Early diagnosis of sepsis, rapid identification of the causative pathogen(s) and prompt initiation of appropriate antibiotic treatment have a combined impact on mortality due to sepsis. In this observational study, a new DNA-based system (LightCycler SeptiFast (LC-SF) test; Roche Diagnostics) allowing detection of 16 pathogens at the species level and four groups of pathogens at the genus level has been evaluated and compared with conventional blood cultures (BCs). One hundred BC and LC-SF results were obtained for 72 patients admitted to the intensive-care unit over a 6-month period for suspected sepsis. Microbiological data were compared with other biological parameters and with clinical data. The positivity rate of BCs for bacteraemia/fungaemia was 10%, whereas the LC-SF test allowed detection of DNA in 15% of cases. The LC-SF performance, based on its clinical relevance, was as follows: sensitivity, 78%; specificity, 99%; positive predictive value, 93%; and negative predictive value, 95%. Management was changed for four of eight (50%) of the patients because organisms were detected by the LC-SF test but not by BC. LC-SF results were obtained in 7-15 h, in contrast to the 24-72 h required for BC. According to the LC-SF results, initial therapy was inadequate in eight patients, and antibiotic treatment was changed. Our results suggest that the LC-SF test may be a valuable complementary tool in the management of patients with clinically suspected sepsis.

HRV were the most frequently detected viruses; their detection was significantly associated with the occurrence of an exacerbation. The reality of viral recombination between HRV was demonstrated in CF patients for the first time, raising the role of viruses in lung microbiota. Further studies are now warranted to decipher virus impact in CF.

A 5 nuclease PCR assay for detection of the Yersinia pestis plasminogen activator (pla) gene in human respiratory specimens with simulated Y. pestis infection was developed. An internal positive control was added to the reaction mixture in order to detect the presence of PCR inhibitors that are often found in biological samples. The assay was 100% specific for Y. pestis. In the absence of inhibitors, a sensitivity of 10 2 CFU/ml of respiratory fluid was obtained. When inhibitors were present, detection of Y. pestis DNA required a longer sample treatment time and an initial concentration of bacteria of at least 10 4 CFU/ml. The test's total turnaround time was less than 5 h. The assay described here is well suited to the rapid diagnosis of pneumonic plague, the form of plague most likely to result from a bioterrorist attack.Protecting the population against an act of bioterrorism is a major concern for many governments. Yersinia pestis, the agent of plague, can be viewed as a potential bioweapon due to its ability to cause high rates of morbidity and mortality in humans. The intentional dissemination of plague by terrorists would most likely occur by aerosolization, causing fulminant pneumonia in exposed individuals. Since pneumonic plague is almost always fatal when untreated, clinical microbiology laboratories play a pivotal role in the early detection of this type of infectious agent, thus allowing rapid implementation of effective preventive measures. Availability of a rapid diagnostic procedure using molecular techniques (such as real-time PCR) is essential in the establishment of coordinated laboratory response systems. Several PCR assays have been developed (1, 3-7, 10, 13): the plasmidic plasminogen activator (pla) gene (located on the Y. pestis-specific pPst/pCP1 plasmid [5,11]) was found to be the most sensitive target, since its copy number can be as high as 186 per bacterium (8). In the present study, we describe a real-time PCR protocol for detection of the Y. pestis pla gene. The assay is based on 5Ј exonuclease assay technology coupled with automated DNA extraction and uses a minor groove binder-conjugated small DNA probe for DNA detection via hybridization-triggered fluorescence: subsequent multiplex development can be thus envisaged. Inclusion of an internal positive control (IPC) in each batch assay enabled the detection of endogenous PCR inhibitors.The primers and the fluorogenic probe for the pla gene (GenBank accession no. M27820) were designed with Primer Express software, version 2.0 (Applied Biosystems, Foster City, Calif.) and were obtained from Applied Biosystems (Warrington, United Kingdom). The nucleotide sequences of the forward and reverse primers were 5Ј-GAAAGGAGTGCGG GTAATAGGTT-3Ј (positions 816 to 838) and 5Ј-AACCAGC GCTTTTCTA-3Ј (positions 869 to 884), respectively. The sequence of the minor groove binder probe was 6-carboxyfluores cein-5Ј-GACTTGCAGGCC-3Ј (positions 840 to 851). PCR amplifications were performed in 25-l reaction volumes including 1ϫ TaqMan Universal Master Mix (A...

ᰔMatrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) allows instant identification of microorganisms by analyzing their total protein content (1-3). In September 2009, we switched from conventional biochemical techniques (mainly Vitek 2 and API strips) to MS (Microflex; Bruker Daltonics, Wissembourg, France) for routine identification of all bacterial isolates except for mycobacteria and mycoplasma. Conventional tests were only kept in case of equipment failure or maintenance (10 days per year) and for diagnosis of Streptococcus pneumoniae, beta-hemolytic streptococci, and Shigella spp., thus entailing a limited stock of identification reagents. After 18 months, we evaluated the benefits in turnaround time and cost in our 3,046-bed acute care university hospital.Our flow chart was as follows: for each isolate, a portion of 1 to 5 colonies were submitted to MS without protein extraction; if the identification score was not acceptable (Ͻ2.0), another round of MS was performed, allowing identification in 97.6% of cases. The accuracy of identification was similar to that reported in the literature (1-4). In our 18-month experience, an average of 3.4 MS tests, each costing $0.12, were required in order to identify one isolate, i.e., with an identification score of Ն2.0, resulting in $0.41 per bacterial identification at the time of study. When MS identification failed, a relevant housekeeping gene sequence (e.g., rrs, recA, sodA, or rpoB) was analyzed (83 isolates a year, compared to 138 in the pre-MS period; P Ͻ 0.05).Changes in time to results and workflow were evaluated first. Although the setup times were identical for MS and biochemical techniques, the time to identification was significantly lower with MS, as 93% and 10% of isolates, respectively, were identified within 24 h after inoculation. Early identification allowed us to select appropriate antibiotics to be tested against clinically significant isolates. Subcultures performed in order to achieve a proper inoculum for conventional techniques were drastically cut after MS was implemented, resulting in technical time and culture medium savings. However, time to susceptibility testing results was not reduced, and implementing MS did not allow us to significantly cut down technical staff. Assessable costs were compared over identical 1-year periods, before and after MS implementation. The overall savings were at least $177,090. MS identifications of 38,624 isolates between October 2009 and September 2010 cost $15,836, and conventional identification of 960 remaining isolates over the same period cost $5,374 (total, $21,210). The year before, a total of $193,754 had been spent for 33,320 isolates identified by Vitek 2 cards and API strips (mean unitary cost, $5.81). In addition, waste disposal decreased from 1,424 kg (cards, pipettes, and suspension media) to 44 kg (MS target cleansing solution and pipette tips), saving $1,794. Other assessable savings resulted from a $1,102 cut in subculture medium expenses and a $1,650...

PurposeMicrobiological diagnosis (MD) of infections remains insufficient. The resulting empirical antimicrobial therapy leads to multidrug resistance and inappropriate treatments. We therefore evaluated the cost-effectiveness of direct molecular detection of pathogens in blood for patients with severe sepsis (SES), febrile neutropenia (FN) and suspected infective endocarditis (SIE).MethodsPatients were enrolled in a multicentre, open-label, cluster-randomised crossover trial conducted during two consecutive periods, randomly assigned as control period (CP; standard diagnostic workup) or intervention period (IP; additional testing with LightCycler®SeptiFast). Multilevel models used to account for clustering were stratified by clinical setting (SES, FN, SIE).ResultsA total of 1416 patients (907 SES, 440 FN, 69 SIE) were evaluated for the primary endpoint (rate of blood MD). For SES patients, the MD rate was higher during IP than during CP [42.6% (198/465) vs. 28.1% (125/442), odds ratio (OR) 1.89, 95% confidence interval (CI) 1.43–2.50; P < 0.001], with an absolute increase of 14.5% (95% CI 8.4–20.7). A trend towards an association was observed for SIE [35.4% (17/48) vs. 9.5% (2/21); OR 6.22 (0.98–39.6)], but not for FN [32.1% (70/218) vs. 30.2% (67/222), P = 0.66]. Overall, turn-around time was shorter during IP than during CP (22.9 vs. 49.5 h, P < 0.001) and hospital costs were similar (median, mean ± SD: IP €14,826, €18,118 ± 17,775; CP €17,828, €18,653 ± 15,966). Bootstrap analysis of the incremental cost-effectiveness ratio showed weak dominance of intervention in SES patients.ConclusionAddition of molecular detection to standard care improves MD and thus efficiency of healthcare resource usage in patients with SES. ClinicalTrials.gov registration number: NCT00709358.Electronic supplementary materialThe online version of this article (doi:10.1007/s00134-017-4766-4) contains supplementary material, which is available to authorized users.