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1.
JNMA J Nepal Med Assoc ; 59(237): 464-467, 2021 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-34508439

RESUMEN

INTRODUCTION: The emergence of severe acute respiratory syndrome coronavirus-2 pandemic is critically challenging the whole world. The real-time reverse transcriptase-polymerase chain reaction is the most widely used confirmatory test for COVID-19 detection. This study aimed to find out the prevalence of COVID-19 infection detected by gold standard reverse transcriptase-polymerase chain reaction test in a tertiary care center of Nepal. METHODS: A descriptive cross-sectional study was conducted in Karnali Academy of Health Sciences from May to August 2020 after taking ethical approval from the Institutional Review Committee of Karnali Academy of Health Sciences, Jumla. Convenient sampling was used. A total of 361 participants enrolled in this study who have done real-time reverse transcriptase-polymerase chain reaction for screening of COVID-19 infection. Also, a designated questionnaire was obtained from persons with a travel history and close contact. Statistical Package for the Social Sciences software was used for the statistical analysis. Point estimate at 95% Confidence Interval was calculated along with frequency and proportion for binary data. RESULTS: The prevalence of COVID-19 was 167 (46.3%) (95% Confidence Interval= 41.16-51.44) by real-time reverse transcriptase-polymerase chain reaction test. Out of 361 samples, 339 (93.9%) were male and 22 (6%) were female. The highest frequency of the participants belongs to the age groups of 20-40 years. CONCLUSIONS: The findings showed a high prevalence of COVID-19 detected by reverse transcriptase-polymerase chain reaction test. Further studies are necessary to improve the precision of prevalence estimations.


Asunto(s)
COVID-19 , Adulto , Estudios Transversales , Femenino , Humanos , Masculino , ADN Polimerasa Dirigida por ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2 , Centros de Atención Terciaria , Adulto Joven
2.
Zhongguo Zhong Yao Za Zhi ; 46(12): 3116-3122, 2021 Jun.
Artículo en Chino | MEDLINE | ID: mdl-34467703

RESUMEN

Screening suitable reference genes is the premise of quantitative Real-time PCR(qRT-PCR)for gene expression analysis. To provide stable reference genes for expression analysis of genes in Aconitum vilmorinianum, this study selected 19 candidate re-ference genes(ACT1, ACT2, ACT3, aTUB1, aTUB2, bTUB, 18S rRNA, UBQ, eIF2, eIF3, eIF4, eIF5, CYP, GAPDH1, GAPDH2, PP2A1, PP2A2, ACP, and EF1α) based on the transcriptome data of A. vilmorinianum. qRT-PCR was conducted to profile the expression of these genes in the root, stem, leaf, and flower of A. vilmorinianum. The Ct values showed that 18S rRNA with high expression level and GAPDH2 with large expression difference among organs were not suitable as the reference genes. NormFinder and geNorm showed similar results of the expression stability of the other candidate reference genes and demonstrated PP2A1, EF1α, and CYP as the highly stable ones. However, BestKeeper suggested EF1α, ACT3, and PP2A1 as the top stable genes. In view of the different results from different softwares, the geometric mean method was employed to analyze the expression stability of the candidate re-ference genes, the results of which indicated that PP2A1, EF1α, and ACT3 were the most stable. Based on the comprehensive analysis results of geNorm, NormFinder, BestKeeper, and geometric mean method, PP2A1 and EF1α presented the most stable expression in different organs of A. vilmorinianum. PP2A1 and EF1α were the superior reference genes for gene expression profiling in different organs of A. vilmorinianum.


Asunto(s)
Aconitum , Perfilación de la Expresión Génica , Genes de Plantas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
3.
Zhongguo Zhong Yao Za Zhi ; 46(15): 3832-3837, 2021 Aug.
Artículo en Chino | MEDLINE | ID: mdl-34472256

RESUMEN

Freshly collected seeds of Amomum tsaoko demonstrate obvious dormancy. Therefore, the selection of stable reference genes during seed dormancy release is very important for the subsequent functional research of related genes. In this study, ten commonly used reference genes(GAPDH, 40S, actin, tubulin, EIF4A-9, EIF2α, UBC, UBCE2, 60S, and UBQ) were selected as candidates for quantitative Real-time polymerase chain reaction(qRT-PCR) of the embryo samples of A. tsaoko at different dormancy release stages. Three kinds of software(BestKeeper, geNorm, and Normfinder) and the Delta CT method were used to evaluate the expression stability of the candidate reference genes, and the RefFinder online tool was employed to integrate the results and generate a comprehensive ranking. The results showed that the expression levels of the ten candidate reference genes differed greatly in different embryo samples. GAPDH and UBC had high expression levels, as manifested by the small Ct values. GeNorm identified 40S and UBCE2 as the most stable genes. NormFinder ranked EIF2α as the most stable gene and UBC as the least stable gene. UBCE2 was found to be the most stable gene and actin the least stable one by BestKeeper. Delta CT analysis suggested that the expression of 40S was most stable. UBCE2 was recommended as the most stably expressed gene by RefFinder. Thus, UBCE2 is the ideal reference gene for qRT-PCR analysis of A. tsaoko seeds at different dormancy release stages. The results may lay a foundation for analyzing the expression of related genes during seed dormancy release of A. tsaoko.


Asunto(s)
Amomum , Perfilación de la Expresión Génica , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semillas/genética
4.
Anticancer Res ; 41(9): 4609-4617, 2021 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-34475089

RESUMEN

BACKGROUND/AIM: This study aimed to assess the yield of an Oncomine comprehensive assay v3 (OCAv3)-based next-generation sequencing (NGS) analysis for detecting anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) fusions in non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: NGS data from 85 NSCLC cases were reviewed. ALK and ROS1 fusion status was compared to conventional tests. RESULTS: ALK or ROS1 fusion reads were detected in 17 NSCLC cases. Results in 10 NSCLC cases showed concordance with conventional tests, high-count fusion reads, a lack of mutually exclusive mutations of ALK or ROS1, and frequent signet-ring cell component. Seven NSCLC cases showing discordant results exhibited low to intermediate fusion read counts and mutations mutually exclusive from ALK or ROS1. CONCLUSION: Cases showing high-count fusion reads in OCAv3-based NGS have a strong possibility of carrying ALK or ROS1 fusion. Cases with low- to intermediate-count fusion reads should be interpreted with caution and may require additional confirmative tests.


Asunto(s)
Quinasa de Linfoma Anaplásico/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Análisis de Secuencia de ARN/métodos , Adulto , Anciano , Anciano de 80 o más Años , Análisis Citogenético , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
5.
Pan Afr Med J ; 39: 89, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34466191

RESUMEN

Coronavirus disease 2019 (COVID-19), a severe acute respiratory syndrome caused by SARS-CoV-2 was declared a global pandemic by the World Health Organization (WHO) in March 2020. As of 21st April 2021, the disease had affected more than 143 million people with more than 3 million deaths worldwide. Urgent effective strategies are required to control the scourge of the pandemic. Rapid sample collection and effective testing of appropriate specimens from patients meeting the suspect case definition for COVID-19 is a priority for clinical management and outbreak control. The WHO recommends that suspected cases be screened for SARS-CoV-2 virus with nucleic acid amplification tests such as real-time Reverse Transcription-Polymerase Chain Reaction (rRT-PCR). Other COVID-19 screening techniques such as serological and antigen tests have been developed and are currently being used for testing at ports of entry and for general surveillance of population exposure in some countries. However, there are limited testing options, equipment, and trained personnel in many African countries. Previously, positive patients have been screened more than twice to determine viral clearance prior to discharge after treatment. In a new policy directive, the WHO now recommends direct discharge after treatment of all positive cases without repeated testing. In this review, we discuss COVID-19 testing capacity, various diagnostic methods, test accuracy, as well as logistical challenges in Africa with respect to the WHO early discharge policy.


Asunto(s)
Prueba de COVID-19/métodos , COVID-19/diagnóstico , Guías de Práctica Clínica como Asunto , África , Humanos , Tamizaje Masivo/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Manejo de Especímenes , Organización Mundial de la Salud
6.
J Assoc Physicians India ; 69(8): 11-12, 2021 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-34472806

RESUMEN

BACKGROUND: Despite global efforts, COVID 19 pandemic is still posing a serious challenge with dearth of effective treatment options. Remdesivir has got FDA approval as the first COVID-19 anti-viral agent in adult and paediatric patients (aged ≥12 years and weighing at least 40 kg) requiring hospitalization. OBJECTIVE: The present prospective, observational, cross sectional study was planned to evaluate the impact of early initiation of Remdesivir on clinical outcomes in moderate to severe COVID 19 patients requiring ICU care. Materials and Methods: In this study, 100 consecutive symptomatic RT-PCR positive COVID 19 patients requiring admission to Intensive care unit based on predefined criteria were included for evaluation. All such moderate to severely ill patients were given Remdesivir intravenously as a 200-mg loading dose on day 1, followed by a 100-mg maintenance dose for the next four days along with other standard care. The main outcome measure analysed was the impact of early initiation of Remdesivir on recovery assessed by number of days in hospital, recovery, biochemical improvements and death. Results: Out of total 100 patients, 84 patients recovered with Remdesivir along with supportive treatment and were discharged from the hospital. Mean age of patients at presentation 53.5± 14.8 years with 5:1 male preponderance. Mean duration of hospital stay was 11.6 ± 5.9 days. D-dimer, CRP, Ferritin, IL-6 decreased significantly post treatment with P values <0.05, <0.001, <0.001, <0.01 respectively when compared to values at admission. No significant side effects were seen with remdesivir infusion. CONCLUSION: This real-world experience suggests that in the subset of hospitalized patients with moderate to severe pneumonia, early use of Remdesivir can lead to better clinical outcomes and help in reduction of associated mortality and morbidity of COVID-19.


Asunto(s)
COVID-19 , Adenosina Monofosfato/análogos & derivados , Adulto , Anciano , Alanina/análogos & derivados , COVID-19/tratamiento farmacológico , Niño , Estudios Transversales , Humanos , India , Unidades de Cuidados Intensivos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2 , Resultado del Tratamiento
7.
J Bras Pneumol ; 47(4): e20210131, 2021.
Artículo en Inglés, Portugués | MEDLINE | ID: mdl-34495176

RESUMEN

OBJECTIVE: To describe baseline characteristics of outpatients with a positive RT-PCR for SARS-CoV-2 and to define whether "red flags" (new-onset fever, dyspnea, and chest pain) can predict clinical worsening during the isolation period. METHODS: This was an epidemiological, observational, descriptive study. Between March and September of 2020, all outpatients who tested positive for SARS-CoV-2 at a tertiary medical center located in Santiago de Chile were included. Demographic variables, comorbidities, red flags, and other symptoms were compiled using follow-up surveys at specific time points. The risk of clinical worsening (hospitalization) and adjusted hazard ratios (HRs) were calculated. RESULTS: A total of 7,108 patients were included. The median age was 38 years (range, 0-101), and 52% were men. At baseline, 77% of the patients reported having characteristic symptoms of SARS-CoV-2 infection. The most prevalent onset symptoms were headache (53%), myalgia (47%), and fever (33%). According to the follow-up surveys, the incidence of symptoms decreased during the isolation period; however, 28% of the patients still presented with symptoms on day 14. The risk of hospitalization for patients with new-onset fever and dyspnea during the follow-up period was HR = 7.43 (95% CI, 3.85-14.3, p<0.01) and HR = 5.27 (95% CI, 1.52-18.30; p < 0.01 for both), respectively. New-onset chest pain showed no association with clinical worsening. CONCLUSIONS: In this sample of outpatients with a recent diagnosis of SARS-CoV-2 infection, a survey-based monitoring of symptoms was useful to identify those at risk of clinical worsening. New-onset fever and dyspnea during the isolation period were considered as red flags associated with clinical worsening and warrants prompt medical evaluation.


Asunto(s)
COVID-19 , SARS-CoV-2 , Adulto , Hospitalización , Humanos , Masculino , Pandemias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
8.
BMC Pulm Med ; 21(1): 278, 2021 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-34465321

RESUMEN

BACKGROUND: There are various reasons for delayed positive nasopharyngeal PCR tests for coronavirus disease 2019 (COVID19) in not only asymptomatic but also severely diseased patients. The pathophysiological attributes are not known. We explore this possibility through a case report. CASE PRESENTATION: A 64-year-old male with history of pulmonary fungal infection, asthma and chronic pulmonary obstructive disease (COPD), diabetes, coronary artery disease presented with shortness of breath, fever and chest image of ground opacity, reticular interstitial thickening, highly suspicious for COVID19. However, nasopharyngeal swab tests were discordantly negative for four times in two weeks, and IgG antibody for COVID19 was also negative. However, serum IgE level was elevated. No other pathogens are identified. His symptoms deteriorated despite corticosteroid, antibiotics and bronchodilator treatment. Bronchoalveolar lavage (BAL) and open lung wedge biopsy were performed for etiology diagnosis. They demonstrated COVID19 viral RNA positive fibrosing organizing pneumonia with respiratory tract damage characterized by suspicious viral cytopathic effect, mixed neutrophilic, lymphoplasmacytic, histiocytic and eosinophilic inflammation and fibrosis besides expected asthma and COPD change. One week later, repeated COVID19 nasopharyngeal tests on day 40 and day 49 became positive. CONCLUSION: Our case and literature review indicate that allergic asthma and associated high IgE level together with corticosteroid inhalation might contribute to the delayed positive nasopharyngeal swab in upper airway; COPD related chronic airways obstruction and the addition of fibrosis induced ventilator dependence and poor prognosis in COVID19 pneumonia, and should be therapeutically targeted besides antiviral therapy.


Asunto(s)
Prueba de Ácido Nucleico para COVID-19 , COVID-19/diagnóstico , Diagnóstico Tardío , Nasofaringe/virología , SARS-CoV-2/aislamiento & purificación , Administración por Inhalación , Corticoesteroides/uso terapéutico , Asma/complicaciones , Asma/tratamiento farmacológico , Asma/patología , Lavado Broncoalveolar , COVID-19/complicaciones , COVID-19/terapia , Resultado Fatal , Humanos , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
9.
Artículo en Inglés | MEDLINE | ID: mdl-34501741

RESUMEN

INTRODUCTION: Rapid antigen tests (RATs) are convenient for SARS-CoV-2 detection because they are simpler and faster than nucleic acid amplification tests (NAATs). This study aimed to assess the accuracy of a locally manufactured test; Rapid Test Ag 2019-nCoV (PROGNOSIS, BIOTECH, Larissa, Greece) in a clinical setting and during mass screening. METHODS: Nasopharyngeal samples from 624 individuals were analyzed. The results of the rapid test were compared to real-time reverse-transcription quantitative polymerase chain reaction (RT-qPCR). At the end of the test's procedure, positive test strips were scanned in an S-Flow reader in order to roughly estimate the antigen concentration. RESULTS: The lower limit of detection of the test was 468.75 genome copies/mL. The PROGNOSIS rapid test displayed a sensitivity of 85.5% (141/165) (95%CI: 79.1-90.5) and a specificity of 99.8% (458/459) (95%CI: 98.8-100.0%). The general inter-rater agreement was 0.89 (95%CI: 85.1-93.3). The regression analysis between the S-flow reader measurements (viral antigen) and the viral load of the positive samples demonstrated a weak correlation (R2 = 0.288, p < 0.001). CONCLUSION: The Rapid Test Ag 2019-nCoV demonstrated sufficient sensitivity, excellent specificity and could be available to be used with low overall cost. Thus, it could be used as point of care test, but also for mass screening for rapid detection of infected persons (e.g., for travelers).


Asunto(s)
COVID-19 , SARS-CoV-2 , Biotecnología , Prueba de Ácido Nucleico para COVID-19 , Prueba Serológica para COVID-19 , Grecia , Hospitales , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad
10.
Mikrobiyol Bul ; 55(3): 311-326, 2021 Jul.
Artículo en Turco | MEDLINE | ID: mdl-34416799

RESUMEN

The SARS-CoV-2 virus, which caused the COVID-19 epidemic, caused more than 55 million cases and nearly 1.5 million deaths worldwide. For the microbiological diagnosis of the disease, the most valid method is detecting the presence of the viral genome by real-time reverse transcription polymerase chain reaction (rRT-PCR). However, due to the nature of the RNA viruses, frequent mutations may affect the sensitivity of the analyses made on the genetic material of the virus, such as PCR. In this study, we aimed to investigate the mutations in the primer-probe binding regions of the rRT-PCR panels used in COVID-19 diagnosis. SARS-CoV-2 whole genome sequence data (n= 194) isolated from COVID-19 cases in Turkey and uploaded on GISAID database from the centers in Istanbul (n= 78), Ankara (n= 58), Kars (n= 47), Bursa (n= 2), Adiyaman (n= 2), Erciyes (n= 1) and Kocaeli (n= 1) between March 17-September 14, 2020 were analyzed. In order to determine the nucleotide changes, SARS-CoV-2 sequences from Turkey were compared to the reference genome sequence (NC_045512.1) present in "GenBank" website. The constructed data set was aligned using the MAFFT program and was checked manually if the sequences were in the same frame by using the AliView program. Primer-probe binding sites of the thirteen SARS-CoV-2 rRT-PCR panels from seven different institutes (US CDC, China CDC, Charite CDC, Pasteur, HKU, Thailand, NIID) that are being used in COVID-19 diagnosis were evaluated in terms of nucleotide changes within the corresponding regions compared to the reference genome. Sequence diversities in the viral genomes were determined via positional nucleotide numerical calculator and entropy calculator modules and nucleotide and entropy changes in primer-probe binding regions for each rRT-PCR panel were examined. Among thirteen different primer-probe panels, nucleotide changes in the target regions of the seven primer-probe panels were determined. When viral sequences with nucleotide changes in the primer-probe binding regions were examined, the most common changes were observed in the "China CDC" N-forward primer and "US CDC" N3-forward primer binding regions. It is important that the kits to be used as diagnostic tests are designed specific to the regions with less nucleotide changes. Nucleotide changes may not be critical for DNA amplification for most PCR panels, but should be carefully monitored as they may affect the sensitivity of the assay. If the risk of alteration of the designed region is high, the primer - probe binding sites should be checked frequently and updated when necessary.


Asunto(s)
COVID-19 , SARS-CoV-2 , Prueba de COVID-19 , Humanos , Nucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Turquia
11.
Mikrobiyol Bul ; 55(3): 426-434, 2021 Jul.
Artículo en Turco | MEDLINE | ID: mdl-34416807

RESUMEN

Human T-lymphotropic virus-I/II (HTLV-I/II) and human immun viruses (HIVs), that have similar genomic characteristics also share the same transmission routes and infect T lymphocytes. Regarding this epidemiological similarity, HIV and HTLV infections can be seen together. HIV and HTLV-I/II coinfection occurs with variable frequencies in different populations and geographic regions. There are not any population-based studies carried out defining the number of individuals coinfected with HIV and HTLV-I/II in Turkey. The aim of this study was to determine the seropositivity rates of HTLV-I/II in patients whose HIV viral load was monitored in Gazi University Faculty of Medicine Medical Virology Laboratory Forty-seven HIV positive cases followed-up in Medical Virology Laboratory for HIV viral load monitoring between May 2017-January 2019 were included in the study. HIV seropositivity of the samples was confirmed by the chemiluminescence microparticle immunoassay method. HIV viral load values of the samples were evaluated by real-time reverse transcriptase polymerase chain reaction. The samples were screened for antibodies against HTLV-I/II using chemiluminescent microparticle immunoassay. The study population range was between 19 to 60 years of age. Among the study population, 39 (83%) patients were male and 8 (17%) patients were female. Of 47 samples, 18 samples (38.3%) had viral load of <1000 copies/ml, 10 samples (21.3%) had viral load of 1000-10000 copies/ml, 19 samples (40.4%) had viral load of ≥10000 copies/ml. HTLV serology was negative in all samples included in the study. CD4+ results were available for 42 patients and the CD4+ results of five patients could not be studied. Co-infection with different retroviruses is a well-known fact which should be thoroughly examined. HTLV-I co-infection leads to faster progression of the disease in HIV-1 positive patients. Although it is known that the co-infection has a significant effect on the progression of the disease, there are very few centers in the world and in our country that routinely perform HTLV testing in HIV-positive patients. We think that in order to evaluate the clinical and microbiological importance of the coinfection of retroviruses with each other and to determine the frequency of these infections together, there is a need for studies involving a larger number of patients, including detailed clinical backgrounds of individuals, and that the importance of this issue should be realized at the same time.


Asunto(s)
Infecciones por VIH , VIH-1 , Virus Linfotrópico T Tipo 1 Humano , Femenino , Infecciones por VIH/complicaciones , Infecciones por VIH/epidemiología , VIH-1/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 2 Humano/genética , Humanos , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
12.
BMC Infect Dis ; 21(1): 828, 2021 Aug 18.
Artículo en Inglés | MEDLINE | ID: mdl-34407759

RESUMEN

BACKGROUND: Lateral flow devices (LFDs) are viral antigen tests for the detection of SARS-CoV-2 that produce a rapid result, are inexpensive and easy to operate. They have been advocated for use by the World Health Organisation to help control outbreaks and break the chain of transmission of COVID-19 infections. There are now several studies assessing their accuracy but as yet no systematic review. Our aims were to assess the sensitivity and specificity of LFDs in a systematic review and summarise the sensitivity and specificity of these tests. METHODS: A targeted search of Pubmed and Medxriv, using PRISMA principles, was conducted identifying clinical studies assessing the sensitivity and specificity of LFDs as their primary outcome compared to reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of SARS-CoV-2. Based on extracted data sensitivity and specificity was calculated for each study. Data was pooled based on manufacturer of LFD and split based on operator (self-swab or by trained professional) and sensitivity and specificity data were calculated. RESULTS: Twenty-four papers were identified involving over 26,000 test results. Sensitivity from individual studies ranged from 37.7% (95% CI 30.6-45.5) to 99.2% (95% CI 95.5-99.9) and specificity from 92.4% (95% CI 87.5-95.5) to 100.0% (95% CI 99.7-100.0). Operation of the test by a trained professional or by the test subject with self-swabbing produced comparable results. CONCLUSIONS: This systematic review identified that the performance of lateral flow devices is heterogeneous and dependent on the manufacturer. Some perform with high specificity but a great range of sensitivities were shown (38.32-99.19%). Test performance does not appear dependent on the operator. Potentially, LFDs could support the scaling up of mass testing to aid track and trace methodology and break the chain of transmission of COVID-19 with the additional benefit of providing individuals with the results in a much shorter time frame.


Asunto(s)
Prueba de COVID-19/normas , COVID-19/diagnóstico , SARS-CoV-2/aislamiento & purificación , Antígenos Virales/análisis , COVID-19/epidemiología , Reacciones Falso Negativas , Reacciones Falso Positivas , Femenino , Humanos , Masculino , Pandemias , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Sensibilidad y Especificidad
13.
J Med Microbiol ; 70(8)2021 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-34369860

RESUMEN

Introduction. The current severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) pandemic has stressed the global supply chain for specialized equipment, including flocked swabs.Hypothesis. Saliva could be a potential alternative specimen source for diagnosis of SARS-CoV-2 infection by reverse-transcriptase PCR (RT-PCR).Aim. To compare the detection efficiency of SARS-CoV-2 RNA in saliva and oro-nasopharyngeal swab (ONPS) specimens.Methodology. Patients recruited from hospital provided paired saliva and ONPS specimens. We performed manual or automated RT-PCR with prior proteinase K treatment without RNA extraction using the Seegene Allplex 2019 nCoV assay.Results. Of the 773 specimen pairs, 165 (21.3 %) had at least one positive sample. Additionally, 138 specimens tested positive by both sampling methods. Fifteen and 12 cases were detected only by nasopharyngeal swab and saliva, respectively. The sensitivity of ONPS (153/165; 92.7 %; 95 % CI: 88.8-96.7) was similar to that of saliva (150/165; 90.9 %; 95 % CI: 86.5-95.3; P=0.5). In patients with symptoms for ≤ 10 days, the sensitivity of ONPS (118/126; 93.7 %; 95 % CI: 89.4-97.9) was similar to that of saliva (122/126; 96.8 %; 95 % CI: 93.8-99.9 %; P=0.9). However, the sensitivity of ONPS (20/22; 95.2 %; 95 % CI: 86.1-100) was higher than that of saliva (16/22; 71.4 %; 95 % CI: 52.1-90.8) in patients with symptoms for more than 10 days.Conclusions. Saliva sampling is an acceptable alternative to ONPS for diagnosing SARS-CoV-2 infection in symptomatic individuals displaying symptoms for ≤ 10 days. These results reinforce the need to expand the use of saliva samples, which are self-collected and do not require swabs.


Asunto(s)
Prueba de Ácido Nucleico para COVID-19 , COVID-19/diagnóstico , Nasofaringe/virología , Orofaringe/virología , Saliva/virología , Adulto , Anciano , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Manejo de Especímenes
14.
Iran J Allergy Asthma Immunol ; 20(4): 394-401, 2021 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-34418893

RESUMEN

Considering the increasing prevalence and burden of coronavirus disease 2019 (COVID-19) disease and false-negative results in routine reverse transcription-polymerase chain reaction (RT-PCR) tests, additional diagnostic methods are needed to diagnose active cases of this disease. This prospective study was conducted on patients, in whom clinical and radiological symptoms/signs were in favor of COVID-19 while their first PCR test was negative. Later on, a second RT-PCR was performed and serological evaluation was carried out and results were compared with each other. Out of 707 patients who had been referred to the hospital and were clinically and radiologically suspicious of disease, 137 patients with negative RT-PCR tests entered the study. RT-PCR assay became positive for the second time in 45 (32.8%). Anti-COVID-19 IgM and IgG antibodies were positive in 83 (60.6%) and 86 (62.8%) patients, respectively. Finally, it was determined that serological test was diagnostic in 73% of patients and the diagnostic yield of serology was significantly higher after the first week of illness (54.8% in the first week and 88% after that). Taking advantage of both serological tests and RT-PCR helps in diagnosing 83.9% of cases. Based on the present study, the serology may be useful as a complementary test and in parallel to RT-PCR assay for diagnosis of COVID-19 among admitted symptomatic cases.


Asunto(s)
Prueba de COVID-19 , COVID-19/diagnóstico , Hospitalización , Adulto , Anciano , Anticuerpos Antivirales/sangre , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , Estudios Prospectivos , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Sensibilidad y Especificidad
15.
Nat Commun ; 12(1): 4898, 2021 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-34385431

RESUMEN

Hedgehog (Hh) signaling is essential during development and in organ physiology. In the canonical pathway, Hh binding to Patched (PTCH) relieves the inhibition of Smoothened (SMO). Yet, PTCH may also perform SMO-independent functions. While the PTCH homolog PTC-3 is essential in C. elegans, worms lack SMO, providing an excellent model to probe non-canonical PTCH function. Here, we show that PTC-3 is a cholesterol transporter. ptc-3(RNAi) leads to accumulation of intracellular cholesterol and defects in ER structure and lipid droplet formation. These phenotypes were accompanied by a reduction in acyl chain (FA) length and desaturation. ptc-3(RNAi)-induced lethality, fat content and ER morphology defects were rescued by reducing dietary cholesterol. We provide evidence that cholesterol accumulation modulates the function of nuclear hormone receptors such as of the PPARα homolog NHR-49 and NHR-181, and affects FA composition. Our data uncover a role for PTCH in organelle structure maintenance and fat metabolism.


Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Colesterol/metabolismo , Homeostasis/genética , Metabolismo de los Lípidos/genética , Receptor Patched-1/genética , Animales , Western Blotting , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/metabolismo , Regulación de la Expresión Génica , Microscopía Electrónica de Transmisión , Receptor Patched-1/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
16.
Nat Commun ; 12(1): 4897, 2021 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-34385432

RESUMEN

Precise control of mammalian gene expression is facilitated through epigenetic mechanisms and nuclear organization. In particular, insulated chromosome structures are important for regulatory control, but the phenotypic consequences of their boundary disruption on developmental processes are complex and remain insufficiently understood. Here, we generated deeply sequenced Hi-C data for human pluripotent stem cells (hPSCs) that allowed us to identify CTCF loop domains that have highly conserved boundary CTCF sites and show a notable enrichment of individual developmental regulators. Importantly, perturbation of such a boundary in hPSCs interfered with proper differentiation through deregulated distal enhancer-promoter activity. Finally, we found that germline variations affecting such boundaries are subject to purifying selection and are underrepresented in the human population. Taken together, our findings highlight the importance of developmental gene isolation through chromosomal folding structures as a mechanism to ensure their proper expression.


Asunto(s)
Diferenciación Celular/genética , Perfilación de la Expresión Génica/métodos , Genoma Humano/genética , Células Madre Embrionarias Humanas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Elementos Reguladores de la Transcripción/genética , Sitios de Unión/genética , Western Blotting , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Línea Celular , Elementos de Facilitación Genéticos/genética , Células Madre Embrionarias Humanas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN/métodos
17.
Nat Commun ; 12(1): 4908, 2021 08 13.
Artículo en Inglés | MEDLINE | ID: mdl-34389711

RESUMEN

C9ORF72 hexanucleotide GGGGCC repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-containing RNA mediates toxicity through nuclear granules and dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG translation. However, it remains unclear how the intron-localized repeats are exported and translated in the cytoplasm. We use single molecule imaging approach to examine the molecular identity and spatiotemporal dynamics of the repeat RNA. We demonstrate that the spliced intron with G-rich repeats is stabilized in a circular form due to defective lariat debranching. The spliced circular intron, instead of pre-mRNA, serves as the translation template. The NXF1-NXT1 pathway plays an important role in the nuclear export of the circular intron and modulates toxic DPR production. This study reveals an uncharacterized disease-causing RNA species mediated by repeat expansion and demonstrates the importance of RNA spatial localization to understand disease etiology.


Asunto(s)
Proteína C9orf72/genética , Núcleo Celular/metabolismo , Intrones/genética , Biosíntesis de Proteínas/genética , ARN/genética , Transporte Activo de Núcleo Celular/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/metabolismo , Línea Celular Tumoral , Núcleo Celular/genética , Expansión de las Repeticiones de ADN/genética , Dipéptidos/genética , Dipéptidos/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Predisposición Genética a la Enfermedad/genética , Células HEK293 , Humanos , Microscopía Fluorescente , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética
18.
Nat Commun ; 12(1): 4917, 2021 08 13.
Artículo en Inglés | MEDLINE | ID: mdl-34389714

RESUMEN

APOBEC3A is a cytidine deaminase driving mutagenesis in tumors. While APOBEC3A-induced mutations are common, APOBEC3A expression is rarely detected in cancer cells. This discrepancy suggests a tightly controlled process to regulate episodic APOBEC3A expression in tumors. In this study, we find that both viral infection and genotoxic stress transiently up-regulate APOBEC3A and pro-inflammatory genes using two distinct mechanisms. First, we demonstrate that STAT2 promotes APOBEC3A expression in response to foreign nucleic acid via a RIG-I, MAVS, IRF3, and IFN-mediated signaling pathway. Second, we show that DNA damage and DNA replication stress trigger a NF-κB (p65/IkBα)-dependent response to induce expression of APOBEC3A and other innate immune genes, independently of DNA or RNA sensing pattern recognition receptors and the IFN-signaling response. These results not only reveal the mechanisms by which tumors could episodically up-regulate APOBEC3A but also highlight an alternative route to stimulate the immune response after DNA damage independently of cGAS/STING or RIG-I/MAVS.


Asunto(s)
Citidina Desaminasa/genética , Daño del ADN , Regulación de la Expresión Génica , Inmunidad/genética , Proteínas/genética , Transducción de Señal/fisiología , Línea Celular , Línea Celular Tumoral , Citidina Desaminasa/metabolismo , Interacciones Huésped-Patógeno , Humanos , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células THP-1 , Factor de Transcripción ReIA/metabolismo , Regulación hacia Arriba , Virus/crecimiento & desarrollo
19.
Nat Chem Biol ; 17(9): 982-988, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34354262

RESUMEN

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. Here, we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT-PCR)-derived cycle threshold (Ct) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.


Asunto(s)
COVID-19/genética , Sistemas CRISPR-Cas/genética , ARN Viral/genética , SARS-CoV-2/genética , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
20.
Nat Commun ; 12(1): 4825, 2021 08 10.
Artículo en Inglés | MEDLINE | ID: mdl-34376658

RESUMEN

Circular RNA (circRNA) is a class of covalently joined non-coding RNAs with functional roles in a wide variety of cellular processes. Their composition shows extensive overlap with exons found in linear mRNAs making it difficult to delineate their composition using short-read RNA sequencing, particularly for long and multi-exonic circRNAs. Here, we use long-read nanopore sequencing of nicked circRNAs (circNick-LRS) and characterize a total of 18,266 and 39,623 circRNAs in human and mouse brain, respectively. We further develop an approach for targeted long-read sequencing of a panel of circRNAs (circPanel-LRS), eliminating the need for prior circRNA enrichment and find >30 circRNA isoforms on average per targeted locus. Our data show that circRNAs exhibit a large number of splicing events such as novel exons, intron retention and microexons that preferentially occur in circRNAs. We propose that altered exon usage in circRNAs may reflect resistance to nonsense-mediated decay in the absence of translation.


Asunto(s)
Encéfalo/metabolismo , Exones/genética , Intrones/genética , Secuenciación de Nanoporos/métodos , ARN Circular/genética , Análisis de Secuencia de ARN/métodos , Animales , Expresión Génica , Humanos , Masculino , Ratones de la Cepa 129 , Isoformas de ARN/genética , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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