Esbl

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Detection of Extended-Spectrum β -Lactamase and Klebsiella pneumoniae Carbapenemase Genes Directly from Blood Cultures by Use of a Nucleic Acid Microarray
Joel T. Fishbain, Oleg Sinyavskiy, Kathleen Riederer, Andrea M. Hujer and Robert A. Bonomo J. Clin. Microbiol. 2012, 50(9):2901. DOI: 10.1128/JCM.01023-12. Published Ahead of Print 20 June 2012.


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Detection of Extended-Spectrum -Lactamase and Klebsiella pneumoniae Carbapenemase Genes Directly from Blood Cultures by Use of a Nucleic Acid Microarray
Downloaded from http://jcm.asm.org/ on January 14, 2013 by UNIVERSIDADE DE SÃOPAULO
Joel T. Fishbain,a Oleg Sinyavskiy,a Kathleen Riederer,a Andrea M. Hujer,b,c and Robert A. Bonomob,c,d,e
Departments of Medicine and Graduate Medical Education, St. John Hospital and Medical Center, Detroit, Michigan, USAa; Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, USAb; Research Service, Louis Stokes Cleveland Veterans Affairs MedicalCenter, Cleveland, Ohio, USAc; and Departments of Molecular Biology and Microbiologyd and Pharmacology,e Case Western Reserve University School of Medicine, Cleveland, Ohio USA

The growing crisis of multidrug-resistant (MDR) Gram-negative bacteria requires that current technologies permit the rapid detection of extended-spectrum -lactamase (blaESBL) and Klebsiella pneumoniae carbapenemase (blaKPC)genes. In the present study, we assessed the performance characteristics of a commercially available nucleic acid microarray system for the detection of blaESBL and blaKPC genes directly from positive blood cultures. Using blood cultures (BCs) that contained Gram-negative bacilli identified by Gram staining, we isolated bacterial DNA using spin columns (BC-C) and rapid water lysis (BC-W). TwentyESBL/KPC-positive and 20 ESBL/KPC-negative blood culture samples, as well as 20 non-lactose-fermenting organisms, were tested. The 20 isolates that were ESBL positive by phenotypic testing were also evaluated on solid medium (SM), and the DNA was extracted by use of a spin column (SM-C). The resulting 140 DNA extractions were assessed for DNA quantity and quality using 260/280-nm absorbance ratios,and DNA microarray analysis was performed in a blinded fashion. Microarray and phenotypic results were concordant for 98.3% of BC-W, 90% of BC-C, and 95% of SM-C samples. Compared to phenotypic testing, the sensitivity and specificity for BC-C samples were 88.9% and 100%, respectively, and for BC-W samples, the sensitivity and specificity were 94.4% and 100%, respectively. BC-W samples yielded thehighest concordance with phenotypic results. Nucleic acid microarrays offer promise in the identification of blaESBL and blaKPC genes directly from blood cultures, thereby reducing the time to identification of these important pathogens.

he prevalence of extended-spectrum -lactamase (ESBL)- and Klebsiella pneumoniae carbapenemase (KPC)-producing Gramnegative bacilli is increasing worldwide, withthe attendant loss of many previously effective antimicrobial therapeutic agents (1, 5). As a result, appropriate empirical antibiotic therapy becomes even more necessary with regard to KPC-producing Klebsiella pneumoniae (13). Mortality has been shown to be markedly higher when inadequate or ineffective antimicrobial therapy is administered as empirical therapy (or delayed by 72 h) pending theresults of phenotype testing (7, 10, 13, 16). The application of rapid molecular diagnostic techniques to the identification and characterization of pathogens promises to improve outcomes as a result of more timely and effective antibiotic therapy. Important advances have recently come from the use of mass spectrometry (MS) in the clinical microbiology laboratory (11, 12, 15). Rapid and inexpensive...
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