scholarly journals Speed, accuracy, sensitivity and quality control choices for detecting clinically relevant microbes in whole blood from patients

2019 ◽  
Author(s):  
James Thornton ◽  
George S. Watts ◽  
Ken Youens-Clark ◽  
Lee D. Cranmer ◽  
Bonnie L. Hurwitz
Keyword(s):  
Quality Control ◽  
Febrile Neutropenia ◽  
Relative Abundance ◽  
Whole Blood ◽  
Computer Time ◽  
Health Concern ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Bacterial Mixtures ◽  
Speed Accuracy

ABSTRACTInfections are a serious health concern worldwide, particularly in vulnerable populations such as the immunocompromised, elderly, and young. Advances in metagenomic sequencing availability, speed, and decreased cost offer the opportunity to supplement or replace culture-based identification of pathogens with DNA sequence-based diagnostics. Adopting metagenomic analysis for clinical use requires that all aspects of the pipeline are optimized and tested, including data analysis. We tested the accuracy, sensitivity, and resource requirements of Centrifuge within the context of clinically relevant bacteria. Binary mixtures of bacteria showed Centrifuge reliably identified organisms down to 0.1% relative abundance. A staggered mock bacterial community showed Centrifuge outperformed CLARK while requiring less computing resources. Shotgun metagenomes obtained from whole blood in three febrile neutropenia patients showed Centrifuge could identify both bacteria and viruses as part of a culture-free workflow. Finally, Centrifuge results changed minimally by eliminating time-consuming read quality control and host screening steps.AUTHOR SUMMARYImmunocompromised patients, such as those with febrile neutropenia (FN), are susceptible to infections, yet cultures fail to identify causative organisms ~80% of the time. High-throughput metagenomic sequencing offers a promising approach for identifying pathogens in clinical samples. Mining through metagenomes can be difficult given the volume of reads, overwhelming human contamination, and lack of well-defined bioinformatics methods. The goal of our study was to assess Centrifuge, a leading tool for the identification and quantitation of microbes, and provide a streamlined bioinformatics workflow real-word data from FN patient blood samples. To ensure the accuracy of the workflow we carefully examined each step using known bacterial mixtures that varied by genetic distance and abundance. We show that Centrifuge reliably identifies microbes present at just 1% relative abundance and requires substantially less computer time and resource than CLARK. Moreover, we found that Centrifuge results changed minimally by quality control and host-screening allowing for further reduction in compute time. Next, we leveraged Centrifuge to identify viruses and bacteria in blood draws for three FN patients, and confirmed suspected pathogens using genome coverage plots. We developed a web-based tool in iMicrobe and detailed protocols to promote re-use.

Speed, accuracy, sensitivity and quality control choices for detecting clinically relevant microbes in whole blood from patients v1 (protocols.io.wjdfci6)

protocols.io ◽  
2018 ◽  
Author(s):  
James Thornton ◽  
George Watts ◽  
Ken Youens ◽  
Lee Crammer ◽  
Bonnie Hurwitz
Keyword(s):  
Quality Control ◽  
Whole Blood ◽  
Speed Accuracy

scholarly journals High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic Nanopore sequencing

2020 ◽  
Author(s):  
Nicholas D Sanderson ◽  
Jeremy Swann ◽  
Leanne Barker ◽  
James Kavanagh ◽  
Sarah Hoosdally ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Health Concern ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Entire Genome ◽  
Culture Independent ◽  
Significant Public Health

AbstractThe rise of antimicrobial resistant Neisseria gonorrhoeae is a significant public health concern. Against this background, rapid culture-independent diagnostics may allow targeted treatment and prevent onward transmission. We have previously shown metagenomic sequencing of urine samples from men with urethral gonorrhoea can recover near-complete N. gonorrhoeae genomes. However, disentangling the N. gonorrhoeae genome from metagenomic samples and robustly identifying antimicrobial resistance determinants from error-prone Nanopore sequencing is a substantial bioinformatics challenge.Here we demonstrate an N. gonorrhoeae diagnostic workflow for analysis of metagenomic sequencing data obtained from clinical samples using R9.4.1 Nanopore sequencing. We compared results from simulated and clinical infections with data from known reference strains and Illumina sequencing of isolates cultured from the same patients. We evaluated three Nanopore variant callers and developed a random forest classifier to filter called SNPs. Clair was the most suitable variant caller after SNP filtering. A minimum depth of 20x reads was required to confidently identify resistant determinants over the entire genome. Our findings show that metagenomic Nanopore sequencing can provide reliable diagnostic information in N. gonorrhoeae infection.

scholarly journals Assessment of Plasmodium antigens and CRP in dried blood spots with multiplex malaria array

2021 ◽  
Author(s):  
Ihn Kyung Jang ◽  
Sara Aranda ◽  
Rebecca Barney ◽  
Andrew Rashid ◽  
Muhammad Helwany ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Whole Blood ◽  
Dried Blood Spots ◽  
Careful Consideration ◽  
Clinical Samples ◽  
Sample Type ◽  
Sample Collection ◽  
Malaria Surveillance ◽  
Inflammatory Biomarker ◽  
Blood Spots

AbstractDried blood spots (DBS) typically prepared on filter papers are an ideal sample type for malaria surveillance by offering easy and cost-effective methods in terms of sample collection, storage, and transport. The objective of this study was to evaluate the applicability of DBS with a commercial multiplex malaria assay, developed to concurrently measure Plasmodium antigens, histidine-rich protein 2 (HRP2), Plasmodium lactate dehydrogenase (pLDH), and a host inflammatory biomarker, C-reactive protein (CRP), in whole blood. The assay conditions were optimized for DBS, and thermal stability for measurement of Plasmodium antigens and CRP in dried blood were determined. Performance of the multiplex assay on matched DBS and whole blood pellet samples was also evaluated using the clinical samples. The results indicate the acceptable performance in multiplex antigen detection using DBS samples. At cutoff levels for DBS, with a diagnostic specificity with a lower 95% confidence bound > 92%, diagnostic sensitivities against polymerase chain reaction (PCR)–confirmed malaria for HRP2, Pf LDH, Pv LDH, and Pan LDH were 93.5%, 80.4%, 21.3%, and 55.6%, respectively. The half-life of pLDH was significantly less than that of HRP2 in thermal stability studies. Results with DBS samples collected from Peru indicate that the uncontrolled storage conditions of DBS can result in inaccurate reporting for infection with P. falciparum parasites with hrp2/3 deletions. With careful consideration that minimizing the unfavorable DBS storage environment is essential for ensuring integrity of heat-labile Plasmodium antigens, DBS samples can be used as an alternative to liquid whole blood to detect P. falciparum with hrp2/3 deletions in malaria surveillance.

scholarly journals Molecular detection of Brucella spp. from broth culture of clinical samples in Nigeria: Its role in vaccine quality control

2009 ◽  
Vol 8 (21) ◽  
pp. 5661-5665 ◽  
Author(s):  
F Antiabong J ◽  
Yakubu B ◽  
A Owolodun O ◽  
Bertu W ◽  
A Ocholi R
Keyword(s):  
Quality Control ◽  
Molecular Detection ◽  
Broth Culture ◽  
Clinical Samples

scholarly journals Use of platelet-rich plasma in regenerative medicine: technical tools for correct quality control

2018 ◽  
Vol 4 (1) ◽  
pp. e000442 ◽  
Author(s):  
Hajer Graiet ◽  
Anna Lokchine ◽  
Pauline Francois ◽  
Melanie Velier ◽  
Fanny Grimaud ◽  
...  
Keyword(s):  
Quality Control ◽  
Whole Blood ◽  
Sports Medicine ◽  
Platelet Rich Plasma ◽  
Systematic Sampling ◽  
Significant Difference ◽  
Platelet Concentration ◽  
Harvesting Method ◽  
Clinical Interest ◽  
The Subject

Background/aimsPlatelet-rich plasma (PRP) injections are used in sports medicine and have been the subject of increased clinical interest. However, there have been very few reports of the composition of initial whole blood and the final PRP product. The objective of this study was to provide technical tools to perform a correct characterisation of platelets, leucocytes and red blood cells (RBCs) from whole blood and PRP.MethodsBlood and PRP were obtained from 26 healthy volunteers and prepared according to the varying parameters encountered within PRP process preparation and quantification (harvesting method, anticoagulant used, sampling method, counting method). Concentrations were measured at t=0, t=1, t=6 and t=24 hours.ResultsSampling of blood in Eppendorf tubes significantly decreased platelet concentration over time, whereas sampling in Microvette EDTA-coated tube kept platelet concentration stable until 24 hours. A non-significant difference was observed in platelet counts in PRP with impedance (median (IQR): 521.8 G/L (505.3–524.7)) and fluorescence (591.5 G/L (581.5–595.8)) methods. Other studied parameters did not influence platelet concentrations in blood or PRP samples. Leucocytes and RBC counts were similar whatever the anticoagulant, sampling, harvesting and counting methods used for both blood and PRP samples.ConclusionsSystematic sampling of blood and PRP in EDTA-coated tubes for quality control is recommended. The use of a validated counter for PRP sample should also be taken into account.

scholarly journals Correction to: Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

Microbiome ◽  
2017 ◽  
Vol 5 (1) ◽  
Author(s):  
Dagmara W. Lewandowska ◽  
Osvaldo Zagordi ◽  
Fabienne-Desirée Geissberger ◽  
Verena Kufner ◽  
Stefan Schmutz ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Clinical Samples ◽  
Metagenomic Sequencing

scholarly journals Benchmark of thirteen bioinformatic pipelines for metagenomic virus diagnostics using datasets from clinical samples

2021 ◽  
Author(s):  
Jutte J.C. de Vries ◽  
Julianne R. Brown ◽  
Nicole Fischer ◽  
Igor A. Sidorov ◽  
Sofia Morfopoulou ◽  
...  
Keyword(s):  
De Novo ◽  
Respiratory Infections ◽  
Limit Of Detection ◽  
Bioinformatic Analysis ◽  
Clinical Samples ◽  
Mixed Infections ◽  
Metagenomic Sequencing ◽  
User Friendliness ◽  
Viral Pathogens ◽  
Clinical Diagnostic

Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories. Methods Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analysed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analysed. Results Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection. Conclusion A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases.

scholarly journals Obesity, Body Image Dissatisfaction, and Sexual Dysfunction: A Narrative Review

Sexes ◽  
2022 ◽  
Vol 3 (1) ◽  
pp. 20-39
Author(s):  
Sean M. McNabney
Keyword(s):  
Body Image ◽  
Sexual Dysfunction ◽  
Sexual Function ◽  
Adult Population ◽  
Body Image Dissatisfaction ◽  
The United States ◽  
The Body ◽  
Narrative Review ◽  
Health Concern ◽  
Clinical Samples

With approximately two-thirds of the United States adult population classified as overweight or obese, obesity remains a critical public health concern. Obesity not only contributes to several health complications including type 2 diabetes mellitus and cardiovascular disease, but the condition is also associated with sexual dysfunction in both women and men. Despite evidence linking obesity and its concomitant pathophysiology to sexual problems, the potential roles of psychosocial factors such as body image are understudied. This narrative review evaluates the research linkages between obesity and sexual dysfunction, with particular attention to the potential effects of body image dissatisfaction. A literature search of biomedical and psychological databases was used to identify research pertaining to obesity, sexual function, and/or body image constructs. The pathophysiological effects of obesity on sexual function are well-documented in mechanistic studies and animal trials, often with corroboration in human clinical samples. However, very few studies examine obesity, body image, and sexual function in tandem. Body image dissatisfaction appears to independently impinge upon the sexual response cycle and mental health outcomes, irrespective of body weight. While obesity is often associated with negative body image appraisal, it is unclear whether these constructs exert additive, synergistic, or antagonistic effects on sexual responsivity. Additionally, overweight/obese individuals who exhibit higher levels of body image satisfaction or self-confidence appear to be protected from the deleterious effects of obesity on sexual satisfaction, at least to some extent. Greater reliance upon conceptual/theoretical models from the body image literature may better clarify the relationships between these constructs.

scholarly journals Multiplexed Detection of Sepsis Markers in Whole Blood using Nanocomposite Coated Electrochemical Sensors

2020 ◽  
Author(s):  
Uroš Zupančič ◽  
Pawan Jolly ◽  
Pedro Estrela ◽  
Despina Moschou ◽  
Donald E. Ingber
Keyword(s):  
Electrochemical Detection ◽  
Whole Blood ◽  
Electrochemical Sensors ◽  
Point Of Care ◽  
Sample Volume ◽  
Nanocomposite Coating ◽  
Cross Reactivity ◽  
Detection Methods ◽  
Clinical Samples ◽  
Undiluted Serum

ABSTRACTSepsis is a leading cause of mortality worldwide that is difficult to diagnose and manage because this requires simultaneous analysis of multiple biomarkers. Electrochemical detection methods could potentially provide a way to accurately quantify multiple sepsis biomarkers in a multiplexed manner as they have very low limits of detection and require minimal sensor instrumentation; however, affinity-based electrochemical sensors are usually hampered by biological fouling. Here we describe development of an electrochemical detection platform that enables detection of multiple sepsis biomarkers simultaneously by incorporating a recently developed nanocomposite coating composed of crosslinked bovine serum albumin containing a network of reduced graphene oxide nanoparticles that prevents biofouling. Using nanocomposite coated planar gold electrodes, we constructed a procalcitonin sensor and demonstrated sensitive PCT detection in undiluted serum and clinical samples, as well as excellent correlation with a conventional ELISA (adjusted r2 = 0.95). Sensors for two additional sepsis biomarkers — C-reactive protein and pathogen-associated molecular patterns — were developed on the same multiplexed platform and tested in whole blood. Due to the excellent antifouling properties of the nanocomposite coating, all three sensors exhibited specific responses within the clinically significant range without any cross-reactivity in the same channel with low sample volume. This platform enables sensitive simultaneous electrochemical detection of multiple analytes in human whole blood, which can be expanded further to any target analyte with an appropriate antibody pair or capturing probe, and thus, may offer a potentially valuable tool for development of clinical point-of-care diagnostics.GRAPHICAL ABSTRACT

scholarly journals The Infant Gut Resistome is Associated with E. coli and Early-life Exposures

2020 ◽  
Author(s):  
Rebecca M. Lebeaux ◽  
Modupe O. Coker ◽  
Erika F. Dade ◽  
Thomas J. Palys ◽  
Hilary G. Morrison ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Relative Risk ◽  
Relative Abundance ◽  
Gut Microbiome ◽  
Early Life ◽  
The United States ◽  
Delivery Mode ◽  
Metagenomic Sequencing ◽  
E Coli ◽  
Early Life Exposures

Abstract Background: Antibiotic resistance is an increasing threat to human health. The human gut microbiome harbors a collection of bacterial antimicrobial resistance genes (ARGs) known as the resistome. The factors associated with establishment of the resistome in early life are not well understood and clarifying these factors would inform strategies to decrease antibiotic resistance. We investigated the early-life exposures and taxonomic signatures associated with resistome development over the first year of life in a large, prospective cohort in the United States. Shotgun metagenomic sequencing was used to profile both microbial composition and ARGs in stool samples collected at 6 weeks and 1 year of age from infants enrolled in the New Hampshire Birth Cohort Study. Negative binomial regression and statistical modeling was used to examine infant factors such as sex, delivery mode, feeding method, gestational age, antibiotic exposure, and infant gut microbiome composition in relation to the diversity and relative abundance of ARGs.Results: Metagenomic sequencing was performed on paired samples from 195 full term (at least 37 weeks’ gestation) and 15 late preterm (33-36 weeks’ gestation) infants. 6-week samples compared to 1-year samples had 4.37 times (95% CI: 3.54-5.39) the rate of harboring ARGs. The majority of ARGs that were at a greater relative abundance at 6 weeks (chi-squared p < 0.01) worked through the mechanism of antibiotic efflux (i.e., by pumping antibiotics out of the cell). The overall relative abundance of the resistome was strongly correlated with Proteobacteria (Spearman correlation = 78.9%) and specifically E. coli (62.2%) relative abundance in the gut microbiome. Among infant characteristics, delivery mode was most strongly associated with the diversity and relative abundance of ARGs. Infants born via cesarean delivery had a higher risk of harboring unique ARGs [relative risk = 1.12 (95% CI: 0.97 – 1.29)] as well as a having an increased risk for overall ARG relative abundance [relative risk = 1.43 (95% CI: 1.12 – 1.84)] at 1 year compared to infants born vaginally. Additionally, 6 specific ARGs were at a greater relative abundance in infants delivered by cesarean section compared to vaginally delivered infants across both time points. Conclusions: Our findings suggest that the developing infant gut resistome may be alterable by early-life exposures. Establishing the extent to which infant characteristics and early-life exposures impact the resistome can ultimately lead to interventions that decrease the transmission of ARGs and thus the possibility of antibiotic resistant life threatening infections.

Sign in / Sign up

Export Citation Format

Share Document