten Hunden und 19 Katzen wurde Giardia Assemblage A in Isolaten von fünf Hunden und zwei Katzen identifiziert. Bei Cryptosporidium-Isolaten aus einem Hund und einer Katze handelte es sich jeweils um C. parvum. In einem Hundeisolat lag eine Mischinfektion von C. parvum und Giardia Assemblage A vor. Diese Ergebnisse liefern einen deutlichen Hinweis darauf, dass die anthropozoonotischen Krankheitserreger Giardia spp. und Cryptosporidium spp. in Haustieren ein mögliches Infektionsreservoir für die Populationen von Mensch und Tier darstellen. Im Vergleich zu anderen molekularbiologischen Methoden ist das LAMP-Verfahren wegen seiner hohen Sensitivität und Spezifität eines der besten molekulardiagnostischen Verfahren zum Nachweis der humanpathogenen Protozoen Cryptosporium und Toxoplasma in Umweltproben, klinischem Material und Nahrungsmitteln. Die Anwendung der LAMP-Technik, kombiniert mit der PCR-Sequenzierung, kann zu einem schnellen Speziesnachweis, zur Differenzierung von für die Bevölkerung gesundheitsrelevanten Spezies sowie zur Identifizierung von Kontaminationsquellen dienen. Die LAMP-Anwendung hat das Potenzial, ein Werkzeug für den schnellen Nachweis dieser wichtigen anthropozoonotischen Spezies, für die menschliche und tierische Wirte anfällig sind, zu sein. Diese Methode kann dazu beitragen, der Kontrolle und Prävention von Krankheiten zu dienen und das Gesundheitsrisiko für die Bevölkerung durch nahrungsmittel- und wasserbürtige (Oo)zysten einzuschätzen

Loop-Mediated Isothermal Amplification: An Advanced Method for the Detection of Giardia

This chapter provides a reliable and quick method for detection of Giardia duodenalis (which causes a dangerous diarrheal disease), prevention of further spreading, identification of the source of contamination, and eventually minimize health risk and economic damage normally caused by an outbreak. The loop-mediated isothermal amplification (LAMP) method is based on the enrichment of parasite-specific nucleotide sequences, similar to PCR, but it is significantly faster and less susceptible to interference. Here, we give an overview of how we developed this method, and using the example of G. duodenalis as a water-associated pathogen, we present an optimized examination scheme for its detection in water. For this purpose, we have analyzed data from extensive electronic libraries PubMed®/MEDLINE®, filtered out relevant articles with a keyword search, and summarized them. The number of publications on LAMP method has generally increased steadily since its first report in 2000. LAMP, used for detection of Giardia, especially surpasses all other methods due to the high specificity, sensitivity, robustness, and cost effectiveness. The ever-increasing number of publications on application of LAMP is similar to the development of PCR in the 1990s of the last century. Certainly, the method will be further developed in future, but it already offers many advantages over other methods for effective detection of G. duodenalis infections and will therefore certainly gain in popularity

Transcriptomic and phenotypic analysis of murine embryonic stem cell derived BMP2+ lineage cells: an insight into mesodermal patterning

Transcriptome analysis of BMP2+ cells in comparison to the undifferentiated BMP2 ES cells and the control population from 7-day old embryoid bodies led to the identification of 479 specifically upregulated and 193 downregulated transcripts

diXa: a data infrastructure for chemical safety assessment

Motivation: The field of toxicogenomics (the application of ‘-omics' technologies to risk assessment of compound toxicities) has expanded in the last decade, partly driven by new legislation, aimed at reducing animal testing in chemical risk assessment but mainly as a result of a paradigm change in toxicology towards the use and integration of genome wide data. Many research groups worldwide have generated large amounts of such toxicogenomics data. However, there is no centralized repository for archiving and making these data and associated tools for their analysis easily available. Results: The Data Infrastructure for Chemical Safety Assessment (diXa) is a robust and sustainable infrastructure storing toxicogenomics data. A central data warehouse is connected to a portal with links to chemical information and molecular and phenotype data. diXa is publicly available through a user-friendly web interface. New data can be readily deposited into diXa using guidelines and templates available online. Analysis descriptions and tools for interrogating the data are available via the diXa portal. Availability and implementation: http://www.dixa-fp7.eu Contact: [email protected]; [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the 'human embryonic stem cell (hESC)- derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (\20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large 'common response' to VPA and MeHg could be distinguished from 'compound-specific' responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.EU/FP7/ESNATSDFGDoerenkamp-Zbinden Foundatio

Molecular identification of

The aim of the present study was to diagnose the presence of Giardia cysts and Cryptosporidium oocysts in household animals using nested polymerase chain reaction (PCR) and sequence analysis. One hundred faecal samples obtained from 81 dogs and 19 cats were investigated. The Cryptosporidium genotypes were determined by sequencing a fragment of the small subunit (SSU) rRNA gene, while the Giardia Assemblages were determined through analysis of the glutamate dehydrogenase (GDH) locus. Isolates from five dogs and two cats were positive by PCR for the presence of Giardia, and their sequences matched the zoonotic Assemblage A of Giardia. Cryptosporidium spp. isolated from one dog and one cat were both found to be C. parvum. One dog isolate harboured a mixed infection of C. parvum and Giardia Assemblage A. These findings support the growing evidence that household animals are potential reservoirs of the zoonotic pathogens Giardia spp. and Cryptosporidium spp. for infections in humans

Molecular identification of Giardia and Cryptosporidium from dogs and cats

The aim of the present study was to diagnose the presence of Giardia cysts and Cryptosporidium oocysts in household animals using nested polymerase chain reaction (PCR) and sequence analysis. One hundred faecal samples obtained from 81 dogs and 19 cats were investigated. The Cryptosporidium genotypes were determined by sequencing a fragment of the small subunit (SSU) rRNA gene, while the Giardia Assemblages were determined through analysis of the glutamate dehydrogenase (GDH) locus. Isolates from five dogs and two cats were positive by PCR for the presence of Giardia, and their sequences matched the zoonotic Assemblage A of Giardia. Cryptosporidium spp. isolated from one dog and one cat were both found to be C. parvum. One dog isolate harboured a mixed infection of C. parvum and Giardia Assemblage A. These findings support the growing evidence that household animals are potential reservoirs of the zoonotic pathogens Giardia spp. and Cryptosporidium spp. for infections in humans

Signaling molecules, transcription growth factors and other regulators revealed from in-vivo and in-vitro models for the regulation of cardiac development

Several in-vivo heart developmental models have been applied to decipher the cardiac developmental patterning encompassing early, dorsal, cardiac and visceral mesoderm as well as various transcription factors such as Gata, Hand, Tin, Dpp, Pnr. The expression of cardiac specific transcription factors, such as Gata4, Tbx5, Tbx20, Tbx2, Tbx3, Mef2c, Hey1 and Hand1 are of fundamental significance for the in-vivo cardiac development. Not only the transcription factors, but also the signaling molecules involved in cardiac development were conserved among various species. Enrichment of the bone morphogenic proteins (BMPs) in the anterior lateral plate mesoderm is essential for the initiation of myocardial differentiation and the cardiac developmental process. Moreover, the expression of a number of cardiac transcription factors and structural genes initiate cardiac differentiation in the medial mesoderm. Other signaling molecules such as TGF-beta, IGF-1/2 and the fibroblast growth factor (FGF) play a significant role in cardiac repair/regeneration, ventricular heart development and specification of early cardiac mesoderm, respectively. The role of the Wnt signaling in cardiac development is still controversial discussed, as in-vitro results differ dramatically in relation to the animal models. Embryonic stem cells (ESC) were utilized as an important in-vitro model for the elucidation of the cardiac developmental processes since they can be easily manipulated by numerous signaling molecules, growth factors, small molecules and genetic manipulation. Finally, in the present review the dynamic role of the long noncoding RNA and miRNAs in the regulation of cardiac development are summarized and discussed. (C) 2015 Elsevier Ireland Ltd. All rights reserved

Human Embryonic and Induced Pluripotent Stem Cell Based Toxicity Testing Models: Future Applications in New Drug Discovery

New drug discovery (NDD) is a fascinating discipline encompassing different facets of medicine, pharmacology, biotechnology and chemistry. NDD is very often restricted by efficacy or safety problems of the new clinical candidate in human patients. Drug regulatory authorities have provided various guidelines for advancement of safe new chemical entities (NCEs) in clinical trials which must be strictly followed. In spite of this, various drugs have failed in clinical trials or withdrawn from market because of human safety issues related to cardiotoxicity, hepatotoxicity, neurotoxicity and teratogenicity. The failure of safety prediction was pointed to species specificity issues, lack of mechanistic toxicity data and inadequate clinical trials. These drugs not only affect human health but also cause loss of resources and time. The species specificity issues are partially addressed by use of primary human cells but their availability is very limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) offer sources for generation of an unlimited number of human somatic cells. The emergence of mechanistic models for toxicity testing with transcriptomics, proteomics along with toxicokinetics readouts based on hESCs and hiPSCs is paving the way to design new human relevant testing strategies. Introduction of these models at the timeframe of lead selection and optimization in parallel with in vitro pharmacokinetic studies will significantly reduce compound attrition rate by selection of safer lead molecules. We focused on upcoming hESCs and hiPSCs based toxicity testing models and their future role to address safety gaps of present drug discovery and development