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1.
Biosens Bioelectron ; 180: 113111, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33743492

RESUMO

Significant barriers to the diagnosis of latent and acute SARS-CoV-2 infection continue to hamper population-based screening efforts required to contain the COVID-19 pandemic in the absence of widely available antiviral therapeutics or vaccines. We report an aptamer-based SARS-CoV-2 salivary antigen assay employing only low-cost reagents ($3.20/test) and an off-the-shelf glucometer. The test was engineered around a glucometer as it is quantitative, easy to use, and the most prevalent piece of diagnostic equipment globally, making the test highly scalable with an infrastructure that is already in place. Furthermore, many glucometers connect to smartphones, providing an opportunity to integrate with contact tracing apps, medical providers, and electronic health records. In clinical testing, the developed assay detected SARS-CoV-2 infection in patient saliva across a range of viral loads - as benchmarked by RT-qPCR - within 1 h, with 100% sensitivity (positive percent agreement) and distinguished infected specimens from off-target antigens in uninfected controls with 100% specificity (negative percent agreement). We propose that this approach provides an inexpensive, rapid, and accurate diagnostic for distributed screening of SARS-CoV-2 infection at scale.


Assuntos
Antígenos Virais/análise , Técnicas Biossensoriais/métodos , /diagnóstico , Testes Imediatos , Saliva/virologia , Adulto , Feminino , Humanos , Masculino , Fosfoproteínas/análise , Técnica de Seleção de Aptâmeros , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/análise
2.
Emerg Infect Dis ; 27(4): 1146-1150, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33754989

RESUMO

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.


Assuntos
/métodos , RNA Viral , Saliva/virologia , /diagnóstico , /virologia , Fortalecimento Institucional/métodos , Humanos , Estabilidade de RNA , RNA Viral/isolamento & purificação , RNA Viral/fisiologia , Reprodutibilidade dos Testes , Alocação de Recursos , /isolamento & purificação , Manejo de Espécimes/economia , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos
4.
Sci Rep ; 11(1): 6264, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33731722

RESUMO

Many educational institutions have partially or fully closed all operations to cope with the challenges of the ongoing COVID-19 pandemic. In this paper, we explore strategies that such institutions can adopt to conduct safe reopening and resume operations during the pandemic. The research is motivated by the University of Illinois at Urbana-Champaign's (UIUC's) SHIELD program, which is a set of policies and strategies, including rapid saliva-based COVID-19 screening, for ensuring safety of students, faculty and staff to conduct in-person operations, at least partially. Specifically, we study how rapid bulk testing, contact tracing and preventative measures such as mask wearing, sanitization, and enforcement of social distancing can allow institutions to manage the epidemic spread. This work combines the power of analytical epidemic modeling, data analysis and agent-based simulations to derive policy insights. We develop an analytical model that takes into account the asymptomatic transmission of COVID-19, the effect of isolation via testing (both in bulk and through contact tracing) and the rate of contacts among people within and outside the institution. Next, we use data from the UIUC SHIELD program and 85 other universities to estimate parameters that describe the analytical model. Using the estimated parameters, we finally conduct agent-based simulations with various model parameters to evaluate testing and reopening strategies. The parameter estimates from UIUC and other universities show similar trends. For example, infection rates at various institutions grow rapidly in certain months and this growth correlates positively with infection rates in counties where the universities are located. Infection rates are also shown to be negatively correlated with testing rates at the institutions. Through agent-based simulations, we demonstrate that the key to designing an effective reopening strategy is a combination of rapid bulk testing and effective preventative measures such as mask wearing and social distancing. Multiple other factors help to reduce infection load, such as efficient contact tracing, reduced delay between testing and result revelation, tests with less false negatives and targeted testing of high-risk class among others. This paper contributes to the nascent literature on combating the COVID-19 pandemic and is especially relevant for educational institutions and similarly large organizations. We contribute by providing an analytical model that can be used to estimate key parameters from data, which in turn can be used to simulate the effect of different strategies for reopening. We quantify the relative effect of different strategies such as bulk testing, contact tracing, reduced infectivity and contact rates in the context of educational institutions. Specifically, we show that for the estimated average base infectivity of 0.025 ([Formula: see text]), a daily number of tests to population ratio T/N of 0.2, i.e., once a week testing for all individuals, is a good indicative threshold. However, this test to population ratio is sensitive to external infectivities, internal and external mobilities, delay in getting results after testing, and measures related to mask wearing and sanitization, which affect the base infection rate.


Assuntos
/prevenção & controle , Pandemias/prevenção & controle , Instituições Acadêmicas/normas , Universidades/normas , Doenças Assintomáticas , Simulação por Computador , Busca de Comunicante/métodos , Humanos , Saliva/virologia
5.
Sci Rep ; 11(1): 5448, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33750853

RESUMO

To safely re-open economies and prevent future outbreaks, rapid, frequent, point-of-need, SARS-CoV-2 diagnostic testing is necessary. However, existing field-deployable COVID-19 testing methods require the use of uncomfortable swabs and trained providers in PPE, while saliva-based methods must be transported to high complexity laboratories for testing. Here, we report the development and clinical validation of High-Performance Loop-mediated isothermal Amplification (HP-LAMP), a rapid, saliva-based, SARS-CoV-2 test with a limit of detection of 1.4 copies of virus per µl of saliva and a sensitivity and specificity with clinical samples of > 96%, on par with traditional RT-PCR based methods using swabs, but can deliver results using only a single fluid transfer step and simple heat block. Testing of 120 patient samples in 40 pools comprised of 5 patient samples each with either all negative or a single positive patient sample was 100% accurate. Thus, HP-LAMP may enable rapid and accurate results in the field using saliva, without need of a high-complexity laboratory.


Assuntos
/diagnóstico , Saliva/virologia , /virologia , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/metabolismo , Sensibilidade e Especificidade , Temperatura
6.
Emerg Infect Dis ; 27(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33755009

RESUMO

We analyzed feasibility of pooling saliva samples for severe acute respiratory syndrome coronavirus 2 testing and found that sensitivity decreased according to pool size: 5 samples/pool, 7.4% reduction; 10 samples/pool, 11.1%; and 20 samples/pool, 14.8%. When virus prevalence is >2.6%, pools of 5 require fewer tests; when <0.6%, pools of 20 support screening strategies.


Assuntos
/métodos , Saliva/virologia , Manejo de Espécimes/métodos , /diagnóstico , Fortalecimento Institucional/métodos , Alocação de Recursos para a Atenção à Saúde , Humanos , Limite de Detecção , Alocação de Recursos/métodos , Sensibilidade e Especificidade , Estados Unidos
7.
BMJ Open Respir Res ; 8(1)2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33627333

RESUMO

BACKGROUND: An outbreak of novel coronavirus (SARS-CoV-2)-associated respiratory infectious diseases (COVID-19) emerged in 2019 and has spread rapidly in humans around the world. The demonstration of in vitro infectiousness of respiratory specimens is an informative surrogate for SARS-CoV-2 transmission from patients with COVID-19; accordingly, viral isolation assays in cell culture are an important aspect of laboratory diagnostics for COVID-19. METHODS: We developed a simple and rapid protocol for isolating SARS-CoV-2 from respiratory specimens using VeroE6/TMPRSS2 cells, a cell line that is highly susceptible to the virus. We also investigated a correlation between isolation of SARS-CoV-2 and viral load detected by real-time RT-PCR (rRT-PCR) using N2 primer/probe set that has been developed for testing of COVID-19 in Japan. RESULTS: The SARS-CoV-2 isolation protocol did not require blind passage of inoculated cells and yielded the results of viral isolation within 7 days after inoculation. Specimens with cycle threshold (Ct) values of <20.2, determined by rRT-PCR, were predicted to be isolation-positive. On the other hand, 6.9% of specimens with Ct values >35 were virus isolation-positive, indicating that low viral loads (high Ct values) in upper respiratory specimens do not always indicate no risk of containing transmissible virus. CONCLUSION: In combination with rRT-PCR, the SARS-CoV-2 isolation protocol provides a means for assessing the potential risk of transmissible virus in upper respiratory specimens.


Assuntos
/transmissão , /patogenicidade , Animais , Linhagem Celular , Chlorocebus aethiops , Efeito Citopatogênico Viral , Humanos , Cavidade Nasal/virologia , Nasofaringe/virologia , Saliva/virologia , Serina Endopeptidases/genética , Manejo de Espécimes , Células Vero
8.
Viruses ; 13(2)2021 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-33562073

RESUMO

The contemporary surge in metagenomic sequencing has transformed knowledge of viral diversity in wildlife. However, evaluating which newly discovered viruses pose sufficient risk of infecting humans to merit detailed laboratory characterization and surveillance remains largely speculative. Machine learning algorithms have been developed to address this imbalance by ranking the relative likelihood of human infection based on viral genome sequences, but are not yet routinely applied to viruses at the time of their discovery. Here, we characterized viral genomes detected through metagenomic sequencing of feces and saliva from common vampire bats (Desmodus rotundus) and used these data as a case study in evaluating zoonotic potential using molecular sequencing data. Of 58 detected viral families, including 17 which infect mammals, the only known zoonosis detected was rabies virus; however, additional genomes were detected from the families Hepeviridae, Coronaviridae, Reoviridae, Astroviridae and Picornaviridae, all of which contain human-infecting species. In phylogenetic analyses, novel vampire bat viruses most frequently grouped with other bat viruses that are not currently known to infect humans. In agreement, machine learning models built from only phylogenetic information ranked all novel viruses similarly, yielding little insight into zoonotic potential. In contrast, genome composition-based machine learning models estimated different levels of zoonotic potential, even for closely related viruses, categorizing one out of four detected hepeviruses and two out of three picornaviruses as having high priority for further research. We highlight the value of evaluating zoonotic potential beyond ad hoc consideration of phylogeny and provide surveillance recommendations for novel viruses in a wildlife host which has frequent contact with humans and domestic animals.


Assuntos
Quirópteros/virologia , Vírus/isolamento & purificação , Zoonoses/virologia , Animais , Reservatórios de Doenças/veterinária , Reservatórios de Doenças/virologia , Fezes/virologia , Genoma Viral/genética , Humanos , Aprendizado de Máquina , Metagenômica , Filogenia , Vírus da Raiva/classificação , Vírus da Raiva/genética , Vírus da Raiva/isolamento & purificação , Saliva/virologia , Vírus/classificação , Vírus/genética
9.
Sci Rep ; 11(1): 4500, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627730

RESUMO

Emerging evidences have shown the utility of saliva for the detection of SARS-CoV-2 by PCR as alternative to nasopharyngeal swab (NPS). However, conflicting results have been reported regarding viral loads between NPS and saliva. We conducted a study to compare the viral loads between NPS and saliva in 42 COVID-19 patients. Viral loads were estimated by the cycle threshold (Ct) values. SARS-CoV-2 was detected in 34 (81%) using NPS with median Ct value of 27.4, and 38 (90%) using saliva with median Ct value of 28.9 (P = 0.79). Kendall's W was 0.82, showing a high degree of agreement, indicating equivalent viral loads in NPS and saliva. After symptom onset, the Ct values of both NPS and saliva continued to increase over time, with no substantial difference. Self-collected saliva has a detection sensitivity comparable to that of NPS and is a useful diagnostic tool with mitigating uncomfortable process and the risk of aerosol transmission to healthcare workers.


Assuntos
/virologia , /genética , Adulto , /métodos , Testes Diagnósticos de Rotina/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Reação em Cadeia da Polimerase/métodos , RNA Viral/genética , Saliva/virologia , Manejo de Espécimes/métodos , Carga Viral/métodos
10.
PLoS One ; 16(2): e0243183, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33621263

RESUMO

BACKGROUND: Sensitive and high throughput molecular detection assays are essential during the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vast majority of the SARS-CoV-2 molecular assays use nasopharyngeal swab (NPS) or oropharyngeal swab (OPS) specimens collected from suspected individuals. However, using NPS or OPS as specimens has apparent drawbacks, e.g. the collection procedures for NPS or OPS specimens can be uncomfortable to some people and may cause sneezing and coughing which in turn generate droplets and/or aerosol particles that are of risk to healthcare workers, requiring heavy use of personal protective equipment. There have been recent studies indicating that self-collected saliva specimens can be used for molecular detection of SARS-CoV-2 and provides more comfort and ease of use for the patients. Here we report the performance of QuantiVirus™ SARS-CoV-2 test using saliva as the testing specimens with or without pooling. METHODS: Development and validation studies were conducted following FDA-EUA and molecular assay validation guidelines. Using SeraCare Accuplex SARS-CoV-2 reference panel, the limit of detection (LOD) and clinical performance studies were performed with the QuantiVirus™ SARS-CoV-2 test. For clinical evaluation, 85 known positive and 90 known negative clinical NPS samples were tested. Additionally, twenty paired NPS and saliva samples collected from recovering COVID-19 patients were tested and the results were further compared to that of the Abbott m2000 SARS-CoV-2 PCR assay. Results of community collected 389 saliva samples for COVID-19 screening by QuantiVirus™ SARS-CoV-2 test were also obtained and analyzed. Additionally, testing of pooled saliva samples was evaluated. RESULTS: The LOD for the QuantiVirus™ SARS-CoV-2 test was confirmed to be 100-200 copies/mL. The clinical performance studies using contrived saliva samples indicated that the positive percentage agreement (PPA) of the QuantiVirus™ SARS-CoV-2 test is 100% at 1xLOD, 1.5xLOD and 2.5xLOD. No cross-reactivity was observed for the QuantiVirus™ SARS-CoV-2 test with common respiratory pathogens. Testing of clinical samples showed a positive percentage agreement (PPA) of 100% (95% CI: 94.6% to 100%) and a negative percentage agreement (NPA) of 98.9% (95% CI: 93.1% to 99.9%). QuantiVirus™ SARS CoV-2 test had 80% concordance rate and no significant difference (p = 0.13) between paired saliva and NPS specimens by Wilcoxon matched pairs signed rank test. Positive test rate was 1.79% for 389 saliva specimens collected from local communities for COVID-19 screening. Preliminary data showed that saliva sample pooling up to 6 samples (1:6 pooling) for SARS-CoV-2 detection is feasible (sensitivity 94.8% and specificity 100%). CONCLUSION: The studies demonstrated that the QuantiVirus™ SARS-CoV-2 test has a LOD of 200 copies/mL in contrived saliva samples. The clinical performance of saliva-based testing is comparable to that of NPS-based testing. Pooling of saliva specimens for SARS-CoV-2 detection is feasible. Saliva based and high-throughput QuantiVirus™ SARS-CoV-2 test offers a highly desirable testing platform during the ongoing COVID-19 pandemic.


Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Saliva/virologia , Manejo de Espécimes/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Adulto Jovem
11.
Allergol Immunopathol (Madr) ; 49(1): 159-164, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33528945

RESUMO

Coronavirus disease 2019 (COVID-19) is a disease caused by a new strain of coronavirus named as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Globally, since the outbreak, more than seven million confirmed cases of COVID-19 have been reported. The rapid spread and increase in the number of new cases is due to person-to-person transmission. To further control its transmission, early laboratory diagnosis of both asymptomatic and symptomatic patients is crucial. Presently, the COVID-19 diagnosis of infected individuals is dependent on computed tomography scanning and real-time polymerase chain reaction (PCR). The latter is considered more sensitive and efficient for early diagnosis. In this review, general comparisons are made (cases, fatality rate, incubation period, clinical features, and reservoirs) and diagnostic laboratory procedures (specimens, extraction methods, and positive rates by real-time PCR) are compared between SARS, Middle East Respiratory Syndrome, and SARS-2. In total, 8982 SARS-2 suspected patients specimen data were retrieved, in which 40.9% (n = 3678) were detected as positive by real-time PCR. The specimen-wise high detection rate was observed from bronchoalveolar lavage, followed by saliva, nasal swabs, and sputum. As the COVID-19 cases are persistently increasing, the selection of appropriate specimens and laboratory assay would help in rapid and timely diagnosis.


Assuntos
/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , /isolamento & purificação , Lavagem Broncoalveolar , /virologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Humanos , Nasofaringe/virologia , Saliva/virologia , Síndrome Respiratória Aguda Grave/diagnóstico , Síndrome Respiratória Aguda Grave/virologia , Escarro/virologia
12.
Sci Rep ; 11(1): 3134, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542443

RESUMO

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.


Assuntos
/diagnóstico , Mucosa Nasal/virologia , RNA Viral/isolamento & purificação , Saliva/virologia , Manejo de Espécimes , Adulto , Estudos de Coortes , Feminino , Humanos , Masculino , Nasofaringe/virologia , /isolamento & purificação , Sensibilidade e Especificidade , Singapura/epidemiologia
13.
Expert Rev Mol Diagn ; 21(1): 31-42, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33523770

RESUMO

Introduction: The unprecedented outbreaks of corona virus disease of 2019 (COVID-19) have highlighted the necessity of readily available, reliable, precise, and faster techniques for its detection. Nasopharyngeal swab has been the gold standard for the diagnosis of COVID-19. However, it is not an ideal screening procedure for massive screening as it implicates the patient's stay in the hospital or at home until diagnosis, thus causing crowding of the specimen at the diagnostic centers. Present study deal with the exploration of potential application of different body fluids using certain highly objective techniques (Optical and e-Nose) for faster detection of molecular markers thereby diagnosing viral infections.Areas covered: This report presents an evaluation of different body fluids, and their advantages for the rapid detection of COVID-19, coupled with highly sensitive optical techniques for the detection of molecular biomarkers.Expert opinion: Tears, saliva, and breath samples can provide valuable information about viral infections. Our brief review strongly recommends the application of saliva/tears and exhaled breath as clinical samples using technics such as high-performance liquid chromatography-laser-induced fluorescence, photoacoustic spectroscopy, and e-Nose, respectively, for the fast diagnosis of viral infections.


Assuntos
/diagnóstico , /isolamento & purificação , Biomarcadores/metabolismo , Líquidos Corporais/virologia , Testes Respiratórios , Cromatografia Líquida , Expiração , Humanos , Lasers , Programas de Rastreamento/métodos , Nanotecnologia , Técnicas Fotoacústicas , Saliva/virologia , Sensibilidade e Especificidade , Lágrimas/virologia
14.
J Clin Virol ; 136: 104760, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33610926

RESUMO

The new coronavirus infection (COVID-19) is a major public health concern, with a high burden and risk for infection among patients and healthcare workers. Saliva droplets containing SARS-COV-2 are a major vector for COVID-19 infection, making saliva a promising alternative for COVID-19 testing using nasopharyngeal swab samples. To diagnose COVID-19 patients in the field, a point-of-care test (POCT) using saliva was conceptualized. We have developed a simple method for extracting RNA from saliva samples using semi-alkaline proteinase, a sputum homogenizer typically used for preparing samples for tuberculosis testing, and a subsequent simple heating step with no need for centrifugation or RNA extraction. Further, we newly developed a triplex reverse transcription loop-mediated isothermal amplification approach (RT-LAMP) which utilizes colorimetric readout using a heat block, with results evaluated with the unaided eye. In 44 clinical patients suspected of having COVID-19 infection, the test took 45 min, and resulted in a diagnostic sensitivity of 82.6% (19/23) and diagnostic specificity of 100% (21/21), compared to the reference standard. The limit of detection was 250 copies/reaction (25,000 copies/mL). Our newly developed POCT approach achieved simple RNA extraction and constant RT-LAMP detection. This POCT has the potential to be used for simple inspection stations in a field setting, helping reduce the risk of infection by simplifying and accelerating testing for COVID-19.


Assuntos
/métodos , Testes Imediatos , RNA Viral/análise , Saliva/virologia , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , /isolamento & purificação
15.
PLoS One ; 16(2): e0246867, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33566873

RESUMO

Widespread diagnostic testing is needed to reduce transmission of COVID-19 and manage the pandemic. Effective mass screening requires robust and sensitive tests that reliably detect the SARS-CoV-2 virus, including asymptomatic and pre-symptomatic infections with a low viral count. Currently, the most accurate tests are based on detection of viral RNA by RT-PCR. We developed a method to process COVID-19 specimens that simplifies and increases the sensitivity of viral RNA detection by direct RT-qPCR, performed without RNA purification. In the method, termed Alkaline-Glycol Processing (AG Processing), a SARS-CoV-2-containing biological specimen, such as saliva or a swab-collected suspension, is processed at pH 12.2 to 12.8 for 5 min at room temperature. An aliquot of the AG-processed specimen is used for detection of SARS-CoV-2 RNA by direct RT-qPCR. AG processing effectively lyses viruses and reduces the effect of inhibitors of RT-PCR that are present in biological specimens. The sensitivity of detecting viral RNA using AG processing is on par with methods that include a viral RNA purification step. One copy of SARS-CoV-2 virus per reaction, equivalent to 300 copies per ml of saliva, is detectable in the AG-processed saliva. The LOD is 300 viral copies per ml of initial saliva specimen. AG processing works with saliva specimens or swab specimens collected into Universal Transport Medium, is compatible with heat treatment of saliva, and was confirmed to work with a range of CDC-approved RT-qPCR products and kits. Detection of SARS-CoV-2 RNA using AG processing with direct RT-qPCR provides a reliable and scalable diagnostic test for COVID-19 that can be integrated into a range of workflows, including automated settings.


Assuntos
/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , RNA Viral/genética , /isolamento & purificação , Humanos , Limite de Detecção , Programas de Rastreamento , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Saliva/virologia , Manejo de Espécimes , Fatores de Tempo
16.
mSphere ; 6(1)2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-33408231

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) environmental contamination occurs through droplets and biological fluids released in the surroundings from patients or asymptomatic carriers. Surfaces and objects contaminated by saliva or nose secretions represent a risk for indirect transmission of coronavirus disease 2019 (COVID-19). We assayed surfaces from hospital and living spaces to identify the presence of viral RNA and the spread of fomites in the environment. Anthropic contamination by droplets and biological fluids was monitored by detecting the microbiota signature using multiplex quantitative real-time PCR (qPCR) on selected species and massive sequencing on 16S amplicons. A total of 92 samples (flocked swabs) were collected from critical areas during the pandemic, including indoor (three hospitals and three public buildings) and outdoor surfaces exposed to anthropic contamination (handles and handrails, playgrounds). Traces of biological fluids were frequently detected in spaces open to the public and on objects that are touched with the hands (>80%). However, viral RNA was not detected in hospital wards or other indoor and outdoor surfaces either in the air system of a COVID hospital but only in the surroundings of an infected patient, in consistent association with droplet traces and fomites. Handled objects accumulated the highest level of multiple contaminations by saliva, nose secretions, and fecal traces, further supporting the priority role of handwashing in prevention. In conclusion, anthropic contamination by droplets and biological fluids is widespread in spaces open to the public and can be traced by qPCR. Monitoring fomites can support evaluation of indirect transmission risks for coronavirus or other flu-like viruses in the environment.IMPORTANCE Several studies have evaluated the presence of SARS-CoV-2 in the environment. Saliva and nasopharyngeal droplets can land on objects and surfaces, creating fomites. A suitable indicator would allow the detection of droplets or biofluids carrying the virus. Therefore, we searched for viral RNA and droplets and fomites on at risk surfaces. We monitored by qPCR or next generation sequencing (NGS) droplets through their microbiota. Although the study was performed during the pandemic, SARS-CoV-2 was not significantly found on surfaces, with the only exception of environmental areas near infectious patients. Conversely, anthropic contamination was frequent, suggesting a role for biofluids as putative markers of indirect transmission and risk assessment. Moreover, all SARS-CoV-2-contaminated surfaces showed droplets' microbiota. Fomite monitoring by qPCR may have an impact on public health strategies, supporting prevention of indirect transmission similarly to what is done for other communicable diseases (e.g., influenza and influenza-like infections).


Assuntos
Exposição Ambiental/análise , Fômites/virologia , Hospitais , Reação em Cadeia da Polimerase em Tempo Real , /fisiologia , /prevenção & controle , /virologia , Humanos , RNA Viral , Saliva/virologia , Propriedades de Superfície
17.
Microb Biotechnol ; 14(1): 307-316, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33497538

RESUMO

In the fight against the recent COVID-19 pandemics, testing is crucial. Nasopharyngeal swabs and real-time RT-PCR are used for the detection of the viral RNA. The collection of saliva is non-invasive, pain-free and does not require trained personnel. An alternative to RT-PCR is loop-mediated isothermal amplification coupled with reverse transcription (RT-LAMP) that is easy to perform, quick and does not require a thermal cycler. The aim of this study was to test whether SARS-CoV-2 RNA can be detected directly in saliva using RT-LAMP. We have tested 16 primer mixes from the available literature in three rounds of sensitivity assays. The selected RT-LAMP primer mix has a limit of detection of 6 copies of viral RNA per reaction in comparison with RT-PCR with 1 copy per reaction. Whole saliva, as well as saliva collected using Salivette collection tubes, interfered with the RT-LAMP analysis. Neither Chelex-100 nor protease treatment of saliva prevented the inhibitory effect of saliva. With the addition of the ribonuclease inhibitor, the sensitivity of the RT-LAMP assay was 12 copies per reaction of RNA in Salivette® saliva samples and 6 copies per reaction of RNA in whole saliva samples. This study shows that it is possible to combine the use of saliva and RT-LAMP for SARS-CoV-2 RNA detection without RNA extraction which was confirmed on a small set of correctly diagnosed clinical samples. Further studies should prove whether this protocol is suitable for point of care testing in the clinical setting.


Assuntos
/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Saliva/virologia , Humanos , Reação em Cadeia da Polimerase em Tempo Real
18.
Viruses ; 13(2)2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33494175

RESUMO

Zika virus (ZIKV) RNA has been found to remain in human semen for up to one year after infection, but the presence of Flavivirus antigens in the different compartments of semen has been largely unexplored. Following the introduction of ZIKV in Nicaragua (2016), a prospective study of patients with clinical symptoms consistent with ZIKV was conducted in León to investigate virus shedding in different fluids. ZIKV infection was confirmed in 16 male subjects (≥18 years of age) by RT-qPCR in either blood, saliva or urine. Of these, three provided semen samples at 7, 14, 21, 28, 60 and 180 days postsymptom onset (DPSO) for Flavivirus antigens and RNA studies. These cases were compared with 19 asymptomatic controls. Flavivirus antigens were examined by immunofluorescence (IF) using the 4G2 Mabs, and confocal microscopy was used to explore fluorescence patterns. The three (100%) symptomatic subjects and 3 (16%) of the 19 asymptomatic subjects had Flavivirus antigens and viral RNA in the spermatozoa fraction. The percentage of IF Flavivirus-positive spermatozoa cells ranged from 1.9% to 25% in specimens from symptomatic subjects, as compared with 0.8% to 3.8% in specimens from asymptomatic controls. A marked IF-pattern in the cytoplasmic droplets and tail of the spermatozoa was observed. The sperm concentrations (45 × 106/mL vs. 63.5 × 106/mL, p = 0.041) and the total motility percentage (54% vs. 75%, p = 0.009) were significantly lower in specimens from ZIKV-positive than in those of ZIKV-negative. In conclusion, this study demonstrated the presence of Flavivirus antigens and RNA within a time frame of 28 DPSO in sperm cells of symptomatic and asymptomatic subjects during the ZIKV epidemic. These findings have implications for public health, in terms of nonarthropod-born, silent transmission facilitated by sperm cells and potential transmission from asymptomatic males to pregnant women, with consequences to the fetus.


Assuntos
Antígenos Virais/análise , Flavivirus/isolamento & purificação , RNA Viral/análise , Espermatozoides/virologia , Infecção por Zika virus/virologia , Zika virus/isolamento & purificação , Adulto , Antígenos Virais/sangue , Antígenos Virais/urina , Flavivirus/genética , Imunofluorescência , Humanos , Masculino , RNA Viral/sangue , RNA Viral/urina , Reação em Cadeia da Polimerase em Tempo Real , Saliva/virologia , Sêmen/virologia , Espermatozoides/química , Eliminação de Partículas Virais , Adulto Jovem , Zika virus/genética
19.
Emerg Microbes Infect ; 10(1): 235-241, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33467982

RESUMO

BACKGROUND: Pediatric COVID-19 studies exploring the relationships between NPS and saliva viral loads, clinical and immunological profiles are lacking. METHODS: Demographics, immunological profiles, nasopharyngeal swab (NPS), and saliva samples collected on admission, and hospital length of stay (LOS) were assessed in children below 18 years with COVID-19. FINDINGS: 91 patients were included between March and August 20 20. NPS and saliva viral loads were correlated (r = 0.315, p = 0.01). Symptomatic patients had significantly higher NPS and saliva viral loads than asymptomatic patients. Serial NPS and saliva viral load measurements showed that the log10 NPS (r = -0.532, p < 0.001) and saliva (r = -0.417, p < 0.001) viral loads for all patients were inversely correlated with the days from symptom onset with statistical significance. Patients with cough, sputum, and headache had significantly higher saliva, but not NPS, viral loads. Higher saliva, but not NPS, viral loads were associated with total lymphopenia, CD3 and CD4 lymphopenia (all p < 0.05), and were inversely correlated with total lymphocyte (r = -0.43), CD3 (r = -0.55), CD4 (r = -0.60), CD8 (r = -0.41), B (r = -0.482), and NK (r = -0.416) lymphocyte counts (all p < 0.05). INTERPRETATION: Saliva viral loads on admission in children correlated better with clinical and immunological profiles than NPS.


Assuntos
/virologia , Saliva/virologia , Carga Viral , Adolescente , /diagnóstico , Criança , Pré-Escolar , Feminino , Humanos , Contagem de Linfócitos , Masculino , Nasofaringe/virologia , /genética
20.
J Am Chem Soc ; 143(4): 1722-1727, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33481575

RESUMO

The development of new methods for direct viral detection using streamlined and ideally reagent-free assays is a timely and important, but challenging, problem. The challenge of combatting the COVID-19 pandemic has been exacerbated by the lack of rapid and effective methods to identify viral pathogens like SARS-CoV-2 on-demand. Existing gold standard nucleic acid-based approaches require enzymatic amplification to achieve clinically relevant levels of sensitivity and are not typically used outside of a laboratory setting. Here, we report reagent-free viral sensing that directly reads out the presence of viral particles in 5 minutes using only a sensor-modified electrode chip. The approach relies on a class of electrode-tethered sensors bearing an analyte-binding antibody displayed on a negatively charged DNA linker that also features a tethered redox probe. When a positive potential is applied, the sensor is transported to the electrode surface. Using chronoamperometry, the presence of viral particles and proteins can be detected as these species increase the hydrodynamic drag on the sensor. This report is the first virus-detecting assay that uses the kinetic response of a probe/virus complex to analyze the complexation state of the antibody. We demonstrate the performance of this sensing approach as a means to detect, within 5 min, the presence of the SARS-CoV-2 virus and its associated spike protein in test samples and in unprocessed patient saliva.


Assuntos
Técnicas Biossensoriais/métodos , /virologia , Técnicas Eletroquímicas/métodos , Vírion/isolamento & purificação , Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Eletrodos , Humanos , Testes Imediatos , Saliva/virologia
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