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1.
Anal Chim Acta ; 1177: 338758, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34482896

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the unprecedented global pandemic of coronavirus disease-2019 (COVID-19). Efforts are needed to develop rapid and accurate diagnostic tools for extensive testing, allowing for effective containment of the infection via timely identification and isolation of SARS-CoV-2 carriers. Current gold standard nucleic acid tests require many separate steps that need trained personnel to operate specialist instrumentation in laboratory environments, hampering turnaround time and test accessibility, especially in low-resource settings. We devised an integrated on-chip platform coupling RNA extraction based on immiscible filtration assisted by surface tension (IFAST), with RNA amplification and detection via colorimetric reverse-transcription loop mediated isothermal amplification (RT-LAMP), using two sets of primers targeting open reading frame 1a (ORF1a) and nucleoprotein (N) genes of SARS-CoV-2. Results were identified visually, with a colour change from pink to yellow indicating positive amplification, and further confirmed by DNA gel electrophoresis. The specificity of the assay was tested against HCoV-OC43 and H1N1 RNAs. The assay based on use of gene N primers was 100% specific to SARS-CoV-2 with no cross-reactivity to HCoV-OC43 nor H1N1. Proof-of-concept studies on water and artificial sputum containing genomic SARS-CoV-2 RNA showed our IFAST RT-LAMP device to be capable of extracting and detecting 470 SARS-CoV-2 copies mL-1 within 1 h (from sample-in to answer-out). IFAST RT-LAMP is a simple-to-use, integrated, rapid and accurate COVID-19 diagnostic platform, which could provide an attractive means for extensive screening of SARS-CoV-2 infections at point-of-care, especially in resource-constrained settings.


Assuntos
COVID-19 , Dispositivos Lab-On-A-Chip , RNA Viral , COVID-19/diagnóstico , Humanos , Vírus da Influenza A Subtipo H1N1 , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/isolamento & purificação , SARS-CoV-2 , Sensibilidade e Especificidade
2.
Ceska Gynekol ; 86(4): 264-272, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34493052

RESUMO

Molecular classification of endometrial carcinoma is becoming an important part of the dia-gnostic process with direct therapeutic implications. Recent international guidelines, including the joint recommendation of the European Society of Gynaecological Oncology, the European Society for Radiotherapy and Oncology and the European Society of Pathology include the molecular classification into standard dia-gnostic algorithms. Molecular testing of endometrial carcinomas is also recommended in the latest (5th edition) of the World Health Organization classification of female genital tumors. Due to the need to implement these recommendations in practice, representatives of four professional societies of the Czech Medical Association of J. E. Purkyně (the Czech Oncological Society, the Oncogynecological Section of the Czech Gynecological and Obstetrical Society, the Society of Radiation Oncology, Biology and Physics, and the Society of Czech Pathologists) organized a meeting focused on this topic. Recommendation for molecular testing of endometrial carcinoma in routine dia-gnostic practice in the Czech Republic.


Assuntos
Neoplasias do Endométrio , Radioterapia (Especialidade) , Biologia , República Tcheca , Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/genética , Feminino , Humanos , Técnicas de Diagnóstico Molecular , Patologistas , Física
3.
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
4.
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
5.
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
6.
Medicine (Baltimore) ; 100(29): e26778, 2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34398057

RESUMO

BACKGROUND: This study aimed to assess whether Xpert MTB/RIF Ultra (Xpert Ultra) can effectively diagnose tuberculosis meningitis (TBM) and to simultaneously compare its effectiveness with Xpert in diagnosing TBM in the same population. METHODS: On August 12, 2020, Wanfang Database, China National Knowledge Infrastructure, Embase, Cochrane Library, and PubMed were searched for studies evaluating the diagnostic accuracy of Xpert Ultra for TBM. Then, we assessed the efficacy of Xpert Ultra against a composite reference standard and culture. If applicable, we also examined the diagnostic efficacy of Xpert in the same population. Heterogeneity was then explored by meta-regression, subgroup, and sensitivity analyses. RESULTS: Six studies containing 601 specimens reported the diagnostic efficacy of Xpert Ultra for TBM, with a composite reference standard. No study had compared the efficacy between Xpert Ultra and culture. The pooled sensitivity of Xpert Ultra was 64% (95% confidence interval [CI]: 45-80), and the I2 value was 86% (95% CI: 76-96); its specificity for TBM was consistently 100%. In the same population, 5 studies compared the diagnostic efficacy between Xpert Ultra and Xpert for TBM. The pooled sensitivity of Xpert Ultra and Xpert was 68% (95% CI: 46-84; I2 = 87%) and 37% (95% CI: 25-50; I2 = 72%), respectively. The studies were significantly heterogeneous in terms of sensitivity but not heterogeneous in specificity. CONCLUSIONS: Xpert Ultra was more sensitive than Xpert, but both were specific (100%). Therefore, Xpert Ultra had an excellent diagnostic efficacy for TBM, and it could be the preferred initial test for TBM.


Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Meníngea/diagnóstico , Testes Diagnósticos de Rotina , Humanos , Técnicas de Diagnóstico Molecular , Mycobacterium tuberculosis/genética , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Tuberculose Meníngea/diagnóstico por imagem
7.
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
8.
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
9.
Anal Chem ; 93(35): 11956-11964, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34424659

RESUMO

Coronavirus diseases such as the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pose serious threats. Portable and accurate nucleic acid detection is still an urgent need to achieve on-site virus screening and timely infection control. Herein, we have developed an on-site, semiautomatic detection system, aiming at simultaneously overcoming the shortcomings suffered by various commercially available assays, such as low accuracy, poor portability, instrument dependency, and labor intensity. Ultrasensitive isothermal amplification [i.e., reverse transcription loop-mediated isothermal amplification (RT-LAMP)] was applied to generate intensified SARS-CoV-2 RNA signals, which were then transduced to portable commercial pregnancy test strips (PTSs) via ultraspecific human chorionic gonadotropin (hCG)-conjugated toehold-mediated strand exchange (TMSE) probes (hCG-P). The entire detection was integrated into a four-channel, palm-size microfluidic device, named the microfluidic point-of-care (POC) diagnosis system based on the PTS (MPSP) detection system. It provides rapid, cost-effective, and sensitive detection, of which the lowest concentration of detection was 0.5 copy/µL of SARS-CoV-2 RNA, regardless of the presence of other similar viruses, even highly similar severe acute respiratory syndrome coronavirus (SARS-CoV). The successful detection of the authentic samples from different resources evaluated the practical application. The commercial PTS provides a colorimetric visible signal, which is instrument- and optimization-free. Therefore, this MPSP system can be immediately used for SARS-CoV-2 emergency detection, and it is worthy of further optimization to achieve full automation and detection for other infectious diseases.


Assuntos
COVID-19 , Testes de Gravidez , Feminino , Humanos , Dispositivos Lab-On-A-Chip , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito , Gravidez , RNA Viral/genética , SARS-CoV-2 , Sensibilidade e Especificidade
10.
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
11.
PLoS One ; 16(8): e0256883, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34464413

RESUMO

BACKGROUND: The GeneXpert diagnostic platform from the US based company Cepheid is an automated molecular diagnostic device that performs sample preparation and pathogen detection within a single cartridge-based assay. GeneXpert devices can enable diagnosis at the district level without the need for fully equipped clinical laboratories, are simple to use, and offer rapid results. Due to these characteristics, the platform is now widely used in low- and middle-income countries for diagnosis of diseases such as TB and HIV. Assays for SARS-CoV-2 are also being rolled out. We aimed to quantify public sector investments in the development of the GeneXpert platform and Cepheid's suite of cartridge-based assays. METHODS: Public funding data were collected from the proprietor company's financial filings, grant databases, review of historical literature concerning key laboratories and researchers, and contacting key public sector entities involved in the technology's development. The value of research and development (R&D) tax credits was estimated based on financial filings. RESULTS: Total public investments in the development of the GeneXpert technology were estimated to be $252 million, including >$11 million in funding for work in public laboratories leading to the first commercial product, $56 million in grants from the National Institutes of Health, $73 million from other U.S. government departments, $67 million in R&D tax credits, $38 million in funding from non-profit and philanthropic organizations, and $9.6 million in small business 'springboard' grants. CONCLUSION: The public sector has invested over $250 million in the development of both the underlying technologies and the GeneXpert diagnostic platform and assays, and has made additional investments in rolling out the technology in countries with high burdens of TB. The key role played by the public sector in R&D and roll-out stands in contrast to the lack of public sector ability to secure affordable pricing and maintenance agreements.


Assuntos
Investimentos em Saúde , Técnicas de Diagnóstico Molecular/economia , COVID-19/diagnóstico , COVID-19/virologia , Bases de Dados Factuais , Infecções por HIV/diagnóstico , História do Século XX , História do Século XXI , Humanos , Técnicas de Diagnóstico Molecular/história , SARS-CoV-2/isolamento & purificação , Tuberculose/diagnóstico , Estados Unidos
12.
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
13.
Front Cell Infect Microbiol ; 11: 581239, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34336708

RESUMO

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has caused the outbreak of coronavirus disease 2019 (COVID-19) all over the world. In the absence of appropriate antiviral drugs or vaccines, developing a simple, rapid, and reliable assay for SARS-CoV-2 is necessary for the prevention and control of the COVID-19 transmission. Methods: A novel molecular diagnosis technique, named multiplex reverse transcription loop-mediated isothermal amplification, that has been linked to a nanoparticle-based lateral flow biosensor (mRT-LAMP-LFB) was applied to detect SARS-CoV-2 based on the SARS-CoV-2 RdRp and N genes, and the mRT-LAMP products were analyzed using nanoparticle-based lateral flow biosensor. The mRT-LAMP-LFB amplification conditions, including the target RNA concentration, amplification temperature, and time were optimized. The sensitivity and specificity of the mRT-LAMP-LFB method were tested in the current study, and the mRT-LAMP-LFB assay was applied to detect the SARS-CoV-2 virus from clinical samples and artificial sputum samples. Results: The SARS-CoV-2 specific primers based on the RdRp and N genes were valid for the establishment of mRT-LAMP-LFB assay to detect the SARS-CoV-2 virus. The multiple-RT-LAMP amplification condition was optimized at 63°C for 30 min. The full process, including reaction preparation, viral RNA extraction, RT-LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the mRT-LAMP-LFB technology was 20 copies per reaction. The specificity of mRT-LAMP-LFB detection was 100%, and no cross-reactions to other respiratory pathogens were observed. Conclusion: The mRT-LAMP-LFB technique developed in the current study is a simple, rapid, and reliable method with great specificity and sensitivity when it comes to identifying SARS-CoV-2 virus for prevention and control of the COVID-19 disease, especially in resource-constrained regions of the world.


Assuntos
Técnicas Biossensoriais , COVID-19 , Nanopartículas Metálicas , Ouro , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , Transcrição Reversa , SARS-CoV-2 , Sensibilidade e Especificidade
14.
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
15.
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
16.
Analyst ; 146(17): 5347-5356, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34323889

RESUMO

Human immunodeficiency virus (HIV) continues to be a major burden on public health globally with on-going increases in the number of new infections each year. Rapid and sensitive point-of-care tests allow timely interventions and are essential to control the spread of the disease. However the highly variable nature of the virus, resulting in the evolution of many subtypes and inter-subtype recombinants, poses important challenges for its diagnosis. Here we describe a variant-tolerant reverse-transcription RT-LAMP amplification of the virus's INT gene, providing a simple to use, rapid (<30 min) in vitro point-of-care diagnostic test with a limit of detection <18 copies/reaction. The assay was first validated in clinical studies of patient samples, using both established RT-LAMP and RT-qPCR assays for reference, with results showing that this new variant-tolerant HIV-1 RT-LAMP diagnostic test is highly sensitive without compromising its high specificity for HIV-1 subtypes. The diagnostic test was subsequently configured within an easy-to-read paper microfluidic lateral flow test and was validated clinically using patient samples, demonstrating its future potential for use in timely, effective, low cost HIV diagnostics in global regions where healthcare resources may be limited.


Assuntos
HIV-1 , HIV-1/genética , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito , Transcrição Reversa , Sensibilidade e Especificidade
17.
ACS Sens ; 6(8): 2902-2910, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34292707

RESUMO

This study introduces a spinning and fully integrated paper-based microdevice that can perform multiple functions, including DNA extraction, amplification, and colorimetric detection, for monitoring two major vancomycin-resistant Enterococci (VREs), which carry the vanA and vanB genes. The spinning microdevice is composed of a stationary part and a spinning part. The square-shaped stationary part has two zones: the lysis and reaction zones. The spinning part, which has a spin wheel-like shape, was inserted perpendicularly into the stationary part so that its two semicircles remained on the upper and lower parts. Sodium hydroxide-treated glass microfiber filter discs, inserted in the upper semicircle, were soaked in the lysis chambers by folding them toward the lysis zone to capture DNA in the lysis chambers. The captured DNA was transferred to the reaction chambers by folding the discs toward the reaction chambers. Water was added to the sodium hydroxide-treated glass microfiber filter discs to elute purified DNA into the reaction chambers. The upper semicircle was then unfolded, and the reaction chambers were sealed for subsequent loop-mediated isothermal amplification (LAMP) for 45 min. After the reaction, the spinning part was spun in the lysis zone direction to bring the lower semicircle, inserted with phenolphthalein-treated glass microfiber filter discs, toward the upper part of the stationary part. By folding it toward the reaction chambers, the lower semicircle came into contact with them and the phenolphthalein-treated glass microfiber filter discs were soaked in the reaction chambers and expressed color after 30 s. Based on the pH change during the LAMP reaction, the phenolphthalein-treated discs remained pink in the absence of target DNA, while those in contact with the positive samples turned colorless. A sensitive detection with a VRE limit of detection of 102 CFU/mL for tap water spiked with VRE carrying the vanA gene was achieved using this microdevice. Both VREs, carrying vanA and vanB genes, were successfully identified from tap water and contaminated equipment surfaces within 75 min. The introduced microdevice demonstrated a rapid, accurate, and sensitive performance for the environmental assessment of VRE contamination in resource-limited regions.


Assuntos
Enterococos Resistentes à Vancomicina , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Vancomicina , Enterococos Resistentes à Vancomicina/genética
18.
Antimicrob Agents Chemother ; 65(9): e0044121, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34228533

RESUMO

Decisions regarding which rapid diagnostic test (RDT) for bloodstream infections to implement remain challenging given the diversity of organisms detected by different platforms. We used the desirability of outcome ranking management of antimicrobial therapy (DOOR-MAT) as a framework to compare two RDT platforms on potential desirability of antimicrobial therapy decisions. An observational study was performed at University of Maryland Medical System comparing Verigene blood culture (BC) to GenMark Dx ePlex blood culture ID (BCID) (research use only) panels on blood cultures from adult patients. Positive percent agreement (PPA) between each RDT platform and Vitek MS was calculated for comparison of on-panel targets. Theoretical antimicrobial decisions were made based on RDT results, taking into consideration patient parameters, antimicrobial stewardship practices, and local infectious diseases epidemiology. DOOR-MAT with a partial credit scoring system was applied to these decisions, and mean scores were compared across platforms using a paired t test. The study consisted of 160 unique patients. The Verigene BC PPA was 98.6% (95% confidence interval [CI], 95.1 to 99.8), and ePlex BCID PPA was 98% (95% CI, 94.3 to 99.6). Among the 31 organisms not on the Verigene BC panels, 61% were identified by the ePlex BCID panels. The mean (standard deviation [SD]) DOOR-MAT score for Verigene BC was 86.8 (28.5), while that for ePlex BCID was 91.9 (23.1) (P = 0.01). Both RDT platforms had high PPA for on-panel targets. The ePlex BCID was able to identify more organisms than Verigene, resulting in higher mean DOOR-MAT scores.


Assuntos
Anti-Infecciosos , Bacteriemia , Sepse , Antibacterianos/uso terapêutico , Bacteriemia/diagnóstico , Bacteriemia/tratamento farmacológico , Hemocultura , Humanos , Técnicas de Diagnóstico Molecular , Sepse/tratamento farmacológico
19.
Tuberk Toraks ; 69(2): 160-166, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34256506

RESUMO

Introduction: This study aimed to evaluate the performance of the Xpert MTB/RIF assay in the identification of M. tuberculosis in pulmonary and extrapulmonary clinical samples by taking the results of the BACTEC MGIT 960TB culture system as a reference. Materials and Methods: A total of 11,341 specimens sent to Sivas Cumhuriyet University Application and Research Hospital Tuberculosis Laboratory for microbiological examination with suspicion of tuberculosis infection between January 2013 and December 2019 were examined, and 6847 clinical specimens that underwent culture (BACTEC MGIT 960TB), Xpert MTB/RIF and AFB (Acid-fast bacilli) testing were selected and included in our study. Of the samples included in the study, 5096 samples were pulmonary, and 1751 were extrapulmonary samples. Result: In our study, sensitivity, specificity, PPV and NPV values of Xpert MTB/ RIF and AFB were calculated by taking TB culture test as reference test. The sensitivity of the Xpert MTB/RIF assay was calculated as 96.1%, specificity as 99.7%, positive predictive value (PPV) as 88.2%, and negative predictive value (NPV) as 99.9%. These values for pulmonary samples were determined as 98.3%, 99.7%, 89.9%, and 99.9%, respectively. For extrapulmonary samples, the sensitivity of the assay was found as 89.4%, specificity as 99.5%, PPV as 82.9%, and NPV as 99.7%. The sensitivity and PPV values for AFBpositive samples were found to be 99.0% and 97.1%, respectively. For AFB negative samples, the sensitivity, specificity, PPV, and NPV values were determined as 90.5%, 99.7%, 73.8%, and 99.9%, respectively. Conclusions: A large number of clinical samples were studied with the Xpert MTB/RIF test in our study. It can be a guide in determining the performance of the test under the conditions of our country. Especially in the diagnosis of extrapulmonary TB, the effectiveness of the Xpert MTB/RIF assay has not been certainly proven in countries having a moderate prevalence of TB, such as Turkey. In most of the published studies, only a small part of the samples is extrapulmonary samples. So, our study provides valuable results in terms of evaluating a large number of extrapulmonary samples.


Assuntos
Técnicas de Diagnóstico Molecular/normas , Kit de Reagentes para Diagnóstico/normas , Tuberculose/diagnóstico , Humanos , Pulmão/microbiologia , Mycobacterium tuberculosis/isolamento & purificação , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose/microbiologia , Turquia
20.
Nat Commun ; 12(1): 4317, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262053

RESUMO

The COVID-19 pandemic exposed difficulties in scaling current quantitative PCR (qPCR)-based diagnostic methodologies for large-scale infectious disease testing. Bottlenecks include lengthy multi-step processes for nucleic acid extraction followed by qPCR readouts, which require costly instrumentation and infrastructure, as well as reagent and plastic consumable shortages stemming from supply chain constraints. Here we report an Oil Immersed Lossless Total Analysis System (OIL-TAS), which integrates RNA extraction and detection onto a single device that is simple, rapid, cost effective, and requires minimal supplies and infrastructure to perform. We validated the performance of OIL-TAS using contrived SARS-CoV-2 viral particle samples and clinical nasopharyngeal swab samples. OIL-TAS showed a 93% positive predictive agreement (n = 57) and 100% negative predictive agreement (n = 10) with clinical SARS-CoV-2 qPCR assays in testing clinical samples, highlighting its potential to be a faster, cheaper, and easier-to-deploy alternative for infectious disease testing.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Teste de Ácido Nucleico para COVID-19/economia , Teste de Ácido Nucleico para COVID-19/instrumentação , Desenho de Equipamento , Humanos , Técnicas de Diagnóstico Molecular , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , RNA Viral/isolamento & purificação , Reprodutibilidade dos Testes , SARS-CoV-2/genética , Sensibilidade e Especificidade , Fatores de Tempo , Vírion/genética , Vírion/isolamento & purificação
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