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1.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445406

RESUMO

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first assays targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level, and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail, and our findings reveal some limitations but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Nevertheless, whilst our data show that some errors can be tolerated, it is always prudent to confirm that the primer and probe sequences complement their intended target, since, when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case, it is unlikely that a mismatch will result in poor specificity or a significant number of false-positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/isolamento & purificação , COVID-19/epidemiologia , COVID-19/virologia , Técnicas de Laboratório Clínico/métodos , Humanos , Pandemias , RNA Viral/genética , RNA Polimerase Dependente de RNA , SARS-CoV-2/genética , Sensibilidade e Especificidade , Temperatura
2.
Microbiol Spectr ; 9(1): e0034221, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34346748

RESUMO

As the COVID-19 pandemic progresses, there is an increasing need for rapid, accessible assays for SARS-CoV-2 detection. We present a clinical evaluation and real-world implementation of the INDICAID COVID-19 rapid antigen test (INDICAID rapid test). A multisite clinical evaluation of the INDICAID rapid test using prospectively collected nasal (bilateral anterior) swab samples from symptomatic subjects was performed. The INDICAID rapid test demonstrated a positive percent agreement (PPA) and negative percent agreement (NPA) of 85.3% (95% confidence interval [95% CI], 75.6% to 91.6%) and 94.9% (95% CI, 91.6% to 96.9%), respectively, compared to laboratory-based reverse transcriptase PCR (RT-PCR) using nasal specimens. The INDICAID rapid test was then implemented at COVID-19 outbreak screening centers in Hong Kong as part of a testing algorithm (termed "dual-track") to screen asymptomatic individuals for prioritization for confirmatory RT-PCR testing. In one approach, preliminary positive INDICAID rapid test results triggered expedited processing for laboratory-based RT-PCR, reducing the average time to confirmatory result from 10.85 h to 7.0 h. In a second approach, preliminary positive results triggered subsequent testing with an onsite rapid RT-PCR, reducing the average time to confirmatory result to 0.84 h. In 22,994 asymptomatic patients, the INDICAID rapid test demonstrated a PPA of 84.2% (95% CI, 69.6% to 92.6%) and an NPA of 99.9% (95% CI, 99.9% to 100%) compared to laboratory-based RT-PCR using combined nasal/oropharyngeal specimens. The INDICAID rapid test has excellent performance compared to laboratory-based RT-PCR testing and, when used in tandem with RT-PCR, reduces the time to confirmatory positive result. IMPORTANCE Laboratory-based RT-PCR, the current gold standard for COVID-19 testing, can require a turnaround time of 24 to 48 h from sample collection to result. The delayed time to result limits the effectiveness of centralized RT-PCR testing to reduce transmission and stem potential outbreaks. To address this, we conducted a thorough evaluation of the INDICAID COVID-19 rapid antigen test, a 20-minute rapid antigen test, in both symptomatic and asymptomatic populations. The INDICAID rapid test demonstrated high sensitivity and specificity with RT-PCR as the comparator method. A dual-track testing algorithm was also evaluated utilizing the INDICAID rapid test to screen for preliminary positive patients, whose samples were then prioritized for RT-PCR testing. The dual-track method demonstrated significant improvements in expediting the reporting of positive RT-PCR test results compared to standard RT-PCR testing without prioritization, offering an improved strategy for community testing and controlling SARS-CoV-2 outbreaks.


Assuntos
Antígenos Virais/análise , Doenças Assintomáticas , Teste para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/imunologia , SARS-CoV-2/isolamento & purificação , Adulto , Técnicas de Laboratório Clínico/métodos , Reações Falso-Negativas , Reações Falso-Positivas , Feminino , Hong Kong , Humanos , Masculino , Programas de Rastreamento/métodos , Pessoa de Meia-Idade , Pandemias , Reação em Cadeia da Polimerase , SARS-CoV-2/genética , Sensibilidade e Especificidade , Manejo de Espécimes , Fatores de Tempo , Adulto Jovem
3.
Microbiol Spectr ; 9(1): e0031221, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34378949

RESUMO

Pooled testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is instrumental for increasing test capacity while decreasing test cost. Pooled testing programs permit sustainable, long-term surveillance measures, which are essential for the early detection of virus resurgence in communities or the emergence of variants of concern. While numerous pooled approaches have been proposed to increase test capacity, uptake by laboratories has been limited. On 9 December 2020, we invited 362 U.S. laboratories that inquired about the Yale School of Public Health SalivaDirect test to participate in a survey to evaluate testing constraints and pooling strategies for SARS-CoV-2 testing. The survey was distributed using Qualtrics, and three reminders were sent. The survey closed on 21 January 2021. Of 93 responses received (25.7% response rate), 90 were from Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories conducting SARS-CoV-2 testing. The remaining three were excluded from the analyses. Responses indicated that the major barriers to the uptake of pooled testing in the United States may not simply be the number of tests a laboratory can process per day, but rather the lack of clear protocols and adequate resources; laboratories are working with fixed physical and human capital constraints. Importantly, laboratories across the country are heterogeneous in infrastructure and workflow. The need for SARS-CoV-2 testing will remain for years to come. Testing programs can be maintained through pooled PCR testing strategies, and while statisticians, operations researchers, and others with expertise in sampling design have important value to add, laboratories require support on how to transition from traditional diagnostic testing to pooled surveillance. IMPORTANCE While numerous pooled SARS-CoV-2 testing approaches have been described in an effort to increase testing capacity and decrease test prices, uptake by laboratories has been limited. Responses to our survey of United States-based laboratories highlight the importance of consulting end-users-those that solutions are being designed for-so challenges can be addressed in a manner tailored to meet the specific needs out in the field. It may be surprising to those designing pooled testing strategies to learn that laboratories view pooling as more time-consuming than testing samples individually, and therefore that it is thought to create delays in test reporting.


Assuntos
Teste para COVID-19/métodos , Teste para COVID-19/estatística & dados numéricos , COVID-19/diagnóstico , Teste para COVID-19/normas , Técnicas de Laboratório Clínico/métodos , Testes Diagnósticos de Rotina , Humanos , Laboratórios/estatística & dados numéricos , RNA Viral , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes , Tempo , Estados Unidos
4.
Int J Lab Hematol ; 43 Suppl 1: 65-70, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34288450

RESUMO

Anemia is a global health problem in all age groups. According to World Health Organization (WHO), approximately 40% of pregnant women are anemic. Iron deficiency anemia (IDA) due to nutritional deficiency is the most common cause. The incidence of IDA varies worldwide depending on the socioeconomic status, but it remains the leading cause even in developed countries. Physiologic anemia of pregnancy due to relatively higher expansion of blood volume in comparison with elevated red blood cell mass also occurs frequently. Complete blood count (CBC) in the first trimester is recommended for all pregnant women to screen for anemia. The screening of pregnant women for IDA in absence of anemia is still debatable. If IDA is suspected, ferritin level of <30 ng/ml is diagnostic. Iron supplementation is recommended for all pregnant women to compensate the increased demand.


Assuntos
Anemia/diagnóstico , Serviços de Laboratório Clínico , Técnicas de Laboratório Clínico , Complicações Hematológicas na Gravidez/diagnóstico , Anemia/etiologia , Anemia Ferropriva/diagnóstico , Anemia Ferropriva/etiologia , Técnicas de Laboratório Clínico/métodos , Diagnóstico Diferencial , Gerenciamento Clínico , Suscetibilidade a Doenças , Feminino , Humanos , Programas de Rastreamento , Gravidez , Complicações Hematológicas na Gravidez/etiologia
5.
Nutrients ; 13(6)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064098

RESUMO

Vitamin D is a micronutrient with pleiotropic effects in humans. Due to sedentary lifestyles and increasing time spent indoors, a growing body of research is revealing that vitamin D deficiency is a global problem. Despite the routine measurement of vitamin D in clinical laboratories and many years of efforts, methods of vitamin D analysis have yet to be standardized and are burdened with significant difficulties. This review summarizes several key analytical and clinical challenges that accompany the current methods for measuring vitamin D. According to an external quality assessment, methods and laboratories still produce a high degree of variability. Structurally similar metabolites are a source of significant interference. Furthermore, there is still no consensus on the normal values of vitamin D in a healthy population. These and other problems discussed herein can be a source of inconsistency in the results of research studies.


Assuntos
Técnicas de Laboratório Clínico/normas , Avaliação Nutricional , Deficiência de Vitamina D/diagnóstico , Vitamina D/análise , Técnicas de Laboratório Clínico/métodos , Humanos , Laboratórios/normas , Padrões de Referência , Valores de Referência , Reprodutibilidade dos Testes
6.
Mol Cell Probes ; 58: 101744, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34089805

RESUMO

To increase the repertoire of PCR based laboratory developed tests (LDTs) for the detection of SARS-CoV-2, we describe a new multiplex assay (SORP), targeting the SARS-CoV-2's, Spike and ORF8 genes. The widely used human RNaseP internal control was modified to specifically co-amplify the RNaseP mRNA. The SORP triplex assay was tested on a cohort (n = 372; POS = 144/NEG = 228) of nasopharyngeal flocked swab (NPFS) specimens, previously tested for the presence of SARS-CoV-2 using a PCR assay targeting E and RdRp genes. The overall sensitivity and specificity of the SORP assay was: 99.31% (95% CI: 96.22-99.98%), 100.0% (95% CI: 98.4-100%) respectively. The SORP assay could also detect a panel of variants of concern (VOC) from the B1.1.7 (UK) and B1.351 (SA) lineage. In summary, access to a repertoire of new SARS-CoV-2 LDT's would assist diagnostic laboratories in developing strategies to overcome some of the testing issues encountered during high-throughput SARS-CoV-2 testing.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Laboratório Clínico/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , COVID-19/virologia , Primers do DNA/genética , Sondas de DNA/genética , Humanos , Técnicas de Diagnóstico Molecular/métodos , Reprodutibilidade dos Testes , Ribonuclease P/genética , SARS-CoV-2/fisiologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais/genética
8.
Clin Biochem ; 95: 73-76, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33989560

RESUMO

BACKGROUND: Non-standard body fluids (NSBFs) can provide essential clinical information otherwise unobtainable with conventional biological specimens. However, as most commercial chemistry reagents are only validated for serum, plasma, and urine by manufacturers, individual laboratories have to validate testing with NSBF to comply with regulatory standards. However, the heightened level of oversight and uncertainty of validation requirements to comply with regulatory standards pose a significant challenge for NSBF testing in clinical laboratories. METHODS: 28 combinations of high-volume chemistry tests requested on NSBF with established clinical utility were selected from retrospective data analysis. Specimens were analyzed with both closed and open channel chemistry reagents on a LABOSPECT 008AS platform (Hitachi High-Tech Co., Tokyo, Japan). Recovery studies were performed using a high concentration serum sample and 5 clinical NSBF samples at varying concentrations for each analyte. Acceptable performance limits were defined as 100 ± 10% of expected recovery. RESULTS: The average percent recovery ranged from 94.5% to 106.6% depending on the analyte/NSBF combination evaluated, and for each of the 28 combinations, the average percent recovery was within the predefined acceptable limits of ± 10%. CONCLUSIONS: The recovery results from this study on the LABOSPECT 008AS platform demonstrates that any systematic matrix interference of high-volume chemistry testing on NSBF samples is well within the defined limits of acceptability. This work also demonstrates recovery studies performed by an individual laboratory are practial and it is feasible to demonstrate compliance with regulatory requirements for accuracy of chemistry testing on NSBF samples.


Assuntos
Líquidos Corporais/química , Testes de Química Clínica/instrumentação , Testes de Química Clínica/métodos , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/métodos , Humanos , Estudos Retrospectivos
9.
Mol Cell Probes ; 58: 101742, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33971264

RESUMO

Point of care detection of SARS-CoV-2 is one pillar in a containment strategy and important to break infection chains. Here we report the sensitive, specific and robust detection of SARS-CoV-2 and respective variants of concern by the ID NOW COVID-19 device.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Sistemas Automatizados de Assistência Junto ao Leito , SARS-CoV-2/genética , COVID-19/virologia , Técnicas de Laboratório Clínico/métodos , Humanos , Reprodutibilidade dos Testes , SARS-CoV-2/fisiologia , Sensibilidade e Especificidade
10.
Sci Rep ; 11(1): 10605, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34012040

RESUMO

Non-pharmaceutical interventions (NPIs) including resource allocation, risk communication, social distancing and travel restriction, are mainstream actions to control the spreading of Coronavirus disease 2019 (COVID-19) worldwide. Different countries implemented their own combinations of NPIs to prevent local epidemics and healthcare system overloaded. Portfolios, as temporal sets of NPIs have various systemic impacts on preventing cases in populations. Here, we developed a probabilistic modeling framework to evaluate the effectiveness of NPI portfolios at the macroscale. We employed a deconvolution method to back-calculate incidence of infections and estimate the effective reproduction number by using the package EpiEstim. We then evaluated the effectiveness of NPIs using ratios of the reproduction numbers and considered them individually and as a portfolio systemically. Based on estimates from Japan, we estimated time delays of symptomatic-to-confirmation and infection-to-confirmation as 7.4 and 11.4 days, respectively. These were used to correct surveillance data of other countries. Considering 50 countries, risk communication and returning to normal life were the most and least effective yielding the aggregated effectiveness of 0.11 and - 0.05 that correspond to a 22.4% and 12.2% reduction and increase in case growth. The latter is quantified by the change in reproduction number before and after intervention implementation. Countries with the optimal NPI portfolio are along an empirical Pareto frontier where mean and variance of effectiveness are maximized and minimized independently of incidence levels. Results indicate that implemented interventions, regardless of NPI portfolios, had distinct incidence reductions and a clear timing effect on infection dynamics measured by sequences of reproduction numbers. Overall, the successful suppression of the epidemic cannot work without the non-linear effect of NPI portfolios whose effectiveness optimality may relate to country-specific socio-environmental factors.


Assuntos
COVID-19/epidemiologia , COVID-19/prevenção & controle , Controle de Doenças Transmissíveis/métodos , Comunicação , Modelos Estatísticos , Algoritmos , Número Básico de Reprodução , COVID-19/economia , COVID-19/transmissão , Técnicas de Laboratório Clínico/métodos , Simulação por Computador , Transmissão de Doença Infecciosa/prevenção & controle , Transmissão de Doença Infecciosa/estatística & dados numéricos , Humanos , Japão/epidemiologia , SARS-CoV-2/isolamento & purificação
11.
Virol J ; 18(1): 95, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947425

RESUMO

The early identification of asymptomatic yet infectious cases is vital to curb the 2019 coronavirus (COVID-19) pandemic and to control the disease in the post-pandemic era. In this paper, we propose a fast, inexpensive and high-throughput approach using painless nasal-swab self-collection followed by direct RT-qPCR for the sensitive PCR detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This approach was validated in a large prospective cohort study of 1038 subjects, analysed simultaneously using (1) nasopharyngeal swabs obtained with the assistance of healthcare personnel and analysed by classic two-step RT-qPCR on RNA isolates and (2) nasal swabs obtained by self-collection and analysed with direct RT-qPCR. Of these subjects, 28.6% tested positive for SARS-CoV-2 using nasopharyngeal swab sampling. Our direct RT-qPCR approach for self-collected nasal swabs performed well with results similar to those of the two-step RT-qPCR on RNA isolates, achieving 0.99 positive and 0.98 negative predictive values (cycle threshold [Ct] < 37). Our research also reports on grey-zone viraemia, including samples with near-cut-off Ct values (Ct ≥ 37). In all investigated subjects (n = 20) with grey-zone viraemia, the ultra-small viral load disappeared within hours or days with no symptoms. Overall, this study underscores the importance of painless nasal-swab self-collection and direct RT-qPCR for mass testing during the SARS-CoV-2 pandemic and in the post-pandemic era.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/prevenção & controle , Programas de Rastreamento/métodos , Autoexame/métodos , Técnicas de Laboratório Clínico/métodos , Testes Diagnósticos de Rotina/métodos , Humanos , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Inquéritos e Questionários , Carga Viral/métodos
12.
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
13.
Am J Clin Pathol ; 156(1): 15-23, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33978164

RESUMO

OBJECTIVES: To report our institutional experience in devising and implementing a pooling protocol and process for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription polymerase chain reaction (RT-PCR) testing over a 3-month period in the fall of 2020. METHODS: The widespread testing implemented in the United States for detecting SARS-CoV-2 infection in response to the coronavirus disease 2019 pandemic has led to a significant shortage of testing supplies and therefore has become a major impediment to the public health response. To date, several institutions have implemented sample pooling, but publications documenting these experiences are sparse. Nasal and nasopharyngeal samples collected from low-positivity (<5%) areas were tested in pools of five on the Roche cobas 6800 analyzer system. Routine SARS-CoV-2 RT-PCR turnaround times between sample collection to result reporting were monitored and compared before and after sample pooling implementation. RESULTS: A total of 4,131 sample pools were tested over a 3-month period (during which 39,770 RT-PCR results were reported from the Roche system), allowing our laboratory to save 13,824 tests, equivalent to a conservation rate of 35%. A 48-hour or less turnaround time was generally maintained throughout the pooling period. CONCLUSIONS: Sample pooling offers a viable means to mitigate shortfalls of PCR testing supplies in the ongoing pandemic without significantly compromising overall turnaround times.


Assuntos
Teste para COVID-19 , COVID-19/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , COVID-19/genética , Técnicas de Laboratório Clínico/métodos , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/patogenicidade , Manejo de Espécimes/métodos
14.
Eur J Protistol ; 79: 125803, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34044354

RESUMO

Insulin activity is generally determined by an in vivo rabbit blood glucose drop assay in research and industriel laboratories. The humane experimental techniques imply the use of alternative invertebrate organisms in place of animals, known as replacement rule of the 3Rs. In this study, we report an alternative in vivo extracellular glucose drop assay using unicellular invertebrate Tetrahymena thermophila to replace the use of rabbit and mouse. This assay has four major steps; growing cells, starving cells, treatment of cells and measurement of glucose drop. In this assay, 0.2 mg/ml of human, porcine and bovine insulins dropped extracellular glucose level to 16%, 14% and 12%, respectively in ten minutes. In addition, mammalian insulins respectively increased the cell area about 19%, 15%, and 16% at 6th hour with statistically significant effect on the cell growth, but not in the cell viability. The results showed that the in vivo Tetrahymena thermophila extracellular glucose drop assay could be used as an alternative assay to replace the mouse or the rabbit insulin blood glucose drop assay.


Assuntos
Alternativas ao Uso de Animais , Técnicas de Laboratório Clínico/métodos , Insulina/análise , Tetrahymena thermophila/metabolismo , Animais , Insulina/metabolismo , Mamíferos , Reprodutibilidade dos Testes
15.
J Appl Lab Med ; 6(4): 998-1004, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-33825844

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays have emerged as a response to the global pandemic, warranting studies evaluating their clinical performance. This study investigated 7 commercially available SARS-CoV-2 serological assays in samples from noninfected individuals and hospitalized patients. METHODS: SARS-CoV-2 qualitative serological assays by Abbott (IgG), Beckman (IgG), DiaSorin (IgG), EUROIMMUN (IgG and IgA), Roche and Bio-Rad (Total) were evaluated using specimens collected pre-December 2019 (n = 393), from nucleic acid amplification testing (NAAT) negative patients (n = 40), and from 53 patients with COVID-19 by NAAT collected 3-21 days post-onset of symptoms (POS) (N = 83). Negative agreement (NA), positive agreement (PA), and positive and negative predictive values (PPV and NPV) at prevalences of 5% and 10% were calculated. RESULTS: The overall %NA; 95% CI in the negative samples were: Roche 99.8%; 99.3-100.2, Beckman 99.8%; 98.7-100.0, Abbott and Bio-Rad 99.3%; 98.0-99.9, DiaSorin 98.4; 97.2-99.6, EUROIMMUN IgG 97.5%; 95.5-98.7, and EUROIMMUN IgA 79.7%; 75.9-83.5), accounting for positive/equivocal results as false positives. The %PA; 95% CI in samples collected 14+ days POS (n = 24) were: Bio-Rad 83.3%; 68.4-98.2, Abbott and Roche 79.2%; 62.9-95.4, EUROIMMUN IgA 70.8%; 52.6-89.0, Beckman 58.3%; 38.6-78.1, DiaSorin 54.2; 34.2-74.1, and EUROIMMUN IgG 50.0%; 30.0-70.0, accounting for negative/equivocal results as false negatives. NPVs ranged from 97.4%-98.9% and 94.7%-97.7% for prevalences 5% and 10%, respectively. PPVs ranged from 15.5%-94.8% and 27.9%-97.4% for prevalences 5% and 10%, respectively. CONCLUSION: The Roche and Beckman assays resulted in fewer false positives, followed by the Bio-Rad and Abbott assays. While the Bio-Rad assay demonstrated higher antibody detection in COVID-19-positive patients, PA claims cannot be established with a high level of confidence in our sample population.


Assuntos
Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Serviços de Laboratório Clínico/estatística & dados numéricos , Técnicas de Laboratório Clínico/métodos , Laboratórios/estatística & dados numéricos , SARS-CoV-2/imunologia , Anticorpos Antivirais/imunologia , COVID-19/sangue , COVID-19/virologia , Estudos de Casos e Controles , Estudos de Coortes , Humanos , Valor Preditivo dos Testes , SARS-CoV-2/isolamento & purificação
16.
Viruses ; 13(5)2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33922716

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. Polymerase chain reaction (PCR)-based diagnostic tests are expensive, reliant on centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests that bind and detect the surface proteins of a virus are rapid and scalable but suffer from high false negative rates. To address this problem, an inexpensive, simple, and robust 60-minute do-it-yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/µL) and specificity (>97% true negative rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed. ALERT (Accessible LAMP-Enabled Rapid Test) incorporates the following features: (1) increased shelf-life and ambient temperature storage, compared to liquid reaction mixes, by using wax layers to isolate enzymes from other reagents; (2) improved specificity compared to other LAMP end-point reporting methods, by using sequence-specific QUASR (quenching of unincorporated amplification signal reporters); (3) increased sensitivity, compared to methods without purification through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal; (4) quality control with a nasopharyngeal-specific mRNA target; and (5) co-detection of other respiratory viruses, such as influenza B, by multiplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for labs and hospitals. With minimal effort, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms, or nucleic acid-based markers.


Assuntos
Teste para COVID-19/métodos , COVID-19/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , Técnicas de Laboratório Clínico/métodos , Humanos , Masculino , Nasofaringe/virologia , Testes Imediatos , RNA Viral/genética , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade
17.
PLoS One ; 16(4): e0245423, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33852576

RESUMO

BACKGROUND: In order to identify an inexpensive yet highly stable SARS-CoV-2 collection device as an alternative to foam swabs stored in transport media, both contrived ("surrogate") CoV-positive and patient-collected spun polyester swabs stored in dry tubes were evaluated for time- and temperature-stability using qPCR. METHODS: Surrogate specimens were prepared by combining multiple, residual SARS-CoV-2-positive clinical specimens and diluting to near-LOD levels in either porcine or human mucus ("matrix"), inoculating foam or polyester nasal swabs, and sealing in dry tubes. Swabs were then subjected to one of three temperature excursions: (1) 4°C for up to 72 hours; (2) 40°C for 12 hours, followed by 32°C for up to 60 hours; or (3) multiple freeze-thaw cycles (-20°C). The stability of extracted SARS-CoV-2 RNA for each condition was evaluated by qPCR. Separate usability studies for the dry polyester swab-based HealthPulse@home COVID-19 Specimen Collection Kit were later conducted in both adult and pediatric populations. RESULTS: Polyester swabs stored dry demonstrated equivalent performance to foam swabs for detection of low and moderate SARS-CoV-2 viral loads. Mimicking warm- and cold- climate shipment, surrogate specimens were stable following either 72 hours of a high-temperature excursion or two freeze-thaw cycles. In addition, usability studies comprised of self-collected patient specimens yielded sufficient material for molecular testing, as demonstrated by RNase P detection. CONCLUSIONS: Polyester nasal swabs stored in dry collection tubes offer a robust and inexpensive self-collection method for SARS-CoV-2 viral load testing, as viral RNA remains stable under conditions required for home collection and shipment to the laboratory.


Assuntos
COVID-19/diagnóstico , COVID-19/virologia , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodos , Animais , Teste para COVID-19/métodos , Técnicas de Laboratório Clínico/métodos , Testes Diagnósticos de Rotina/métodos , Humanos , Técnicas de Diagnóstico Molecular , Nasofaringe/virologia , Poliésteres , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Suínos
18.
Viruses ; 13(4)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33915875

RESUMO

Coronavirus (CoV) spillover events from wildlife reservoirs can result in mild to severe human respiratory illness. These spillover events underlie the importance of detecting known and novel CoVs circulating in reservoir host species and determining CoV prevalence and distribution, allowing improved prediction of spillover events or where a human-reservoir interface should be closely monitored. To increase the likelihood of detecting all circulating genera and strains, we have modified primers published by Watanabe et al. in 2010 to generate a semi-nested pan-CoV PCR assay. Representatives from the four coronavirus genera (α-CoVs, ß-CoVs, γ-CoVs and δ-CoVs) were tested and all of the in-house CoVs were detected using this assay. After comparing both assays, we found that the updated assay reliably detected viruses in all genera of CoVs with high sensitivity, whereas the sensitivity of the original assay was lower. Our updated PCR assay is an important tool to detect, monitor and track CoVs to enhance viral surveillance in reservoir hosts.


Assuntos
Coronavirus/classificação , Coronavirus/genética , Coronavirus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , Animais Selvagens , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Reservatórios de Doenças/virologia , Genoma Viral , Especificidade de Hospedeiro , Humanos , Limite de Detecção , Pandemias , Filogenia , RNA Viral
19.
J Med Virol ; 93(7): 4242-4246, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33710634

RESUMO

Coronavirus disease 2019 (COVID-19) has brought a huge impact on global health and the economy. Early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is essential for epidemic prevention and control. The detection of SARS-CoV-2 antibodies is an important criterion for diagnosing COVID-19. However, SARS-CoV-2 antibody testing also has certain false positives causing confusion in clinical diagnosis. This article summarizes the causes of false-positive detection of SARS-CoV-2 antibodies in clinical practice. The results indicate that the most common endogenous interferences include rheumatoid factor, heterophile antibodies, human anti-animal antibodies, lysozyme, complement, and cross-antigens. The exogenous interference is mainly incomplete coagulation of the specimen, contamination of the specimen, and insufficient optimization of the diagnostic kit's reaction system.


Assuntos
Anticorpos Antivirais/sangue , Teste para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/imunologia , Técnicas de Laboratório Clínico/métodos , Reações Falso-Positivas , Humanos , Testes Imunológicos/métodos
20.
PLoS One ; 16(3): e0248042, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33657176

RESUMO

A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).


Assuntos
COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genética , COVID-19/genética , Teste para COVID-19/métodos , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Primers do DNA/genética , Humanos , Proteínas do Nucleocapsídeo/genética , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcrição Reversa/genética , SARS-CoV-2/patogenicidade , Sensibilidade e Especificidade
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