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The Omicron variant of concern (VOC) has surged in many countries and replaced the previously reported VOC. To identify different Omicron strains/sublineages on a rapid, convenient, and precise platform, we report a novel multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) method in one tube based on the Omicron lineage sequence variants' information. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subvariants were used in a PCR-based assay for rapid identification of Omicron sublineage genotyping in 1000 clinical samples. Several characteristic mutations were analyzed using specific primers and probes for the spike gene, del69-70, and F486V. To distinguish Omicron sublineages (BA.2, BA.4, and BA.5), the NSP1:141-143del in the ORF1a region and D3N mutation in membrane protein occurring outside the spike protein region were analyzed. Results from the real-time PCR assay for one-tube accuracy were compared to those of whole genome sequencing. The developed PCR assay was used to analyze 400 SARS-CoV-2 positive samples. Ten samples determined as BA.4 were positive for NSP1:141-143del, del69-70, and F486V mutations; 160 BA.5 samples were positive for D3N, del69-70, and F486V mutations, and 230 BA.2 samples were without del69-70. Screening these samples allowed the identification of epidemic trends at different time intervals. Our novel one-tube multiplex PCR assay was effective in identifying Omicron sublineages.
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COVID-19 , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , COVID-19/diagnóstico , COVID-19/epidemiologia , SARS-CoV-2/genética , Pandemias , Teste para COVID-19 , Reação em Cadeia da Polimerase Multiplex , Glicoproteína da Espícula de CoronavírusRESUMO
BACKGROUND: The viral load (VL) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals is critical for improving clinical treatment strategies, care, and decisions. Several studies have reported that the initial SARS-CoV-2 VL is associated with disease severity and mortality. Cycle threshold (Ct) values and/or copies/mL are often used to quantify VL. However, a multitude of platforms, primer/probe sets of different SARS-CoV-2 target genes, and reference material manufacturers may cause inconsistent interlaboratory interpretations. The first International Standard for SARS-CoV-2 RNA quantitative assays has allowed diagnostic laboratories to transition SARS-CoV-2 VL results into international units per milliliter (IU/mL). The Cobas SARS-CoV-2 Duo quantitative assay provides VL results expressed in IU/mL. MATERIALS AND METHODS: We enrolled 145 and 50 SARS-CoV-2-positive, hospitalized and 50-negative individuals at the Tri-Service General Hospital, Taiwan from January to May 2022. Each participant's electronic medical record was reviewed to determine asymptomatic, mild, moderate, and severe cases. Nasopharyngeal swabs were collected using universal transport medium. We investigated the association of SARS-CoV-2 VL with disease severity using the Cobas SARS-CoV-2 Duo quantitative assay and its functionality in clinical assessment and decision making to further improve clinical treatment strategies. Limit of detection (LOD) was assessed. RESULTS: All 50 SARS-CoV-2-negative samples confirmed negative for SARS-CoV-2, demonstrating 100 % specificity of the Cobas SARS-CoV-2 Duo assay. Patients with severe symptoms had longer hospital stays, and the length of hospital stay (30.56 days on average) positively correlated with the VL (8.22 ± 1.21 log10 IU/mL). Asymptomatic patients had the lowest VL (5.54 ± 2.06 log10 IU/mL) at admission and the shortest hospital stay (14.1 days on average). CONCLUSIONS: VL is associated with disease severity and duration of hospitalization; therefore, its quantification should be considered when making clinical care decisions and treatment strategies. The Cobas SARS-CoV-2 Duo assay provides a commutable unitage IU/mL for interlaboratory interpretations.
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COVID-19 , Progressão da Doença , SARS-CoV-2 , Carga Viral , Humanos , COVID-19/diagnóstico , COVID-19/virologia , SARS-CoV-2/isolamento & purificação , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Idoso , RNA Viral/análiseRESUMO
Purpose: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major healthcare threat worldwide. Since it was first identified in November 2021, the Omicron (B.1.1.529) variant of SARS-CoV-2 has evolved into several lineages, including BA.1, BA.2-BA.4, and BA.5. SARS-CoV-2 variants might increase transmissibility, pathogenicity, and resistance to vaccine-induced immunity. Thus, the epidemiological surveillance of circulating lineages using variant phenotyping is essential. The aim of the current study was to characterize the clinical outcome of Omicron BA.2 infections among hospitalized COVID-19 patients and to perform an immunological assessment of such cases against SARS-CoV-2. Patients and Methods: We evaluated the analytical and clinical performance of the BioIC SARS-CoV-2 immunoglobulin (Ig)M/IgG detection kit, which was used for detecting antibodies against SARS-CoV-2 in 257 patients infected with the Omicron variant. Results: Poor prognosis was noted in 38 patients, including eight deaths in patients characterized by comorbidities predisposing them to severe COVID-19. The variant-of-concern (VOC) typing and serological analysis identified time-dependent epidemic trends of BA.2 variants emerging in the outbreak of the fourth wave in Taiwan. Of the 257 specimens analyzed, 108 (42%) and 24 (9.3%) were positive for anti-N IgM and IgG respectively. Conclusion: The VOC typing of these samples allowed for the identification of epidemic trends by time intervals, including the B.1.1.529 variant replacing the B.1.617.2 variant. Moreover, antibody testing might serve as a complementary method for COVID-19 diagnosis. The combination of serological testing results with the reverse transcription-polymerase chain reaction cycle threshold value has potential value in disease prognosis, thereby aiding in epidemic investigations conducted by clinicians or the healthcare department.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Teste para COVID-19 , Algoritmos , Anticorpos Antivirais , Imunoglobulina G , Imunoglobulina MRESUMO
OBJECTIVES: We have established a novel 5-in-1 VOC assay to rapidly detect SARS-CoV-2 and immediately distinguish whether positive samples represent variants of concern (VOCs). METHODS: This assay could distinguish among five VOCs: Alpha, Beta, Gamma, Delta, and Omicron, in a single reaction tube. The five variants exhibit different single nucleotide polymorphisms (SNPs) in their viral genome, which can be used to distinguish them. We selected target SNPs in the spike gene, including N501Y, P681R, K417N, and deletion H69/V70 for the assay. RESULTS: The limit of detection of each gene locus was 80 copies per polymerase chain reaction. We observed a high consistency among the results when comparing the performance of our 5-in-1 VOC assay, whole gene sequencing, and the Roche VirSNiP SARS-CoV-2 test in retrospectively analyzing 150 clinical SARS-CoV-2 variant positive samples. The 5-in-1 VOC assay offers an alternative and rapid high-throughput test for most diagnostic laboratories in a flexible sample-to-result platform. CONCLUSION: The assay can also be applied in a commercial platform with the completion of the SARS-CoV-2 confirmation test and identification of its variant within 2.5 hours.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Estudos Retrospectivos , COVID-19/diagnóstico , Reação em Cadeia da Polimerase , DNA Polimerase Dirigida por RNA , Teste para COVID-19RESUMO
PURPOSE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent behind coronavirus disease-2019 (COVID-19). Single-plex reverse transcription-polymerase chain reaction (RT-PCR)-based assays are widely used for COVID-19 detection but exhibit decreased sensitivity and specificity in detecting the rapidly spreading SARS-CoV-2 variants; in contrast, multiplex RT-PCR reportedly yields better results. Here, we aimed at comparatively analyzing the clinical performance of the LabTurboTM AIO COVID-19 RNA testing kit, a multiplex quantitative RT-PCR kit, including a three-target (E, N1, and RNase P), single-reaction, triplex assay used for SARS-CoV-2 detection, with that of the WHO-recommended RT-PCR assay. MATERIALS AND METHODS: Residual, natural, nasopharyngeal swabs obtained from universal transport medium specimens at SARS-CoV-2 testing centers (n = 414) were collected from May to October 2021. For SARS-CoV-2 qRT-PCR, total viral nucleic acid was extracted. The limit of detection (LOD) and the comparative clinical performances of the LabTurboTM AIO COVID-19 RNA kit and the WHO-recommended RT-PCR assay were assessed. Statistical analysis of the correlation was performed and results with R2 values >0.9 were considered to be highly correlated. RESULTS: The LOD of the LabTurboTM AIO COVID-19 RNA kit was 9.4 copies/reaction for the target genes N1 and E. The results obtained from 102 SARS-CoV-2-positive and 312 SARS-CoV-2-negative samples showed 100% correlation with previous WHO-recommended RT-PCR assay results. CONCLUSION: Multiplex qRT-PCR is a critical tool for detecting unknown pathogens and employs multiple target genes. The LabTurboTM AIO COVID-19 RNA testing kit provides an effective and efficient assay for SARS-CoV-2 detection and is highly compatible with SARS-CoV-2 variants.
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OBJECTIVES: With the emergence of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) B.1.1.7 lineage in the ongoing coronavirus disease 2019 (COVID-19) pandemic, Taiwan confronted a COVID-19 flare up in May 2021. Large-scale, accurate, affordable and rapid diagnostic tests such as the lateral flow assay can help to prevent community transmission, but their performance characteristics in real-world conditions and relevant subpopulations remain unclear. METHODS: The COVID-19 Antigen Rapid Test Kit (Eternal Materials, New Taipei City, Taiwan) was used in a high-throughput community testing site; the paired reverse transcription polymerase chain reaction (RT-PCR) results served as a reference for sensitivity and specificity calculations. RESULTS: Of 2096 specimens tested using the rapid antigen test, 70 (3.33%) were positive and 2026 (96.7%) were negative. This clinical performance was compared with the RT-PCR results. The sensitivity and specificity of the rapid antigen test were 76.39% [95% confidence interval (CI) 64.91-85.60%] and 99.26% (95% CI 98.78-99.58%), respectively, with high sensitivity in subjects with cycle threshold values ≤24. Further, the rapid antigen test detected the SARS-CoV-2 B.1.1.7 lineage effectively. CONCLUSIONS: Considering the short turnaround times and lower costs, this simple SARS-CoV-2 antigen detection test for rapid screening combined with RT-PCR as a double confirmatory screening tool can facilitate the prevention of community transmission during COVID-19 emergencies.
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COVID-19 , SARS-CoV-2 , Antígenos Virais , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Taiwan/epidemiologiaRESUMO
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread worldwide. Many variants of SARS-CoV-2 have been reported, some of which have increased transmissibility and/or reduced susceptibility to vaccines. There is an urgent need for variant phenotyping for epidemiological surveillance of circulating lineages. Whole-genome sequencing is the gold standard for identifying SARS-CoV-2 variants, which constitutes a major bottleneck in developing countries. Methodological simplification could increase epidemiological surveillance feasibility and efficiency. We designed a novel multiplex real-time reverse transcriptase PCR (RT-PCR) to detect SARS-CoV-2 variants with S gene mutations. This multiplex PCR typing method was established to detect 9 mutations with specific primers and probes (ΔHV 69/70, K417T, K417N, L452R, E484K, E484Q, N501Y, P681H, and P681R) against the receptor-binding domain of the spike protein of SARS-CoV-2 variants. In silico analyses showed high specificity of the assays. Variants of concern (VOC) typing results were found to be highly specific for our intended targets, with no cross-reactivity observed with other upper respiratory viruses. The PCR-based typing methods were further validated using whole-genome sequencing and a commercial kit that was applied to clinical samples of 250 COVID-19 patients from Taiwan. The screening of these samples allowed the identification of epidemic trends by time intervals, including B.1.617.2 in the third Taiwan wave outbreak. This PCR typing strategy allowed the detection of five major variants of concern and also provided an open-source PCR assay which could rapidly be deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, P.1, and B.1.617.2 variants and of four Omicron mutations on the spike protein (ΔHV 69/70, K417N, N501Y, P681H). IMPORTANCE COVID-19 has spread globally. SARS-CoV-2 variants of concern (VOCs) are leading the next waves of the COVID-19 pandemic. Previous studies have pointed out that these VOCs may have increased infectivity, have reduced vaccine susceptibility, change treatment regimens, and increase the difficulty of epidemic prevention policy. Understanding SARS-CoV-2 variants remains an issue of concern for all local government authorities and is critical for establishing and implementing effective public health measures. A novel SARS-CoV-2 variant identification method based on a multiplex real-time RT-PCR was developed in this study. Five SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, and Omicron) were identified simultaneously using this method. PCR typing can provide rapid testing results with lower cost and higher feasibility, which is well within the capacity for any diagnostic laboratory. Characterizing these variants and their mutations is important for tracking SAR-CoV-2 evolution and is conducive to public infection control and policy formulation strategies.
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COVID-19/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/classificação , COVID-19/epidemiologia , Monitoramento Epidemiológico , Humanos , Mutação , Pandemias , Saúde Pública , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética , Taiwan , Sequenciamento Completo do GenomaRESUMO
Since the late 2020, the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern has been characterized by the emergence of spike protein mutations, and these variants have become dominant worldwide. The gold standard SARS-CoV-2 diagnosis protocol requires two complex processes, namely, RNA extraction and real-time reverse transcriptase polymerase chain reaction (RT-PCR). There is a need for a faster, simpler, and more cost-effective detection strategy that can be utilized worldwide, especially in developing countries. We propose the novel use of direct RT-qPCR, which does not require RNA extraction or a preheating step. For the detection, retrospectively, we used 770 clinical nasopharyngeal swabs, including positive and negative samples. The samples were subjected to RT-qPCR in the N1 and E genes using two different thermocyclers. The limit of detection was 30 copies/reaction for N1 and 60 copies/reaction for E. Analytical sensitivity was assessed for the developed direct RT-qPCR; the sensitivity was 95.69%, negative predictive value was 99.9%, accuracy of 99.35%, and area under the curve was 0.978. This novel direct RT-qPCR diagnosis method without RNA extraction is a reliable and high-throughput alternative method that can significantly save cost, labor, and time during the coronavirus disease 2019 pandemic.
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COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Teste para COVID-19 , Técnicas de Laboratório Clínico/métodos , Análise Custo-Benefício , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estudos Retrospectivos , SARS-CoV-2/genética , Sensibilidade e EspecificidadeRESUMO
BACKGROUND/PURPOSE: Mass screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important to prevent the spread of coronavirus disease 2019 (COVID-19). Pooling samples can increase the number of tests processed. LabTurbo AIO 48 is an automated platform that allows ribonucleic acid extraction and sample analysis on the same instrument. We created a novel pooling assay on this platform for SARS-CoV-2 detection and demonstrated that the pooling strategy increases testing capacity without affecting accuracy and sensitivity. METHODS: Comparative limit of detection (LoD) assessment was performed on the LabTurbo AIO 48 platform and the current standard detection system based on real-time reverse transcription polymerase chain reaction (rRT-PCR) using 55 clinically positive samples. An additional 330 primary clinical samples were assessed. RESULTS: Six samples pooled into one reaction tube were detected in approximately 2.5 h using the World Health Organization rRT-PCR protocol. LabTurbo AIO 48 also demonstrated a higher throughput than our reference rRT-PCR assay, with an LoD of 1000 copies/mL. The overall percentage agreement between the methods for the 330 samples was 100%. CONCLUSION: We created a novel multi-specimen pooling assay using LabTurbo AIO 48 for the robust detection of SARS-CoV-2, allowing high-throughput results; this assay will aid in better control and prevention of COVID-19. The diagnostic assay was cost-effective and time-efficient; thus, the pooling strategy is a practical and effective method for diagnosing large quantities of specimens without compromising precision.
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COVID-19 , SARS-CoV-2 , Humanos , COVID-19/diagnóstico , Teste para COVID-19 , Manejo de Espécimes/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , RNA Viral/genéticaRESUMO
PURPOSE: Accurate molecular diagnostic assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, are needed for epidemiology studies and to support infection-control measures. We evaluated the analytical sensitivity and clinical performance of three sample-to-answer molecular-diagnostics systems for detecting SARS-CoV-2 using 325 nasopharyngeal swab clinical samples from symptomatic patients. METHODS: The BioFire Respiratory Panel 2.1 (RP2.1), cobas Liat SARS-CoV-2 and Influenza A/B, and Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV platforms, which have been granted emergency-use authorization by the US FDA, were tested and compared. RESULTS: The positive percent agreement, negative percent agreement, and overall percent agreement among the three point of care testing systems were 98-100%, including for the wild-type SARS-CoV-2 (non-B.1.1.7) and a variant of concern (B.1.1.7). Notably, the BioFire RP2.1 may fail to detect the SARS-CoV-2 S gene in the B.1.1.7 lineage because of the spike protein mutation. CONCLUSION: All three point of care testing platforms provided highly sensitive, robust, and almost accurate results for rapidly detecting SARS-CoV-2. These automated molecular diagnostic assays can increase the effectiveness of control and prevention measures for infectious diseases.
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BACKGROUND: There is a global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Information on viral genomics is crucial for understanding global dispersion and for providing insight into viral pathogenicity and transmission. Here, we characterized the SARS-CoV-2 genomes isolated from five travelers who returned to Taiwan from the United States of America (USA) between March and April 2020. METHODS: Haplotype network analysis was performed using genome-wide single-nucleotide variations to trace potential infection routes. To determine the genetic variations and evolutionary trajectory of the isolates, the genomes of isolates were compared to those of global virus strains from GISAID. Pharyngeal specimens were confirmed to be SARS-CoV-2-positive by RT-PCR. Direct whole-genome sequencing was performed, and viral assemblies were subsequently uploaded to GISAID. Comparative genome sequence and single-nucleotide variation analyses were performed. RESULTS: The D614G mutation was identified in imported cases, which separated into two clusters related to viruses originally detected in the USA. Our findings highlight the risk of spreading SARS-CoV-2 variants through air travel and the need for continued genomic tracing for the epidemiological investigation and surveillance of SARS-CoV-2 using viral genomic data. CONCLUSIONS: Continuous genomic surveillance is warranted to trace virus circulation and evolution in different global settings during future outbreaks.
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Since the Coronavirus 19 (COVID-19) pandemic, several SARS-CoV-2 variants of concern (SARS-CoV-2 VOC) have been reported. The B.1.1.7 variant has been associated with increased mortality and transmission risk. Furthermore, cluster and possible co-infection cases could occur in the next influenza season or COVID-19 pandemic wave, warranting efficient diagnosis and treatment decision making. Here, we aimed to detect SARS-CoV-2 and other common respiratory viruses using multiplex RT-PCR developed on the LabTurbo AIO 48 open system. We performed a multicenter study to evaluate the performance and analytical sensitivity of the LabTurbo AIO 48 system for SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV) using 652 nasopharyngeal swab clinical samples from patients. The LabTurbo AIO 48 system demonstrated a sensitivity of 9.4 copies/per PCR for N2 of SARS-CoV-2; 24 copies/per PCR for M of influenza A and B; and 24 copies/per PCR for N of RSV. The assay presented consistent performance in the multicenter study. The multiplex RT-PCR applied on the LabTurbo AIO 48 open platform provided highly sensitive, robust, and accurate results and enabled high-throughput detection of B.1.1.7, influenza A/B, and RSV with short turnaround times. Therefore, this automated molecular diagnostic assay could enable streamlined testing if COVID-19 becomes a seasonal disease.
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Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Influenza Humana/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Infecções por Vírus Respiratório Sincicial/diagnóstico , Adulto , Idoso , COVID-19/virologia , Feminino , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Influenza Humana/virologia , Betainfluenzavirus/genética , Betainfluenzavirus/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/isolamento & purificação , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Adulto JovemRESUMO
The COVID-19 pandemic has caused a global public health crisis. Taiwan experienced two waves of imported cases of coronavirus disease 2019 (COVID-19), first from China in January to late February, 2020 then from other countries starting in early March. As of Dec 14, 2020, 733 cases have been reported in Taiwan, with cases of entire families being infected. This study aimed to investigate the clinical characteristics and differentiation of genetic variation among isolates from a cluster of familial COVID-19 infection. The parents had pneumonia (Case 14, father, and Case 15, mother), the elder son (Case 17) had mild cough, and the younger son (Case 18) was asymptomatic. In this study, four full viral genomes were sequenced by Illumina sequencing directly from specimens. Phylogenetic tree analysis revealed that these sequences came from Italy, not China, indicating that no major strain has been circulating in Taiwan. Several novel mutations were observed in the asymptomatic patient, such as nsp2, nsp12, and nsp14. These mutations may be associated with the severity of COVID-19 infection.
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BACKGROUND AND AIMS: Immediate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for preventing the spread of coronavirus disease 2019 (COVID-19). The LabTurbo AIO 48 system is an automated platform that allows nucleic acid extraction and sample analysis on the same instrument, producing faster results without affecting their accuracy. We aimed to independently evaluate the LabTurbo AIO 48 (all-in-one system) for SARS-CoV-2 detection. MATERIALS AND METHODS: Comparative limit of detection (LOD) was assessed on both the LabTurbo AIO 48 and current standard detection system based on real-time reverse transcriptase polymerase chain reaction (RT-PCR), using SARS-CoV-2 RNA control. Additional 125 primary clinical samples were assessed using both the protocols in parallel. RESULTS: The turnaround time from sample to results for 48 samples analyzed on LabTurbo AIO 48 was approximately 2.5 h, whereas that analyzed using the in-house RT-PCR protocol was 4.8 h. LabTurbo AIO 48 also demonstrated higher sensitivity than our reference RT-PCR assay, with a LOD of 9.4 copies/reaction. The overall percentage agreement between both the methods for 125 samples was 100%. CONCLUSION: LabTurbo AIO 48 is a robust detection option for SARS-CoV-2, allowing faster results and, consequently, aiding in better control and prevention of COVID-19.
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Teste para COVID-19/métodos , Ensaios de Triagem em Larga Escala/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , COVID-19/diagnóstico , Humanos , Limite de Detecção , RNA Viral/química , Padrões de Referência , Sensibilidade e Especificidade , Carga ViralRESUMO
SARS-CoV-2 has spread rapidly, causing deaths worldwide. In this study, we evaluated the performance of the BD MAX Open System module for identifying viral pathogens, including SARS-CoV-2, in nasopharyngeal specimens from individuals with symptoms of upper respiratory tract infection. We developed and validated a rapid total nucleic acid extraction method based on real-time reverse transcription-polymerase chain reaction (RT-PCR) for the reliable, high-throughput simultaneous detection of common cold viral pathogens using the BD MAX Platform. The system was evaluated using 205 nasopharyngeal swab clinical samples. For assessment of the limit of detection (LoD), we used SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV) RNA standards. The BD MAX dual multiplex real-time RT-PCR panel demonstrated a sensitivity comparable to that of the World Health Organization-recommended SARS-CoV-2 assay with an LoD of 50 copies/PCR. The LoD of influenza A/B and RSV was 100-200 copies/PCR. The overall percent agreement between the BD MAX panel and laboratory-developed RT-PCR test on 55 SARS-CoV-2-positive clinical samples was 100%. Among the 55 positive cases of COVID-19 analysed, no coinfection was detected. The BD MAX rapid multiplex PCR provides a highly sensitive, robust, and accurate assay for the rapid detection of SARS-CoV-2, influenza A/B, and RSV.
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COVID-19/diagnóstico , Influenza Humana/diagnóstico , Reação em Cadeia da Polimerase/métodos , Infecções por Vírus Respiratório Sincicial/diagnóstico , SARS-CoV-2/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Teste para COVID-19 , Coinfecção/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adulto JovemRESUMO
Coronavirus disease 2019 has become a worldwide pandemic. By April 7, 2020, approximately 1,279,722 confirmed cases were reported worldwide including those in Asia, European Region, African Region and Region of the Americas. Rapid and accurate detection of Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2) is critical for patient care and implementing public health measures to control the spread of infection. In this study, we developed and validated a rapid total nucleic acid extraction method based on real-time RT-PCR for reliable, high-throughput identification of SARS-CoV-2 using the BD MAX platform. For clinical validation, 300 throat swab and 100 sputum clinical samples were examined by both the BD MAX platform and in-house real-time RT-PCR methods, which showed 100% concordant results. This BD MAX protocol is fully automated and the turnaround time from sample to results is approximately 2.5 h for 24 samples compared to 4.8 h by in-house real-time RT-PCR. Our developed BD MAX RT-PCR assay can accurately identify SARS-CoV-2 infection and shorten the turnaround time to increase the effectiveness of control and prevention measures for this emerging infectious disease.
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Epstein-Barr nuclear antigen 1 (EBNA-1) is consistently expressed in all EBV-associated gastric carcinomas. We explored its biological effects in gastric carcinoma cells by expressing the protein in two Epstein-Barr virus (EBV)-negative gastric carcinoma cell lines (SCM1 and TMC1). EBNA1-expressing SCM1 and TMC1 cells displayed no significant differences in growth rates, respectively, compared to those of vector-transfected SCM1 and TMC1 cells in vitro. However, EBNA1 was able to enhance tumorigenicity, the growth rate and the malignant histopathological grade in a xenograft nude mice test. We also evaluated whether EBNA1 caused EBNA1-expressing cells to have enhanced tumorigenicity in an immunocompetent host. We showed that EBNA1-expressing LL/2 cells (derived from lung carcinoma of a Swiss mouse) had enhanced tumorigenicity and growth ability in the immunocompetent allograft Balb/c mice test. These results support the expression of EBNA1 in EBV-associated gastric carcinoma being able to provide advantages of EBV-mediated cell growth and transformation, and to enhance the malignant potential in vivo. In a clonogenic assay, we showed that EBNA1 could reduce the sensitivity of gastric carcinoma cells (SCM1 cells) harboring wild-type p53 to cisplatin, but this was not found in mutant p53-bearing TMC1 cells. In addition, we demonstrated that EBNA1-expressing SCM1 cells, but not EBNA1-expressing TMC1 cells, were associated with reduced expression levels of p53. These findings are compatible with EBNA1 efficiently competing with p53 for binding to ubiquitin-specific protease 7, which causes p53 to degrade by the ubiquitin/proteasome system. These findings suggest that EBNA1 expression is able to reduce the p53 protein level, resulting in the inhibition of its functional activities. Finally, our results suggest that EBV infection with EBNA1 expression in gastric carcinomas provides advantages for host cell survival, growth ability and transformation potential involving escape from immunosurveillance and a reduction in the sensitivity to DNA damage or other apoptotic stress stimuli mediated by suppression of the wild-type p53 protein level; these are distinct from the pathogenesis of EBV-negative gastric carcinomas.