SUMMARY Sepsis, a leading cause of morbidity and mortality throughout the world, is a clinical syndrome with signs and symptoms relating to an infectious event and the consequent important inflammatory response. From a clinical point of view, sepsis is a continuous process ranging from systemic inflammatory response syndrome (SIRS) to multiple-organ-dysfunction syndrome (MODS). Blood cultures are the current “gold standard” for diagnosis, and they are based on the detection of viable microorganisms present in blood. However, on some occasions, blood cultures have intrinsic limitations in terms of sensitivity and rapidity, and it is not expected that these drawbacks will be overcome by significant improvements in the near future. For these principal reasons, other approaches are therefore needed in association with blood culture to improve the overall diagnostic yield for septic patients. These considerations have represented the rationale for the development of highly sensitive and fast laboratory methods. This review addresses non-culture-based techniques for the diagnosis of sepsis, including molecular and other non-culture-based methods. In particular, the potential clinical role for the sensitive and rapid detection of bacterial and fungal DNA in the development of new diagnostic algorithms is discussed.
Successful transplantation requires the prevention of allograft rejection and, in the case of transplantation to treat autoimmune disease, the suppression of autoimmune responses. The standard immunosuppressive treatment regimen given to patients with autoimmune type 1 diabetes who have received an islet transplant results in the loss of T cells. In many other situations, the immune system responds to T cell loss through cytokine-dependant homeostatic proliferation of any remaining T cells. Here we show that T cell loss after islet transplantation in patients with autoimmune type 1 diabetes was associated with both increased serum concentrations of IL-7 and IL-15 and in vivo proliferation of memory CD45RO + T cells, highly enriched in autoreactive glutamic acid decarboxylase 65-specific T cell clones. Immunosuppression with FK506 and rapamycin after transplantation resulted in a chronic homeostatic expansion of T cells, which acquired effector function after immunosuppression was removed. In contrast, the cytostatic drug mycophenolate mofetil efficiently blocked homeostatic T cell expansion. We propose that the increased production of cytokines that induce homeostatic expansion could contribute to recurrent autoimmunity in transplanted patients with autoimmune disease and that therapy that prevents the expansion of autoreactive T cells will improve the outcome of islet transplantation.
The rapid diagnosis of an infectious cause in the course of fever of unknown origin plays a pivotal role in the correct management of neutropenic patients. In this study, blood samples from febrile oncohaematological patients were tested using a novel commercial real-time PCR assay (LightCycler SeptiFast; Roche Molecular Systems) and blood culture (BacT/Alert 3D; bioMé rieux). Twenty-one (20.4 %) and 34 (33 %) of the 103 samples under study tested positive by blood culture and PCR, respectively. The analysis of concordance evidenced a low correlation between the two approaches (83 %), mainly due to samples that tested negative by culture but positive using the molecular approach. Among 14 discordant cases negative by culture but positive by PCR, 12 were observed in sequential samples of patients with initial concordant results on samples drawn before the administration of a specific antimicrobial therapy. Moreover, DNA of a fastidious organism, Aspergillus fumigatus, not easily detectable by the cultural approach was rapidly detected in the two remaining discordant cases. Overall, the characteristics featured by the molecular method could be of interest in the development of new algorithms for the diagnosis of sepsis in critical patients.
The immunoregulatory function of NKT cells is crucial for prevention of autoimmunity. The prototypical NKT cell Ag alpha-galactosylceramide is not present in mammalian cells, and little is known about the mechanism responsible for NKT cell recruitment and activation. Up-regulation of CD1d, the NKT cell restriction molecule, expressed on mononuclear cells infiltrating the target organ, could represent the physiological trigger for NKT cells to self-contain T cell immunity and to prevent autoimmune disease. Recognition of CD1d, either by itself or bound to self-ligands (selfCD1d), could drive NKT cells toward an immunoregulatory phenotype. Hence, ineffective NKT cell-mediated immunoregulation in autoimmune-prone individuals including nonobese diabetic (NOD) mice could be related to defective signals that regulate CD1d expression at time and site of autoimmunity. To test this hypothesis, we transgenically overexpressed CD1d molecules under the control of the insulin promoter within the pancreatic islets of NOD mice (insCD1d). Recognition of overexpressed CD1d molecules rescued NKT cell immunoregulatory function and prevented autoimmune diabetes in insCD1d transgenic NOD mice. Protection from diabetes was associated with a biased IL-4-secreting cytokine phenotype of NKT cells and alteration of the cytokine microenvironment in the pancreatic lymph nodes of transgenic mice. The net effect was a reduced development of the autoimmune T cell repertoire. Our findings suggest that up-regulation of CD1d expression during inflammation is critical to maintain T cell homeostasis and to prevent autoimmunity.
We evaluated the Verigene Gram-negative blood culture (BC-GN) test, a microarray that detects Gram-negative bacteria and several resistance genes. A total of 102 positive blood cultures were tested, and the BC-GN test correctly identified 97.9% of the isolates within its panel. In cases of sepsis, timely microbiological diagnosis, including data on antimicrobial susceptibility, is crucial for prompt initiation of targeted drug therapy (1). This is not possible with currently used methods, thus causing a significant delay in specific treatment and the empirical use of broad-spectrum antimicrobials (2-4). Nucleic acid-based assays are considered to be a potential adjuvant tool for improving the microbiological diagnosis of sepsis (5-7). These assays may be classified into one of two groups (5-7): (i) those using positive blood cultures, which are potentially useful but burdened by the usual culture-associated drawbacks (i.e., interfering effect of ongoing antibiotics, long time to positivity, and the presence of fastidious pathogens), and (ii) those using blood samples, which are promising but still not developed for the sensitive detection of resistance markers (5-7).In this pilot study, we evaluated the Verigene Gram-negative blood culture (BC-GN) test (Nanosphere, Northbrook, IL, USA), a microarray-based, almost fully automated, and random-access system allowing for bacterial identification (Table 1) and detection of several resistance genes (Table 2) from positive blood cultures. The turnaround time is 2 h, with a hands-on time of Ͻ10 min. The BC-GN test has been approved for clinical use in Europe and is currently under submission for use in the United States.Several papers have already evaluated the Verigene panel dedicated to Gram-positive bacteria (8-12), but this is the first one on the BC-GN test. To investigate its potential clinical usefulness, we evaluated the following parameters: (i) the concordance of identification and of antibiotic susceptibility data with those obtained with the traditional blood culture flowchart, (ii) the time to definitive results, and (iii) the impact of the BC-GN test results on ongoing empirical therapy, evidencing the rate of potential BC-GN-induced antibiotic changes. In this analysis, the following phases of the standard management of blood cultures were considered: time from blood sampling to the loading of bottles into the bioMérieux BacT/Alert system, time to positivity, and time from positivity to Gram stain and subculturing on solid medium (positive bottles are downloaded every 2 h, from 8:00 a.m. to 6:00 p.m. Monday to Friday, 8:00 a.m. to 2:00 p.m. on Saturday, and 9:00 a.m. to 1:00 p.m. on Sunday).Our study prospectively included all blood cultures positive for Gram-negative pathogens submitted to our center from June to September 2013, but only one positive bottle was considered per patient. Antibiotic susceptibility was phenotypically evaluated by disk diffusion from positive blood culture broth (preliminary antibiotic susceptibility testing [pAST]) and by au...
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