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1.
J Clin Virol ; 131: 104609, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32866811

RESUMO

INTRODUCTION: IgG immunoassays have been developed and used widely for clinical samples and serosurveys for SARS-CoV2, with most detecting antibodies against the spike/receptor-binding-domain or nucleocapsid. Limited information is available on comparative evaluation of IgG immunoassays against a clinical reference standard, i.e., RT-PCR positivity with >20 days of illness. This study addresses the need for comparing clinical performance of IgG immunoassays with respect to this alternate reference standard. METHODS: We compared the performance of three immunoassays, an in-house RBD assay, and two commercial assays, the Diasorin LIAISON SARS-CoV-2 S1/S1 IgG CLIA which detects antibodies against S1/S2 domains of the Spike protein and the Zydus Kavach assay based on inactivated virus using a well-characterized panel of sera. 379 sera and plasma samples from RTPCR positive individuals >20 days of illness in symptomatic or RT-PCR positivity in asymptomatic individuals and 184 samples collected prior to 2019 were used for assay evaluation. RESULTS: The sensitivity of the assays were 84.7 (95 %CI 80.6-88.1), 82.6 (95 %CI 78.3-86.2) and 75.7 (95 %CI 71.0-79.9) respectively for RBD, LIAISON and Kavach. Kavach and the in-house RBD ELISA showed a specificity of 99.5 % and 100 %, respectively. The RBD and LIAISON (S1/S2) assays showed high agreement (94.7 %; 95 %CI: 92.0, 96.6) and were able to correctly identify more positive sera/plasma than Kavach. CONCLUSION: Independent comparisons support the evaluation of performance characteristics of immunoassays. All three assays are suitable for serosurveillance studies, but in low prevalence sites, estimation of exposure may require adjustment based on our findings.


Assuntos
Anticorpos Antivirais/sangue , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/imunologia , Imunoensaio/métodos , Imunoglobulina G/sangue , Pneumonia Viral/imunologia , Automação Laboratorial , Betacoronavirus , Infecções por Coronavirus/diagnóstico , Humanos , Índia , Estudos Longitudinais , Medições Luminescentes , Pandemias , Estudos Prospectivos , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade
2.
J Clin Virol ; 130: 104549, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32763809

RESUMO

BACKGROUND: The global market for SARS-CoV-2-immunoassays is becoming ever more crowded with antibody-tests of various formats, targets and technologies, careful evaluation is crucial for understanding the implications of individual test results. Here, we evaluate the clinical performance of five automated immunoassays on a set of clinical samples. METHODS: Serum/plasma samples of 75 confirmed COVID-19 patients and 320 pre-pandemic serum samples of healthy blood donors were subjected to two IgG and three total antibody SARS-CoV-2-immunoassays. All test setups were automated workflows. RESULTS: Positivity of assays (onset of symptoms > 10 days) ranged between 68.4 % and 81.6 % (Diasorin 68.4 %, Euroimmun 70.3 %, Siemens 73.7 %, Roche 79.0 % and Wantai 81.6 %). All examined assays demonstrated high specificity of >99 % (Euroimmun, Diasorin: 99.1 %, Wantai: 99.4 %) but only two reached levels above 99.5 % (Roche: 99.7 %, Siemens 100 %). Interestingly, there was no overlap in false positive results between the assays. The strongest correlation of quantitative results was observed between the Diasorin and Euroimmun IgG tests (r2 = 0.76). Overall, we observed no difference in the distribution of test results between female and male patients (p-values: 0.18-0.87). A significant difference between severely versus critically ill patients was demonstrated for the Euroimmun, Diasorin, Wantai and Siemens assays (p-values:0.041). CONCLUSION: All assays showed good clinical performance. Our data confirm that orthogonal test strategies as recommended by the CDC can enhance clinical specificity. However, the suboptimal rates of test positivity found at time of hospitalization in this cohort underline the importance of molecular diagnostics to rule out/confirm active infection with SARS-CoV-2.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Automação Laboratorial , Betacoronavirus , Técnicas de Laboratório Clínico , Estudos de Coortes , Infecções por Coronavirus/imunologia , Reações Falso-Positivas , Feminino , Hospitalização/estatística & dados numéricos , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/imunologia , Sensibilidade e Especificidade , Adulto Jovem
3.
J Clin Virol ; 130: 104578, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32777761

RESUMO

The SARS-CoV-2 pandemic has challenged molecular microbiology laboratories to quickly implement and validate diagnostic assays and to expand testing capacity in a short timeframe. Multiple molecular diagnostic methods received FDA emergency use authorization (EUA) and were promptly validated for use nationwide. Several studies reported the analytical and/ or clinical evaluation of these molecular assays, however differences in the viral materials used for these evaluations complicated direct comparison of their analytical performance. In this study, we compared the analytical sensitivity (lower limit of detection, LOD) of seven commonly used qualitative SARS-CoV-2 molecular assays: the Abbott Molecular RealTime SARS-CoV-2 assay, the NeuMoDx™ SARS-CoV-2 assay, the Roche Cobas®SARS-CoV-2 assay, the BD SARS-CoV-2 reagents for BD MAX™ system, the Hologic Aptima® SARS-CoV-2 assay, the Xpert Xpress SARS-CoV-2 test, and the GenMark ePlex SARS-CoV-2 test. The comparison was performed utilizing a single positive clinical specimen that was serially diluted in viral transport media and quantified by the EUA approved SARS-CoV-2 droplet digital PCR (ddPCR) assay. Replicate samples were prepared and evaluated for reproducibility across different molecular assays with multiple replicates per assay. Our data demonstrated that the seven assays could detect 100 % of replicates at a nucleocapsid gene concentration of (N1 = 1,267 and N2 = 1,392) copies/mL. At a one log less concentration, the Abbott, the Roche, and the Xpert Xpress assays detected 100 % of the tested replicates.


Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Automação Laboratorial , Betacoronavirus , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular/métodos , Pandemias , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
4.
J Clin Virol ; 130: 104583, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32791382

RESUMO

The SARS-CoV-2 virus has caused millions of confirmed COVID-19 cases worldwide and hundreds of thousands of deaths in less than 6 months. Mitigation measures including social distancing were implemented to control disease spread, however, thousands of new cases continue to be diagnosed daily. To resume some suspended social activities, early diagnosis and contact tracing are essential. To meet this required diagnostic and screening capacity, high throughput diagnostic assays are needed. The NeuMoDx™ SARS-CoV-2 assay, performed on a NeuMoDx molecular system, is a rapid, fully automated, qualitative real-time RT-PCR diagnostic test with throughput of up to 288 tests in an 8 -h shift. The assay received emergency use authorization from the FDA and is used in some large testing centers in the US. This paper describes the analytical and clinical performance of the assay at three centers: Johns Hopkins Hospital, St. Jude Children's Research Hospital, and the Wadsworth Center.


Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Ensaios de Triagem em Larga Escala/métodos , Pneumonia Viral/diagnóstico , Automação Laboratorial , Betacoronavirus , Infecções por Coronavirus/virologia , Humanos , Pandemias , Pneumonia Viral/virologia , Sensibilidade e Especificidade , Manejo de Espécimes
5.
J Clin Microbiol ; 58(10)2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32727828

RESUMO

The COVID-19 pandemic caused by the new SARS-CoV-2 coronavirus has imposed severe challenges on laboratories in their effort to achieve sufficient diagnostic testing capability for identifying infected individuals. In this study, we report the analytical and clinical performance characteristics of a new, high-throughput, fully automated nucleic acid amplification test system for the detection of SARS-CoV-2. The assay utilizes target capture, transcription-mediated amplification, and acridinium ester-labeled probe chemistry on the automated Panther system to directly amplify and detect two separate target sequences in the open reading frame 1ab (ORF1ab) region of the SARS-CoV-2 RNA genome. The probit 95% limit of detection of the assay was determined to be 0.004 50% tissue culture infective dose (TCID50)/ml using inactivated virus and 25 copies/ml (c/ml) using synthetic in vitro transcript RNA targets. Analytical sensitivity (100% detection) was confirmed to be 83 to 194 c/ml using three commercially available SARS-CoV-2 nucleic acid controls. No cross-reactivity or interference was observed with testing of six related human coronaviruses, as well as 24 other viral, fungal, and bacterial pathogens, at high titers. Clinical nasopharyngeal swab specimen testing (n = 140) showed 100%, 98.7%, and 99.3% positive, negative, and overall agreement, respectively, with a validated reverse transcription-PCR nucleic acid amplification test (NAAT) for SARS-CoV-2 RNA. These results provide validation evidence for a sensitive and specific method for pandemic-scale automated molecular diagnostic testing for SARS-CoV-2.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Automação Laboratorial , Betacoronavirus/genética , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Humanos , Nasofaringe/virologia , RNA Viral/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteínas Virais/genética
6.
PLoS One ; 15(7): e0234959, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32663230

RESUMO

The economic and social impacts due to diseases transmitted by mosquitoes in the latest years have been significant. Currently, no specific treatment or commercial vaccine exists for the control and prevention of arboviruses, thereby making entomological characterization fundamental in combating diseases such as dengue, chikungunya, and Zika. The morphological identification of mosquitos includes a visual exam of the samples. It is time consuming and requires adequately trained professionals. Accordingly, the development of a new automated method for realizing mosquito-perception and -classification is becoming increasingly essential. Therefore, in this study, a computational model based on a convolutional neural network (CNN) was developed to extract features from the images of mosquitoes and then classify the species Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus. In addition, the model was trained to detect the mosquitoes of the genus Aedes. To train CNNs to perform the automatic morphological classification of mosquitoes, a dataset, which included 7,561 images of the target mosquitoes and 1,187 images of other insects, was acquired. Various neural networks, such as Xception and DenseNet, were used for developing the automatic-classification model based on images. A structured optimization process of random search and grid search was developed to select the hyperparameters set and increase the accuracy of the model. In addition, strategies to eliminate overfitting were implemented to increase the generalization of the model. The optimized model, during the test phase, obtained the balanced accuracy (BA) of 93.5% in classifying the target mosquitoes and other insects and the BA of 97.3% in detecting the mosquitoes of the genus Aedes in comparison to Culex. The results provide fundamental information for performing the automatic morphological classification of mosquito species. Using a CNN-embedded entomological tool is a valuable and accessible resource for health workers and non-taxonomists for identifying insects that can transmit infectious diseases.


Assuntos
Arbovirus/classificação , Culicidae/classificação , Processamento de Imagem Assistida por Computador/métodos , Aedes/virologia , Animais , Automação Laboratorial/métodos , Febre de Chikungunya/transmissão , Vírus Chikungunya/genética , Culex/virologia , Culicidae/genética , Dengue/transmissão , Vírus da Dengue/genética , Feminino , Masculino , Mosquitos Vetores/virologia , Zika virus/genética , Infecção por Zika virus/transmissão
7.
PLoS One ; 15(7): e0229967, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645012

RESUMO

Phthalic acid esters (phthalates) are male reproductive toxicants, which exert their most potent toxicity during fetal development. In the fetal rat, exposure to phthalates reduces testosterone biosynthesis, alters the development of seminiferous cords and other male reproductive tissues, and induces the formation of abnormal multinucleated germ cells (MNGs). Identification of MNGs is a time-intensive process, and it requires specialized training to identify MNGs in histological sections. As a result, MNGs are not routinely quantified in phthalate toxicity experiments. In order to speed up and standardize this process, we have developed an improved method for automated detection of MNGs. Using hand-labeled histological section images with human-identified MNGs, we trained a convolutional neural network with a U-Net architecture to identify MNGs on unlabeled images. With unseen hand-labeled images not used in model training, we assessed the performance of the model, using five different configurations of the data. On average, the model reached near human accuracy, and in the best model, it exceeded it. The use of automated image analysis will allow data on this histopathological endpoint to be more readily collected for analysis of phthalate toxicity. Our trained model application code is available for download at github.com/brown-ccv/mngcount.


Assuntos
Automação Laboratorial/métodos , Núcleo Celular , Separação Celular/métodos , Células Germinativas/patologia , Processamento de Imagem Assistida por Computador , Animais , Feminino , Masculino , Redes Neurais de Computação , Ácidos Ftálicos/toxicidade , Ratos Sprague-Dawley , Testículo/patologia
8.
J Clin Microbiol ; 58(9)2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32580948

RESUMO

In the coronavirus (CoV) disease 2019 (COVID-19) pandemic, highly selective serological testing is essential to define exposure to severe acute respiratory syndrome CoV 2 (SARS-CoV-2). Many tests have been developed, yet with variable speeds to first results, and are of unknown quality, particularly when considering the prediction of neutralizing capacity. The LIAISON SARS-CoV-2 S1/S2 IgG assay was designed to measure antibodies against the SARS-CoV-2 native S1/S2 proteins in a standardized automated chemiluminescence assay. The clinical and analytical performances of the test were validated in an observational study using residual samples (>1,500) with a positive or negative COVID-19 diagnosis. The LIAISON SARS-CoV-2 S1/S2 IgG assay proved to be highly selective and specific and offered semiquantitative measures of serum or plasma levels of anti-S1/S2 IgG with neutralizing activity. The assay's diagnostic sensitivities were 91.3% and 95.7% at >5 or ≥15 days from diagnosis, respectively, and 100% when assessed against a neutralizing assay. The assay's specificity ranged between 97% and 98.5%. The average imprecision of the assay was a <5% coefficient of variation. Assay performance at 2 different cutoffs was evaluated to optimize predictive values. The automated LIAISON SARS-CoV-2 S1/S2 IgG assay brings efficient, sensitive, specific, and precise serological testing to the laboratory, with the capacity to test large amounts of samples per day; first results are available within 35 min, with a throughput of 170 tests/hour. The semiquantitative results provided by the test also associate with the presence of neutralizing antibodies and may provide a useful tool for the large-scale screening of convalescent-phase plasma for safe therapeutic use.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Testes Sorológicos , Anticorpos Neutralizantes/sangue , Automação Laboratorial , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/imunologia , Humanos , Imunoglobulina G/sangue , Pandemias , Pneumonia Viral/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Testes Sorológicos/normas , Testes Sorológicos/estatística & dados numéricos , Glicoproteína da Espícula de Coronavírus/imunologia
9.
J Clin Virol ; 129: 104474, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32504946

RESUMO

BACKGROUND: High-throughput assays for the SARS-CoV-2 virus are critical to increasing test capacity and slowing the spread of COVID-19. Abbott Molecular developed and received emergency use authorization (EUA) to deploy the new RealTime SARS-CoV-2 assay, run on the automated m2000sp/rt system. OBJECTIVE: To evaluate analytical and clinical performance of the RealTime SARS-CoV-2 assay compared to the SARS-CoV-2 CDC-based laboratory developed test (LDT) in clinical use by the University of Washington Clinical Virology Laboratory (UW Virology). METHODS: RealTime SARS-CoV-2 assay limit of detection (LOD) was evaluated by testing two dilution panels of 60 replicates each. Cross-reactivity was evaluated by testing 24 clinical samples positive for various non‒SARS-CoV-2 respiratory viruses. Clinical performance was evaluated using 30 positive and 30 negative SARS-CoV-2 clinical samples previously tested using the UW Virology SARS-CoV-2 LDT. RESULTS: Exceeding the 100 copies/mL LOD reported in the RealTime SARS-CoV-2 assay EUA product insert, 19 of 20 replicates were detected at 50 copies/mL and 16 of 20 replicates were detected at 25 copies/mL. All clinical samples positive for 24 non‒SARS-CoV-2 respiratory viruses were SARS-CoV-2 negative on the RealTime SARS-CoV-2 assay. The assay had high sensitivity (93%) and specificity (100%) for detecting SARS-CoV-2 in clinical samples. Two positive samples that tested negative with the RealTime SARS-CoV-2 assay had cycle numbers of 35.94 or greater and required dilution prior to testing. One of these samples was also inconclusive on the SARS-CoV-2 LDT. CONCLUSION: The RealTime SARS-CoV-2 assay is acceptable for clinical use. With the high-throughput, fully automated m2000 system, this assay will accelerate the pace of SARS-CoV-2 testing.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/diagnóstico , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Automação Laboratorial/métodos , Betacoronavirus/genética , Ensaios de Triagem em Larga Escala/métodos , Hospitais Universitários , Humanos , Pandemias , RNA Viral/genética , Sensibilidade e Especificidade , Washington
10.
J Clin Virol ; 129: 104480, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32505777

RESUMO

Serological SARS-CoV-2 assays are urgently needed for diagnosis, contact tracing and for epidemiological studies. So far, there is limited data on how recently commercially available, high-throughput immunoassays, using different recombinant SARS-CoV-2 antigens, perform with clinical samples. Focusing on IgG and total antibodies, we demonstrate the performance of four automated immunoassays (Abbott Architect™ i2000 (N protein-based)), Roche cobas™ e 411 analyzer (N protein-based, not differentiating between IgA, IgM or IgG antibodies), LIAISON®XL platform (S1 and S2 protein-based), VIRCLIA® automation system (S1 and N protein-based) in comparison to two ELISA assays (Euroimmun SARS-CoV-2 IgG (S1 protein-based) and Virotech SARS-CoV-2 IgG ELISA (N protein-based)) and an in-house developed plaque reduction neutralization test (PRNT). We tested follow up serum/plasma samples of individuals PCR-diagnosed with COVID-19. When calculating the overall sensitivity, in a time frame of 49 days after first PCR-positivity, the PRNT as gold standard, showed the highest sensitivity with 93.3% followed by the dual-target assay for the VIRCLIA® automation system with 89%. The overall sensitivity in the group of N protein-based assays ranged from 66.7 to 77.8% and in the S protein-based-assays from 71.1 to 75.6%. Five follow-up samples of three individuals were only detected in either an S and/or N protein-based assay, indicating an individual different immune response to SARS-CoV-2 and the influence of the used assay in the detection of IgG antibodies. This should be further analysed. The specificity of the examined assays was ≥ 97%. However, because of the low or unknown prevalence of SARS-CoV-2, the examined assays in this study are currently primarily eligible for epidemiological investigations, as they have limited information in individual testing.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Ensaios de Triagem em Larga Escala/métodos , Imunoglobulina G/sangue , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos , Automação Laboratorial/métodos , Humanos , Pandemias , Sensibilidade e Especificidade
11.
J Clin Virol ; 129: 104512, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32563180

RESUMO

There is an urgent need for reliable high-throughput serological assays for the management of the ongoing COVID-19 pandemic. Preferably, the performance of serological tests for a novel virus should be determined with clinical specimens against a gold standard, i.e. virus neutralisation. We compared the performance of six commercial immunoassays for the detection of SARS-COV-2 IgG, IgA and IgM antibodies, including four automated assays [Abbott SARS-COV-2 IgG (CE marked), Diasorin Liaison® SARS-COV-2 S1/S2 IgG (research use only, RUO), and Euroimmun SARS-COV-2 IgG and IgA (CE marked)], and two rapid lateral flow (immunocromatographic) tests [Acro Biotech 2019-nCoV IgG/IgM (CE marked) and Xiamen Biotime Biotechnology SARS-COV-2 IgG/IgM (CE marked)] with a microneutralisation test (MNT). Two specimen panels from serum samples sent to Helsinki University Hospital Laboratory (HUSLAB) were compiled: the patient panel (N=70) included sera from PCR confirmed COVID-19 patients, and the negative panel (N=81) included sera sent for screening of autoimmune diseases and respiratory virus antibodies in 2018 and 2019. The MNT was carried out for all COVID-19 samples (70 serum samples, 62 individuals) and for 53 samples from the negative panel. Forty-one out of 62 COVID-19 patients showed neutralising antibodies.The specificity and sensitivity values of the commercial tests against MNT, respectively, were as follows: 95.1 %/80.5 % (Abbott Architect SARS-CoV-2 IgG), 94.9 %/43.8 % (Diasorin Liaison SARS-CoV-2 IgG; RUO), 68.3 %/87.8 % (Euroimmun SARS-CoV-2 IgA), 86.6 %/70.7 % (Euroimmun SARS-CoV-2 IgG), 74.4 %/56.1 % (Acro 2019-nCoV IgG), 69.5 %/46.3 % (Acro 2019-nCoV IgM), 97.5 %/71.9 % (Xiamen Biotime SARS-CoV-2 IgG), and 88.8 %/81.3 % (Xiamen Biotime SARS-CoV-2 IgM). This study shows variable performance values. Laboratories should carefully consider their testing process, such as a two-tier approach, in order to optimize the overall performance of SARS- CoV-2 serodiagnostics.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Testes de Neutralização/métodos , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Automação Laboratorial/métodos , Criança , Pré-Escolar , Feminino , Hospitais , Humanos , Imunoensaio/métodos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Pandemias , Sensibilidade e Especificidade , Adulto Jovem
12.
J Clin Virol ; 129: 104511, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32593133

RESUMO

BACKGROUND: The emergence of new SARS-CoV-2 has promoted the development of new serological tests that could be complementary to RT-PCR. Nevertheless, the assessment of clinical performances of available tests is urgently required as their use has just been initiated for diagnose. OBJECTIVES: The aim of this study was to assess the performance of three immunoassays for the detection of SARS-CoV-2 antibodies. METHODS: Two automated immunoassays (Abbott SARS-CoV-2 CLIA IgG and Euroimmun Anti-SARS-CoV-2 ELISA IgG/IgA assays) and one lateral flow immunoassay (LFIA NG-Test® IgG-IgM COVID-19) were tested. 293 specimens were analyzed from patients with a positive RT-PCR response, from patients with symptoms consistent with COVID-19 but exhibiting a negative response to the RT-PCR detection test, and from control group specimens. Days since symptoms onset were collected from clinical information sheet associated with respiratory tract samples. RESULTS: Overall sensitivity for IgG was equivalent (around 80 %) for CLIA, ELISA and LFIA. Sensitivity for IgG detection, >14 days after onset of symptoms, was 100.0 % for all assays. Overall specificity for IgG was greater for CLIA and LFIA (more than 98 %) compared to ELISA (95.8 %). Specificity was significantly different between IgA ELISA (78.9 %) and IgM LFIA (95.8 %) (p < 0.05). The best agreement was observed between CLIA and LFIA assays (97 %; k = 0.936). CONCLUSION: Excellent sensitivity for IgG detection was obtained >14 days after onset of symptoms for all immunoassays. Specificity was also excellent for IgG CLIA and IgG LFIA. Our study shows that NG-Test® is reliable and accurate for routine use in clinical laboratories.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Imunoensaio/métodos , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos , Idoso , Automação Laboratorial/métodos , Feminino , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Pandemias , Estudos Retrospectivos , Sensibilidade e Especificidade , Fatores de Tempo
13.
PLoS One ; 15(6): e0232869, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579562

RESUMO

Automated colony counting methods have long been known in Microbiology. Numerous methods for automated image analysis have been described and a wide range of commercial products exists. Known advantages are saving cost by reducing enumeration time, automatic documentation, reproducibility, and operator independence. Still, even today the realization of all advantages of automated image analysis makes it necessary to either invest in an expensive, high performance commercial system, or to acquire expert knowledge in image processing. This is a considerable obstacle for many laboratories, and the reason why manual colony counting is still done frequently. This article describes an easy to apply automatic colony counting system-including suggestions for sample preparation-that can be put into operation with basic knowledge of image processing and low budget.


Assuntos
Automação Laboratorial/métodos , Contagem de Colônia Microbiana/métodos , Processamento de Imagem Assistida por Computador/métodos , Ágar , Automação Laboratorial/instrumentação , Contagem de Colônia Microbiana/instrumentação , Escherichia coli , Processamento de Imagem Assistida por Computador/instrumentação , Reconhecimento Automatizado de Padrão/métodos , Software
14.
APMIS ; 128(8): 497-505, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32562292

RESUMO

Primary high-risk human papillomavirus (hrHPV) DNA testing has been introduced in several countries worldwide, including The Netherlands. The objective of this study was to compare three automated workflow procedures for hrHPV testing of which the hrHPV detection assays meet the international guidelines for HPV testing. To mimic a realistic screening situation, we aimed to process 15 000 residual PreservCyt cervical samples in a period of 3 months. During a 3 months period, four technicians were involved in processing 5000 specimens per month on three automated platforms, (1) Qiagen Digene® HC2 HPV DNA test (HC2, signal amplification); (2) Roche Cobas® HPV test (DNA amplification), and (3) Hologic Aptima® HPV test (RNA amplification). We measured and scored general aspects (time-to-results, hands-on-time (HOT)), maintenance, pre-run, run and post-run aspects, inventory (orders, storage), and number of errors on a scale from 1 to 10. As determined for one complete workflow each, maximum processing capacity and HOT were 296 samples and 2 h:55 m, 282 samples and 3 h:20 m, and 264 samples and 4 h:15 m for Aptima, Cobas, and HC2, respectively. The mean throughput time per run was 5 h:51 m for Cobas in which 94 samples could be processed. For Aptima, the mean throughput time per run was 6 h:30 m for 60 samples. Mean throughput time for HC2 is longer since results were provided on day 2. In this study, the fully automated Aptima workflow scores best with a 7.2, followed by Cobas with a score of 7.1 and HC2 with a score of 5.8. Although all HPV tests used in this comparison meet the international test guidelines, the performance (workflow) characteristics of the assays vary widely. A specific choice of a laboratory for high-throughput testing can be different based on the laboratory's demands, but also hands-on-time, time-to-results/ # samples, maintenance, pre-run, run and post-run parameters, consumables, technical support, and number of errors are important operational factors for the selection of a fully automated workflow for hrHPV testing.


Assuntos
Automação Laboratorial/métodos , Ensaios de Triagem em Larga Escala , Testes de DNA para Papilomavírus Humano/métodos , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Fluxo de Trabalho , Feminino , Humanos , Países Baixos , Papillomaviridae/classificação , Papillomaviridae/genética , Estudos Retrospectivos , Fatores de Tempo
15.
Emerg Microbes Infect ; 9(1): 1489-1496, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32543298

RESUMO

In December 2019, Wuhan, China suffered a serious outbreak of a novel coronavirus infectious disease (COVID) caused by novel severe acute respiratory syndrome-related coronavirus (SARS-CoV 2). To quickly identify the pathogen, we designed and screened primer sets, and established a sensitive and specific qRT-PCR assay for SARS-CoV 2; the lower limit of detection (LOD) was 14.8 (95% CI: 9.8-21) copies per reaction. We combined this qRT-PCR assay with an automatic integration system for nucleic acid extraction and amplification, thereby establishing an automatic integrated gene detection system (AIGS) for SARS-CoV 2. Cross reactive analysis performed in 20 other respiratory viruses and 37 nasopharyngeal swabs confirmed a 100% specificity of the assay. Using two fold diluted SARS-CoV 2 culture, the LOD of AIGS was confirmed to be 365 copies/ml (95% CI: 351-375), which was Comparable to that of conventional q RT-PCR (740 copies/ml, 95% CI: 689-750). Clinical performances of AIGS assay were assessed in 266 suspected COVID-19 clinical respiratory tract samples tested in parallel with a commercial kit. The clinical sensitivity of the AIGS test was 97.62% (95% CI: 0.9320-0.9951) based on the commercial kit test result, and concordance analysis showed a high agreement in SARS-CoV-2 detection between the two assays, Pearson R was 0.9623 (95% CI: 0.9523-0.9703). The results indicated that this AIGS could be used for rapid detection of SARS-CoV 2. With the advantage of simple operation and less time consuming, AIGS could be suitable for SARS-CoV2 detection in primary medical institutions, thus would do a great help to improve detection efficiency and control the spread of COVID-19.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Automação Laboratorial , China , Primers do DNA , Humanos , Limite de Detecção , Pandemias , RNA Viral/análise , Sensibilidade e Especificidade , Cultura de Vírus
16.
Euro Surveill ; 25(18)2020 05.
Artigo em Inglês | MEDLINE | ID: covidwho-197012

RESUMO

Antibody-screening methods to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) need to be validated. We evaluated SARS-CoV-2 IgG and IgA ELISAs in conjunction with the EUROLabworkstation (Euroimmun, Lübeck, Germany). Overall specificities were 91.9% and 73.0% for IgG and IgA ELISAs, respectively. Of 39 coronavirus disease patients, 13 were IgG and IgA positive and 11 IgA alone at sampling. IgGs and IgAs were respectively detected at a median of 12 and 11 days after symptom onset.


Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Pneumonia Viral/diagnóstico , Kit de Reagentes para Diagnóstico/normas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Automação Laboratorial , Betacoronavirus , Criança , Pré-Escolar , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/epidemiologia , Finlândia/epidemiologia , Humanos , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/epidemiologia , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto Jovem
17.
Indian J Med Res ; 151(2 & 3): 210-215, 2020.
Artigo em Inglês | MEDLINE | ID: covidwho-181979

RESUMO

Background & objectives: Nearly 5,500 tests for coronavirus disease 2019 (COVID-19) had been conducted on March 31, 2020 across the Indian Council of Medical Research (ICMR)-approved public and private laboratories in India. Given the need to rapidly increase testing coverage, we undertook an exercise to explore and quantify interventions to increase the daily real-time reverse transcription-polymerase chain reaction (qRT-PCR)-based testing capacity over the next few months. The objective of this exercise was to prepare a potential plan to scale-up COVID-19 testing in India in the public sector. Methods: Potential increase in daily testing capacity of the existing public laboratories was calculated across the three base scenarios of shifts (9, 16 and 24 h). Additional testing capacity was added for each shift scenario based on interventions ranging from procurement of additional qRT-PCR machines, leveraging spare capacity on available qRT-PCR machines not drafted into COVID-19 testing, to in-laboratory process optimization efforts. Results: Moving to a 24 h working model in the existing approved laboratories can enhance the daily testing capacity to 40,464 tests/day. The capacity can be further bolstered by leveraging qRT-PCR and nucleic acid amplification test (NAAT)-based machines available with the Multidisciplinary Research Units (MRUs), National AIDS Control Organisation (NACO) and National Tuberculosis Elimination Programme (NTEP). Using combination/multiplex kits, and provision of automated RNA extraction platforms at all laboratories could also optimize run time and contribute to capacity increase by 1.5-2 times. Interpretation & conclusions: Adopting these interventions could help increase public sector's daily testing capacity to nearly 100,000-120,000 tests/day. It is important to note that utilization of the scaled-up testing capacity will require deployment of additional workforce, procurement of corresponding commodities for testing and scale-up of sample collection and transportation efforts.


Assuntos
Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Planejamento Estratégico , Automação Laboratorial , Betacoronavirus , Técnicas de Laboratório Clínico , Ensaios de Triagem em Larga Escala , Humanos , Índia , Técnicas de Amplificação de Ácido Nucleico , Pandemias , Setor Público , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Indian J Med Res ; 151(2 & 3): 210-215, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32362646

RESUMO

Background & objectives: Nearly 5,500 tests for coronavirus disease 2019 (COVID-19) had been conducted on March 31, 2020 across the Indian Council of Medical Research (ICMR)-approved public and private laboratories in India. Given the need to rapidly increase testing coverage, we undertook an exercise to explore and quantify interventions to increase the daily real-time reverse transcription-polymerase chain reaction (qRT-PCR)-based testing capacity over the next few months. The objective of this exercise was to prepare a potential plan to scale-up COVID-19 testing in India in the public sector. Methods: Potential increase in daily testing capacity of the existing public laboratories was calculated across the three base scenarios of shifts (9, 16 and 24 h). Additional testing capacity was added for each shift scenario based on interventions ranging from procurement of additional qRT-PCR machines, leveraging spare capacity on available qRT-PCR machines not drafted into COVID-19 testing, to in-laboratory process optimization efforts. Results: Moving to a 24 h working model in the existing approved laboratories can enhance the daily testing capacity to 40,464 tests/day. The capacity can be further bolstered by leveraging qRT-PCR and nucleic acid amplification test (NAAT)-based machines available with the Multidisciplinary Research Units (MRUs), National AIDS Control Organisation (NACO) and National Tuberculosis Elimination Programme (NTEP). Using combination/multiplex kits, and provision of automated RNA extraction platforms at all laboratories could also optimize run time and contribute to capacity increase by 1.5-2 times. Interpretation & conclusions: Adopting these interventions could help increase public sector's daily testing capacity to nearly 100,000-120,000 tests/day. It is important to note that utilization of the scaled-up testing capacity will require deployment of additional workforce, procurement of corresponding commodities for testing and scale-up of sample collection and transportation efforts.


Assuntos
Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Planejamento Estratégico , Automação Laboratorial , Betacoronavirus , Técnicas de Laboratório Clínico , Ensaios de Triagem em Larga Escala , Humanos , Índia , Técnicas de Amplificação de Ácido Nucleico , Pandemias , Setor Público , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
Euro Surveill ; 25(18)2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32400364

RESUMO

Antibody-screening methods to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) need to be validated. We evaluated SARS-CoV-2 IgG and IgA ELISAs in conjunction with the EUROLabworkstation (Euroimmun, Lübeck, Germany). Overall specificities were 91.9% and 73.0% for IgG and IgA ELISAs, respectively. Of 39 coronavirus disease patients, 13 were IgG and IgA positive and 11 IgA alone at sampling. IgGs and IgAs were respectively detected at a median of 12 and 11 days after symptom onset.


Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Pneumonia Viral/diagnóstico , Kit de Reagentes para Diagnóstico/normas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Automação Laboratorial , Betacoronavirus , Criança , Pré-Escolar , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/epidemiologia , Finlândia/epidemiologia , Humanos , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/epidemiologia , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto Jovem
20.
J Clin Microbiol ; 58(8)2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32366669

RESUMO

Nucleic acid amplification tests (NAATs) are the primary means of identifying acute infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Accurate and fast test results may permit more efficient use of protective and isolation resources and allow rapid therapeutic interventions. We evaluated the analytical and clinical performance characteristics of the Xpert Xpress SARS-CoV-2 (Xpert) test, a rapid, automated molecular test for SARS-CoV-2. Analytical sensitivity and specificity/interference were assessed with infectious SARS-CoV-2; other infectious coronavirus species, including SARS-CoV; and 85 nasopharyngeal swab specimens positive for other respiratory viruses, including endemic human coronaviruses (hCoVs). Clinical performance was assessed using 483 remnant upper- and lower-respiratory-tract specimens previously analyzed by standard-of-care (SOC) NAATs. The limit of detection of the Xpert test was 0.01 PFU/ml. Other hCoVs, including Middle East respiratory syndrome coronavirus, were not detected by the Xpert test. SARS-CoV, a closely related species in the subgenus Sarbecovirus, was detected by a broad-range target (E) but was distinguished from SARS-CoV-2 (SARS-CoV-2-specific N2 target). Compared to SOC NAATs, the positive agreement of the Xpert test was 219/220 (99.5%), and the negative agreement was 250/261 (95.8%). A third tie-breaker NAAT resolved all but three of the discordant results in favor the Xpert test. The Xpert test provided sensitive and accurate detection of SARS-CoV-2 in a variety of upper- and lower-respiratory-tract specimens. The high sensitivity and short time to results of approximately 45 min may impact patient management.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Automação Laboratorial/métodos , Betacoronavirus/genética , Criança , Pré-Escolar , Infecções por Coronavirus/virologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Pandemias , Pneumonia Viral/virologia , Sensibilidade e Especificidade , Adulto Jovem
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