18 research outputs found
Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics
Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 104 copies/ml (corresponding to CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.</p
Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics
Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 104 copies/ml (corresponding to CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.</p
Staphylococcus aureus endocarditis: Identifying prognostic factors using a method derived from morbidity and mortality conferences
ObjectivesLethality of Staphylococcus aureus (Sa) infective endocarditis (IE) is high and might be due to yet unidentified prognostic factors. The aim of this study was to search for new potential prognostic factors and assess their prognostic value in SaIE.Materials and methodsWe used a two-step exploratory approach. First, using a qualitative approach derived from mortality and morbidity conferences, we conducted a review of the medical records of 30 patients with SaIE (15 deceased and 15 survivors), randomly extracted from an IE cohort database (NCT03295045), to detect new factors of possible prognostic interest. Second, we collected quantitative data for these factors in the entire set of SaIE patients and used multivariate Cox models to estimate their prognostic value.ResultsA total of 134 patients with modified Duke definite SaIE were included, 64 of whom died during follow-up. Of the 56 candidate prognostic factors identified at the first step, 3 had a significant prognostic value in multivariate analysis: the prior use of non-steroidal anti-inflammatory drugs [aHR 3.60, 95% CI (1.59–8.15), p = 0.002]; the non-performance of valve surgery when indicated [aHR 1.85, 95% CI (1.01–3.39), p = 0.046]; and the decrease of vegetation size on antibiotic treatment [aHR 0.34, 95% CI (0.12–0.97), p = 0.044].ConclusionWe identified three potential SaIE prognostic factors. These results, if externally validated, might eventually help improve the management of patients with SaIE
Genome-wide distribution of histone trimethylation reveals a global impact of bisphenol A on telomeric binding proteins and histone acetyltransferase factors: a pilot study with human and in vitro data
International audienceObjective To assess the genetic and epigenetic effects promoted by Bisphenol A (BPA) exposure in adolescent males from the Spanish INMA-Granada birth cohort, and in human cells. Methods DNA methylation was analysed using MEDIP. Repeat number variation in genomic DNA was evaluated, along with the analysis of H3K4me3 by using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). Analyses were performed with material extracted from whole blood of the adolescents, complemented by in vitro assessments of human (HeLa) cells exposed to 10 nM BPA, specifically, immunofluorescence evaluation of protein levels, gene expression analysis and ChIP‒qPCR analysis. Results Adolescents in the high urinary BPA levels group presented a higher level of Satellite A (SATA) repetitive region copy numbers compared to those in the low BPA group and a tendency towards increase in telomere length. We also observed decreased DNA methylation at the promoters of the imprinted genes H19, KCNQ1, and IGF2 ; at LINE1 retroelements; and at the ARID2, EGFR and ESRRA and TERT genes. Genome-wide sequencing revealed increased H3K4me3 occupancy at the promoters of genes encoding histone acetyltransferases, telomeric DNA binding factors and DNA repair genes. Results were supported in HeLa cells exposed to 10 nM BPA in vitro. In accordance with the data obtained in blood samples, we observed higher H3K4me3 occupancy and lower DNA methylation at some specific targets in HeLa cells. In exposed cells, changes in the expression of genes encoding DNA repair factors ( ATM, ARID2, TRP53 ) were observed, and increased expression of several genes encoding telomeric DNA binding factors ( SMG7, TERT, TEN1, UPF1, ZBTB48 ) were also found. Furthermore, an increase in ESR1/ERa was observed in the nuclei of HeLa cells along with increased binding of ESR1 to KAT5, KMT2E and TERF2IP promoters and decreased ESR1 binding at the RARA promoter. The DNA damage marker p53/TP53 was also increased. Conclusion In this pilot study, genome-wide analysis of histone trimethylation in adolescent males exposed to BPA revealed a global impact on the expression of genes encoding telomeric binding proteins and histone acetyltransferase factors with similar results in HeLa cells. Nevertheless, larger studies should confirm our findings
Staphylococcus aureus endocarditis: Identifying prognostic factors using a method derived from morbidity and mortality conferences
International audienceObjectives: Lethality of Staphylococcus aureus (Sa) infective endocarditis (IE) is high and might be due to yet unidentified prognostic factors. The aim of this study was to search for new potential prognostic factors and assess their prognostic value in SaIE.Materials and methods: We used a two-step exploratory approach. First, using a qualitative approach derived from mortality and morbidity conferences, we conducted a review of the medical records of 30 patients with SaIE (15 deceased and 15 survivors), randomly extracted from an IE cohort database (NCT03295045), to detect new factors of possible prognostic interest. Second, we collected quantitative data for these factors in the entire set of SaIE patients and used multivariate Cox models to estimate their prognostic value.Results: A total of 134 patients with modified Duke definite SaIE were included, 64 of whom died during follow-up. Of the 56 candidate prognostic factors identified at the first step, 3 had a significant prognostic value in multivariate analysis: the prior use of non-steroidal anti-inflammatory drugs [aHR 3.60, 95% CI (1.59-8.15), p = 0.002]; the non-performance of valve surgery when indicated [aHR 1.85, 95% CI (1.01-3.39), p = 0.046]; and the decrease of vegetation size on antibiotic treatment [aHR 0.34, 95% CI (0.12-0.97), p = 0.044].Conclusion: We identified three potential SaIE prognostic factors. These results, if externally validated, might eventually help improve the management of patients with SaIE