Rapid differentiation of Francisella species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA

<p>Abstract</p> <p>Background</p> <p><it>Francisella (F.) tularensis </it>is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, <it>F. tularensis </it>was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR) have been developed but are restricted to reference laboratories.</p> <p>Results</p> <p>The complete 23S rRNA genes of representative strains of <it>F. philomiragia </it>and all subspecies of <it>F. tularensis </it>were sequenced. Single nucleotide polymorphisms on species and subspecies level were confirmed by partial amplification and sequencing of 24 additional strains. Fluorescent In Situ Hybridization (FISH) assays were established using species- and subspecies-specific probes.</p> <p>Different FISH protocols allowed the positive identification of all 4 <it>F. philomiragia </it>strains, and more than 40 <it>F. tularensis </it>strains tested. By combination of different probes, it was possible to differentiate the <it>F. tularensis </it>subspecies <it>holarctica, tularensis, mediasiatica </it>and <it>novicida</it>. No cross reactivity with strains of 71 clinically relevant bacterial species was observed. FISH was also successfully applied to detect different <it>F. tularensis </it>strains in infected cells or tissue samples. In blood culture systems spiked with <it>F. tularensis</it>, bacterial cells of different subspecies could be separated within single samples.</p> <p>Conclusion</p> <p>We could show that FISH targeting the 23S rRNA gene is a rapid and versatile method for the identification and differentiation of <it>F. tularensis </it>isolates from both laboratory cultures and clinical samples.</p

Static overscreening and nonlinear response in the Hubbard Model

We investigate the static charge response for the Hubbard model. Using the Slave-Boson method in the saddle-point approximation we calculate the charge susceptibility. We find that RPA works quite well close to half-filling, breaking, of course, down close to the Mott transition. Away from half filling RPA is much less reliable: Already for very small values of the Hubbard interaction U, the linear response becomes much more efficient than RPA, eventually leading to overscreening already beyond quite moderate values of U. To understand this behavior we give a simple argument, which implies that the response to an external perturbation at large U should actually be strongly non-linear. This prediction is confirmed by the results of exact diagonalization.Comment: 10 pages, 7 figures, RevTe

Phylogeography of Francisella tularensis subsp. holarctica, Europe

Francisella tularensis subsp. holarctica isolates from Austria, Germany, Hungary, Italy, and Romania were placed into an existing phylogeographic framework. Isolates from Italy were assigned to phylogenetic group B.FTNF002–00; the other isolates, to group B.13. Most F. tularensis subsp. holarctica isolates from Europe belong to these 2 geographically segregated groups

Re-emergence of tularemia in Germany: Presence of <it>Francisella tularensis </it>in different rodent species in endemic areas

<p>Abstract</p> <p>Background</p> <p>Tularemia re-emerged in Germany starting in 2004 (with 39 human cases from 2004 to 2007) after over 40 years of only sporadic human infections. The reasons for this rise in case numbers are unknown as is the possible reservoir of the etiologic agent <it>Francisella (F.) tularensis</it>. No systematic study on the reservoir situation of <it>F. tularensis </it>has been published for Germany so far.</p> <p>Methods</p> <p>We investigated three areas six to ten months after the initial tularemia outbreaks for the presence of <it>F. tularensis </it>among small mammals, ticks/fleas and water. The investigations consisted of animal live-trapping, serologic testing, screening by real-time-PCR and cultivation.</p> <p>Results</p> <p>A total of 386 small mammals were trapped. <it>F. tularensis </it>was detected in five different rodent species with carrier rates of 2.04, 6.94 and 10.87% per trapping area. None of the ticks or fleas (n = 432) tested positive for <it>F. tularensis</it>. We were able to demonstrate <it>F. tularensis-</it>specific DNA in one of 28 water samples taken in one of the outbreak areas.</p> <p>Conclusion</p> <p>The findings of our study stress the need for long-term surveillance of natural foci in order to get a better understanding of the reasons for the temporal and spatial patterns of tularemia in Germany.</p

Investigating an Airborne Tularemia Outbreak, Germany

Infectious aerosols can contribute to the transmission of tularemia during processing of dead hares

Genetic predisposition to in situ and invasive lobular carcinoma of the breast.

Invasive lobular breast cancer (ILC) accounts for 10-15% of all invasive breast carcinomas. It is generally ER positive (ER+) and often associated with lobular carcinoma in situ (LCIS). Genome-wide association studies have identified more than 70 common polymorphisms that predispose to breast cancer, but these studies included predominantly ductal (IDC) carcinomas. To identify novel common polymorphisms that predispose to ILC and LCIS, we pooled data from 6,023 cases (5,622 ILC, 401 pure LCIS) and 34,271 controls from 36 studies genotyped using the iCOGS chip. Six novel SNPs most strongly associated with ILC/LCIS in the pooled analysis were genotyped in a further 516 lobular cases (482 ILC, 36 LCIS) and 1,467 controls. These analyses identified a lobular-specific SNP at 7q34 (rs11977670, OR (95%CI) for ILC = 1.13 (1.09-1.18), P = 6.0 × 10(-10); P-het for ILC vs IDC ER+ tumors = 1.8 × 10(-4)). Of the 75 known breast cancer polymorphisms that were genotyped, 56 were associated with ILC and 15 with LCIS at P<0.05. Two SNPs showed significantly stronger associations for ILC than LCIS (rs2981579/10q26/FGFR2, P-het = 0.04 and rs889312/5q11/MAP3K1, P-het = 0.03); and two showed stronger associations for LCIS than ILC (rs6678914/1q32/LGR6, P-het = 0.001 and rs1752911/6q14, P-het = 0.04). In addition, seven of the 75 known loci showed significant differences between ER+ tumors with IDC and ILC histology, three of these showing stronger associations for ILC (rs11249433/1p11, rs2981579/10q26/FGFR2 and rs10995190/10q21/ZNF365) and four associated only with IDC (5p12/rs10941679; rs2588809/14q24/RAD51L1, rs6472903/8q21 and rs1550623/2q31/CDCA7). In conclusion, we have identified one novel lobular breast cancer specific predisposition polymorphism at 7q34, and shown for the first time that common breast cancer polymorphisms predispose to LCIS. We have shown that many of the ER+ breast cancer predisposition loci also predispose to ILC, although there is some heterogeneity between ER+ lobular and ER+ IDC tumors. These data provide evidence for overlapping, but distinct etiological pathways within ER+ breast cancer between morphological subtypes

REQUITE: A prospective multicentre cohort study of patients undergoing radiotherapy for breast, lung or prostate cancer

Purpose: REQUITE aimed to establish a resource for multi-national validation of models and biomarkers that predict risk of late toxicity following radiotherapy. The purpose of this article is to provide summary descriptive data. Methods: An international, prospective cohort study recruited cancer patients in 26 hospitals in eight countries between April 2014 and March 2017. Target recruitment was 5300 patients. Eligible patients had breast, prostate or lung cancer and planned potentially curable radiotherapy. Radiotherapy was prescribed according to local regimens, but centres used standardised data collection forms. Pre-treatment blood samples were collected. Patients were followed for a minimum of 12 (lung) or 24 (breast/prostate) months and summary descriptive statistics were generated. Results: The study recruited 2069 breast (99% of target), 1808 prostate (86%) and 561 lung (51%) cancer patients. The centralised, accessible database includes: physician-(47,025 forms) and patient-(54,901) reported outcomes; 11,563 breast photos; 17,107 DICOMs and 12,684 DVHs. Imputed genotype data are available for 4223 patients with European ancestry (1948 breast, 1728 prostate, 547 lung). Radiation-induced lymphocyte apoptosis (RILA) assay data are available for 1319 patients. DNA (n = 4409) and PAXgene tubes (n = 3039) are stored in the centralised biobank. Example prevalences of 2-year (1-year for lung) grade >= 2 CTCAE toxicities are 13% atrophy (breast), 3% rectal bleeding (prostate) and 27% dyspnoea (lung). Conclusion: The comprehensive centralised database and linked biobank is a valuable resource for the radiotherapy community for validating predictive models and biomarkers. Patient summary: Up to half of cancer patients undergo radiation therapy and irradiation of surrounding healthy tissue is unavoidable. Damage to healthy tissue can affect short-and long-term quality-of-life. Not all patients are equally sensitive to radiation "damage" but it is not possible at the moment to identify those who are. REQUITE was established with the aim of trying to understand more about how we could predict radiation sensitivity. The purpose of this paper is to provide an overview and summary of the data and material available. In the REQUITE study 4400 breast, prostate and lung cancer patients filled out questionnaires and donated blood. A large amount of data was collected in the same way. With all these data and samples a database and biobank were created that showed it is possible to collect this kind of information in a standardised way across countries. In the future, our database and linked biobank will be a resource for research and validation of clinical predictors and models of radiation sensitivity. REQUITE will also enable a better understanding of how many people suffer with radiotherapy toxicity

Evaluation of genetic AIDS vaccines in rhesus monkeys

0\. Titelblatt und Inhaltsverzeichnis 1\. Einleitung 2\. Material und Methoden 3\. Ergebnisse 4\. Diskussion 5\. Zusammenfassung 6\. Summary 7\. Abkürzungsverzeichnis 8\. Literaturverzeichnis 9\. Danksagung 10\. Lebenslauf 11\. Publikationsliste 12\. ErklärungDas vom European Network for Vaccine Evaluation in Primates (ENVEP) ausgearbeitete EU Projekt diente der Evaluation von rekombinaten Impfstoffkandidaten in Rhesusaffen zur Entwicklung eines AIDS Impfstoffes. An dem horizontalen und vertikalen Netzwerk waren im Rahmen dieser Studie acht europäische Institute beteiligt, von denen jedes eine andere Kombination verschiedener Impfstoffkandidaten zur Immunisierung der Affen verwendete. Nach der Immunisierung wurden die Tiere mit dem in Rhesusaffen AIDS-verursachenden SIVmac infiziert, um einen durch die Impfstoffkandidaten vermittelten Schutz vor der Infektion bzw. dem Krankheitsausbruch zu prüfen. Ziel hierbei war, diejenige Kombination rekombinanter Impfstoffkandidaten zu identifizieren, welche den besten Schutz vermittelte. Darüber hinaus sollten die erhobenen Daten auf einen Zusammenhang zwischen einer Protektion der Tiere vor SIV/AIDS und ihrer Immunantwort untersucht werden. Unsere Kombination begann zunächst mit einer DNA-Immunisierung, gefolgt von einer Immunisierung mit rekombinatem Modified Vaccinia Ankara (rMVA) und zwei Immunisierungen mit rekombinantem Semliki Forest Virus (rSFV). Alle Immunisierungen erfolgten im Abstand von jeweils acht Wochen, die Impfstoffe trugen dabei folgende SIVmac Gene: gag, pol, env, tat, rev und nef. Zur Kontrolle wurde eine andere Gruppe nach dem gleichen Schema mit leeren Vektoren (ohne SIV Gene) immunisiert, eine weitere Kontrollgruppe verblieb unbehandelt. Die Untersuchung der humoralen und zellulären Immunantworten gegen SIV in diesem Zeitraum ergab eine deutliche Antikörper- und T-Helferzellantwort nach der zweiten rSFV-Immunisierung sowie eine T-Zellantwort im IFN(-ELISpot vor allem nach den ersten beiden Immunisierungen (DNA und rMVA). Acht Wochen nach der letzten SFV Immunisierung erfolgte die rektale Belastung aller Tiere mit SIVmac. Die zellulären und humoralen Immunantworten, die Viruslast sowie die Anzahl der CD4+ und CD8+ T-Zellen wurden im Zeitraum danach gemessen. Zwar konnte keines der immunisierten Tiere vor einer Infektion mit SIV geschützt werden, dennoch wies diese Gruppe gegenüber den Kontrollgruppen eine signifikant niedrigere Spitzenviruslast zwei Wochen nach der Belastung auf. Ein immunisiertes Tier konnte sogar die Virusreplikation sehr schnell kontrollieren und blieb danach virusnegativ. Bis zum Ende der Studie konnte zwischen den immunisierten Tieren und den Kontrolltieren kein Unterschied in der Menge an CD4+ und CD8+ T-Zellen beobachtet werden.Nach der Belastung entwickelten sich in allen Tieren robuste humorale und zelluläre Immunreaktionen gegen SIV. Einige der immunisierten Affen zeigten im Gegensatz zu den Kontrolltieren anamnestische Reaktionen bezüglich der SIV-spezifischen Antikörper und der IFN(-produzierenden Zellen im ELISpot. Der Vergleich unserer Ergebnisse mit denen anderer Teilnehmer dieser Studie zeigt, dass die Effektivität der unterschiedlichen Kombinationen nicht gleich ist, die Wahl der Kombination also Einfluss auf die Immunogenität und die Kontrolle der Infektion hat.The EU project "European Network for Vaccine Evaluation in Primates (ENVEP)" was designed to evaluate recombinant vaccine candidates in rhesus macaques as part of ongoing efforts to develop an AIDS vaccine. Eight European institutes were involved in this horizontal and vertical network, each of which used a different combination of the vaccine candidates for immunising the monkeys. Following immunisation, the animals were challenged with the simian AIDS- inducing virus SIVmac to evaluate the protection against infection or disease development induced by the vaccine candidates. The aim was to identify the combination of recombinant vaccine candidates able to induce the best level of protection. In addition, the data collected would allow the correlation between the animal's protection from SIV/AIDS and the corresponding immune responses to be investigated. The combination used in our study was an initial DNA immunisation followed by an immunisation with recombinant Modified Vaccinia Ankara (rMVA) and two immunisations with recombinant Semliki Forest Virus (rSFV). All immunisations were given at eight-week intervals and all constructs carried the following SIVmac genes: gag, pol, env, tat, rev and nef. In addition, animals of one control group were immunised with empty vectors (without SIV genes) using the same schedule while a second control group remained untreated. Analysis of the SIV-specific humoral and cellular immune responses during this period revealed a clear antibody and T-helper cell response following the second rSFV immunisation as well as a T-cell response in IFN( ELISpot, particularly after the first two immunisations (DNA and rMVA). The animals were challenged with SIVmac eight weeks after the final immunisation. The cellular and humoral immune responses, the virus loads and the numbers of CD4+ and CD8+ T-cells were continuously measured thereafter. Although none of the immunised animals were protected from SIV infection, this group did show a significantly reduced peak virus load two weeks after challenge. One immunised animal was even able to rapidly control the virus replication and remained virus-negative from then on. However, no difference between the vaccinated and the control animals with regard to the number of CD4+ and CD8+ T-cells was observed during the study. Following challenge, all animals developed robust humoral and cellular immune responses against SIV. Some of the immunised monkeys showed, in comparison to the others, distinct anamnestic responses in terms of SIV-specific antibodies and IFN(-secreting cells in ELISpot. By comparing our results with those of the other participants it is clear that the efficacy of the different combinations was not equal and that the choice of combination can influence the immunogenicity and subsequent control of infection