The results of the present study suggest that ASA score ≤ 2 and use of rifampin-combination therapy are two independent factors associated with favorable outcome of patients treated for total hip or knee prosthetic infections due to S. aureus.

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...

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BackgroundOutcome of patients with streptococcal prosthetic joint infections (PJIs) is not well known.MethodsWe performed a retrospective multicenter cohort study that involved patients with total hip/knee prosthetic joint (THP/TKP) infections due to Streptococcus spp. from 2001 through 2009.ResultsNinety-five streptococcal PJI episodes (50 THP and 45 TKP) in 87 patients of mean age 69.1 ± 13.7 years met the inclusion criteria. In all, 55 out of 95 cases (57.9 %) were treated with debridement and retention of the infected implants with antibiotic therapy (DAIR). Rifampicin-combinations, including with levofloxacin, were used in 52 (54.7 %) and 28 (29.5 %) cases, respectively. After a mean follow-up period of 895 days (IQR: 395–1649), the remission rate was 70.5 % (67/95). Patients with PJIs due to S. agalactiae failed in the same proportion as in the other patients (10/37 (27.1 %) versus 19/58 (32.7 %); p = .55). In the univariate analysis, antibiotic monotherapy, DAIR, antibiotic treatments other than rifampicin-combinations, and TKP were all associated with a worse outcome. The only independent variable significantly associated with the patients’ outcomes was the location of the prosthesis (i.e., hip versus knee) (OR = 0.19; 95 % CI 0.04–0.93; p value 0.04).ConclusionsThe prognosis of streptococcal PJIs may not be as good as previously reported, especially for patients with an infected total knee arthroplasty. Rifampicin combinations, especially with levofloxacin, appear to be suitable antibiotic regimens for these patients.

Detection of microorganisms by blood cultures (BCs) is essential in managing patients with bacteraemia. Rather than the number of punctures, the volume of blood drawn is considered paramount in efficient and reliable detection of microorganisms. We performed a 1-year prospective multicentre study in adult emergency departments of three French university hospitals comparing two methods for BCs: a unique blood culture (UBC) collecting a large volume of blood (40 mL) and the standard method of multiple blood cultures (MBC). The performances of both methods for bacterial contamination and efficient microbial detection were compared, each patient serving as his own control. Amongst the 2314 patients included, three hundred were positive for pathogens (n=245) or contaminants (n=55). Out of the 245 patients, 11 were positive for pathogens by UBC but negative by MBC and seven negative by UBC but positive by MBC (p 0.480). In the subgroup of 137 patients with only two BCs, UBC was superior to MBC (p 0.044). Seven and 17 patients had contaminated BCs by UBC and MBC only, respectively (p 0.062). Considering the sums of pathogens missed and contaminants, UBC significantly outperformed MBC (p 0.045). Considering the complete picture of cost savings, efficient detection of microorganisms and decrease in contaminations, UBC offers an interesting alternative to MBC.

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...

Level IV. Retrospective cohort study.