Multiplexed Signal Ion Emission Reactive Release Amplification (SIERRA) Assay for the Culture-Free Detection of Gram-Negative and Gram-Positive Bacteria and Antimicrobial Resistance Genes.

The global prevalence of antibiotic-resistant bacteria has increased the risk of dangerous infections, requiring rapid diagnosis and treatment. The standard method for diagnosis of bacterial infections remains dependent on slow culture-based methods, carried out in central laboratories, not easily extensible to rapid identification of organisms, and thus not optimal for timely treatments at the point-of-care (POC). Here, we demonstrate rapid detection of bacteria by combining electrochemical immunoassays (EC-IA) for pathogen identification with confirmatory quantitative mass spectral immunoassays (MS-IA) based on signal ion emission reactive release amplification (SIERRA) nanoparticles with unique mass labels. This diagnostic method uses compatible reagents for all involved assays and standard fluidics for automatic sample preparation at POC. EC-IA, based on alkaline phosphatase-conjugated pathogen-specific antibodies, quantified down to 104 bacteria per sample when testing Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa lysates. EC-IA quantitation was also obtained for wound samples. The MS-IA using nanoparticles against S. aureus, E. coli, Klebsiella pneumoniae, and P. aeruginosa allowed selective quantitation of ∼105 bacteria per sample. This method preserves bacterial cells allowing extraction and amplification of 16S ribosomal RNA genes and antibiotic resistance genes, as was demonstrated through identification and quantitation of two strains of E. coli, resistant and nonresistant due to ß-lactamase cefotaximase genes. Finally, the combined immunoassays were compared against culture using remnant deidentified patient urine samples. The sensitivities for these immunoassays were 83, 95, and 92% for the prediction of S. aureus, P. aeruginosa, and E. coli or K. pneumoniae positive culture, respectively, while specificities were 85, 92, and 97%. The diagnostic platform presented here with fluidics and combined immunoassays allows for pathogen isolation within 5 min and identification in as little as 15 min to 1 h, to help guide the decision for additional testing, optimally only on positive samples, such as multiplexed or resistance gene assays (6 h).

Multicenter Evaluation of the BD Phoenix CPO Detect Test for Detection and Classification of Carbapenemase-Producing Organisms in Clinical Isolates.

Limited treatment options contribute to high morbidity/mortality rates with carbapenem-resistant, Gram-negative bacterial infections. New approaches for carbapenemase-producing organism (CPO) detection may help inform clinician decision-making on patient treatment and infection control. BD Phoenix CPO detect (CPO detect) detects and classifies carbapenemases in Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa during susceptibility testing. The clinical performance of CPO detect is reported here. Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa isolates were evaluated across three sites using CPO detect and a composite reference method (RM); the latter was comprised of the modified carbapenem inactivation method and a MIC screen for ertapenem, imipenem, and meropenem. Multiplex PCR testing was also utilized for Ambler class determination. Positive and negative percentages of agreement (PPA and NPA, respectively) between CPO detect and the RM were determined. The PPA and NPA for Enterobacterales were 98.5% (confidence intervals, 96.6%, 99.4%) and 97.2% (95.8%, 98.2%), respectively. The A. baumannii PPA and NPA, respectively, were 97.1% (90.2%, 99.2%) and 97.1% (89.9%, 99.2%). The P. aeruginosa PPA and NPA, respectively, were 95.9% (88.6%, 98.6%) and 92.3% (86.7%, 95.6%). The PPA values for carbapenemase class designations for all organisms combined and Enterobacterales alone, respectively, were 95.3% (90.2%, 97.8%) and 94.6% (88.8%, 97.5%) for class A, 94.0% (88.7%, 96.6%) and 96.4% (90.0%, 98.8%) for class B, and 95.0% (90.1%, 97.6%) and 99.0% (94.4%, 99.8%) for class D carbapenemases. NPA values for all organisms and Enterobacterales alone ranged from 98.5% to 100%. CPO detect provided accurate detection and classification of CPOs for the majority of isolates of Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa tested.

A dual-center evaluation of platelet culture vials to detect the presence of microorganisms in platelets.

BACKGROUND: Microorganism contamination of platelets results in a high risk of transfusion-related sepsis. Here, the ability of culture vials (BD BACTEC Platelet Aerobic/F and Platelet Anaerobic/F vials, Becton, Dickinson and Company) to detect microorganisms in leukoreduced apheresis platelets (LRAPs) and leukoreduced whole blood platelet concentrates (LRWBPCs) was assessed. METHODS: LRAPs or LRWBPCs were inoculated into Aerobic/F and Anaerobic/F vials and placed in a blood culturing system (BD BACTEC FX System, Becton, Dickinson and Company) for growth/monitoring over 7 days to detect preexisting contamination during false-positive testing. Subsequently, platelets were seeded with microorganisms at approximately 10 CFU/mL or approximately 1 CFU/mL to simulate contamination. Aerobic/F and Anaerobic/F vials were inoculated with platelets (sets of 12). Microorganism growth was detected in the BACTEC FX instrument over 7 days. Overall, 2925 vials were tested. RESULTS: Of the 1905 vials included in the microorganism detection phase, 63 (3.3%) Aerobic/F and 16 (0.8%) Anaerobic/F vials were both BACTEC FX and subculture negative. From the remaining 1827 vials, two (0.1%) Anaerobic/F vials were false positive; no false positives were observed in Aerobic/F vials, and no false negatives occurred in either vial type. Of the remaining 1825 vials (99.9%), 955 Aerobic/F and 870 Anaerobic/F vials were true positives. The mean-time-to-detection range was 8.5 to 77 hours. All true-positive Aerobic/F and Anaerobic/F vials showed 100% agreement with subculture for positive identification of seeded microorganisms. CONCLUSION: Aerobic/F and Anaerobic/F vials facilitate contamination detection in LRAPs and LRWBPCs down to approximately 1 CFU/mL. These results support the use of Aerobic/F and Anaerobic/F vials for quality control testing of platelets before transfusion.

Mechanism of Microbicidal Action of E-101 Solution, a Myeloperoxidase-Mediated Antimicrobial, and Its Oxidative Products.

E-101 solution is a first-in-class myeloperoxidase-mediated antimicrobial developed for topical application. It is composed of porcine myeloperoxidase (pMPO), glucose oxidase (GO), glucose, sodium chloride, and specific amino acids in an aqueous solution. Once activated, the reactive species hydrogen peroxide (H2O2), hypochlorous acid, and singlet oxygen are generated. We evaluated the treatment effects of E-101 solution and its oxidative products on ultrastructure changes and microbicidal activity against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli Time-kill and transmission electron microscopy studies were also performed using formulations with pMPO or GO omitted. The glutathione membrane protection assay was used to study the neutralization of reactive oxygen species. The potency of E-101 solution was also measured in the presence of serum and whole blood by MIC and minimal bactericidal concentration (MBC) determinations. E-101 solution demonstrated rapid bactericidal activity and ultracellular changes in MRSA and E. coli cells. When pMPO was omitted, high levels of H2O2 generated from GO and glucose demonstrated slow microbicidal activity with minimal cellular damage. When GO was omitted from the formulation, no antimicrobial activity or cellular damage was observed. Protection from exposure to E-101 solution reactive oxygen species in the glutathione protection assay was competitive and temporary. E-101 solution maintained its antimicrobial activity in the presence of inhibitory substances, such as serum and whole blood. E-101 solution is a potent myeloperoxidase enzyme system with multiple oxidative mechanisms of action. Our findings suggest that the primary site where E-101 solution exerts microbicidal action is the cell membrane, by inactivation of essential cell membrane components.

Antimicrobial Susceptibility of Streptococcus pneumoniae from North America, Europe, Latin America, and the Asia-Pacific Region: Results From 20 Years of the SENTRY Antimicrobial Surveillance Program (1997-2016).

BACKGROUND: The SENTRY Antimicrobial Surveillance Program monitors the frequency of occurrence and antimicrobial susceptibility of organisms from various infection types worldwide. In this investigation, we evaluated the antimicrobial susceptibility of Streptococcus pneumoniae isolates collected worldwide over 20 years (1997-2016). METHODS: A total of 65 993 isolates were consecutively collected (1 per infection episode) from North America (NA; n = 34 626; 2 nations), Europe (EUR; n = 19 123; 23 nations), the Asia-Pacific region (APAC; n = 7111; 10 nations), and Latin America (LATAM; n = 5133; 7 nations) and tested for susceptibility using reference broth microdilution methods. Resistant subgroups included multidrug-resistant (MDR; nonsusceptible to ≥3 classes of agents) and extensively drug-resistant (XDR; nonsusceptible to ≥5 classes). RESULTS: The isolates were collected primarily from respiratory tract infections (77.3%), and 25.4% were from pediatric patients. Penicillin susceptibility (≤0.06 mg/L) rates varied from 70.7% in EUR to 52.4% in APAC for all years combined. In NA, there was a slight improvement in susceptibility for the first few years of the program, from 66.5% in 1997-1998 to 69.4% in 1999-2000, followed by a decline until 2011-2012 (57.0%). Similar declines in penicillin susceptibility rates were observed in all regions, with the lowest rates of 67.3% in EUR (2011-2012), 41.6% in the APAC region (2007-2008), and 48.2% in LATAM (2013-2014). These declines were followed by improved susceptibility rates in all regions in later program years, with susceptibility rates of 55.6% to 71.8% in 2015-2016 (65.8% overall). Susceptibility rates to ceftriaxone, erythromycin, clindamycin, tetracycline, and trimethoprim-sulfamethoxazole followed a similar pattern, with a decrease in the first 12-14 years and a continued increase in the last 6-8 years of the program. MDR and XDR frequencies were highest in APAC (49.8% and 17.3% overall, respectively) and lowest in LATAM (10.8% and 1.9% overall, respectively). The most active agents for MDR/XDR isolates were ceftaroline (99.7%/99.1% susceptible), tigecycline (96.8%/95.9% susceptible), linezolid (100.0%/100.0% susceptible), and vancomycin (100.0%/100.0% susceptible). CONCLUSIONS: S. pneumoniae susceptibility to many antibiotics increased in all regions in the last few years, and these increases may be related to PCV13 immunization, which was introduced in 2010.

Validation of the Bio-Response Solutions Human-28 Low-Temperature Alkaline Hydrolysis System.

Introduction: High temperature alkaline hydrolysis (AH) is recognized as an alternative method for sterilization and disposition of animal carcasses and human remains. The aim of this study is to validate the low temperature (LT) AH process specific to its use in the Bio-Response Solutions, Inc. Human-28 LT System. Methods: A 313-lb pig was processed using the manufacturers recommended cycle parameters. Stainless steel sample vials containing spore suspensions of Geobacillus stearothermophilus were implanted into the pig's deep tissue to validate the efficacy of the process conditions. Spore suspensions of Bacillus thuringiensis were suspended in the vessel headspace to validate sterilization. The spore challenge was greater than the recommended 106 log used to determine sterilization. MALDI-TOF mass spectrometry analysis was used to validate the destruction of prion-sized particles in processed effluent. Results: Complete inactivation of spores and digestion of animal tissue were achieved after processing in the Bio-Response Solutions Human-28 LT Alkaline Hydrolysis System. Complete inactivation of spores was achieved when exposed to heat in the animal carcass and headspace. No peptide fragments larger than 2500 Da were observed in the treatment effluent. Discussion: The Bio-Response Solutions, Inc. Human-28 LT Alkaline Hydrolysis System was as effective as high-temperature alkaline hydrolysis for use on animal and human tissue. Conclusion: LT AH for tissue and bodies exceeded the sterility assurance level III of the US State and Territorial Association on Alternative Treatment Technologies and sterility requirements for animal biosafety level-3 and -4 facilities. LT AH process validated destruction of prion-sized particles.

Multicenter Evaluation of the Etest Gradient Diffusion Method for Ceftolozane-Tazobactam Susceptibility Testing of Enterobacteriaceae and Pseudomonas aeruginosa.

Ceftolozane-tazobactam (C/T) is a novel beta-lactam-beta-lactamase inhibitor combination antibiotic approved by the U.S. Food and Drug Administration in 2014 for the treatment of complicated intra-abdominal infections (in combination with metronidazole) and complicated urinary tract infections. In this study, we evaluated the performance of the C/T Etest, a gradient diffusion method. C/T Etest was compared to broth microdilution (BMD) for 51 Enterobacteriaceae challenge isolates and 39 Pseudomonas aeruginosa challenge isolates at three clinical sites. Essential agreement (EA) between the methods ranged from 47 to 49/51 (92.2 to 96.1%) for the Enterobacteriaceae, and categorical agreement (CA) ranged from 49 to 51/51 (96.1 to 100.0%). EA and CA for P. aeruginosa were 100% at all sites. The C/T Etest was also compared to BMD for susceptibility testing on 966 clinical isolates (793 Enterobacteriaceae, including 167 Klebsiella pneumoniae and 159 Escherichia coli isolates, in addition to 173 P. aeruginosa isolates) collected at four clinical sites. EA between Etest and BMD was 96.9% for Enterobacteriaceae isolates and 98.8% for P. aeruginosa isolates. Within the Enterobacteriaceae, isolates from each species examined had >96% CA. For the clinical isolates, no very major errors were identified but two major errors were found (one for K. pneumoniae and one for Providencia rettgeri). By BMD, 47.0% of Enterobacteriaceae and 46.2% of P. aeruginosa challenge strains were nonsusceptible to C/T by CLSI breakpoint criteria; 8.2% of clinical Enterobacteriaceae isolates and 12.1% of clinical P. aeruginosa isolates were nonsusceptible to C/T by CLSI breakpoint criteria. In conclusion, Etest is accurate and reproducible for C/T susceptibility testing of Enterobacteriaceae and P. aeruginosa.

Rapid Detection and Differentiation of Clinically Relevant Candida Species Simultaneously from Blood Culture by Use of a Novel Signal Amplification Approach.

Fungal bloodstream infections are a significant problem in the United States, with an attributable mortality rate of up to 40%. An early diagnosis to direct appropriate therapy has been shown to be critical to reduce mortality rates. Conventional phenotypic methods for fungal detection take several days, which is often too late to impact outcomes. Herein, we describe a cost-effective multiplex assay platform for the rapid detection and differentiation of major clinically relevant Candida species directly from blood culture. This approach utilizes a novel biotin-labeled polymer-mediated signal amplification process combined with targeting rRNA to exploit phylogenetic differences for sensitive and unambiguous species identification; this assay detects seven pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. lusitaniae, and C. guilliermondii) simultaneously with very high specificity to the species level in less than 80 min with the limits of detection at 1 × 103 to 10 × 103 CFU/ml or as few as 50 CFU per assay. The performance of the described assay was verified with 67 clinical samples (including mixed multiple-species infections as well), with an overall 100% agreement with matrix-assisted laser desorption ionization (MALDI) mass spectrometry-based reference results. By providing a species identity rapidly, the clinician is aided with information that may direct appropriate therapy sooner and more accurately than current approaches, including PCR-based tests.

Unusual Escherichia coli PBP 3 Insertion Sequence Identified from a Collection of Carbapenem-Resistant Enterobacteriaceae Tested In Vitro with a Combination of Ceftazidime-, Ceftaroline-, or Aztreonam-Avibactam.

Carbapenemase-producing Enterobacteriaceae isolates (n = 110) from health care centers in central Indiana (from 2010 to 2013) were tested for susceptibility to combinations of avibactam (4 µg/ml) with ceftazidime, ceftaroline, or aztreonam. MIC50/MIC90 values were 1/2 µg/ml (ceftazidime-avibactam), 0.5/2 µg/ml (ceftaroline-avibactam), and 0.25/0.5 µg/ml (aztreonam-avibactam.) A ß-lactam MIC of 8 µg/ml was reported for the three combinations against one Escherichia coli isolate with an unusual TIPY insertion following Tyr344 in penicillin-binding protein 3 (PBP 3) as the result of gene duplication.

Parallel Evaluation of the MALDI Sepsityper and Verigene BC-GN Assays for Rapid Identification of Gram-Negative Bacilli from Positive Blood Cultures.

Rapid identification of microorganisms from positive blood cultures has improved clinical management and antimicrobial stewardship. The advent of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has reduced the time to identification of cultured isolates and is now often the definitive method used in the clinical microbiology laboratory. The commercial in vitro diagnostic MALDI Sepsityper (Sepsityper) kit has the potential for standardization and clinical routine use for the rapid identification of a broad range of bacteria from positive blood cultures. In this study, we performed a parallel evaluation of the Sepsityper (Bruker Daltonics, Billerica, MA) and the Verigene BC-GN (BC-GN) assays (Nanosphere, Inc., Northfield, IL) for the identification of Gram-negative bacilli. A total of 210 Bactec bottles demonstrating Gram-negative bacilli were prospectively enrolled for this study. Among these, 200 monomicrobial cultures were included in the comparative analysis. For monomicrobial cultures, the BC-GN detected 85% (170/200) compared to that detected by routine culture while the Sepsityper detected 94% (188/200) and 91% (181/200) to the genus and species levels, respectively. Comparable positive percentage agreement and negative percentage agreement were observed between the Sepsityper (96.5% and 98.8%, respectively) and the BC-GN (99.4% and 99.8%, respectively) when only (n = 170, 85%) organisms targeted by the latter test were included in the analysis. In conclusion, the two methods evaluated in this study showed excellent performance characteristics for the identification of Gram-negative bacilli commonly isolated from blood cultures. The Sepsityper showed a broader identification range capability that may further improve clinical management and antimicrobial stewardship in patients with less frequent Gram-negative bacilli bloodstream infections.

Multicenter Evaluation of the Portrait Staph ID/R Blood Culture Panel for Rapid Identification of Staphylococci and Detection of the mecA Gene.

Bloodstream infections are a leading cause of morbidity and mortality in the United States and are associated with increased health care costs. We evaluated the Portrait Staph ID/R blood culture panel (BCP) multiplex PCR assay (Great Basin Scientific, Salt Lake City, UT) for the rapid and simultaneous identification (ID) of Staphylococcus aureus, Staphylococcus lugdunensis, and Staphylococcus species to the genus level and the detection of the mecA gene directly from a positive blood culture bottle. A total of 765 Bactec bottles demonstrating Gram-positive cocci in singles or clusters were tested during the prospective trial at 3 clinical sites. The Portrait Staph ID/R BCP results were compared with results from conventional biochemical and cefoxitin disk methods performed at an independent laboratory. Discordant ID and mecA results were resolved by rpoB gene sequencing and mecA gene sequencing, respectively. A total of 658 Staphylococcus species isolates (S. aureus, 211 isolates; S. lugdunensis, 3 isolates; and Staphylococcus spp., 444 isolates) were recovered from monomicrobial and 33 polymicrobial blood cultures. After discrepant analysis, the overall ratios of Portrait Staph ID/R BCP positive percent agreement and negative percent agreement were 99.4%/99.9% for Staphylococcus ID and 99.7%/99.2% for mecA detection.

Propionibacterium acnes, Coagulase-Negative Staphylococcus, and the "Biofilm-like" Intervertebral Disc.

STUDY DESIGN: Patients scheduled for spinal surgery were screened prospectively for a microbial presence associated with intervertebral disc specimens. Inclusion was limited to patients requiring surgery for any of five conditions: study patients with cervical spine intervertebral herniation (IVH), lumbar spine IVH, lumbar spine discogenic pain, and control patients with idiopathic scoliosis/Scheurermann's kyphosis or trauma/neuromuscular deformity. Exclusion criteria included ongoing systemic infection, abnormal pre-operative white cell counts, documented or suspected spinal infection, or previous surgery to the involved disc. OBJECTIVE: The aim of this study was to test for an association between the presence of a bacterial entity in operated discs and a diagnosis of pathologic disc disease. SUMMARY OF BACKGROUND DATA: An association has been described between microbial colonization and progressive intervertebral disc degeneration in 36 herniation patients undergoing microdiscectomies. A total of 19 patients had positive cultures on long-term incubation, with Propionibacterium acnes present in 84% of discs. MATERIALS AND METHODS: Discs were harvested during surgery, using strict sterile technique. Each disc was divided, with half the sample sealed in a sterile, commercially prepared anaerobic culture transport container, and half fixed in formalin. Live specimens were cultured for bacteria at a university-affiliated laboratory in a blinded fashion. Fixed pathologic specimens were gram-stained and read by a board-certified pathologist. RESULTS: A total of 169 intervertebral discs from 87 patients were evaluated (46 males, 41 females). Positive cultures were noted in 76 of 169 discs (45%), with 34 discs positive for P. acnes and 30 discs positive for Staphylococcus. No pathologic evidence was seen of microorganisms, acute or chronic inflammation, or infection. Pooling the IVH and discogenic pain patients and contrasting them with control patients showed a significant association of IVH with positive bacterial cultures (χ = 15.37; P = 0.000088). CONCLUSION: Endemic bacterial biofilms are significantly associated with IVH and discogenic pain. LEVEL OF EVIDENCE: N/A.

Linezolid-resistant Staphylococcus aureus strain 1128105, the first known clinical isolate possessing the cfr multidrug resistance gene.

The Cfr methyltransferase confers resistance to six classes of drugs which target the peptidyl transferase center of the 50S ribosomal subunit, including some oxazolidinones, such as linezolid (LZD). The mobile cfr gene was identified in European veterinary isolates from the late 1990s, although the earliest report of a clinical cfr-positive strain was the 2005 Colombian methicillin-resistant Staphylococcus aureus (MRSA) isolate CM05. Here, through retrospective analysis of LZD(r) clinical strains from a U.S. surveillance program, we identified a cfr-positive MRSA isolate, 1128105, from January 2005, predating CM05 by 5 months. Molecular typing of 1128105 revealed a unique pulsed-field gel electrophoresis (PFGE) profile most similar to that of USA100, spa type t002, and multilocus sequence type 5 (ST5). In addition to cfr, LZD resistance in 1128105 is partially attributed to the presence of a single copy of the 23S rRNA gene mutation T2500A. Transformation of the ∼37-kb conjugative p1128105 cfr-bearing plasmid from 1128105 into S. aureus ATCC 29213 background strains was successful in recapitulating the Cfr antibiogram, as well as resistance to aminoglycosides and trimethoprim. A 7-kb cfr-containing region of p1128105 possessed sequence nearly identical to that found in the Chinese veterinary Proteus vulgaris isolate PV-01 and in U.S. clinical S. aureus isolate 1900, although the presence of IS431-like sequences is unique to p1128105. The cfr gene environment in this early clinical cfr-positive isolate has now been identified in Gram-positive and Gram-negative strains of clinical and veterinary origin and has been associated with multiple mobile elements, highlighting the versatility of this multidrug resistance gene and its potential for further dissemination.

Antibiotic resistance in nosocomial respiratory infections.

Nosocomial respiratory infections are the most common acquired infections in patients with severe underlying conditions and are responsible for high morbidity and mortality in this patient population. Multidrug-resistant (MDR) pathogens are associated with hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP). This article describes the etiology, epidemiology, pathogenesis, diagnosis, and treatment of HAP and VAP associated with antibiotic-resistant bacterial pathogens.

Five-year longitudinal assessment (2008 to 2012) of E-101 solution activity against clinical target and antimicrobial-resistant pathogens.

This study summarizes the topical E-101 solution susceptibility testing results for 760 Gram-positive and Gram-negative target pathogens collected from 75 U.S. sites between 2008 and 2012 and 103 ESKAPE pathogens. E-101 solution maintained potent activity against all bacterial species studied for each year tested, with MICs ranging from <0.008 to 0.25 µg porcine myeloperoxidase (pMPO)/ml. These results confirm that E-101 solution retains its potent broad-spectrum activity against U.S. clinical isolates and organisms with challenging resistance phenotypes.

Portrait Toxigenic Clostridium difficile assay, an isothermal amplification assay detects toxigenic C. difficile in clinical stool specimens.

The Portrait Toxigenic Clostridium difficile assay is a rapid, qualitative assay for the detection of the tcdB gene of C. difficile in stool specimens from patients suspected of C. difficile infections, and received 510(k) clearance by the US FDA in March 2012. The Portrait Toxigenic C. difficile assay combines novel blocked-primer-mediated helicase-dependent multiplex amplification (bpHDA) technology and chip-based detection in an automated sample-to-result format. The assay requires minimal sample preparation and results are available within 90 min. In a multicenter evaluation, the Portrait Toxigenic C. difficile assay had a sensitivity of 98.2% and specificity of 92.8% compared with toxigenic culture. A comparative study between the Portrait Toxigenic C. difficile assay and three FDA-cleared molecular assays for the detection of toxigenic C. difficile exhibited a high degree of agreement (93.8-97.5%). The Portrait Toxigenic C. difficile assay provides a simple, cost-effective method with broad applicability to panel-based approaches, potentially simplifying workflow.

Antimicrobial susceptibility among gram-negative isolates collected in the USA between 2005 and 2011 as part of the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.).

BACKGROUND: The Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) was designed to monitor in vitro antimicrobial susceptibility to tigecycline and comparator agents. We present susceptibility data on Gram-negative organisms collected between 2005 and 2011 from nine United States census regions. METHODS: T.E.S.T. was conducted using standardized CLSI methodologies or FDA-approved breakpoints. RESULTS: Tigecycline was highly active (MIC90 ≤ 2 mg/L) against Enterobacteriaceae irrespective of species or region of collection (N = 25011). The isolates were also highly susceptible to the carbapenems when all regional data are combined, except for ESBL-producing Klebsiella pneumoniae (MIC90 16 mg/L) and Acinetobacter baumannii (MIC90 ≥ 32 mg/L). In addition, 883 (30%) of 2900 A. baumannii isolates were classified as multidrug-resistant (MDR): these MDR organisms were most susceptible to tigecycline (MIC90 2 mg/L) and minocycline (MIC90 8 mg/L) when all regional data are considered together. Susceptibility patterns also varied widely among the regions CONCLUSIONS: The findings highlight the importance of monitoring antimicrobial susceptibility patterns and implementing effective methods to curb increased resistance and also confirm that additional studies to determine the efficacy of tigecycline in vivo, especially for treating infections with MDR organisms, are warranted.

Three-way comparison of BBL CHROMagar MRSA II, MRSASelect, and spectra MRSA for detection of methicillin-resistant Staphylococcus aureus isolates in nasal surveillance cultures.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired and life-threatening infections. Active surveillance programs for MRSA utilize either molecular or culture-based methods. A prospective study was performed to compare the performance of selective and differential chromogenic media, BBL CHROMagar MRSA II (CMRSA II; BD Diagnostics, Sparks, MD), MRSASelect (Bio-Rad Laboratories, Redmond, WA), and Spectra MRSA (Remel, Lenexa, KS), for the detection of MRSA in nasal swab specimens. A total of 515 compliant remnant nasal swab specimens were sequentially used to inoculate BBL Trypticase soy agar with 5% sheep blood (TSA II) and each chromogenic medium. After 24 h of incubation, colony color reactions and morphology on chromogenic media were compared to suspicious colonies on nonselective TSA II. MRSA on TSA II was confirmed by Gram staining, a coagulase test, and a cefoxitin disk test. The overall prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) on TSA II was 12.4% (64/515) and 9.7% (50/515), respectively. When each chromogenic medium was compared to the standard culture method, the sensitivity and specificity, respectively, were as follows: CMRSA II, 87.7% and 98.6%; MRSASelect, 89.0% and 93.4%; and Spectra MRSA, 83.6% and 92.1%. The positive predictive values were highest for CMRSA II (91.4%), followed by MRSASelect (69.1%) and Spectra MRSA (63.5%). False-positive results on chromogenic media were mainly due to color interpretation. The negative predictive values for all three media were greater than 97%. In conclusion, CMRSA II gave the best overall results for detecting MRSA from nasal specimens.

Multicenter clinical evaluation of the portrait toxigenic C. difficile assay for detection of toxigenic Clostridium difficile strains in clinical stool specimens.

We compared the Portrait Toxigenic C. difficile Assay, a new semiautomated sample-to-result molecular test, to a toxigenic bacterial culture/cell cytotoxin neutralization assay (TBC/CCNA) for the detection of toxigenic Clostridium difficile in 549 stool specimens. Stool specimens were also tested by one of three alternative FDA-cleared molecular tests for toxigenic C. difficile (Xpert C. difficile, Illumigene C. difficile, or GeneOhm Cdiff). The sensitivities and specificities of the molecular tests compared to TBC/CCNA were as follows: 98.2% and 92.8% for the Portrait assay, 100% and 91.7% for the Xpert assay, 93.3% and 95.1% for the Illumigene assay, and 97.4% and 98.5% for the GeneOhm assay, respectively. The majority of Portrait false-positive results (20/31; 64.5%) were also positive for C. difficile by an alternative molecular test, suggesting an increased sensitivity compared to the culture-based "gold standard" method. The Portrait test detected an assay input of 30 CFU in 100% of spiked samples and detected an input of 10 CFU in 96.7% of samples tested.

Automated detection of toxigenic Clostridium difficile in clinical samples: isothermal tcdB amplification coupled to array-based detection.

Clostridium difficile can carry a genetically variable pathogenicity locus (PaLoc), which encodes clostridial toxins A and B. In hospitals and in the community at large, this organism is increasingly identified as a pathogen. To develop a diagnostic test that combines the strengths of immunoassays (cost) and DNA amplification assays (sensitivity/specificity), we targeted a genetically stable PaLoc region, amplifying tcdB sequences and detecting them by hybridization capture. The assay employs a hot-start isothermal method coupled to a multiplexed chip-based readout, creating a manual assay that detects toxigenic C. difficile with high sensitivity and specificity within 1 h. Assay automation on an electromechanical instrument produced an analytical sensitivity of 10 CFU (95% probability of detection) of C. difficile in fecal samples, along with discrimination against other enteric bacteria. To verify automated assay function, 130 patient samples were tested: 31/32 positive samples (97% sensitive; 95% confidence interval [CI], 82 to 99%) and 98/98 negative samples (100% specific; 95% CI, 95 to 100%) were scored correctly. Large-scale clinical studies are now planned to determine clinical sensitivity and specificity.