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.

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.

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.

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.

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.

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.

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.

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.

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.

In vitro antibacterial activity of E-101 Solution, a novel myeloperoxidase-mediated antimicrobial, against Gram-positive and Gram-negative pathogens.

OBJECTIVES: E-101 Solution (E-101) is a novel myeloperoxidase-mediated antimicrobial. It is composed of porcine myeloperoxidase (pMPO), glucose oxidase, glucose as the substrate and specific amino acids in an aqueous vehicle. E-101 is being developed for topical application directly into surgical wounds to prevent surgical site infections (SSIs). The in vitro activity of E-101 was investigated. METHODS: MIC, MBC, time-kill and antimicrobial combination experiments were performed according to CLSI guidelines with modifications. Resistance selection studies were performed using a serial passage method. RESULTS: E-101 showed MIC(90) values of 0.03, 0.5 and 0.5 mg pMPO/L for staphylococci (n = 140), streptococci (n = 95) and enterococci (n = 55), respectively. MIC(90) values ranged between 0.03-0.5 and ≤ 0.004-0.12 mg pMPO/L for Enterobacteriaceae (n = 148) and Gram-negative non-Enterobacteriaceae (n = 92) strains, respectively. There was no antimicrobial tolerance to E-101 for Staphylococcus aureus, Streptococcus agalactiae or Streptococcus pyogenes. Time-kill studies demonstrated a rapid (<30 min) bactericidal effect against S. aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa in a concentration-dependent and time-dependent manner. There was no evidence of stable resistance to E-101 among staphylococci, enterococci, E. coli or P. aeruginosa strains and no evidence of E-101 interaction with antibiotics commonly used in clinical medicine. Conclusions E-101 shows potent and broad-spectrum in vitro activity against bacteria that are the causative pathogens of SSIs, thereby providing the impetus to test its clinical utility in the prevention of SSIs.

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.

Distribution of resistant gram-positive organisms across the census regions of the United States and in vitro activity of tigecycline, a new glycylcycline antimicrobial.

BACKGROUND: Gram-positive isolates were collected from 76 medical centers within the 9 census regions across the United States. METHODS: Antimicrobial susceptibilities were determined according to Clinical and Laboratory Standards Institute guidelines. RESULTS: Vancomycin resistance among Enterococcus faecium isolates varied from 45.5% (New England) to 85.3% (East South Central). The lowest concentrations at which 90% of the isolates were inhibited (MIC90) were for tigecycline (0.06-0.12 microg/mL) and for linezolid (2-4 microg/mL). Methicillin-resistant Staphylococcus aureus (MRSA) varied from 27.4% (New England) to 62.4% (East South Central). All MRSA were susceptible to tigecycline, linezolid, and vancomycin. Penicillin-nonsusceptible Streptococcus pneumoniae ranged from 23.3% in the Pacific region to 54.5% in the East South Central region. Tigecycline, imipenem, levofloxacin, linezolid, and vancomycin all maintained MIC90 of < or =1 microg/mL against penicillin-nonsusceptible S pneumoniae in vitro, irrespective of region. CONCLUSION: This study demonstrates the variable rate of antimicrobial-resistant gram-positive organisms in the United States. Tigecycline may make a useful addition to the antimicrobial armamentarium.

Comparative in vitro and bactericidal activities of telithromycin against penicillin-nonsusceptible, levofloxacin-resistant, and macrolide-resistant Streptococcus pneumoniae by time-kill methodology.

Broth microdilution MICs were determined for 14 antimicrobial agents against 296 clinical, non-duplicate isolates of Streptococcus pneumoniae collected at Methodist Hospital (Indianapolis, Indiana, USA) from January 2001 to December 2003. Isolates were categorized as susceptible, intermediate, or resistant using Clinical and Laboratory Standards Institute breakpoints. Time-kill studies were performed to evaluate the bactericidal activity of telithromycin at 1, 2, 4, and 8x MIC against 10 penicillin-nonsusceptible, levofloxacin-resistant, and macrolide-resistant (7 M-phenotype, 3 MLS(B)-phenotype) strains. Bactericidal activity was defined as a >/=3-log(10) reduction in CFU/mL. The prevalence of resistance was highest for the macrolides (32%), followed by penicillin (16.2%), clindamycin (10.8%), amoxicillin+/-clavulanate (4.4%), levofloxacin (3.0%), gatifloxacin and moxifloxacin (2.4%), ceftriaxone and cefotaxime (2.0%), and gemifloxacin (1.4%). None of the isolates tested were resistant to telithromycin. At 24h, telithromycin was bactericidal for 0/10, 2/10, 7/10, and 7/10 isolates at 1x MIC, 2x MIC, 4x MIC, and 8x MIC, respectively. At 4-8x MIC, telithromycin was bactericidal for 7/7 M-phenotype isolates and 0/3 MLS(B)-phenotype isolates. For the MLS(B)-phenotype isolates, colony counts were decreased by 1.3-2.1log(10) colony-forming units/mL after 24h at 8x MIC. Overall, telithromycin was highly active against 296 isolates of S. pneumoniae from our institution and demonstrated bactericidal activity at clinically achievable concentrations for 7 of 10 penicillin-nonsusceptible, levofloxacin-resistant, and macrolide-resistant S. pneumoniae. However, telithromycin was bacteriostatic for the MLS(B)-phenotype isolates.

Selection of a gyrA mutation and treatment failure with gatifloxacin in a patient with Streptococcus pneumoniae with a preexisting parC mutation.

An 81-year-old woman had pneumonia caused by Streptococcus pneumoniae (levofloxacin Etest minimum inhibitory concentration [MIC] 1.5 microg/ml) and was treated with intravenous gatifloxacin 200 mg/day. After 3 days of therapy, repeat sputum cultures were positive for S. pneumoniae, which was resistant to levofloxacin (Etest MIC > 32 microg/ml). The isolate obtained before therapy showed a preexisting parC mutation of aspartic acid-83 to asparagine (Asp83-->Asn), and the isolate obtained during therapy showed an acquired gyrA mutation from serine-81 to phenylalanine (Ser81-->Phe) and a second parC mutation from lysine-137 to Asn (Lys137-->Asn). Both isolates were the same strain, as determined with pulsed-field gel electrophoresis. This case demonstrates the potential for resistance to emerge during 8-methoxy fluoroquinolone therapy for fluoroquinolone-susceptible S. pneumoniae with a preexisting parC mutation. Additional clinical failures with a fluoroquinolone may occur unless these first-step parC mutants can be identified to assist clinicians in selecting appropriate antimicrobial therapy.

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.

Azithromycin treatment failure in community-acquired pneumonia caused by Streptococcus pneumoniae resistant to macrolides by a 23S rRNA mutation.

In this report, we describe an azithromycin treatment failure in community-acquired pneumonia. During the first three days of azithromycin, the patient's symptoms worsened, and she was subsequently admitted to the hospital. Blood cultures were positive for a penicillin-susceptible, macrolide-resistant S. pneumoniae. DNA sequencing revealed an A2059G mutation in domain V of the 23S rRNA. To our knowledge, this is the first clinical report of an azithromycin failure in the treatment of S. pneumoniae resistant to macrolides by this mechanism.

Levofloxacin treatment failure in a patient with fluoroquinolone-resistant Streptococcus pneumoniae pneumonia.

The frequency of fluoroquinolone-resistant Streptococcus pneumoniae has increased as fluoroquinolone administration for treatment of respiratory tract infections has increased. Levofloxacin treatment failed in a patient who had pneumococcal pneumonia and had received three previous courses of levofloxacin therapy. Susceptibility testing revealed high-level resistance to levofloxacin (minimum inhibitory concentration [MIC] > 32 microg/ml), and cross-resistance to moxifloxacin (MIC 4 microg/ml), trovafloxacin (6 microg/ml), and gatifloxacin (12 microg/ml). Sequencing of the quinolone-resistance determining region revealed a mutation of serine-81 to phenylalanine (Ser81-->Phe) in the gyrA region of DNA gyrase and a Ser79-->Phe mutation in the parC region of topoisomerase IV The patient was treated successfully with intravenous ceftriaxone followed by oral cefprozil. Clinicians must be aware of local resistance patterns and the potential for fluoroquinolone treatment failures in patients with infections caused by S. pneumoniae.

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.