Detection of AmpC beta-lactamase in Escherichia coli: comparison of three phenotypic confirmation assays and genetic analysis.

Two mechanisms account for AmpC activity in Escherichia coli, namely, mutations in the ampC promoter and attenuator regions resulting in ampC overexpression and acquisition of plasmid-carried ampC genes. In this study, we analyzed 51 clinical E. coli isolates with reduced susceptibility to amoxicillin-clavulanic acid, piperacillin-tazobactam, or extended-spectrum cephalosporins for the presence of AmpC production. Three phenotypic AmpC confirmation assays (cefoxitin-cloxacillin disk diffusion test, cefoxitin-EDTA disk diffusion test, and AmpC Etest) were compared for the detection of AmpC activity. All 51 isolates were characterized genetically by mutational analysis of the chromosomal ampC promoter/attenuator region and by PCR detection of plasmid-carried ampC genes. Altogether, 21/51 (41%) E. coli isolates were considered true AmpC producers. AmpC activity due to chromosomal ampC promoter/attenuator mutations was found in 12/21 strains, and plasmid-carried ampC genes were detected in 8/21 isolates. One strain contained both ampC promoter mutations and a plasmid-carried ampC gene. All three phenotypic tests were able to detect the majority (>90%) of AmpC-positive strains correctly. Cefoxitin resistance was found to be a discriminative parameter, detecting 20/21 AmpC-producing strains. Susceptibility to extended-spectrum cephalosporins, e.g., ceftriaxone, ceftazidime, and cefotaxime, was found in 9 of the 21 AmpC-positive strains. Considering the elevated zone diameter breakpoints of the 2010 CLSI guidelines, 2/21 AmpC-positive strains were categorized as susceptible to extended-spectrum cephalosporins.

Detection and identification of Mycobacterium spp. in clinical specimens by combining the Roche Cobas Amplicor Mycobacterium tuberculosis assay with Mycobacterium genus detection and nucleic acid sequencing.

We have recently developed a PCR assay for detection of Mycobacterium spp. at the genus level based on the Cobas Amplicor platform. The sensitivities for smear-positive and smear-negative specimens were found to be 100% and 47.9%, respectively. The specificity was 97.7%, the positive predictive value 84.6%, and the negative predictive value 93.1%. In a follow-up study, we have systematically evaluated the Mycobacterium genus assay in parallel with the Cobas Amplicor Mycobacterium tuberculosis assay on 2,169 clinical specimens, including respiratory and nonrespiratory specimens. Based on the genus assay, nontuberculous mycobacteria were readily detected and identified to the species level by PCR-mediated sequencing. In addition, our data point to a limited specificity of the Cobas Amplicor M. tuberculosis assay.

Development and evaluation of a molecular assay for detection of nontuberculous mycobacteria by use of the cobas amplicor platform.

We have developed and evaluated a semiautomated assay for detection of nontuberculous mycobacteria (NTM) from clinical samples based on the Cobas Amplicor Mycobacterium tuberculosis test (Roche Diagnostics, Switzerland). A capture probe, specific for mycobacteria at the genus level, was linked to magnetic beads and used for the detection of amplification products obtained by the Cobas Amplicor M. tuberculosis assay. We demonstrate that the analytical sensitivity of the genus assay is similar to that of Cobas Amplicor M. tuberculosis detection. Four hundred sixteen clinical specimens were evaluated for the presence of NTM DNA. Sensitivities for smear-positive and smear-negative specimens were found to be 100% and 47.9%, respectively. Specificity was 97.7%, the positive predictive value 84.6%, and the negative predictive value 93.1%. The genus assay is easy to perform, produces reliable results, and was found to be a valuable diagnostic tool for rapid diagnosis of infections with NTM. The genus assay has the potential to detect NTM not routinely recovered by culture and to discover new mycobacterial species.