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1.
J Nanobiotechnology ; 19(1): 273, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34496881

RESUMO

The control of contagious or refractory diseases requires early, rapid diagnostic assays that are simple, fast, and easy-to-use. Here, easy-to-implement CRISPR/Cas12a-based diagnostic platform through Raman transducer generated by Raman enhancement effect, term as SERS-CRISPR (S-CRISPR), are described. The S-CRISPR uses high-activity noble metallic nanoscopic materials to increase the sensitivity in the detection of nucleic acids, without amplification. This amplification-free platform, which can be performed within 30-40 min of incubation time, is then used for detection of SARS-CoV-2 derived nucleic acids in RNA extracts obtained from nasopharyngeal swab specimens (n = 112). Compared with the quantitative reverse transcription polymerase chain reaction (RT-qPCR), the sensitivity and specificity of S-CRISPR reaches 87.50% and 100%, respectively. In general, the S-CRISPR can rapidly identify the RNA of SARS-CoV-2 RNA without amplification and is a potential strategy for nucleic acid point of care test (POCT).


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Amplificação de Ácido Nucleico , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Análise Espectral Raman , COVID-19/diagnóstico , COVID-19/virologia , Regulação Fúngica da Expressão Gênica , Genes Virais , Humanos , RNA Viral/análise , Sensibilidade e Especificidade
2.
Sci Rep ; 11(1): 15997, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362977

RESUMO

Simple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA (RNA Isothermal Co-assisted and Coupled Amplification) reaction, that consists of a simple one-pot format of 'sample-in and result-out' with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/µL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/µL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/µL with a total assay time of 15-30 min.


Assuntos
COVID-19/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Vírus de RNA/genética , RNA Viral/genética , SARS-CoV-2/genética , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/instrumentação , Teste de Ácido Nucleico para COVID-19/métodos , Desenho de Equipamento , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Vírus de RNA/isolamento & purificação , RNA Viral/análise , SARS-CoV-2/isolamento & purificação , Saliva/virologia
3.
PLoS One ; 16(8): e0255999, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34379690

RESUMO

BACKGROUND: The primary goal of the presented cross-sectional observational study was to determine the clinical and demographic risk factors for adverse coronavirus disease 2019 (COVID-19) outcomes in the Pakistani population. METHODS: We examined the individuals (n = 6331) that consulted two private diagnostic centers in Lahore, Pakistan, for COVID-19 testing between May 1, 2020, and November 30, 2020. The attending nurse collected clinical and demographic information. A confirmed case of COVID-19 was defined as having a positive result through real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay of nasopharyngeal swab specimens. RESULTS: RT-PCR testing was positive in 1094 cases. Out of which, 5.2% had severe, and 20.8% had mild symptoms. We observed a strong association of COVID-19 severity with the number and type of comorbidities. The severity of the disease intensified as the number of comorbidities increased. The most vulnerable groups for the poor outcome are patients with diabetes and hypertension. Increasing age was also associated with PCR positivity and the severity of the disease. CONCLUSIONS: Most cases of COVID-19 included in this study developed mild symptoms or were asymptomatic. Risk factors for adverse outcomes included older age and the simultaneous presence of comorbidities.


Assuntos
COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/patologia , COVID-19/virologia , Comorbidade , Demografia , Complicações do Diabetes/patologia , Humanos , Hipertensão/complicações , Paquistão/epidemiologia , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença
4.
Iran J Allergy Asthma Immunol ; 20(4): 394-401, 2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34418893

RESUMO

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.


Assuntos
Teste para COVID-19 , COVID-19/diagnóstico , Hospitalização , Adulto , Idoso , Anticorpos Antivirais/sangue , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade
5.
PLoS One ; 16(8): e0256378, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34403446

RESUMO

Saliva is a matrix which may act as a vector for pathogen transmission and may serve as a possible proxy for SARS-CoV-2 contagiousness. Therefore, the possibility of detection of intracellular SARS-CoV-2 in saliva by means of fluorescence in situ hybridization is tested, utilizing probes targeting the antisense or sense genomic RNA of SARS-CoV-2. This method was applied in a pilot study with saliva samples collected from healthy persons and those presenting with mild or moderate COVID-19 symptoms. In all participants, saliva appeared a suitable matrix for the detection of SARS-CoV-2. Among the healthy, mild COVID-19-symptomatic and moderate COVID-19-symptomatic persons, 0%, 90% and 100% tested positive for SARS-CoV-2, respectively. Moreover, the procedure allows for simultaneous measurement of viral load ('presence', sense genomic SARS-CoV-2 RNA) and viral replication ('activity', antisense genomic SARS-CoV-2 RNA) and may yield qualitative results. In addition, the visualization of DNA in the cells in saliva provides an additional cytological context to the validity and interpretability of the test results. The method described in this pilot study may be a valuable diagnostic tool for detection of SARS-CoV-2, distinguishing between 'presence' (viral load) and 'activity' (viral replication) of the virus. Moreover, the method potentially gives more information about possible contagiousness.


Assuntos
COVID-19/diagnóstico , Hibridização in Situ Fluorescente/métodos , RNA Viral/análise , SARS-CoV-2/genética , Saliva/virologia , COVID-19/patologia , COVID-19/virologia , Estudos de Casos e Controles , Genômica , Humanos , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia , Índice de Gravidade de Doença , Carga Viral , Replicação Viral
6.
PLoS One ; 16(8): e0256352, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34403456

RESUMO

Rapid tests for SARS-COV-2 infection are important tools for pandemic control, but current rapid tests are based on proprietary designs and reagents. We report clinical validation results of an open-access lateral flow assay (OA-LFA) design using commercially available materials and reagents, along with RT-qPCR and commercially available comparators (BinaxNOW® and Sofia®). Adult patients with suspected COVID-19 based on clinical signs and symptoms, and with symptoms ≤7 days duration, underwent anterior nares (AN) sampling for the OA-LFA, Sofia®, BinaxNOW ™, and RT-qPCR, along with nasopharyngeal (NP) RT-qPCR. Results indicate a positive predictive agreement with NP sampling as 69% (60% -78%) OA-LFA, 74% (64% - 82%) Sofia®, and 82% (73% - 88%) BinaxNOW™. The implication for these results is that we provide an open-access LFA design that meets the minimum WHO target product profile for a rapid test, that virtually any diagnostic manufacturer could produce.


Assuntos
Antígenos Virais/análise , COVID-19/diagnóstico , Imunoensaio , SARS-CoV-2/metabolismo , Área Sob a Curva , COVID-19/virologia , Humanos , Nasofaringe/virologia , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/análise , RNA Viral/metabolismo , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade
7.
Sci Rep ; 11(1): 16740, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408169

RESUMO

The coronavirus pandemic, which appeared in Wuhan, China, in December 2019, rapidly spread all over the world in only a few weeks. Faster testing techniques requiring less resources are key in managing the pandemic, either to enable larger scale testing or even just provide developing countries with limited resources, particularly in Africa, means to perform tests to manage the crisis. Here, we report an unprecedented, rapid, reagent-free and easy-to-use screening spectroscopic method for the detection of SARS-CoV-2 on RNA extracts. This method, validated on clinical samples collected from 280 patients with quantitative predictive scores on both positive and negative samples, is based on a multivariate analysis of FTIR spectra of RNA extracts. This technique, in agreement with RT-PCR, achieves 97.8% accuracy, 97% sensitivity and 98.3% specificity while reducing the testing time post RNA extraction from hours to minutes. Furthermore, this technique can be used in several laboratories with limited resources.


Assuntos
Teste para COVID-19/métodos , RNA Viral/análise , Espectroscopia de Infravermelho com Transformada de Fourier , Humanos , RNA Viral/química , RNA Viral/isolamento & purificação , Fatores de Tempo
8.
Biomed Res Int ; 2021: 5516344, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34368349

RESUMO

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic of pneumonia spreading around the world, leading to serious threats to public health and attracting enormous attention. There is an urgent need for sensitive diagnostic testing implementation to control and manage SARS-CoV-2 in public health laboratories. The quantitative reverse transcription PCR (RT-qPCR) assay is the gold standard method, but the sensitivity and specificity of SARS-CoV-2 testing are dependent on a number of factors. Methods: We synthesized RNA based on the genes published to estimate the concentration of inactivated virus samples in a biosafety level 3 laboratory. The limit of detection (LOD), linearity, accuracy, and precision were evaluated according to the bioanalytical method validation guidelines. Results: We found that the LOD reached around 3 copies/reaction. Furthermore, intra-assay precision, accuracy, and linearity met the accepted criterion with an RSD for copies of less than 25%, and linear regression met the accepted R 2 of 0.98. Conclusions: We suggest that synthesized RNA based on the database of the NCBI gene bank for estimating the concentration of inactivated virus samples provides a potential opportunity for reliable testing to diagnose coronavirus disease 2019 (COVID-19) as well as limit the spread of the disease. This method may be relatively quick and inexpensive, and it may be useful for developing countries during the pandemic era. In the long term, it is also applicable for evaluation, verification, validation, and external quality assessment.


Assuntos
COVID-19/virologia , Técnicas de Diagnóstico Molecular/normas , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/genética , Países em Desenvolvimento/estatística & dados numéricos , Humanos , Técnicas de Diagnóstico Molecular/métodos , Pandemias , RNA Viral/análise , SARS-CoV-2/isolamento & purificação , Estudos de Validação como Assunto
9.
Viruses ; 13(8)2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34452415

RESUMO

The emergence and ensuing dominance of COVID-19 on the world stage has emphasized the urgency of efficient animal models for the development of therapeutics for and assessment of immune responses to SARS-CoV-2 infection. Shortcomings of current animal models for SARS-CoV-2 include limited lower respiratory disease, divergence from clinical COVID-19 disease, and requirements for host genetic modifications to permit infection. In this study, n = 12 specific-pathogen-free domestic cats were infected intratracheally with SARS-CoV-2 to evaluate clinical disease, histopathologic lesions, and viral infection kinetics at 4 and 8 days post-inoculation; n = 6 sham-inoculated cats served as controls. Intratracheal inoculation of SARS-CoV-2 produced a significant degree of clinical disease (lethargy, fever, dyspnea, and dry cough) consistent with that observed in the early exudative phase of COVID-19. Pulmonary lesions such as diffuse alveolar damage, hyaline membrane formation, fibrin deposition, and proteinaceous exudates were also observed with SARS-CoV-2 infection, replicating lesions identified in people hospitalized with ARDS from COVID-19. A significant correlation was observed between the degree of clinical disease identified in infected cats and pulmonary lesions. Viral loads and ACE2 expression were also quantified in nasal turbinates, distal trachea, lungs, and other organs. Results of this study validate a feline model for SARS-CoV-2 infection that results in clinical disease and histopathologic lesions consistent with acute COVID-19 in humans, thus encouraging its use for future translational studies.


Assuntos
COVID-19 , Gatos , Modelos Animais de Doenças , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/patologia , COVID-19/fisiopatologia , COVID-19/virologia , Feminino , Genoma Viral , Humanos , Pulmão/enzimologia , Pulmão/patologia , Pulmão/virologia , Linfonodos/virologia , Masculino , RNA Viral/análise , SARS-CoV-2/genética , Organismos Livres de Patógenos Específicos , Traqueia/enzimologia , Traqueia/virologia , Conchas Nasais/enzimologia , Conchas Nasais/virologia
10.
Viruses ; 13(8)2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34452462

RESUMO

We aimed to assess the duration of nasopharyngeal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA persistence in adults self-confined at home after acute infection; and to identify the associations of SARS-CoV-2 persistence with respiratory virus co-detection and infection transmission. A cross-sectional intra-household study was conducted in metropolitan Barcelona (Spain) during the time period of April to June 2020. Every adult who was the first family member reported as SARS-CoV-2-positive by reverse transcription polymerase chain reaction (RT-PCR) as well as their household child contacts had nasopharyngeal swabs tested by a targeted SARS-CoV-2 RT-PCR and a multiplex viral respiratory panel after a 15 day minimum time lag. Four-hundred and four households (404 adults and 708 children) were enrolled. SARS-CoV-2 RNA was detected in 137 (33.9%) adults and 84 (11.9%) children. Rhinovirus/Enterovirus (RV/EV) was commonly found (83.3%) in co-infection with SARS-CoV-2 in adults. The mean duration of SARS-CoV-2 RNA presence in adults' nasopharynx was 52 days (range 26-83 days). The persistence of SARS-CoV-2 was significantly associated with RV/EV co-infection (adjusted odds ratio (aOR) 9.31; 95% CI 2.57-33.80) and SARS-CoV-2 detection in child contacts (aOR 2.08; 95% CI 1.24-3.51). Prolonged nasopharyngeal SARS-CoV-2 RNA persistence beyond the acute infection phase was frequent in adults quarantined at home during the first epidemic wave; which was associated with RV/EV co-infection and could enhance intra-household infection transmission.


Assuntos
COVID-19/complicações , COVID-19/virologia , Coinfecção , Infecções por Enterovirus/complicações , Infecções por Picornaviridae/complicações , SARS-CoV-2/isolamento & purificação , Adolescente , Adulto , Anticorpos Antivirais/sangue , COVID-19/epidemiologia , COVID-19/transmissão , Teste de Ácido Nucleico para COVID-19 , Criança , Pré-Escolar , Estudos Transversais , Enterovirus/genética , Enterovirus/isolamento & purificação , Saúde da Família , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Quarentena , RNA Viral/análise , Rhinovirus/genética , Rhinovirus/isolamento & purificação , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Fatores de Tempo , Adulto Jovem
11.
Viruses ; 13(8)2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34452537

RESUMO

The post-acute phase of SARS-CoV-2 infection was investigated in rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis). During the acute phase of infection, SARS-CoV-2 was shed via the nose and throat, and viral RNA was occasionally detected in feces. This phase coincided with a transient change in systemic immune activation. Even after the alleged resolution of the infection, computed tomography (CT) and positron emission tomography (PET)-CT revealed pulmonary lesions and activated tracheobronchial lymph nodes in all animals. Post-mortem histological examination of the lung tissue revealed mostly marginal or resolving minimal lesions that were indicative of SARS-CoV-2 infection. Evidence for SARS-CoV-2-induced histopathology was also found in extrapulmonary tissue samples, such as conjunctiva, cervical, and mesenteric lymph nodes. However, 5-6 weeks after SARS-CoV-2 exposure, upon necropsy, viral RNA was still detectable in a wide range of tissue samples in 50% of the macaques and included amongst others the heart, the respiratory tract and surrounding lymph nodes, salivary gland, and conjunctiva. Subgenomic messenger RNA was detected in the lungs and tracheobronchial lymph nodes, indicative of ongoing virus replication during the post-acute phase. These results could be relevant for understanding the long-term consequences of COVID-19 in humans.


Assuntos
COVID-19/patologia , COVID-19/virologia , Pulmão/patologia , SARS-CoV-2/fisiologia , Animais , Anticorpos Antivirais/sangue , COVID-19/imunologia , Citocinas/sangue , Modelos Animais de Doenças , Humanos , Pulmão/virologia , Linfonodos/patologia , Linfonodos/fisiopatologia , Macaca fascicularis , Macaca mulatta , RNA Mensageiro/análise , RNA Viral/análise , Sistema Respiratório/patologia , Sistema Respiratório/virologia , SARS-CoV-2/imunologia , Replicação Viral
12.
ACS Appl Mater Interfaces ; 13(35): 41445-41453, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34428374

RESUMO

Airborne transmission of exhaled virus can rapidly spread, thereby increasing disease progression from local incidents to pandemics. Due to the COVID-19 pandemic, states and local governments have enforced the use of protective masks in public and work areas to minimize the disease spread. Here, we have leveraged the function of protective face coverings toward COVID-19 diagnosis. We developed a user-friendly, affordable, and wearable collector. This noninvasive platform is integrated into protective masks toward collecting airborne virus in the exhaled breath over the wearing period. A viral sample was sprayed into the collector to model airborne dispersion, and then the enriched pathogen was extracted from the collector for further analytical evaluation. To validate this design, qualitative colorimetric loop-mediated isothermal amplification, quantitative reverse transcription polymerase chain reaction, and antibody-based dot blot assays were performed to detect the presence of SARS-CoV-2. We envision that this platform will facilitate sampling of current SARS-CoV-2 and is potentially broadly applicable to other airborne diseases for future emerging pandemics.


Assuntos
Testes Respiratórios/instrumentação , Teste para COVID-19/instrumentação , Máscaras , SARS-CoV-2/isolamento & purificação , Microbiologia do Ar , Anticorpos Antivirais/imunologia , Testes Respiratórios/métodos , Teste para COVID-19/métodos , Colódio/química , Colorimetria/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Cimento de Policarboxilato/química , Porosidade , Estudo de Prova de Conceito , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/química , Proteínas Virais/análise , Proteínas Virais/imunologia
13.
Cell ; 184(18): 4713-4733.e22, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34352228

RESUMO

SARS-CoV-2 infection can cause severe respiratory COVID-19. However, many individuals present with isolated upper respiratory symptoms, suggesting potential to constrain viral pathology to the nasopharynx. Which cells SARS-CoV-2 primarily targets and how infection influences the respiratory epithelium remains incompletely understood. We performed scRNA-seq on nasopharyngeal swabs from 58 healthy and COVID-19 participants. During COVID-19, we observe expansion of secretory, loss of ciliated, and epithelial cell repopulation via deuterosomal cell expansion. In mild and moderate COVID-19, epithelial cells express anti-viral/interferon-responsive genes, while cells in severe COVID-19 have muted anti-viral responses despite equivalent viral loads. SARS-CoV-2 RNA+ host-target cells are highly heterogenous, including developing ciliated, interferon-responsive ciliated, AZGP1high goblet, and KRT13+ "hillock"-like cells, and we identify genes associated with susceptibility, resistance, or infection response. Our study defines protective and detrimental responses to SARS-CoV-2, the direct viral targets of infection, and suggests that failed nasal epithelial anti-viral immunity may underlie and precede severe COVID-19.


Assuntos
COVID-19/imunologia , COVID-19/virologia , Imunidade , SARS-CoV-2/fisiologia , Índice de Gravidade de Doença , Adulto , Idoso , Efeito Espectador , COVID-19/genética , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/patologia , Nasofaringe/virologia , RNA Viral/análise , RNA Viral/genética , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Transcrição Genética , Carga Viral
15.
Cells ; 10(8)2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34440669

RESUMO

Multiorgan tropism of SARS-CoV-2 has previously been shown for several major organs. We have comprehensively analyzed 25 different formalin-fixed paraffin-embedded (FFPE) tissues/organs from autopsies of fatal COVID-19 cases (n = 8), using histopathological assessment, detection of SARS-CoV-2 RNA using polymerase chain reaction and RNA in situ hybridization, viral protein using immunohistochemistry, and virus particles using transmission electron microscopy. SARS-CoV-2 RNA was mainly localized in epithelial cells across all organs. Next to lung, trachea, kidney, heart, or liver, viral RNA was also found in tonsils, salivary glands, oropharynx, thyroid, adrenal gland, testicles, prostate, ovaries, small bowel, lymph nodes, skin and skeletal muscle. Viral RNA was predominantly found in cells expressing ACE2, TMPRSS2, or both. The SARS-CoV-2 replicating RNA was also detected in these organs. Immunohistochemistry and electron microscopy were not suitable for reliable and specific SARS-CoV-2 detection in autopsies. These findings were validated using in situ hybridization on external COVID-19 autopsy samples (n = 9). Apart from the lung, correlation of viral detection and histopathological assessment did not reveal any specific alterations that could be attributed to SARS-CoV-2. In summary, SARS-CoV-2 and its replication could be observed across all organ systems, which co-localizes with ACE2 and TMPRSS2 mainly in epithelial but also in mesenchymal and endothelial cells. Apart from the respiratory tract, no specific (histo-)morphologic alterations could be assigned to the SARS-CoV-2 infection.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/metabolismo , Células Endoteliais/metabolismo , RNA Viral/análise , SARS-CoV-2/fisiologia , Serina Endopeptidases/genética , Idoso , Autopsia , COVID-19/genética , COVID-19/patologia , COVID-19/virologia , Células Endoteliais/patologia , Células Endoteliais/virologia , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos , Tropismo
16.
Microbiol Spectr ; 9(1): e0006221, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34431689

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has challenged clinical diagnostic operations due to supply shortages and high staffing needs to collect nasopharyngeal (NP) swab samples. Saliva is an easily accessible alternative specimen type to overcome some of these challenges. In this study, we first used paired saliva and NP swab specimens (n = 128) to compare test performance characteristics with three RNA extraction platforms, i.e., Maxwell RSC (Promega), MagNA Pure 96 (Roche), and KingFisher Flex (Thermo Fisher Scientific), together with two PCR chemistries, i.e., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (2019-nCoV) Centers for Disease Control and Prevention (CDC) quantitative PCR (qPCR) probe assay (Integrated DNA Technologies) and TagPath COVID-19 combination kit (Thermo Fisher Scientific). This study demonstrated that both saliva and NP swab specimens performed well, with 97% agreement when tested by the CDC qPCR chemistry using Maxwell and MagNA Pure RNA extraction platforms. We then compared 12 weeks of saliva and NP swab testing results using two independent asymptomatic populations, including a community surveillance program using saliva samples only (n = 466) and preoperative screening using NP swab samples only (n = 8,461). The positive detection rates among participants with either saliva or NP swab samples were 1.07% and 1.12%, respectively, which confirms the low pretest probabilities for COVID-19 infections in asymptomatic populations. Notably, there was no increased proportion of low-titer cases (inconclusive results) reported in the asymptomatic groups, compared with the all-comers groups (0.21% and 0.66%, respectively, in the community population and 0.25% and 0.49%, respectively, in the preoperative population); this suggests that low-viral-titer carriers can be found similarly in both groups with saliva or NP swab specimens. In summary, saliva can be considered a good alternative for noninvasive but well-instructed self-collection. IMPORTANCE Our study shows that saliva is a noninvasive respiratory secretion sample type that contains equal or more host materials (RNase P), compared with those contained in the corresponding NP swab specimens, in 103 paired samples. SARS-CoV-2 detection with two RNA extraction platforms, Maxwell and MagNA Pure, with CDC qPCR chemistry showed similar test sensitivities for paired specimens. We then analyzed SARS-CoV-2 detections rates in two independent groups of asymptomatic participants, i.e., a group at a community screening station with supervised saliva collection only (n = 466) and a preoperative screening group (n = 8,461) with NP swabbing only. Similar detection rates of 1.07% for the community group and 1.12% for the preoperative group supported the similar test performances in these groups predicted to have low pretest probabilities of infection. With mindful preparation, saliva can be considered for schools and clinical participants when adequate collection education can be provided and compliance can be established.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Nasofaringe/virologia , Reação em Cadeia da Polimerase/métodos , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Pandemias , RNA Viral/análise , Manejo de Espécimes/métodos , Adulto Jovem
18.
J Med Microbiol ; 70(8)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34369860

RESUMO

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.


Assuntos
Teste de Ácido Nucleico para COVID-19 , COVID-19/diagnóstico , Nasofaringe/virologia , Orofaringe/virologia , Saliva/virologia , Adulto , Idoso , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes
19.
Sci Rep ; 11(1): 15715, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344959

RESUMO

Key elements for viral pathogenesis include viral strains, viral load, co-infection, and host responses. Several studies analyzing these factors in the function of disease severity of have been published; however, no studies have shown how all of these factors interplay within a defined cohort. To address this important question, we sought to understand how these four key components interplay in a cohort of COVID-19 patients. We determined the viral loads and gene expression using high throughput sequencing and various virological methods. We found that viral loads in the upper respiratory tract in COVID-19 patients at an early phase of infection vary widely. While the majority of nasopharyngeal (NP) samples have a viral load lower than the limit of detection of infectious viruses, there are samples with an extraordinary amount of SARS-CoV-2 RNA and a high viral titer. No specific viral factors were identified that are associated with high viral loads. Host gene expression analysis showed that viral loads were strongly correlated with cellular antiviral responses. Interestingly, however, COVID-19 patients who experience mild symptoms have a higher viral load than those with severe complications, indicating that naso-pharyngeal viral load may not be a key factor of the clinical outcomes of COVID-19. The metagenomics analysis revealed that the microflora in the upper respiratory tract of COVID-19 patients with high viral loads were dominated by SARS-CoV-2, with a high degree of dysbiosis. Finally, we found a strong inverse correlation between upregulation of interferon responses and disease severity. Overall our study suggests that a high viral load in the upper respiratory tract may not be a critical factor for severe symptoms; rather, dampened antiviral responses may be a critical factor for a severe outcome from the infection.


Assuntos
COVID-19/patologia , Interferons/metabolismo , SARS-CoV-2/genética , Adulto , Idoso , COVID-19/virologia , Disbiose/etiologia , Feminino , Humanos , Masculino , Metagenômica , Microbiota/genética , Pessoa de Meia-Idade , Nasofaringe/virologia , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real , Sistema Respiratório/microbiologia , Sistema Respiratório/virologia , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença , Transcriptoma , Regulação para Cima , Carga Viral
20.
PLoS One ; 16(8): e0256142, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34437579

RESUMO

Long-COVID-19 is a proposed syndrome negatively affecting the health of COVID-19 patients. We present data on self-rated health three to eight months after laboratory confirmed COVID-19 disease compared to a control group of SARS-CoV-2 negative patients. We followed a cohort of 8786 non-hospitalized patients who were invited after SARS-CoV-2 testing between February 1 and April 15, 2020 (794 positive, 7229 negative). Participants answered online surveys at baseline and follow-up including questions on demographics, symptoms, risk factors for SARS-CoV-2, and self-rated health compared to one year ago. Determinants for a worsening of self-rated health as compared to one year ago among the SARS-CoV-2 positive group were analyzed using multivariate logistic regression and also compared to the population norm. The follow-up questionnaire was completed by 85% of the SARS-CoV-2 positive and 75% of the SARS-CoV-2 negative participants on average 132 days after the SARS-CoV-2 test. At follow-up, 36% of the SARS-CoV-2 positive participants rated their health "somewhat" or "much" worse than one year ago. In contrast, 18% of the SARS-CoV-2 negative participants reported a similar deterioration of health while the population norm is 12%. Sore throat and cough were more frequently reported by the control group at follow-up. Neither gender nor follow-up time was associated with the multivariate odds of worsening of self-reported health compared to one year ago. Age had an inverted-U formed association with a worsening of health while being fit and being a health professional were associated with lower multivariate odds. A significant proportion of non-hospitalized COVID-19 patients, regardless of age, have not returned to their usual health three to eight months after infection.


Assuntos
COVID-19/complicações , COVID-19/patologia , Adolescente , Adulto , Idoso , COVID-19/etiologia , COVID-19/virologia , Fadiga/etiologia , Feminino , Febre/etiologia , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , RNA Viral/análise , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Autorrelato , Inquéritos e Questionários , Fatores de Tempo , Adulto Jovem
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