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1.
Molecules ; 26(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207320

RESUMO

We evaluated mycophenolic acid (MPA) limited sampling strategies (LSSs) established using multiple linear regression (MLR) in children with nephrotic syndrome treated with mycophenolate mofetil (MMF). MLR-LSS is an easy-to-determine approach of therapeutic drug monitoring (TDM). We assessed the practicability of different LSSs for the estimation of MPA exposure as well as the optimal time points for MPA TDM. The literature search returned 29 studies dated 1998-2020. We applied 53 LSSs (n = 48 for MPA, n = 5 for free MPA [fMPA]) to predict the area under the time-concentration curve (AUCpred) in 24 children with nephrotic syndrome, for whom we previously determined MPA and fMPA concentrations, and compare the results with the determined AUC (AUCtotal). Nine equations met the requirements for bias and precision ±15%. The MPA AUC in children with nephrotic syndrome was predicted the best by four time-point LSSs developed for renal transplant recipients. Out of five LSSs evaluated for fMPA, none fulfilled the ±15% criteria for bias and precision probably due to very high percentage of bound MPA (99.64%). MPA LSS for children with nephrotic syndrome should include blood samples collected 1 h, 2 h and near the second MPA maximum concentration. MPA concentrations determined with the high performance liquid chromatography after multiplying by 1.175 may be used in LSSs based on MPA concentrations determined with the immunoassay technique. MPA LSS may facilitate TDM in the case of MMF, however, more studies on fMPA LSS are required for children with nephrotic syndrome.


Assuntos
Ácido Micofenólico/metabolismo , Síndrome Nefrótica/metabolismo , Adolescente , Coleta de Amostras Sanguíneas/métodos , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Humanos , Transplante de Rim/métodos , Modelos Lineares , Masculino , Análise Multivariada , Manejo de Espécimes/métodos
2.
BMC Infect Dis ; 21(1): 648, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34225656

RESUMO

BACKGROUND: A considerable amount of evidence demonstrates the potential of saliva in the diagnosis of COVID-19. Our aim was to determine the sensitivity of saliva versus swabs collected by healthcare workers (HCWs) and patients themselves to assess whether saliva detection can be offered as a cost-effective, risk-free method of SARS-CoV-2 detection. METHODS: This study was conducted in a hospital involving outpatients and hospitalized patients. A total of 3018 outpatients were tested. Of these, 200 qRT-PCR-confirmed SARS-CoV-2-positive patients were recruited for further study. In addition, 101 SARS-CoV-2-positive hospitalized patients with symptoms were also enrolled in the study. From outpatients, HCWs collected nasopharyngeal swabs (NPS), saliva were obtained. From inpatients, HCWs collected swabs, patient-collected swabs, and saliva were obtained. qRT-PCR was performed to detect SARS-CoV-2 by TAQPATH assay to determine the sensitivity of saliva detection. Sensitivity, specificity and positive/negative predictive values (PPV, NPV) of detecting SARS-CoV-2 were calculated using MedCalc. RESULTS: Of 3018 outpatients (asymptomatic: 2683, symptomatic: 335) tested by qRT-PCR, 200 were positive (males: 140, females: 60; aged 37.9 ± 12.8 years; (81 asymptomatic, 119 symptomatic). Of these, saliva was positive in 128 (64%); 39 of 81 asymptomatic (47%),89 of 119 symptomatic patients (74.8%). Sensitivity of detection was 60.9% (55.4-66.3%, CI 95%), with a negative predictive value of 36%(32.9-39.2%, CI 95%).Among 101 hospitalized patients (males:65, females: 36; aged 53.48 ± 15.6 years), with HCW collected NPS as comparator, sensitivity of saliva was 56.1% (47.5-64.5, CI 95%), specificity 63.5%(50.4-75.3, CI95%) with PPV of 77.2% and NPV of 39.6% and that of self-swab was 52.3%(44-60.5%, CI95%), specificity 56.6% (42.3-70.2%, CI95%) with PPV 77.2% and NPV29.7%. Comparison of positivity with the onset of symptoms revealed highest detection in saliva on day 3 after onset of symptoms. Additionally, only saliva was positive in 13 (12.8%) hospitalized patients. CONCLUSION: Saliva which is easier to collect than nasopharyngeal swab is a viable alternate to detect SARS-COV-2 in symptomatic patients in the early stage of onset of symptoms. Although saliva is currently not recommended for screening asymptomatic patients, optimization of collection and uniform timing of sampling might improve the sensitivity enabling its use as a screening tool at community level.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Manejo de Espécimes/métodos , Adulto , Idoso , Estudos de Coortes , Feminino , Pessoal de Saúde , Humanos , Masculino , Pessoa de Meia-Idade
3.
Sci Rep ; 11(1): 13592, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193912

RESUMO

With global demand for SARS-CoV-2 testing ever rising, shortages in commercially available viral transport media pose a serious problem for laboratories and health care providers. For reliable diagnosis of SARS-CoV-2 and other respiratory viruses, executed by Real-time PCR, the quality of respiratory specimens, predominantly determined by transport and storage conditions, is crucial. Therefore, our aim was to explore the reliability of minimal transport media, comprising saline or the CDC recommended Viral Transport Media (HBSS VTM), for the diagnosis of SARS-CoV-2 and other respiratory viruses (influenza A, respiratory syncytial virus, adenovirus, rhinovirus and human metapneumovirus) compared to commercial products, such as the Universal Transport Media (UTM). We question the assumptions, that the choice of medium and temperature for storage and transport affect the accuracy of viral detection by RT-PCR. Both alternatives to the commercial transport medium (UTM), HBSS VTM or saline, allow adequate detection of SARS-CoV-2 and other respiratory viruses, regardless of storage temperatures up to 28 °C and storage times up to 28 days. Our study revealed the high resilience of SARS-CoV-2 and other respiratory viruses, enabling proper detection in clinical specimens even after long-time storage at high temperatures, independent of the transport medium's composition.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Meios de Cultura/química , Preservação Biológica/métodos , SARS-CoV-2/genética , Manejo de Espécimes/métodos , Virologia/métodos , Temperatura Baixa , Humanos , Reagentes de Laboratório/química , Reprodutibilidade dos Testes , Fatores de Tempo
4.
Med Microbiol Immunol ; 210(4): 235-244, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34196781

RESUMO

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In this study, we evaluated lysis buffers that are commonly used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. In addition, viral stability in cell culture media at 4 °C and on display glass and plastic surfaces used in laboratory environment was analyzed. Furthermore, we evaluated chemical and non-chemical inactivation methods including heat inactivation, UV-C light, addition of ethanol, acetone-methanol, and PFA, which might be used as a subsequent inactivation step in the case of insufficient inactivation. We infected susceptible Caco-2 and Vero cells with pre-treated SARS-CoV-2 and determined the tissue culture infection dose 50 (TCID50) using crystal violet staining and microscopy. In addition, lysates of infected cells and virus containing supernatant were subjected to RT-qPCR analysis. We have found that guanidine thiocyanate and most of the tested detergent containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate the virus. In conclusion, careful evaluation of the used inactivation methods is required especially for non-denaturing buffers. Additional inactivation steps might be necessary before removal of lysed viral samples from BSL-3.


Assuntos
Anti-Infecciosos/farmacologia , COVID-19/prevenção & controle , COVID-19/virologia , Guanidinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Tiocianatos/farmacologia , Inativação de Vírus , Animais , Células CACO-2 , Linhagem Celular , Chlorocebus aethiops , Contenção de Riscos Biológicos , Humanos , RNA Viral , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/fisiologia , Manejo de Espécimes/métodos , Fatores de Tempo , Células Vero
6.
Nat Commun ; 12(1): 4423, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285218

RESUMO

The Cancer Genome Atlas (TCGA) is one of the largest biorepositories of digital histology. Deep learning (DL) models have been trained on TCGA to predict numerous features directly from histology, including survival, gene expression patterns, and driver mutations. However, we demonstrate that these features vary substantially across tissue submitting sites in TCGA for over 3,000 patients with six cancer subtypes. Additionally, we show that histologic image differences between submitting sites can easily be identified with DL. Site detection remains possible despite commonly used color normalization and augmentation methods, and we quantify the image characteristics constituting this site-specific digital histology signature. We demonstrate that these site-specific signatures lead to biased accuracy for prediction of features including survival, genomic mutations, and tumor stage. Furthermore, ethnicity can also be inferred from site-specific signatures, which must be accounted for to ensure equitable application of DL. These site-specific signatures can lead to overoptimistic estimates of model performance, and we propose a quadratic programming method that abrogates this bias by ensuring models are not trained and validated on samples from the same site.


Assuntos
Biomarcadores Tumorais/análise , Aprendizado Profundo , Processamento de Imagem Assistida por Computador/métodos , Neoplasias/patologia , Manejo de Espécimes/métodos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Análise Mutacional de DNA/métodos , Confiabilidade dos Dados , Perfilação da Expressão Gênica/métodos , Humanos , Mutação , Estadiamento de Neoplasias , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/mortalidade , Medição de Risco/métodos
7.
Nat Commun ; 12(1): 4400, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285229

RESUMO

Rapid and widespread testing of severe acute respiratory coronavirus 2 (SARS-CoV-2) is essential for an effective public health response aimed at containing and mitigating the coronavirus disease 2019 (COVID-19) pandemic. Successful health policy implementation relies on early identification of infected individuals and extensive contact tracing. However, rural communities, where resources for testing are sparse or simply absent, face distinctive challenges to achieving this success. Accordingly, we report the development of an academic, public land grant University laboratory-based detection assay for the identification of SARS-CoV-2 in samples from various clinical specimens that can be readily deployed in areas where access to testing is limited. The test, which is a quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based procedure, was validated on samples provided by the state laboratory and submitted for FDA Emergency Use Authorization. Our test exhibits comparable sensitivity and exceeds specificity and inclusivity values compared to other molecular assays. Additionally, this test can be re-configured to meet supply chain shortages, modified for scale up demands, and is amenable to several clinical specimens. Test development also involved 3D engineering critical supplies and formulating a stable collection media that allowed samples to be transported for hours over a dispersed rural region without the need for a cold-chain. These two elements that were critical when shortages impacted testing and when personnel needed to reach areas that were geographically isolated from the testing center. Overall, using a robust, easy-to-adapt methodology, we show that an academic laboratory can supplement COVID-19 testing needs and help local health departments assess and manage outbreaks. This additional testing capacity is particularly germane for smaller cities and rural regions that would otherwise be unable to meet the testing demand.


Assuntos
Teste de Ácido Nucleico para COVID-19/instrumentação , COVID-19/diagnóstico , Kit de Reagentes para Diagnóstico , Serviços de Saúde Rural/organização & administração , COVID-19/epidemiologia , COVID-19/prevenção & controle , COVID-19/virologia , Controle de Doenças Transmissíveis/métodos , Controle de Doenças Transmissíveis/organização & administração , Desenho de Equipamento , Humanos , Limite de Detecção , Nasofaringe/virologia , Pandemias/prevenção & controle , Impressão Tridimensional , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos
8.
BMC Infect Dis ; 21(1): 504, 2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34058992

RESUMO

BACKGROUND: HPV self-sampling has been widely supported by the scientific community following a strong body of literature on the subject. Self-sampling is important in cervical cancer screening as it has been shown to improve participation. It is well documented that HPV-testing has proven superior to cytology with regards to sensitivity in detection of CIN and cancer. The value of self-collected samples is reliant on the quality of the molecular testing performed, as well as the patients' preference in sampling procedure and compliance to follow up on positive test results. Due to the incompatibility of self-samples and cytology, triage of HPV-DNA positives by testing for molecular biomarkers is highly warranted. METHODS: Our objective was to compare the detection rate of genital Human Papillomavirus (HPV) infection in self- and clinician-collected samples by a 14-type HPV-DNA test and a 7-type mRNA E6/E7 test. RESULTS: Five hundred five women were recruited. Each study participant had two sample collection procedures performed upon the same visit, alternating order in execution of the self-collection or the clinician-taken procedure first or second, 1010 samples in total. HPV-DNA prevalence was 22.8% in self-collected versus 19.2% in clinician-collected samples (P = 0.19). Overexpression of mRNA E6/E7 from 7 HPV types was 7.1 and 6.3%, respectively (P = 0.71). The difference between HPV-DNA and HPV-mRNA positivity rates were statistically significant in both self-collected (22.8% versus 7.1%, P < 0.001) and clinician-collected samples (19.2% versus 6.3%, P < 0.001). Overall agreement between the two collection methods was fair, with a concordance rate of 78.2% (390/505), k = 0.34 (95% CI: 0.25-0.44), P < 0.001, for the HPV-DNA test and 92.5% (467/505), k = 0.40 (95% CI, 0.25-0.56), P < 0.001, for the mRNA test, respectively. 96.8% of the participants reported they felt confident carrying out the self-collection themselves, and 88.8% reported no discomfort at all performing the procedure. CONCLUSIONS: This comparative study of two sampling methods reports fair agreement of HPV positivity rates between the self-collected and clinician-collected specimens using Abbott hrHPV and PreTect HPV-Proofer'7 tests. Only one third of HPV-DNA positive women had overexpression of mRNA E6/E7. TRIAL REGISTRATION: ISRCTN77337300 .


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Manejo de Espécimes/métodos , Adulto , DNA Viral/genética , Detecção Precoce de Câncer/métodos , Feminino , Humanos , Pessoa de Meia-Idade , Papillomaviridae/genética , RNA Mensageiro/genética , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologia
9.
Viruses ; 13(5)2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066046

RESUMO

The aim of this study was to determine whether self-collected pure saliva (SCPS) is comparable to nasopharyngeal (NP) swabs in the quantitative detection of SARS-CoV-2 by RT-PCR in asymptomatic, mild patients with confirmed COVID-19. Thirty-one patients aged from 18 to 85 years were included between 9 June and 11 December 2020. A SCPS sample and a NP sample were taken for each patient. Quantitative PCR was performed to detect SARS-CoV-2 viral load. Results of SCPS vs. NP samples testing were compared. Statistical analyses were performed. Viral load was significantly correlated (r = 0.72). The concordance probability was estimated at 73.3%. In symptomatic adults, SCPS performance was similar to that of NP swabs (Percent Agreement = 74.1%; p = 0.11). Thus, the salivary test based on pure oral saliva samples easily obtained by noninvasive techniques has a fair agreement with the nasopharyngeal one in asymptomatic, mild patients with a confirmed diagnosis of COVID-19.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Nasofaringe/virologia , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Adulto , Idoso , Doenças Assintomáticas , Testes Diagnósticos de Rotina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral , Reação em Cadeia da Polimerase em Tempo Real , Manejo de Espécimes/métodos , Carga Viral/métodos , Adulto Jovem
10.
Sci Rep ; 11(1): 11899, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099796

RESUMO

The pandemic caused by SARS-CoV-2 resulted in increasing demands for diagnostic tests, leading to a shortage of recommended testing materials and reagents. This study reports on the performance of self-sampled alternative swabbing material (ordinary Q-tips tested against flocked swab and rayon swab), of reagents for classical RNA extraction (phenol/guanidine-based protocol against a commercial kit), and of intercalating dye-based one-step quantitative reverse transcription real-time PCRs (RT-qPCR) compared against the gold standard hydrolysis probe-based assays for SARS-CoV-2 detection. The study found sampling with Q-tips, RNA extraction with classical protocol and intercalating dye-based RT-qPCR as a reliable and comparably sensitive strategy for detection of SARS-CoV-2-particularly valuable in the current period with a resurgent and dramatic increase in SARS-CoV-2 infections and growing shortage of diagnostic materials especially for regions limited in resources.


Assuntos
Teste para COVID-19 , COVID-19/diagnóstico , RNA Viral/genética , SARS-CoV-2/patogenicidade , Manejo de Espécimes , Teste para COVID-19/métodos , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcrição Reversa/fisiologia , Manejo de Espécimes/métodos , Fatores de Tempo
11.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069402

RESUMO

The total damage inflicted on the liver before transplantation is associated with several surgical manipulations, such as organ recovery, washout of the graft, cold conservation in organ preservation solutions (UW, Celsior, HTK, IGL-1), and rinsing of the organ before implantation. Polyethylene glycol 35 (PEG35) is the oncotic agent present in the IGL-1 solution, which is an alternative to UW and Celsior solutions in liver clinical transplantation. In a model of cold preservation in rats (4 °C; 24 h), we evaluated the effects induced by PEG35 on detoxifying enzymes and nitric oxide, comparing IGL-1 to IGL-0 (which is the same as IGL-1 without PEG). The benefits were also assessed in a new IGL-2 solution characterized by increased concentrations of PEG35 (from 1 g/L to 5 g/L) and glutathione (from 3 mmol/L to 9 mmol/L) compared to IGL-1. We demonstrated that PEG35 promoted the mitochondrial enzyme ALDH2, and in combination with glutathione, prevented the formation of toxic aldehyde adducts (measured as 4-hydroxynonenal) and oxidized proteins (AOPP). In addition, PEG35 promoted the vasodilator factor nitric oxide, which may improve the microcirculatory disturbances in steatotic grafts during preservation and revascularization. All of these results lead to a reduction in damage inflicted on the fatty liver graft during the cold storage preservation. In this communication, we report on the benefits of IGL-2 in hypothermic static preservation, which has already been proved to confer benefits in hypothermic oxygenated dynamic preservation. Hence, the data reported here reinforce the fact that IGL-2 is a suitable alternative to be used as a unique solution/perfusate when hypothermic static and preservation strategies are used, either separately or combined, easing the logistics and avoiding the mixture of different solutions/perfusates, especially when fatty liver grafts are used. Further research regarding new therapeutic and pharmacological insights is needed to explore the underlying mitochondrial mechanisms exerted by PEG35 in static and dynamic graft preservation strategies for clinical liver transplantation purposes.


Assuntos
Transplante de Fígado/métodos , Preservação de Órgãos/métodos , Polietilenoglicóis/farmacologia , Alanina Transaminase/metabolismo , Aldeído-Desidrogenase Mitocondrial/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Criopreservação/métodos , Fígado Gorduroso/metabolismo , Glutationa/metabolismo , Fígado/citologia , Masculino , Microcirculação/efeitos dos fármacos , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , Soluções para Preservação de Órgãos/farmacologia , Ratos , Ratos Zucker , Manejo de Espécimes/métodos
12.
Nat Commun ; 12(1): 3541, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112790

RESUMO

Technical advancements significantly improve earlier diagnosis of cervical cancer, but accurate diagnosis is still difficult due to various factors. We develop an artificial intelligence assistive diagnostic solution, AIATBS, to improve cervical liquid-based thin-layer cell smear diagnosis according to clinical TBS criteria. We train AIATBS with >81,000 retrospective samples. It integrates YOLOv3 for target detection, Xception and Patch-based models to boost target classification, and U-net for nucleus segmentation. We integrate XGBoost and a logical decision tree with these models to optimize the parameters given by the learning process, and we develop a complete cervical liquid-based cytology smear TBS diagnostic system which also includes a quality control solution. We validate the optimized system with >34,000 multicenter prospective samples and achieve better sensitivity compared to senior cytologists, yet retain high specificity while achieving a speed of <180s/slide. Our system is adaptive to sample preparation using different standards, staining protocols and scanners.


Assuntos
Inteligência Artificial , Manejo de Espécimes/métodos , Neoplasias do Colo do Útero/diagnóstico , Esfregaço Vaginal/métodos , Simulação por Computador , Aprendizado Profundo , Detecção Precoce de Câncer , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Estudos Prospectivos , Estudos Retrospectivos , Neoplasias do Colo do Útero/diagnóstico por imagem , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/fisiopatologia
13.
Rev Bras Ginecol Obstet ; 43(5): 377-383, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34182582

RESUMO

OBJECTIVE: The coronavirus disease 2019 (COVID-19) is a pandemic viral disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The impact of the disease among the obstetric population remains unclear, and the study of the placenta can provide valuable information. Adequate sampling of the placental tissue can help characterize the pathways of viral infections. METHODS: A protocol of placental sampling is proposed, aiming at guaranteeing representativity of the placenta and describing the adequate conservation of samples and their integrity for future analysis. The protocol is presented in its complete and simplified versions, allowing its implementation in different complexity settings. RESULTS: Sampling with the minimum possible interval from childbirth is the key for adequate sampling and storage. This protocol has already been implemented during the Zika virus outbreak. CONCLUSION: A protocol for adequate sampling and storage of placental tissue is fundamental for adequate evaluation of viral infections on the placenta. During the COVID-19 pandemic, implementation of this protocol may help to elucidate critical aspects of the SARS-CoV-2 infection.


Assuntos
COVID-19/virologia , Placenta/virologia , Manejo de Espécimes/métodos , Manejo de Espécimes/normas , Feminino , Humanos , Gravidez , Virologia/métodos , Virologia/normas , Viroses/virologia
14.
Sci Rep ; 11(1): 12676, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135391

RESUMO

Regular PCR testing of nasopharyngeal swabs from symptomatic individuals for SARS-CoV-2 virus has become the established method by which health services are managing the COVID-19 pandemic. Businesses such as AstraZeneca have also prioritised voluntary asymptomatic testing to keep workplaces safe and maintain supply of essential medicines to patients. We describe the development of an internal automated SARS-CoV-2 testing programme including the transformative introduction of saliva as an alternative sample type.


Assuntos
Doenças Assintomáticas/epidemiologia , Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/epidemiologia , Pandemias/prevenção & controle , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Saliva/virologia , Recursos Humanos , COVID-19/virologia , Testes Diagnósticos de Rotina/métodos , Humanos , Nasofaringe/virologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Manejo de Espécimes/métodos
15.
Methods Mol Biol ; 2276: 357-382, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060055

RESUMO

Untargeted metabolomics has rapidly become a profiling method of choice in many areas of research, including mitochondrial biology. Most commonly, untargeted metabolomics is performed with liquid chromatography/mass spectrometry because it enables measurement of a relatively wide range of physiochemically diverse molecules. Specifically, to assess energy pathways that are associated with mitochondrial metabolism, hydrophilic interaction liquid chromatography (HILIC) is often applied before analysis with a high-resolution accurate mass instrument. The workflow produces large, complex data files that are impractical to analyze manually. Here, we present a protocol to perform untargeted metabolomics on biofluids such as plasma, urine, and cerebral spinal fluid with a HILIC separation and an Orbitrap mass spectrometer. Our protocol describes each step of the analysis in detail, from preparation of solvents for chromatography to selecting parameters during data processing.


Assuntos
Líquido Cefalorraquidiano/química , Metabolômica/métodos , Mitocôndrias/metabolismo , Plasma/química , Manejo de Espécimes/métodos , Urina/química , Líquido Cefalorraquidiano/metabolismo , Cromatografia Líquida/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas/métodos , Metaboloma , Plasma/metabolismo
16.
PLoS One ; 16(6): e0253007, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34111196

RESUMO

BACKGROUND: Diagnosis of COVID-19 in symptomatic patients and screening of populations for SARS-CoV-2 infection require access to straightforward, low-cost and high-throughput testing. The recommended nasopharyngeal swab tests are limited by the need of trained professionals and specific consumables and this procedure is poorly accepted as a screening method In contrast, saliva sampling can be self-administered. METHODS: In order to compare saliva and nasopharyngeal/oropharyngeal samples for the detection of SARS-CoV-2, we designed a meta-analysis searching in PubMed up to December 29th, 2020 with the key words "(SARS-CoV-2 OR COVID-19 OR COVID19) AND (salivary OR saliva OR oral fluid)) NOT (review[Publication Type]) NOT (PrePrint[Publication Type])" applying the following criteria: records published in peer reviewed scientific journals, in English, with at least 15 nasopharyngeal/orapharyngeal swabs and saliva paired samples tested by RT-PCR, studies with available raw data including numbers of positive and negative tests with the two sampling methods. For all studies, concordance and sensitivity were calculated and then pooled in a random-effects model. FINDINGS: A total of 377 studies were retrieved, of which 50 were eligible, reporting on 16,473 pairs of nasopharyngeal/oropharyngeal and saliva samples. Meta-analysis showed high concordance, 92.5% (95%CI: 89.5-94.7), across studies and pooled sensitivities of 86.5% (95%CI: 83.4-89.1) and 92.0% (95%CI: 89.1-94.2) from saliva and nasopharyngeal/oropharyngeal swabs respectively. Heterogeneity across studies was 72.0% for saliva and 85.0% for nasopharyngeal/oropharyngeal swabs. INTERPRETATION: Our meta-analysis strongly suggests that saliva could be used for frequent testing of COVID-19 patients and "en masse" screening of populations.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Nasofaringe/virologia , RNA Viral/análise , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Humanos , RNA Viral/genética , SARS-CoV-2/genética , Sensibilidade e Especificidade , Manejo de Espécimes/métodos
17.
PLoS One ; 16(6): e0252687, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34115762

RESUMO

BACKGROUND: Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. METHODS: We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. RESULTS: SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. CONCLUSION: eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Guanidina/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Saliva/efeitos dos fármacos , Saliva/virologia , Manejo de Espécimes/métodos , Inativação de Vírus/efeitos dos fármacos , Animais , COVID-19/virologia , Chlorocebus aethiops , Meios de Cultura , Voluntários Saudáveis , Humanos , RNA Viral/genética , Células Vero
18.
Viruses ; 13(5)2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-34067983

RESUMO

The primary approach to controlling the spread of the pandemic SARS-CoV-2 is to diagnose and isolate the infected people quickly. Our paper aimed to investigate the efficiency and the reliability of a hierarchical pooling approach for large-scale PCR testing for SARS-CoV-2 diagnosis. To identify the best conditions for the pooling approach for SARS-CoV-2 diagnosis by RT-qPCR, we investigated four manual methods for both RNA extraction and PCR assessment targeting one or more of the RdRp, N, S, and ORF1a genes, by using two PCR devices and an automated flux for SARS-CoV-2 detection. We determined the most efficient and accurate diagnostic assay, taking into account multiple parameters. The optimal pool size calculation included the prevalence of SARS-CoV-2, the assay sensitivity of 95%, an assay specificity of 100%, and a range of pool sizes of 5 to 15 samples. Our investigation revealed that the most efficient and accurate procedure for detecting the SARS-CoV-2 has a detection limit of 2.5 copies/PCR reaction. This pooling approach proved to be efficient and accurate in detecting SARS-CoV-2 for all samples with individual quantification cycle (Cq) values lower than 35, accounting for more than 94% of all positive specimens. Our data could serve as a comprehensive practical guide for SARS-CoV-2 diagnostic centers planning to address such a pooling strategy.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/genética , COVID-19/sangue , COVID-19/genética , Ensaios de Triagem em Larga Escala/métodos , Humanos , Pandemias/prevenção & controle , RNA Viral/sangue , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , SARS-CoV-2/patogenicidade , Sensibilidade e Especificidade , Manejo de Espécimes/métodos
19.
Sci Prog ; 104(2): 368504211026152, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34143699

RESUMO

The most common method for SARS-CoV-2 testing is throat or nasal swabbing by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. In South Korea, drive-through swab test is used for screening system and community treatment centers (CTCs), which admit and treat confirmed COVID-19 patients with mild symptoms, are being used. This retrospective study was conducted on patients admitted to a CTC on March 6, 2020. A total of 313 patients were admitted. The nasal and throat swabs were collected from the upper respiratory tract, and a sputum test was performed to obtain lower respiratory samples. The positive rate of the first set of test, sputum test was higher than that of the swab test (p = 0.011). In the second set of test, 1 week after the first ones, the rate of positive swab tests was relatively high (p = 0.026). In the first set of test, 66 of 152 (43.4%) patients showed 24-h consecutive negative swab test results, when the sputum test results were considered together, that number fell to 29 patients (19.1%) (p < 0.001). Also, in the second set of test, 63 of 164 (38.4%) patients met the discharge criteria only when the swab test was considered; that number fell to 30 (18.3%) when the sputum test results were also considered (p < 0.001). Using the swab test alone is insufficient for screening test and discharge decision. Patients who may have positive result in the sputum test can be missed.


Assuntos
Teste de Ácido Nucleico para COVID-19/normas , COVID-19/diagnóstico , Alta do Paciente/estatística & dados numéricos , SARS-CoV-2/genética , Manejo de Espécimes/métodos , Adulto , Doenças Assintomáticas , COVID-19/epidemiologia , COVID-19/virologia , Centros Comunitários de Saúde/organização & administração , Feminino , Humanos , Masculino , Programas de Rastreamento/métodos , Nasofaringe/virologia , Faringe/virologia , Quarentena/métodos , República da Coreia/epidemiologia , Estudos Retrospectivos , Índice de Gravidade de Doença , Escarro/virologia
20.
Virol J ; 18(1): 99, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001180

RESUMO

BACKGROUND: Sensitive, rapid, and accessible diagnostics continue to be critical to track the COVID-19 pandemic caused by the SARS-CoV-2 virus. RT-qPCR is the gold standard test, and comparison of methodologies and reagents, utilizing patient samples, is important to establish reliable diagnostic pipelines. METHODS: Here, we assessed indirect methods that require RNA extraction with direct RT-qPCR on patient samples. Four different RNA extraction kits (Qiagen, Invitrogen, BGI and Norgen Biotek) were compared. For detection, we assessed two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). We also tested and optimized direct, extraction-free detection using these RT-qPCR systems and performed a cost analysis of the different methods evaluated here. RESULTS: Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system provided overall superior performance (lower detection limit, lower Ct values and higher sensitivity), generating comparable results to original clinical diagnostic data, and identifying samples ranging from 65 copies to 2.1 × 105 copies of viral genome/µl. However, the BGI detection system is more expensive than other options tested here. With direct RT-qPCR, simply adding an RNase inhibitor greatly improved detection, without the need for any other treatments (e.g. lysis buffers or boiling). The best direct methods detected ~ 10 fold less virus than indirect methods, but this simplified approach reduced sample handling, as well as assay time and cost. CONCLUSIONS: With extracted RNA, the BGI RT-qPCR detection system exhibited superior performance over the Norgen system, matching initial clinical diagnosis with the Seegene Allplex assay. The BGI system was also suitable for direct, extraction-free analysis, providing 78.4% sensitivity. The Norgen system, however, still accurately detected samples with a clinical Ct < 33 from extracted RNA, provided significant cost savings, and was superior to SYBR green assays that exhibited reduced specificity.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Kit de Reagentes para Diagnóstico , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodos , Humanos , Nasofaringe/virologia , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade
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