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1.
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
3.
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
4.
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
5.
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 , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction/standards , Reproducibility of Results , Sputum/virology , United States
6.
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 , Coronavirus Infections/diagnosis , Coronavirus Infections/genetics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/genetics , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/standards , Reverse Transcriptase Polymerase Chain Reaction/standards , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Clinical Laboratory Techniques/standards , Humans , Pandemics , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/methods , Sputum/chemistry , Sputum/virology
8.
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
9.
Indian J Med Res ; 151(2 & 3): 216-225, 2020.
Article in English | MEDLINE | ID: covidwho-32576

ABSTRACT

Background & objectives: An outbreak of respiratory illness of unknown aetiology was reported from Hubei province of Wuhan, People's Republic of China, in December 2019. The outbreak was attributed to a novel coronavirus (CoV), named as severe acute respiratory syndrome (SARS)-CoV-2 and the disease as COVID-19. Within one month, cases were reported from 25 countries. In view of the novel viral strain with reported high morbidity, establishing early countrywide diagnosis to detect imported cases became critical. Here we describe the role of a countrywide network of VRDLs in early diagnosis of COVID-19. Methods: The Indian Council of Medical Research (ICMR)-National Institute of Virology (NIV), Pune, established screening as well as confirmatory assays for SARS-CoV-2. A total of 13 VRDLs were provided with the E gene screening real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay. VRDLs were selected on the basis of their presence near an international airport/seaport and their past performance. The case definition for testing included all individuals with travel history to Wuhan and symptomatic individuals with travel history to other parts of China. This was later expanded to include symptomatic individuals returning from Singapore, Japan, Hong Kong, Thailand and South Korea. Results: Within a week of standardization of the test at NIV, all VRDLs could initiate testing for SARS-CoV-2. Till February 29, 2020, a total of 2,913 samples were tested. This included both 654 individuals quarantined in the two camps and others fitting within the case definition. The quarantined individuals were tested twice - at days 0 and 14. All tested negative on both occasions. Only three individuals belonging to different districts in Kerala were found to be positive. Interpretation & conclusions: Sudden emergence of SARS-CoV-2 and its potential to cause a pandemic posed an unsurmountable challenge to the public health system of India. However, concerted efforts of various arms of the Government of India resulted in a well-coordinated action at each level. India has successfully demonstrated its ability to establish quick diagnosis of SARS-CoV-2 at NIV, Pune, and the testing VRDLs.


Subject(s)
Clinical Laboratory Techniques/standards , Coronavirus Infections/diagnosis , Mass Screening/organization & administration , Pneumonia, Viral/diagnosis , Adolescent , Adult , Aged , Betacoronavirus , Child , Child, Preschool , Female , Humans , India , Infant , Male , Middle Aged , Pandemics , Quality Control , Real-Time Polymerase Chain Reaction/standards , Reverse Transcriptase Polymerase Chain Reaction/standards , Specimen Handling , Young Adult
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