scholarly journals The Platelet Fraction Is a Novel Reservoir to Detect Lyme Borrelia in Blood

Biology ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 366
Author(s):  
Victoria P. Sanderson ◽  
Iain L. Mainprize ◽  
Lisette Verzijlenberg ◽  
Cezar M. Khursigara ◽  
Melanie K. B. Wills
Keyword(s):  
Pathogen Detection ◽  
Bacterial Culture ◽  
Direct Detection ◽  
Laboratory Culture ◽  
Detection Methods ◽  
Clinical Samples ◽  
Blood Collection ◽  
Processing Conditions ◽  
Proof Of Concept ◽  
Clinical Detection

Serological diagnosis of Lyme disease suffers from considerable limitations. Yet, the technique cannot currently be replaced by direct detection methods, such as bacterial culture or molecular analysis, due to their inadequate sensitivity. The low bacterial burden in vasculature and lack of consensus around blood-based isolation of the causative pathogen, Borrelia burgdorferi, are central to this challenge. We therefore addressed methodological optimization of Borrelia recovery from blood, first by analyzing existing protocols, and then by using experimentally infected human blood to identify the processing conditions and fractions that increase Borrelia yield. In this proof-of-concept study, we now report two opportunities to improve recovery and detection of Borrelia from clinical samples. To enhance pathogen viability and cultivability during whole blood collection, citrate anticoagulant is superior to more commonly used EDTA. Despite the widespread reliance on serum and plasma as analytes, we found that the platelet fraction of blood concentrates Borrelia, providing an enriched resource for direct pathogen detection by microscopy, laboratory culture, Western blot, and PCR. The potential for platelets to serve as a reservoir for Borrelia and its diagnostic targets may transform direct clinical detection of this pathogen.

scholarly journals Reporter Phage-Based Detection of Bacterial Pathogens: Design Guidelines and Recent Developments

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 944 ◽  
Author(s):  
Susanne Meile ◽  
Samuel Kilcher ◽  
Martin J. Loessner ◽  
Matthew Dunne
Keyword(s):  
Viral Genome ◽  
Pathogen Detection ◽  
Bacterial Pathogens ◽  
Design Guidelines ◽  
Detection Methods ◽  
Clinical Samples ◽  
Cell Infection ◽  
Reporter Protein ◽  
Detection Systems ◽  
Conventional Culture

Fast and reliable detection of bacterial pathogens in clinical samples, contaminated food products, and water supplies can drastically improve clinical outcomes and reduce the socio-economic impact of disease. As natural predators of bacteria, bacteriophages (phages) have evolved to bind their hosts with unparalleled specificity and to rapidly deliver and replicate their viral genome. Not surprisingly, phages and phage-encoded proteins have been used to develop a vast repertoire of diagnostic assays, many of which outperform conventional culture-based and molecular detection methods. While intact phages or phage-encoded affinity proteins can be used to capture bacteria, most phage-inspired detection systems harness viral genome delivery and amplification: to this end, suitable phages are genetically reprogrammed to deliver heterologous reporter genes, whose activity is typically detected through enzymatic substrate conversion to indicate the presence of a viable host cell. Infection with such engineered reporter phages typically leads to a rapid burst of reporter protein production that enables highly sensitive detection. In this review, we highlight recent advances in infection-based detection methods, present guidelines for reporter phage construction, outline technical aspects of reporter phage engineering, and discuss some of the advantages and pitfalls of phage-based pathogen detection. Recent improvements in reporter phage construction and engineering further substantiate the potential of these highly evolved nanomachines as rapid and inexpensive detection systems to replace or complement traditional diagnostic approaches.

scholarly journals Prevalence of metallo-β-lactamase genes among Pseudomonas aeruginosa isolated from various clinical samples in China

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Wei Wang ◽  
Xiaoya Wang
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Susceptibility ◽  
Ethylenediaminetetraacetic Acid ◽  
Carbapenem Resistance ◽  
Opportunistic Pathogen ◽  
Detection Methods ◽  
Clinical Samples ◽  
Routine Practice ◽  
Carbapenem Resistant ◽  
High Prevalence

AbstractBackgroundPseudomonas aeruginosa is an opportunistic pathogen which is associated with nosocomial infections and causes various diseases including urinary tract infection, pneumonia, soft-tissue infection and sepsis. The emergence of P. aeruginosa-acquired metallo-β-lactamase (MBL) is most worrisome and poses a serious threat during treatment and infection control. The objective of this study was to identify antibiotic susceptibility, phenotypic detection of MBL production and to determine the prevalence of MBL genes in carbapenem-resistant P. aeruginosa isolated from different clinical samples.MethodsA total of 329 non-duplicate P. aeruginosa isolated from various clinical samples from two hospitals in China between September 2017 and March 2019 were included in this study. Phenotypic detection of MBL was performed by the combined detection method using imipenem and imipenem-ethylenediaminetetraacetic acid (EDTA) discs. MBL-encoding genes including blaVIM-1, blaVIM-2, blaIMP-1, blaIMP-2, blaSPM-1, blaSIM, blaNDM-1 and blaGIM were detected by polymerase chain reaction (PCR).ResultsOf the 329 P. aeruginosa, majority of the isolates were resistant to imipenem (77.5%) followed by meropenem (64.7%). Of the 270 P. aeruginosa isolates tested, 149 (55.2%) isolates were found to be positive for MBL detection. Of the different samples, 57.8% (n = 26) of P. aeruginosa isolated from blood were found to be positive for MBL production. Of the various MBL genes, blaIMP-1 (28.2%) was the most predominant gene detected followed by blaVIM-2 (18.8%), blaVIM-1 (16.1%), blaNDM-1 (9.4%), blaIMP-2 (6.7%), blaSIM (6.0%), blaSPM-1 (4.0%) and blaGIM (1.3%) genes.ConclusionsThe high resistance of P. aeruginosa toward imipenem and meropenem and the high prevalence of blaIMP-1 and blaVIM-2 set the alarm on the increasing, perhaps the increased, carbapenem resistance. In addition to routine antibiotic susceptibility testings, our results emphasize the importance of both the phenotypic and genotypic MBL detection methods in routine practice for early detection of carbapenem resistance and to prevent further dissemination of this resistant pathogen.

scholarly journals Review of Electrochemical DNA Biosensors for Detecting Food Borne Pathogens

Sensors ◽  
2019 ◽  
Vol 19 (22) ◽  
pp. 4916 ◽  
Author(s):  
Qiaoyun Wu ◽  
Yunzhe Zhang ◽  
Qian Yang ◽  
Ning Yuan ◽  
Wei Zhang
Keyword(s):  
Pathogen Detection ◽  
Low Cost ◽  
Electrochemical Techniques ◽  
Ease Of Use ◽  
Detection Methods ◽  
Future Research ◽  
Dna Biosensors ◽  
Food Borne Pathogens ◽  
Chip Technology ◽  
Food Borne

The vital importance of rapid and accurate detection of food borne pathogens has driven the development of biosensor to prevent food borne illness outbreaks. Electrochemical DNA biosensors offer such merits as rapid response, high sensitivity, low cost, and ease of use. This review covers the following three aspects: food borne pathogens and conventional detection methods, the design and fabrication of electrochemical DNA biosensors and several techniques for improving sensitivity of biosensors. We highlight the main bioreceptors and immobilizing methods on sensing interface, electrochemical techniques, electrochemical indicators, nanotechnology, and nucleic acid-based amplification. Finally, in view of the existing shortcomings of electrochemical DNA biosensors in the field of food borne pathogen detection, we also predict and prospect future research focuses from the following five aspects: specific bioreceptors (improving specificity), nanomaterials (enhancing sensitivity), microfluidic chip technology (realizing automate operation), paper-based biosensors (reducing detection cost), and smartphones or other mobile devices (simplifying signal reading devices).

Abstract 11119: Real-Time Nanopore Sequencing for Bacterial Pneumonia After Out-of-Hospital Cardiac Arrest, a Pilot Proof-of-Concept Study

Circulation ◽  
2021 ◽  
Vol 144 (Suppl_2) ◽  
Author(s):  
Alexandra Weissman ◽  
Mariam Bramah Lawani ◽  
Thomas Rohan ◽  
Clifton W CALLAWAY
Keyword(s):  
Patient Care ◽  
Pathogen Detection ◽  
Bacterial Pathogen ◽  
Improve Patient Care ◽  
Antibiotic Administration ◽  
Nanopore Sequencing ◽  
Proof Of Concept ◽  
Chi Square ◽  
Significant Difference ◽  
Improve Patient

Introduction: Pneumonia is common after OHCA but is difficult to diagnose in the first 72 hours following ROSC, this results in early untargeted antibiotic administration based on non-specific imaging and laboratory findings. Antibiotic resistance is rising, is influenced by untargeted antibiotic administration, and can increase patient morbidity and mortality as well as healthcare costs. Precision methods of bacterial pathogen detection in OHCA patients are needed to improve patient care. This proof-of-concept pilot study aimed to assess feasibility of bacterial pathogen sequencing and comparability of sequencing results to clinical culture after OHCA. Methods: Blood and bronchoalveolar lavage (BAL) were obtained from residual clinical specimens collected within 12 hours of ROSC. Bacterial DNA was extracted using the Qiagen PowerLyzer PowerSoil DNA kit, sequenced using the MinION nanopore sequencer, and analyzed with Oxford Nanopore Technologies’ EPI2ME bioinformatics software. Sequencing results were compared to culture results using McNemar’s chi-square statistic. Study-defined pneumonia was based on presence of at least two characteristics within 72 hours of ROSC: fever (temperature ≥38°C); persistent leukocytosis >15,000 or leukopenia <3,500 for 48 hours; persistent chest radiography infiltrates for 48 hours per clinical radiology read; bacterial pathogen cultured. Results: We enrolled 38 consecutive OHCA subjects: mean age 61.8 years (18.0); 16 (42%) female; 25 (66%) White, 7 (18%) Black, 6 (16%) “Other” race; 7 subjects (18%) survived and 31 (82%) died; 16 (42%) subjects had pneumonia. Sequencing results were available in 12 hours while culture results were available in 48-72 hours after collection. There was a non-significant difference in the proportion of the same pathogens identified for each method per McNemar’s chi-square: p = 0.38, difference of 0.095 (-0.095, 0.286). Conclusions: Nanopore sequencing detects pathogenic bacteria comparable to clinical microbiologic culture and in less time. This technology can produce a paradigm shift in early bacterial pathogen detection in OHCA survivors, which can improve patient care. The technology is applicable to other patient populations and for viral and fungal pathogens.

scholarly journals Artifacts associated with mithramycin fluorescence in the clinical detection and quantitation of aneuploidy by flow cytometry.

1982 ◽  
Vol 30 (4) ◽  
pp. 317-322 ◽  
Author(s):  
R E Cunningham ◽  
K S Skramstad ◽  
A E Newburger ◽  
S E Shackney
Keyword(s):  
Flow Cytometry ◽  
Room Temperature ◽  
Human Melanoma ◽  
Time Dependent ◽  
Clinical Samples ◽  
Fixation Time ◽  
C Cells ◽  
Temperature Dependent ◽  
Clinical Detection

Ethanol-fixed cells stored at 4 degrees C exhibit fixation time-dependent hyperchromatism in comparison with freshly fixed cells when stained with mithramycin and examined by flow cytometry. This hyperchromatism has been found to be temperature-dependent, developing fully within 72 hr at room temperature, and within 2 hr at 37 degrees C. Cells from normal donors that are stained with mithramycin exhibit spurious aneuploid peaks. These spurious aneuploid peaks can be eliminated by incubating ethanol-fixed cells at 37 degrees C for 2 hr prior to staining; true aneuploidy is not affected by this procedure. In rare instances, cytoplasmic fluorescence can be observed in mithramycin-stained cells. In addition, unexplained hypochromatism and hyperchromatism can be observed in some clinical samples, particularly in human melanoma. The effects of these unexplained staining artifacts can be minimized or eliminated by adopting strict criteria for the clinical detection of aneuploidy by flow cytometry.

scholarly journals ANTIMICROBIAL SUSCEPTIBILITY AND DETECTION METHODS FOR THE EXTENDED‑SPECTRUM β-LACTAMASES PRODUCING ENTEROBACTERIACEAE FROM CLINICAL SAMPLES

2018 ◽  
Vol 11 (5) ◽  
pp. 139
Author(s):  
Kaviyarasan G ◽  
Rajamanikandan Kcp ◽  
Sabarimuthu M ◽  
Ramya S ◽  
Arvind Prasanth D
Keyword(s):  
Infection Control ◽  
Bacterial Infections ◽  
Control Measures ◽  
Detection Methods ◽  
Clinical Samples ◽  
Healthcare Settings ◽  
Infection Control Measures ◽  
Extended Spectrum ◽  
Drug Choice ◽  
Synergy Test

Objectives: Detection of extended-spectrum β-lactamases (ESBLs) is crucial for the infection control and antibiotic choice in healthcare settings. The aim of this study is to develop a standardized, inexpensive, and simple approach that is able to detect ESBL-producing Enterobacteriaceae isolates.Methods: Isolates those were resistant to at least one of the three indicator cephalosporins (cefotaxime, cefpodoxime, and ceftazidime) were tested for ESBL production using the double disc synergy test (DDST), combined disc synergy test (CDST) test and genotypic detection of the responsible gene for the ESBL.Result: From 64 isolates, 28 were resistant to cephalosporins. In 28 isolates, 23 were positive in CDST but in the DDST 18 were showing ESBL positive. 10 were positive in both CDST and DDST.Conclusion: Resistance to cephalosporins, which are the drug choice to treat mixed bacterial infections by the Enterobacteriaceae of which disseminate rapidly being plasmid mediated. Hence, it is necessary that rapid detection of ESBL should be done and immediate infection control measures should be implemented to prevent their dissemination.

scholarly journals SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

scholarly journals Multiplexed Detection of COVID-19 with Single-Molecule Technology

2021 ◽  
Author(s):  
Noa Furth ◽  
Shay Shilo ◽  
Niv Cohen ◽  
Nir Erez ◽  
Vadim Fedyuk ◽  
...  
Keyword(s):  
Immune Response ◽  
Viral Infection ◽  
Single Molecule ◽  
Direct Detection ◽  
Genetic Material ◽  
Diagnostic Tools ◽  
Proof Of Concept ◽  
Multiplexed Detection ◽  
Diagnostic Approaches ◽  
Quantitative Nature

The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for multiplexed detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.

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

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