Real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples

Two real-time PCR assays for detection of Mycolasma hyopneumoniae (Mhyop) in clinical lung samples were established and validated in parallel. One is targeting a repetitive DNA element (REP assay) the other a putative ABC transporter gene (ABC assay). The two assays were shown to be 100% specific when testing pig lungs from defined negative farms. When investigating defined positive farms the REP assay tested with a sensitivity of about 50%, the ABC assay with 90%. The two assays together, however detected 100% of positive farms. Within a single positive farm on average 90% of the samples tested positive with the REP or ABC assay. Analysing a set of 41 lungs from infected pigs from routine diagnostic the REP assay detected 50% and the ABC assay 70%, while both assays together had a sensitivity of 85%

The Prognostic Value of a Single, Randomly Timed Circulating Tumor DNA Measurement in Patients with Metastatic Melanoma

Simple Summary In this study, we investigated the associations of circulating tumor DNA (ctDNA), measured at a random time point during the patient’s treatment, with tumor progression and routine blood markers (protein S100, lactate dehydrogenase (LDH), and C-reactive protein (CRP)) in a cohort of patients with metastatic melanoma. Detectable ctDNA was associated with the presence of extracerebral disease, tumor progression, and poorer overall survival (OS). Elevated S100 and CRP was correlated with detectable ctDNA, whereas LDH was not. Our results further support the use of ctDNA in the clinical management of patients with metastatic melanoma. Abstract Melanoma currently lacks validated blood-based biomarkers for monitoring and predicting treatment efficacy. Circulating tumor DNA (ctDNA), originating from tumor cells and detectable in plasma, has emerged as a possible biomarker in patients with metastatic melanoma. In this retrospective, single-center study, we collected 129 plasma samples from 79 patients with stage IIIB–IV melanoma as determined by the American Joint Committee on Cancer (AJCC, 8th edition). For the determination of ctDNA levels, we used eight different assays of droplet digital polymerase chain reaction (ddPCR) to detect the most common hotspot mutations in the BRAF and NRAS genes. The aim of the study was to investigate the association of the detectability of ctDNA at a non-prespecified time point in a patient’s treatment with tumor progression, and to correlate ctDNA with commonly used biomarkers (protein S100, LDH, and CRP). Patients with detectable ctDNA progressed more frequently in PET-CT within 12 months than those without detectable ctDNA. Detectability of ctDNA was associated with shorter OS in univariate and multivariate analyses. ctDNA was detectable in a statistically significantly larger proportion of patients with distant metastases (79%) than in patients with no distant metastases or only intracranial metastases (32%). Elevated protein S100 and CRP correlated better with detectable ctDNA than LDH. This study supports the potential of ctDNA as a prognostic biomarker in patients with metastatic melanoma. However, additional prospective longitudinal studies with quantitative assessments of ctDNA are necessary to investigate the limitations and strengths of ctDNA as a biomarker. Keywords: ctDNA; melanoma; tumor progression; PET-CT; S100; biomarke

Hanbury Brown Twiss effects in channel mixing normal-superconducting systems

An investigation of the role of the proximity effect in current cross correlations in multiterminal, channel-mixing, normal-superconducting systems is presented. The proposed experiment is an electrical analog of the optical Hanbury Brown Twiss intensity cross correlation experiment. A chaotic quantum dot is connected via quantum point contacts to two normal and one superconducting reservoir. For dominating coupling of the dot to the superconducting reservoir, a magnetic flux of the order of a flux quantum in the dot suppresses the proximity effect and reverses the sign of the cross correlations, from positive to negative. In the opposite limit, for a dominating coupling to the normal reservoirs, the proximity effect is weak and the cross correlation are positive for a nonideal contact between the dot and the superconducting reservoir. We show that in this limit the correlations can be explained with particle counting arguments.Comment: Invited talk at LT2

Virulence Factors for Hemolytic Uremic Syndrome, Denmark1

We present an analysis of strain and patient factors associated with the development of bloody diarrhea and hemolytic uremic syndrome (HUS) among Shiga toxin-producing Escherichia coli (STEC) patients registered in Denmark in a 6-year period. Of 343 STEC patients, bloody diarrhea developed in 36.4% and HUS in 6.1%. In a multivariate logistic regression model, risk factors for bloody diarrhea were the eae and stx2 genes, O groups O157 and O103, and increasing age. Risk factors for HUS were presence of the stx2 (odds ratio [OR] 18.9) and eae (OR undefined) genes, being a child, and having bloody diarrhea. O group O157, although associated with HUS in a univariate analysis (OR 4.0), was not associated in the multivariate analysis (OR 1.1). This finding indicates that, rather than O group, the combined presence of the eae and stx2 genes is an important predictor of HUS

A single-dose comparison of the acute effects between the new somatostatin analog SOM230 and octreotide in acromegalic patients

Treatment with the somatostatin receptor (sst) subtype 2 predominant analogs octreotide and lanreotide induces clinical and biochemical cure in approximately 65% of acromegalic patients. GH-secreting pituitary adenomas, which are not controlled, also express sst(5). We compared the acute effects of octreotide and SOM230, a new somatostatin analog with high affinity for sst(1,2,3,5) on hormone release in acromegalic patients. In a single-dose, proof-of-concept study, 100 microg octreotide and 100 and 250 microg SOM230 were given s.c. to 12 patients with active acromegaly. Doses of 100 and 250 microg SOM230 dose-dependently suppressed GH levels from 2-8 h after administration (-38 +/- 7.7 vs. -61 +/- 6.7%, respectively; P < 0.01). A comparable suppression of GH levels by octreotide and 250 microg SOM230 was observed in eight patients (-65 +/- 7 vs. -72 +/- 7%, respectively). In three patients, the acute GH-lowering effect of 250 microg SOM230 was significantly superior to that of octreotide (-70 +/- 2 vs. -17 +/- 15%, respectively; P < 0.01). In one patient, the GH-lowering effect of octreotide was better than that of SOM230. Tolerability for SOM230 was good. Glucose levels were initially slightly elevated after octreotide and SOM230, compared with control day, whereas insulin levels were only significantly suppressed by octreotide. We conclude that SOM230 is an effective GH-lowering drug in acromegalic patients with the potential to increase the number of patients controlled during long-term medical treatment

Occurrence of virulence genes associated with diarrheagenic pathotypes in Escherichia coli isolates from surface water

Escherichia coli isolates (n=300) collected from six sites in subtropical Brisbane, Australia, prior to and after storm events were tested for the presence of 11 virulence genes (VGs) specific to diarrheagenic pathotypes. The presence of eaeA, stx, stx, and ehxA genes specific for the enterohemorrhagic E. coli (EHEC) pathotype was detected in 56%, 6%, 10%, and 13% of isolates, respectively. The VGs astA (69%) and aggR (29%), carried by enteroaggregative (EAEC) pathotypes, were frequently detected in E. coli isolates. The enteropathogenic E. coli (EPEC) gene bfp was detected in 24% of isolates. In addition, enteroinvasive E. coli (EIEC) VG ipaH was also detected in 14% of isolates. During dry periods, isolates belonging to the EAEC pathotype were most commonly detected (23%), followed by EHEC (11%) and EPEC (11%). Conversely, a more uniform prevalence of pathotypes, EPEC (14%), EAEC (12%), EIEC (10%), EHEC (7%), and ETEC (7%), was observed after the storm events. The results of this study highlight the widespread occurrence of potentially diarrheagenic pathotypes in the urban aquatic ecosystems. While the presence of VGs in E. coli isolates alone is insufficient to determine pathogenicity, the presence of diarrheagenic E. coli pathotypes in high frequency after the storm events could lead to increased health risks if untreated storm water were to be used for nonpotable purposes and recreational activities

Towards a Pathogenic Escherichia coli Detection Platform Using Multiplex SYBR®Green Real-Time PCR Methods and High Resolution Melting Analysis

Escherichia coli is a group of bacteria which has raised a lot of safety concerns in recent years. Five major intestinal pathogenic groups have been recognized amongst which the verocytotoxin or shiga-toxin (stx1 and/or stx2) producing E. coli (VTEC or STEC respectively) have received a lot of attention recently. Indeed, due to the high number of outbreaks related to VTEC strains, the European Food Safety Authority (EFSA) has requested the monitoring of the “top-five” serogroups (O26, O103, O111, O145 and O157) most often encountered in food borne diseases and addressed the need for validated VTEC detection methods. Here we report the development of a set of intercalating dye Real-time PCR methods capable of rapidly detecting the presence of the toxin genes together with intimin (eae) in the case of VTEC, or aggregative protein (aggR), in the case of the O104:H4 strain responsible for the outbreak in Germany in 2011. All reactions were optimized to perform at the same annealing temperature permitting the multiplex application in order to minimize the need of material and to allow for high-throughput analysis. In addition, High Resolution Melting (HRM) analysis allowing the discrimination among strains possessing similar virulence traits was established. The development, application to food samples and the flexibility in use of the methods are thoroughly discussed. Together, these Real-time PCR methods facilitate the detection of VTEC in a new highly efficient way and could represent the basis for developing a simple pathogenic E. coli platform