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2.
Emerg Infect Dis ; 27(4): 1146-1150, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33754989

RESUMEN

The expense of saliva collection devices designed to stabilize severe acute respiratory syndrome coronavirus 2 RNA is prohibitive to mass testing. However, virus RNA in nonsupplemented saliva is stable for extended periods and at elevated temperatures. Simple plastic tubes for saliva collection will make large-scale testing and continued surveillance easier.


Asunto(s)
/métodos , ARN Viral , Saliva/virología , /diagnóstico , /virología , Creación de Capacidad/métodos , Humanos , Estabilidad del ARN , ARN Viral/aislamiento & purificación , ARN Viral/fisiología , Reproducibilidad de los Resultados , Asignación de Recursos , /aislamiento & purificación , Manejo de Especímenes/economía , Manejo de Especímenes/instrumentación , Manejo de Especímenes/métodos
3.
BMJ Case Rep ; 14(3)2021 Mar 03.
Artículo en Inglés | MEDLINE | ID: mdl-33658220

RESUMEN

Asymptomatic individuals positive for SARS-CoV-2 RNA constitute a significant proportion of the infected population and play a role in the transmission of the virus. We describe a healthcare worker who presented with fever and malaise and was diagnosed with mild COVID-19. The symptoms resolved within 4 days but there was persistent positivity of viral RNA in the upper respiratory tract for more than 58 days, which is the longest reported duration of persistence of SARS-CoV-2 in a healthcare worker. In this case report, we discuss clinical and administrative issues such as the role of asymptomatic cases in the transmission of the virus to patients and coworkers as an occupational hazard, interpretation of persistent positivity of nucleic acid test, duration of isolation and return-to-work guidelines pertinent to researchers and global health policymakers.


Asunto(s)
Infecciones Asintomáticas , Personal de Salud , /aislamiento & purificación , /diagnóstico , Humanos , Nasofaringe/virología , Ácidos Nucleicos , ARN Viral/aislamiento & purificación , Carga Viral , Virión
4.
Med J (Ft Sam Houst Tex) ; (PB 8-21-01/02/03): 8-11, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33666905

RESUMEN

The recent emergence of SARS-CoV-2 has led to a global pandemic of unprecedented proportions. Current diagnosis of COVID-19 relies on the detection of SARS-CoV-2 RNA by reverse transcription polymerase chain reaction (RT-PCR) in upper and lower respiratory specimens. While sensitive and specific, these RT-PCR assays require considerable supplies and reagents, which are often limited during global pandemics and surge testing. Here, we show that a nasopharyngeal swab pooling strategy can detect a single positive sample in pools of up to 10 samples without sacrificing RT-PCR sensitivity and specificity. We also report that this pooling strategy can be applied to rapid, moderate complexity assays, such as the BioFire COVID-19 test. Implementing a pooling strategy can significantly increase laboratory testing capacity while simultaneously reducing turnaround times for rapid identification and isolation of positive COVID-19 cases in high risk populations.


Asunto(s)
/diagnóstico , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Manejo de Especímenes , Humanos , Nasofaringe/virología , Sensibilidad y Especificidad
5.
Med J (Ft Sam Houst Tex) ; (PB 8-21-01/02/03): 37-49, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33666911

RESUMEN

SARS-CoV-2 has highlighted the requirement for a drastic change in pandemic response. While cases continue to rise, there is an urgent need to deploy sensitive and rapid testing in order to identify potential outbreaks before there is an opportunity for further community spread. Currently, reverse transcription quantitative polymerase chain reaction (RT-qPCR) is considered the gold standard for diagnosing an active infection, using a nasopharyngeal swab; however, it can take days after symptoms develop to properly identify and trace the infection. While many civilian jobs can be performed remotely, the Department of Defense (DOD) is by nature a very fluid organization which requires in-person interaction and a physical presence to maintain effectiveness. In this commentary, we examine several current and emergent technologies and their ability to identify both active and previous SARS-CoV-2 infection, possibly in those without symptoms. Further, we will explore an ongoing study at the Air Force Research Laboratory, utilizing Reverse Transcription Loop-mediated isothermal amplification (RT-LAMP), next-generation sequencing, and the presence of SARS-CoV-2 antibodies through Lateral Flow Immunoassays. The ability to identify SARS-CoV-2 through volatile organic compound biomarker identification will also be explored. By exploring and validating multiple testing strategies, and contributing to Operation Warp Speed, the DOD is postured to respond to SARS-CoV-2, and future pandemics.


Asunto(s)
/diagnóstico , Personal Militar , /aislamiento & purificación , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , ARN Viral/aislamiento & purificación , Sensibilidad y Especificidad , Estados Unidos
6.
J Appl Lab Med ; 6(2): 421-428, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33674879

RESUMEN

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse transcription PCR is the primary method to diagnose coronavirus disease 2019 (COVID-19). However, the analytic sensitivity required is not well defined and it is unclear how available assays compare. METHODS: For the Abbott RealTime SARS-CoV-2 assay (m2000; Abbott Molecular), we determined that it could detect viral concentrations as low as 26 copies/mL, we defined the relationship between cycle number and viral concentrations, and we tested naso- and oropharyngeal swab specimens from 8538 consecutive individuals. Using the m2000 as a reference assay method, we described the distribution of viral concentrations in these patients. We then used selected clinical specimens to determine the positive percent agreement of 2 other assays with more rapid turnaround times [Cepheid Xpert Xpress (GeneXpert; Cepheid); n = 27] and a laboratory developed test on the Luminex ARIES system [ARIES LDT (Luminex); n = 50] as a function of virus concentrations, from which we projected their false-negative rates in our patient population. RESULTS: SARS-CoV-2 was detected in 27% (95% CI: 26%-28%) of all specimens. Estimated viral concentrations were widely distributed, and 17% (95% CI: 16%-19%) of positive individuals had viral concentrations <845 copies/mL. Positive percent agreement was strongly related to viral concentration, and reliable detection (i.e., ≥95%) was observed at concentrations >100 copies/mL for the GeneXpert but not the ARIES LDT, corresponding to projected false-negative rates of 4% (95% CI: 0%-21%) and 27% (95% CI: 11%-46%), respectively. CONCLUSIONS: Substantial proportions of clinical specimens have low to moderate viral concentrations and may be missed by methods with less analytic sensitivity.


Asunto(s)
/instrumentación , Juego de Reactivos para Diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/instrumentación , /aislamiento & purificación , Adulto , Anciano , Anciano de 80 o más Años , Reacciones Falso Negativas , Femenino , Humanos , Límite de Detección , Masculino , Persona de Mediana Edad , ARN Viral/aislamiento & purificación , Reproducibilidad de los Resultados , Estudios Retrospectivos , /genética
7.
BMJ Open ; 11(3): e046800, 2021 03 24.
Artículo en Inglés | MEDLINE | ID: mdl-33762247

RESUMEN

OBJECTIVES: In Italy, the pandemic of COVID-19 resulted in congestion of hospitals and laboratories and probably determined an underestimation of the number of infected subjects, as the molecular diagnosis of SARS-CoV-2 infection was mainly performed on hospitalised patients. Therefore, limited data are available about the number of asymptomatic/paucisymptomatic subjects in the general population across time. To understand SARS-CoV-2 infection in the general population, we have developed a cross-sectional study (the 'UNIversity against CORoNavirus study') to investigate infection trends in asymptomatic/paucisymptomatic subjects in Milan (Italy), between March and June 2020. PARTICIPANTS: The study population included 2023 subjects asymptomatic at the enrolment. PRIMARY OUTCOME MEASURES: A nasal mid-turbinate swab for the detection of SARS-CoV-2 RNA and blood specimen for testing serum antibodies (immunoglobulin M (IgM) and IgG) were collected. RESULTS: Subjects showing positivity for the SARS-CoV-2 RNA and/or for anti-SARS-CoV-2 Ig is 237 (11.7%). Only 1.2% (n=25) of the total population had a positive nasal swab for SARS-CoV-2 and the large majority (21/25) of them were observed in March. A total of 226 subjects (11%) had IgM (n=19; 0.9%), IgG (n=155; 7.7%) or both (n=52; 2.6%) against SARS-CoV-2. Subjects with a present or past SARS-CoV-2 infection did not differ from other subjects as regards the number of cohabiting family members, travels, fever and upper and lower respiratory infection episodes. CONCLUSIONS: Results from the present study support the hypothesis that the actual spread of the virus in Lombardy was underestimated in the official records. However, as it is not known how long Ig persist, numbers should be taken cautiously.


Asunto(s)
Anticuerpos Antivirales/aislamiento & purificación , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina M/aislamiento & purificación , ARN Viral/aislamiento & purificación , Adulto , Estudios Transversales , Femenino , Humanos , Italia/epidemiología , Masculino , Persona de Mediana Edad
8.
PLoS One ; 16(3): e0248371, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33755704

RESUMEN

Since its emergence in China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide including Pakistan. During the pandemic, whole genome sequencing has played an important role in understanding the evolution and genomic diversity of SARS-CoV-2. Although an unprecedented number of SARS-CoV-2 full genomes have been submitted in GISAID and NCBI, data from Pakistan is scarce. We report the sequencing, genomic characterization, and phylogenetic analysis of five SARS-CoV-2 strains isolated from patients in Pakistan. The oropharyngeal swabs of patients that were confirmed positive for SARS-CoV-2 through real-time RT-PCR at National Institute of Health, Pakistan, were selected for whole-genome sequencing. Sequencing was performed using NEBNext Ultra II Directional RNA Library Prep kit for Illumina (NEW ENGLAND BioLabs Inc., MA, US) and Illumina iSeq 100 instrument (Illumina, San Diego, US). Based on whole-genome analysis, three Pakistani SARS-CoV-2 strains clustered into the 20A (GH) clade along with the strains from Oman, Slovakia, United States, and Pakistani strain EPI_ISL_513925. The two 19B (S)-clade strains were closely related to viruses from India and Oman. Overall, twenty-nine amino acid mutations were detected in the current study genome sequences, including fifteen missense and four novel mutations. Notably, we have found a D614G (aspartic acid to glycine) mutation in spike protein of the sequences from the GH clade. The G614 variant carrying the characteristic D614G mutation has been shown to be more infectious that lead to its rapid spread worldwide. This report highlights the detection of GH and S clade strains and G614 variant from Pakistan warranting large-scale whole-genome sequencing of strains prevalent in different regions to understand virus evolution and to explore their genetic diversity.


Asunto(s)
Variación Genética , Glicoproteína de la Espiga del Coronavirus/genética , Anciano de 80 o más Años , /virología , Femenino , Eliminación de Gen , Humanos , Masculino , Persona de Mediana Edad , Mutación Missense , Orofaringe/virología , Pakistán , Filogenia , ARN Viral/química , ARN Viral/aislamiento & purificación , ARN Viral/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , /aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/metabolismo , Secuenciación Completa del Genoma , Adulto Joven
9.
PLoS One ; 16(2): e0247767, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33635923

RESUMEN

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has revealed the global public health importance of robust diagnostic testing. To overcome the challenge of nucleic acid (NA) extraction and testing kit availability, an efficient method is urgently needed. OBJECTIVES: To establish an efficient, time and resource-saving and cost-effective methods, and to propose an ad hoc pooling approach for mass screening of SARS-CoV-2. METHODS: We evaluated pooling approach on both direct clinical and NA samples. The standard reverse transcriptase polymerase chain reaction (RT-PCR) test of the SARS CoV-2 was employed targeting the nucleocapsid (N) and open reading frame (ORF1ab) genomic region of the virus. The experimental pools were created using SARS CoV-2 positive clinical samples and extracted RNA spiked with up to 9 negative samples. For the direct clinical samples viral NA was extracted from each pool to a final extraction volume of 200µL, and subsequently both samples tested using the SARS CoV-2 RT-PCR assay. RESULTS: We found that a single positive sample can be amplified and detected in pools of up to 7 samples depending on the cycle threshold (Ct) value of the original sample, corresponding to high, and low SARS CoV-2 viral copies per reaction. However, to minimize false negativity of the assay with pooling strategies and with unknown false negativity rate of the assay under validation, we recommend pooling of 4/5 in 1 using the standard protocols of the assay, reagents and equipment. The predictive algorithm indicated a pooling ratio of 5 in 1 was expected to retain accuracy of the test irrespective of the Ct value samples spiked, and result in a 137% increase in testing efficiency. CONCLUSIONS: The approaches showed its concept in easily customized and resource-saving manner and would allow expanding of current screening capacities and enable the expansion of detection in the community. We recommend clinical sample pooling of 4 or 5 in 1. However, we don't advise pooling of clinical samples when disease prevalence is greater than 7%; particularly when sample size is large.


Asunto(s)
/métodos , ARN Viral/aislamiento & purificación , /aislamiento & purificación , Algoritmos , /economía , Humanos , Tamizaje Masivo/economía , Tamizaje Masivo/métodos , ARN Viral/genética , Sensibilidad y Especificidad , Manejo de Especímenes/economía , Manejo de Especímenes/métodos
10.
J Prim Care Community Health ; 12: 2150132720987711, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33525985

RESUMEN

SARS-CoV-2 initially emerged in Wuhan, China in late 2019. It has since been recognized as a pandemic and has led to great social and economic disruption globally. The Reverse Transcriptase Real-Time Polymerase Chain Reaction (rtRT-PCR) has become the primary method for COVID-19 testing worldwide. The method requires a specialized laboratory set up. Long-term persistence of SARS-CoV-2 RNA in nasopharyngeal secretion after full clinical recovery of the patient is regularly observed nowadays. This forces the patients to spend a longer period in isolation and test repeatedly to obtain evidence of viral clearance. Repeated COVID-19 testing in asymptomatic or mildly symptomatic cases often leads to extra workload for laboratories that are already struggling with a high specimen turnover. Here, we present 5 purposively selected cases with different patterns of clinical presentations in which nasopharyngeal shedding of SARS-CoV-2 RNA was observed in patients for a long time. From these case studies, we emphasized the adoption of a symptom-based approach for discontinuing transmission-based precautions over a test-based strategy to reduce the time spent by asymptomatic and mildly symptomatic COVID-19 patients in isolation. A symptom-based approach will also help reduce laboratory burden for COVID-19 testing as well as conserve valuable resources and supplies utilized for rtRT-PCR testing in an emerging lower-middle-income setting. Most importantly, it will also make room for critically ill COVID-19 patients to visit or avail COVID-19 testing at their convenience.


Asunto(s)
/métodos , Asignación de Recursos para la Atención de Salud/métodos , Evaluación de Síntomas , Adulto , /estadística & datos numéricos , Países en Desarrollo , Femenino , Humanos , Laboratorios/estadística & datos numéricos , Masculino , Aislamiento de Pacientes/estadística & datos numéricos , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , /aislamiento & purificación , Adulto Joven
11.
BMJ Open ; 11(2): e044224, 2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33637549

RESUMEN

PURPOSE: The Mother and Child COVID-19 study is a cohort recruiting pregnant women and their children in Cantabria, North of Spain, during COVID-19 pandemic in order to ascertain consequences of SARS-CoV-2 infection on pregnant women and their descendants. This article reports the cohort profile and preliminary results as recruitment is still open. PARTICIPANTS: Three subcohorts can be identified at recruitment. Subcohort 1 includes women giving birth between 23 March and 25 May 2020; they have been retrospectively recruited and could have been exposed to COVID-19 only in their third trimester of pregnancy. Subcohort 2 includes women giving birth from 26 May 2020 on; they are being prospectively recruited and could have been exposed to COVID-19 in both their second and third trimesters of pregnancy. Subcohort 3 includes women in their 12 week of pregnancy prospectively recruited from 26 May 2020 on; they could have been exposed to COVID-19 anytime in their pregnancy. All women are being tested for SARS-CoV-2 infection using both RT-PCR for RNA detection and ELISA for anti-SARS-CoV-2 antibodies. All neonates are being tested for antibodies using immunochemoluminiscency tests; if the mother is tested positive for SARS-CoV-2 RNA, a nasopharyngeal swab is also obtained from the child for RT-PCR analysis. FINDINGS TO DATE: As of 22 October, 1167 women have been recruited (266, 354 and 547 for subcohorts 1, 2 and 3, respectively). Fourteen women tested positive to SARS-CoV-2 RNA by the day of delivery. All 14 children born from these women tested negative for SARS-CoV-2 RNA. FUTURE PLANS: Children from women included in subcohort 3 are expected to be recruited by the end of 2020. Children will be followed-up for 1 year in order to ascertain the effect that COVID-19 on their development.


Asunto(s)
/diagnóstico , Transmisión Vertical de Enfermedad Infecciosa , Complicaciones Infecciosas del Embarazo , Femenino , Humanos , Lactante , Recién Nacido , Pandemias , Embarazo , Complicaciones Infecciosas del Embarazo/epidemiología , Complicaciones Infecciosas del Embarazo/virología , Estudios Prospectivos , ARN Viral/aislamiento & purificación , Estudios Retrospectivos , España/epidemiología
12.
PLoS One ; 16(2): e0247115, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33596239

RESUMEN

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance, and determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding, and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for the detection of SARS-CoV-2, with an additional advantage of enabling the genetic epidemiology of SARS-CoV-2.


Asunto(s)
/epidemiología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , /aislamiento & purificación , /genética , Genoma Viral/genética , Humanos , India/epidemiología , Epidemiología Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Pandemias , Filogenia , ARN Viral/genética , ARN Viral/aislamiento & purificación , Sensibilidad y Especificidad
13.
J Vis Exp ; (168)2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33616108

RESUMEN

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.


Asunto(s)
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Impresión Tridimensional , Transcripción Reversa/genética , /aislamiento & purificación , /genética , Recursos en Salud , Humanos , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos
14.
Sci Rep ; 11(1): 3459, 2021 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-33568738

RESUMEN

During the COVID-19 pandemic it is essential to test as many people as possible, in order to detect early outbreaks of the infection. Present testing solutions are based on the extraction of RNA from patients using oropharyngeal and nasopharyngeal swabs, and then testing with real-time PCR for the presence of specific RNA filaments identifying the virus. This approach is limited by the availability of reactants, trained technicians and laboratories. One of the ways to speed up the testing procedures is a group testing, where the swabs of multiple patients are grouped together and tested. In this paper we propose to use the group testing technique in conjunction with an advanced replication scheme in which each patient is allocated in two or more groups to reduce the total numbers of tests and to allow testing of even larger numbers of people. Under mild assumptions, a 13 ×  average reduction of tests can be achieved compared to individual testing without delay in time.


Asunto(s)
/métodos , ARN Viral/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , /aislamiento & purificación , Humanos , Pandemias
15.
ACS Chem Neurosci ; 12(4): 573-580, 2021 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-33538586

RESUMEN

Long-COVID is a postviral illness that can affect survivors of COVID-19, regardless of initial disease severity or age. Symptoms of long-COVID include fatigue, dyspnea, gastrointestinal and cardiac problems, cognitive impairments, myalgia, and others. While the possible causes of long-COVID include long-term tissue damage, viral persistence, and chronic inflammation, the review proposes, perhaps for the first time, that persistent brainstem dysfunction may also be involved. This hypothesis can be split into two parts. The first is the brainstem tropism and damage in COVID-19. As the brainstem has a relatively high expression of ACE2 receptor compared with other brain regions, SARS-CoV-2 may exhibit tropism therein. Evidence also exists that neuropilin-1, a co-receptor of SARS-CoV-2, may be expressed in the brainstem. Indeed, autopsy studies have found SARS-CoV-2 RNA and proteins in the brainstem. The brainstem is also highly prone to damage from pathological immune or vascular activation, which has also been observed in autopsy of COVID-19 cases. The second part concerns functions of the brainstem that overlap with symptoms of long-COVID. The brainstem contains numerous distinct nuclei and subparts that regulate the respiratory, cardiovascular, gastrointestinal, and neurological processes, which can be linked to long-COVID. As neurons do not readily regenerate, brainstem dysfunction may be long-lasting and, thus, is long-COVID. Indeed, brainstem dysfunction has been implicated in other similar disorders, such as chronic pain and migraine and myalgic encephalomyelitis or chronic fatigue syndrome.


Asunto(s)
Encefalopatías/fisiopatología , Tronco Encefálico/fisiopatología , Inflamación/fisiopatología , Trombosis/fisiopatología , /metabolismo , Encefalopatías/metabolismo , Encefalopatías/virología , Tronco Encefálico/irrigación sanguínea , Tronco Encefálico/metabolismo , Tronco Encefálico/virología , /fisiopatología , Humanos , Inflamación/metabolismo , Inflamación/virología , Neuropilina-1/metabolismo , ARN Viral/aislamiento & purificación , ARN Viral/metabolismo , /genética , Trombosis/metabolismo , Trombosis/virología , Tropismo Viral
16.
Genet Test Mol Biomarkers ; 25(2): 85-101, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33596144

RESUMEN

Coronavirus disease 2019 (COVID-19) displays a broad spectrum of clinical presentations ranging from lack of symptoms to severe multiorgan system complications and death. Various laboratory assays have been employed in the diagnosis of COVID-19, including: nucleic acid-based tests; antigen tests; and serum testing for anti-severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies. The disease can also be diagnosed based on suggestive clinical features and radiological findings. Until now, remdesivir is the only medication approved for the treatment of COVID-19 by the U.S. Food and Drug Administration (FDA); however, it is anticipated that several anti-SARS-CoV-2 monoclonal antibodies will gain soon approval. Other methods of treatment include supportive care directed toward treating the symptoms. Nevertheless, many studies have recently emerged, showing controversial preliminary results with the off-label medication hydroxychloroquine. Given that all results are still preliminary, including those seen by remdesivir, additional evidence and research are required to identify effective medications that are broadly effective and well tolerated. Importantly, two RNA-based vaccines have recently gained approval from Pfizer and Moderna, with many others still in clinical trials. This article reviews various aspects of COVID-19, including its epidemiology; its evolution and mutational spectrum; and its clinical dynamics, symptoms and complications, diagnosis, and treatment.


Asunto(s)
Carga Global de Enfermedades/estadística & datos numéricos , Pandemias/estadística & datos numéricos , /patogenicidad , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/uso terapéutico , Alanina/análogos & derivados , Alanina/uso terapéutico , Antivirales/uso terapéutico , /tratamiento farmacológico , /terapia , /métodos , Ensayos Clínicos como Asunto , Evolución Molecular , Humanos , Hidroxicloroquina/uso terapéutico , Mutación , Uso Fuera de lo Indicado , Pandemias/prevención & control , ARN Viral/genética , ARN Viral/aislamiento & purificación , /inmunología , Índice de Severidad de la Enfermedad
17.
BMJ Case Rep ; 14(2)2021 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-33542020

RESUMEN

We present a case of a patient who had a history of severe coronavirus disease (COVID-19) 4 months prior to this current presentation and, after a long asymptomatic period, subsequently tested positive for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) by a RNA PCR assay, after several interval negative SARS-CoV-2 RNA tests. We present this potential case of SARS-CoV-2 reinfection in order to incite discussion around differentiating persistent infection with intermittent viral shedding and reinfection, as well as to discuss evolving knowledge and approaches to the clinical management, follow-up molecular testing and treatment of COVID-19 reinfection.


Asunto(s)
/diagnóstico , /virología , Esparcimiento de Virus , Adulto , Anticuerpos Monoclonales Humanizados/uso terapéutico , /virología , Humanos , Unidades de Cuidados Intensivos , Masculino , ARN Viral/aislamiento & purificación , Radiografía/métodos , Resultado del Tratamiento
18.
PLoS One ; 16(2): e0246647, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33534838

RESUMEN

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. This study evaluates several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, isopropanol precipitation is shown to provide an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, direct addition of NP swab samples to RT-qPCRs is evaluated without an RNA extraction step. A simple, inexpensive swab collection solution suitable for direct addition is validated using contrived swab samples. Third, an open-source master mix for RT-qPCR is described that permits detection of viral RNA in NP swab samples with a limit of detection of approximately 50 RNA copies per reaction. Quantification cycle (Cq) values for purified RNA from 30 known positive clinical samples showed a strong correlation (r2 = 0.98) between this homemade master mix and commercial TaqPath master mix. Lastly, end-point fluorescence imaging is found to provide an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, open-source methods has the potential to reduce the time and expense of COVID-19 testing.


Asunto(s)
/diagnóstico , ARN Viral/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , /genética , /virología , Precipitación Química , Humanos , Límite de Detección , Nasofaringe/virología , Fosfoproteínas/genética , ARN Viral/aislamiento & purificación , ARN Viral/metabolismo , /aislamiento & purificación
19.
Sci Rep ; 11(1): 3122, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33542424

RESUMEN

Sample pooling strategy was intended to determine the optimal parameters for group testing of pooled specimens for the detection of SARS-CoV-2 and process them without significant loss of test usability. Standard molecular diagnostic laboratory equipment, and commercially available centrifugal filters, RNA isolation kits and SARS Cov2 PCR tests were used. The basic idea was to combine and concentrate several samples to the maximal volume, which can be extracted with the single extraction column. Out of 16 tested pools, 12 were positive with cycle threshold (Ct) values within 0.5 and 3.01 Ct of the original individual specimens. The analysis of 112 specimens determined that 12 pools were positive, followed by identification of 6 positive individual specimens among the 112 tested. This testing was accomplished with the use of 16 extractions/PCR tests, resulting in saving of 96 reactions but adding the 40 centrifugal filters. The present study demonstrated that pool testing could detect even up to a single positive sample with Ct value as high as 34. According to the standard protocols, reagents and equipment, this pooling method can be applied easily in current clinical testing laboratories.


Asunto(s)
/métodos , ARN Viral/aislamiento & purificación , Manejo de Especímenes/métodos , Humanos , /aislamiento & purificación , Sensibilidad y Especificidad
20.
Sci Rep ; 11(1): 3134, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33542443

RESUMEN

We aimed to test the sensitivity of naso-oropharyngeal saliva and self-administered nasal (SN) swab compared to nasopharyngeal (NP) swab for COVID-19 testing in a large cohort of migrant workers in Singapore. We also tested the utility of next-generation sequencing (NGS) for diagnosis of COVID-19. Saliva, NP and SN swabs were collected from subjects who presented with acute respiratory infection, their asymptomatic roommates, and prior confirmed cases who were undergoing isolation at a community care facility in June 2020. All samples were tested using RT-PCR. SARS-CoV-2 amplicon-based NGS with phylogenetic analysis was done for 30 samples. We recruited 200 subjects, of which 91 and 46 were tested twice and thrice respectively. In total, 62.0%, 44.5%, and 37.7% of saliva, NP and SN samples were positive. Cycle threshold (Ct) values were lower during the earlier period of infection across all sample types. The percentage of test-positive saliva was higher than NP and SN swabs. We found a strong correlation between viral genome coverage by NGS and Ct values for SARS-CoV-2. Phylogenetic analyses revealed Clade O and lineage B.6 known to be circulating in Singapore. We found saliva to be a sensitive and viable sample for COVID-19 diagnosis.


Asunto(s)
/diagnóstico , Mucosa Nasal/virología , ARN Viral/aislamiento & purificación , Saliva/virología , Manejo de Especímenes , Adulto , Estudios de Cohortes , Femenino , Humanos , Masculino , Nasofaringe/virología , /aislamiento & purificación , Sensibilidad y Especificidad , Singapur/epidemiología
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