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1.
Genes (Basel) ; 11(10)2020 10 12.
Article in English | MEDLINE | ID: covidwho-905037

ABSTRACT

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.


Subject(s)
Automation, Laboratory/standards , Clinical Laboratory Techniques/standards , Real-Time Polymerase Chain Reaction/standards , Reverse Transcriptase Polymerase Chain Reaction/standards , Automation, Laboratory/methods , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Humans , Limit of Detection , Real-Time Polymerase Chain Reaction/methods , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/methods
2.
Euro Surveill ; 25(27)2020 07.
Article in English | MEDLINE | ID: covidwho-845124

ABSTRACT

Laboratory preparedness with quality-assured diagnostic assays is essential for controlling the current coronavirus disease (COVID-19) outbreak. We conducted an external quality assessment study with inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples to support clinical laboratories with a proficiency testing option for molecular assays. To analyse SARS-CoV-2 testing performance, we used an online questionnaire developed for the European Union project RECOVER to assess molecular testing capacities in clinical diagnostic laboratories.


Subject(s)
Clinical Laboratory Techniques/methods , Clinical Laboratory Techniques/standards , Coronavirus Infections/diagnosis , Coronavirus/isolation & purification , Molecular Diagnostic Techniques/methods , Pandemics , Pneumonia, Viral/diagnosis , Betacoronavirus , Clinical Laboratory Services , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Disease Outbreaks , Europe , Humans , Pandemics/prevention & control , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction/standards , Reverse Transcriptase Polymerase Chain Reaction/standards , Sensitivity and Specificity , Surveys and Questionnaires
3.
EBioMedicine ; 61: 103036, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-844322

ABSTRACT

BACKGROUND: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. FINDINGS: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. INTERPRETATION: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. FUNDING: Detailed funding information is available at the end of the manuscript.


Subject(s)
Bacterial Proteins/metabolism , Betacoronavirus/genetics , CRISPR-Associated Proteins/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Endodeoxyribonucleases/metabolism , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Coronavirus Infections/pathology , Coronavirus Infections/virology , Humans , Limit of Detection , Nasal Cavity/virology , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Pandemics , Phosphoproteins , Pneumonia, Viral/diagnosis , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Polyproteins , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/standards , Reference Standards , Viral Proteins/genetics , Viral Proteins/metabolism
4.
Ann Oncol ; 31(10): 1320-1335, 2020 10.
Article in English | MEDLINE | ID: covidwho-804478

ABSTRACT

We established an international consortium to review and discuss relevant clinical evidence in order to develop expert consensus statements related to cancer management during the severe acute respiratory syndrome coronavirus 2-related disease (COVID-19) pandemic. The steering committee prepared 10 working packages addressing significant clinical questions from diagnosis to surgery. During a virtual consensus meeting of 62 global experts and one patient advocate, led by the European Society for Medical Oncology, statements were discussed, amended and voted upon. When consensus could not be reached, the panel revised statements until a consensus was reached. Overall, the expert panel agreed on 28 consensus statements that can be used to overcome many of the clinical and technical areas of uncertainty ranging from diagnosis to therapeutic planning and treatment during the COVID-19 pandemic.


Subject(s)
Betacoronavirus , Consensus , Coronavirus Infections/therapy , Medical Oncology/standards , Neoplasms/therapy , Pneumonia, Viral/therapy , Societies, Medical/standards , Coronavirus Infections/epidemiology , Coronavirus Infections/immunology , Disease Management , Europe/epidemiology , Granulocyte Colony-Stimulating Factor/pharmacology , Granulocyte Colony-Stimulating Factor/therapeutic use , Humans , Medical Oncology/methods , Neoplasms/epidemiology , Neoplasms/immunology , Pandemics/prevention & control , Pneumonia, Viral/epidemiology , Pneumonia, Viral/immunology , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology , Telemedicine/methods , Telemedicine/standards
5.
J Clin Lab Anal ; 34(10): e23554, 2020 Oct.
Article in English | MEDLINE | ID: covidwho-796047

ABSTRACT

BACKGROUND: To compare the diagnostic efficacy between two different real-time reverse transcription polymerase chain reaction (RT-PCR) test kits for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection and provide references for laboratories. METHODS: Throat swab samples from 18 hospitalized patients were clinically diagnosed with coronavirus disease 2019 (COVID-19) and 100 hospitalized patients without COVID-19 were collected. SARS-CoV-2 nucleic acid was detected in throat swab samples with RT-PCR test kits from Sansure Biotech Inc (Hunan, China) and Shanghai BioGerm Medical Biotechnology Co., Ltd.(Shanghai, China). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and kappa value were analyzed, and three parallel tests were performed with three weakly positive samples. RESULTS: The sensitivity, specificity, PPV, NPV, and kappa value of the Sansure PCR kit were 0.833, 1.000, 1.000, 0.971, and 0.894, respectively, and the sensitivity, specificity, PPV, NPV, and kappa value of the BioGerm PCR kit were 0.944, 1.000, 1.000, 0.990, and 0.966, respectively. For the three parallel tests, the coefficient of variation value of the BioGerm PCR kit in all three samples was the smallest for both the ORF1ab and N gene. CONCLUSION: The detection efficacy of the BioGerm PCR kit for SARS-CoV-2 nucleic acid detection was relatively higher than that of the Sansure PCR kit.


Subject(s)
Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction , Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Clinical Laboratory Techniques/standards , Humans , Pandemics , Pharynx/virology , Predictive Value of Tests , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards
6.
J Bras Nefrol ; 42(2 suppl 1): 4-8, 2020 Aug 26.
Article in English, Portuguese | MEDLINE | ID: covidwho-740457

ABSTRACT

The Covid-19 pandemic brought several challenges to the healthcare system: diagnosis, treatment and measures to prevent the spread of the disease. With the greater availability and variety of diagnostic tests, it is essential to properly interpret them. This paper intends to help dialysis units concerning the use of clinical criteria and diagnostic tests for decision making regarding the discontinuation of isolation of patients with suspected or confirmed Covid-19, as well as the return to work activities for employees with suspected or confirmed Covid-19.


Subject(s)
Betacoronavirus , Clinical Laboratory Techniques/standards , Coronavirus Infections/diagnosis , Nephrology/standards , Pneumonia, Viral/diagnosis , Renal Dialysis , Return to Work , Algorithms , Brazil , Checklist , Clinical Decision-Making , Clinical Laboratory Techniques/methods , Coronavirus Infections/epidemiology , Humans , Occupational Diseases/diagnosis , Pandemics , Patient Isolation , Pneumonia, Viral/epidemiology , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Societies, Medical/standards , Urology Department, Hospital/standards
7.
Int J Mol Sci ; 21(16)2020 Aug 07.
Article in English | MEDLINE | ID: covidwho-714483

ABSTRACT

Sensitive molecular assays are critical for coronavirus disease 2019 (COVID-19) diagnosis. Here, we designed and evaluated two single-tube nested (STN) real-time RT-PCR assays, targeting SARS-CoV-2 RdRp/Hel and N genes. Both STN assays had a low limit of detection and did not cross react with other human coronaviruses and respiratory viruses. Using 213 initial respiratory specimens from suspected COVID-19 patients, the sensitivity of both the STN COVID-19-RdRp/Hel and the STN COVID-19-N assays was 100% (99/99), while that of the comparator non-nested N assay was 95% (94/99). Among 108 follow-up specimens from confirmed COVID-19 patients who tested negative by the non-nested COVID-19-RdRp/Hel assay, 28 (25.9%) were positive for SARS-CoV-2 by the STN COVID-19-RdRp/Hel or the STN COVID-19-N assay. To evaluate the performance of our novel STN assays in pooled specimens, we created four sample pools, with each pool consisting of one low positive specimen and 49 negative specimens. While the non-nested COVID-19-RdRp/Hel assay was positive in only one of four sample pools (25%), both of the STN assays were positive in two of four samples pools (50%). In conclusion, the STN assays are highly sensitive and specific for SARS-CoV-2 detection. Their boosted sensitivity offers advantages in non-traditional COVID-19 testing algorithms such as saliva screening and pooled sample screening during massive screening.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Molecular Diagnostic Techniques/methods , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/pathogenicity , Coronavirus Infections/diagnosis , Humans , Molecular Diagnostic Techniques/standards , Nucleocapsid Proteins/genetics , Pandemics , Phosphoproteins , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/standards , Respiratory Mucosa/virology , Sensitivity and Specificity , Viral Nonstructural Proteins/genetics
8.
Ann Oncol ; 31(10): 1320-1335, 2020 10.
Article in English | MEDLINE | ID: covidwho-704911

ABSTRACT

We established an international consortium to review and discuss relevant clinical evidence in order to develop expert consensus statements related to cancer management during the severe acute respiratory syndrome coronavirus 2-related disease (COVID-19) pandemic. The steering committee prepared 10 working packages addressing significant clinical questions from diagnosis to surgery. During a virtual consensus meeting of 62 global experts and one patient advocate, led by the European Society for Medical Oncology, statements were discussed, amended and voted upon. When consensus could not be reached, the panel revised statements until a consensus was reached. Overall, the expert panel agreed on 28 consensus statements that can be used to overcome many of the clinical and technical areas of uncertainty ranging from diagnosis to therapeutic planning and treatment during the COVID-19 pandemic.


Subject(s)
Betacoronavirus , Consensus , Coronavirus Infections/therapy , Medical Oncology/standards , Neoplasms/therapy , Pneumonia, Viral/therapy , Societies, Medical/standards , Coronavirus Infections/epidemiology , Coronavirus Infections/immunology , Disease Management , Europe/epidemiology , Granulocyte Colony-Stimulating Factor/pharmacology , Granulocyte Colony-Stimulating Factor/therapeutic use , Humans , Medical Oncology/methods , Neoplasms/epidemiology , Neoplasms/immunology , Pandemics/prevention & control , Pneumonia, Viral/epidemiology , Pneumonia, Viral/immunology , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology , Telemedicine/methods , Telemedicine/standards
9.
Emerg Infect Dis ; 26(10): 2353-2360, 2020 10.
Article in English | MEDLINE | ID: covidwho-691167

ABSTRACT

External quality assessment (EQA) is essential for ensuring reliable test results, especially when laboratories are using assays authorized for emergency use for newly emerging pathogens. We developed an EQA panel to assess the quality of real-time reverse transcription PCR assays being used in South Korea to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the participation of 23 public health organization laboratories and 95 nongovernmental laboratories involved in SARS-CoV-2 testing, we conducted qualitative and semiquantitative performance assessments by using pooled respiratory samples containing different viral loads of SARS-CoV-2 or human coronavirus OC43. A total of 110 (93.2%) laboratories reported correct results for all qualitative tests; 29 (24.6%) laboratories had >1 outliers according to cycle threshold values. Our EQA panel identified the potential weaknesses of currently available commercial reagent kits. The methodology we used can provide practical experience for those planning to conduct evaluations for testing of SARS-CoV-2 and other emerging pathogens in the future.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/standards , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/standards , Reverse Transcriptase Polymerase Chain Reaction/standards , Humans , Laboratory Proficiency Testing , Pandemics , Quality Assurance, Health Care , Reagent Kits, Diagnostic/standards , Real-Time Polymerase Chain Reaction/methods , Republic of Korea , Respiratory System/virology , Reverse Transcriptase Polymerase Chain Reaction/methods
10.
Virulence ; 11(1): 964-967, 2020 12.
Article in English | MEDLINE | ID: covidwho-690823

ABSTRACT

Currently, testing for coronavirus is performed with time and personnel consuming PCR assays. The aim of this study was to evaluate the sensitivity, specificity and capacity of a fully automated, random access high-throughput real-time PCR-based diagnostic platform for the detection of SARS-CoV-2. The NeuMoDx N96 system displayed an equal or better detection rate for SARS-CoV-2 compared with the LightCycler 480II system and showed a specificity of 100%. The median PCR run time for all 28 PCR runs was 91 (IQR 84-97) minutes. The capacity of the NeuMoDx N96 could easily surpass the capacity of most currently used molecular test systems and significantly reduce the turn-around time.


Subject(s)
Betacoronavirus/isolation & purification , High-Throughput Nucleotide Sequencing/methods , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/genetics , High-Throughput Nucleotide Sequencing/instrumentation , High-Throughput Nucleotide Sequencing/standards , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/instrumentation , Real-Time Polymerase Chain Reaction/standards , Reproducibility of Results , Sensitivity and Specificity , Time Factors
11.
Euro Surveill ; 25(27)2020 07.
Article in English | MEDLINE | ID: covidwho-652787

ABSTRACT

Laboratory preparedness with quality-assured diagnostic assays is essential for controlling the current coronavirus disease (COVID-19) outbreak. We conducted an external quality assessment study with inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples to support clinical laboratories with a proficiency testing option for molecular assays. To analyse SARS-CoV-2 testing performance, we used an online questionnaire developed for the European Union project RECOVER to assess molecular testing capacities in clinical diagnostic laboratories.


Subject(s)
Clinical Laboratory Techniques/methods , Clinical Laboratory Techniques/standards , Coronavirus Infections/diagnosis , Coronavirus/isolation & purification , Molecular Diagnostic Techniques/methods , Pandemics , Pneumonia, Viral/diagnosis , Betacoronavirus , Clinical Laboratory Services , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Disease Outbreaks , Europe , Humans , Pandemics/prevention & control , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction/standards , Reverse Transcriptase Polymerase Chain Reaction/standards , Sensitivity and Specificity , Surveys and Questionnaires
12.
J Clin Virol ; 129: 104537, 2020 08.
Article in English | MEDLINE | ID: covidwho-633879

ABSTRACT

BACKGROUND: Broad and decentralised testing of SARS-CoV-2 RNA genomes is a WHO-recommended strategy to contain the SARS-CoV-2 pandemic by identifying infected cases in order to minimize onward transmission. With the need to increase the test capacities in Austria, nation-wide numerous laboratories rapidly implemented assays for molecular detection of SARS-CoV-2 based on real-time RT-PCR assays. The objective of this study was to monitor reliability of the laboratory results for SARS-CoV-2 RNA detection through an external quality assessment (EQA) scheme. METHODS: For this, the Center for Virology, Medical University of Vienna was tasked by the Federal Ministry of Social Affairs, Health, Care and Consumer Protection to perform the first Austrian EQA on SARS-CoV-2 which was organised in cooperation with the Austrian Association for Quality Assurance and Standardization of Medical and Diagnostic Tests (ÖQUASTA). Data were analysed on the basis of qualitative outcome of testing in relation to the nucleic acid (NA) extraction and detection methods used. RESULTS AND CONCLUSION: A total of 52 laboratories participated, contributing results from 67 test panels comprising 42 distinct combinations of NA extraction and PCR reagents. By testing 3 positive (CT values: S1, 28.4; S2, 33.6; S3, 38.5) and 1 negative sample, no false-positive results were obtained by any of the laboratories. Otherwise, 40/67 tests (60 %) detected all positive samples correctly as positive, but 25/67 tests (37 %) did not detect the weakest positive sample (S3), and 3 % reported S2 and S3 as false-negative. Improvement in test sensitivity by focusing on NA extraction and/or PCR-based detection is recommended.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Clinical Laboratory Techniques/standards , Coronavirus Infections/diagnosis , Laboratory Proficiency Testing/organization & administration , Molecular Diagnostic Techniques/methods , Molecular Diagnostic Techniques/standards , Pneumonia, Viral/diagnosis , Austria , Diagnostic Errors/statistics & numerical data , Humans , Pandemics , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Sensitivity and Specificity
14.
Emerg Microbes Infect ; 9(1): 1175-1179, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-361278

ABSTRACT

Different primers/probes sets have been developed all over the world for the nucleic acid detection of SARS-CoV-2 by quantitative real time polymerase chain reaction (qRT-PCR) as a standard method. In our recent study, we explored the feasibility of droplet digital PCR (ddPCR) for clinical SARS-CoV-2 nucleic acid detection compared with qRT-PCR using the same primer/probe sets issued by Chinese Center for Disease Control and Prevention (CDC) targeting viral ORF1ab or N gene, which showed that ddPCR could largely minimize the false negatives reports resulted by qRT-PCR [Suo T, Liu X, Feng J, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv [Internet]. 2020;2020.02.29.20029439. Available from: https://medrxiv.org/content/early/2020/03/06/2020.02.29.20029439.abstract]. Here, we further stringently compared the performance of qRT-PCR and ddPCR for 8 primer/probe sets with the same clinical samples and conditions. Results showed that none of 8 primer/probe sets used in qRT-PCR could significantly distinguish true negatives and positives with low viral load (10-4 dilution). Moreover, false positive reports of qRT-PCR with UCDC-N1, N2 and CCDC-N primers/probes sets were observed. In contrast, ddPCR showed significantly better performance in general for low viral load samples compared to qRT-PCR. Remarkably, the background readouts of ddPCR are relatively lower, which could efficiently reduce the production of false positive reports.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Multiplex Polymerase Chain Reaction , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction , DNA Primers , DNA Probes , Humans , Multiplex Polymerase Chain Reaction/methods , Pandemics , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Sensitivity and Specificity , Viral Load
15.
Emerg Microbes Infect ; 9(1): 1233-1237, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-291196

ABSTRACT

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay on anal swabs was recently reported to be persistently positive even after throat testing was negative during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, data about the consistent performance of RT-PCR assay on throat and anal swabs remain limited in paediatric patients. Here, we retrospectively reviewed RT-PCR-testing results of 212 paediatric patients with suspected SARS-CoV-2 infection at Wuhan Children's Hospital. The diagnostic potential of these two types of specimens showed significant difference (positive rate: 78.2% on throat swabs vs. 52.6% on anal swabs, McNemar Test P = 0.0091) and exhibited a weak positive consistency (Kappa value was 0.311, P < 0.0001) in paediatric patients. Furthermore, viral loads detected on both throat and anal swabs also showed no significant difference (P = 0.9511) and correlation (Pearson r = 0.0434, P = 0.8406), and exhibited an inconsistent kinetic change through the course of SARS-CoV-2 infection. Besides, viral loads in the throat and anal swabs were correlated with different types of immune states, immune-reactive phase, and the resolution phase/immunologic tolerance, respectively. These findings revealed that RT-PCR-testing on throat and anal swabs showed significant difference for monitoring SARS-CoV-2 infection and correlated with different immune state in paediatric patients.


Subject(s)
Anal Canal/virology , Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Pharynx/virology , Pneumonia, Viral/virology , Viral Load , Betacoronavirus/genetics , Child , China/epidemiology , Coronavirus Infections/diagnosis , Coronavirus Infections/epidemiology , Female , Humans , Male , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/epidemiology , Real-Time Polymerase Chain Reaction/standards , Retrospective Studies
16.
Emerg Infect Dis ; 26(8)2020 Aug.
Article in English | MEDLINE | ID: covidwho-245493

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the etiologic agent associated with coronavirus disease, which emerged in late 2019. In response, we developed a diagnostic panel consisting of 3 real-time reverse transcription PCR assays targeting the nucleocapsid gene and evaluated use of these assays for detecting SARS-CoV-2 infection. All assays demonstrated a linear dynamic range of 8 orders of magnitude and an analytical limit of detection of 5 copies/reaction of quantified RNA transcripts and 1 x 10-1.5 50% tissue culture infectious dose/mL of cell-cultured SARS-CoV-2. All assays performed comparably with nasopharyngeal and oropharyngeal secretions, serum, and fecal specimens spiked with cultured virus. We obtained no false-positive amplifications with other human coronaviruses or common respiratory pathogens. Results from all 3 assays were highly correlated during clinical specimen testing. On February 4, 2020, the Food and Drug Administration issued an Emergency Use Authorization to enable emergency use of this panel.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Nucleocapsid Proteins/genetics , Pneumonia, Viral/diagnosis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Biomarkers/analysis , Centers for Disease Control and Prevention, U.S. , Coronavirus Infections/virology , DNA Primers/chemical synthesis , DNA Primers/genetics , Feces/virology , Fluoresceins/chemistry , Fluorescent Dyes/chemistry , Humans , Limit of Detection , Nasopharynx/virology , Pandemics , Phosphoproteins , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction/standards , Reproducibility of Results , Sputum/virology , United States
17.
Am J Forensic Med Pathol ; 41(3): 143-151, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-215983

ABSTRACT

As a result of the 2019 novel human coronavirus (COVID-19) global spread, medical examiner/coroner offices will inevitably encounter increased numbers of COVID-19-infected decedents at autopsy. While in some cases a history of fever and/or respiratory distress (eg, cough or shortness of breath) may suggest the diagnosis, epidemiologic studies indicate that the majority of individuals infected with COVID-19 develop mild to no symptoms. Those dying with-but not of-COVID-19 may still be infectious, however. While multiple guidelines have been issued regarding autopsy protocol in cases of suspected COVID-19 deaths, there is some variability in the recommendations. Additionally, limited recommendations to date have been issued regarding scene investigative protocol, and there is a paucity of publications characterizing COVID-19 postmortem gross and histologic findings. A case of sudden unexpected death due to COVID-19 is presented as a means of illustrating common autopsy findings, as well as diagnostic and biosafety considerations. We also review and summarize the current COVID-19 literature in an effort to provide practical evidence-based biosafety guidance for medical examiner-coroner offices encountering COVID-19 at autopsy.


Subject(s)
Autopsy/standards , Betacoronavirus/isolation & purification , Containment of Biohazards/standards , Coronavirus Infections/mortality , Coronavirus Infections/pathology , Pneumonia, Viral/mortality , Pneumonia, Viral/pathology , Betacoronavirus/genetics , Centers for Disease Control and Prevention, U.S. , Female , Humans , Middle Aged , Mortuary Practice/methods , Mortuary Practice/standards , Pandemics , Real-Time Polymerase Chain Reaction/standards , Triage , United States
18.
Clin Chim Acta ; 507: 139-142, 2020 Aug.
Article in English | MEDLINE | ID: covidwho-130086

ABSTRACT

BACKGROUND: The detection of SARS-CoV-2 RNA by real-time reverse transcription-polymerase chain reaction (rRT-PCR) is used to confirm the clinical diagnosis of COVID-19 by molecular diagnostic laboratories. We developed a multiplex rRT-PCR methodology for the detection of SARS-CoV-2 RNA. METHODS: Three genes were used for multiplex rRT-PCR: the Sarbecovirus specific E gene, the SARS-CoV-2 specific N gene, and the human ABL1 gene as an internal control. RESULTS: Good correlation of Cq values was observed between the simplex and multiplex rRT-PCR methodologies. Low copies (<25 copies/reaction) of SARS-CoV-2 RNA were detected by the novel multiplex rRT-PCR method. CONCLUSION: The proposed multiplex rRT-PCR methodology will enable highly sensitive detection of SARS-CoV-2 RNA, reducing reagent use and cost, and time required by clinical laboratory technicians.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/genetics , Multiplex Polymerase Chain Reaction , Pneumonia, Viral/diagnosis , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/standards , Humans , Pandemics , Pneumonia, Viral/genetics , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/standards , Sputum/chemistry , Sputum/virology
20.
West J Emerg Med ; 21(3): 470-472, 2020 Apr 13.
Article in English | MEDLINE | ID: covidwho-72826

ABSTRACT

Many public officials are calling for increased testing for the 2019 novel coronavirus disease (COVID-19), and some governments have taken extraordinary measures to increase the availability of testing. However, little has been published about the sensitivity and specificity of the reverse transcriptase-polymerase chain reaction (RT-PCR) nasopharyngeal swabs that are commonly used for testing. This narrative review evaluates the literature regarding the accuracy of these tests, and makes recommendations based on this literature. In brief, a negative RT-PCR nasopharyngeal swab test is insufficient to rule out COVID-19. Thus, over-reliance on the results of the test may be dangerous, and the push for widespread testing may be overstated.


Subject(s)
Betacoronavirus , Clinical Laboratory Techniques/standards , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Coronavirus , Diagnostic Tests, Routine , False Negative Reactions , Humans , Nasal Cavity , Pandemics , Real-Time Polymerase Chain Reaction/standards , Sensitivity and Specificity , Specimen Handling
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