Titel Deelnemers "Abcès hépatique amibien contract‚ en Inde avec confirmation du diagnostic par PCR" "G. Ho, M.M. Arenas Sanchez, P. Léonard, Marjan Van Esbroeck, M.P. Hayette" "Suivi du premier cas d'infection à Mycobacterium ulcerans confirmé par culture, PCR et génotypage en République du Congo-Brazzaville" "Anatole Kibadi Kapay, Pieter Stragier, Jean-Jacques Muyembe, J Pedrosa, Françoise Portaels" "Suivi du premier cas d'infection à Mycobacterium ulcerans confirmé par culture, PCR et génotypage en République du Congo-Brazzaville" "K Kibadi, Pieter Stragier, J Muyembe-Tamfum, J Pedrosa, Françoise Portaels" "Evaluation and comparison of the Amplicor HIV-1 PCR kit with an 'in house' nested PCR" "K. Fransen, I Van Kerckhoven, P. Piot, Guido Van Der Groen" "Comparison of Leishmania OligoC-TesT PCR with conventional and real-time PCR for the detection of canine Leishmania infection" "C Carson, RJ Quinnell, J Holden, LM Garcez, Stijn Deborggraeve, O Courtenay" "There is a need for standardization and simplification of the existing methods for molecular detection of Leishmania infantum in the canine reservoir host. The commercially available OligoC-TesT kit incorporates standardized PCR reagents with rapid oligochromatographic dipstick detection of PCR products, and is highly sensitive in humans, but not yet independently validated in dogs. Here we compare the sensitivity of the OligoC-TesT with that of nested kinetoplast DNA (kDNA) PCR, nested internal transcribed spacer-1 (ITS-1) PCR, and a PCR/hybridization protocol, in longitudinal naturally infected canine bone marrow samples for which parasite burdens were measured by real-time quantitative PCR (qPCR). Sensitivity of the OligoC-TesT in infected dogs was 70% (95% C.I. 63-78%), similar to that of kDNA PCR (72%; 95% C.I. 65-80%; P=0.69), but significantly greater than PCR/hybridization (61%; 95% C.I. 53-69%; P=0.007) and ITS-1 nested PCR (54%; 95% C.I. 45-62%; P" "Prospective multicenter comparison of urine culture with PCR on dried blood spots using 2 different extraction and PCR methods in neonates suspected for congenital cytomegalovirus infection" "Koen Vercauteren, Annelies Keymeulen, Ludo Mahieu, Veerle Cossey, Alexandra Casaer, Christine Van Mol, Koenraad Smets, Elizaveta Padalko" "To evaluate the potential of dried blood spots (DBS) as a congenital cytomegalovirus (cCMV) testing specimen, the laboratory diagnostic accuracy of polymerase chain reaction (PCR) on DBS was compared to viral urine cultures from neonates suspected for cCMV. Two different extraction methods (EasyMAG, bioMerieux versus Qiagen) and 2 real-time PCR protocols (in-house versus Argene) were compared. We were able to collect both DBS and urine samples in 6 Belgian neonatal units from 276 neonates suspected for cCMV registered in CMVREG (an online neonatal registry system). Forty-eight neonates (17.4%) were positive by viral culture in urine. Laboratory diagnostic accuracy parameters of DBS-PCR were both extraction method and PCR protocol dependent. Not all DBS-CMV-PCR methods successfully detected urine-culture-positive neonates born after first-trimester seroconversions. Interestingly, however, all urine-culture-positive neonates having clinical signs of cCMV did consistently score positive. Keywords: Cytomegalovirus Congenital Dried blood spot testing (C) 2020 Elsevier Inc. All rights reserved." "PCR based target enrichment for variant confirmation, gene panels and multiplex PCR sample tracking in a whole exome sequencing workflow" "Frauke Coppieters, Thalia Van Laethem, Matthias De Smet, Paul Coucke, Elfride De Baere, Kathleen Claes, Björn Menten, Jo Vandesompele, Steve Lefever" "Background: Targeted PCR-based resequencing is an important application in clinical diagnostics. Using our best-in-class primer design tool primerXL, we have designed almost one million PCR assays for both fresh frozen and formalin-fixed paraffin-embedded samples, covering the entire human exome. Over 6200 assays for hundreds of clinically relevant genes in total were wet-lab validated. In addition, over 5000 patient-specific variants, from exome sequencing, were confirmed using pxlence PCR assays. All singleplex PCR assays work under universal PCR conditions and result in equimolar sequencing coverage. As a latest addition, we present the compatibility of pxlence assays with multiplex PCR applications. As a first product, we designed and validated a cost-effective and flexible sample tracking test. This primer pool enables fast identification of sample swapping or contamination which may occur in laborious library preparation workflows. Methods: Thirty SNPs were selected based on their minor allele frequency, exonic location and overlap with the capture region of exome enrichment kits. We evaluated three different mastermixes for multiplex PCR and two library preparation methods, followed by 150 bp paired-end sequencing on a MiSeq instrument (Illumina). Results: The SsoAdvanced PreAmp Supermix (Bio-Rad) resulted in superior homogenous coverage following multiplex PCR of all SNP assays (pxlence). No significant difference in coverage uniformity was observed between the Nextera DNA Flex and the NexteraXT DNA library prep method (both Illumina). In virtually all tested DNA samples (n=393), 86.29% of the SNPs had a uniform coverage within 2-fold of the mean. Based on the SNP genotypes, DNA samples could unambiguously be discriminated. Conclusion: In conclusion, pxlence provides high-quality and versatile PCR assays for various targeted resequencing applications. Here, we designed and validated a novel sample tracking test for whole exome or whole genome sequencing, involving a straightforward single multiplex PCR reaction followed by DNA sequencing library prep. In principle, our strategy could also be used to design gene panel-specific sample tracking solutions." "Comparison of SPF10 real-time PCR and conventional PCR in combination with the INNO-LiPA HPV Genotyping Extra assay for the detection and typing of human papillomavirus in cervical samples" "Maria Isabel Micalessi, Gaëlle Boulet, S. Pillet, J. Jacquet, B. Pozzetto, John-Paul Bogers, T. Bourlet" "The novel SPF10 real-time PCR assay allows the simultaneous amplification and detection of the HPV target. That way, LiPA analysis of the HPV-negative samples can be avoided, reducing workload and cost. This study aims to evaluate the performance of the SPF10 real-time PCR in combination with the LiPA assay for HPV detection and typing in cervical samples. Thirty-nine cervical samples were subjected to the SPF10 conventional PCR in combination with the LiPA assay. Subsequently, the SPF10 real-time PCR was performed to enable the comparison between the SPF10 conventional and the real-time PCR results. In case of discrepancy, the samples were subjected to the CLAR(R) HPV2 assay. As a result, 27 out of 39 samples were identified as HPV-positive by the SPF10 real-time PCR and were genotyped further by the LiPA assay. Twenty samples (74.1%) showed an absolute agreement between the conventional and real-time SPF10 PCR (concordant), three (11.1%) displayed additional or fewer types (compatible), two (7.4%) did not show any similarity between both assays (discordant) and the remaining two (7.4%) were LiPA-negative. The two assays showed an excellent strength of agreement for individual (k = 0.932) and multiple genotype detection (k = 0.834). In conclusion, the two SPF10 PCR methods are comparable. Therefore, the SPF10 real-time PCR with subsequent LiPA could be used for the detection and genotyping of HPV in cervical samples. (C) 2013 Elsevier B.V. All rights reserved." "The clinical performance of Aspergillus PCR when testing serum and plasma- a study by the European Aspergillus PCR Initiative" "Katrien Lagrou" "Aspergillus PCR testing of serum provides technical simplicity, but with potentially reduced sensitivity compared to whole blood testing. For a disease where screening to exclude disease provides an optimal strategy sensitivity is paramount. The associated analytical study confirmed that DNA concentrations were greater in plasma compared to serum. The aim of this current investigation was to confirm the analytical findings by comparing the performance of Aspergillus PCR testing of plasma and serum in the clinical setting.Standardised Aspergillus PCR was performed on plasma and serum samples concurrently obtained from haematology patients, as a multi-centre retrospective anonymous case-control study with cases diagnosed according to EORTC/MSG consensus definitions (19 proven/probable cases and 42 controls). Clinical performance and clinical utility (time to positivity) were calculated for both samples.The sensitivity and specificity for Aspergillus PCR when testing serum were 68.4% and 76.2%, and plasma were 94.7% and 83.3%, respectively. Eighty-five percent of serum and plasma PCR results were concordant. On average plasma PCR was positive 16.8 days before diagnosis and was the earliest indicator of infection in 13 cases, combined with other biomarkers in five cases. On average serum PCR was positive 10.8 days before diagnosis and was the earliest indicator of infection in six cases, combined with other biomarkers in three cases." "Technical and clinical validation of three commercial real-time PCR kits for the diagnosis of neuroborreliosis in cerebrospinal fluid on three different real-time PCR platforms" "Liesbeth Maes, Vicky De Preter, Sven Ignoul, Leen Braeken" "This study reports the evaluation of the technical and clinical validation of the O-DiaBorburg kit (DIA), Borrelia burgdorferi PCR kit, ISEX (GENE), and Borrelia burgdorferi sensu lato Real-TM (SAC) for the diagnosis of neuroborreliosis in cerebrospinal fluid based on both Borrelia DNA and CSF samples from patients with clinical suspicion of neuroborreliosis. This validation study was done by analysing the kits on the Rotorgene Q (RGQ), CFX96, and LightCycler480 (LC480). For all kits, the linear range was larger on RGQ than on CFX96 and LC480. A good reproducibility was obtained for all assays on all instruments. Storage at -20 °C resulted in a decreased reproducibility for SAC. Results of the limit of detection (LOD95) experiments indicated a better sensitivity than described in the kit insert for all kits on all PCR platforms. No cross-reactivity was found for genetically related organisms nor for other pathogens which may be present in CSF. All species of the Borrelia burgdorferi sensu lato complex were detected with the GENE and SAC kits. The DIA kit failed to detect B. lusitaniae. The results seemed to indicate a better overall performance for the GENE kit on RGQ. However, its diagnostic value could not be confirmed in the clinical validation study, wherein none of the 103 CSF samples from clinical neuroborreliosis cases showed a positive real-time PCR result with the GENE kit analysed on RGQ."