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.

One of the regions responsible for the stable inheritance of the broad-host-range plasmid RK2 is contained within the PstI C fragment, located from coordinates 30.8 to 37.0 kb (P. N. Saurugger, 0. Hrabak, H. Schwab, and R. M. Lafferty, J. Biotechnol. 4:333-343, 1986). Genetic analysis of this 6.2-kb region demonstrated that no function was present that stabilized by selectively killing plasmid-free segregants. The sequence from 36.0 to 37.0 kb mediated a twofold increase in plasmid copy number, but this region was not required for stabilization activity. The PstI C fragment was shown to encode a multimer resolution system from 33.1 to 35.3 kb. The resolution cis-acting site was mapped to 140 bp, sequenced, and observed to contain two directly repeated sequences of 6 and 7 bases and two perfect inverted repeats of 6 and 8 bases. The trans-acting factor(s) was mapped and functionally determined to encode a resolvase capable of catalyzing recombination at high frequency between cis-acting sites in either direct or inverted orientation. Multimer resolution alone did not account for complete plasmid stabilization by the PstI C fragment, since removal of regions adjacent to the 35.3-kb border of the minimal mrs locus dramatically reduced stabilization. The minimal region required for complete stabilization, from 32.8 to 35.9 kb, was capable of fully stabilizing plasmids independently of the replicon or the recA proficiency of the host. Stabilization activity was also fully expressed in several diverse gram-negative bacteria, whereas the F plasmid par locus functioned only in Escherichia coli. On the basis of these observations, we conclude that under the growth conditions used, the minimal stabilization locus encodes both an mrs activity and a stabilization activity that has the properties of a par locus.

Identification of anti-hepatitis C virus (anti-HCV) human antibody clones with broad neutralizing activity is important for a better understanding of the interplay between the virus and host and for the design of an effective passive immunotherapy and an effective vaccine. We report the identification of a human monoclonal Fab (e137) able to bind the HCV E2 glycoprotein of all HCV genotypes but genotype 5. The results of antibody competition assays and testing the reactivity to alanine mutant E2 proteins confirmed that the e137 epitope includes residues (T416, W420, W529, G530, and D535) highly conserved across all HCV genotypes. Fab e137 neutralized HCV pseudoparticles bearing genotype 1a, 1b, and 4 E1-E2 proteins and to a lesser extent, genotype 2b. Fab e137 was also able to inhibit cell culture-grown HCV (genotype 2a). These data indicate that broadly cross-reacting and cross-neutralizing antibodies are generated during HCV infection.

Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.

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.