Direct molecular detection of pathogens in blood as specific rule-in diagnostic biomarker in patients with presumed sepsis: our experience on a heterogeneous cohort of patients with signs of infective systemic inflammatory response syndrome.

The practical value of blood cultures in the diagnosis of sepsis is impaired by a delay in the turnaround time to result and by the fact that blood culture positive can be found for only about 30% of these patients. Conventional laboratory signs of sepsis and acute phase protein biomarkers are sensitive and easy to use, but often also very nonspecific. Molecular diagnostic reflects currently the most promising avenue to decrease time to result and to influence decision making for antibiotic therapy in the septic host. In this study, we wish to highlight the impact of the LightCycler SeptiFast, a multipathogen probe-based real-time polymerase chain reaction, in the rapid etiological diagnosis of sepsis in patients with clinical and laboratory signs of bloodstream infections. We have evaluated prospectively 830 adult patients with suspected bloodstream infection and at least two criteria of systemic inflammatory response syndrome. In more than 50% of critically ill patients strongly suspected of having sepsis, we arrived to an etiological diagnosis only by the molecular method in a median time of 15 h, with specificity and predictive positive values of 96% and 94%, respectively. We highlight the role of DNAemia as time-critical, high-specificity, etiological, non-culture-based rule-in diagnostic biomarker in patients with presumed sepsis.

Molecular identification of bloodstream pathogens in patients presenting to the emergency department with suspected sepsis.

The rapid detection of pathogens in blood is critical for a favorable outcome of patients with suspected sepsis. Although blood culture (BC) is considered the criterion standard for diagnosis of bloodstream infection, it often takes several days to detect the causative organism. In this study, we compared BC with a commercially available multiplex real-time polymerase chain reaction (PCR) assay to detect bacteria and fungi in blood samples from 144 patients admitted to the emergency department with suspected sepsis. Of 144 blood samples examined, 91 (63%) were negative by both methods and 53 (37%) were positive by at least one of the two methods. In 30 among all positive cases (56.6%),both methods identified the same organisms, in 13 cases (24.5%), BC identified organisms not detected by real-time PCR,and in 10 cases (18.9%), SeptiFast PCR assay gave positive results, whereas the BC was negative. In this study, we wished to compare SeptiFast results obtained by standard procedures, but future clinical studies are necessary to define SeptiFast PCR as support for BC in the early diagnosis of severe bloodstream infections.

[Evaluation of Vitek 2 performance for identifying extended spectrum beta-lactamases in Enterobacteriaceae "other than Escherichia coli, Proteus mirabilis and Klebsiella spp"]. / Valutazione della performance di Vitek 2 per la rilevazione delle beta-lattamasi a spettro esteso in Enterobacteriaceae "other than Escherichia coli, Proteus mirabilis e Klebsiella spp".

Production of beta-lactamases is the main resistance mechanism of gram-negative bacteria against beta-lactam antibiotics. Extended spectrum beta-lactamases (ESBLs) have the ability to hydrolyze a broader spectrum of beta-lactam drugs. Hence rapid, accurate detection of this resistance mechanism is extremely important to guide proper patient antimicrobial therapy. These enzymes are most commonly produced by Klebsiella spp. and Escherichia coli, but may also occur widely in other gram-negative bacteria, including Enterobacter spp., Proteus spp., Morganella morganii, Providencia stuartii, Serratia marcescens and others that also produce other chromosomal and plasmid-mediated enzymes, like AmpC beta-lactamases. The main problem is that no CLSI (Clinical and Laboratory Standards Institute) recommendations exist for ESBL detection for Enterobacteriaceae other than E. coli, Proteus mirabilis and Klebsiella spp and for detecting plasmid-mediated AmpC beta-lactamases. We carried out an evaluation of Vitek 2 Advanced Expert System (AES) performance for identifying ESBL in Enterobacteriaceae, also other than E. coli, Proteus mirabilis and Klebsiella spp, comparing results obtained with Etest and double disk data. Seventy isolates of Enterobacteriaceae were tested for the production of extended-spectrum beta-lactamases (ESBLs) by using Vitek 2, Etest and the double disk method. The use of Etest was performed as a gold standard method by comparing interpretation results of Vitek 2 Advanced Expert System (AES). In comparison with the Etest method, AES produced 19 ESBL warnings, of which only 5 were classified as major misunderstandings, especially for Enterobacteriaceae other than E. coli, Proteus mirabilis and Klebsiella spp which produced plasmid-mediated AmpC beta-lactamases. The Etest, together with the cefoxitin sensibility test, was found to be the best method to confirm ESBLs and distinguish AmpC from ESBLs.