From May until July 2011 a large outbreak of infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) occurred in Germany. More than 800 patients suffered from hemolytic uremic syndrome (HUS), and 49 fatal cases were reported. Obviously, a mandatory requirement for such a clinical situation is the availability of rapid and reliable STEC tests from the investigating laboratory. The standard methods like enzyme immunoassay (EIA), vero cell cytotoxicity assay (VCA), and microbiological culture are, however, hampered by a lack of sensitivity and specificity unless a prior, time consuming broth enrichment step is employed. In order to acelerate the laboratory diagnosis, we evaluated an in-house real-time PCR assay for the detection of the Stx genes (stx1 and stx2) directly from stool specimens without the need of broth enrichment procedures.754 faecal samples were collected from 481 predominantly hospitalised patients with diarrhea from May 23 to June 10, 2011 at the Medical Laboratory Bremen, Germany. The samples were analysed with a direct stx real-time PCR and compared to EIA, VCA and culturing on enterohemolysin, ESBL, and CPS agar after broth enrichment. In addition, artificial samples (n = 12) from three official EHEC/STEC PCR quality proficiency panels (INSTAND, Germany, September 2006, September 2007, and April 2008) were analysed by real-time PCR only.The real-time PCR produced reliable, distinct melting profiles with characteristic peaks for the stx1 and stx2 PCR products. The quality proficiency panels revealed a detection limit of 10 CFU/PCR per reaction. 112, 86, 99, and 122 of 754 clinical samples were positive for culture, EIA, VCA, and real-time PCR, respectively. 121 of 122 PCR samples were positive only for stx2. Compared to culture as the gold standard, sensitivities of EIA, VCA, and real-time PCR were 76.8 %, 83.9%, and 96.4% and specificities were 99.4%, 99.2%, and 97.8%, respectively.The direct fecal stx real-time PCR proved superior to enrichment based VCA and EIA and can be recommended as a quick and sensitive tool for the early diagnosis of STEC infection in addition to microbiological culture.
Objectives: One of the most common clinical complications in patients with an implanted left ventricular assist device (LVAD) is recurrent nonsurgical bleeding (NSB). Nonphysiological shear stress caused by LVADs may contribute to platelet receptor shedding and thereby facilitate platelet dysfunction which is an important cause of major bleeding during LVAD support. We investigated the device-induced alterations of platelets to identify patients at risk of bleeding after LVAD implantation.
Hedgehog (Hh) morphogens are essential regulators of growth and patterning at considerable distances from their source, despite being produced as double lipidated proteins that attach to the outer plasma membrane (PM) leaflet of producing cells. While mechanisms to overcome this limitation include the 12-pass transmembrane protein Dispatched1 (Disp), the soluble glycoprotein Scube2, proteolytic processing of lipidated peptide termini, and soluble lipoproteins (LPPs) acting as Hh acceptors and transporters, the molecular interplay of these release factors for effective Hh solubilization from the PM remains controversial. In this work, we used in vivo functional knockdown together with in vitro experiments to show that A Disintegrin and Metalloproteinase 10, Scube2 and Disp act synergistically to remove Hh from the PM and transfer it to LPP acceptors. We also show that Scube2 requirement for Hh transfer depends on LPP acceptor levels: low LPP levels in media require Scube2 for efficient Hh release, whereas increasing LPP levels render Scube2 function increasingly dispensable. Biochemical analysis explained this observation by showing that Scube2 binds high-density LPP (HDL) via its 9 EGF domains and associates with the PM via interactions with cell surface heparan sulfate (HS), revealing a previously unknown role for Scube2 in bridging low-abundance soluble carriers for Hh with HS-rich Hh release sites.
