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OBJECTIVES Venoarterial extracorporeal life support (ECLS) has emerged as a potentially life-saving treatment option in therapy-refractory cardiocirculatory failure, but longer-term outcome is poorly defined. Here, we present a comprehensive follow-up analysis covering all major organ systems. METHODS From February 2012 to December 2016, 180 patients were treated with ECLS for therapy-refractory cardiogenic shock or cardiac arrest. The 30-day survival was 43.9%, and 30-day survivors (n = 79) underwent follow-up analysis with the assessment of medium-term survival, quality of life, neuropsychological, cardiopulmonary and end-organ status. RESULTS After a median of 1.9 (1.1–3.6) years (182.4 patient years), 45 of the 79 patients (57.0%) were alive, 35.4% had died and 7.6% were lost to follow-up. Follow-up survival estimates were 78.0% at 1, 61.2% at 3 and 55.1% at 5 years. NYHA class at follow-up was ≤II for 83.3%. The median creatinine was 1.1 (1.0–1.4) mg/dl, and the median bilirubin was 0.8 (0.5–1.0) mg/dl. No patient required dialysis. Overall, 94.4% were free from moderate or severe disability, although 11.1% needed care. Full re-integration into social life was reported by 58.3%, and 39.4% were working. Quality of life was favourable for mental components, but a subset showed deficits in physical aspects. While age was the only peri-implantation parameter significantly predicting medium-term survival, adverse events and functional status at discharge or 30 days were strong predictors. CONCLUSIONS This study demonstrates positive medium-term outcome with high rates of independence in daily life and self-care but a subset of 10–20% suffered from sustained impairments. Our results indicate that peri-implantation parameters lack predictive power but downstream morbidity and functional status at discharge or 30 days can help identify patients at risk for poor recovery.

Lassa virus is endemic in West Africa and can cause severe hemorrhagic fever. The viral L protein transcribes and replicates the RNA genome via its RNA-dependent RNA polymerase activity. Here, we present nine cryo-EM structures of the L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved 3’ and 5’ promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and early elongation states, the endonuclease is inhibited by two distinct L protein peptides, whereas in the pre-initiation state it is uninhibited. In the early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide and the full C-terminal region of the L protein, including the putative cap-binding domain, is well-ordered. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated., The L protein of segmented, negative strand RNA viruses contains the RNA-dependent RNA polymerase essential for virus amplification. Here, the authors report cryoEM structures of the Lassa virus L protein in active, RNA-bound states, and provide mechanistic insights.

Despite multimodal treatment of patients with cardiogenic shock (CS) complicating acute myocardial infarction (AMI), the outcome remains poor. Large retrospective analyses and animal models suggest that extracorporeal life support (ECLS) in CS-complicating AMI improves outcome [(1)][1]. However, to

Background Surgical treatment of constrictive pericarditis (CP) is particularly challenging because of the increased risk of right heart failure. The necessity of postoperative extracorporeal life support (ECLS) can result in mortality rates of 100%. Preemptive implantation of ECLS may improve postoperative outcomes; however, no data are currently available on its use. We conducted a retrospective study to evaluate the feasibility of our strategy. Methods Between September 2012 and June 2016, ECLS was established percutaneously through the groin vessels in 12 individually selected patients with high-risk CP immediately before pericardiectomy in the operating theater as part of the surgical strategy. Prolonged weaning was performed in the intensive care unit. Demographic characteristics, perioperative data, and survival were analyzed. Results The median patient age was 61.5 years (first quartile, third quartile: 51.3, 68.5 years), with a preoperative central venous pressure of 24 mm Hg (first quartile, third quartile: 21, 28 mm Hg). Furthermore, the pulmonary artery pressure was greater than 60 mm Hg in 50% of patients and a dip plateau sign existed in 75% before surgery. The median duration of ECLS therapy was 132 hours (first quartile, third quartile: 96, 168 hours) with a length of stay on the intensive care unit of 10 days (first quartile, third quartile: 7.0, 16.8 days). There was no intraoperative death. The cumulative 30-day, 1-year, and 5-year survival rates were 83% ± 11%, 75% ± 13%, and 75% ± 13%, respectively. Conclusions From our real-world data, preemptive use of perioperative ECLS, assigned by individual team decision in selected patients with severe CP, is a feasible and safe strategy.

Objectives Diagnosis of prosthetic graft infection after cardiac and proximal aortic surgery is a challenge. Besides technical considerations, redo surgery is associated with substantial morbidity and mortality. Therefore, an accurate diagnosis is mandatory. We report on our experience with hybrid 18-fluorodeoxyglucose positron emission tomography ((18)F-FDG PET)/computed tomography (CT) imaging, which is increasingly used to diagnose infections in the detection of graft infection after cardiac surgery. Methods Twenty-six patients who underwent (18)F-FDG PET/CT imaging after cardiac surgery between February 2010 and September 2014 for suspected graft infection were retrospectively analysed (81% male, age 54.3 ± 13.7 years). PET/CT imaging was performed 36.5 ± 70.5 (0.5-300) months after surgery. 2 patients (8%) had undergone aortic valve replacement (concomitant ascending and proximal arch replacement in 1), 1 (4%) aortic root reconstruction, 9 (35%) aortic root replacement (concomitant partial arch in 4, arch replacement and postoperative TEVAR in 1), 2 (8%) ascending aortic and partial arch replacement and 2 (8%) ascending aortic replacement along with frozen elephant trunk. In 10 (38%), more than one previous cardiac surgical procedure had been performed. Maximum standardized uptake values (SUVmax) were obtained for all patients. If the patients were reoperated on, the final diagnosis was derived from intraoperative findings and/or microbiological results. Otherwise, the longest clinical follow-up available served as a reference. Results Conventional CT was positive for infection in 13 cases (50%). In 22 (85%), PET was indicative of infection (SUVmax 10.5 ± 4.1). PET did not only confirm true-positive CT results in all but 1 case; in almost 30%, it provided substantial additional diagnostic information in comparison with CT alone. Receiver operating characteristic analysis identified an SUVmax of 7.25 to achieve maximum sensitivity (89%) and specificity (100%) in prediction of infection. Twelve patients (46%) required redo surgery for graft infection; in 1 additional patient (4%), sternal re-fixation was necessary. Furthermore, 2 patients had to be reoperated on for torn-out anastomosis and paraprosthetic perfusion (8%). Conclusions PET provides functional data, confirms a CT diagnosis and may even increase diagnostic sensitivity in comparison with CT alone in selected cases. Specificity can be compromised by postoperative changes or chronic inflammatory reactions induced by the graft. CT and/or echocardiography should remain the first diagnostic step in case of a suspected infection because of their broad and fast availability. If confirmation is needed or diagnosis is not achievable using conventional methods, PET might be chosen as the next modality to gain additional information in experienced centres.

No guidelines for mechanical circulatory support in patients with therapy-refractory cardiogenic shock and multiorgan failure including ongoing cardiopulmonary resuscitation (CPR) exist. To achieve immediate cardiopulmonary stabilization, we established an interdisciplinary concept with on-site percutaneous extracorporeal life support (ECLS) implantation. From February 2012 to November 2014, 96 patients were deemed eligible for ECLS implantation. Establishing ECLS was successful in 87 patients (mean age 54 +/- 13 years, 16% female, initial flow 4.4 +/- 0.9 l/min). Aetiologies included acute coronary syndromes (n = 52, 60%), cardiomyopathies (n = 25, 29%) and other pathologies. Fifty-nine patients (68%) had been resuscitated, and in 27 (31%), implantation was performed during CPR;11 patients (13%) were awake at implantation and 20 (23%) underwent implantation in the referring hospital. Metabolic parameters differed in non-survivors versus survivors before ECLS implantation (pH 7.15 +/- 0.23 vs. 7.27 +/- 0.18, P = 0.007;lactate levels 10.90 +/- 6.00 mmol/l vs. 8.79 +/- 5.78 mmol/l, P = 0.091) and 6 h postimplantation (pH 7.27 +/- 0.11 vs. 7.37 +/- 0.11, P < 0.001;lactate levels 10.19 +/- 5.52 mmol/l vs. 5.52 +/- 4.17 mmol/l, P < 0.001). Altogether 44 patients could be weaned, and 9 were bridged to assist device implantation and 1 to heart transplantation. The mean time of support was 6 days, and the 30-day survival rate was 47% (n = 41). ECLS serves as a bridge-to-decision and bridge-to-treatment device. Our interdisciplinary ECLS programme achieved acceptable survival of critically ill patients despite a substantial percentage of patients having been resuscitated and no absolute exclusion criteria. Further studies defining inclusion- and exclusion criteria might additionally improve outcome.

A significant subset of Veterans residing in in VA Community Living Centers (CLCs) demonstrate challenging dementia-related behaviors that affect their quality of life, stress the caregiving staff, and interfere with successful placement in the community. The Behavioral Recovery Outreach (BRO) team was created at the VA Central Iowa Healthcare System to: (1) treat challenging dementia-related behaviors while Veterans reside in the CLC, and (2) ensure successful placements post-discharge. This team, comprised of a psychologist, nurse, social worker, and recreational therapist, facilitates successful placements by communicating effective behavior plans at discharge, providing on-site visits at 1, 3, 6, and 12 months post-discharge, and providing as-needed consultation. The team, working with 66 Veterans to date, has demonstrated 89% success in community placements. The BRO team implementation process and outcomes will be discussed.

The reattachment of the supra-aortic vessels during hybrid arch repair using a branched prosthesis is time consuming and sometimes technically challenging. Here, we describe the surgical technique of bridging the end-to-end anastomoses between the graft branches and the supra-aortic vessels by self-expanding covered stents to reduce suturing time, avoid anastomotic bleeding, enhance true lumen remodeling, and improve vessel alignment to the hybrid graft.

Objectives Different revascularization strategies for patients with acute myocardial infarction (AMI) exist. It remains unclear whether ventricular unloading using cardiopulmonary bypass (CPB) or extracorporeal life support (ECLS) has an impact on early postischaemic ventricular function. Here, we report on the results of an approach using a miniaturized CPB in a well-established animal model of AMI. Methods In a randomized fashion, 30 male Wistar rats were assigned to temporary left anterior descending (LAD) ligation (30 min) followed by 180 min of reperfusion either with or without 60 min of CPB (70 ml/min, 36°C). The CPB circuit consisted of a venous reservoir, a peristaltic roller pump and a membrane oxygenator with heat exchanger. Cardiac function was measured at 60 and 120 min after reperfusion (F60, F120) using a conductance catheter. Results The mortality rate was 37% (11/30). Thus, 19 animals could be included into the analysis (8 CPB). The mean cardiac output did not differ between the groups at F60 [63 ± 29 vs 54 ± 25 ml/min (CPB), P = 0.56] and F120 [73 ± 27 vs 53 ± 24 ml/min (CPB), P = 0.21]. During reperfusion, the mean left ventricular ejection fraction (LVEF) was stable in both the control (F60 37 ± 5% vs F120 33 ± 8%, P = 0.42) and the CPB groups (F60 52 ± 11% vs F120 51 ± 13%, P = 0.71). CPB animals had a significantly better LVEF after reperfusion (F60 P = 0.007, F120 P = 0.01). Conclusions In this animal model of AMI, the establishment of CPB resulted in a significantly better LVEF in comparison with conventional reperfusion only. This beneficial effect may have an impact on revascularization strategies and timing in patients presenting with AMI in the future.

OBJECTIVES: Therapy refractory cardiogenic shock is associated with dismal outcome. Percutaneous implantation of an extracorporeal life support (ECLS) system achieves immediate cardiopulmonary stabilization, sufficient end-organ perfusion and reduction of subsequent multiorgan failure (MOF). METHODS: Forty-one patients undergoing percutaneous ECLS implantation for cardiogenic shock from February 2012 until August 2013 were retrospectively analysed. Mean age was 52 ± 13 years, 6 (15%) were female. Mean pH values obtained before ECLS implantation were 7.15 ± 0.24, mean lactate concentration was 11.7 ± 6.4 mmol/l. Levels obtained 6 h after ECLS implantation were 7.30 ± 0.14 and 8.7 ± 5.0 mmol/l, respectively. In 23 patients (56%) cardiogenic shock resulted from an acute coronary syndrome in 13 (32%) from cardiomyopathy, in 5 (12%) from other causes. Twenty-seven (66%) had been resuscitated, in 14 (34%) implantation was performed under ongoing cardiopulmonary resuscitation (CPR). Of note, 97% of the acute coronary syndrome patients underwent percutaneous coronary intervention (PCI) either before ECLS implantation or under ECLS support. Extracorporeal life support implantation was performed on scene (Emergency Department, Cath Lab, Intensive Care Unit) by a senior cardiac surgeon and a trained perfusionist, in 8 cases (20%) in the referring hospital. RESULTS: Thirty-day mortality was 51% [21 patients, due to MOF (n= 14), cerebral complications (n= 6) and heart failure (n= 1)]. Logistic regression analysis identified 6-h pH values as an independent risk factor of 30-day mortality (P< 0.001, OR = 0.000, 95% CI 0.000–0.042). Neither CPR nor implantation under ongoing CPR resulted in significant differences. In 26 cases (63%), the ECLS system could be explanted, after mean support of 169 ± 67 h. Seven of these patients received cardiac surgery [ventricular assist device implantation (n= 4), heart transplantation (n= 1), other procedures (n= 2)]. CONCLUSIONS: Due to the evolution of transportable ECLS systems and percutaneous techniques implantation on scene is feasible. Extracorporeal life support may serve as a bridge-to-decision and bridge-to-treatment device. Neurological evaluation before ventricular assist device implantation and PCI under stable conditions are possible. Despite substantial mortality, ECLS implantation in selected patients by an experienced team offers additional support to conventional therapy as well as CPR and allows survival in patients that otherwise most likely would have died. This concept has to be implemented in cardiac survival networks in the future.

Highlights ► EVA is a globally available virus collection serving academia, public health and industry. ► EVA is a unique, networked, quality-controlled non-profit virus archive that benefits science. ► EVA provides wide-ranging access to virus collections held in laboratories worldwide. ► Laboratories in developing countries contribute to the pool of quality-controlled reagents., The European Virus Archive (EVA) was conceived as a direct response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry, initially within Europe, but ultimately throughout the world. Although scientists worldwide have accumulated virus collections since the early twentieth century, the quality of the collections and the viruses collected may vary according to the personal interests and agenda of the scientists. Moreover, when laboratories are re-organised or closed, collections are no longer maintained and gradually cease to exist. The tragedy of 9/11 and other disruptive activities have also meant that some previously available biological reagents are no longer openly exchanged between countries. In 2008, funding under the FP7–EU infrastructure programme enabled the initiation of the EVA. Within three years, it has developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. There is every reason to believe that EVA will continue to expand and ultimately exist as a globally networked, quality-controlled non-profit archive for the benefit of science. Organizations or individuals who would like to be considered as contributors are invited to contact the EVA coordinator, Jean–Louis Romette, at jean-louis.romette@univmed.fr.

Reciprocal coevolution between host and pathogen is widely seen as a major driver of evolution and biological innovation. Yet, to date, the underlying genetic mechanisms and associated trait functions that are unique to rapid coevolutionary change are generally unknown. We here combined experimental evolution of the bacterial biocontrol agent Bacillus thuringiensis and its nematode host Caenorhabditis elegans with large-scale phenotyping, whole genome analysis, and functional genetics to demonstrate the selective benefit of pathogen virulence and the underlying toxin genes during the adaptation process. We show that: (i) high virulence was specifically favoured during pathogen–host coevolution rather than pathogen one-sided adaptation to a nonchanging host or to an environment without host; (ii) the pathogen genotype BT-679 with known nematocidal toxin genes and high virulence specifically swept to fixation in all of the independent replicate populations under coevolution but only some under one-sided adaptation; (iii) high virulence in the BT-679-dominated populations correlated with elevated copy numbers of the plasmid containing the nematocidal toxin genes; (iv) loss of virulence in a toxin-plasmid lacking BT-679 isolate was reconstituted by genetic reintroduction or external addition of the toxins. We conclude that sustained coevolution is distinct from unidirectional selection in shaping the pathogen's genome and life history characteristics. To our knowledge, this study is the first to characterize the pathogen genes involved in coevolutionary adaptation in an animal host–pathogen interaction system., A combination of experimental evolution with large-scale phenotyping, genomics and functional genetics reveals the specific role of virulence and toxin genes during the evolutionary adaptation of a pathogen to an animal host., Author Summary Evolution can be extremely fast and dramatic, especially when infectious disease agents such as bacterial pathogens engage in a continuous arms race with their host organism. Rounds of novel pathogen attack strategies and associated host counterdefenses conspire to drive host–pathogen coevolution and biological innovation. To better understand the underlying genetic mechanisms and the exact trait characteristics under selection, we conducted experimental evolution using a simple host–pathogen model system (nematode versus bacterium) under controlled laboratory conditions. We analysed the associated adaptive changes in real time using large-scale phenotyping, population whole genome sequencing, and genetic analysis of the identified candidate genes. We show that coevolution (rather than one-sided adaptation) particularly favors and maintains pathogen virulence, and that two specific toxin genes significantly influence this virulence during coevolution.

This paper presents the design of a sensor packaging for a Fibre Bragg Grating (FBG) based fibre optic humidity sensor. The evaluation of the developed fibre optic sensor was performed under experimental conditions and verified its capability to withstand highly alkaline environments. Therefore, the sensor can be applied to monitor the concrete humidity level and thus to indicate the maintenance of concrete structures.

International audience; Antimicrobial resistance is a global challenge that impacts both human and veterinary health care. The resilience of microbes is reflected in their ability to adapt and survive in spite of our best efforts to constrain their infectious capabilities. As science advances, many of the mechanisms for microbial survival and resistance element transfer have been identified. During the 2012 meeting of Antimicrobial Agents in Veterinary Medicine (AAVM), experts provided insights on such issues as use vs. resistance, the available tools for supporting appropriate drug use, the importance of meeting the therapeutic needs within the domestic animal health care, and the requirements associated with food safety and food security. This report aims to provide a summary of the presentations and discussions occurring during the 2012 AAVM with the goal of stimulating future discussions and enhancing the opportunity to establish creative and sustainable solutions that will guarantee the availability of an effective therapeutic arsenal for veterinary species.

Background The number of patients presenting with acute myocardial infarction (AMI) and being untreatable by interventional cardiologists increased during the last years. Previous experience in emergency coronary artery bypass grafting (CABG) in these patients spurred us towards a more liberal acceptance for surgery. Following a prospective protocol, patients were operated on and further analysed. Methods Within a two year interval, 127 patients (38 female, age 68±12 years, EuroScore (ES) II 6.7±7.2%) presenting with AMI (86 non-ST-elevated myocardial infarction (NSTEMI), 41 STEMI) were immediately accepted for emergency CABG and operated on within six hours after cardiac catheterisation (77% three-vessel-disease, 47% left main stem stenosis, 11% cardiogenic shock, 21% preoperative intraaortic balloon pump (IABP), left ventricular ejection fraction 48±15%). Results 30-day-mortality was 6% (8 patients, 2 NSTEMI (2%) 6 STEMI (15%), p=0.014). Complete revascularisation could be achieved in 80% of the patients using 2±1 grafts and 3±1 distal anastomoses. In total, 66% were supported by IABP, extracorporal life support (ECLS) systems were implanted in two patients. Logistic regression analysis revealed the ES II as an independent risk factor for mortality (p Conclusions Quo ad vitam, results of emergency CABG for patients presenting with NSTEMI can be compared with those of elective revascularisation. Complete revascularisation obviously offers a clear benefit for the patients. Mortality in patients presenting with STEMI and cardiogenic shock is substantially high. For these patients, other concepts regarding timing of surgical revascularisation and bridging until surgery need to be taken into consideration.

T he ability to identify and map areas cattle are expected to utilize heavily and areas cattle may avoid will reduce the time required to establish realistic objectives. There are many factors that cause cattle to typically utilize forage in an uneven manner. Distance to water and steepness of slope are two site factors that are known to strongly affect uniform utilization of forage. A GIS/Analysis system was used to develop maps based on a combination of slope and distance to water that predicts the level of forage utilization by cattle. The maps have been effectively used to facilitate several planning and analysis tasks that are often time consuming.

Role of [Ca2+]iin the ATP-induced heat sensitization process of rat nociceptive neurons. In inflamed tissue, nociceptors show increased sensitivity to noxious heat, which may account for heat hyperalgesia. In unmyelinated nociceptive afferents in rat skin in vitro, a drop of heat threshold and an increase in heat responses were induced by experimental elevation of intracellular calcium ([Ca2+]i) levels with the calcium ionophore ionomycin (10 μM). Similar results were obtained in experiments employing [Ca2+]irelease from preloaded “caged calcium” (NITR-5/AM) via UV photolysis. In both cases, sensitization was prevented by preventing rises in [Ca2+]iwith the membrane-permeant calcium chelator BAPTA-AM (1 mM). No pronounced change of mechanical sensitivity was observed. Heat-induced membrane currents ( Iheat) were investigated with patch-clamp recordings, and simultaneous calcium measurements were performed in small sensory neurons isolated from adult rat dorsal root ganglia (DRG). Ionomycin-induced rises in [Ca2+]iresulted in reversible sensitization of Iheat. In the same subset of DRG neurons, the endogenous algogen ATP (100 μM) was used to elevate [Ca2+]i, which again resulted in significant sensitization of Iheat. In correlative recordings from the skin–nerve preparation, ATP induced heat sensitization of nociceptors, which again could be blocked by preincubation with BAPTA-AM. Rises in [Ca2+]iin response to inflammatory mediators, e.g., ATP, thus appear to play a central role in plastic changes of nociceptors, which may account for hypersensitivity of inflamed tissue.

Viral hemorrhagic fevers (VHF) are acute infections with high case fatality rates, associated with the risk of nosocomial transmission (3). A rapid confirmation of the clinical diagnosis is therefore required by methods such as antigen capture enzyme immunoassay and serologic detection of immunoglobulin M. With the development of PCR technology, it has become possible to rapidly test for viruses that cause VHF (4, 5, 10). We have recently confirmed a case of acute yellow fever with fulminating hepatic failure by reverse transcription-PCR (RT-PCR) (2). In spite of the very high viral RNA concentration in the plasma sample, it initially tested negative. A confirmation by PCR would have been missed if we had not tested a duplicate sample, inoculated with in vitro-transcribed yellow fever virus RNA, to detect substances that are inhibitory to RT-PCR (5). When this control reaction failed, we diluted the plasma in log10 intervals, reprepared RNA from the diluted material, and tested it by RT-PCR. The virus was clearly detectable in the patient's plasma diluted 1:100, 1:1,000, and 1:10,000 (Fig. ​(Fig.1).1). Again, no virus was detectable in the undiluted sample, and detection was also partially inhibited in the 1:10 dilution. Quantitative real-time RT-PCR with the diluted material yielded a projected concentration of >106 copies of viral RNA per ml of the undiluted plasma (Fig. ​(Fig.1).1). Interestingly, we have observed a similarly strong inhibition phenomenon with plasma from a moribund patient with acute Ebola hemorrhagic fever from Gulu, Uganda (Fig. ​(Fig.2).2). The viral RNA concentration in this case was 6.9 × 108 copies/ml. No hemolysis, which often causes inhibition of PCR (1), was observed in plasma from the two patients. In both cases, however, the patients were suffering from severe organ manifestation of their disease (aspartate aminotransferase in yellow fever sample, 15,000 IU; alanine aminotransferase, 6,000 IU). FIG. 1. Results of quantitative real-time yellow fever virus RT-PCR (5) with original plasma and prediluted plasma. Closed data points depict the viral RNA concentration in plasma, as determined by testing of each dilution step of the patient sample (y axis). ... FIG. 2. Result of agarose gel electrophoresis after qualitative Ebola virus RT-PCR (5) with original plasma and prediluted plasma from a patient with acute Ebola fever. The dilution factor is depicted above each gel lane. No amplification was possible when the ... Inhibition of RT-PCR in plasma samples has been reported to occur at a frequency of 0.34 to 2.1% of tests (patients infected with human immunodeficiency virus type 1 or hepatitis C virus, respectively [6, 9]). It can usually be circumvented by using standard ETDA sampling tubes (as done in both cases described here) and preparation procedures based on silica column purification (7, 8, 11). Furthermore, at least with plasma, inhibition is usually reversible upon repetition of extraction of nucleic acids from the same sample (6, 9). In these cases, however, the concentration in plasma of substances interfering with PCR appeared to be extraordinarily high: amplification was repeatedly inhibited in a second extraction, even though a reliable silica column purification method (viral RNA kit; Qiagen, Hilden, Germany) was applied. Moreover, partial inhibition occurred even in 1:10-diluted material. Two important consequences can be drawn from these observations. (i) False-negative RT-PCR results are likely to occur for patients with severe viral hemorrhagic fevers, especially in the acute phase of the disease where a rapid confirmation is required. Their plasma may contain large amounts of RT-PCR inhibitors, probably resulting from the decay of tissue. These inhibitors can be detected by control reactions with spiked samples (low copy numbers of control RNA, 1 log10 above detection limit of the PCR) (5). Control reactions to detect inhibitors of RT-PCR are mandatory for a safe diagnosis for patients with suspected VHF. (ii) When PCR is used for diagnosis of viral hemorrhagic fevers, dilutions of the test sample should be tested in parallel with the original sample. The high viral RNA concentration in samples from acute VHF cases is likely to facilitate the diagnosis in spite of the dilution factor.

We tested the feasibility of a reduced-intensity conditioning regimen for hematopoietic stem cell transplantation (HSCT) perspectively allowing an adapted conditioning for performance and disease status. Treatment consisted of 2–4 Gy total body irradiation (TBI), 120mg/m2 fludarabine, 120mg/kg cyclophosphamide, 0–40 mg/kg anti-thymocyte globulin (ATG) and immunosuppression by ciclosporin-A and mycophenolate mofetil. Up to date, 10 patients (median age 59 years) ineligible for conventional conditioning due to age (>50 years), concomitant disease or previous toxicities have been transplanted on an observational basis after obtaining written informed consent. 2 patients received grafts from related donors, 1 patient an unrelated graft with HLA-C mismatch in GvHD vector and all others unrelated matched grafts. 9 patients (90%) received 2 Gy TBI and ≥30mg/kg ATG. 1 patient received 4 Gy TBI and no ATG since presenting with progressiv disease at time of HSCT and receiving an related graft. Median pre-transplant adapted Charlson index was 2,5 (range 0–6), number of previous chemotherapy protocols 2 (range 1–5). No obvious graft rejection occurred. Of the patients evaluable beyond day +30 (N = 8) 8 patients (100%) achieved consistent neutrophil counts >500/μl and 6 (75%) consistent platelet counts of >25,000/μl. Median duration for neutropenia was 25 days (range 15 – 44 days) and for thrombocytopenia 23 days (range 7–55 days). 14 units (range 2–58) packed red cells and of 7 units (range 1–62) platelets had to be given. Median hospital stay was 49 days (range 34–66 days). Of 8 patients with a follow up beyond day +30, 6 (75%) showed a chimerism of >90% around day +30. 1 patient showed a chimerism of 50% due to parallel relapse while 1 patient was not evaluable due to sepsis. All patients evaluable for toxicity (N=8) had CTC grade 4 hematological toxicity. 6 patients (75%) experienced toxicities CTC ≥3, mainly infections. Two patients (25%) died of treatment-related infections. No gastrointestinal toxicity CTC ≥3° and no case of veno-occlusive disease was observed. Hemorrhagic cystitis CTC 2° was observed in 1 patient (10%). Of the patients with a Charlson index ≥3 (N=5), 2 (40%) died of infections and 3 (60%) experienced only toxicities of ≤3. Of the evaluable patients with neutrophil recovery (N=8), 1 (12,5%) presented with acute GvHD of the skin 2° and 2 (25%) respectively with acute GvHD 1° of the skin or gut. 4 patients (50%) showed no signs of acute GvHD. Of the 2 patients evaluable for day >+100, one has chronic limited GvHD. All cases of GvHD required no therapy. Of the three patients evaluable day +100, one suffered from relapse and two are alive in complete remission. Of the patients evaluable day +30 (N=8) two (25%) presented with relapse or progressive disease around day +30, 1 (12,5%) respectively showed stable disease or further remission and 4 patients (50%) showed persisting complete remission. We conclude that the presented regimen is applicable to patients not elegibile for conventional regimens due to its tolerable overall toxicity and particularly low gastrointestinal side effects. Moreover, due to the use of 3 cytotoxic agents and 3 immunosuppressive substances it offers the option for a conditioning adapted to commorbidity, disease status and graft characteristics. A respective stratification will be presented at the meeting.

Information is presented to aid communities tailor an energy conservation program specifically to themselves. Existing and new buildings, procurement, employee transportation programs, street lighting systems, and energy resource recovery are discussed. Examples are given on what can and has been done in communities. (ERA citation 06:022248)