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Molecular-based detection of bacterial pathogens directly from clinical specimens permits rapid initiation of effective antimicrobial treatment and adequate patient management. Broad-range polymerase chain reaction (PCR) amplification of the 16S rRNA gene (16S rDNA qPCR) is used in many diagnostic laboratories as a complement to cultural identification of bacterial pathogens. However, efforts for automation of 16S rDNA PCR workflows are needed in order to reduce turnaround times and to enhance reproducibility and standardization of the technique. In this retrospective method evaluation study, clinical specimens (N = 499) from patients with suspected bacterial infections were used to evaluate 2 diagnostic semiautomated workflows for rapid bacterial pathogen detection. The workflows included automated DNA extraction (QIASymphony), 16S rDNA qPCR, fragment or melting curve analysis, and amplicon sequencing. Our results support the use of the 16S rDNA qPCR and fragment analysis workflow as it enabled rapid and accurate identification of bacterial pathogens in clinical specimens., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Acute bacterial meningitis is a medical emergency, and delays in initiating effective antimicrobial therapy result in increased morbidity and mortality. Culture-based methods, thus far considered the "gold standard" for identifying bacterial microorganisms, require 24 to 48 h to provide a diagnosis. In addition, antimicrobial therapy is often started prior to clinical sample collection, thereby decreasing the probability of confirming the bacterial pathogen by culture-based methods. To enable a fast and accurate detection of the most important bacterial pathogens causing meningitis, namely, Streptococcus pneumoniae , Haemophilus influenzae , Neisseria meningitidis , Streptococcus agalactiae , and Listeria monocytogenes , we evaluated a commercially available multiplex LightMix real-time PCR (RT-PCR) in 220 cerebrospinal fluid (CSF) specimens. The majority of CSF samples were collected by lumbar puncture, but we also included some CSF samples from patients with symptoms of meningitis from the neurology department that were recovered from shunts. CSF samples were analyzed by multiplex RT-PCR enabling a first diagnosis within a few hours after sample arrival at our institute. In contrast, bacterial identification took between 24 and 48 h by culture. Overall, a high agreement of bacterial identification between culture and multiplex RT-PCR was observed (99%). Moreover, multiplex RT-PCR enabled the detection of pathogens, S. pneumoniae ( n = 2), S. agalactiae ( n = 1), and N. meningitidis ( n = 1), in four culture-negative samples. As a complement to classical bacteriological CSF culture, the LightMix RT-PCR assay proved to be valuable by improving the rapidity and accuracy of the diagnosis of bacterial meningitis., (Copyright © 2018 Wagner et al.)

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.