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1.
Int J Lab Hematol ; 43(6): 1334-1340, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34596329

RESUMO

INTRODUCTION: Coronavirus disease 2019 (COVID-19) caused by SARS-CoV2 can present from mild flu-like symptoms to acute respiratory distress syndrome. There is multi-organ involvement; particularly, hematopoietic system can be associated with morphological changes in blood cells of COVID-19 patients. METHOD: We conducted a cross-sectional study on a cohort of 50 COVID-19 patients, confirmed on RT-PCR with documented cycle threshold (Ct) value. Peripheral blood sample of these patients was collected and examined for complete blood counts (CBC) on automated haematological analyser as well as Leishman-stained blood smears to look for morphological changes in blood cells. Morphological changes were evaluated with reference to clinical severity and Ct value. Additionally, association between Ct value and clinical severity was also performed. Statistical tests were performed, and P value <.05 was considered significant. RESULTS: Mean age of our study group was 42.16 ± 15.55 years, with male preponderance. Most commonly observed peripheral blood changes were hypolobation (P value = .002) and toxic granules (P value = .005) in neutrophils, atypical granules with nucleolar prominence in lymphocytes, cytoplasmic granulation with clumped nuclear chromatin in monocytes, giant platelets and thrombocytopenia and normocytic normochromic anaemia. CONCLUSION: No association was found between clinical severity and Ct value as well as peripheral blood morphological changes with Ct value. We conclude that examination of peripheral smear coupled with complete blood count (CBC) is only partially supportive of disease pathogenesis and to assess the viral load other parameters should be utilised instead of relying solely on Ct value.


Assuntos
Células Sanguíneas/ultraestrutura , Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/sangue , SARS-CoV-2/isolamento & purificação , Carga Viral , Viremia/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Contagem de Células Sanguíneas , COVID-19/virologia , Forma Celular , Tamanho Celular , Estudos Transversais , Grânulos Citoplasmáticos/ultraestrutura , Feminino , Hematopoese , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Orofaringe/virologia , Estudos Prospectivos , RNA Viral/sangue , Índice de Gravidade de Doença , Adulto Jovem
2.
Emerg Microbes Infect ; 10(1): 2090-2097, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34689717

RESUMO

Since December 2019, coronavirus disease 2019 (COVID-19) caused by SARS coronavirus 2 (SARS-CoV-2) has spread and threatens public health worldwide. The recurrence of SARS-CoV-2 RNA detection in patients after discharge from hospital signals a risk of transmission from such patients to the community and challenges the current discharge criteria of COVID-19 patients. A wide range of clinical specimens has been used to detect SARS-CoV-2. However, to date, a consensus has not been reached regarding the most appropriate specimens to use for viral RNA detection in assessing COVID-19 patients for discharge. An anal swab sample was proposed as the standard because of prolonged viral detection. In this retrospective longitudinal study of viral RNA detection in 60 confirmed COVID-19 patients, we used saliva, oropharyngeal/nasopharyngeal swab (O/N swab) and anal swab procedures from admission to discharge. The conversion times of saliva and anal swab were longer than that of O/N swab. The conversion time of hyper sensitive-CRP was the shortest and correlated with that of CT scanning and viral detection. Some patients were found to be RNA-positive in saliva while RNA-negative in anal swab while the reverse was true in some other patients, which indicated that false negatives were inevitable if only the anal swab is used for evaluating suitability for discharge. These results indicated that double-checking for viral RNA using multiple and diverse specimens was essential, and saliva could be a candidate to supplement anal swabs to reduce false-negative results and facilitate pandemic control.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Adulto , Canal Anal/virologia , Reações Falso-Negativas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Orofaringe/virologia , Alta do Paciente , RNA Viral/análise , Estudos Retrospectivos , Adulto Jovem
3.
Mikrochim Acta ; 188(10): 335, 2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-34505191

RESUMO

A practical colorimetric assay was developed for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For this purpose, magnetic γ Fe2O3 nanoparticles were synthesized and used as a peroxidase-like mimic activity molecule. In the presence of γ Fe2O3 nanoparticles, the color change of H2O2 included 3,3',5,5'-tetramethylbenzidine was monitored at the wavelength of 654 nm when spike protein interacted with angiotensin-converting enzyme 2 receptor. This oxidation-reduction reaction was examined both spectroscopically and by using electrochemical techniques. The experimental parameters were optimized and the analytical characteristics investigated. The developed assay was applied to real SARS-CoV-2 samples, and very good results that were in accordance with the real time polymerase chain reaction were obtained.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Colorimetria/métodos , Nanopartículas Magnéticas de Óxido de Ferro/química , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Enzima de Conversão de Angiotensina 2/metabolismo , Benzidinas/química , Técnicas Biossensoriais/métodos , Teste para COVID-19/instrumentação , Catálise , Compostos Cromogênicos/química , Cisteína/química , Humanos , Peróxido de Hidrogênio/química , Limite de Detecção , Nasofaringe/virologia , Orofaringe/virologia , Oxirredução , Peroxidase/química , Glicoproteína da Espícula de Coronavírus/metabolismo
4.
PLoS One ; 16(9): e0257350, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34555073

RESUMO

SARS-CoV-2 has spread worldwide and has become a global health problem. As a result, the demand for inputs for diagnostic tests rose dramatically, as did the cost. Countries with inadequate infrastructure experience difficulties in expanding their qPCR testing capacity. Therefore, the development of sensitive and specific alternative methods is essential. This study aimed to develop, standardize, optimize, and validate conventional RT-PCR targeting the N gene of SARS-CoV-2 in naso-oropharyngeal swab samples compared to qPCR. Using bioinformatics tools, specific primers were determined, with a product expected to be 519 bp. The reaction conditions were optimized using a commercial positive control, and the detection limit was determined to be 100 fragments. To validate conventional RT-PCR, we determined a representative sampling of 346 samples from patients with suspected infection whose diagnosis was made in parallel with qPCR. A sensitivity of 92.1% and specificity of 100% were verified, with an accuracy of 95.66% and correlation coefficient of 0.913. Under current Brazilian conditions, this method generates approximately 60% savings compared to qPCR costs. Conventional RT-PCR, validated herein, showed sufficient results for the detection of SARS-CoV-2 and can be used as an alternative for epidemiological studies and interspecies correlations.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Nariz/virologia , Proteínas do Nucleocapsídeo/genética , Orofaringe/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Adolescente , Brasil , COVID-19/virologia , Primers do DNA/genética , Feminino , Humanos , Masculino , Técnicas de Diagnóstico Molecular/métodos , RNA Viral/genética , Padrões de Referência , Sensibilidade e Especificidade , Manejo de Espécimes/métodos
5.
Diagn Microbiol Infect Dis ; 101(4): 115519, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34571354

RESUMO

To improve laboratory safety we thermally treated naso-oropharyngeal samples before testing with the cobas SARS-CoV-2 assay. This study aimed to determine if thermal treatment significantly affects the qualitative detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the quantitative measurement of cobas SARS-CoV-2 ORF1a and E-gene target copy number using an in-house quantitative method. A collection of positive (n = 238) and negative samples (n = 196) was tested in parallel comparing thermal treatment (75 °C for 15 minutes) to room-temperature. There were no significant differences in the final qualitative outcomes for thermal treatment versus room-temperature (99.8% agreement) despite a statistically significant reduction (P < 0.05) in target copy number following thermal treatment. The median ORF1a and E-gene reduction in target copy number was -0.07 (1.6%) and -0.22 (4.2%) log10 copies/mL respectively. The standard curves for both ORF1a and E-gene targets were highly linear (r2 = 0.99). Good correlation was observed for ORF1a (r2 = 0.96) and E-gene (r2 = 0.98) comparing thermal treatment to room-temperature control.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Nasofaringe/virologia , Orofaringe/virologia , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodos , Temperatura Alta , Humanos , RNA Viral/isolamento & purificação , Inativação de Vírus
6.
PLoS One ; 16(8): e0256316, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34407126

RESUMO

Efficient and effective viral detection methodologies are a critical piece in the global response to COVID-19, with PCR-based nasopharyngeal and oropharyngeal swab testing serving as the current gold standard. With over 100 million confirmed cases globally, the supply chains supporting these PCR testing efforts are under a tremendous amount of stress, driving the need for innovative and accurate diagnostic solutions. Herein, the utility of a direct-to-PCR method of SARS-CoV-2 detection grounded in mechanical homogenization is examined for reducing resources needed for testing while maintaining a comparable sensitivity to the current gold standard workflow of nasopharyngeal and oropharyngeal swab testing. In a head-to-head comparison of 30 patient samples, this initial clinical validation study of the proposed homogenization-based workflow demonstrated significant agreeability with the current extraction-based method utilized while cutting the total resources needed in half.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/instrumentação , Teste de Ácido Nucleico para COVID-19/instrumentação , Estudos de Viabilidade , Humanos , Nasofaringe/virologia , Orofaringe/virologia , Estudos Prospectivos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , Sensibilidade e Especificidade , Fluxo de Trabalho
7.
Viruses ; 13(8)2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34452319

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people globally since its first detection in late 2019. Besides humans, cats and, to some extent, dogs were shown to be susceptible to SARS-CoV-2, highlighting the need for surveillance in a One Health context. Seven veterinary clinics from regions with high incidences of coronavirus disease (COVID-19) were recruited during the early pandemic (March to July 2020) for the screening of patients. A total of 2257 oropharyngeal and nasal swab specimen from 877 dogs and 260 cats (including 18 animals from COVID-19-affected households and 92 animals with signs of respiratory disease) were analyzed for the presence of SARS-CoV-2 RNA using reverse transcriptase real-time polymerase chain reaction (RT-qPCR) targeting the viral envelope (E) and RNA dependent RNA polymerase (RdRp) genes. One oropharyngeal swab from an Italian cat, living in a COVID-19-affected household in Piedmont, tested positive in RT-qPCR (1/260; 0.38%, 95% CI: 0.01-2.1%), and SARS-CoV-2 infection of the animal was serologically confirmed six months later. One oropharyngeal swab from a dog was potentially positive (1/877; 0.1%, 95% CI: 0.002-0.63%), but the result was not confirmed in a reference laboratory. Analyses of convenience sera from 118 animals identified one dog (1/94; 1.1%; 95% CI: 0.02-5.7%) from Lombardy, but no cats (0/24), as positive for anti-SARS-CoV-2 receptor binding domain (RBD) antibodies and neutralizing activity. These findings support the hypothesis that the prevalence of SARS-CoV-2 infection in pet cat and dog populations, and hence, the risk of zoonotic transmission to veterinary staff, was low during the first wave of the pandemic, even in hotspot areas.


Assuntos
COVID-19/veterinária , Doenças do Gato/virologia , Doenças do Cão/virologia , SARS-CoV-2/isolamento & purificação , Animais , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/virologia , Doenças do Gato/diagnóstico , Doenças do Gato/epidemiologia , Gatos , Doenças do Cão/diagnóstico , Doenças do Cão/epidemiologia , Cães , Feminino , Alemanha/epidemiologia , Itália/epidemiologia , Masculino , Orofaringe/virologia , Pandemias , RNA Viral/genética , SARS-CoV-2/genética
8.
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
9.
Viruses ; 13(7)2021 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-34372545

RESUMO

Human papillomavirus (HPV) imposes an increased risk of developing cervical, anal and oropharyngeal cancer. In the Western world, HPV infection is currently the major cause of oropharyngeal cancer. The effectiveness of HPV vaccines for oral or oropharyngeal HPV infection is yet to be determined. This study conducted a systematic literature search in Pubmed and Embase. Studies investigating the impact of HPV vaccines on oral or oropharyngeal HPV infection were enrolled. This review reports the relative prevention percentage (RPP), including a risk of bias assessment as well as a quality assessment study. Nine studies were included (48,777 participants): five cross-sectional studies; one randomized community trial study (RCT); one longitudinal cohort study; and two case-control studies. A significant mean RPP of 83.9% (66.6-97.8%) was calculated from the cross-sectional studies, 82.4% in the included RCT and 83% in the longitudinal cohort study. Further, two case-control studies that measured antibody response in participants immunized with HPV vaccines were included. Respectively, 100% and 93.2% of participants developed HPV-16 Immunoglobulin G (IgG) antibodies in oral fluids post-vaccination. Analysis of the studies identified a significant decrease in vaccine-type oral or oropharyngeal HPV infections in study participants immunized with HPV vaccines across study designs and heterogenous populations. Further, a significant percentage of participants developed IgG antibodies in oral fluid post-vaccination.


Assuntos
Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/farmacologia , Alphapapillomavirus/patogenicidade , Estudos de Casos e Controles , Estudos Transversais , Feminino , Humanos , Estudos Longitudinais , Masculino , Boca/virologia , Neoplasias Bucais/prevenção & controle , Neoplasias Bucais/virologia , Neoplasias Orofaríngeas/prevenção & controle , Neoplasias Orofaríngeas/virologia , Orofaringe/virologia , Papillomaviridae/imunologia , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/metabolismo , Vacinas contra Papillomavirus/metabolismo , Profilaxia Pré-Exposição/métodos , Vacinação
10.
J Med Virol ; 93(12): 6837-6840, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34324212

RESUMO

BACKGROUND: Gargle samples have been proposed as a noninvasive method for detection of SARS-CoV-2 RNA. The clinical performance of gargle specimens diluted in Cobas® PCR Media and in Cobas® Omni Lysis Reagent was compared to oropharyngeal/nasopharyngeal swab (ONPS) for the detection of SARS-CoV-2 RNA. STUDY DESIGN: Participants were recruited prospectively in two COVID-19 screening clinics. In addition to the ONPS, participants gargled with 5 ml of natural spring water split in the laboratory as follows: 1 ml was added to 4.3 ml of polymerase chain reaction (PCR) media and 400 µl was added to 200 µl of lysis buffer. Testing was performed with the Cobas® SARS-CoV-2 test on the Cobas® 6800 or 8800 platforms. RESULTS: Overall, 134/647 (20.7%) participants were considered infected because the ONPS or at least one gargle test was positive. ONPS had, respectively, a sensitivity of 96.3% (95% confidence interval [CI]: 91.3-98.5); both gargle processing methods were slightly less but equally sensitive (90.3% [95% CI: 83.9-94.3]). When ONPS and gargle specimens were both positive, the mean cycle threshold (Ct ) was significantly higher for gargles, suggesting lower viral loads. CONCLUSION: Gargle specimens directly added in PCR Media provide a similar clinical sensitivity to chemical lysis, both having a slightly, not significantly, lower sensitivity to ONPS.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/virologia , Nasofaringe/virologia , Orofaringe/virologia , SARS-CoV-2/genética , Testes Diagnósticos de Rotina/métodos , Humanos , Programas de Rastreamento/métodos , Estudos Prospectivos , RNA Viral/genética , Saliva/virologia , Manejo de Espécimes/métodos , Carga Viral/genética
11.
Ann Intern Med ; 174(9): 1261-1269, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34251903

RESUMO

BACKGROUND: New treatment modalities are urgently needed for patients with COVID-19. The World Health Organization (WHO) Solidarity trial showed no effect of remdesivir or hydroxychloroquine (HCQ) on mortality, but the antiviral effects of these drugs are not known. OBJECTIVE: To evaluate the effects of remdesivir and HCQ on all-cause, in-hospital mortality; the degree of respiratory failure and inflammation; and viral clearance in the oropharynx. DESIGN: NOR-Solidarity is an independent, add-on, randomized controlled trial to the WHO Solidarity trial that included biobanking and 3 months of clinical follow-up (ClinicalTrials.gov: NCT04321616). SETTING: 23 hospitals in Norway. PATIENTS: Eligible patients were adults hospitalized with confirmed SARS-CoV-2 infection. INTERVENTION: Between 28 March and 4 October 2020, a total of 185 patients were randomly assigned and 181 were included in the full analysis set. Patients received remdesivir (n = 42), HCQ (n = 52), or standard of care (SoC) (n = 87). MEASUREMENTS: In addition to the primary end point of WHO Solidarity, study-specific outcomes were viral clearance in oropharyngeal specimens, the degree of respiratory failure, and inflammatory variables. RESULTS: No significant differences were seen between treatment groups in mortality during hospitalization. There was a marked decrease in SARS-CoV-2 load in the oropharynx during the first week overall, with similar decreases and 10-day viral loads among the remdesivir, HCQ, and SoC groups. Remdesivir and HCQ did not affect the degree of respiratory failure or inflammatory variables in plasma or serum. The lack of antiviral effect was not associated with symptom duration, level of viral load, degree of inflammation, or presence of antibodies against SARS-CoV-2 at hospital admittance. LIMITATION: The trial had no placebo group. CONCLUSION: Neither remdesivir nor HCQ affected viral clearance in hospitalized patients with COVID-19. PRIMARY FUNDING SOURCE: National Clinical Therapy Research in the Specialist Health Services, Norway.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/virologia , Hidroxicloroquina/uso terapêutico , Carga Viral/efeitos dos fármacos , Monofosfato de Adenosina/uso terapêutico , Alanina/uso terapêutico , Anticorpos Antivirais/sangue , Biomarcadores/sangue , COVID-19/complicações , COVID-19/mortalidade , Causas de Morte , Feminino , Mortalidade Hospitalar , Humanos , Inflamação/virologia , Masculino , Pessoa de Meia-Idade , Noruega/epidemiologia , Orofaringe/virologia , Insuficiência Respiratória/virologia , SARS-CoV-2/imunologia , Índice de Gravidade de Doença , Padrão de Cuidado , Resultado do Tratamento
12.
Med J Aust ; 215(6): 273-278, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34287935

RESUMO

OBJECTIVE: To compare the concordance and acceptability of saliva testing with standard-of-care oropharyngeal and bilateral deep nasal swab testing for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in children and in general practice. DESIGN: Prospective multicentre diagnostic validation study. SETTING: Royal Children's Hospital, and two general practices (cohealth, West Melbourne; Cirqit Health, Altona North) in Melbourne, July-October 2020. PARTICIPANTS: 1050 people who provided paired saliva and oropharyngeal-nasal swabs for SARS-CoV-2 testing. MAIN OUTCOME MEASURES: Numbers of cases in which SARS-CoV-2 was detected in either specimen type by real-time polymerase chain reaction; concordance of results for paired specimens; positive percent agreement (PPA) for virus detection, by specimen type. RESULTS: SARS-CoV-2 was detected in 54 of 1050 people with assessable specimens (5%), including 19 cases (35%) in which both specimens were positive. The overall PPA was 72% (95% CI, 58-84%) for saliva and 63% (95% CI, 49-76%) for oropharyngeal-nasal swabs. For the 35 positive specimens from people aged 10 years or more, PPA was 86% (95% CI, 70-95%) for saliva and 63% (95% CI, 45-79%) for oropharyngeal-nasal swabs. Adding saliva testing to standard-of-care oropharyngeal-nasal swab testing increased overall case detection by 59% (95% CI, 29-95%). Providing saliva was preferred to an oropharyngeal-nasal swab by most participants (75%), including 141 of 153 children under 10 years of age (92%). CONCLUSION: In children over 10 years of age and adults, saliva testing alone may be suitable for SARS-CoV-2 detection, while for children under 10, saliva testing may be suitable as an adjunct to oropharyngeal-nasal swab testing for increasing case detection.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodos , Adolescente , Adulto , Fatores Etários , Idoso , COVID-19/virologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Orofaringe/virologia , Estudos Prospectivos , RNA Viral/isolamento & purificação , SARS-CoV-2/genética , Saliva/virologia , Adulto Jovem
13.
Eur J Clin Microbiol Infect Dis ; 40(12): 2489-2496, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34224033

RESUMO

Easy access to screening for timely identification and isolation of infectious COVID-19 patients remains crucial in sustaining the international efforts to control COVID-19 spread. A major barrier limiting broad-based screening is the lack of a simple, rapid, and cost-effective COVID-19 testing method. We evaluated the feasibility and utility of facemask sampling in a cohort of 42 COVID-19-positive and 36 COVID-19-negative patients. We used a prototype of Steri-Strips™ (3 M) applied to the inner surface of looped surgical facemasks (Assure), which was worn by patients for a minimum wear time of 3 h, then removed and sent for SARS-CoV-2 PCR testing. Baseline demographics and symptomatology were also collected. Facemask sampling positivity was highest within the first 5 days of symptomatic presentation. Patients with nasopharyngeal and/or oropharyngeal swab SARS-CoV-2 PCR Ct values < 25.09 had SARS-CoV-2 detected on facemask sampling, while patients with Ct values ≥ 25.2 had no SARS-CoV-2 detected on facemask sampling. Facemask sampling can identify patients with COVID-19 during the early symptomatic phase or those with high viral loads, hence allowing timely identification and isolation of those with the highest transmission risk. Given the widespread use of facemasks, this method can potentially be easily applied to achieve broad-based, or even continuous, population screening.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/virologia , Máscaras/virologia , SARS-CoV-2/isolamento & purificação , Adulto , Idoso , COVID-19/diagnóstico , COVID-19/epidemiologia , Teste de Ácido Nucleico para COVID-19/instrumentação , Estudos de Coortes , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Orofaringe/virologia , Pandemias , SARS-CoV-2/classificação , SARS-CoV-2/genética , Adulto Jovem
14.
Am J Pathol ; 191(10): 1822-1836, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34214507

RESUMO

Human papillomavirus (HPV) is a ubiquitous human pathogen that can be cleared by host immunity. Nonetheless, a small percentage of the patients develop persistent infection with oncogenic HPV, which poses an increased risk of developing HPV-associated malignancy. Although cell-mediated immunity is a known systemic factor, local factors that influence persistent HPV infection have not been fully investigated. HPV-related head/neck cancers have a strong site preference for the oropharynx, suggesting the existence of unique local factors that promote HPV-induced oncogenesis. The human oropharynx often harbors anaerobic bacteria that produce a variety of byproducts, including butyrate. Because butyrate is a potent epigenetic modulator, it could be an environmental factor influencing the development of HPV-positive oropharyngeal malignancy. In this study, we showed that butyrate treatment changed the property of HPV16 E6/E7-immortalized keratinocytes. In vitro, the treatment increased the cells' migration ability, slowed the growth, and increased the genotoxic resistance. When implanted in the syngeneic mice, the treated keratinocytes survived longer and exhibited a different growth pattern. The survival advantage obtained after butyrate exposure may increase the susceptibility of HPV-infected oropharyngeal keratinocytes to further malignant transformation. These results suggest that fermentation products of tonsillar bacteria may play an important role in the long-term persistence of high-risk HPV infection, which is a critical risk factor for developing HPV-positive oropharyngeal malignancy.


Assuntos
Bactérias/metabolismo , Butiratos/metabolismo , Queratinócitos/patologia , Queratinócitos/virologia , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Repressoras/metabolismo , Animais , Animais Recém-Nascidos , Morte Celular , Diferenciação Celular , Linhagem Celular , Linhagem Celular Transformada , Proliferação de Células , Sobrevivência Celular , Feminino , Masculino , Camundongos , Orofaringe/patologia , Orofaringe/virologia
15.
Int J Biol Sci ; 17(8): 2080-2088, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34131407

RESUMO

Coronavirus disease 2019 (COVID-19), an infectious disease caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed a persistent global threat. The transmission of SARS-CoV-2 is wide and swift. Rapid detection of the viral RNA and effective therapy are imperative to prevent the worldwide spread of the new infectious disease. Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)- CRISPR-associated protein (Cas) system is an RNA-directed adaptive immune system, and it has been transformed into a gene editing tool. Applications of CRISPR-Cas system involves in many fields, such as human gene therapy, drug discovery and disease diagnosis. Under the background of COVID-19 pandemic, CRISPR-Cas system shows hidden capacity to fight the emergency in many aspects. This review will focus on the role of gene editing in COVID-19 diagnosis and treatment. We will describe the potential use of CRISPR-Cas-based system in combating COVID-19, from diagnosis to treatment. Furthermore, the limitation and perspectives of this novel technology are also evaluated.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/terapia , Sistemas CRISPR-Cas , Edição de Genes/métodos , Regulação Viral da Expressão Gênica/genética , RNA Viral/análise , SARS-CoV-2/genética , Animais , Fluorometria/métodos , Previsões , Técnicas de Inativação de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Transgênicos , Modelos Animais , Terapia de Alvo Molecular , Nasofaringe/virologia , Orofaringe/virologia , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade
16.
Laryngoscope ; 131(12): E2865-E2873, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34076275

RESUMO

OBJECTIVE: To analyze the patterns, risk factors, and salvage outcomes for locoregional recurrences (LRR) after treatment with transoral robotic surgery (TORS) for HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC). STUDY DESIGN: Retrospective analysis of HPV+ OPSCC patients completing primary TORS, neck dissection, and NCCN-guideline-compliant adjuvant therapy at a single institution from 2007 to 2017. METHODS: Features associated with LRR, detailed patterns of LRR, and outcomes of salvage therapy were analyzed. Disease-free survival (DFS) and overall survival (OS) were calculated for subgroups of patients receiving distinct adjuvant treatments. RESULTS: Of 541 patients who completed guideline-indicated therapy, the estimated 5-year LRR rate was 4.5%. There were no identifiable clinical or pathologic features associated with LRR. Compared to patients not receiving adjuvant therapy, those who received indicated adjuvant radiation alone had a lower risk of LRR (HR 0.28, 95% CI [0.09-0.83], P = .023), but there was no difference in DFS (P = .21) and OS (P = .86) between adjuvant therapy groups. The 5-year OS for patients who developed LRR was 67.1% vs. 93.9% for those without LRR (P < .001). Patients who initially received adjuvant chemoradiation and those suffering local, in-field, and/or retropharyngeal node recurrences had decreased disease control after salvage therapy. CONCLUSION: LRR rates are low for HPV+ OPSCCs completing TORS and guideline-compliant adjuvant therapy. Patients without indication for adjuvant therapy more often suffer LRR, but these recurrences are generally controllable by salvage therapy. Improved understanding of the patterns of recurrence most amenable to salvage therapy may guide treatment decisions, counseling, and adjuvant therapy de-escalation trials. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:E2865-E2873, 2021.


Assuntos
Recidiva Local de Neoplasia/epidemiologia , Neoplasias Orofaríngeas/terapia , Infecções por Papillomavirus/terapia , Procedimentos Cirúrgicos Robóticos/estatística & dados numéricos , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Idoso , Alphapapillomavirus/isolamento & purificação , Quimiorradioterapia Adjuvante/estatística & dados numéricos , Inibidor p16 de Quinase Dependente de Ciclina/análise , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Cirurgia Endoscópica por Orifício Natural/métodos , Recidiva Local de Neoplasia/prevenção & controle , Recidiva Local de Neoplasia/virologia , Neoplasias Orofaríngeas/mortalidade , Neoplasias Orofaríngeas/patologia , Neoplasias Orofaríngeas/virologia , Orofaringe/patologia , Orofaringe/cirurgia , Orofaringe/virologia , Infecções por Papillomavirus/mortalidade , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Radioterapia Adjuvante/estatística & dados numéricos , Estudos Retrospectivos , Procedimentos Cirúrgicos Robóticos/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia
17.
Biomed Res Int ; 2021: 6653950, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34124257

RESUMO

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 µL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 µL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49 ng/uL, which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.


Assuntos
COVID-19/diagnóstico , Proteínas do Envelope de Coronavírus/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , 2-Propanol/química , Biomarcadores/análise , COVID-19/virologia , Primers do DNA/síntese química , Primers do DNA/genética , Humanos , Nasofaringe/virologia , Orofaringe/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
18.
Curr Protoc ; 1(5): e145, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34004070

RESUMO

Since December 2019, SARS-CoV-2 has spread extensively throughout the world, with more than 117 million reported cases and 2.6 million deaths (Johns Hopkins coronavirus resource center, https://coronavirus.jhu.edu/map.html). Detecting the virus is the first step in diagnosing the infection, followed by quarantine to prevent transmission. Nasopharyngeal/oropharyngeal swabs (NP/OP) and saliva are two specimen types that are most often analyzed to detect SARS-CoV-2 by molecular tests that detect viral RNA or by antigen/antibody tests that detect viral proteins and/or the host immune response against the virus. Compared to antigen/antibody tests, molecular tests are highly sensitive and specific for detecting the virus. A significant drawback is that specimen collection requirements are specific to each test and cannot be interchanged with another test. Some tests are qualified to be used on NP swabs or saliva, but not both specimen types. Even with NP swabs, a test may be qualified to detect the virus only with swabs collected in viral transport medium (VTM) but not in other media. These restrictive pre-analytic steps are disadvantageous in that a lab would have to develop and validate different tests for SARS-CoV-2 depending on the specimen type and collection media, with added setup cost, infrastructure, and training requirements. To overcome these problems, we developed and validated a cost-effective multiplex reverse-transcription real-time PCR assay that can be used to detect SARS-CoV-2 in different specimen types. The assay is highly sensitive and specific, can be used to detect the virus in saliva as well as NP swabs collected in different media such as VTM, saline, and commercial preservative fluid, and serves as one test for all applications. The protocol also describes an optimal laboratory setup and unidirectional workflow for detecting SARS-CoV-2 by RT-qPCR. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Manual viral nucleic acid extraction from NP/OP swabs collected in different media, and from saliva Alternate Protocol 1: Low-throughput automated extraction on the Qiagen EZ1 Advanced XL machine (1-14 samples) Alternate Protocol 2: High-throughput automated extraction on the Kingfisher Flex machine (1-96 samples) Basic Protocol 2: Multiplex RT-qPCR protocol to detect SARS-CoV-2 Alternate Protocol 3: Multiplex one-step RT-qPCR protocol to detect SARS-CoV-2 with S and E gene probes labeled with the same fluorochrome.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Nasofaringe/virologia , Orofaringe/virologia , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Teste de Ácido Nucleico para COVID-19/economia , Humanos , Reação em Cadeia da Polimerase Multiplex/economia , Reação em Cadeia da Polimerase Multiplex/métodos , RNA Viral/análise , RNA Viral/isolamento & purificação
19.
J Virol Methods ; 295: 114184, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34029634

RESUMO

With increasing demands for SARS-CoV-2 testing, as well as the shortages for testing supplies, collection devices, and trained healthcare workers (HCWs) to collect specimens, self-collection is an attractive prospect to reduce the need for HCWs and expenditure of personal protective equipment. Apart from the traditional nasopharyngeal swab used for SARS-CoV-2 detection, alternative specimens have been validated such as a combined swabs of the oropharynx and anterior nares (OP/N), or throat samples using saline gargles. Both the alternative specimen types are amenable to self-collection. Objectives. This study aimed to compare the sensitivity of HCW-collected (OP/N) swabs, self-collected OP/N swabs, and self-collected saline gargles. Among 38 individuals previously testing positive for SARS-CoV-2 (or their close contacts), two self-collected specimen types (OP/N and saline gargles) were compared to HCW-collected OP/N swabs. SARS-CoV-2 testing was performed on three molecular assays: a laboratory-developed test (LDT), and two commercial assays on automated platforms: Cobas 6800 (Roche Diagnostics) and Panther (Hologic). The sensitivity of self-collected OP/N swabs was equivalent to healthcare worker (HCW)-collected OP/N swabs at 100.0 % [92.6%-100.0%] for all three molecular tests. The sensitivity of saline gargles was not significantly different than HCW-collected OP/N swabs, but varied slightly between instruments at 93.8 % [85.9%-93.8%] for the LDT, 96.8 % [88.6%-96.8%] for the Cobas assay, and 96.7 % [89.2%-96.9%] for the Panther assay. Overall, self-collection using OP/N swabs or saline gargles are reasonable alternatives to HCW-based collections for SARS-CoV-2 detection, and could facilitate broader surveillance strategies.


Assuntos
COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodos , Teste de Ácido Nucleico para COVID-19 , Pessoal de Saúde , Humanos , Cavidade Nasal/virologia , Orofaringe/virologia , SARS-CoV-2/genética , Saliva/virologia , Sensibilidade e Especificidade
20.
Laryngorhinootologie ; 100(7): 517-525, 2021 07.
Artigo em Alemão | MEDLINE | ID: mdl-34010974

RESUMO

Since the beginning of the SARS-CoV-2 pandemic, swabs or other samples have increasingly been taken from the upper aero-digestive tract, since high viral loads exist here, especially in the early stages of the disease. As diagnostic options, swabs from the anterior nose, from the nasopharynx, from the oropharynx or the extraction of throat rinse water or saliva are possible. The laboratory methods available are antigen tests that can be read in a few minutes or more lengthy RT-PCR methods in a lab. Swabs are carried out by physicians, medical staff, laypeople and in the self-test, in each case according to prior instructions. Many of these factors therefore have an influence on the informative value and the sensitivity of the entire diagnostic process. The PCR laboratory method is more sensitive than the antigen method; the swabs from the nasopharynx are considered the most valid smear site; correct execution of a test can be achieved even with non-professional individuals with good instructions. Complications with such swabs are reported very rarely, given the assumed number of procedures performed. Short-term nosebleeds after traumatic smears can be assumed without publications about it being found. Broken parts of swabs had to be removed by an ENT doctor. There are only very few reports on injuries to the skullbase with CSF-leaks, including 2 times with anomalies such as meningoceles. The choice of a suitable diagnostic medium depends on many parameters such as availability, the timing of the result, a smear test by knowledgeable staff or a self-test, and a number of other practical considerations.


Assuntos
COVID-19 , Nasofaringe , Orofaringe , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , Teste para COVID-19 , Humanos , Nasofaringe/virologia , Orofaringe/virologia , Pandemias
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