Preeti Pancholi

An Antimicrobial Stewardship Program's Impact with Rapid Polymerase Chain Reaction Methicillin-Resistant Staphylococcus aureus/S. aureus Blood Culture Test in Patients with S. aureus Bacteremia

Rapid organism detection of Staphylococcus aureus bacteremia and communication to clinicians expedites antibiotic optimization. We evaluated clinical and economic outcomes of a rapid polymerase chain reaction methicillin‐resistant S. aureus/S. aureus blood culture test (rPCR). This single‐center study compared inpatients with S. aureus bacteremia admitted from 1 September 2008 through 31 December 2008 (pre‐rPCR) and those admitted from 10 March 2009 through 30 June 2009 (post‐rPCR). An infectious diseases pharmacist was contacted with results of the rPCR; effective antibiotics and an infectious diseases consult were recommended. Multivariable regression assessed clinical and economic outcomes of the 156 patients. Mean time to switch from empiric vancomycin to cefazolin or nafcillin in patients with methicillin‐susceptible S. aureus bacteremia was 1.7 days shorter post‐rPCR (P = .002). In the post‐rPCR methicillin‐susceptible and methicillin‐resistant S. aureus groups, the mean length of stay was 6.2 days shorter (P = .07) and the mean hospital costs were

Multicenter Evaluation of the Cepheid Xpert Methicillin-Resistant Staphylococcus aureus (MRSA) Test as a Rapid Screening Method for Detection of MRSA in Nares

21,387 less (P = .02). rPCR allows rapid differentiation of S. aureus bacteremia, enabling timely, effective therapy and is associated with decreased length of stay and health care costs.

Performance of the Cepheid CT/NG Xpert Rapid PCR Test for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae

The first U.S. multicenter clinical trial to assess the performance of the Cepheid Xpert MRSA assay (Xpert MRSA) was conducted. The assay is a qualitative test designed for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) directly from nares swabs. This novel test combines integrated nucleic acid extraction and automated real-time PCR for the detection of a MRSA-specific signature sequence. A total of 1,077 nares specimens were collected from seven geographically distinct health care sites across the United States with prevalence rates ranging from 5.2% to 44%. Nares specimens were tested by (i) the Xpert MRSA assay, (ii) direct culture on CHROMagar MRSA medium (direct CM culture), and (iii) broth-enriched culture (Trypticase soy broth with 6.5% sodium chloride) followed by plating onto CHROMagar MRSA medium (broth-enriched CM culture). When direct CM culture was designated the reference method, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Xpert MRSA assay were 94.3%, 93.2%, 73.0%, and 98.8%, respectively. When broth-enriched CM culture was used as the reference method, the clinical sensitivity, specificity, PPV, and NPV of the Xpert MRSA assay were 86.3%, 94.9%, 80.5%, and 96.6%, respectively. The BD GeneOhm MRSA (BDGO) assay was performed as a comparative molecular method. No statistical performance differences were observed between the Xpert MRSA and BDGO assays when they were compared to culture methods. From this large-scale, multicenter clinical comparison, we conclude that the Xpert MRSA assay is a simple, rapid, and accurate method for performing active surveillance for MRSA in a variety of health care populations.

Synergy Testing by Etest, Microdilution Checkerboard, and Time-Kill Methods for Pan-Drug-Resistant Acinetobacter baumannii

ABSTRACT Tests for Chlamydia trachomatis and Neisseria gonorrhoeae, which can provide results rapidly to guide therapeutic decision-making, offer patient care advantages over laboratory-based tests that require several days to provide results. We compared results from the Cepheid GeneXpert CT/NG (Xpert) assay to results from two currently approved nucleic acid amplification assays in 1,722 female and 1,387 male volunteers. Results for chlamydia in females demonstrated sensitivities for endocervical, vaginal, and urine samples of 97.4%, 98.7%, and 97.6%, respectively, and for urine samples from males, a sensitivity of 97.5%, with all specificity estimates being ≥99.4%. Results for gonorrhea in females demonstrated sensitivities for endocervical, vaginal, and urine samples of 100.0%, 100.0%, and 95.6%, respectively, and for urine samples from males, a sensitivity of 98.0%, with all estimates of specificity being ≥99.8%. These results indicate that this short-turnaround-time test can be used to accurately test patients and to possibly do so at the site of care, thus potentially improving chlamydia and gonorrhea control efforts.

Evaluation of commercial ResPlex II v2.0, MultiCode-PLx, and xTAG respiratory viral panels for the diagnosis of respiratory viral infections in adults.

ABSTRACT Pan-drug-resistant (PDR) Acinetobacter baumannii is an important nosocomial pathogen that poses therapeutic challenges. Tigecycline alone or in combination with agents such as colestimethate, imipenem, and/or amikacin is being used clinically to treat PDR A. baumannii infections. The purpose of this study was to compare in vitro susceptibility testing by epsilometric (Etest) methods and the checkerboard (CB) method with testing by time-kill analysis. PDR A. baumannii clinical strains representing eight unique pulsed-field gel electrophoresis clones selected from a total of 32 isolates were tested in vitro with tigecycline, colestimethate, imipenem, and amikacin in single- and two-drug combinations by using two different methods of Etest (with a fixed ratio method [method 1] and with the incorporation of the active drug in medium [method 2]) and by using CB. The three-drug combination of imipenem, tigecycline, and amikacin was also tested by CB. These results were compared to time-kill results. Synergy was consistently detected with the imipenem plus colestimethate and tigecycline plus imipenem combinations. The Etest method with active drug incorporated into the agar allowed us to detect synergy even in the presence of the active drug and was more comparable to CB and time-kill tests. Synergy was detected with the three-drug combination of imipenem, tigecycline, and amikacin by both CB and time-kill methods among several tested clones. These findings indicate the utility of synergy testing to predict activity of specific antibiotic combinations against PDR A. baumannii.

Detection of Toxigenic Clostridium difficile: Comparison of the Cell Culture Neutralization, Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile Assays

Abstract Background Commercial multiplex PCR panels for respiratory viruses (PRV) have been recently developed. ResPlex II Panel v2.0 (Qiagen), MultiCode®-PLx (EraGen Biosciences), and xTAG® (Luminex) PRVs were studied. All assays detect influenza A and B, adenovirus, parainfluenza 1–3, respiratory syncytial virus A and B, human metapneumovirus and human rhinovirus. The ResPlex II additionally detects coronavirus (229E, OC43, NL63, HKU1), coxsackie/echo virus, bocavirus and differentiates adenoviruses (B, E). The MultiCode-PLX assay detects 229E, OC43, and NL63, differentiates parainfluenza 4a, 4b and adenoviruses (B, C, E). The xTAG additionally subtypes influenza A as seasonal H1 and H3. Study design 202 specimens collected from adult patients with signs of respiratory infection from November, 2008 to May, 2009 were used for evaluating the performance of the three commercial PRV assays. Viral culture and xTAG were used as the standards to assess sensitivity and specificity. Results The PRV assays detected more viruses than culture. When compared to culture, the xTAG PRV showed a sensitivity and specificity of 100% and 91%, compared to MultiCode-PLx with 89% and 87%, and ResPlex II with 89% and 94%, respectively. Co-infection was detected in a small subset of patient specimens. Each panel showed differences in sensitivities for individual viruses. Conclusions While the ResPlex II and MultiCode-PLx offer a broader virus detection range and greater ease of use, the xTAG PRV showed increased sensitivity to common viral targets represented in the assays, and also had the ability to differentiate human from non-human influenza A H1.

Identification of Mycobacteria from Solid and Liquid Media by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry in the Clinical Laboratory

ABSTRACT Clostridium difficile is the most important cause of nosocomial diarrhea. Several laboratory techniques are available to detect C. difficile toxins or the genes that encode them in fecal samples. We evaluated the Xpert C. difficile and Xpert C. difficile/Epi (Cepheid, CA) that detect the toxin B gene (tcdB) and tcdB, cdt, and a deletion in tcdC associated with the 027/NAP1/BI strain, respectively, by real-time PCR, and the Illumigene C. difficile (Meridian Bioscience, Inc.) that detects the toxin A gene (tcdA) by loop-mediated isothermal amplification in stool specimens. Toxigenic culture was used as the reference method for discrepant stool specimens. Two hundred prospective and fifty retrospective diarrheal stool specimens were tested simultaneously by the cell cytotoxin neutralization assay (CCNA) and the Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays. Of the 200 prospective stools tested, 10.5% (n = 23) were determined to be positive by CCNA, 17.5% (n = 35) were determined to be positive by Illumigene C. difficile, and 21.5% (n = 43) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 50 retrospective stools, previously determined to be positive by CCNA, 94% (n = 47) were determined to be positive by Illumigene C. difficile and 100% (n = 50) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 11 discrepant results (i.e., negative by Illumigene C. difficile but positive by Xpert C. difficile and Xpert C. difficile/Epi), all were determined to be positive by the toxigenic culture. A total of 21% of the isolates were presumptively identified by the Xpert C. difficile/Epi as the 027/NAP1/BI strain. The Xpert C. difficile and Xpert C. difficile/Epi assays were the most sensitive, rapid, and easy-to use assays for the detection of toxigenic C. difficile in stool specimens.

Genetic relatedness and molecular characterization of multidrug resistant Acinetobacter baumannii isolated in central Ohio, USA

ABSTRACT Mycobacteria cause significant morbidity in humans. Rapid and accurate mycobacterial identification is important for improvement of patient outcomes. However, identification may be challenging due to the slow and fastidious growth of mycobacteria. Several diagnostic methods, such as biochemical, sequencing, and probe methods, are used for mycobacterial identification. We compared the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) Biotyper system (Bruker Daltonics) to 16S rRNA/hsp65 sequencing and/or DNA probes (Gen-Probe) for mycobacterial identification. One hundred seventy-eight mycobacterial isolates grown on solid and/or broth medium were included in the study. MALDI-TOF MS identified 93.8% of the mycobacteria isolates accurately to the species level and 98.3% to the genus level, independent of the type of medium used for isolation. The identification of mycobacteria directly from cultures using MALDI-TOF MS allows for precise identification in an hour compared to traditional biochemical and phenotypic methods that can take weeks or probes and sequencing that may take a few hours. Identification by MALDI-TOF MS potentially reduces the turnaround time and cost, thereby saving resources within the health care system.

Molecular Methods and Platforms for Infectious Diseases Testing: A Review of FDA-Approved and Cleared Assays

BackgroundOver the last decade, nosocomial infections due to Acinetobacter baumannii have been described with an increasing trend towards multidrug resistance, mostly in intensive care units. The aim of the present study was to determine the clonal relatedness of clinical isolates and to elucidate the genetic basis of imipenem resistance.MethodsA. baumannii isolates (n = 83) originated from two hospital settings in central Ohio were used in this study. Pulsed-field gel electrophoresis genotyping and antimicrobial susceptibility testing for clinically relevant antimicrobials were performed. Resistance determinants were characterized by using different phenotypic (accumulation assay for efflux) and genotypic (PCR, DNA sequencing, plasmid analysis and electroporation) approaches.ResultsThe isolates were predominantly multidrug resistant (>79.5%) and comprised of thirteen unique pulsotypes, with genotype VII circulating in both hospitals. The presence of blaOXA-23 in 13% (11/83) and ISAba1linked blaOXA-66 in 79.5% (66/83) of clinical isolates was associated with high level imipenem resistance. In this set of OXA producing isolates, multidrug resistance was bestowed by blaADC-25, class 1 integron-borne aminoglycoside modifying enzymes, presence of sense mutations in gyrA/parC and involvement of active efflux (with evidence for the presence of adeB efflux gene).ConclusionThis study underscores the major role of carbapenem-hydrolyzing class D β-lactamases, and in particular the acquired OXA-23, in the dissemination of imipenem-resistant A. baumannii. The co-occurrence of additional resistance determinant could also be a significant threat.

Identification of Gram-Negative Bacteria and Genetic Resistance Determinants from Positive Blood Culture Broths by Use of the Verigene Gram-Negative Blood Culture Multiplex Microarray-Based Molecular Assay

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 Administrations Summary of Safety and Effectiveness Data or the Office of In Vitro Diagnostic Device Evaluation and Safetys 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.