scholarly journals Highly Sensitive and Rapid Quantitative Detection of Plasmodium falciparum Using an Image Cytometer

2020 ◽  
Vol 8 (11) ◽  
pp. 1769
Author(s):  
Muneaki Hashimoto ◽  
Kazumichi Yokota ◽  
Kazuaki Kajimoto ◽  
Musashi Matsumoto ◽  
Atsuro Tatsumi ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Limit Of Detection ◽  
Malaria Diagnosis ◽  
Image Cytometry ◽  
Quantitative Detection ◽  
Economic Costs ◽  
Infected People ◽  
Highly Sensitive ◽  
Filter Unit ◽  
Syringe Filter

The gold standard for malaria diagnosis is microscopic examination of blood films by expert microscopists. It is important to detect submicroscopic and asymptomatic Plasmodium infections in people, therefore the development of highly sensitive devices for diagnosing malaria is required. In the present study, we investigated whether an imaging cytometer was useful for the highly sensitive quantitative detection of parasites. Whole blood samples were prepared from uninfected individuals spiked with Plasmodium falciparum-infected erythrocytes. Thereafter, erythrocytes were purified using a push column comprising of a syringe filter unit with SiO2-nanofiber filters. After adding the erythrocytes, stained with nuclear stain, to a six-well plate, quantitative detection of the parasites was performed using an image cytometer, CQ1. Imaging of 2.6 × 106 erythrocytes was completed in 3 min, and the limit of detection indicated parasitemia of 0.00010% (≈5 parasites/μL of blood). In addition to rapid, highly sensitive, and quantitative detection, the ease of application and economic costs, image cytometry could be efficiently applied to diagnose submicroscopic parasites in infected people from endemic countries.

scholarly journals 1832. Development of an Ultrasensitive Field-Applicable Plasmodium falciparum Assay for Malaria Diagnosis and Eradication

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S42-S43
Author(s):  
Rose Lee ◽  
Helena De Puig Guixe ◽  
Jeffrey Dvorin ◽  
James Collins
Keyword(s):  
Plasmodium Falciparum ◽  
Nucleic Acid ◽  
Genomic Dna ◽  
Limit Of Detection ◽  
Malaria Diagnosis ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
One Pot ◽  
Minimal Sample ◽  
Isothermal Assay

Abstract Background Malaria control and eradication have been hampered by asymptomatic carriage which serves as a parasite reservoir. Low-density infections (< 100 parasites/microliter) frequently fall below the limit of detection (LOD) of microscopy and rapid diagnostic tests (RDT) which are antigen-based tests. Molecular methods such as polymerase chain reaction are capable of higher sensitivity yet remain impractical for resource-limited settings. We describe development of an isothermal assay using the nucleic acid detection platform SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing), which may also be increasingly important as there has been rising detection of histidine-rich protein 2 (HRP2) gene deletions in Plasmodium spp. HRP2 is the most commonly used antigen in RDTs and deletion of this gene would render many RDTs obsolete. Methods SHERLOCK leverages the endonucleases of CRISPR-associated microbial adaptive immunity. Cas12a is an RNA-guided, DNA-cleaving enzyme, which can be programmed with guide RNAs to cleave nontarget reporter ssDNA. We exploit the nonspecific degradation of labeled ssDNA to detect the presence of the dsDNA target that activated Cas12a (Figure 1). Recombinase polymerase amplification (RPA) coupled with Cas12a detection enables a lower LOD. Plasmodium falciparum whole genomic DNA was compared with parasites cultured in red blood cells (RBCs) with known parasitemia and boiled at 95°C for 5 minutes for lysis of RBCs/parasites then diluted 1:2.5 to prevent solidification. Results This SHERLOCK assay detected simulated Plasmodium falciparum infection at attomolar LODs when applied to whole genomic DNA and simulated samples of infected RBCs spiked into whole blood. The genomic assay detected down to 0.2 parasites/microliter and the simulated sample detected to 10 parasites/microliter in the final reaction volume. In comparison, LODs from the initial input volume was 5aM and 250aM, respectively (Figure 2). Conclusion We demonstrate an isothermal nucleic acid detection platform capable of diagnosis in 60 minutes in a one-pot assay requiring minimal sample preparation and reaching an LOD recommended by the WHO for malaria eradication. In summary, we illustrate the utility of the SHERLOCK platform in application to malaria and global health. Disclosures All Authors: No reported Disclosures.

scholarly journals A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Aline Lamien-Meda ◽  
Hans-Peter Fuehrer ◽  
David Leitsch ◽  
Harald Noedl
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Limit Of Detection ◽  
Plasmodium Species ◽  
Malaria Diagnosis ◽  
Epidemiological Studies ◽  
Taqman Probe ◽  
Species Differentiation ◽  
Resource Limited ◽  
Highly Sensitive

Abstract Background The use of highly sensitive molecular tools in malaria diagnosis is currently largely restricted to research and epidemiological settings, but will ultimately be essential during elimination and potentially eradication. Accurate diagnosis and differentiation down to species levels, including the two Plasmodium ovale species and zoonotic variants of the disease, will be important for the understanding of changing epidemiological patterns of the disease. Methods A qPCR-high resolution melting (HRM) method was to detect and differentiate all human Plasmodium species with one forward and one reverse primer set. The HRM detection method was further refined using a hydrolysis probe to specifically discriminate Plasmodium falciparum. Results Out of the 113 samples tested with the developed HRM-qPCR- P. falciparum probe assay, 96 (85.0 %) single infections, 12 (10.6 %) mixed infections, and 5 (4.4 %) were Plasmodium negative. The results were concordant with those of the nested PCR at 98.2 %. The assay limit of detection was varied from 21.47 to 46.43 copies /µl, equivalent to 1–2.11 parasites/µl. All P. falciparum infections were confirmed with the associated Taqman probe. Conclusions Although the dependence on qPCR currently limits its deployment in resource-limited environments, this assay is highly sensitive and specific, easy to perform and convenient for Plasmodium mono-infection and may provide a novel tool for rapid and accurate malaria diagnosis also in epidemiological studies.

scholarly journals Specific, highly sensitive and simple spectrofluorimetric method for quantification of daclatasvir in HCV human plasma patients and in tablets dosage form

2019 ◽  
Vol 17 (1) ◽  
pp. 116-126
Author(s):  
Ramadan Ali ◽  
Mohamed M Elsutohy
Keyword(s):  
Limit Of Detection ◽  
Degradation Products ◽  
Antiviral Drug ◽  
Pharmaceutical Preparations ◽  
Linear Range ◽  
Pure Form ◽  
Quantitative Detection ◽  
Plasma Samples ◽  
Spectrofluorimetric Method ◽  
Highly Sensitive

AbstractA simple, selective and highly sensitive spectrofluorimetric method for the quantitative detection of an antiviral drug, daclatasvir (DCV) has been developed and analytically validated in its pure form, in its commercially available pharmaceutical preparations, and in hepatitis-C (HCV) patient’s plasma. The method was based on recording the native fluorescence of DCV that exhibit an emission wavelength maximum at 380 nm upon excitation at 320 nm. Many factors affecting the fluorescence intensity and the method sensitivity including pH, type of surfactant and solvent have been studied and optimized. In addition, a stability-indicating study was performed in accordance with the guidelines of the International Conference on Harmonization (ICH), to detect the drug in the presence of its degradation products which validates the method for application in quality control laboratories. In addition, extraction of DCV from the plasma proteins was performed using a simple technique that was based on using methanol and borate buffer (pH 9) that gave a recovery of ~95%. The results showed that DCV could be detected using this method in the pure form (with a linear range of 2-1000 ng/mL), commercially available tablets and in plasma samples (with a linear range of 5-1000 ng/mL), without any interferences. Furthermore, the method was also analytically and clinically validated according to the Food and Drug Administration (FDA) guidelines, with limit of detection (LOD) of 0.3 and 0.5 ng/mL for DCV in the pure form and plasma samples, respectively.

Detection of Glucose in Human Serum Based on Silicon Dot Probe

2020 ◽  
Vol 16 (6) ◽  
pp. 744-752
Author(s):  
Kuan Luo ◽  
Xinyu Jiang
Keyword(s):  
Quantum Dots ◽  
Blood Glucose ◽  
Human Serum ◽  
Organic Dyes ◽  
Limit Of Detection ◽  
Fasting Blood Glucose ◽  
Glucose Biosensor ◽  
Metal Nanoclusters ◽  
Glucose Levels ◽  
Highly Sensitive

Background: Diabetes Mellitus (DM) is a major public metabolic disease that influences 366 million people in the world in 2011, and this number is predicted to rise to 552 million in 2030. DM is clinically diagnosed by a fasting blood glucose that is equal or greater than 7 mM. Therefore, the development of effective glucose biosensor has attracted extensive attention worldwide. Fluorescence- based strategies have sparked tremendous interest due to their rapid response, facile operation, and excellent sensitivity. Many fluorescent compounds have been employed for precise analysis of glucose, including quantum dots, noble metal nanoclusters, up-converting nanoparticles, organic dyes, and composite fluorescent microspheres. Silicon dot as promising quantum dots materials have received extensive attention, owing to their distinct advantages such as biocompatibility, low toxicity and high photostability. Methods: MnO2 nanosheets on the Si nanoparticles (NPs) surface serve as a quencher. Si NPs fluorescence can make a recovery by the addition of H2O2, which can reduce MnO2 to Mn2+, and the glucose can thus be monitored based on the enzymatic conversion of glucose by glucose oxidase to generate H2O2. Therefore, the glucose concentration can be derived by recording the fluorescence recovery spectra of the Si NPs. Results: This probe enabled selective detection of glucose with a linear range of 1-100 μg/mL and a limit of detection of 0.98 μg/mL. Compared with the commercial glucometer, this method showed favorable results and convincing reliability. Conclusion: We have developed a novel method based on MnO2 -nanosheet-modified Si NPs for rapid monitoring of blood glucose levels. By combining the highly sensitive H2O2/MnO2 reaction with the excellent photostability of Si NPs, a highly sensitive, selective, and cost-efficient sensing approach for glucose detection has been designed and applied to monitor glucose levels in human serum with satisfactory results.

scholarly journals Computational Design of a Molecularly Imprinted Polymer for the Biomonitoring of the Organophosphorous Metabolite Chlorferron

Biosensors ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 192
Author(s):  
Bakhtiyar Qader ◽  
Issam Hussain ◽  
Mark Baron ◽  
Rebeca Jiménez-Pérez ◽  
Guzmán Gil-Ramírez ◽  
...  
Keyword(s):  
Biological Samples ◽  
Limit Of Detection ◽  
Computational Design ◽  
Signal Change ◽  
Highly Sensitive ◽  
Limit Of Quantitation ◽  
Molecularly Imprinting Polymers ◽  
Mip Sensor ◽  
Parasitic Mites

Coumaphos is an organophosphorus compound used as insecticide and frequently used by beekeepers for the management of parasitic mites. The most important metabolite, chlorferron (CFN), has been identified in biological samples and foodstuff. The need to quickly identify the presence of typical metabolites, as an indication of interaction with coumaphos has driven the need to produce a highly sensitive electrochemical method for chlorferron analysis, based on molecularly imprinting polymers (MIP) technology. It showed irreversible behaviour with mixed diffusion/adsorption-controlled reactions at the electrode surface. A monoelectronic mechanism of reaction for oxidation has also been suggested. The linear range observed was from 0.158 to 75 µM. Median precision in terms of %RSD around 3% was also observed. For DPV, the limit of detection (LOD) and the limit of quantitation (LOQ) for the CFN-MIP were 0.158 µM and 0.48 µM, respectively. The obtained median % recovery was around 98%. The results were also validated to reference values obtained using GC-MS. Urine and human synthetic plasma spiked with CFN were used to demonstrate the usability of the method in biological samples, showing the potential for biomonitoring. The developed imprinted sensor showed maximum signal change less than 16.8% when related metabolites or pesticide were added to the mix, suggesting high selectivity of the MIP sensor toward CFN molecules. The results from in vitro metabolism of CMP analysed also demonstrates the potential for detection and quantification of CFN in environmental samples. The newly developed CFN-MIP sensor offers similar LoDs than chromatographic methods with shorter analysis time.

scholarly journals No evidence of false-negative Plasmodium falciparum rapid diagnostic results in Monrovia, Liberia

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Mandella King ◽  
Alexander E. George ◽  
Pau Cisteró ◽  
Christine K. Tarr-Attia ◽  
Beatriz Arregui ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
False Negative ◽  
Malaria Diagnosis ◽  
Rapid Diagnostic Tests ◽  
Molecular Testing ◽  
False Negatives ◽  
Gene Deletions ◽  
History Of ◽  
Catholic Hospital ◽  
Or History

Abstract Background Malaria diagnosis in many malaria-endemic countries relies mainly on the use of rapid diagnostic tests (RDTs). The majority of commercial RDTs used in Africa detect the Plasmodium falciparum histidine-rich protein 2 (PfHRP2). pfhrp2/3 gene deletions can therefore lead to false-negative RDT results. This study aimed to evaluate the frequency of PCR-confirmed, false-negative P. falciparum RDT results in Monrovia, Liberia. Methods PfHRP2-based RDT (Paracheck Pf®) and microscopy results from 1038 individuals with fever or history of fever (n = 951) and pregnant women at first antenatal care (ANC) visit (n = 87) enrolled in the Saint Joseph’s Catholic Hospital (Monrovia) from March to July 2019 were used to assess the frequency of false-negative RDT results. True–false negatives were confirmed by detecting the presence of P. falciparum DNA by quantitative PCR in samples from individuals with discrepant RDT and microscopy results. Samples that were positive by 18S rRNA qPCR but negative by PfHRP2-RDT were subjected to multiplex qPCR assay for detection of pfhrp2 and pfhrp3. Results One-hundred and eighty-six (19.6%) and 200 (21.0%) of the 951 febrile participants had a P. falciparum-positive result by RDT and microscopy, respectively. Positivity rate increased with age and the reporting of joint pain, chills and shivers, vomiting and weakness, and decreased with the presence of coughs and nausea. The positivity rate at first ANC visit was 5.7% (n = 5) and 8% (n = 7) by RDT and microscopy, respectively. Out of 207 Plasmodium infections detected by microscopy, 22 (11%) were negative by RDT. qPCR confirmed absence of P. falciparum DNA in the 16 RDT-negative but microscopy-positive samples which were available for molecular testing. Among the 14 samples that were positive by qPCR but negative by RDT and microscopy, 3 only amplified pfldh, and among these 3 all were positive for pfhrp2 and pfhrp3. Conclusion There is no qPCR-confirmed evidence of false-negative RDT results due to pfhrp2/pfhrp3 deletions in this study conducted in Monrovia (Liberia). This indicates that these deletions are not expected to affect the performance of PfHRP2-based RDTs for the diagnosis of malaria in Liberia. Nevertheless, active surveillance for the emergence of PfHRP2 deletions is required.

scholarly journals A rapid, accurate, scalable, and portable testing system for COVID-19 diagnosis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Guanhua Xun ◽  
Stephan Thomas Lane ◽  
Vassily Andrew Petrov ◽  
Brandon Elliott Pepa ◽  
Huimin Zhao
Keyword(s):  
Limit Of Detection ◽  
Low Cost ◽  
High Volume ◽  
N Gene ◽  
Testing System ◽  
Target Sequence ◽  
One Pot ◽  
Sequence Detection ◽  
Highly Sensitive ◽  
Speed Accuracy

AbstractThe need for rapid, accurate, and scalable testing systems for COVID-19 diagnosis is clear and urgent. Here, we report a rapid Scalable and Portable Testing (SPOT) system consisting of a rapid, highly sensitive, and accurate assay and a battery-powered portable device for COVID-19 diagnosis. The SPOT assay comprises a one-pot reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) followed by PfAgo-based target sequence detection. It is capable of detecting the N gene and E gene in a multiplexed reaction with the limit of detection (LoD) of 0.44 copies/μL and 1.09 copies/μL, respectively, in SARS-CoV-2 virus-spiked saliva samples within 30 min. Moreover, the SPOT system is used to analyze 104 clinical saliva samples and identified 28/30 (93.3% sensitivity) SARS-CoV-2 positive samples (100% sensitivity if LoD is considered) and 73/74 (98.6% specificity) SARS-CoV-2 negative samples. This combination of speed, accuracy, sensitivity, and portability will enable high-volume, low-cost access to areas in need of urgent COVID-19 testing capabilities.

scholarly journals PERFORMANCE CHARACTERISTICS OF A FRET-BASED IMMUNOASSAY FOR QUANTITATION OF INFLIXIMAB ON A POINT-OF-CARE INSTRUMENT SYSTEM

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S57-S57
Author(s):  
Edgar Ong ◽  
Ruo Huang ◽  
Richard Kirkland ◽  
Michael Hale ◽  
Larry Mimms
Keyword(s):  
Whole Blood ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Quantitative Detection ◽  
Therapeutic Drug ◽  
Analytical Sensitivity ◽  
Sample Type ◽  
Hook Effect ◽  
Limit Of Quantitation ◽  
Assay Precision

Abstract Introduction A fast (&lt;5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassay was developed for the quantitative detection of infliximab (IFX) and biosimilars for use in therapeutic drug monitoring using only 20 µL of fingerstick whole blood or serum at the point-of-care. The Procise IFX assay and ProciseDx analyzer are CE-marked. Studies were performed to characterize analytical performance of the Procise IFX assay on the ProciseDx analyzer. Methods Analytical testing was performed by spiking known amounts of IFX into negative serum and whole blood specimens. Analytical sensitivity was determined using limiting concentrations of IFX. Linearity was determined by testing IFX across the assay range. Hook effect was assessed at IFX concentrations beyond levels expected to be found within a patient. Testing of assay precision, cross-reactivity and potential interfering substances, and biosimilars was performed. The Procise IFX assay was also compared head-to-head with another CE-marked assay: LISA-TRACKER infliximab ELISA test (Theradiag, France). The accuracy of the Procise IFX assay is established through calibrators and controls traceable to the WHO 1st International Standard for Infliximab (NIBSC code: 16/170). Results The Procise IFX assay shows a Limit of Blank, Limit of Detection, and Lower Limit of Quantitation (LLoQ) of 0.1, 0.2, and 1.1 µg/mL in serum and 0.6, 1.1, and 1.7 µg/mL in whole blood, respectively. The linear assay range was determined to be 1.7 to 77.2 µg/mL in serum and whole blood. No hook effect was observed at an IFX concentration of 200 µg/mL as the value reported as “&gt;ULoQ”. Assay precision testing across 20 days with multiple runs and reagent lots showed an intra-assay coefficient of variation (CV) of 2.7%, an inter-assay CV of &lt;2%, and a total CV of 3.4%. The presence of potentially interfering/cross-reacting substances showed minimal impact on assay specificity with %bias within ±8% of control. Testing of biosimilars (infliximab-dyyb and infliximab-abda) showed good recovery. A good correlation to the Theradiag infliximab ELISA was obtained for both serum (slope=1.01; r=0.99) and whole blood (slope=1.01; r=0.98) samples (Figure 1). Conclusion Results indicate that the Procise IFX assay is sensitive, specific, and precise yielding results within 5 minutes from both whole blood and serum without the operator needing to specify sample type. Additionally, it shows very good correlation to a comparator assay that takes several hours and sample manipulation to yield results. This makes the Procise IFX assay ideal for obtaining fast and accurate IFX quantitation, thus allowing for immediate drug level dosing decisions to be made by the physician during patient treatment.

Rapid, highly sensitive and quantitative detection of interleukin 6 based on SERS magnetic immunoassay

2021 ◽  
Vol 13 (15) ◽  
pp. 1823-1831
Author(s):  
Xiaomei Wang ◽  
Li Ma ◽  
Shijiao Sun ◽  
Tingwei Liu ◽  
Hao Zhou ◽  
...  
Keyword(s):  
Interleukin 6 ◽  
High Sensitivity ◽  
Quantitative Detection ◽  
Detection Time ◽  
Highly Sensitive ◽  
Sandwich Method

We have developed a SERS magnetic immunoassay method based on the principle of sandwich method for rapid and quantitative detection of IL-6. The developed SERS method has the advantages of high sensitivity and detection time is only 15 min.

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