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1.
Sci Rep ; 11(1): 14204, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244543

RESUMO

The pandemic of 2019 caused by the novel coronavirus (SARS-CoV-2) is still rapidly spreading worldwide. Nucleic acid amplification serves as the gold standard method for confirmation of COVID-19 infection. However, challenges faced for diagnostic laboratories from undeveloped countries includes shortage of kits and supplies to purify viral RNA. Therefore, it is urgent to validate alternative nucleic acid isolation methods for SARS-CoV-2. Our results demonstrate that a concentrated viral lysis amplification buffer (vLAB) prepared with the nonionic detergent IGEPAL enables qualitative detection of SARS-CoV-2 by direct Reverse Transcriptase-Polymerase Chain Reaction (dRT-PCR). Furthermore, vLAB was effective in inactivating SARS-CoV-2. Since this method is inexpensive and no RNA purification equipment or additional cDNA synthesis is required, this dRT-PCR with vLAB should be considered as an alternative method for qualitative detection of SARS-CoV-2.


Assuntos
Teste de Ácido Nucleico para COVID-19 , COVID-19 , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , Manejo de Espécimes , COVID-19/diagnóstico , COVID-19/genética , Humanos , Sensibilidade e Especificidade
2.
BMC Infect Dis ; 21(1): 678, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34256733

RESUMO

BACKGROUND: In this case report, we presented a rare case of maternal death with massive vulvar edema and Covid-19 diagnosis. CASE PRESENTATION: The case was a 20-year-old woman who was referred to with pain and massive vulvar edema by passing 7 days from her labor. The laboratory tests showed leukocytosis, lymphopenia, and elevated C-reactive protein levels. The high-resolution computed tomography was in favor of Covid-19 changes. Finally, she died because of respiratory distress, ON the 8th day postpartum. CONCLUSION: Given the increasing prevalence of Covid-19, it is important and vital to be aware of its potential complications and then to try prevent and manage them, especially during high-risk periods such as pregnancy and postpartum.


Assuntos
COVID-19/complicações , Edema/complicações , Edema/diagnóstico por imagem , Morte Materna , Período Pós-Parto , SARS-CoV-2/genética , Doenças da Vulva/complicações , Doenças da Vulva/diagnóstico por imagem , COVID-19/sangue , COVID-19/virologia , Teste para COVID-19/métodos , Evolução Fatal , Feminino , Humanos , Linfopenia , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tomografia Computadorizada por Raios X/métodos , Adulto Jovem
3.
Pediatr Infect Dis J ; 40(8): e300-e304, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34250969

RESUMO

BACKGROUND: Saliva reverse transcriptase-Polymerase chain reaction (RT-PCR) is an attractive alternative for the detection of severe acute respiratory syndrome coronavirus 2 in adults with less known in children. METHODS: Children with coronavirus disease 2019 symptoms were prospectively enrolled in a 1-month comparative clinical trial of saliva and nasopharyngeal (NP) RT-PCR. Detection rates and sensitivities of saliva and NP RT-PCR were compared as well as discordant NP and saliva RT-PCR findings including viral loads (VLs). RESULTS: Of 405 patients enrolled, 397 patients had 2 tests performed. Mean age was 12.7 years (range, 1.2-17.9). Sensitivity of saliva was 85.2% (95% confidence interval: 78.2%-92.1%) when using NP as the standard; sensitivity of NP was 94.5% (89.8%-99.2%) when saliva was considered as the standard. For a NP RT-PCR VL threshold of ≥103 and ≥104 copies/mL, sensitivity of saliva increases to 88.7% and 95.2%, respectively. Sensitivity of saliva and NP swabs was, respectively, 89.5% and 95.3% in patient with symptoms less than 4 days (P = 0.249) and 70.0% and 95.0% in those with symptoms ≥4-7 days (P = 0.096). The 15 patients who had an isolated positive NP RT-PCR were younger (P = 0.034), had lower NP VL (median 5.6 × 103 vs. 3.9 × 107, P < 0.001), and could not drool saliva at the end of the sampling (P = 0.002). VLs were lower with saliva than with NP RT-PCR (median 8.7 cp/mL × 104; interquartile range 1.2 × 104-5.2 × 105; vs. median 4.0 × 107 cp/mL; interquartile range, 8.6 × 105-1 × 108; P < 0.001). CONCLUSIONS: While RT-PCR testing on saliva performed more poorly in younger children and likely after longer duration of symptoms, saliva remains an attractive alternative to NP swabs in children.


Assuntos
Teste para COVID-19 , COVID-19/diagnóstico , COVID-19/virologia , Nasofaringe/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Criança , Pré-Escolar , Testes Diagnósticos de Rotina , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Estudos Prospectivos , SARS-CoV-2/genética , Sensibilidade e Especificidade , Manejo de Espécimes , Carga Viral
4.
BMC Infect Dis ; 21(1): 688, 2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34271860

RESUMO

BACKGROUND: Being able to use COVID-19 RT-PCR Ct values as simple clinical markers of disease outcome or prognosis would allow for the easy and proactive identification and triaging of high-risk cases. This study's objective was thus to explore whether a correlation exists between COVID-19 viral loads, as indicated by RT-PCR Ct values, and disease severity, as indicated by respiratory indices. RESULTS: A multi-centre cross-sectional retrospective study was conducted, using data obtained from Bahrain's National COVID-19 Task force's centralised database. The study period ranged from May 2, 2020 to July 31, 2020. A multivariable logistic regression was used to assess for a correlation using data from a total of 1057 admitted COVID-19 cases. The covariates adjusted for included sex, age, presentation, and comorbidities. In our cohort, Ct value showed no statistical significance for an association with requirement for oxygenation on admission (Odds ratio 1.046; 95%CI 0.999 to 1.096, p = 0.054). CONCLUSION: Viral load, as indicated by Ct values, did not seem to be associated with requirement for oxygenation on admission in our cohort. We postulate however that time since onset of symptom may have acted as an unaccounted-for confounder. As such, RT-PCR Ct values may not be a useful prognostic clinical tool in isolation.


Assuntos
COVID-19/diagnóstico , COVID-19/patologia , SARS-CoV-2/fisiologia , Carga Viral/fisiologia , Adulto , Idoso , Barein/epidemiologia , COVID-19/epidemiologia , COVID-19/virologia , Estudos de Coortes , Comorbidade , Estudos Transversais , Feminino , Hospitalização/estatística & dados numéricos , Humanos , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Testes Sorológicos , Índice de Gravidade de Doença , Carga Viral/estatística & dados numéricos
5.
BMJ ; 374: n1637, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230058

RESUMO

OBJECTIVE: To assess the performance of the SARS-CoV-2 antigen rapid lateral flow test (LFT) versus polymerase chain reaction testing in the asymptomatic general population attending testing centres. DESIGN: Observational cohort study. SETTING: Community LFT pilot at covid-19 testing sites in Liverpool, UK. PARTICIPANTS: 5869 asymptomatic adults (≥18 years) voluntarily attending one of 48 testing sites during 6-29 November 2020. INTERVENTIONS: Participants were tested using both an Innova LFT and a quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) test based on supervised self-administered swabbing at testing sites. MAIN OUTCOME MEASURES: Sensitivity, specificity, and predictive values of LFT compared with RT-qPCR in an epidemic steady state of covid-19 among adults with no classic symptoms of the disease. RESULTS: Of 5869 test results, 22 (0.4%) LFT results and 343 (5.8%) RT-qPCR results were void (that is, when the control line fails to appear within 30 minutes). Excluding the void results, the LFT versus RT-qPCR showed a sensitivity of 40.0% (95% confidence interval 28.5% to 52.4%; 28/70), specificity of 99.9% (99.8% to 99.99%; 5431/5434), positive predictive value of 90.3% (74.2% to 98.0%; 28/31), and negative predictive value of 99.2% (99.0% to 99.4%; 5431/5473). When the void samples were assumed to be negative, a sensitivity was observed for LFT of 37.8% (26.8% to 49.9%; 28/74), specificity of 99.6% (99.4% to 99.8%; 5431/5452), positive predictive value of 84.8% (68.1% to 94.9%; 28/33), and negative predictive value of 93.4% (92.7% to 94.0%; 5431/5814). The sensitivity in participants with an RT-qPCR cycle threshold (Ct) of <18.3 (approximate viral loads >106 RNA copies/mL) was 90.9% (58.7% to 99.8%; 10/11), a Ct of <24.4 (>104 RNA copies/mL) was 69.4% (51.9% to 83.7%; 25/36), and a Ct of >24.4 (<104 RNA copies/mL) was 9.7% (1.9% to 23.7%; 3/34). LFT is likely to detect at least three fifths and at most 998 in every 1000 people with a positive RT-qPCR test result with high viral load. CONCLUSIONS: The Innova LFT can be useful for identifying infections among adults who report no symptoms of covid-19, particularly those with high viral load who are more likely to infect others. The number of asymptomatic adults with lower Ct (indicating higher viral load) missed by LFT, although small, should be considered when using single LFT in high consequence settings. Clear and accurate communication with the public about how to interpret test results is important, given the chance of missing some cases, even at high viral loads. Further research is needed to understand how infectiousness is reflected in the viral antigen shedding detected by LFT versus the viral loads approximated by RT-qPCR.


Assuntos
Teste de Ácido Nucleico para COVID-19 , Teste Sorológico para COVID-19 , COVID-19/diagnóstico , Portador Sadio/diagnóstico , Portador Sadio/virologia , Adulto , COVID-19/complicações , Estudos de Coortes , Feminino , Humanos , Masculino , Projetos Piloto , Valor Preditivo dos Testes , Curva ROC , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Reino Unido
6.
Front Public Health ; 9: 649524, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34249831

RESUMO

Background: COVID-19 outbursts have been registered worldwide within care homes with asymptomatic transmission combined with shortage/inaccuracy of diagnostic tests undermining the efforts at containment of the disease. Nursing facilities in Lombardy (Italy) were left with no, or limited, access to testing for 8 weeks after the outbreak of COVID-19. Methods: This study includes 246 residents and 286 workers of three different nursing homes in Brescia-Lombardy. Clinical questionnaires and rapid serology tests were devised to integrate the data of the first available RT-PCR screening. Follow-up serology after 60-days was performed on 67 of 86 workers with positive serology or clinically suspicious. Findings: Thirty-seven residents and 18 workers had previous positive RT-PCR. Thorough screening disclosed two additional RT-PCR-positive workers. Serology screening revealed antibodies in 59 residents and 48 workers, including 32/37 residents and all workers previously positive at RT-PCR. Follow up serology disclosed antibodies in two additional workers with recent symptoms at the time of screening. The professionals in close contact with residents had more infections (47/226-20.79% vs. 1/60-1.66%; p = 0.00013 Fisher exact-test). A suspicious clinical score was present in 44/64 residents and in 41/50 workers who tested positive with either method with totally asymptomatic disease more frequent among residents 28.1 vs. 10.0% (p = 0.019 Fisher exact-test). Interpretation: Based on the available RT-PCR ± results at the time of symptoms/contacts, our integrated clinical and serological screening demonstrated sensitivity 89% and specificity 87%. This multimodal assessment proved extremely useful in understanding the viral spread in nursing homes, in defining its stage and in implementing protective measures. Rapid serology tests demonstrated efficient and particularly suited for older people less able to move/cooperate.


Assuntos
COVID-19 , Sistemas Automatizados de Assistência Junto ao Leito , Idoso , Humanos , Itália/epidemiologia , Casas de Saúde , Projetos Piloto , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2
8.
Ann Intern Med ; 174(7): JC82, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34224255

RESUMO

SOURCE CITATION: Drain PK, Ampajwala M, Chappel C, et al. A rapid, high-sensitivity SARS-CoV-2 nucleocapsid immunoassay to aid diagnosis of acute COVID-19 at the point of care: a clinical performance study. Infect Dis Ther. 2021;10:753-61 33629225.


Assuntos
COVID-19 , Adulto , Antígenos Virais , Criança , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2 , Sensibilidade e Especificidade
9.
Ann Intern Med ; 174(7): JC83, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34224272

RESUMO

SOURCE CITATION: Möckel M, Corman VM, Stegemann MS, et al. SARS-CoV-2 antigen rapid immunoassay for diagnosis of COVID-19 in the emergency department. Biomarkers. 2021;26:213-20. 33455451.


Assuntos
COVID-19 , Adulto , Antígenos Virais , Criança , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2 , Sensibilidade e Especificidade
10.
PLoS One ; 16(7): e0252509, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34260637

RESUMO

The current global pandemic due to the SARS-CoV-2 has pushed the limits of global health systems across all aspects of clinical care, including laboratory diagnostics. Supply chain disruptions and rapidly-shifting markets have resulted in flash-scarcity of commercial laboratory reagents; this has motivated health care providers to search for alternative workflows to cope with the international increase in demand for SARS-CoV-2 testing. The aim of this study is to present a reproducible workflow for real time RT-PCR SARS-CoV-2 testing using OT-2 open-source liquid-handling robots (Opentrons, NY). We have developed a framework that includes a code template which is helpful for building different stand-alone robotic stations, capable of performing specific protocols. Such stations can be combined together to create a complex multi-stage workflow, from sample setup to real time RT-PCR. Using our open-source code, it is easy to create new stations or workflows from scratch, adapt existing templates to update the experimental protocols, or to fine-tune the code to fit specific needs. Using this framework, we developed the code for two different workflows and evaluated them using external quality assessment (EQA) samples from the European Molecular Genetics Quality Network (EMQN). The affordability of this platform makes automated SARS-CoV-2 PCR testing accessible for most laboratories and hospitals with qualified bioinformatics personnel. This platform also allows for flexibility, as it is not dependent on any specific commercial kit, and thus it can be quickly adapted to protocol changes, reagent, consumable shortages, or any other temporary material constraints.


Assuntos
Teste de Ácido Nucleico para COVID-19/instrumentação , SARS-CoV-2/isolamento & purificação , Codificação Clínica , Diagnóstico Precoce , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Robótica , SARS-CoV-2/genética , Fluxo de Trabalho
11.
PLoS One ; 16(7): e0253941, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34242243

RESUMO

Accurate SARS-CoV-2 diagnosis is essential to guide prevention and control of COVID-19. Here we examine SARS-CoV-2 molecular-based test performance characteristics and summarize case-level data related to COVID-19 diagnosis. From January 11 through April 22, 2020, Public Health Ontario conducted SARS-CoV-2 testing of 86,942 specimens collected from 80,354 individuals, primarily using real-time reverse-transcription polymerase chain reaction (rRT-PCR) methods. We analyzed test results across specimen types and for individuals with multiple same-day and multi-day collected specimens. Nasopharyngeal compared to throat swabs had a higher positivity (8.8% vs. 4.8%) and an adjusted estimate 2.9 Ct lower (SE = 0.5, p<0.001). Same-day specimens showed high concordance (98.8%), and the median Ct of multi-day specimens increased over time. Symptomatic cases had rRT-PCR results with an adjusted estimate 3.0 Ct (SE = 0.5, p<0.001) lower than asymptomatic/pre-symptomatic cases. Overall test sensitivity was 84.6%, with a negative predictive value of 95.5%. Molecular testing is the mainstay of SARS-CoV-2 diagnosis and testing protocols will continue to be dynamic and iteratively modified as more is learned about this emerging pathogen.


Assuntos
Teste de Ácido Nucleico para COVID-19 , COVID-19 , Pandemias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , Adolescente , Adulto , Idoso , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/genética , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Ontário/epidemiologia
12.
Clinics (Sao Paulo) ; 76: e2913, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34287482

RESUMO

OBJECTIVES: To test conjunctival swabs from patients with laboratory-confirmed severe forms of coronavirus disease 2019 (COVID-19) for the presence of SARS-CoV-2 on real-time reverse-transcription polymerase chain reaction (rRT-PCR). METHODS: Fifty conjunctival swabs were collected from 50 in-patients with laboratory-confirmed severe forms of COVID-19 at the largest teaching hospital and referral center in Brazil (HCFMUSP, São Paulo, SP). The samples were tested for SARS-CoV-2 on rRT-PCR with the primers and probes described in the CDC protocol which amplify the region of the nucleocapsid N gene (2019_nCoV_N1 and 2019_nCoV_N2) of SARS-CoV-2 RNA and compared with naso/oropharyngeal swabs collected within 24 hours of the conjunctival swabs. RESULTS: Five conjunctival samples (10%) tested positive (amplification of the N1 and N2 primer/probe sets) while two conjunctival samples (4%) yielded inconclusive results (amplification of the N1 primer/probe set only). The naso/oropharyngeal swabs were positive for SARS-CoV-2 on rRT-PCR in 34 patients (68%), negative in 14 (28%) and inconclusive in 2 (4%). The 5 patients with positive conjunctival swabs had positive (n=2), negative (n=2) or inconclusive (n=1) naso/oropharyngeal swabs on rRT-PCR. Patients with negative or inconclusive naso/oropharyngeal swabs had the diagnosis of COVID-19 confirmed by previous positive rRT-PCR results or by serology. CONCLUSION: This is the first study to present conjunctival swab rRT-PCR results for SARS-CoV-2 in a Brazilian population. In our sample of 50 patients with severe forms of COVID-19, 10% had positive conjunctival swabs, most of which were correlated with positive naso/oropharyngeal rRT-PCR results.


Assuntos
COVID-19 , Brasil , Humanos , RNA Viral , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa , SARS-CoV-2
13.
Mymensingh Med J ; 30(3): 796-802, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34226470

RESUMO

This study was aimed to find out the socio-demographic profiles as well as difficulties of Reverse transcription polymerase chain reaction (RT-PCR) testing faced by Coronavirus Disease 2019 (COVID-19) patients. This cross-sectional study was conducted from 10th August to 7th September 2020. Data were collected by telephone interview using a pre-designed questionnaire after taking verbal consent from the participants. Out of 281 COVID-19 patients, 279 were diagnosed by RT-PCR; 10.3% were asymptomatic. Off them 67.6%were males, 24% were hospital staffs. Majority (66.2%) were from the non-city corporation area. History of recent contact with known COVID-19 patients was present in 56.9% cases. Fever (78.3%) and cough (58%) were the most common symptoms. One third of the patients faced difficulty to test RT-PCR for COVID-19. Sixteen percent patients complained of difficulty of getting serial for testing, the maximum waiting time was one week before giving samples. Thirty patients (10.8%) had to wait longer than usual time after reaching the center before giving sample. Hospital staffs were unable to co-operate in 2.5% of the patients while difficulty of managing transport to the hospital for suspected COVID-19 patient was an issue in 2.2% of the patients. Though testing was more difficult in city corporation areas (p=0.028), delay of getting test result was less (p<0.001). Maximum delay of getting test result was 10 days. Finding out the difficulties of COVID-19 testing will help to point out the issues behind these and will help to take necessary steps to tackle this matter. Testing rate can be increased to contain this highly contagious virus in this densely populated country.


Assuntos
Teste para COVID-19 , COVID-19 , Estudos Transversais , Demografia , Humanos , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2
14.
Clin Transl Gastroenterol ; 12(6): e00363, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34106090

RESUMO

INTRODUCTION: Mounting evidence demonstrates potential for fecal-oral transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The US Food and Drug Administration now requires SARS-CoV-2 testing of potential feces donors before the use of stool manufactured for fecal microbiota transplantation. We sought to develop and validate a high-sensitivity SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) procedure for testing stool specimens. METHODS: A modified extraction method was used with an RT-PCR assay adapted from the Centers for Disease Control and Prevention PCR protocol for respiratory specimens. Contrived specimens were created using pre-COVID-19 banked stool specimens and spiking in known concentrations of SARS-CoV-2-specific nucleic acid. The highest transcript concentration at which 2/2 or 1/2 SARS-CoV-2 targets were detected in 9/10 replicates was defined as the dual-target limit and single-target limit of detection, respectively. The clinical performance of the assay was evaluated with stool samples collected from 17 nasopharyngeal swab RT-PCR-positive patients and 14 nasopharyngeal RT-PCR-negative patients. RESULTS: The dual-target and single-target limit of detection were 56 copies/µL and 3 copies/µL, respectively. SARS-CoV-2 was detected at concentrations as low as 0.6 copies/µL. Clinical stool samples from known COVID-19-positive patients demonstrated the detection of SARS-CoV-2 in stool up to 29 days from symptom onset with a high agreement with nasopharyngeal swab tests (kappa statistic of 0.95, P value < 0.001). DISCUSSION: The described RT-PCR test is a sensitive and flexible approach for the detection of SARS-CoV-2 in stool specimens. We propose an integrated screening approach that incorporates this stool test to support continuation of fecal microbiota transplantation programs.


Assuntos
Teste para COVID-19/métodos , COVID-19/transmissão , Transplante de Microbiota Fecal/métodos , Fezes/virologia , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/virologia , Teste para COVID-19/estatística & dados numéricos , Centers for Disease Control and Prevention, U.S./normas , Transplante de Microbiota Fecal/estatística & dados numéricos , Humanos , Nasofaringe/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/isolamento & purificação , Doadores de Tecidos/provisão & distribuição , Estados Unidos
15.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071104

RESUMO

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a "cholinergic locus", and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.


Assuntos
Colina O-Acetiltransferase/biossíntese , Neurônios Colinérgicos/metabolismo , Gânglios Espinais/citologia , Proteínas do Tecido Nervoso/biossíntese , Células Receptoras Sensoriais/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/biossíntese , Acetilcolina/metabolismo , Processamento Alternativo , Animais , Colina O-Acetiltransferase/genética , Neurônios Colinérgicos/citologia , Gânglios Espinais/embriologia , Gânglios Espinais/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Neurogênese , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Receptoras Sensoriais/citologia , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/genética
16.
Clin Lab ; 67(6)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34107642

RESUMO

BACKGROUND: Rapid and accurate diagnosis of influenza virus (Flu) and respiratory syncytial virus (RSV) is important for managing both the patient and laboratory. We compared the cobas Influenza A/B & RSV assay (cobas Liat) with the Simplexa Flu A/B & RSV assay (Simplexa) to evaluate which test method is more advantageous considering the resources of the laboratory and results of test performance. METHODS: A total of 236 respiratory specimens from patients referred for respiratory virus testing were retrospectively evaluated; 53 specimens tested positive for each of Flu A, Flu B, and RSV, and 77 specimens tested negative based on the results of the reference method, i.e., the Seegene Allplex Respiratory Panel 1/2/3 (Seegene, Seoul, Korea). The turnaround time (TAT) was 20 minutes per specimen for cobas Liat and 78 minutes per eight speci-mens for Simplexa. The total hands-on time was around one minute per specimen for both tests. The specimen volume required for testing was 200 µL for cobas Liat and 50 µL for Simplexa. Seegene Allplex Respiratory Panel 1/2/3 was used as the reference method. RESULTS: The number of invalid results was 1 (0.4%) for cobas Liat and 10 (4.2%) for Simplexa (p < 0.05). All results were consistent with those of the reference method in cobas Liat. The sensitivity and specificity for Flu A, Flu B, and RSVA were 100% with Simplexa. However, the sensitivity for RSVB was 80.0% with Simplexa, which was a statistically significant difference with the finding for cobas Liat (p < 0.05). Comparison of the cycle threshold (Ct) values of RSV for Simplexa with the reference method showed correlation as continuous variables (p < 0.001) with a higher propensity for obtaining Ct values with Simplexa, the exception being the six false negative results; their Ct values were more than 30 in the reference method. CONCLUSIONS: Cobas Liat showed accurate performance with a rapid TAT and a good workflow efficiency. Cobas Liat is more efficient than Simplexa as a point-of-care test for the detection of RSV.


Assuntos
Vírus da Influenza A , Influenza Humana , Infecções por Vírus Respiratório Sincicial , Humanos , Vírus da Influenza A/genética , Vírus da Influenza B/genética , Influenza Humana/diagnóstico , Masculino , Técnicas de Diagnóstico Molecular , Nasofaringe , República da Coreia , Infecções por Vírus Respiratório Sincicial/diagnóstico , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
17.
BMC Infect Dis ; 21(1): 566, 2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34126960

RESUMO

BACKGROUND: Vitamin D deficiency has been suggested to favor a poorer outcome of Coronavirus disease-19 (COVID-19). We aimed to assess if 25-hydroxyvitamin-D (25OHD) levels are associated with interleukin 6 (IL-6) levels and with disease severity and mortality in COVID-19. METHODS: We prospectively studied 103 in-patients admitted to a Northern-Italian hospital (age 66.1 ± 14.1 years, 70 males) for severely-symptomatic COVID-19. Fifty-two subjects with SARS-CoV-2 infection but mild COVID-19 symptoms (mildly-symptomatic COVID-19 patients) and 206 subjects without SARS-CoV-2 infection were controls. We measured 25OHD and IL-6 levels at admission and focused on respiratory outcome during hospitalization. RESULTS: Severely-symptomatic COVID-19 patients had lower 25OHD levels (18.2 ± 11.4 ng/mL) than mildly-symptomatic COVID-19 patients and non-SARS-CoV-2-infected controls (30.3 ± 8.5 ng/mL and 25.4 ± 9.4 ng/mL, respectively, p < 0.0001 for both comparisons). 25OHD and IL-6 levels were respectively lower and higher in severely-symptomatic COVID-19 patients admitted to intensive care Unit [(ICU), 14.4 ± 8.6 ng/mL and 43.0 (19.0-56.0) pg/mL, respectively], than in those not requiring ICU admission [22.4 ± 1.4 ng/mL, p = 0.0001 and 16.0 (8.0-32.0) pg/mL, p = 0.0002, respectively]. Similar differences were found when comparing COVID-19 patients who died in hospital [13.2 ± 6.4 ng/mL and 45.0 (28.0-99.0) pg/mL] with survivors [19.3 ± 12.0 ng/mL, p = 0.035 and 21.0 (10.5-45.9) pg/mL, p = 0.018, respectively). 25OHD levels inversely correlated with: i) IL-6 levels (ρ - 0.284, p = 0.004); ii) the subsequent need of the ICU admission [relative risk, RR 0.99, 95% confidence interval (95%CI) 0.98-1.00, p = 0.011] regardless of age, gender, presence of at least 1 comorbidity among obesity, diabetes, arterial hypertension, creatinine, IL-6 and lactate dehydrogenase levels, neutrophil cells, lymphocytes and platelets count; iii) mortality (RR 0.97, 95%CI, 0.95-0.99, p = 0.011) regardless of age, gender, presence of diabetes, IL-6 and C-reactive protein and lactate dehydrogenase levels, neutrophil cells, lymphocytes and platelets count. CONCLUSION: In our COVID-19 patients, low 25OHD levels were inversely correlated with high IL-6 levels and were independent predictors of COVID-19 severity and mortality.


Assuntos
COVID-19/sangue , COVID-19/mortalidade , SARS-CoV-2/genética , Índice de Gravidade de Doença , Vitamina D/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/complicações , COVID-19/epidemiologia , Calcifediol/administração & dosagem , Comorbidade , Diabetes Mellitus/epidemiologia , Feminino , Humanos , Hipertensão/epidemiologia , Unidades de Terapia Intensiva , Interleucina-6/sangue , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Obesidade/epidemiologia , Admissão do Paciente , Estudos Prospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vitamina D/sangue , Deficiência de Vitamina D/complicações , Vitaminas/administração & dosagem
18.
Methods Mol Biol ; 2298: 185-195, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085246

RESUMO

Technological advances in high-throughput sequencing in combination with antibody enrichment and/or induced nucleotide-specific chemical modifications have accelerated the mapping of epitranscriptomic modifications. However, site-specific detection and quantification of m6A are still technically challenging. Here, we describe a simple RT-QPCR-based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues.


Assuntos
RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Metilação , Processamento Pós-Transcricional do RNA/genética , Transcriptoma/genética
19.
Methods Mol Biol ; 2298: 197-216, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085247

RESUMO

The post-transcriptional modification of tRNAs at the wobble position plays a critical role in proper mRNA decoding and efficient protein synthesis. In particular, certain wobble uridines in eukaryotes are converted to 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The mcm5s2U modification modulates decoding during translation by increasing the stringency of the wobble uridine to base pair with its canonical nucleotide partner, thereby restricting decoding to its cognate codon. Here, we outline a technique to monitor wobble uridine status in mcm5s2U-containing tRNAs using the gamma-toxin endonuclease from the yeast Kluyveromyces lactis that naturally cleaves tRNAs containing the mcm5s2U modification. This technique is coupled to Northern blotting or reverse transcription-PCR to enable rapid and sensitive detection of changes in mcm5s2U modification state.


Assuntos
Endonucleases/metabolismo , Tiouridina/análogos & derivados , Códon/genética , Proteínas Fúngicas/metabolismo , Kluyveromyces/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tiouridina/metabolismo
20.
Sci Rep ; 11(1): 12425, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127708

RESUMO

Saliva has significant advantages as a test medium for detection of SARS-CoV-2 infection in patients, such as ease of collection, minimal requirement of supplies and trained personnel, and safety. Comprehensive validation in a large cohort of prospectively collected specimens with unknown SARS-CoV-2 status should be performed to evaluate the potential and limitations of saliva-based testing. We developed a saliva-based testing pipeline for detection of SARS-CoV-2 nucleic acids using real-time reverse transcription PCR (RT-PCR) and droplet digital PCR (ddPCR) readouts, and measured samples from 137 outpatients tested at a curbside testing facility and 29 inpatients hospitalized for COVID-19. These measurements were compared to the nasal swab results for each patient performed by a certified microbiology laboratory. We found that our saliva testing positively detects 100% (RT-PCR) and 93.75% (ddPCR) of curbside patients that were identified as SARS-CoV-2 positive by the Emergency Use Authorization (EUA) certified nasal swab testing assay. Quantification of viral loads by ddPCR revealed an extremely wide range, with 1 million-fold difference between individual patients. Our results demonstrate for both community screening and hospital settings that saliva testing reliability is on par with that of the nasal swabs in detecting infected cases, and has potential for higher sensitivity when combined with ddPCR in detecting low-abundance viral loads that evade traditional testing methods.


Assuntos
COVID-19/diagnóstico , SARS-CoV-2/genética , Saliva/virologia , Adulto , COVID-19/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/análise , RNA Viral/genética , RNA Viral/metabolismo , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/isolamento & purificação , Carga Viral
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