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1.
J Infect Chemother ; 27(1): 117-119, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32994136

RESUMO

The novel coronavirus disease 2019 (COVID-19) is diagnosed by positive result of reverse transcription polymerase chain reaction (RT-PCR) for the novel coronavirus. We concluded that cycle threshold value (Ct-value) of real-time RT-PCR (rRT-PCR) assay could decrease as patients recover. Results of rRT-PCR assay could remain positive among asymptomatic patients for longer than 2 weeks. The discharge criteria of COVID-19 patients using a negative result of rRT-PCR should be reconsidered.


Assuntos
Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adolescente , Adulto , Idoso de 80 Anos ou mais , Doenças Assintomáticas , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Pandemias , Alta do Paciente , Pneumonia Viral/virologia , Índice de Gravidade de Doença , Carga Viral , Adulto Jovem
2.
PLoS One ; 15(11): e0241959, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33166373

RESUMO

The coronavirus disease 2019 (Covid-19) pandemic, caused by SARS-CoV-2, has resulted in a global testing supply shortage. In response, pooled testing has emerged as a promising strategy that can immediately increase testing capacity. In pooled sample testing, multiple samples are combined (or pooled) together and tested as a single unit. If the pool is positive, the individual samples can then be individually tested to identify the positive case(s). Here, we provide support for the adoption of sample pooling with the point-of-care Cepheid Xpert® Xpress SARS-CoV-2 molecular assay. Corroborating previous findings, the limit of detection of this assay was comparable to laboratory-developed reverse-transcription quantitative PCR SARS-CoV-2 tests, with observed detection below 100 copies/mL. The Xpert® Xpress assay detected SARS-CoV-2 after samples with minimum viral loads of 461 copies/mL were pooled in groups of six. Based on these data, we recommend the adoption of pooled testing with the Xpert® Xpress SARS-CoV-2 assay where warranted based on public health needs. The suggested number of samples per pool, or the pooling depth, is unique for each point-of-care testing site and can be determined by the positive test rates. To statistically determine appropriate pooling depth, we have calculated the pooling efficiency for numerous combinations of pool sizes and test rates. This information is included as a supplemental dataset that we encourage public health authorities to use as a guide to make recommendations that will maximize testing capacity and resource conservation.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Humanos , Pandemias , Pneumonia Viral/virologia , Testes Imediatos , RNA Viral/genética , Kit de Reagentes para Diagnóstico , Manejo de Espécimes , Carga Viral
3.
BMC Med ; 18(1): 346, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33143712

RESUMO

BACKGROUND: Tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral ribonucleic acid (RNA) using reverse transcription polymerase chain reaction (RT-PCR) are pivotal to detecting current coronavirus disease (COVID-19) and duration of detectable virus indicating potential for infectivity. METHODS: We conducted an individual participant data (IPD) systematic review of longitudinal studies of RT-PCR test results in symptomatic SARS-CoV-2. We searched PubMed, LitCOVID, medRxiv, and COVID-19 Living Evidence databases. We assessed risk of bias using a QUADAS-2 adaptation. Outcomes were the percentage of positive test results by time and the duration of detectable virus, by anatomical sampling sites. RESULTS: Of 5078 studies screened, we included 32 studies with 1023 SARS-CoV-2 infected participants and 1619 test results, from - 6 to 66 days post-symptom onset and hospitalisation. The highest percentage virus detection was from nasopharyngeal sampling between 0 and 4 days post-symptom onset at 89% (95% confidence interval (CI) 83 to 93) dropping to 54% (95% CI 47 to 61) after 10 to 14 days. On average, duration of detectable virus was longer with lower respiratory tract (LRT) sampling than upper respiratory tract (URT). Duration of faecal and respiratory tract virus detection varied greatly within individual participants. In some participants, virus was still detectable at 46 days post-symptom onset. CONCLUSIONS: RT-PCR misses detection of people with SARS-CoV-2 infection; early sampling minimises false negative diagnoses. Beyond 10 days post-symptom onset, lower RT or faecal testing may be preferred sampling sites. The included studies are open to substantial risk of bias, so the positivity rates are probably overestimated.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Betacoronavirus/genética , Técnicas de Laboratório Clínico , Infecções por Coronavirus/genética , Humanos , Estudos Longitudinais , Pandemias , Pneumonia Viral/genética
4.
Sci Rep ; 10(1): 18899, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144632

RESUMO

Severe acute respiratory coronavirus 2 (SARS-CoV-2) testing reagents are expected to become scarce worldwide. However, little is known regarding whether pooling of samples accurately detects SARS-CoV-2. To validate the feasibility of pooling samples, serial dilution analysis and spike-in experiments were conducted using synthetic DNA and nucleic acids extracted from SARS-CoV-2-positive and -negative patients. Furthermore, we studied 1000 individuals, 667 of whom were "healthy" individuals (195 healthcare workers and 472 hospitalized patients with disorders other than COVID-19 infection), and 333 infection-suspected patients with cough and fever. Serial dilution analysis showed a limit of detection of around 10-100 viral genome copies according to the protocol of the National Institute of Infectious Diseases, Japan. Spike-in experiments demonstrated that RT-qPCR detected positive signals in pooled samples with SARS-CoV-2-negative and -positive patients at 5-, 10-, 20-fold dilutions. By screening with this pooling strategy, by the end of April 2020 there were 12 SARS-CoV-2-positive patients in 333 infection-suspected patients (3.6%) and zero in 667 "healthy" controls. We obtained these results with a total of 538 runs using the pooling strategy, compared with 1000 standard runs. In a prospective study, we successfully detected SARS-CoV-2 using 10- to 20-fold diluted samples of nasopharyngeal swabs from eighteen COVID-19 patients with wide ranges of viral load. Pooling sample is feasible for conserving test reagents and detecting SARS-CoV-2 in clinical settings. This strategy will help us to research the prevalence infected individuals and provide infected-status information to prevent the spread of the virus and nosocomial transmission.


Assuntos
Técnicas de Laboratório Clínico/métodos , Programas de Rastreamento/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Humanos , Limite de Detecção , Programas de Rastreamento/normas , Reprodutibilidade dos Testes , Mucosa Respiratória/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas
5.
Sci Rep ; 10(1): 19004, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33149153

RESUMO

Ecuador is one of the most affected countries, with the coronavirus disease 2019 (COVID-19) infection, in Latin America derived from an ongoing economic crisis. One of the most important methods for COVID-19 detection is the use of techniques such as real time RT-PCR based on a previous extraction/purification of RNA procedure from nasopharyngeal cells using functionalized magnetic nanoparticles (MNP). This technique allows the processing of ~ 10,000 tests per day in private companies and around hundreds per day at local Universities guaranteeing to reach a wide range of the population. However, the main drawback of this method is the need for specialized MNP with a strong negative charge for the viral RNA extraction to detect the existence of the SARS-CoV-2 virus. Here we present a simplified low cost method to produce 10 g of nanoparticles in 100 mL of solution that was scaled to one litter by parallelizing the process 10 times in just two days and allowing for the possibility of making ~ 50,000 COVID-19 tests. This communication helps in reducing the cost of acquiring MNP for diverse biomolecular applications supporting developing country budgets constraints and chemical availability specially during the COVID-19 International Health Emergency.


Assuntos
Técnicas de Laboratório Clínico/métodos , Custos e Análise de Custo , Nanopartículas de Magnetita/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecções por Coronavirus/diagnóstico , Países em Desenvolvimento , Humanos , Nanopartículas de Magnetita/economia , RNA Viral/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia
6.
PLoS One ; 15(11): e0241740, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33137168

RESUMO

SARS-CoV-2 is spreading globally with unprecedented consequences for modern societies. The early detection of infected individuals is a pre-requisite to contain the virus. Currently, purification of RNA from patient samples followed by RT-PCR is the gold standard to assess the presence of this single-strand RNA virus. However, these procedures are time consuming, require continuous supply of specialized reagents, and are prohibitively expensive in resource-poor settings. Here, we report an improved nucleic-acid-based approach to detect SARS-CoV-2 with the ability to detect as little as five viral genome equivalents. The approach delivers results without the need to purify RNA, reduces handling steps, minimizes costs, and allows evaluation by non-specialized equipment. The use of unprocessed swap samples is enabled by employing a heat-stable RNA- and DNA-dependent DNA polymerase, which performs the double task of stringent reverse transcription of RNA at elevated temperatures as well as PCR amplification of a SARS-CoV-2 specific target gene. As results are obtained within 2 hours and can be read-out by a hand-held LED-screen, this novel protocol will be of particular importance for large-scale virus surveillance in economically constrained settings.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Humanos , Nasofaringe/virologia , Pandemias , Pneumonia Viral/virologia , RNA Viral/genética , Temperatura
7.
Trials ; 21(1): 881, 2020 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-33106170

RESUMO

OBJECTIVES: The BCG vaccine, widely used in Brazil in new-borns, induces adjuvant protection for several diseases, including childhood virus infections. BCG activates monocytes and innate memory NK cells which are crucial for the antiviral immune response. Therefore, strategies to prevent COVID-19 in health workers (HW) should be carried out to prevent them becoming unwell so that they can continue to work during the pandemic. The hypothesis is that BCG will improve the innate immune response and prevent symptomatic infection or COVID-19 severity. The primary objective is to verify the effectiveness and safety of the BCG vaccine to prevent or reduce incidence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in the city of Goiânia (Brazil) among HW previously vaccinated with BCG and also its severity and mortality during the pandemic of the disease. Secondary objectives are to estimate the incidence of COVID-19 among these professionals and the innate immune response elicited to BCG. TRIAL DESIGN: This a phase II trial for repositioning BCG as a preventive strategy against COVID-19. The trial is an open-label, parallel-group randomised clinical trial, comparing HW vaccinated with BCG and HW not vaccinated. PARTICIPANTS: The trial will recruit 800 HW of Goiânia - Goiás, Brazil to reach a total of 400 HW included after comorbidities questioning and laboratorial evaluation. Eligibility criteria: Any HW presenting BCG vaccination scar with direct contact with suspected COVID-19 patients for at least 8 hours per week, whether in hospital beds, ICU, or in transportation or admission (nurses, doctors, physiotherapists, nutritionists, receptionists, etc.) who have negative IgM and IgG COVID-19 test. Participants with any of the following characteristics will be excluded: - Have had in the last fifteen days any signs or symptoms of virus infection, including COVID-19; - Have had fever in the last fifteen days; - Have been vaccinated fifteen days before the inclusion; - Have a history or confirmation of any immunosuppressive disease such as HIV, presented solid tumour in the last two years or autoimmune diseases; - Are under preventive medication with antibiotics, steroid anti-inflammatories, or chemotherapy; - Have less than 500 neutrophils per mL of blood; - Have previously been diagnosed with tuberculosis; - Are breastfeeding or pregnant; - Are younger than 18 years old; - Are participating as an investigator in this clinical trial. INTERVENTION AND COMPARATOR: HW will be randomized into the BCG vaccinated group or the BCG unvaccinated control group. The BCG vaccinated group will receive in the right arm, intradermally, a one off dose of 0.1 mL corresponding to approximately 2 x105 to 8 x105 CFU of live, freeze-dried, attenuated BCG Moscow 361-I, Bacillus Calmette Guerin vaccine (Serum Institute of India PVT. LTD.). The unvaccinated control group will not be vaccinated. The HW allocated in both groups will be followed up at specific times points until 180 days post inclusion. The vaccinated and control groups will be compared according to COVID-19 related outcomes. MAIN OUTCOMES: The primary outcomes are the incidence coefficient of infection by SARS-CoV-2 determined by RT-PCR of naso-oropharyngeal swab specimen or rapid lateral flow IgG and IgM test, and presence of general COVID-19 symptoms, disease severity and admission to hospital during the 180 days of follow up. The secondary outcome is the innate immune response elicited 15-20 days after vaccination. RANDOMISATION: The vaccine vial contains approximately 10 doses. In order to optimize the vaccine use, the randomisation was performed in blocks of 20 participants using the platform randomization.com [ http://www.jerrydallal.com/random/permute.htm ]. The randomization was prepared before any HW inclusion. The results were printed and inserted in sealed envelopes that were numbered with BCG-001 to BCG-400. The printed results as well the envelopes had the same numbers. At the time of the randomisation, each participant that meets the inclusion criteria will receive a consecutive participant number [BCG-001-BCG-400]. The sealed envelope with the assigned number, blinded to the researchers, will be opened in front of the participant and the arm allocation will be known. BLINDING (MASKING): There is no masking for the participants or for the healthcare providers. The study will be blinded to the laboratory researchers and to those who will be evaluating the outcomes and performing the statistical analyses. In this case, only the participant identification number will be available. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): Four hundred heath workers will be randomised in two groups. Two hundred participants will be vaccinated, and 200 participants will not be vaccinated. TRIAL STATUS: The protocol approved by the Brazilian Ethical Committee is the seventh version, number CAAE: 31783720.0.0000.5078. The trial has been recruiting since September 20th, 2020. The clinical trial protocol was registered on August 5th, 2020. It is estimated that recruitment will finish by March 2021. TRIAL REGISTRATION: The protocol number was registered on August 5th, 2020 at REBEC (Registro Brasileiro de Ensaios Clínicos). Register number: RBR-4kjqtg and WHO trial registration number UTN: U1111-1256-3892. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.


Assuntos
Vacina BCG/administração & dosagem , Infecções por Coronavirus/prevenção & controle , Imunidade Inata/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Betacoronavirus/imunologia , Brasil/epidemiologia , Estudos de Casos e Controles , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Proteção Cruzada/imunologia , Seguimentos , Pessoal de Saúde/estatística & dados numéricos , Hospitalização/estatística & dados numéricos , Humanos , Imunização Secundária/métodos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Incidência , Injeções Intradérmicas , Células Matadoras Naturais/imunologia , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Segurança , Resultado do Tratamento
8.
BMC Infect Dis ; 20(1): 749, 2020 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-33050892

RESUMO

BACKGROUND: Two months after the outbreak of coronavirus disease 2019 (COVID-19) in Wuhan, China, tens of thousands of hospitalized patients had recovered, and little is known about the follow-up of the recovered patients. METHODS: The clinical characteristics, reverse transcriptase-polymerase chain reaction (RT-PCR) results from throat swab specimens and the results of serological COVID-19 rapid diagnostic test (RDT) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were retrospectively reviewed for a total of 758 recovered patients who were previously hospitalized in 17 hospitals and quarantined at 32 rehabilitation stations in Wuhan, China. RESULTS: In total, 59 patients (7.78%) had recurrent positive findings for COVID-19 on RT-PCR from throat swabs. With regard to antibody detection, 50/59 (84.75%) and 4/59 (6.78%) patients had positive IgG or dual positive IgG/IgM RDT results, respectively. CONCLUSIONS: Some patients who had been quarantined and had subsequently recovered from COVID-19 had recurrent positive RT-PCR results for SARS-CoV-2, and the possibility of transmission of the virus by recovered patients needs further investigation. TRIAL REGISTRATION: Current Controlled Trials ChiCTR2000033580 , Jun 6th 2020. Retrospectively registered.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/epidemiologia , Pneumonia Viral/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Síndrome Respiratória Aguda Grave/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Criança , China/epidemiologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/fisiopatologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Feminino , Seguimentos , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/fisiopatologia , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Estudos Retrospectivos , Fatores de Risco , Síndrome Respiratória Aguda Grave/fisiopatologia , Síndrome Respiratória Aguda Grave/virologia , Adulto Jovem
9.
PLoS One ; 15(10): e0240076, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33022019

RESUMO

Public health emergency of SARS-CoV-2 has facilitated diagnostic testing as a related medical countermeasure against COVID-19 outbreak. Numerous serologic antibody tests have become available through an expedited federal emergency use only process. This paper highlights the analytical characteristic of an ELISA based assay by AnshLabs and three random access immunoassay (RAIA) by DiaSorin, Roche, and Abbott that have been approved for emergency use authorization (EUA), at a tertiary academic center in a low disease-prevalence area. The AnshLabs gave higher estimates of sero-prevalence, over the three RAIA methods. For positive results, AnshLabs had 93.3% and 100% agreement with DiaSorin or Abbott and Roche respectively. For negative results, AnshLabs had 74.3% and 78.3% agreement with DiaSorin and Roche or Abbott respectively. All discrepant samples that were positive by AnshLabs and negative by RAIA tested positive by all-in-one step SARS-CoV-2 Total (COV2T) assay performed on the automated Siemens Advia Centaur XPT analyzer. None of these methods, however, are useful in early diagnosis of SARS-CoV-2.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos , Infecções por Coronavirus/virologia , Testes Diagnósticos de Rotina , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/imunologia
10.
Bol Med Hosp Infant Mex ; 77(5): 228-233, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33064678

RESUMO

Background: Diagnostic testing for coronavirus disease (COVID)-19 is performed using nasopharyngeal swabs. This type of sampling is uncomfortable for the patient, dangerous for health workers, and its high demand has led to a global shortage of swabs. One of the alternative specimens is saliva. However, the optimal conditions for the test have not been established. Methods: Reverse transcription-polymerase chain reaction was used to detect the viral genome in saliva samples kept at room temperature, in the fridge or frozen for 2 days. In addition, the influence of brushing teeth and feeding on the detection of the virus in saliva was addressed. Finally, the efficiency of saliva in revealing the presence of the virus during the hospitalization period was determined in children. Results: The viral genome was consistently detected regardless of the storage conditions of saliva samples. Brushing teeth and feeding did not influence the sensitivity of the test. In hospitalized children, positive results were obtained only during the early days. Conclusions: These results support the idea of the use of saliva as an alternative specimen for diagnostic testing for COVID-19. The viral genome is stable and endures perturbations in the oral cavity. However, clearance of the virus from the mouth during the infection may limit the use of the test only to the early stages of the disease.


Assuntos
Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Saliva/virologia , Adolescente , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Pré-Escolar , Infecções por Coronavirus/virologia , Feminino , Genoma Viral , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/virologia , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Temperatura , Fatores de Tempo
11.
J Breath Res ; 14(4): 042003, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33021206

RESUMO

Diagnosis of SARS-COV-2 infection (COVID-19) is currently based on detection of the viral RNA in nasopharyngeal swab samples by reverse transcription polymerase chain reaction (RT-PCR). However, sampling via nasopharyngeal swabs frequently provokes sneezing or coughing, which results in increased risk of the viral dissemination and environmental contamination. Furthermore, the sensitivity associated with the PCR tests s limited to 60%-70%, which is mainly attributable to technical deficiency in sampling. Given that the disease is transmitted via exhaled aerosol and droplets, and that the exhaled breath condensate (EBC) is the established modality for sampling exhaled aerosol, detection of the viral RNA in EBC is a promising approach for safe and efficient diagnosis of the disease. Subjects are those patients who are diagnosed with COVID-19 by positive nasopharyngeal swab PCR test and admitted to Saitama Medical Center, Japan. EBC samples will be collected using an R-tube® or R-tubeVent® device. Collected EBC samples will be introduced into a nucleic acid purifier. The purified nucleic acids will undergo amplification through RT-PCR for detection and quantification of SARS-COV-2 RNA. To date we have collected eight samples from seven subjects. Among them, two samples from two subjects tested positive for SARS-COV-2 RNA by the RT-PCR. Reflecting the second wave of COVID-19 prevalence in Japan, new admissions of COVID-19 patients to the Saitama Medical Center are increasing, and we are expecting to collect at least 50 EBC samples from 25 patients before the end of this year.


Assuntos
Testes Respiratórios/instrumentação , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Aerossóis/análise , Betacoronavirus , Técnicas de Laboratório Clínico , Tosse , Expiração , Humanos , Japão , Pandemias , RNA Viral/análise , Projetos de Pesquisa , Manejo de Espécimes , Carga Viral
12.
J Vet Sci ; 21(5): e71, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33016018

RESUMO

BACKGROUND: Feline calicivirus (FCV) is a major and highly infectious pathogen in cats worldwide. However, there have been limited studies about the status of FCV infections in Korea. OBJECTIVES: To investigate the current status of FCV infections in stray cats in Korea. METHODS: A novel reverse transcription polymerase chain reaction (RT-PCR) assay was developed based on the conserved nucleotide sequences of reported FCV strains. Field swab samples were collected from 122 cats (2 hospital admitted cats and 120 stray cats) in 2016 and 2017. All the samples were tested by virus isolation and 2 different RT-PCRs, including the novel RT-PCR, for the detection of FCV. RESULTS: The novel RT-PCR assay showed no cross-reactivity to the nucleic acids of the other feline pathogens tested, and the limit of detection was calculated as 10° TCID50/mL based on an in vitro assessment. The novel RT-PCR assay detected 5 positive samples from the 122 field samples, which showed perfect agreement with the results of the virus isolation method. In contrast, another RT-PCR assay used in a previous study in Korea detected no positive samples. The prevalence of FCV infection in stray cats was 2.5% (3/120) based on the results of virus isolation and the novel RT-PCR assays. CONCLUSIONS: The current study is the first report of the detection and prevalence of FCV in stray cats in Korea. The novel RT-PCR assay developed in this study showed high sensitivity and specificity, which indicates a useful diagnostic assay to identify FCV infection in cats.


Assuntos
Infecções por Caliciviridae/veterinária , Calicivirus Felino/isolamento & purificação , Doenças do Gato/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Animais , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Doenças do Gato/virologia , Gatos , Prevalência , República da Coreia/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade
13.
Genes (Basel) ; 11(10)2020 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-33053675

RESUMO

WHO declared the novel coronavirus (COVID-19) outbreak a global pandemic on 11 March 2020. The establishment of standardized RT-qPCR protocols for respiratory secretions testing, as well as sharing of specimens, data, and information became critical. Here, we investigate the analytical performance of two interim RT-qPCR protocols (Charité and Centers for Disease Control (CDC)) for the qualitative detection of SARS-CoV-2 executed in a fully automated platform. Analytical specificity, PCR amplification efficiency, analytical sensitivity (limit of detection), and cross-reactivity were evaluated using contrived samples. The on-going accuracy was evaluated by retrospective analysis of our test results database (real clinical samples). N1, E, and a modified version of RdRP assays presented adequate analytical specificity, amplification efficiency, and analytical sensitivity using contrived samples. The three assays were applied to all individuals who requested the SARS-CoV-2 molecular test assay in our laboratory and it was observed that N1 gave more positive results than E, and E gave more positive results than RdRP (modified). The RdRP and E were removed from the test and its final version, based on N1 assay only, was applied to 30,699 Brazilian individuals (from 19 February 2020 to 8 May 2020). The aggregated test results available in the database were also presented.


Assuntos
Automação Laboratorial/normas , Técnicas de Laboratório Clínico/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Automação Laboratorial/métodos , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
14.
PLoS One ; 15(10): e0241029, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33079951

RESUMO

The SARS-CoV-2 pandemic has resulted in shortages of both critical reagents for nucleic acid purification and highly trained staff as supply chains are strained by high demand, public health measures and frequent quarantining and isolation of staff. This created the need for alternate workflows with limited reliance on specialised reagents, equipment and staff. We present here the validation and implementation of such a workflow for preparing samples for downstream SARS-CoV-2 RT-PCR using liquid handling robots. The rapid sample preparation technique evaluated, which included sample centrifugation and heating prior to RT-PCR, showed a 97.37% (95% CI: 92.55-99.28%) positive percent agreement and 97.30% (95% CI: 90.67-99.52%) negative percent agreement compared to nucleic acid purification-based testing. This method was subsequently adopted as the primary sample preparation method in the Groote Schuur Hospital Virology Diagnostic Laboratory in Cape Town, South Africa.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Laboratórios Hospitalares , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Robótica/métodos , Infecções por Coronavirus/virologia , Humanos , Pandemias , Pneumonia Viral/virologia , RNA Viral/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , África do Sul/epidemiologia , Manejo de Espécimes
15.
Biomed Res Int ; 2020: 7610678, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33029522

RESUMO

Background: There is a shortage of chemical reagents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis and a surge of SARS-CoV-2 cases, especially in limited-resource settings. Therefore, the combination of an optimal assay kit is necessary. Methods: We compared the ability to screen SARS-CoV-2 among three primer-probe sets in two different master mixes, Invitrogen™ SuperScript™ III One-Step RT-PCR and LightCycler Multiplex RNA Virus Master. Results: The assay with TIB-Molbiol, IDT, and Phu Sa sets for LightCycler Multiplex RNA Virus Master or Invitrogen™ SuperScript™ III One-Step RT-PCR showed positive results from a single reaction of triplicate in the three days of 4.8 copies per reaction. R squared and amplification efficiency were 0.97 and ranged from 107 to 108%, respectively. Conclusions: Our findings indicated that TIB-Molbiol, IDT, and Phu Sa primer-probe sets could be beneficial for the laboratory screening of SARS-CoV-2 by RT-qPCR assay of E gene. There is a need to consider the combination of these reagent sets as a new strategy to increase the testing capacity of screening programs for COVID-19.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Primers do DNA/genética , Pneumonia Viral/diagnóstico , Sondas RNA/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/estatística & dados numéricos , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/estatística & dados numéricos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sensibilidade e Especificidade
16.
PLoS One ; 15(9): e0239522, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32960934

RESUMO

The genus Vitivirus in the family Betaflexiviridae includes eleven viruses known to infect grapevine: grapevine vitiviruses A, B, D, E, F, G, H, I, J, L and M (GVA-GVM). Three of these viruses, GVA, GVB and GVD, have been associated with the etiology of rugose wood disease in grapevine and cause agronomically significant losses. The other vitiviruses were more recently discovered and their effects on grapevine are undetermined. To certify grape material for propagation as virus tested, an updated reverse transcription PCR (RT-PCR) assay to detect all known vitiviruses is desirable. To accomplish this, multiple grapevine vitivirus sequences were aligned at the amino acid level to search for conserved motifs. Two highly conserved motifs were found at an ideal distance for RT-PCR detection in the RNA-dependent RNA polymerase region of the replicase protein. The amino acid motifs were back translated to create degenerate primers and used to successfully amplify all eleven grapevine vitiviruses. The RT-PCR primers were used to test a panel of vitivirus-infected vines for inclusivity as well as vines infected with closely related viruses in the Betaflexiviridae family (i.e. grapevine pinot gris virus and grapevine rupestris stem pitting-associated virus) for exclusivity. Broader use of these primers to detect vitiviruses in other plant hosts was investigated. In summary, an end-point RT-PCR assay that detects all the known grapevine vitiviruses and potentially other members of the genus Vitivirus has been developed. The universal assay represents an alternative to individual assays to reduce the work associated with the diagnosis of vitiviruses, including for regulatory purposes.


Assuntos
Flexiviridae/genética , Doenças das Plantas/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Vitis/virologia , Primers do DNA/genética
17.
Diagn Microbiol Infect Dis ; 98(3): 115161, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32947206

RESUMO

In a Clinical Laboratory Improvement Amendments laboratory setting, we evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG detection with 4 lateral flow immunoassays [LFIAs; 2 iterations from BTNX Inc. (n = 457) and 1 each from ACON Laboratories (n = 200) and SD BIOSENSOR (n = 155)]. In a cohort of primarily hospitalized, reverse-transcription polymerase chain reaction-confirmed coronavirus disease 2019 cases, sensitivity at ≥14 days from symptom onset was: BTNX kit 1, 95%; BTNX kit 2, 91%; ACON, 95%; and SD, 92%. All assays showed good concordance with the Abbott SARS-CoV-2 IgG assay at ≥14 days from symptom onset: BTNX kit 1, 99%; BTNX kit 2, 94%; ACON, 99%; and SD, 100%. Specificity, measured using specimens collected prior to SARS-CoV-2 circulation in the United States and "cross-reactivity challenge" specimens, was 98% for BTNX kit 1 and ACON and 100% for BTNX kit 2 and SD. These results suggest that LFIAs may provide adequate results for rapid detection of SARS-CoV-2.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Imunoensaio/métodos , Imunoglobulina G/sangue , Pneumonia Viral/diagnóstico , Humanos , Pandemias , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade
18.
Anal Bioanal Chem ; 412(28): 7977-7988, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32951064

RESUMO

Motivated by the current COVID-19 health crisis, we consider data analysis for quantitative polymerase chain-reaction (qPCR) measurements. We derive a theoretical result specifying the conditions under which all qPCR amplification curves (including their plateau phases) are identical up to an affine transformation, i.e. a multiplicative factor and horizontal shift. We use this result to develop a data analysis procedure for determining when an amplification curve exhibits characteristics of a true signal. The main idea behind this approach is to invoke a criterion based on constrained optimization that assesses when a measurement signal can be mapped to a master reference curve. We demonstrate that this approach: (i) can decrease the fluorescence detection threshold by up to a decade; and (ii) simultaneously improve confidence in interpretations of late-cycle amplification curves. Moreover, we demonstrate that the master curve is transferable reference data that can harmonize analyses between different labs and across several years. Application to reverse-transcriptase qPCR measurements of a SARS-CoV-2 RNA construct points to the usefulness of this approach for improving confidence and reducing limits of detection in diagnostic testing of emerging diseases. Graphical Abstract Left: a collection of qPCR amplification curves. Right: Example of data collapse after affine transformation.


Assuntos
Algoritmos , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Humanos , Pandemias , Pneumonia Viral/diagnóstico , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos
19.
Pediatr Infect Dis J ; 39(11): e369-e372, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32868745

RESUMO

From March 2, 2020, to April 26, 2020, 52,588 reverse transcription polymerase chain reaction (RT-PCR) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were performed in France, 6490 in children and 46,098 in adults. The rate of RT-PCR-positive SARS-CoV-2 tests for children (5.9%) was always less than that for adults (20.3%) but vary according to the epidemic stage. The risk ratio of RT-PCR-positive SARS-CoV-2 tests for adults compared with children was 3.5 (95% confidence interval: 3.2-3.9) for the whole study period.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Adulto , Fatores Etários , Betacoronavirus/isolamento & purificação , Criança , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , França/epidemiologia , Humanos , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
20.
Expert Rev Mol Diagn ; 20(9): 971-983, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32896179

RESUMO

INTRODUCTION: The starting months of 2020 witnessed a global pandemic of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection. The first case of Coronavirus Disease 2019 (COVID-19) was reported in December, 2019 in Wuhan, China and millions of cases and thousands of deaths were reported within five months. Currently, reverse transcription-polymerase chain reaction (RT-PCR) and computed tomography (CT) scanning are clinically prescribed for COVID-19 detection across the globe. AREAS COVERED: This systematic review is focused on currently used diagnostic methods for COVID-19 detection and their future prospects. Online searches on Google Scholar, PubMed and online resources were conducted on the period of year 2017 to mid-2020. Studies investigating laboratory examinations, radiographical analysis, and potential sensors for COVID-19 detection were included. Along with this, the current status of commercially available kits for SARS-CoV-2 coronavirus detection is discussed. EXPERT OPINION: The search has identified the potential applications of nucleic acid technology, diagnostics radiology examinations, and in-vitro diagnostic kits in detection of COVID-19 infections. Despite having their own limitations of each technology, the emerging diagnostic technologies for COVID-19 detection along with undergoing clinical trials are summarized suggesting more collaborations and funding are required for fast track clinical trials.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Técnicas Biossensoriais , Sistemas CRISPR-Cas , Infecções por Coronavirus/diagnóstico por imagem , Prova Pericial , Humanos , Pandemias , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tomografia Computadorizada por Raios X/métodos , Ultrassonografia/métodos
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