Contemporary Considerations for Establishing Reference Methods for Antibacterial Susceptibility Testing.

Antibacterial susceptibility testing (AST) is performed to guide therapy, perform resistance surveillance studies, and support development of new antibacterial agents. For 5 decades, broth microdilution (BMD) has served as the reference method to assess in vitro activity of antibacterial agents against which both novel agents and diagnostic tests have been measured. BMD relies on in vitro inhibition or killing of bacteria. It is associated with several limitations: it is a poor mimic of the in vivo milieu of bacterial infections, requires multiple days to perform, and is associated with subtle, difficult to control variability. In addition, new reference methods will soon be needed for novel agents whose activity cannot be evaluated by BMD (e.g., those that target virulence). Any new reference methods must be standardized, correlated with clinical efficacy and be recognized internationally by researchers, industry, and regulators. Herein, we describe current reference methods for in vitro assessment of antibacterial activity and highlight key considerations for the generation of novel reference methods.

Prevalence and Characterization of the Cefazolin Inoculum Effect in North American Methicillin-Susceptible Staphylococcus aureus Isolates.

Antistaphylococcal penicillins and cefazolin remain the primary treatments for infections with methicillin-susceptible Staphylococcus aureus (MSSA). The cefazolin inoculum effect (CzIE) causes the cefazolin MIC to be elevated in proportion to the number of bacteria in the inoculum. The objective of this multicenter study was to evaluate the prevalence of the CzIE in North American MSSA isolates. Clinical MSSA isolates from six microbiology laboratories in the United States and one microbiology laboratory in Canada were screened for the CzIE by broth microdilution at a standard inoculum (~5 × 105 CFU/mL) and a high inoculum (~5 × 107 CFU/mL). Genome sequencing was performed to further characterize the MSSA isolates. The CzIE was present in 57/305 (18.6%) MSSA isolates, ranging from 0% to 27.9% across study sites. More of the CzIE-positive isolates (29.8%) had standard inoculum cefazolin MICs of 1.0 µg/mL than the CzIE-negative isolates did (3.2%) (P < 0.0001). Conversely, more CzIE-negative isolates (39.5%) had standard inoculum MICs of 0.25 µg/mL than the CzIE positive isolates did (5.3%) (P < 0.0001). The most common BlaZ ß-lactamase types found in the CzIE-positive strains were type C (53.7%) and type A (44.4%). ST8 and ST30 were the most common sequence types among CzIE-positive isolates and correlated with BlaZ type C and A, respectively. The CzIE was present in up to a quarter of clinical MSSA isolates from North American clinical laboratories. Further studies to determine the impact of the presence of the CzIE on clinical outcomes are needed.

Performance of Five Commercial Identification Platforms for Identification of Staphylococcus delphini.

The Staphylococcus intermedius group (SIG) is a collection of coagulase-positive staphylococci consisting of four distinct species, namely, Staphylococcus cornubiensis, Staphylococcus delphini, Staphylococcus intermedius, and Staphylococcus pseudintermedius SIG members are animal pathogens and rare causes of human infection. Accurate identification of S. pseudintermedius has important implications for interpretation of antimicrobial susceptibility testing data and may be important for other members of the group. Therefore, we sought to evaluate the performance of five commercially available identification platforms with 21 S. delphini isolates obtained from a variety of animal and geographic sources. Here, we show that automated biochemical platforms were unable to identify S. delphini to the species level, a function of its omission from their databases, but could identify isolates to the SIG level with various degrees of success. However, all automated systems misidentified at least one isolate as Staphylococcus aureus One matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system was able to identify S. delphini to the species level, suggesting that MALDI-TOF MS is the best option for distinguishing members of the SIG. With the exception of S. pseudintermedius, it is unclear if other SIG members should be routinely identified to the species level; however, as our understanding of their role in animal and human diseases increases, it may be necessary and important to do so.

Direct-from-Blood-Culture Disk Diffusion To Determine Antimicrobial Susceptibility of Gram-Negative Bacteria: Preliminary Report from the Clinical and Laboratory Standards Institute Methods Development and Standardization Working Group.

The performance of a disk diffusion test using broth from positive blood cultures as inoculum (direct disk diffusion [dDD]) was evaluated for a collection of 20 challenge isolates of Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa Isolates seeded into human blood were inoculated into Bactec Plus Aerobic/F, VersaTREK Redox 1, and BacT/Alert FA Plus bottles and incubated in the respective automated blood culture systems. Disk diffusion results were compared to reference disk diffusion results. Categorical agreement (CA) values for dDD, after removal of random errors due to natural MIC variation, were 87.8%, 88.4%, and 92.2% for the BacT/Alert, Bactec, and VersaTREK systems, respectively. No very major errors (VME) were observed, and major error (ME) rates were 3.0%, 2.3%, and 1.7%, respectively. Incubation of the dDD test samples for 6 h compared to incubation for 16 to 18 h resulted in 19.9% of tests having too light of growth to allow reading of zones of inhibition. Among the evaluable dDD tests, CA values were 58.9%, 76.6%, and 73.2% for the isolates seeded into the BacT/Alert, Bactec, and VersaTREK systems, respectively. VME rates for isolates seeded into these systems were 2.2%, 1.8%, and 3.0%, respectively, and ME rates were 25.4%, 6.1%, and 2.8%, respectively, at the 6-h reading. The best performance of dDD was found for blood cultures with bacterial concentrations in the range of 7.6 × 107 to 5.0 × 108 CFU/ml; CA values ranged from 94.7 to 96.2% for these concentrations after 18 h of incubation and from 76.9 to 84.1% after 6 h of incubation. These preliminary data demonstrate the potential accuracy of dDD testing by the clinical laboratory.

Health economic aspects of breast cancer treatment: the compulsory health insurances' view.

Due to the impact of rising expenditures for the delivery of high-standard health care, further efforts supporting evidence-based, cost-efficient and patient-centered management in oncology are advised. This also concerns the treatment of patients with breast cancer. Reimbursement of diagnostic and/or therapeutic innovations in oncologic health care within the compulsory health insurances (CHIs) in Germany requests their evidence-based proof of benefit and medical need. Using selected examples in pharmacotherapy, recommendations to improve outpatient breast cancer care are discussed.

Molecular methods and platforms for infectious diseases testing a review of FDA-approved and cleared assays.

The superior sensitivity and specificity associated with the use of molecular assays has greatly improved the field of infectious disease diagnostics by providing clinicians with results that are both accurate and rapidly obtained. Herein, we review molecularly based infectious disease diagnostic tests that are Food and Drug Administration approved or cleared and commercially available in the United States as of December 31, 2010. We describe specific assays and their performance, as stated in the Food and Drug Administration's Summary of Safety and Effectiveness Data or the Office of In Vitro Diagnostic Device Evaluation and Safety's decision summaries, product inserts, or peer-reviewed literature. We summarize indications for testing, limitations, and challenges related to implementation in a clinical laboratory setting for a wide variety of common pathogens. The information presented in this review will be particularly useful for laboratories that plan to implement or expand their molecular offerings in the near term.

Diagnostics as essential tools for containing antibacterial resistance.

Antibacterial drugs are overused and often inappropriately selected. This exacerbates drug resistance and exacts a high burden from acute respiratory tract, bloodstream, sexually-transmitted, diarrheal and other infections. Appropriate use of existing diagnostic tests, and developing better ones, could avert these costs and would avoid selective pressure from unnecessary antibacterial use. Product profiles of resistance-averting tests would specify WHO 'ASSURED' (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free and Deliverable) criteria and request susceptibility as well as etiological information. Advances in genomics, nanoscience, microfluidics and bioengineering, as well as innovative funding paradigms can help to overcome research and development barriers for such diagnostics if they are deliberately and forcefully applied. Rapid uptake of new tests requires timely translation of research on cost-benefit analyses into policy, value-based subsidies and reimbursements, as well as behavioral change of health care providers and users.

Correlation of cefoxitin MICs with the presence of mecA in Staphylococcus spp.

This report describes the results of an 11-laboratory study to determine if a cefoxitin broth microdilution MIC test could predict the presence of mecA in staphylococci. Using breakpoints of < or = 4 microg/ml for mecA-negative and > or = 6 or 8 microg/ml for mecA-positive isolates, sensitivity and specificity based on mecA or presumed mecA for Staphylococcus aureus at 18 h of incubation were 99.7 to 100% in three cation-adjusted Mueller-Hinton broths tested. For coagulase-negative strains at 24 h of incubation, breakpoints of < or = 2 microg/ml for mecA-negative and > or = 4 microg/ml for mecA-positive isolates gave sensitivity and specificity of 94 to 99% and 69 to 80%, respectively.

Detection of inducible clindamycin resistance in staphylococci by broth microdilution using erythromycin-clindamycin combination wells.

A study conducted by 11 laboratories investigated the ability of four combinations of erythromycin (ERY) and clindamycin (CC) (ERY and CC at 4 and 0.5, 6 and 1, 8 and 1.5, and 0.5 and 2 microg/ml) in a single well of a broth microdilution panel to predict the presence of inducible CC resistance. Each laboratory tested approximately 30 Staphylococcus aureus isolates and 20 coagulase-negative staphylococcus (CoNS) isolates in a panel using cation-adjusted Mueller-Hinton broth from three different manufacturers. Only the strains resistant to ERY and those susceptible or intermediate to CC were included in the analysis (S. aureus, n = 333; CoNS, n = 97). Results of the D-zone test were used as the gold standard. After an 18-h incubation, the combination of 4 microg/ml ERY and 0.5 microg/ml CC performed the best, with 98 to 100% sensitivity and 100% specificity for both organism groups. After a 24-h incubation, the ERY-CC combinations of 4 and 0.5, 6 and 1, and 8 and 1.5 microg/ml correlated well with the D-zone test.

Biochemical properties of a newly described Escherichia species, Escherichia albertii.

Five strains of a newly described Escherichia species, Escherichia albertii, were extensively characterized by conventional biochemical methods and by commercial identification panels. E. albertii is an indole-negative species that ferments D-mannitol but not D-xylose. Because these strains are not included in the databases of commercial systems at present, they were most often identified as Hafnia, Salmonella, Escherichia coli, or, on one system (MicroScan dried overnight panels), Yersinia ruckeri.

BetalasEN: microdilution panel for identifying beta-lactamases present in isolates of Enterobacteriaceae.

A dried investigational use-only microdilution panel named betalasEN (a short named derived from the panel's purpose, to identify beta-lactamases in Enterobacteriaceae) containing 10 beta-lactam drugs with and without beta-lactamase inhibitors was developed to identify beta-lactamases among clinical isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri, Citrobacter freundii group, Enterobacter spp., and Serratia marcescens. The MICs obtained with a collection of 383 organisms containing well-characterized beta-lactamases were used to develop numeric codes and logic pathways for computerized analysis of results. The resultant logic pathways and betalasEN panel were then used to test and identify beta-lactamases among 885 isolates of Enterobacteriaceae recovered in cultures obtained at six different hospital laboratories across the United States. beta-Lactamases present in 801 (90.5%) of the 885 isolates were identified by betalasEN by using the existing logic pathways and codes or after minor modifications were made to the existing codes. The 84 strains that gave codes that betalasEN could not identify were collected, reidentified, and retested by using betalasEN. Three strains had been misidentified, 54 strains gave different codes upon repeat testing that could be identified by betalasEN, and 27 strains repeated new codes. The beta-lactamases in these strains were identified, and the new codes were added to the betalasEN logic pathways. These results indicate that betalasEN can identify clinically important beta-lactamases among most isolates of Enterobacteriaceae. The results also show that good quality control and attention to proper performance of the tests are essential to the correct performance of betalasEN.

Smoking Relapse During Postpartum.

PURPOSE: While many women quit smoking when learning of their pregnancy, the majority relapses within one year of giving birth. This paper provides an integrative review of studies that assess the contributing factors to maternal smoking relapse within the first year of giving birth. The framework for this review is the relapse prevention model, which seeks to uncover the contributing factors, or high-risk situations, that lead to relapse. Additionally, this paper examines existing practice interventions aimed at reducing postpartum relapse and provides recommendations for best practice. CONCLUSIONS: Research evidence to date shows that two main factors are associated with smoking relapse among postpartum women: (1) their association with other smokers and (2) their choosing not to breastfeed. Since maternal smoking results in poor health outcomes for both mothers and children, relapse prevention is a health issue worthy of examination. Nurses can use this information in planning, implementing, and evaluating smoking prevention and cessation programs for pregnant and postpartum women.