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Almost a decade ago our diagnostic laboratory implemented an in-house real-time PCR for the detection of Plasmodium DNA to diagnose malaria in parallel with conventional diagnostics, i.e., microscopy (thick and thin smears), quantitative buffy coat microscopy (QBC), and a rapid diagnostic test (RDT). Here we report our experiences and make a comparison between the different diagnostic procedures used in this non-endemic setting. All patients during the period February 2009-December 2017 suspected of malaria were prospectively tested at the moment of sample collection. Both PCR and conventional malaria diagnostics were carried out on a total of 839 specimens from 825 patients. In addition, three Plasmodium falciparum (Pf) patients were closely followed by real-time PCR and microscopy after treatment. Overall, 56 samples (55 patients) tested positive by real-time PCR, of which six were missed by microscopy and seven by QBC. RDT showed fairly good results in detecting Pf, whereas specificity was not optimal. RDT failed to detect 10 of 17 non-Pf PCR positive specimens. One Plasmodium malariae patient would have been missed if only conventional diagnostic tests had been used. The high sensitivity of the PCR was confirmed by the number of PCR positive, microscopy negative post-treatment samples. In conclusion, within our routine diagnostic setting, malaria real-time PCR not only showed a high level of agreement with the conventional methods used, but also showed higher sensitivity and better specificity. Still, for complete replacement of the conventional procedures in a non-endemic setting, the time-to-results of the real-time PCR is currently too long.

Background: Human astroviruses (HAstV) comprise three phylogenetically compact and non-adjacent groups of species including classical HAstV (HAstV-C) and the novel ones (HAstV-VA/HMO and HAstV-MLB). Of these, HAstV-C is known to be responsible for gastroenteritis while the novel HAstV are associated with cases of neurological disorders. Accurate detection of all known variants by (real-time) PCR is challenging because of the high intra- and intergroup genetic divergence of HAstV., Objectives: To evaluate published HAstV PCR assays in silico, design de novo real-time PCR assays that can detect and discriminate three groups of HAstV, and apply those to patient samples to analyse the prevalence of HAstV in stool and cerebrospinal fluid (CSF) specimens., Study Design: In silico evaluation of published PCR assays and design of real-time PCR assays for detection of different subsets of HAstV was conducted within a common computational framework that used all astrovirus full genome sequences from GenBank. The newly designed real-time PCR assays were evaluated in vitro and applied to faecal samples (collected in January-May 2016) and cerebrospinal fluid specimens (2010-2016) from patients in the Netherlands., Results: Quantitative in silico evaluation of published PCRs is provided. The newly designed real-time PCR assays can reliably assign all available HAstV genome sequences to one of the three phylogenetic groups in silico, and differentiate among HAstV-specific controls in vitro. A total of 556 samples were tested using these PCR assays. Fourteen fecal samples (2.5%) tested positive for HAstV, 3 of which could be identified as the novel HAstV-MLB variants. No novel HAstV were found in CSF specimens., Conclusion: Newly designed real-time PCR assays with improved detection of all known HAstV allowed the first-time identification of novel astroviruses from stool samples in the Netherlands., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Infections of the respiratory tract can be caused by a diversity of pathogens, both viral and bacterial. Rapid microbiological diagnosis ensures appropriate antimicrobial therapy as well as effective implementation of isolation precautions. The ePlex respiratory pathogen panel (RP panel) is a novel molecular biology-based assay, developed by GenMark Diagnostics, Inc. (Carlsbad, CA), to be performed within a single cartridge for the diagnosis of 25 respiratory pathogens (viral and bacterial). The objective of this study was to compare the performance of the RP panel with those of laboratory-developed real-time PCR assays, using a variety of previously collected clinical respiratory specimens. A total of 343 clinical specimens as well as 29 external quality assessment (EQA) specimens and 2 different Middle East respiratory syndrome coronavirus isolates have been assessed in this study. The RP panel showed an agreement of 97.4% with the real-time PCR assay regarding 464 pathogens found in the clinical specimens. All pathogens present in clinical samples and EQA samples with a threshold cycle ( C T ) value of <30 were detected correctly using the RP panel. The RP panel detected 17 additional pathogens, 7 of which could be confirmed by discrepant testing. In conclusion, this study shows excellent performance of the RP panel in comparison to real-time PCR assays for the detection of respiratory pathogens. The ePlex system provided a large amount of useful diagnostic data within a short time frame, with minimal hands-on time, and can therefore potentially be used for rapid diagnostic sample-to-answer testing, in either a laboratory or a decentralized setting., (Copyright © 2017 Nijhuis et al.)

Rapid identification of patients colonized with carbapenemase-producing Enterobacteriaceae (CPE) is essential to prevent introduction and the spread of CPE in the hospital. This article presents the results of a new screening method to detect patients colonized with CPE within 24h after hospital admission. From high-risk patients rectal and throat swabs were collected and incubated overnight, after which DNA was isolated and tested for the most prevalent CPE genes (KPC, NDM, OXA-48, VIM and IMP) by a ligation-mediated real-time polymerase chain reaction. In 14 months 454 patients were screened; in six patients CPE were detected (carriage rate 1.3%)., (Copyright © 2014 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

To prevent the spread of carbapenemase-producing bacteria, a fast and accurate detection of patients carrying these bacteria is extremely important. The Check-Direct CPE assay (Check-Points, Wageningen, The Netherlands) is a new multiplex real-time PCR assay, which has been developed to detect and differentiate between the most prevalent carbapenemase genes encountered in Enterobacteriaceae (blaKPC, blaOXA-48, blaVIM, and blaNDM) directly from rectal swabs. Evaluation of this assay using 83 non-duplicate isolates demonstrated 100% sensitivity and specificity and the correct identification of the carbapenemase gene(s) present in all carbapenemase-producing isolates. Moreover, the limit of detection (LoD) of the real-time PCR assay in spiked rectal swabs was determined and showed comparable LoDs with the ChromID CARBA agar. With an excellent performance on clinical isolates and spiked rectal swabs, this assay appeared to be an accurate and rapid method to detect blaKPC, blaOXA-48, blaVIM, and blaNDM genes directly from a rectal screening swab., (© 2013.)

Extended-spectrum β-lactamases (ESBLs) are emerging worldwide, making rapid and adequate ESBL detection crucial for infection control measures as well as for the choice of correct antimicrobial therapy. The aim of this study was to compare the performance of a novel rapid ligation-mediated real-time PCR (LM-PCR) with a combination disc test (CDT). In total, 172 prospective putative ESBL-positive Enterobacteriaceae isolates from clinical specimens based on VITEK2 results were included in this study and tested with the phenotypic CDT and the LM-PCR. Positive ESBL results were obtained in 100 and 95 isolates using CDT and LM-PCR, respectively. The sensitivity, specificity, negative predictive value and positive predictive value of the LM-PCR were 99.0, 92.2, 98.6 and 94.0 %, respectively, compared with the CDT. The LM-PCR technique provides an important reduction in turnaround time (~4.5 h versus overnight incubation using CDT) for ESBL confirmation. As a consequence, all ESBL results are available within the same day, making this assay an important tool for rapid and accurate ESBL detection.