Effect of Anemia on Prognosis in Patients on Extracorporeal Membrane Oxygenation.

Anemia is a component of the pathological triangle in cardiorenal anemia syndrome and is a risk factor for mortality in acute respiratory distress syndrome. This study assessed the predictive value of anemia for outcomes in critically ill patients receiving extracorporeal membrane oxygenation (ECMO) support. This retrospective study analyzed patients who received ECMO support at the cardiovascular surgery intensive care unit in the study institute between July 2003 and March 2012. Patient data, such as demographic information, etiologies of ECMO implementation, clinical parameters, and in-hospital and 6-month mortality rates, were statistically analyzed. The overall in-hospital mortality rate among the enrolled 295 patients was 55.6%. Multivariate logistical regression analysis indicated that age, albumin levels, sequential organ failure assessment (SOFA) score, and hemoglobin (Hb) level on ECMO day 1 exhibited independent prognostic significance for predicting in-hospital mortality rate. The SOFA score exhibited the highest areas under the receiver operating characteristic curve value (0.812 ± 0.025). The Hb level on ECMO day 1 exhibited satisfactory calibration and discriminatory power. The cumulative 6-month survival rates differed significantly between patients with Hb levels less than and more than 8.85 g/dL (30.6 vs. 54.0%, respectively, P < 0.001). This study indicated that old age, low albumin levels, low Hb levels, and higher SOFA scores on ECMO day 1 increased the risk of mortality. The Hb level is a readily measurable parameter and with good predictive power for critical patients on ECMO.

Genomic analysis of Staphylococcus phage Stau2 isolated from medical specimen.

Stau2 is a lytic myophage of Staphylococcus aureus isolated from medical specimen. Exhibiting a broad host range against S. aureus clinical isolates, Stau2 is potentially useful for topical phage therapy or as an additive in food preservation. In this study, Stau2 was firstly revealed to possess a circularly permuted linear genome of 133,798 bp, with low G + C content, containing 146 open reading frames, but encoding no tRNA. The genome is organized into several modules containing genes for packaging, structural proteins, replication/transcription and host-cell-lysis, with the structural proteins and DNA polymerase modules being organized similarly to that in Twort-like phages of Staphylococcus. With the encoded DNA replication genes, Stau2 can possibly use its own system for replication. In addition, analysis in silico found several introns in seven genes, including those involved in DNA metabolism, packaging, and structure, while one of them (helicase gene) is experimentally confirmed to undergo splicing. Furthermore, phylogenetic analysis suggested Stau2 to be most closely related to Staphylococcus phages SA11 and Remus, members of Twort-like phages. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed 14 structural proteins of Stau2 and N-terminal sequencing identified three of them. Importantly, this phage does not encode any proteins which are known or suspected to be involved in toxicity, pathogenicity, or antibiotic resistance. Therefore, further investigations of feasible therapeutic application of Stau2 are needed.

The kSORT assay to detect renal transplant patients at high risk for acute rejection: results of the multicenter AART study.

BACKGROUND: Development of noninvasive molecular assays to improve disease diagnosis and patient monitoring is a critical need. In renal transplantation, acute rejection (AR) increases the risk for chronic graft injury and failure. Noninvasive diagnostic assays to improve current late and nonspecific diagnosis of rejection are needed. We sought to develop a test using a simple blood gene expression assay to detect patients at high risk for AR. METHODS AND FINDINGS: We developed a novel correlation-based algorithm by step-wise analysis of gene expression data in 558 blood samples from 436 renal transplant patients collected across eight transplant centers in the US, Mexico, and Spain between 5 February 2005 and 15 December 2012 in the Assessment of Acute Rejection in Renal Transplantation (AART) study. Gene expression was assessed by quantitative real-time PCR (QPCR) in one center. A 17-gene set--the Kidney Solid Organ Response Test (kSORT)--was selected in 143 samples for AR classification using discriminant analysis (area under the receiver operating characteristic curve [AUC] = 0.94; 95% CI 0.91-0.98), validated in 124 independent samples (AUC = 0.95; 95% CI 0.88-1.0) and evaluated for AR prediction in 191 serial samples, where it predicted AR up to 3 mo prior to detection by the current gold standard (biopsy). A novel reference-based algorithm (using 13 12-gene models) was developed in 100 independent samples to provide a numerical AR risk score, to classify patients as high risk versus low risk for AR. kSORT was able to detect AR in blood independent of age, time post-transplantation, and sample source without additional data normalization; AUC = 0.93 (95% CI 0.86-0.99). Further validation of kSORT is planned in prospective clinical observational and interventional trials. CONCLUSIONS: The kSORT blood QPCR assay is a noninvasive tool to detect high risk of AR of renal transplants. Please see later in the article for the Editors' Summary.

Identification of gene microarray expression profiles in patients with chronic graft-versus-host disease following allogeneic hematopoietic cell transplantation.

Chronic graft-versus-host disease (GVHD) results in significant morbidity and mortality, limiting the benefit of allogeneic hematopoietic cell transplantation (HCT). Peripheral blood gene expression profiling of the donor immune repertoire following HCT may provide associated genes and pathways thereby improving the pathophysiologic understanding of chronic GVHD. We profiled 70 patients and identified candidate genes that provided mechanistic insight in the biologic pathways that underlie chronic GVHD. Our data revealed that the dominant gene signature in patients with chronic GVHD represented compensatory responses that control inflammation and included the interleukin-1 decoy receptor, IL-1 receptor type II, and genes that were profibrotic and associated with the IL-4, IL-6 and IL-10 signaling pathways. In addition, we identified three genes that were important regulators of extracellular matrix. Validation of this discovery phase study will determine if the identified genes have diagnostic, prognostic or therapeutic implications.

Wide host range and strong lytic activity of Staphylococcus aureus lytic phage Stau2.

In searching for an alternative antibacterial agent against multidrug-resistant Staphylococcus aureus, we have isolated and characterized a lytic staphylophage, Stau2. It possesses a double-stranded DNA genome estimated to be about 134.5 kb and a morphology resembling that of members of the family Myoviridae. With an estimated latency period of 25 min and a burst size of 100 PFU/infected cell, propagation of Stau2 in liquid culture gave a lysate of ca. 6 × 10(10) PFU/ml. It was stable at pH 5 to 13 in normal saline at room temperature for at least 4 weeks and at -85°C for more than 2 years, while 1 × 10(9) out of 2 × 10(12) PFU/ml retained infectivity after 36 months at 4°C. Stau2 could lyse 80% of the S. aureus isolates (164/205) obtained from hospitals in Taiwan, with complete lysis of most of the isolates tested within 3 h; however, it was an S. aureus-specific phage because no lytic infection could be found in the coagulase-negative staphylococci tested. Its host range among S. aureus isolates was wider than that of polyvalent phage K (47%), which can also lyse many other staphylococcal species. Experiments with mice demonstrated that Stau2 could provide 100% protection from lethal infection when a multiplicity of infection of 10 was administered immediately after a challenge with S. aureus S23. Considering these results, Stau2 could be considered at least as a candidate for topical phage therapy or an additive in the food industry.

Interference of globin genes with biomarker discovery for allograft rejection in peripheral blood samples.

Microarray technology is a powerful tool in the discovery of new biomarkers for disease. After solid organ transplantation, where the detection of rejection is usually made on invasive biopsies, it could be hypothesized that noninvasive transcriptional profiling of peripheral blood will reveal rejection-specific expression patterns from circulating immune cells. However, in kidney transplant rejection, the analysis of gene expression data in whole blood has proven difficult for detecting significant genes specific for acute graft rejection. Previous studies have demonstrated that the abundance of globin genes in whole blood may mask the underlying biological differences between whole blood samples. In the present study, we compared the gene expression profiles of peripheral blood of nine stable renal allograft recipients with seven matched patients having an ongoing acute renal transplant rejection, using four different protocols of preparation, amplification, and synthesis of cRNA or cDNA and hybridization on the Affymetrix platform. We demonstrated that the globin reduction method is not sufficient to unmask clinically relevant rejection-specific transcriptome profiles in whole blood. Applying an additional mathematical depletion of the globin genes improves the efficacy of globin reduction but cannot remove the confounding influence of globin gene hybridization. Sampling of peripheral blood leukocytes alone, without the confounding influence of globin mRNA, provides sensitive and specific peripheral signatures for graft rejection, with many of these signals overlapping with rejection-driven tissue (kidney)-specific signatures from matched biopsies. Similar applications may exist for array-based biomarker discovery for other diseases associated with changes in leukocyte trafficking, activation, or function.

A combined biomarker and clinical panel for chronic graft versus host disease diagnosis.

Whilst many chronic graft versus host disease (cGVHD) biomarkers have been previously reported, few have been verified in an independent cGVHD cohort. We aimed to verify the diagnostic accuracy of previously reported markers of cGVHD in a multi-centre Chronic GVHD Consortium. A total of 42 RNA and 18 protein candidate biomarkers were assessed amongst 59 cGVHD cases and 33 matched non-GVHD controls. Total RNA was isolated from PBMC, and RNA markers were quantified using PCR. Serum protein markers were quantified using ELISA. A combined 3 RNA biomarker (IRS2, PLEKHF1 and IL1R2) and 2 clinical variables (recipient CMV serostatus and conditioning regimen intensity) panel accurately (AUC 0.81) segregated cGVHD cases from controls. Other studied RNA and protein markers were not confirmed as accurate cGVHD diagnostic biomarkers. The studied markers failed to segregate higher risk cGVHD (per overall NIH 0-3 score, and overlap versus classic cGVHD status). These data support the need for multiple independent verification studies for the ultimate clinical application of cGVHD diagnostic biomarkers.

Importance of amino acid alterations and expression of penicillin-binding protein 5 to ampicillin resistance of Enterococcus faecium in Taiwan.

The importance of the amino acid sequence in the C-terminal domain of penicillin-binding protein 5 (PBP5) and the levels of PBP5 expression to ampicillin resistance of Taiwan clinical isolates of Enterococcus faecium were studied. Sequence data revealed the existence of 12 amino acid sequence variants within the C-terminal domain of PBP5 in the 33 tested isolates (ampicillin minimum inhibitory concentrations (MICs) 1 mg/L to >256 mg/L). Western blot analyses of the levels of PBP5 showed that, with few exceptions, lower amounts of PBP5 were present in the susceptible strains than in the resistant strains. More importantly, a significant correlation (P=0.004, Fisher's exact test) between the expression of PBP5 and ampicillin resistance was detected. Point mutations in PBP5, including addition of aspartic acid or serine after position 466 and change of methionine to alanine or threonine at position 485, alanine or isoleucine to threonine at position 499 and glutamate to valine at position 629, were found to be significantly associated with ampicillin resistance. A significant correlation was obtained for the combined mutation (alleles 10 and 11), suggesting that combined mutation of PBP5 can be a marker for ampicillin resistance of E. faecium.

Transplant reno-vascular stenoses associated with early erythropoietin use.

BACKGROUND AND OBJECTIVES: This report describes an unusual presentation of severe hypertension (HTN) in a subset of pediatric kidney recipients treated with a steroid avoidance pediatric renal transplantation protocol. The HTN was secondary to atypical, reno-vascular abnormalities (RVA) of the transplanted vasculature, temporally associated with erythropoietin (EPO) use. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: To investigate the clinical significance underlying this event, a retrospective clinical study of 100 pediatric renal transplants was undertaken (50 steroid-free and 50 matched steroid-based controls), with peripheral blood transcriptional analysis of four RVA patients and controls. RESULTS: Regardless of a higher observed incidence of anemia (p < 0.001) and greater overall EPO usage in the first post-transplant year in steroid-free patients, the incidence of new-onset HTN at one yr was significantly less in the steroid-free cohort (p = 0.03). Nevertheless, early EPO (first week post-transplant) was significantly associated with the combinatory findings of new-onset HTN (p = 0.03) and RVA (p = 0.007). Molecular mechanisms of RVA injury were investigated further by peripheral blood cDNA microarray gene expression profiling. A panel of 42 transcripts differentiated patients with RVA and HTN from three sets of matched controls, with and without HTN and EPO use, with 100% concordance (p < 0.001). The biological processes governed by these significant genes suggest a role for EPO regulation of growth factor receptor ubiquitination as a putative mechanism for renal vascular injury. CONCLUSION: This study cautions against the use early post-transplant use of EPO in immunosuppression regimens with steroid minimization/avoidance, which may have an increased incidence of post-transplant anemia.