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1.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200331

RESUMO

The coronavirus disease 2019 (COVID-19) has caused a large global outbreak. It is accordingly important to develop accurate and rapid diagnostic methods. The polymerase chain reaction (PCR)-based method including reverse transcription-polymerase chain reaction (RT-PCR) is the most widely used assay for the detection of SARS-CoV-2 RNA. Along with the RT-PCR method, digital PCR has emerged as a powerful tool to quantify nucleic acid of the virus with high accuracy and sensitivity. Non-PCR based techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) are considered to be rapid and simple nucleic acid detection methods and were reviewed in this paper. Non-conventional molecular diagnostic methods including next-generation sequencing (NGS), CRISPR-based assays and nanotechnology are improving the accuracy and sensitivity of COVID-19 diagnosis. In this review, we also focus on standardization of SARS-CoV-2 nucleic acid testing and the activity of the National Metrology Institutes (NMIs) and highlight resources such as reference materials (RM) that provide the values of specified properties. Finally, we summarize the useful resources for convenient COVID-19 molecular diagnostics.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/isolamento & purificação , Animais , COVID-19/virologia , Sistemas CRISPR-Cas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Técnicas de Diagnóstico Molecular , Nanotecnologia , Reação em Cadeia da Polimerase , RNA Viral , Recombinases , Transcrição Reversa , Sensibilidade e Especificidade
2.
J Med Microbiol ; 70(7)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34251298

RESUMO

Carbapenem-resistant Gram-negative bacteria (CR-GNB) are a major source of nosocomial infections worldwide. In this study, the ability of a loop-mediated isothermal amplification (LAMP)-based method (Isoplex CRE-ART) to rapidly detect carbapenemase-encoding genes bla OXA-48-like, bla OXA-23-like, bla OXA-24-like, bla KPC, bla VIM, bla NDM and bla IMP in 231 carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii isolates was investigated. The accuracy of the LAMP test was compared to results of molecular isolate characterization using a Laboratory Developed Test multiplex carbapenemase PCR assay. The LAMP test correctly identified the presence of on-panel carbapenemases with a sensitivity of 99.16 % [95 % confidence interval (CI): 95.39-99.96 %] and a specificity of 98.21 % (95 % CI: 93.72-99.68 %) in 60 min. Our findings suggest that the Isoplex CRE-ART assay is able to rapidly identify carbapenemase genes in CR-GNB and improves options for pathogen characterization in the context of clinical microbiological and infection control diagnostics.


Assuntos
Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Bactérias Gram-Negativas/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pseudomonas aeruginosa/genética , beta-Lactamases/genética , Genes Bacterianos
3.
Viruses ; 13(5)2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-34068899

RESUMO

The use of rapid antigenic tests (Ag-RDTs) to diagnose a SARS-CoV-2 infection has become a common practice recently. This study aimed to evaluate performance of Abbott PanbioTM Ag-RDTs with regard to nucleic acid amplification testing (NAAT) in the early stages of the disease. A cohort of 149,026 infected symptomatic patients, reported in Catalonia from November 2020 to January 2021, was selected. The positivity rates of the two tests were compared with respect to the dates of symptom onset. Ag-RDTs presented positivity rates of 84% in the transmission phases of the disease and 31% in the pre-symptomatic period, compared to 93% and 91%, respectively, for NAAT. The detection of many false negatives with Ag-RDTs during the pre-symptomatic period demonstrates the risk of virus dissemination with this diagnostic technique if used outside the symptomatic period.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Virais/imunologia , Doenças Assintomáticas , Criança , Pré-Escolar , Estudos de Coortes , Reações Falso-Negativas , Reações Falso-Positivas , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico/métodos , Sensibilidade e Especificidade , Espanha , Adulto Jovem
4.
Viruses ; 13(5)2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069710

RESUMO

SARS-CoV-2 is the coronavirus responsible for COVID-19, which has spread worldwide, affecting more than 200 countries, infecting over 140 million people in one year. The gold standard to identify infected people is RT-qPCR, which is highly sensitive, but needs specialized equipment and trained personnel. The demand for these reagents has caused shortages in certain countries. Isothermal nucleic acid techniques, such as loop-mediated isothermal amplification (LAMP) have emerged as an alternative or as a complement to RT-qPCR. In this study, we developed and evaluated a multi-target RT-LAMP for the detection of SARS-CoV-2. The method was evaluated against an RT-qPCR in 152 clinical nasopharyngeal swab samples. The results obtained indicated that both assays presented a "good concordance" (Cohen's k of 0.69), the RT-LAMP was highly specific (99%) but had lower sensitivity compared to the gold standard (63.3%). The calculated low sensitivity was associated with samples with very low viral load (RT-qPCR Cq values higher than 35) which may be associated with non-infectious individuals. If an internal Cq threshold below 35 was set, the sensitivity and Cohen's k increased to 90.9% and 0.92, respectively. The interpretation of the Cohen's k for this was "very good concordance". The RT-LAMP is an attractive approach for frequent individual testing in decentralized setups.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Humanos , Técnicas de Diagnóstico Molecular/métodos , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Carga Viral , Proteínas Virais/genética
5.
Cochrane Database Syst Rev ; 5: CD012972, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-34097769

RESUMO

BACKGROUND: The World Health Organization (WHO) recommends Xpert MTB/RIF in place of smear microscopy to diagnose tuberculosis (TB), and many countries have adopted it into their diagnostic algorithms. However, it is not clear whether the greater accuracy of the test translates into improved health outcomes. OBJECTIVES: To assess the impact of Xpert MTB/RIF on patient outcomes in people being investigated for tuberculosis. SEARCH METHODS: We searched the following databases, without language restriction, from 2007 to 24 July 2020: Cochrane Infectious Disease Group (CIDG) Specialized Register; CENTRAL; MEDLINE OVID; Embase OVID; CINAHL EBSCO; LILACS BIREME; Science Citation Index Expanded (Web of Science), Social Sciences citation index (Web of Science), and Conference Proceedings Citation Index - Social Science & Humanities (Web of Science). We also searched the WHO International Clinical Trials Registry Platform, ClinicalTrials.gov, and the Pan African Clinical Trials Registry for ongoing trials. SELECTION CRITERIA: We included individual- and cluster-randomized trials, and before-after studies, in participants being investigated for tuberculosis. We analysed the randomized and non-randomized studies separately.  DATA COLLECTION AND ANALYSIS: For each study, two review authors independently extracted data, using a piloted data extraction tool. We assessed the risk of bias using Cochrane and Effective Practice and Organisation of Care (EPOC) tools. We used random effects meta-analysis to allow for heterogeneity between studies in setting and design.  The certainty of the  evidence in the randomized trials was assessed by GRADE. MAIN RESULTS: We included 12 studies: eight were randomized controlled trials (RCTs), and four were before-and-after studies. Most included RCTs had a low risk of bias in most domains of the Cochrane 'Risk of bias' tool. There was inconclusive evidence of an effect of Xpert MTB/RIF on all-cause mortality, both overall (risk ratio (RR) 0.89, 95% confidence interval (CI) 0.75 to 1.05; 5 RCTs, 9932 participants, moderate-certainty evidence), and restricted to studies with six-month follow-up (RR 0.98, 95% CI 0.78 to 1.22; 3 RCTs, 8143 participants; moderate-certainty evidence). There was probably a reduction in mortality in participants known to be infected with HIV (odds ratio (OR) 0.80, 95% CI 0.67 to 0.96; 5 RCTs, 5855 participants; moderate-certainty evidence). It is uncertain whether Xpert MTB/RIF has no or a modest effect on the proportion of participants starting tuberculosis treatment who had a successful treatment outcome (OR) 1.10, 95% CI 0.96 to 1.26; 3RCTs, 4802 participants; moderate-certainty evidence). There was also inconclusive evidence of an effect on the  proportion of participants who were treated for tuberculosis (RR 1.10, 95% CI 0.98 to 1.23; 5 RCTs, 8793 participants; moderate-certainty evidence). The proportion of participants treated for tuberculosis who had bacteriological confirmation was probably higher in the Xpert MTB/RIF group (RR 1.44, 95% CI 1.29 to 1.61; 6 RCTs, 2068 participants; moderate-certainty evidence). The proportion of participants with bacteriological confirmation who were lost to follow-up pre-treatment was probably reduced (RR 0.59, 95% CI 0.41 to 0.85; 3 RCTs, 1217 participants; moderate-certainty evidence). AUTHORS' CONCLUSIONS: We were unable to confidently rule in or rule out the effect on all-cause mortality of using Xpert MTB/RIF rather than smear microscopy. Xpert MTB/RIF probably reduces mortality among participants known to be infected with HIV. We are uncertain whether Xpert MTB/RIF has a modest effect or not on the proportion treated or, among those treated, on the proportion with a successful outcome. It probably does not have a substantial effect on these outcomes. Xpert MTB/RIF probably increases both the proportion of treated participants who had bacteriological confirmation, and the proportion with a laboratory-confirmed diagnosis who were treated. These findings may inform decisions about uptake alongside evidence on cost-effectiveness and implementation.


Assuntos
Antibióticos Antituberculose/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Rifampina/farmacologia , Tuberculose Pulmonar/diagnóstico , Viés , Intervalos de Confiança , Estudos Controlados Antes e Depois , Farmacorresistência Bacteriana , Infecções por HIV/mortalidade , Humanos , Mycobacterium tuberculosis/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Razão de Chances , Ensaios Clínicos Controlados Aleatórios como Assunto , Kit de Reagentes para Diagnóstico , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/mortalidade
6.
Biosensors (Basel) ; 11(6)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073756

RESUMO

Despite collaborative efforts from all countries, coronavirus disease 2019 (COVID-19) pandemic has been continuing to spread globally, forcing the world into social distancing period, making a special challenge for public healthcare system. Before vaccine widely available, the best approach to manage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is to achieve highest diagnostic accuracy by improving biosensor efficacy. For SARS-CoV-2 diagnostics, intensive attempts have been made by many scientists to ameliorate the drawback of current biosensors of SARS-CoV-2 in clinical diagnosis to offer benefits related to platform proposal, systematic analytical methods, system combination, and miniaturization. This review assesses ongoing research efforts aimed at developing integrated diagnostic tools to detect RNA viruses and their biomarkers for clinical diagnostics of SARS-CoV-2 infection and further highlights promising technology for SARS-CoV-2 specific diagnosis. The comparisons of SARS-CoV-2 biomarkers as well as their applicable biosensors in the field of clinical diagnosis were summarized to give scientists an advantage to develop superior diagnostic platforms. Furthermore, this review describes the prospects for this rapidly growing field of diagnostic research, raising further interest in analytical technology and strategic plan for future pandemics.


Assuntos
Técnicas Biossensoriais/instrumentação , Teste para COVID-19/instrumentação , SARS-CoV-2/isolamento & purificação , Animais , Técnicas Biossensoriais/métodos , Teste para COVID-19/métodos , Colorimetria/instrumentação , Colorimetria/métodos , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Ensaio de Imunoadsorção Enzimática/instrumentação , Ensaio de Imunoadsorção Enzimática/métodos , Desenho de Equipamento , Humanos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Testes Imediatos
7.
Sci Rep ; 11(1): 13378, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183720

RESUMO

The highly infectious nature of SARS-CoV-2 necessitates the use of widespread testing to control the spread of the virus. Presently, the standard molecular testing method (reverse transcriptase-polymerase chain reaction, RT-PCR) is restricted to the laboratory, time-consuming, and costly. This increases the turnaround time for getting test results. This study sought to develop a rapid, near-patient saliva-based test for COVID-19 (Saliva-Dry LAMP) with similar accuracy to that of standard RT-PCR tests. A lyophilized dual-target reverse transcription-loop-mediated isothermal amplification (RT-LAMP) test with fluorometric detection by the naked eye was developed. The assay relies on dry reagents that are room temperature stable. A device containing a centrifuge, heat block, and blue LED light system was manufactured to reduce the cost of performing the assay. This test has a limit of detection of 1 copy/µL and achieved a positive percent agreement of 100% [95% CI 88.43% to 100.0%] and a negative percent agreement of 96.7% [95% CI 82.78-99.92%] relative to a reference standard test. Saliva-Dry LAMP can be completed in 105 min. Precision, cross-reactivity, and interfering substances analysis met international regulatory standards. The combination of ease of sample collection, dry reagents, visual detection, low capital equipment cost, and excellent analytical sensitivity make Saliva-Dry LAMP particularly useful for resource-limited settings.


Assuntos
COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/análise , Saliva/virologia , COVID-19/virologia , Fluorometria , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/normas , RNA Viral/normas , Padrões de Referência , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Temperatura
8.
Biochem Biophys Res Commun ; 567: 195-200, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34166918

RESUMO

Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a target DNA sequence with a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA were used as targets. After evaluating the concentration of each component, the uvsY, gp32, and ATP concentrations appeared to be rate-determining factors. In particular, the balance between the binding and dissociation of uvsX and DNA primer was precisely adjusted. Under the optimized condition, 60 copies of the target DNA were specifically detected. Detection of 60 copies of RNA was also achieved. Our results prove the fabrication flexibility of RPA reagents, leading to an expansion of the use of RPA in various fields.


Assuntos
DNA Viral/análise , DNA Polimerase Dirigida por DNA/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/normas , RNA Viral/análise , Recombinases/metabolismo , SARS-CoV-2/genética , Estatística como Assunto , Primers do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , SARS-CoV-2/isolamento & purificação , Proteínas Virais/metabolismo
9.
Chem Commun (Camb) ; 57(56): 6871-6874, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34169944

RESUMO

The trans-cleavage activity of the target-activated CRISPR/Cas12a liberated an RNA crosslinker from a molecular transducer, which facilitated the assembly of gold nanoparticles. Integration of the molecular transducer with isothermal amplification and CRISPR/Cas12a resulted in visual detection of the N gene and E gene of SARS-CoV-2 in 45 min.


Assuntos
COVID-19/diagnóstico , Sistemas CRISPR-Cas , Genes Virais/genética , Ouro/química , Nanopartículas Metálicas/química , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genética , COVID-19/virologia , Colorimetria , Reagentes para Ligações Cruzadas , RNA/química
10.
PLoS One ; 16(6): e0252757, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34161355

RESUMO

BACKGROUND: A number of nucleic acid amplification tests (NAATs) for SARS-CoV-2 with different reagents have been approved for clinical use in Japan. These include research kits approved under emergency use authorization through simplified process to stabilize the supply of the reagents. Although these research kits have been increasingly used in clinical practice, limited data is available for the diagnostic performance in clinical settings. METHODS: We compared sensitivity, specificity, and cycle threshold (Ct) values obtained by NAATs using 10 kits approved in Japan including eight kits those receiving emergency use authorization using 69 frozen-stored clinical samples including 23 positive samples with various Ct values and 46 negative samples. RESULTS: Viral copy number of the frozen-stored samples determined with LightMix E-gene test ranged from 0.6 to 84521.1 copies/µL. While no false-positive results were obtained by any of these tests (specificity: 100% [95% CI, 88.9%-100%]), sensitivity of the nine tests ranged from 68.2% [95% CI, 45.1%-86.1%] to 95.5% [95% CI, 77.2%-99.9%] using LightMix E-gene test as the gold standard. All tests showed positive results for all samples with ≥100 copies/µL. Significant difference of Ct values even among tests amplifying the same genetic region (N1-CDC, N2) was also observed. CONCLUSION: Difference in the diagnostic performance was observed among NAATs approved in Japan. Regarding diagnostic kits for emerging infectious diseases, a system is needed to ensure both rapidity of reagent supply and accuracy of diagnosis. Ct values, which are sometimes regarded as a marker of infectivity, are not interchangeable when obtained by different assays.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Calibragem , Proteínas do Envelope de Coronavírus/genética , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade
11.
Pan Afr Med J ; 38: 294, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34178213

RESUMO

Introduction: quantification of hepatitis B virus DNA, a key element in the management of chronic hepatitis B, allows a more direct and reliable measurement of viral replication and monitoring of the virological response to therapy. Polymerase chain reaction (PCR) platforms performing this quantification and adaptable to intermediate laboratories have been developed. Thus, this study was conducted to evaluate the on-site performance of the AMPLIX® hepatitis B virus (HBV) real-time PCR technique in comparison with the COBAS AmpliPrep™ technique. Methods: performance of the AMPLIX® HBV real-time PCR technique was evaluated with repeatability and intermediate precision (reproducibility) determined. The comparison with COBAS Taqman was performed by testing, in parallel, 42 plasma samples. The statistical analysis using Meth Val® software was focused on correlation and concordance determination. Results: AMPLIX® real-time PCR assay showed good reproducibility for the low (CV=6.65%) and high (CV=3.15%) control levels but also good repeatability for both the low (CV=2.12%) and high (CV=1.60%) concentration levels. Accuracy obtained in our study were less than acceptability limit fixed to 5%. Viral load measurements between Amplix and COBAS Taqman correlated strongly with a correlation coefficient of 0.97%. Concordance analysis gave an average of the differences of 0.54 log IU/L between the viral load measurements of the 2 techniques. Conclusion: based on these results, the Amplix real-time PCR platform for the quantification of HBV DNA can be considered as a reliable system for the monitoring of chronic hepatitis B and also a system adapted to intermediate laboratories.


Assuntos
Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA Viral/sangue , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Senegal , Carga Viral
12.
Food Chem ; 358: 129894, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33933968

RESUMO

Listeria monocytogenes is an important foodborne pathogen, which imposes great burdens on public health. The current methods for detecting L. monocytogene are limited in several ways such as time consuming and lab equipment dependent. In this study, we developed a new electrochemical assay to improve the efficacy. This assay allows us to generate numerous G-quadruplex sequences while loop-mediated isothermal amplification happens. Then, these G-quadruplex sequences form DNAzyme to produce a color change and an electrochemical signal by oxidizing tetramethylbenzidine. This assay could be finished in 2 h, which significantly reduced the detection time. Also, we confirmed the limit of detection of this assay at 6.8 CFU/mL according to 3σ criterion. Our assay shows good sensitivity to detect bacteria range from 52.5 to 5.25 × 104 CFU/mL. This assay's reliability was also confirmed by detecting artificially contaminated pork samples. Thus, we propose this electrochemical assay for rapid and sensitive detection of L. monocytogenes in food.


Assuntos
Técnicas Eletroquímicas/métodos , Microbiologia de Alimentos/métodos , Listeria monocytogenes/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Carne de Porco/microbiologia , Animais , Benzidinas/química , DNA Catalítico/química , DNA Catalítico/metabolismo , Quadruplex G , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
13.
Ann Clin Microbiol Antimicrob ; 20(1): 38, 2021 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-34022903

RESUMO

BACKGROUND: SARS-CoV-2 is a newly emerged coronavirus, causing the coronavirus disease 2019 (COVID-19) outbreak in December, 2019. As drugs and vaccines of COVID-19 remain in development, accurate virus detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kits have been reliably used for detection of SARS-CoV-2 RNA since the beginning of the COVID-19 outbreak, whereas isothermal nucleic acid amplification-based point-of-care automated kits have also been considered as a simpler and rapid alternative. However, as these kits have only been developed and applied clinically within a short timeframe, their clinical performance has not been adequately evaluated to date. We describe a comparative study between a newly developed cross-priming isothermal amplification (CPA) kit (Kit A) and five RT-qPCR kits (Kits B-F) to evaluate their sensitivity, specificity, predictive values and accuracy. METHODS: Fifty-two clinical samples were used including throat swabs (n = 30), nasal swabs (n = 7), nasopharyngeal swabs (n = 7) and sputum specimens (n = 8), comprising confirmed (n = 26) and negative cases (n = 26). SARS-CoV-2 detection was simultaneously performed on each sample using six nucleic acid amplification kits. The sensitivity, specificity, positive/negative predictive values (PPV/NPV) and the accuracy for each kit were assessed using clinical manifestation and molecular diagnoses as the reference standard. Reproducibility for RT-qPCR kits was evaluated in triplicate by three different operators using a SARS-CoV-2 RNA-positive sample. On the basis of the six kits' evaluation results, CPA kit (Kit A) and two RT-qPCR Kits (Kit B and F) were applied to the SARS-CoV-2 detection in close-contacts of COVID-19 patients. RESULTS: For Kit A, the sensitivity, specificity, PPV/NPV and accuracy were 100%. Among the five RT-qPCR kits, Kits B, C and F had good agreement with the clinical diagnostic reports (Kappa ≥ 0.75); Kits D and E were less congruent (0.4 ≤ Kappa < 0.75). Differences between all kits were statistically significant (P < 0.001). The reproducibility of RT-qPCR kits was determined using a coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. CONCLUSIONS: This is the first comparative study to evaluate CPA and RT-qPCR kits' specificity and sensitivity for SARS-CoV-2 detection, and could serve as a reference for clinical laboratories, thus informing testing protocols amid the rapidly progressing COVID-19 pandemic.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Kit de Reagentes para Diagnóstico , SARS-CoV-2/genética , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
14.
Viruses ; 13(5)2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947014

RESUMO

The availability of simple SARS-CoV-2 detection methods is crucial to contain the COVID-19 pandemic. This study examined whether a commercial LAMP assay can reliably detect SARS-CoV-2 genomes directly in respiratory samples without having to extract nucleic acids (NA) beforehand. Nasopharyngeal swabs (NPS, n = 220) were tested by real-time reverse transcription (RT)-PCR and with the LAMP assay. For RT-PCR, NA were investigated. For LAMP, NA from 26 NPS in viral transport medium (VTM) were tested. The other 194 NPS were analyzed directly without prior NA extraction (140 samples in VTM; 54 dry swab samples stirred in phosphate buffered saline). Ten NPS were tested directly by LAMP using a sous-vide cooking unit. The isothermal assay demonstrated excellent specificity (100%) but moderate sensitivity (68.8%), with a positive predictive value of 1 and a negative predictive value of 0.65 for direct testing of NPS in VTM. The use of dry swabs, even without NA extraction, improved the analytical sensitivity; up to 6% of samples showed signs of inhibition. LAMP could be performed successfully with a sous-vide cooking unit. This technique is very fast, requires little laboratory resources, and can replace rapid antigen tests or verify reactive rapid tests on-site.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , DNA Viral/análise , Técnicas de Diagnóstico Molecular/métodos , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/isolamento & purificação , Humanos , Sensibilidade e Especificidade , Manejo de Espécimes
15.
Clin Microbiol Rev ; 34(3)2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-33980687

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory disease coronavirus 2 (SARS-CoV-2), has led to millions of confirmed cases and deaths worldwide. Efficient diagnostic tools are in high demand, as rapid and large-scale testing plays a pivotal role in patient management and decelerating disease spread. This paper reviews current technologies used to detect SARS-CoV-2 in clinical laboratories as well as advances made for molecular, antigen-based, and immunological point-of-care testing, including recent developments in sensor and biosensor devices. The importance of the timing and type of specimen collection is discussed, along with factors such as disease prevalence, setting, and methods. Details of the mechanisms of action of the various methodologies are presented, along with their application span and known performance characteristics. Diagnostic imaging techniques and biomarkers are also covered, with an emphasis on their use for assessing COVID-19 or monitoring disease severity or complications. While the SARS-CoV-2 literature is rapidly evolving, this review highlights topics of interest that have occurred during the pandemic and the lessons learned throughout. Exploring a broad armamentarium of techniques for detecting SARS-CoV-2 will ensure continued diagnostic support for clinicians, public health, and infection prevention and control for this pandemic and provide advice for future pandemic preparedness.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico por imagem , COVID-19/diagnóstico , SARS-CoV-2/genética , Técnicas Biossensoriais , Genoma Viral/genética , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Testes Imediatos , SARS-CoV-2/imunologia , Manejo de Espécimes/métodos
16.
PLoS One ; 16(5): e0250202, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33951060

RESUMO

Diagnosis of any infectious disease is vital for opportune treatment and to prevent dissemination. RT-qPCR tests for detection of SARS-CoV-2, the causative agent for COVID-19, are ideal in a hospital environment. However, mass testing requires cheaper and simpler tests, especially in settings that lack sophisticated machinery. The most common current diagnostic method is based on nasopharyngeal sample collection, RNA extraction, and RT-qPCR for amplification and detection of viral nucleic acids. Here, we show that samples obtained from nasopharyngeal swabs in VTM and in saliva can be used with or without RNA purification in an isothermal loop-mediated amplification (LAMP)-based assay, with 60-93% sensitivity for SARS-CoV-2 detection as compared to standard RT-qPCR tests. A series of simple modifications to standard RT-LAMP published methods to stabilize pH fluctuations due to salivary acidity resulted in a significant improvement in reliability, opening new avenues for efficient, low-cost testing of COVID-19 infection.


Assuntos
COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/análise , SARS-CoV-2/genética , Saliva/química , COVID-19/virologia , Reações Falso-Positivas , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Nasofaringe/virologia , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade
17.
mSphere ; 6(3)2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011690

RESUMO

Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva.IMPORTANCE One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accurate and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genética , Saliva/virologia , Humanos , Testes Imediatos , RNA Viral/genética , SARS-CoV-2/isolamento & purificação
18.
J Virol Methods ; 294: 114182, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33984397

RESUMO

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires fast and accurate high-throughput diagnostic tools. To evaluate the analytical performance of the Hologic Aptima transcription-mediated amplification (TMA) assay for detection of SARS-CoV-2 RNA from respiratory samples we analysed 103 clinical and proficiency panel samples pre-tested by real-time RT-PCR (Altona, RealStar) and found a positive percent agreement (sensitivity) of 95.7 % and a negative percent agreement (specificity) of 100 %. The limit of detection of the Aptima test was 150 copies/mL determined as 95 % detection probability. To further assess the Aptima assay's specificity we prospectively analysed 7545 clinical specimens from the upper and lower respiratory tract sent for the purpose of routine SARS-CoV-2 screening. SARS-CoV-2 RNA was detected in 16/7545 (0.2 %) samples by the TMA assay and confirmed independently by the Xpert SARS-CoV-2 RT-PCR (Cepheid); in one case a previous discrepant result was confirmed as true SARS-CoV-2 infection in a subsequent sample from the same patient. Results from the Aptima SARS-CoV-2 TMA assay agreed well with RT-PCR and showed an excellent specificity in a large number of routine specimens despite the low prevalence at that time of the pandemic, indicating that this assay can be used even for screening purposes.


Assuntos
Teste para COVID-19/normas , Técnicas de Amplificação de Ácido Nucleico/normas , RNA Viral/genética , Kit de Reagentes para Diagnóstico/normas , SARS-CoV-2/genética , Infecções Assintomáticas , Teste para COVID-19/métodos , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Humanos , Limite de Detecção , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Estudos Prospectivos , Estudos Retrospectivos , Sensibilidade e Especificidade
19.
Biotechniques ; 70(4): 218-225, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33820475

RESUMO

Evaluation of the performance of a new set of primers defined from the ORF1ab sequence, and its combination with a previously published set of primers from the N sequence, to detect SARS-CoV-2 RNA by the loop-mediated isothermal amplification technique is presented. The ORF1ab primer set enables visual detection of SARS-CoV-2 RNA in 16 min. In addition, a simultaneous reaction with both ORF1ab and N primers allows for higher sensitivity of detection, particularly when low numbers of copies are present (250 viral RNA copies). Further, the protocol is able to detect viral RNA in saliva samples. The procedure reported could be easily implemented in the generation of a new and sensitive rapid point-of care device for SARS-CoV-2 RNA visual detection.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Colorimetria/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genética , COVID-19/virologia , Primers do DNA , Humanos , Poliproteínas/genética , Proteínas Virais/genética
20.
Food Microbiol ; 98: 103664, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33875195

RESUMO

Vibrio vulnificus is an important foodborne pathogenic bacterium that mainly contaminates seafood. Rapid and accurate technologies that suitable for on-site detection are critical for effective control of its spreading. Conventional detection methods and polymerase chain reaction (PCR)-based and qPCR-based approaches have application limitations in on-site scenarios. Application of loop-mediated isothermal amplification (LAMP) technology was a good step towards the on-site detection. In this study, a recombinase polymerase amplification (RPA)-based detection method for V. vulnificus was developed combining with lateral flow strip (LFS) for visualized signal. The method targeted the conservative empV gene encoding the extracellular metalloproteinase, and finished detection in 35 min at a conveniently low temperature of 37 °C. It showed good specificity and an excellent sensitivity of 2 copies of the genome or 10-1 colony forming unit (CFU) per reaction, or 1 CFU/10 g in spiked food samples with enrichment. The method tolerated unpurified templates directly from sample boiling, which added the convenience of the overall procedure. Application of the RPA-LFS method for clinical samples showed accurate and consistent detection results compared to bioassay and quantitative PCR. This RPA-LFS combined method is well suited for on-site detection of V. vulnificus.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Alimentos Marinhos/microbiologia , Vibrio vulnificus/isolamento & purificação , Animais , Contaminação de Alimentos/análise , Recombinases/química , Recombinases/metabolismo , Alimentos Marinhos/análise , Sensibilidade e Especificidade , Vibrio vulnificus/classificação , Vibrio vulnificus/genética
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