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1.
Blood Coagul Fibrinolysis ; 35(5): 265-270, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38874904

RESUMO

Heparin-induced thrombocytopenia (HIT) is a prothrombotic condition induced by platelet-activating IgG antibodies that recognize PF4/heparin complexes. Diagnosis of HIT relies on enzyme immunologic assays (EIAs) and functional assays [serotonin release assay (SRA)]. Our institution uses a latex immunoturbidimetric assay (LIA), which has shown a positive-predictive value (PPV) of 55.6%, and a negative-predictive value (NPV) of 99.7%. The low PPV of EIAs/LIAs, in combination with the clinical delay in obtaining results of a SRA, commonly leads to a false-positive diagnosis of HIT and inappropriate treatment. We performed a single-institution retrospective study at a large tertiary center to assess patient management decisions and economic costs following a false-positive HIT (LIA) test. This study found an 89.5% incidence of false-positive HIT (LIA) tests. 97.4% of patients underwent anticoagulation changes. 69.6% of patients were switched to argatroban. Of patients with a false-positive HIT immunoassay (LIA), 42 (40.7%) patients were on a prophylactic dose of anticoagulation at the time of HIT (LIA) positivity, of which 22 (52.4%) were switched to full anticoagulation with either argatroban or fondaparinux. Of the 22 patients switched to full anticoagulation, 15 (68%) had low-probability 4T scores. Seven (8.8%) of patients had bleeding events after HIT (LIA) positivity. All seven patients were switched to argatroban from a full-dose heparin anticoagulation. Five of the seven patients were considered major bleeds. Utilization of argatroban incurred substantial costs, estimated at approximately $73 000 for false-positive HIT cases. False-positive HIT (LIA) tests contribute to unwarranted anticoagulation changes, increased bleeding risks, and substantial healthcare costs. Incorporating the 4T score into diagnostic algorithms may help mitigate these risks by guiding appropriate clinical decisions. Future research should focus on refining diagnostic approaches and standardizing management strategies to improve patient outcomes and cost-effectiveness in HIT diagnosis and management.


Assuntos
Anticoagulantes , Heparina , Trombocitopenia , Humanos , Trombocitopenia/induzido quimicamente , Trombocitopenia/diagnóstico , Trombocitopenia/economia , Heparina/efeitos adversos , Reações Falso-Positivas , Estudos Retrospectivos , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , Anticoagulantes/efeitos adversos , Anticoagulantes/uso terapêutico , Anticoagulantes/economia , Imunoensaio/economia , Imunoensaio/métodos , Arginina/análogos & derivados , Ácidos Pipecólicos/uso terapêutico , Ácidos Pipecólicos/economia , Sulfonamidas/economia , Sulfonamidas/uso terapêutico
2.
Food Chem ; 449: 139050, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38581779

RESUMO

Ensuring the safety of animal-derived foods requires the reliable and swift identification of enrofloxacin residues to monitor the presence of antibiotics. In this regard, we synthesized, tuned, and investigated the optical properties of a bimetallic metal-organic framework (Ce/Zr-UiO 66). The investigation was facilitated by employing a polydopamine-coated pipette tip with high adsorption efficiency, serving as an immunoreactive carrier. Subsequently, an immunofunctionalized variant of Ce/Zr-UiO 66, referred to as Ce/Zr-UiO 66@ Bovine serum albumin-enrofloxacin, was developed as an optical probe for the rapid and sensitive identification of enrofloxacin across a variety of samples. The method can accurately detect enrofloxacin at concentrations as low as 0.12 ng/mL, with a determination time of under 15 min; furthermore, it demonstrates exceptional efficacy when applied to food, environmental, and clinical samples. The implementation of this methodology offers a valuable means for cost-effective, rapid, and on-site enrofloxacin determination.


Assuntos
Antibacterianos , Enrofloxacina , Contaminação de Alimentos , Estruturas Metalorgânicas , Leite , Enrofloxacina/análise , Estruturas Metalorgânicas/química , Animais , Leite/química , Contaminação de Alimentos/análise , Antibacterianos/análise , Bovinos , Imunoensaio/métodos , Imunoensaio/instrumentação , Imunoensaio/economia , Técnicas Biossensoriais/instrumentação , Limite de Detecção
3.
Anal Bioanal Chem ; 414(8): 2607-2618, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35091761

RESUMO

The lateral flow assay (LFA) is one of the most popular technologies on the point-of-care diagnostics market due to its low cost and ease of use, with applications ranging from pregnancy to environmental toxins to infectious disease. While the use of these tests is relatively straightforward, significant development time and effort are required to create tests that are both sensitive and specific. Workflows to guide the LFA development process exist but moving from target selection to an LFA that is ready for field testing can be labor intensive, resource heavy, and time consuming. To reduce the cost and the duration of the LFA development process, we introduce a novel development platform centered on the flexibility, speed, and throughput of an automated robotic liquid handling system. The system comprises LFA-specific hardware and software that enable large optimization experiments with discrete and continuous variables such as antibody pair selection or reagent concentration. Initial validation of the platform was demonstrated during development of a malaria LFA but was readily expanded to encompass development of SARS-CoV-2 and Mycobacterium tuberculosis LFAs. The validity of the platform, where optimization experiments are run directly on LFAs rather than in solution, was based on a direct comparison between the robotic system and a more traditional ELISA-like method. By minimizing hands-on time, maximizing experiment size, and enabling improved reproducibility, the robotic system improved the quality and quantity of LFA assay development efforts.


Assuntos
COVID-19/diagnóstico , Imunoensaio/instrumentação , Malária/diagnóstico , Testes Imediatos , Tuberculose/diagnóstico , Teste Sorológico para COVID-19/economia , Teste Sorológico para COVID-19/instrumentação , Desenho de Equipamento , Humanos , Imunoensaio/economia , Mycobacterium tuberculosis/isolamento & purificação , Plasmodium/isolamento & purificação , Testes Imediatos/economia , Reprodutibilidade dos Testes , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Fatores de Tempo
4.
Anal Bioanal Chem ; 413(29): 7251-7263, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34622322

RESUMO

Supply shortage for the development and production of preventive, therapeutic, and diagnosis tools during the COVID-19 pandemic is an important issue affecting the wealthy and poor nations alike. Antibodies and antigens are especially needed for the production of immunological-based testing tools such as point-of-care tests. Here, we propose a simple and quick magnetic nanoparticle (MNP)-based separation/isolation approach for the repurposing of infected human samples to produce specific antibodies and antigen cocktails. Initially, an antibody cocktail was purified from serums via precipitation and immunoaffinity chromatography. Purified antibodies were conjugated onto MNPs and used as an affinity matrix to separate antigens. The characterization process was performed by ELISA, SDS-PAGE, electrochemistry, isothermal titration calorimetry, and LC-Q-TOF-MS/MS analyses. The MNP-separated peptides can be used for mass spectrometry-based as well as paper-based lateral flow assay diagnostic. The exploitation of the current workflow for the development of efficient diagnostic tools, specific treatments, and fundamental research can significantly impact the present or eventual pandemic. This workflow can be considered as a two birds, one stone-like strategy.


Assuntos
Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/isolamento & purificação , COVID-19/diagnóstico , Análise Custo-Benefício , Imunoensaio/economia , SARS-CoV-2/isolamento & purificação , Viremia/virologia , Anticorpos Antivirais/sangue , Antígenos Virais/sangue , COVID-19/virologia , Calorimetria , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , SARS-CoV-2/imunologia , Manejo de Espécimes , Espectrometria de Massas em Tandem , Viremia/sangue , Fluxo de Trabalho
5.
J Immunol Methods ; 498: 113134, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34464606

RESUMO

Immune checkpoint Inhibitors (ICIs) are effective immunno-therapeutic agents for cancer. Rapid and sensitive determination of the blocking activity of ICIs is important for ICIs development and immunological research. Among various immune checkpoint (IC) binding assays, cell-based binding assays are widely regarded, and the functional ELISA is a convenient alternative. However, these methodologies are limited by time-consuming preparation of cell lines stably expressing IC molecules, or long turnaround time with high cost. In this study, two magnetic bead based binding assays were developed to evaluate activity of ICIs, which was determined by a soluble ligand/bead immobilized receptor based binding assay (sL/bR binding assay) that assessed efficacy to block binding of one soluble IC ligand on its cognate receptor immobilized beads, or by a soluble receptor/bead immobilized ligand based binding assay (sR/bL binding assay) that assessed efficacy to block binding of soluble IC receptor on its cognate ligand immobilized beads. Half maximal inhibitory concentration (IC50) values of ICIs were calculated to determine ICIs activity. The sL/bR binding assay accurately determined the activity of two TIGIT blocking antibodies, since the relative blocking activity of two TIGIT antibodies determined by the sL/bR binding assay established in this study and that by the cell based binding assay were almost identical. In contrast, the sR/bL binding assay showed significantly improved sensitivity to determine activity of two PD-1 blocking antibodies than the sL/bR binding assay that was tested in this study and previous reports. Moreover, both amount of the used recombinant protein of ICI receptor/ligand and turnaround time of the two binding assays were more than 10 times less than those of the functional ELISA. These data indicate that the two magnetic bead based binding assays are sensitive, rapid and cost-effective methods to determine blocking activity of ICIs.


Assuntos
Inibidores de Checkpoint Imunológico/farmacologia , Imunoensaio/economia , Nivolumabe/farmacologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores Imunológicos/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Células CHO , Linhagem Celular Tumoral , Redução de Custos , Análise Custo-Benefício , Cricetulus , Ensaio de Imunoadsorção Enzimática/economia , Citometria de Fluxo/economia , Células HEK293 , Humanos , Camundongos , Valor Preditivo dos Testes , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Reprodutibilidade dos Testes , Fatores de Tempo , Fluxo de Trabalho
6.
J Med Virol ; 93(12): 6512-6518, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34241912

RESUMO

There is a great demand for more rapid tests for SARS-CoV-2 detection to reduce waiting time, boost public health strategies for combating disease, decrease costs, and prevent overwhelming laboratory capacities. This study was conducted to assess the performance of 10 lateral flow device viral antigen immunoassays for the detection of SARS-CoV-2 in nasopharyngeal swab specimens. We analyzed 231 nasopharyngeal samples collected from October 2020 to December 2020, from suspected COVID-19 cases and contacts of positive cases at Biotechnology Research Center laboratories, Tripoli, Libya. The performance of 10 COVID-19 Antigen (Ag) rapid test devices for the detection of SARS-CoV-2 antigen was compared to a quantitative reverse transcription-polymerase chain reaction (RT-qPCR). In this study, 161 cases had symptoms consistent with COVID-19. The mean duration from symptom onset was 6.6 ± 4.3 days. The median cycle threshold (Ct ) of positive samples was 25. Among the 108 positive samples detected by RT-qPCR, the COVID-19 antigen (Ag) tests detected 83 cases correctly. All rapid Ag test devices used in this study showed 100% specificity. While tests from six manufacturers had an overall sensitivity range from 75% to 100%, the remaining four tests had a sensitivity of 50%-71.43%. Sensitivity during the first 6 days of symptoms and in samples with high viral loads (Ct < 25), was 100% in all but two of the test platforms. False-negative samples had a median Ct of 34 and an average duration of onset of symptoms of 11.3 days (range = 5-20 days). Antigen test diagnosis has high sensitivity and specificity in early disease when patients present less than 7 days of symptom onset. Patients are encouraged to test as soon as they get COVID-19-related symptoms within 1 week and to seek medical advice within 24 h if they develop disturbed smell/taste. The use of rapid antigen tests is important for controlling the COVID-19 pandemic and reducing the burden on molecular diagnostic laboratories.


Assuntos
Antígenos Virais/análise , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Imunoensaio/métodos , Adulto , Teste Sorológico para COVID-19/economia , Reações Falso-Negativas , Feminino , Humanos , Imunoensaio/economia , Masculino , Nasofaringe/virologia , Estudos Prospectivos , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Fatores de Tempo , Carga Viral
7.
Anaerobe ; 70: 102390, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34058377

RESUMO

BACKGROUND: Availability of several commercial tests with different Clostridioides difficile targets contributes to uncertainty and controversies around the optimal diagnostic algorithm. While numerous studies have estimated the financial impact of C. difficile infection, models to guide testing strategies decisions in developing countries, where economic value significantly impacts clinical practice, are currently not available. AIM: To determine the cost of illness of different C. difficile infection (CDI) diagnostic strategies in developing countries. METHODS: Cost-comparison analysis was performed to compare eleven different algorithms of CDI diagnosis. The basis of calculation was a hypothetical cohort of 1000 adult inpatients suspected of CDI. We analyzed turnaround time of test results (i.e., time from taking sample to results emission), test performance (i.e., sensitivity and specificity) and testing costs. Patients were divided in true positive, false positive, true negative and false negative in order to integrate test performance and economics effects. Additional medical costs were calculated: costs of hygiene, medication, length of stay and intensive care unit costs, based on a Brazilian University Hospital costs. CDI prevalence was considered 22.64%. FINDINGS: From laboratory-assisted tests, simultaneous glutamate dehydrogenase (GDH) and toxin A/B rapid immunoassay arbitrated by nucleic acid amplification test (NAAT) presented the lowest cost of illness (450,038.70 USD), whereas standalone NAAT had the highest (523,709.55 USD). Empirical diagnosis only presented the highest overall cost (809,605.44 USD). CONCLUSION: The two-step algorithm with simultaneous GDH and toxin A/B rapid immunoassay arbitrated by NAAT seems to be the best strategy for CDI diagnosis in developing countries.


Assuntos
Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/economia , Imunoensaio/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Algoritmos , Antibacterianos/economia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Toxinas Bacterianas/análise , Brasil , Clostridioides difficile/genética , Clostridioides difficile/fisiologia , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/microbiologia , Efeitos Psicossociais da Doença , Países em Desenvolvimento/economia , Reações Falso-Negativas , Glutamato Desidrogenase/genética , Humanos , Imunoensaio/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos
8.
Proc Natl Acad Sci U S A ; 118(18)2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33945500

RESUMO

Novel technologies are needed to facilitate large-scale detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific antibodies in human blood samples. Such technologies are essential to support seroprevalence studies and vaccine clinical trials, and to monitor quality and duration of immunity. We developed a microfluidic nanoimmunoassay (NIA) for the detection of anti-SARS-CoV-2 IgG antibodies in 1,024 samples per device. The method achieved a specificity of 100% and a sensitivity of 98% based on the analysis of 289 human serum samples. To eliminate the need for venipuncture, we developed low-cost, ultralow-volume whole blood sampling methods based on two commercial devices and repurposed a blood glucose test strip. The glucose test strip permits the collection, shipment, and analysis of 0.6 µL of whole blood easily obtainable from a simple finger prick. The NIA platform achieves high throughput, high sensitivity, and specificity based on the analysis of 289 human serum samples, and negligible reagent consumption. We furthermore demonstrate the possibility to combine NIA with decentralized and simple approaches to blood sample collection. We expect this technology to be applicable to current and future SARS-CoV-2 related serological studies and to protein biomarker analysis in general.


Assuntos
Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , COVID-19/sangue , Teste Sorológico para COVID-19/economia , Teste em Amostras de Sangue Seco , Ensaios de Triagem em Larga Escala/economia , Humanos , Imunoensaio/economia , Imunoglobulina G/sangue , Técnicas Analíticas Microfluídicas/economia , Reprodutibilidade dos Testes , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Manejo de Espécimes
9.
Anal Bioanal Chem ; 413(14): 3725-3735, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33851226

RESUMO

In this study, for the first time, we propose a sensitive colloidal gold-based lateral flow immunoassay (LFIA) that can be used to detect carbendazim residues in functional foods. The adoption of inline cleanup LFIA strips effectively improved background interference to reduce misjudgment of results. First, the hapten 2-(methylamino)-1H-benzo[d]imidazole-5-carboxylic acid was used to establish the carbendazim immunoassay method. Subsequently, colloidal gold-mAb preparation and LFIA detection conditions were systematically optimized. For root and fruit samples (ginseng, ginger, jujube, and Chinese wolfberry), the designed strips had a cutoff value of 8 ng/mL. For flower and seed samples (chrysanthemum, coix seed, and malt), the cutoff value was 12 ng/mL. Even in a complex matrix, the established LFIA method demonstrates satisfactory sensitivity and anti-interference ability. This method was successfully applied in detection of carbendazim residues in complex functional foods, and the assay results are consistent with those obtained via liquid chromatography-tandem mass spectrometry. In short, the proposed method is fast and sensitive and has strong anti-interference ability. Furthermore, it provides a new technical method highly relevant to the on-site rapid detection of carbendazim residues in complex sample matrix.


Assuntos
Benzimidazóis/análise , Carbamatos/análise , Contaminação de Alimentos/análise , Alimento Funcional/análise , Fungicidas Industriais/análise , Coloide de Ouro/química , Imunoensaio/métodos , Desenho de Equipamento , Imunoensaio/economia , Imunoensaio/instrumentação , Limite de Detecção , Fatores de Tempo
10.
Anal Bioanal Chem ; 413(14): 3683-3693, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33825917

RESUMO

How to achieve simultaneous and rapid detection of various mycotoxins in food has important practical significance in the field of food processing and safety. In this paper, a smartphone immunoassay system based on hydrogel microspheres has been constructed to quickly detect two mycotoxins at the same time. The rapid detection system was reflected in the following three processes: (1) rapid separation of free matter after direct competition reaction based on hydrogel solid-phase carrier particles; (2) rapid detection process based on efficient catalytic function of enzymes; (3) fast capture and analysis of images based on smartphone software. Ochratoxin A (OTA) and zearalenone (ZEN) are secondary toxic metabolites of fungi that can contaminate a wide range of foods and feeds. OTA and ZEN were used as detection model molecules to verify the feasibility of the intelligent rapid detection system. The entire detection process was within 30 min, and the results were analyzed in only 10 s. Detection limits of mycotoxins OTA and ZEN are 0.7711 ng L-1 and 1.0391 ng L-1. The recoveries of both mycotoxins ranged from 76.72 to 122.05%. This study provides a universal rapid detection method for on-site application of large-scale food security testing. Schematic diagram of the construction of the smartphone detection system: The system is divided into three parts: detection, image capture and analysis, and result.


Assuntos
Imunoensaio/métodos , Micotoxinas/análise , Ocratoxinas/análise , Smartphone , Zearalenona/análise , Contaminação de Alimentos/análise , Imunoensaio/economia , Imunoensaio/instrumentação , Limite de Detecção , Fatores de Tempo
11.
Eur J Clin Microbiol Infect Dis ; 40(10): 2235-2241, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33782783

RESUMO

We report evaluation of 30 assays' (17 rapid tests (RDTs) and 13 automated/manual ELISA/CLIA assay (IAs)) clinical performances with 2594 sera collected from symptomatic patients with positive SARS-CoV-2 rRT-PCR on a respiratory sample, and 1996 pre-epidemic serum samples expected to be negative. Only 4 RDT and 3 IAs fitted both specificity (> 98%) and sensitivity (> 90%) criteria according to French recommendations. Serology may offer valuable information during COVID-19 pandemic, but inconsistent performances observed among the 30 commercial assays evaluated, which underlines the importance of independent evaluation before clinical implementation.


Assuntos
Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/sangue , Imunoensaio/métodos , SARS-CoV-2/imunologia , COVID-19/virologia , Humanos , Imunoensaio/economia , Imunoglobulina M/sangue , Kit de Reagentes para Diagnóstico , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade
12.
Food Chem ; 352: 129415, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33711728

RESUMO

Furazolidone (FZD) and its metabolite called 3-amino-2-oxazolidinone (AOZ) would induce carcinogenic and mutagenic effects to human. In this work, to develop a novel, stable, and simple point of care testing (POCT) with a potential to social applied for FZD detection, we utilized the aspect of protein staining of coomassie brilliant blue (CBB) to exploit a new CBB-LFIA strategy free of NPs. Only one mixing step is needed during the probe manufacturing process, which requires just 2 h and is a great time saving strategy compared with other methods (requiring 4-33 h for probe preparation). Besides, the cost of CBB-LFIA is 300 times lesser than other LFIA with respect to obtaining the label. The developed CBB-LFIA was successfully applied to detect AOZ with a detection limit of 2 ng mL-1, without any influence from other potential interfering compounds. The proposed CBB-LFIA exhibited prominent practical application, and possesses considerable utilization potential in the related field.


Assuntos
Custos e Análise de Custo , Furazolidona/análise , Furazolidona/química , Imunoensaio/economia , Imunoensaio/métodos , Sistemas Automatizados de Assistência Junto ao Leito/economia , Corantes de Rosanilina/química , Humanos , Limite de Detecção , Fatores de Tempo
13.
Anal Bioanal Chem ; 413(18): 4635-4644, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33735408

RESUMO

Pd-Ir nanocubes are promising peroxidase-mimicking nanozymes for immunoassays, enabled by their excellent stability, relatively high catalytic activity, and reproducible performance. A key step involved in the preparation of Pd-Ir nanocubes is the synthesis of Pd nanocubes. However, the traditional method to synthesize Pd nanocubes requires sophisticated and expensive equipment to precisely control the reaction temperature and highly skilled technicians to achieve satisfactory and reproducible product yields. Herein, we report a simple, cost-effective, high-yield (> 99%) and one-pot strategy to synthesize Pd nanocubes with sizes of 7, 18, and 51 nm for the preparation of Pd-Ir nanocubes. The resulting 18 nm Pd-Ir nanocubes display three orders of magnitude higher peroxidase activity compared to horseradish peroxidase, leading to a significantly increased detection sensitivity when applied in the immunoassay of nucleocapsid protein from SARS-CoV-2. Due to the simplicity in both material synthesis and assaying procedures and the excellent detection sensitivity, our method should allow for the generalized application of Pd-Ir nanocube-based immunoassays for the diagnosis of human diseases.


Assuntos
COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/química , Imunoensaio/métodos , Irídio/química , Paládio/química , SARS-CoV-2 , Anticorpos Antivirais , Análise Custo-Benefício , Humanos , Imunoensaio/economia , Estrutura Molecular , Nanoestruturas/química , Nanoestruturas/economia , Fosfoproteínas/química
14.
Biosens Bioelectron ; 171: 112715, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33099241

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), a newly emerging human infectious disease. Because no specific antiviral drugs or vaccines are available to treat COVID-19, early diagnostics, isolation, and prevention are crucial for containing the outbreak. Molecular diagnostics using reverse transcription polymerase chain reaction (RT-PCR) are the current gold standard for detection. However, viral RNAs are much less stable during transport and storage than proteins such as antigens and antibodies. Consequently, false-negative RT-PCR results can occur due to inadequate collection of clinical specimens or poor handling of a specimen during testing. Although antigen immunoassays are stable diagnostics for detection of past infection, infection progress, and transmission dynamics, no matched antibody pair for immunoassay of SARS-CoV-2 antigens has yet been reported. In this study, we designed and developed a novel rapid detection method for SARS-CoV-2 spike 1 (S1) protein using the SARS-CoV-2 receptor ACE2, which can form matched pairs with commercially available antibodies. ACE2 and S1-mAb were paired with each other for capture and detection in a lateral flow immunoassay (LFIA) that did not cross-react with SARS-CoV Spike 1 or MERS-CoV Spike 1 protein. The SARS-CoV-2 S1 (<5 ng of recombinant proteins/reaction) was detected by the ACE2-based LFIA. The limit of detection of our ACE2-LFIA was 1.86 × 105 copies/mL in the clinical specimen of COVID-19 Patients without no cross-reactivity for nasal swabs from healthy subjects. This is the first study to detect SARS-CoV-2 S1 antigen using an LFIA with matched pair consisting of ACE2 and antibody. Our findings will be helpful to detect the S1 antigen of SARS-CoV-2 from COVID-19 patients.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais/instrumentação , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Peptidil Dipeptidase A/química , Pneumonia Viral/diagnóstico , Glicoproteína da Espícula de Coronavírus/análise , Enzima de Conversão de Angiotensina 2 , Anticorpos Monoclonais/química , Técnicas Biossensoriais/economia , COVID-19 , Teste para COVID-19 , Técnicas de Laboratório Clínico/economia , Técnicas de Laboratório Clínico/instrumentação , Infecções por Coronavirus/economia , Desenho de Equipamento , Humanos , Imunoensaio/economia , Imunoensaio/instrumentação , Imunoconjugados/química , Pandemias , SARS-CoV-2 , Sensibilidade e Especificidade , Fatores de Tempo
15.
PLoS One ; 15(11): e0242255, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33196653

RESUMO

BACKGROUND: Our objective was to assess the cost-effectiveness of novel rapid diagnostic tests: rapid influenza diagnostic tests (RIDT), digital immunoassays (DIA), rapid nucleic acid amplification tests (NAAT), and other treatment algorithms for influenza in high-risk patients presenting to hospital with influenza-like illness (ILI). METHODS: We developed a decision-analytic model to assess the cost-effectiveness of diagnostic test strategies (RIDT, DIA, NAAT, clinical judgement, batch polymerase chain reaction) preceding treatment; no diagnostic testing and treating everyone; and not treating anyone. We modeled high-risk 65-year old patients from a health payer perspective and accrued outcomes over a patient's lifetime. We reported health outcomes, quality-adjusted life years (QALYs), healthcare costs, and net health benefit (NHB) to measure cost-effectiveness per cohort of 100,000 patients. RESULTS: Treating everyone with no prior testing was the most cost-effective strategy, at a cost-effectiveness threshold of $50,000/QALY, in over 85% of simulations. This strategy yielded the highest NHB of 15.0344 QALYs, but inappropriately treats all patients without influenza. Of the novel rapid diagnostics, NAAT resulted in the highest NHB (15.0277 QALYs), and the least number of deaths (1,571 per 100,000). Sensitivity analyses determined that results were most impacted by the pretest probability of ILI being influenza, diagnostic test sensitivity, and treatment effectiveness. CONCLUSIONS: Based on our model, treating high-risk patients presenting to hospital with influenza-like illness, without performing a novel rapid diagnostic test, resulted in the highest NHB and was most cost-effective. However, consideration of whether treatment is appropriate in the absence of diagnostic confirmation should be taken into account for decision-making by clinicians and policymakers.


Assuntos
Análise Custo-Benefício , Influenza Humana/diagnóstico , Testes Imediatos/economia , Idoso , Canadá , Serviço Hospitalar de Emergência/economia , Feminino , Custos de Cuidados de Saúde , Humanos , Imunoensaio/economia , Influenza Humana/mortalidade , Influenza Humana/terapia , Masculino , Técnicas de Amplificação de Ácido Nucleico/economia , Anos de Vida Ajustados por Qualidade de Vida
16.
Sci Rep ; 10(1): 20593, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33244066

RESUMO

Tristeza is a highly destructive disease of citrus caused by the phloem-limited, flexuous filamentous Citrus tristeza virus (CTV) in the genus Closterovirus and the family Closteroviridae. It has been a major constraint for higher productivity and has destroyed millions of citrus trees globally. CTV is graft transmissible and spread through use of virus infected nursery plants. Therefore, virus detection by using specific and reliable diagnostic tools is very important to mitigate disease outbreaks. Currently, the standard molecular techniques for CTV detection include RT-PCR and RT-qPCR. These diagnostic methods are highly sensitive but time consuming, labor intensive and require sophisticated expensive instruments, thus not suitable for point-of-care use. In the present study, we report the development of a rapid, sensitive, robust, reliable, and highly specific reverse transcription-RPA technique coupled with a lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA). RT-RPA technique was standardized to amplify the coat protein gene of CTV (CTV-p25) and detect double labeled amplicons on a sandwich immunoassay by designing specific labeled primer pair and probe combinations. The optimally performing primer set (CTRPA-F1/CTRPA-R9-Btn) and the corresponding TwistAmp nfo probe (CTRPA-Probe) was optimized for temperature and reaction time using purified cDNA and viral RNA as template. The sensitivity of the developed assay was compared with other detection techniques using in vitro-transcribed RNA. The efficacy and specificity of the assay was evaluated using CTV positive controls, healthy samples, field grown citrus plants of unknown status, and other virus and bacterial pathogens that infect citrus plants. The RT-RPA-LFICA was able to detect ≤ 141 fg of RNA when cDNA used as a template. The assay detected ≤ 0.23 ng/µl of CTV RNA when directly used as template without cross-reactivity with other citrus pathogens. Best results were achieved at the isothermal temperature of 40 °C within 15-20 min. The study demonstrated that RT-RPA-LFICA has potential to become an improved detection technique for end users in bud-wood certification and quarantine programs and a promising platform for rapid point-of-care diagnostics for citrus farmers and small nurseries in low resource settings.


Assuntos
Citrus/virologia , Closterovirus/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças das Plantas/virologia , RNA Viral/análise , Closterovirus/genética , Imunoensaio/economia , Imunoensaio/métodos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/economia , RNA Viral/genética , Transcrição Reversa , Fatores de Tempo
17.
Int J Infect Dis ; 101: 382-390, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33039612

RESUMO

Timely and accurate laboratory testing is essential for managing the global COVID-19 pandemic. Reverse transcription polymerase chain reaction remains the gold-standard for SARS-CoV-2 diagnosis, but several practical issues limit the test's use. Immunoassays have been indicated as an alternative for individual and mass testing. OBJECTIVES: To access the performance of 12 serological tests for COVID-19 diagnosis. METHODS: We conducted a blind evaluation of six lateral-flow immunoassays (LFIAs) and six enzyme-linked immunosorbent assays (ELISAs) commercially available in Brazil for detecting anti-SARS-CoV-2 antibodies. RESULTS: Considering patients with seven or more days of symptoms, the sensitivity ranged from 59.5% to 83.1% for LFIAs and from 50.7% to 92.6% for ELISAs. For both methods, the sensitivity increased with clinical severity and days of symptoms. The agreement among LFIAs performed with digital blood and serum was moderate. Specificity was, in general, higher for LFIAs than for ELISAs. Infectious diseases prevalent in the tropics, such as HIV, leishmaniasis, arboviruses, and malaria, represent conditions with the potential to cause false-positive results with these tests, which significantly compromises their specificity. CONCLUSION: The performance of immunoassays was only moderate, affected by the duration and clinical severity of the disease. Absence of discriminatory power between IgM/IgA and IgG has also been demonstrated, which prevents the use of acute-phase antibodies for decisions on social isolation.


Assuntos
COVID-19/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Imunoensaio/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Brasil , COVID-19/sangue , COVID-19/virologia , Infecções por Coronavirus/epidemiologia , Ensaio de Imunoadsorção Enzimática/economia , Feminino , Humanos , Imunoensaio/economia , Masculino , Pessoa de Meia-Idade , Pandemias , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Adulto Jovem
18.
Biomed Microdevices ; 22(2): 39, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32436002

RESUMO

Currently, most HIV tests are performed with blood samples, or alternatively saliva samples are used for HIV testing. Simple HIV tests need to be performed in hospitals or other medical agencies instead of more invasive HIV blood tests. To enable point-of-care (POC) HIV diagnostics, based on a recently developed lateral flow strip for HIV urine testing, a microfluidic immunoassay cassette with a handheld optical reader is developed. Based on lateral flow strip with gold colloid reporter, the integrated immunoassay cassette can perform sample introduction, metering, discharging, applying and detection which simplifies HIV testing. An indicator is incorporated into the cassette to guide sample introduction based on color change, and further, the excess test sample is stored inside the sealed cassette to avoid any contamination. The low-cost handheld optical reader can provide a test result within a few seconds, which is useful for simple, sensitive and affordable HIV onsite detection. Instead of using normal white LEDs, a customized back light module embedded with green LEDs is adopted to illuminate the lateral flow strip with an appropriate working current to achieve optimal performance. Compared to the standard lateral flow strips using a benchtop reader, with the disposable immunoassay cassette assisted by the handheld optical reader, more convenient, easier-to-operate, and more affordable HIV urine testing can be achieved in POC diagnostics.


Assuntos
Infecções por HIV/urina , Imunoensaio/instrumentação , Testes Imediatos , Urinálise/instrumentação , Custos e Análise de Custo , Infecções por HIV/diagnóstico , Humanos , Imunoensaio/economia , Urinálise/classificação , Urinálise/economia
19.
J Agric Food Chem ; 68(7): 2193-2200, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-31976658

RESUMO

Various mycotoxins widely co-exist in agro-products, and their combined effects cause toxicity and potential carcinogenicity to humans and animals. In this work, we developed an economical and sensitive quantum dots (QDs)/QD microbead (QDs/QB)-based multiplex immunochromatographic assay (mICA) for the rapid detection of fumonisin B1 (FB1), zearalenone (ZEN), and ochratoxin A (OTA) without the building-up process of mycotoxin conjugates. QDs and QBs were selected as fluorescent reporters and conjugated with antimycotoxin monoclonal antibodies for improving sensitivity. Furthermore, phage-displayed FB1, ZEN, and OTA mimotope peptide-based soluble and monovalent fusions to maltose-binding protein (MBP) were applied onto the test line of the mICA as the mimetic coating antigen. Under the optimized conditions, the visual detection limits (vLODs) of peptide-MBP-based mICA could be obtained as 0.25 ng/mL for FB1, 3.0 ng/mL for ZEN, and 0.5 ng/mL for OTA within 10 min. The results for spiked real sample detection indicate good accuracy, reproducibility, and practicability. In addition, the proposed mICA was comparable with ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) in terms of reliability in detecting FB1, ZEN, and OTA using natural samples. From the point of promoting commercial production, these time-saving and low-cost peptide-MBP antigens applied in ICA might provide promising potential for promoting productivity and decreasing the cost of production.


Assuntos
Fumonisinas/análise , Imunoensaio/métodos , Ocratoxinas/análise , Zearalenona/análise , Contaminação de Alimentos/análise , Imunoensaio/economia , Imunoensaio/instrumentação , Limite de Detecção , Proteínas Ligantes de Maltose/química , Pontos Quânticos/química
20.
Blood ; 135(14): 1171-1184, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-31945147

RESUMO

Prompt diagnostic evaluation of suspected heparin-induced thrombocytopenia (HIT) is critical for guiding initial patient management. We assessed the performance of 3 immunoassays detecting anti-platelet factor 4 (PF4)/heparin antibodies, derived a diagnostic algorithm with a short analytical turnaround time (TAT), and prospectively validated the algorithm. Plasma samples were analyzed by Zymutest-HIA-IgG, HemosIL-AcuStar-HIT-IgG, and ID-H/PF4-PaGIA in retrospective (n = 221) and prospective (n = 305) derivation cohorts. We calculated likelihood ratios of result intervals and cutoff values with 100% negative (NPV) and positive (PPV) predictive values for a positive gold standard functional assay (heparin-induced platelet activation [HIPA]). A diagnostic algorithm was established based on the Bayesian combination of pretest probability and likelihood ratios of first- and second-line immunoassays. Cutoffs with 100% PPV for positive HIPA were >3.0 U/mL (HemosIL-AcuStar-HIT-IgG) and titer ≥16 (ID-H/PF4-PaGIA); cutoffs with 100% NPV were <0.13 U/mL and ≤1, respectively. During the prospective validation of the derived algorithm (n = 687), HemosIL-AcuStar-HIT-IgG was used as unique testing in 566 (82.4%) of 687 cases (analytical TAT, 30 minutes). In 121 (17.6%) of 687 unresolved cases, ID-H/PF4-PaGIA was used as second-line testing (additional TAT, 30 minutes). The algorithm accurately predicted HIT in 51 (7.4%) of 687 patients and excluded it in 604 (87.9%) of 687 patients, leaving only 20 (2.9%) cases unresolved. We also identified 12 (1.7%) of 687 positive predictions not confirmed by HIPA: 10 patients with probable HIT despite negative HIPA and 2 possible false-positive algorithm predictions. The combination of pretest probability with first- and second-line immunoassays for anti-PF4/heparin antibodies is accurate for ruling in or out HIT in ≥95% of cases within 60 minutes. This diagnostic approach improves initial management of patients with suspected HIT.


Assuntos
Anticorpos/sangue , Anticoagulantes/efeitos adversos , Heparina/efeitos adversos , Trombocitopenia/induzido quimicamente , Trombocitopenia/diagnóstico , Idoso , Anticorpos/imunologia , Anticoagulantes/imunologia , Teorema de Bayes , Ensaio de Imunoadsorção Enzimática/economia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Heparina/imunologia , Humanos , Imunoensaio/economia , Imunoensaio/métodos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Fator Plaquetário 4/imunologia , Estudos Prospectivos , Estudos Retrospectivos , Sensibilidade e Especificidade , Trombocitopenia/sangue , Fatores de Tempo
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