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1.
ACS Appl Mater Interfaces ; 13(35): 41445-41453, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34428374

RESUMO

Airborne transmission of exhaled virus can rapidly spread, thereby increasing disease progression from local incidents to pandemics. Due to the COVID-19 pandemic, states and local governments have enforced the use of protective masks in public and work areas to minimize the disease spread. Here, we have leveraged the function of protective face coverings toward COVID-19 diagnosis. We developed a user-friendly, affordable, and wearable collector. This noninvasive platform is integrated into protective masks toward collecting airborne virus in the exhaled breath over the wearing period. A viral sample was sprayed into the collector to model airborne dispersion, and then the enriched pathogen was extracted from the collector for further analytical evaluation. To validate this design, qualitative colorimetric loop-mediated isothermal amplification, quantitative reverse transcription polymerase chain reaction, and antibody-based dot blot assays were performed to detect the presence of SARS-CoV-2. We envision that this platform will facilitate sampling of current SARS-CoV-2 and is potentially broadly applicable to other airborne diseases for future emerging pandemics.


Assuntos
Testes Respiratórios/instrumentação , Teste para COVID-19/instrumentação , Máscaras , SARS-CoV-2/isolamento & purificação , Microbiologia do Ar , Anticorpos Antivirais/imunologia , Testes Respiratórios/métodos , Teste para COVID-19/métodos , Colódio/química , Colorimetria/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Cimento de Policarboxilato/química , Porosidade , Estudo de Prova de Conceito , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/química , Proteínas Virais/análise , Proteínas Virais/imunologia
2.
Sci Rep ; 11(1): 16193, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376716

RESUMO

We have optimised a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from extracted RNA for clinical application. We improved the stability and reliability of the RT-LAMP assay by the addition of a temperature-dependent switch oligonucleotide to reduce self- or off-target amplification. We then developed freeze-dried master mix for single step RT-LAMP reaction, simplifying the operation for end users and improving long-term storage and transportation. The assay can detect as low as 13 copies of SARS-CoV2 RNA per reaction (25-µL). Cross reactivity with other human coronaviruses was not observed. We have applied the new RT-LAMP assay for testing clinical extracted RNA samples extracted from swabs of 72 patients in the UK and 126 samples from Greece and demonstrated the overall sensitivity of 90.2% (95% CI 83.8-94.7%) and specificity of 92.4% (95% CI 83.2-97.5%). Among 115 positive samples which Ct values were less than 34, the RT-LAMP assay was able to detect 110 of them with 95.6% sensitivity. The specificity was 100% when RNA elution used RNase-free water. The outcome of RT-LAMP can be reported by both colorimetric detection and quantifiable fluorescent reading. Objective measures with a digitized reading data flow would allow for the sharing of results for local or national surveillance.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Teste de Ácido Nucleico para COVID-19/normas , Humanos , Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificação de Ácido Nucleico/normas , Sensibilidade e Especificidade
3.
Sci Rep ; 11(1): 16201, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376765

RESUMO

Optical spectroscopic techniques have been commonly used to detect the presence of biofilm-forming pathogens (bacteria and fungi) in the agro-food industry. Recently, near-infrared (NIR) spectroscopy revealed that it is also possible to detect the presence of viruses in animal and vegetal tissues. Here we report a platform based on visible and NIR (VNIR) hyperspectral imaging for non-contact, reagent free detection and quantification of laboratory-engineered viral particles in fluid samples (liquid droplets and dry residue) using both partial least square-discriminant analysis and artificial feed-forward neural networks. The detection was successfully achieved in preparations of phosphate buffered solution and artificial saliva, with an equivalent pixel volume of 4 nL and lowest concentration of 800 TU·[Formula: see text]L-1. This method constitutes an innovative approach that could be potentially used at point of care for rapid mass screening of viral infectious diseases and monitoring of the SARS-CoV-2 pandemic.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Infecções por Lentivirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Células HEK293 , Humanos , Processamento de Imagem Assistida por Computador/normas , Lentivirus/isolamento & purificação , Lentivirus/patogenicidade , Infecções por Lentivirus/virologia , Técnicas de Diagnóstico Molecular/normas , Sistemas Automatizados de Assistência Junto ao Leito , Saliva/virologia , Sensibilidade e Especificidade , Espectroscopia de Luz Próxima ao Infravermelho/normas
4.
Sci Rep ; 11(1): 16430, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385527

RESUMO

Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.


Assuntos
Teste para COVID-19/métodos , COVID-19/virologia , Colorimetria/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , Humanos , Pandemias , Portugal/epidemiologia , RNA Viral/genética , SARS-CoV-2/genética , Saliva/química , Saliva/virologia , Sensibilidade e Especificidade
5.
Sci Immunol ; 6(62)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376480

RESUMO

The IMmunoPhenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective longitudinal study designed to enroll 1000 hospitalized patients with COVID-19 (NCT04378777). IMPACC collects detailed clinical, laboratory and radiographic data along with longitudinal biologic sampling of blood and respiratory secretions for in depth testing. Clinical and lab data are integrated to identify immunologic, virologic, proteomic, metabolomic and genomic features of COVID-19-related susceptibility, severity and disease progression. The goals of IMPACC are to better understand the contributions of pathogen dynamics and host immune responses to the severity and course of COVID-19 and to generate hypotheses for identification of biomarkers and effective therapeutics, including optimal timing of such interventions. In this report we summarize the IMPACC study design and protocols including clinical criteria and recruitment, multi-site standardized sample collection and processing, virologic and immunologic assays, harmonization of assay protocols, high-level analyses and the data sharing plans.


Assuntos
Biomarcadores , COVID-19/imunologia , COVID-19/virologia , Imunofenotipagem , SARS-CoV-2/imunologia , Biomarcadores/sangue , COVID-19/diagnóstico , COVID-19/epidemiologia , Biologia Computacional/métodos , Análise de Dados , Perfilação da Expressão Gênica , Hospitalização , Humanos , Estudos Longitudinais , Técnicas de Diagnóstico Molecular/métodos , Estudos Prospectivos , Proteômica/métodos , Estados Unidos/epidemiologia
6.
Virol J ; 18(1): 178, 2021 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-34461941

RESUMO

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 pandemic, has infected more than 179 million people worldwide. Testing of infected individuals is crucial for identification and isolation, thereby preventing further spread of the disease. Presently, Taqman™ Reverse Transcription Real Time PCR is considered gold standard, and is the most common technique used for molecular testing of COVID-19, though it requires sophisticated equipments, expertise and is also relatively expensive. OBJECTIVE: Development and optimization of an alternate molecular testing method for the diagnosis of COVID-19, through a two step Reverse Transcription Loop-mediated isothermal AMPlification (RT-LAMP). RESULTS: Primers for LAMP were carefully designed for discrimination from other closely related human pathogenic coronaviruses. Care was also taken that primer binding sites are present in conserved regions of SARS-CoV2. Our analysis shows that the primer binding sites are well conserved in all the variants of concern (VOC) and variants of interest (VOI), notified by World Health Organization (WHO). These lineages include B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.427/B.1.429, P.2, B.1.525, P.3, B.1.526 and B.1.617.1. Various DNA polymerases with strand displacement activity were evaluated and conditions were optimized for LAMP amplification and visualization. Different LAMP primer sets were also evaluated using synthetic templates as well as patient samples. CONCLUSION: In a double blind study, the RT-LAMP assay was validated on more than 150 patient samples at two different sites. The RT-LAMP assay appeared to be 89.2% accurate when compared to the Taqman™ rt-RT-PCR assay.


Assuntos
Teste para COVID-19/métodos , COVID-19/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genética , COVID-19/diagnóstico , Humanos , Transcrição Reversa , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia , Sensibilidade e Especificidade
7.
Biomed Res Int ; 2021: 5516344, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34368349

RESUMO

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic of pneumonia spreading around the world, leading to serious threats to public health and attracting enormous attention. There is an urgent need for sensitive diagnostic testing implementation to control and manage SARS-CoV-2 in public health laboratories. The quantitative reverse transcription PCR (RT-qPCR) assay is the gold standard method, but the sensitivity and specificity of SARS-CoV-2 testing are dependent on a number of factors. Methods: We synthesized RNA based on the genes published to estimate the concentration of inactivated virus samples in a biosafety level 3 laboratory. The limit of detection (LOD), linearity, accuracy, and precision were evaluated according to the bioanalytical method validation guidelines. Results: We found that the LOD reached around 3 copies/reaction. Furthermore, intra-assay precision, accuracy, and linearity met the accepted criterion with an RSD for copies of less than 25%, and linear regression met the accepted R 2 of 0.98. Conclusions: We suggest that synthesized RNA based on the database of the NCBI gene bank for estimating the concentration of inactivated virus samples provides a potential opportunity for reliable testing to diagnose coronavirus disease 2019 (COVID-19) as well as limit the spread of the disease. This method may be relatively quick and inexpensive, and it may be useful for developing countries during the pandemic era. In the long term, it is also applicable for evaluation, verification, validation, and external quality assessment.


Assuntos
COVID-19/virologia , Técnicas de Diagnóstico Molecular/normas , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/genética , Países em Desenvolvimento/estatística & dados numéricos , Humanos , Técnicas de Diagnóstico Molecular/métodos , Pandemias , RNA Viral/análise , SARS-CoV-2/isolamento & purificação , Estudos de Validação como Assunto
8.
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
9.
Ann Glob Health ; 87(1): 71, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34327118

RESUMO

Despite the pandemic, 34,154 migrants, refugees or asylum-seekers landed in Sicily (Italy) in 2020, representing the main point of entry by sea into Europe. The SARS-CoV-2 surveillance program among migrants arriving to Sicily via the Mediterranean Sea, made by the combination of clinical examination and molecular testing, has been integrated by full-genome sequencing strains using the NGS technology from the last week of February. To date, more than one hundred full-genome strains have been sequenced and 8 different lineages have been identified mostly belonging to the lineages B.1.1.7 and B.1.525. As global access to COVID-19 vaccines should be ensured, the need to provide more detailed information to inform policies and to drive the possible re-engineering of vaccines needed to deal with the challenge of new and future variants should be highlighted.


Assuntos
COVID-19 , Genoma Viral , SARS-CoV-2 , Migrantes/estatística & dados numéricos , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/classificação , Vacinas contra COVID-19/normas , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Sicília/epidemiologia
10.
Curr Issues Mol Biol ; 43(2): 728-748, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34287238

RESUMO

The ongoing coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a severe threat to human health and the global economy and has resulted in overwhelming stress on health care systems worldwide. Despite the global health catastrophe, especially in the number of infections and fatalities, the COVID-19 pandemic has also revolutionized research and discovery with remarkable success in diagnostics, treatments, and vaccine development. The use of many diagnostic methods has helped establish public health guidelines to mitigate the spread of COVID-19. However, limited information has been shared about these methods, and there is a need for the scientific community to learn about these technologies, in addition to their sensitivity, specificity, and limitations. This review article is focused on providing insights into the major methods used for SARS-CoV-2 detection. We describe in detail the core principle of each method, including molecular and serological approaches, along with reported claims about the rates of false negatives and false positives, the types of specimens needed, and the level of technology and the time required to perform each test. Although this study will not rank or prioritize these methods, the information will help in the development of guidelines and diagnostic protocols in clinical settings and reference laboratories.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Teste Sorológico para COVID-19/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ensaio de Imunoadsorção Enzimática/métodos , Coloide de Ouro , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunoensaio/métodos , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação
11.
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
12.
Viruses ; 13(6)2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208336

RESUMO

Development of High-Throughput Sequencing (HTS), also known as next generation sequencing, revolutionized diagnostic research of plant viruses. HTS outperforms bioassays and molecular diagnostic assays that are used to screen domestic and quarantine grapevine materials in data throughput, cost, scalability, and detection of novel and highly variant virus species. However, before HTS-based assays can be routinely used for plant virus diagnostics, performance specifications need to be developed and assessed. In this study, we selected 18 virus-infected grapevines as a test panel for measuring performance characteristics of an HTS-based diagnostic assay. Total nucleic acid (TNA) was extracted from petioles and dormant canes of individual samples and constructed libraries were run on Illumina NextSeq 500 instrument using a 75-bp single-end read platform. Sensitivity was 98% measured over 264 distinct virus and viroid infections with a false discovery rate (FDR) of approximately 1 in 5 positives. The results also showed that combining a spring petiole test with a fall cane test increased sensitivity to 100% for this TNA HTS assay. To evaluate extraction methodology, these results were compared to parallel dsRNA extractions. In addition, in a more detailed dilution study, the TNA HTS assay described here consistently performed well down to a dilution of 5%. In that range, sensitivity was 98% with a corresponding FDR of approximately 1 in 5. Repeatability and reproducibility were assessed at 99% and 93%, respectively. The protocol, criteria, and performance levels described here may help to standardize HTS for quality assurance and accreditation purposes in plant quarantine or certification programs.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Doenças das Plantas/virologia , Vírus de Plantas/genética , Vitis/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , RNA Viral , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
13.
Przegl Epidemiol ; 75(1): 14-26, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34328283

RESUMO

INTRODUCTION: Since the SARS-CoV-2 emergence in 2019/2020, at least 158 million infections with this pathogen have been recorded, of which 3.29 million infected people have died. Due to the non-specific symptoms of SARS-CoV-2 infection, laboratory tests based on RT-PCR (reverse transcription and polymerase chain reaction) are mainly used in the diagnosis of COVID-19 disease. AIM: The aim of this study is to compare the molecular tests available on the Polish market for the diagnosis of SARS-CoV2 infection. RESULTS: Based on the data provided by the manufacturers and the performed laboratory analyses, we have shown that the available diagnostic kits differ mainly in the sensitivity and duration of the reaction. CONCLUSION: Due to the ongoing COVID-19 pandemic, the indicated parameters are key to effective control of the spread of SARS-CoV2, and therefore should be mainly taken into account when choosing and purchasing by diagnostic centres.


Assuntos
COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Carga Viral , Humanos , Polônia , Sensibilidade e Especificidade
14.
J Clin Lab Anal ; 35(8): e23876, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34132419

RESUMO

BACKGROUND: Pooling of samples for SARS-CoV-2 testing in low-prevalence settings has been used as an effective strategy to expand testing capacity and mitigate challenges with the shortage of supplies. We evaluated two automated molecular test systems for the detection of SARS-CoV-2 RNA in pooled specimens. METHODS: Pooled nasopharyngeal and saliva specimens were tested by Qiagen QIAstat-Dx Respiratory SARS-CoV-2 Panel (QIAstat) or Cepheid Xpert Xpress SARS-CoV-2 (Xpert), and the results were compared to that of standard RT-qPCR tests without pooling. RESULTS: In nasopharyngeal specimens, the sensitivity/specificity of the pool testing approach, with 5 and 10 specimens per pool, were 77%/100% (n = 105) and 74.1%/100% (n = 260) by QIAstat, and 97.1%/100% (n = 250) and 100%/99.5% (n = 200) by Xpert, respectively. Pool testing of saliva (10 specimens per pool; n = 150) by Xpert resulted in 87.5% sensitivity and 99.3% specificity compared to individual tests. Pool size of 5 or 10 specimens did not significantly affect the difference of RT-qPCR cycle threshold (CT ) from standard testing. RT-qPCR CT values obtained with pool testing by both QIAstat and Xpert were positively correlated with that of individual testing (Pearson's correlation coefficient r = 0.85 to 0.99, p < 0.05). However, the CT values from Xpert were significantly stronger (p < 0.01, paired t test) than that of QIAstat in a subset of SARS-CoV-2 positive specimens, with mean differences of -4.3 ± 2.43 and -4.6 ± 2 for individual and pooled tests, respectively. CONCLUSION: Our results suggest that Xpert SARS-CoV-2 can be utilized for pooled sample testing for COVID-19 screening in low-prevalence settings providing significant cost savings and improving throughput without affecting test quality.


Assuntos
Teste para COVID-19/métodos , Nasofaringe/virologia , Saliva/virologia , Automação Laboratorial , Teste de Ácido Nucleico para COVID-19/métodos , Humanos , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade
15.
Diagn Microbiol Infect Dis ; 101(2): 115441, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34186320

RESUMO

To meet the testing demands and overcome supply chain issues during the SARS-CoV-2 pandemic, many clinical laboratories validated multiple SARS-CoV-2 molecular testing platforms. Here, we compare three different molecular assays for SARS-CoV-2 that received emergency use authorization (EUA) from the U.S. Food and Drug Administration. In order to determine the agreement among Roche cobas® SARS-CoV-2 Test (Cobas), Abbott RealTime SARS-CoV-2 assay (ART), and Mayo Clinic Laboratory SARS-CoV-2 Molecular Detection Assay (Mayo LDT), 100 each of anterior nares (AN), nasopharyngeal (NP), oropharyngeal (OP), and NP+OP swabs were tested on each platform. The consensus result was defined as agreement by 2 or more methods. Furthermore, 30 positive NP swabs from each molecular platform (n = 90 total) were tested on the three platforms to determine the PPA among positive samples. ART platform called more specimens positive than the other two platforms. All three assays performed with greater than 90% agreement for NP specimens throughout the study.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , SARS-CoV-2/isolamento & purificação , Humanos , Nasofaringe/virologia , Nariz/virologia , Pandemias , Reação em Cadeia da Polimerase , Sistema Respiratório/virologia , Sensibilidade e Especificidade , Manejo de Espécimes/métodos
16.
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
17.
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
19.
Virus Res ; 302: 198484, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34146608

RESUMO

Novel corona virus SARS-CoV-2, causing coronavirus disease 2019 (COVID-19), has become a global health challenge particularly for developing countries like Pakistan where overcrowded cities, inadequate sanitation, little health awareness and poor socioeconomic conditions exist. The SARS-CoV-2 has been known to spread primarily through direct contact and respiratory droplets. However, detection of SARS-CoV-2 in stool and sewage have raised the possibility of fecal-oral mode of transmission. Currently, quantitative reverse-transcriptase PCR (qRT-PCR) is the only method being used for SARS-CoV-2 detection, which requires expensive instrumentation, dedicated laboratory setup, highly skilled staff, and several hours to report results. Considering the high transmissibility and rapid spread, a robust, sensitive, specific and cheaper assay for rapid SARS-CoV-2 detection is highly needed. Herein, we report a novel colorimetric RT-LAMP assay for naked-eye detection of SARS-COV-2 in clinical as well as sewage samples. Our SARS-CoV-2 RdRp-based LAMP assay could successfully detect the virus RNA in 26/28 (93%) of RT-PCR positive COVID-19 clinical samples with 100% specificity (n = 7) within 20 min. We also tested the effect of various additives on the performance of LAMP assay and found that addition of 1 mg/ml bovine serum albumin (BSA) could increase the sensitivity of assay up to 101 copies of target sequence. Moreover, we also successfully applied this assay to detect SARS-CoV-2 in sewage waters collected from those areas of Lahore, a city of Punjab province of Pakistan, declared as virus hotspots by local government. Our optimized LAMP assay could provide a sensitive first tier strategy for SARS-CoV-2 screening and can potentially help diagnostic laboratories in better handling of high sample turnout during pandemic situation. By providing rapid naked-eye SARS-CoV-2 detection in sewage samples, this assay may support pandemic readiness and emergency response to any possible virus outbreaks in future.


Assuntos
COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , SARS-CoV-2/isolamento & purificação , Esgotos/virologia , COVID-19/virologia , Teste para COVID-19 , Colorimetria , Fezes/virologia , Humanos , Programas de Rastreamento , Paquistão/epidemiologia , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/genética , Sensibilidade e Especificidade
20.
Medicine (Baltimore) ; 100(25): e26403, 2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34160425

RESUMO

INTRODUCTION: Sepsis and septic shock are the most severe forms of infection affecting predominantly elderly people, preterm and term neonates, and young infants. Even in high-income countries sepsis causes about 8% of admissions to pediatric intensive care units (PICUs). Early diagnosis, rapid anti-infective treatment, and prompt hemodynamic stabilization are crucial for patient survival. In this context, it is essential to identify the causative pathogen as soon as possible to optimize antimicrobial treatment. To date, culture-based diagnostic procedures (e.g., blood cultures) represent the standard of care. However, they have 2 major problems: on the one hand, in the case of very small sample volumes (and thus usually in children), they are not sufficiently sensitive. On the other hand, with a time-to-result of 2 to 5 days, blood cultures need a relatively long time for the anti-infective therapy to be calculated. To overcome these problems, culture-independent molecular diagnostic procedures such as unbiased sequence analysis of circulating cell-free DNA (cfDNA) from plasma samples of septic patients by next-generation sequencing (NGS) have been tested successfully in adult septic patients. However, these results still need to be transferred to the pediatric setting. METHODS: The Next GeneSiPS-Trial is a prospective, observational, non-interventional, multicenter study used to assess the diagnostic performance of an NGS-based approach for the identification of causative pathogens in (preterm and term) neonates (d1-d28, n = 50), infants (d29 to <1 yr, n = 50), and toddlers (1 yr to <5 yr, n = 50) with suspected or proven severe sepsis or septic shock (according to the pediatric sepsis definition) by the use of the quantitative sepsis indicating quantifier (SIQ) score in comparison to standard of care (culture-based) microbiological diagnostics. Potential changes in anti-infective treatment regimens based on these NGS results will be estimated retrospectively by a panel of 3 independent clinical specialists. DISCUSSION: Neonates, infants, and young children are significantly affected by sepsis. Fast and more sensitive diagnostic approaches are urgently needed. This prospective, observational, non-interventional, multicenter study seeks to evaluate an NGS-based approach in critically ill children suffering from sepsis. TRIAL REGISTRATION: DRKS-ID: DRKS00015705 (registered October 24, 2018). https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00015705.


Assuntos
Bacteriemia/diagnóstico , Bactérias/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas de Diagnóstico Molecular/métodos , Choque Séptico/diagnóstico , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Bactérias/genética , Hemocultura , Pré-Escolar , DNA Bacteriano/isolamento & purificação , Diagnóstico Precoce , Feminino , Humanos , Lactente , Recém-Nascido , Unidades de Terapia Intensiva Pediátrica , Masculino , Estudos Multicêntricos como Assunto , Estudos Observacionais como Assunto , Estudos Prospectivos , Estudos Retrospectivos , Índice de Gravidade de Doença , Choque Séptico/sangue , Choque Séptico/tratamento farmacológico , Choque Séptico/microbiologia
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