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1.
Viruses ; 14(2)2022 02 02.
Article in English | MEDLINE | ID: covidwho-1667351

ABSTRACT

Efficient, wide-scale testing for SARS-CoV-2 is crucial for monitoring the incidence of the infection in the community. The gold standard for COVID-19 diagnosis is the molecular analysis of epithelial secretions from the upper respiratory system captured by nasopharyngeal (NP) or oropharyngeal swabs. Given the ease of collection, saliva has been proposed as a possible substitute to support testing at the population level. Here, we used a novel saliva collection device designed to favour the safe and correct acquisition of the sample, as well as the processivity of the downstream molecular analysis. We tested 1003 nasopharyngeal swabs and paired saliva samples self-collected by individuals recruited at a public drive-through testing facility. An overall moderate concordance (68%) between the two tests was found, with evidence that neither system can diagnose the infection in 100% of the cases. While the two methods performed equally well in symptomatic individuals, their discordance was mainly restricted to samples from convalescent subjects. The saliva test was at least as effective as NP swabs in asymptomatic individuals recruited for contact tracing. Our study describes a testing strategy of self-collected saliva samples, which is reliable for wide-scale COVID-19 screening in the community and is particularly effective for contact tracing.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19 Nucleic Acid Testing/standards , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Saliva/virology , COVID-19/diagnosis , COVID-19/virology , Female , Humans , Male , Mass Screening , Nasopharynx/virology , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , Specimen Handling/methods
2.
Anal Bioanal Chem ; 414(5): 1773-1785, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1653430

ABSTRACT

Nucleic acid tests to detect the SARS-CoV-2 virus have been performed worldwide since the beginning of the COVID-19 pandemic. For the quality assessment of testing laboratories and the performance evaluation of molecular diagnosis products, reference materials (RMs) are required. In this work, we report the production of a lentiviral SARS-CoV-2 RM containing approximately 12 kilobases of its genome including common diagnostics targets such as RdRp, N, E, and S genes. The RM was measured with multiple assays using two different digital PCR platforms. To measure the homogeneity and stability of the lentiviral SARS-CoV-2 RM, reverse transcription droplet digital PCR (RT-ddPCR) was used with in-house duplex assays. The copy number concentration of each target gene in the extracted RNA solution was then converted to that of the RM solution. Their copy number values are measured to be from 1.5 × 105 to 2.0 × 105 copies/mL. The RM has a between-bottle homogeneity of 4.80-8.23% and is stable at 4 °C for 1 week and at -70 °C for 6 months. The lentiviral SARS-CoV-2 RM closely mimics real samples that undergo identical pre-analytical processes for SARS-CoV-2 molecular testing. By offering accurate reference values for the absolute copy number of viral target genes, the developed RM can be used to improve the reliability of SARS-CoV-2 molecular testing.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Genome, Viral , RNA, Viral/genetics , Reagent Kits, Diagnostic/standards , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Nucleic Acid Testing/standards , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/metabolism , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Gene Dosage , Gene Expression , Humans , Jurkat Cells , Lentivirus/genetics , Lentivirus/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , RNA, Viral/metabolism , RNA, Viral/standards , Reagent Kits, Diagnostic/supply & distribution , Reference Standards , Reproducibility of Results , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Genome Packaging
3.
PLoS One ; 17(1): e0262733, 2022.
Article in English | MEDLINE | ID: covidwho-1643277

ABSTRACT

This study aims at establishing specimens pooling approach for the detection of SARS-CoV-2 using the RT-PCR BGI and Sansure-Biotech kits used in Gabon. To validate this approach, 14 positive samples, stored at -20°C for three to five weeks were analyzed individually (as gold standard) and in pools of five, eight and ten in the same plate. We created 14 pools of 5, 8 and 10 samples using 40 µL from each of the selected positive samples mixed with 4, 7 and 9 confirmed negative counterparts in a total volume of 200 µL, 320 µL and 400 µL for the pools of 5, 8 and 10 respectively. Both individual and pooled samples testing was conducted according to the BGI and Sansure-Biotech RT-PCR protocols used at the Professor Daniel Gahouma Laboratory (PDGL). Furthermore, the pooling method was also tested by comparing results of 470 unselected samples tested in 94 pools and individually. Results of our experiment showed that using a BGI single positive sample with cycle threshold (Ct) value of 28.42, confirmed by individual testing, detection occurred in all the pools. On the contrary samples with Ct >31 were not detected in pools of 10 and for these samples (Ct value as high as 37.17) their detection was possible in pool of 8. Regarding the Sansure-Biotech kit, positive samples were detected in all the pool sizes tested, irrespective of their Ct values. The specificity of the pooling method was 100% for the BGI and Sansure-Biotech RT-PCR assays. The present study found an increase in the Ct values with pool size for the BGI and Sansure-Biotech assays. This trend was statistically significant (Pearson's r = 0.978; p = 0,022) using the BGI method where the mean Ct values were 24.04±1.1, 26.74±1.3, 27.91±1.1 and 28.32±1.1 for the individual, pool of 5, 8 and 10 respectively. The testing of the 470 samples showed that one of the 94 pools had a positive test similar to the individual test using the BGI and Sansure-Biotech kits. The saving of time and economizing test reagents by using the pooling method were demonstrated in this study. Ultimately, the pooling method could be used for the diagnosis of SARS-CoV-2 without modifying the accuracy of results in Gabon. We recommend a maximum pool size of 8 for the BGI kit. For the Sansure-Biotech kit, a maximum pool size of 10 can be used without affecting its accuracy compared to the individual testing.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , RNA, Viral/genetics , SARS-CoV-2/genetics , Specimen Handling/methods , COVID-19/epidemiology , Gabon/epidemiology , Health Services , Humans , Reagent Kits, Diagnostic/standards , SARS-CoV-2/classification , Sensitivity and Specificity
4.
PLoS One ; 16(11): e0260087, 2021.
Article in English | MEDLINE | ID: covidwho-1528723

ABSTRACT

The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Real-Time Polymerase Chain Reaction/methods , Adult , COVID-19 Nucleic Acid Testing/standards , Female , Humans , Male , Nucleic Acid Denaturation , Oligonucleotides/chemistry , Real-Time Polymerase Chain Reaction/standards , Respiratory Mucosa/virology , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Saliva/virology , Sensitivity and Specificity
5.
Transplant Proc ; 53(4): 1126-1131, 2021 May.
Article in English | MEDLINE | ID: covidwho-1525970

ABSTRACT

Coronavirus disease 2019 drastically impacted solid organ transplantation. Lacking scientific evidence, a very stringent but safer policy was imposed on liver transplantation (LT) early in the pandemic. Restrictive transplant guidelines must be reevaluated and adjusted as data become available. Before LT, the prevailing policy requires a negative severe acute respiratory syndrome coronavirus 2 real-time polymerase chain reaction (RT-PCR) of donors and recipients. Unfortunately, prolonged viral RNA shedding frequently hinders transplantation. Recent data reveal that positive test results for viral genome are frequently due to noninfectious and prolonged convalescent shedding of viral genome. Moreover, studies demonstrated that the cycle threshold of quantitative RT-PCR could be leveraged to inform clinical transplant decision-making. We present an evidence-adjusted and significantly less restrictive policy for LT, where risk tolerance is tiered to recipient acuity. In addition, we delineate the pretransplant clinical decision-making, intra- and postoperative management, and early outcome of 2 recipients of a liver graft performed while their RT-PCR of airway swabs remained positive. Convalescent positive RT-PCR results are common in the transplant arena, and the proposed policy permits reasonably safe LT in many circumstances.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , Health Policy , Liver Transplantation/legislation & jurisprudence , SARS-CoV-2/genetics , COVID-19/prevention & control , COVID-19 Nucleic Acid Testing/methods , Female , Humans , Infection Control/legislation & jurisprudence , Infection Control/methods , Liver Transplantation/adverse effects , Male , Middle Aged , Postoperative Complications/prevention & control , Postoperative Complications/virology , Preoperative Care/legislation & jurisprudence , Preoperative Care/methods , Reference Values , Tissue Donors , Virus Shedding
6.
J Clin Lab Anal ; 35(11): e23998, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1525445

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), is detected using real-time RT-PCR. However, there are limitations pertaining to quality control, particularly with respect to establishing quality control measures for extraction of viral nucleic acids. Here, we investigated the quality control measures for the various processes using an extrinsic quality control substance and quality control charts. METHODS: An extrinsic quality control substance was added to the sample, and then, real-time RT-PCR was performed. Samples with negative test results and the corresponding data were analyzed; a quality control chart was created and examined. RESULTS: Data analysis and the quality control charts indicated that SARS-CoV-2 could be reliably detected using real-time RT-PCR, even when different nucleic acid extraction methods were used or when different technicians were employed. CONCLUSION: With the use of quality control substances, it is possible to achieve quality control throughout the process-from nucleic acid extraction to nucleic acid detection-even upon using varying extraction methods. Further, generating quality control charts would guarantee the stable detection of SARS-CoV-2.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , Nucleic Acids/isolation & purification , Quality Control , SARS-CoV-2/genetics , Humans , Retrospective Studies , SARS-CoV-2/isolation & purification
7.
J Infect Dis ; 224(8): 1287-1293, 2021 10 28.
Article in English | MEDLINE | ID: covidwho-1505875

ABSTRACT

BACKGROUND: Previous studies demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be detected for weeks after infection. The significance of this finding is unclear and, in most patients, does not represent active infection. Detection of subgenomic RNA has been proposed to represent productive infection and may be a useful marker for monitoring infectivity. METHODS: We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR) to quantify total and subgenomic nucleocapsid (sgN) and envelope (sgE) transcripts in 185 SARS-CoV-2-positive nasopharyngeal swab samples collected on hospital admission and to relate to symptom duration. RESULTS: We find that all transcripts decline at the same rate; however, sgE becomes undetectable before other transcripts. The median duration of symptoms to a negative test is 14 days for sgE and 25 days for sgN. There is a linear decline in subgenomic compared to total RNA, suggesting that subgenomic transcript copy number is dependent on copy number of total transcripts. The mean difference between total and sgN is 16-fold and the mean difference between total and sgE is 137-fold. This relationship is constant over duration of symptoms, allowing prediction of subgenomic copy number from total copy number. CONCLUSIONS: Subgenomic RNA may be no more useful in determining infectivity than a copy number threshold determined for total RNA.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , RNA, Viral/isolation & purification , SARS-CoV-2/isolation & purification , Viral Load , Aged , COVID-19/transmission , COVID-19/virology , COVID-19 Nucleic Acid Testing/standards , COVID-19 Nucleic Acid Testing/statistics & numerical data , Coronavirus Envelope Proteins/genetics , Coronavirus Nucleocapsid Proteins/genetics , Feasibility Studies , Female , Humans , Male , Middle Aged , Nasopharynx/pathology , Nasopharynx/virology , Phosphoproteins/genetics , Real-Time Polymerase Chain Reaction/statistics & numerical data , Reference Values , Retrospective Studies , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity
8.
Medicine (Baltimore) ; 100(41): e27478, 2021 Oct 15.
Article in English | MEDLINE | ID: covidwho-1501203

ABSTRACT

ABSTRACT: The COVID-19 pandemic has challenged institutions' diagnostic processes worldwide. The aim of this study was to assess the feasibility of an artificial intelligence (AI)-based software tool that automatically evaluates chest computed tomography for findings of suspected COVID-19.Two groups were retrospectively evaluated for COVID-19-associated ground glass opacities of the lungs (group A: real-time polymerase chain reaction positive COVID patients, n = 108; group B: asymptomatic pre-operative group, n = 88). The performance of an AI-based software assessment tool for detection of COVID-associated abnormalities was compared with human evaluation based on COVID-19 reporting and data system (CO-RADS) scores performed by 3 readers.All evaluated variables of the AI-based assessment showed significant differences between the 2 groups (P < .01). The inter-reader reliability of CO-RADS scoring was 0.87. The CO-RADS scores were substantially higher in group A (mean 4.28) than group B (mean 1.50). The difference between CO-RADS scoring and AI assessment was statistically significant for all variables but showed good correlation with the clinical context of the CO-RADS score. AI allowed to predict COVID positive cases with an accuracy of 0.94.The evaluated AI-based algorithm detects COVID-19-associated findings with high sensitivity and may support radiologic workflows during the pandemic.


Subject(s)
Artificial Intelligence/standards , COVID-19/diagnosis , Lung/diagnostic imaging , Aged , Aged, 80 and over , COVID-19/epidemiology , COVID-19 Nucleic Acid Testing/standards , Feasibility Studies , Female , Humans , Lung/pathology , Male , Middle Aged , Pandemics , Retrospective Studies , SARS-CoV-2 , Tomography, X-Ray Computed
9.
Mayo Clin Proc ; 96(11): 2856-2860, 2021 11.
Article in English | MEDLINE | ID: covidwho-1492385

ABSTRACT

Although there have been several case reports and simulation models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission associated with air travel, there are limited data to guide testing strategy to minimize the risk of SARS-CoV-2 exposure and transmission onboard commercial aircraft. Among 9853 passengers with a negative SARS-CoV-2 polymerase chain reaction test performed within 72 hours of departure from December 2020 through May 2021, five (0.05%) passengers with active SARS-CoV-2 infection were identified with rapid antigen tests and confirmed with rapid molecular test performed before and after an international flight from the United States to Italy. This translates to a case detection rate of 1 per 1970 travelers during a time of high prevalence of active infection in the United States. A negative molecular test for SARS-CoV-2 within 72 hours of international airline departure results in a low probability of active infection identified on antigen testing during commercial airline flight.


Subject(s)
Air Travel , COVID-19 Testing/standards , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , COVID-19/transmission , COVID-19 Nucleic Acid Testing/standards , Humans , Italy , Risk Assessment , United States
10.
Clin Chem ; 68(1): 153-162, 2021 12 30.
Article in English | MEDLINE | ID: covidwho-1462309

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA quantities, measured by reverse transcription quantitative PCR (RT-qPCR), have been proposed to stratify clinical risk or determine analytical performance targets. We investigated reproducibility and how setting diagnostic cutoffs altered the clinical sensitivity of coronavirus disease 2019 (COVID-19) testing. METHODS: Quantitative SARS-CoV-2 RNA distributions [quantification cycle (Cq) and copies/mL] from more than 6000 patients from 3 clinical laboratories in United Kingdom, Belgium, and the Republic of Korea were analyzed. Impact of Cq cutoffs on clinical sensitivity was assessed. The June/July 2020 INSTAND external quality assessment scheme SARS-CoV-2 materials were used to estimate laboratory reported copies/mL and to estimate the variation in copies/mL for a given Cq. RESULTS: When the WHO-suggested Cq cutoff of 25 was applied, the clinical sensitivity dropped to about 16%. Clinical sensitivity also dropped to about 27% when a simulated limit of detection of 106 copies/mL was applied. The interlaboratory variation for a given Cq value was >1000 fold in copies/mL (99% CI). CONCLUSION: While RT-qPCR has been instrumental in the response to COVID-19, we recommend Cq (cycle threshold or crossing point) values not be used to set clinical cutoffs or diagnostic performance targets due to poor interlaboratory reproducibility; calibrated copy-based units (used elsewhere in virology) offer more reproducible alternatives. We also report a phenomenon where diagnostic performance may change relative to the effective reproduction number. Our findings indicate that the disparities between patient populations across time are an important consideration when evaluating or deploying diagnostic tests. This is especially relevant to the emergency situation of an evolving pandemic.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19 , Nucleic Acids , Belgium , COVID-19/diagnosis , Humans , Nucleic Acids/analysis , RNA, Viral/analysis , Reproducibility of Results , Republic of Korea , SARS-CoV-2 , Sensitivity and Specificity , United Kingdom
11.
Nat Commun ; 12(1): 5753, 2021 10 01.
Article in English | MEDLINE | ID: covidwho-1447302

ABSTRACT

Patients with COVID-19 shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. This may be significant for patient health, epidemiology, and diagnosis. However, methods to preserve stool, and to extract and quantify viral RNA are not standardized. We test the performance of three preservative approaches at yielding detectable SARS-CoV-2 RNA: the OMNIgene-GUT kit, Zymo DNA/RNA shield kit, and the most commonly applied, storage without preservative. We test these in combination with three extraction kits: QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. We also test the utility of ddPCR and RT-qPCR for the reliable quantification of SARS-CoV-2 RNA from stool. We identify that the Zymo DNA/RNA preservative and the QiaAMP extraction kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR. Taken together, we recommend a comprehensive methodology for preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , Feces/virology , SARS-CoV-2/isolation & purification , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/genetics , Humans , Phosphoproteins/genetics , Preservation, Biological/standards , RNA, Viral/analysis , RNA, Viral/genetics , Reagent Kits, Diagnostic , Reference Standards , SARS-CoV-2/genetics , Specimen Handling/standards , Viral Load/standards
12.
PLoS One ; 16(9): e0257169, 2021.
Article in English | MEDLINE | ID: covidwho-1405341

ABSTRACT

A prospective study was conducted among different intra and extra-hospital populations of French Guiana to evaluate the performance of saliva testing compared to nasopharyngeal swabs. Persons aged 3 years and older with mild symptoms suggestive of COVID-19 and asymptomatic persons with a testing indication were prospectively enrolled. Nasopharyngeal and salivary samples were stored at 4°C before analysis. Both samples were analyzed with the same Real-time PCR amplification of E gene, N gene, and RdRp gene. Between July 22th and October 28th, 1159 persons were included, of which 1028 were analyzed. When only considering as positives those with 2 target genes with Ct values <35, the sensitivity of RT-PCR on saliva samples was 100% relative to nasopharyngeal samples. Specificity positive and negative predictive values were above 90%. Across a variety of cultures and socioeconomic conditions, saliva tests were generally much preferred to nasopharyngeal tests and persons seemed largely confident that they could self-sample. For positive patients defined as those with the amplification of 2 specific target genes with Ct values below 35, the sensitivity and specificity of RT-PCR on saliva samples was similar to nasopharyngeal samples despite the broad range of challenging circumstances in a tropical environment.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Saliva/virology , Adolescent , Adult , Aged , COVID-19 Nucleic Acid Testing/standards , Child , Child, Preschool , Female , French Guiana , Hospitals/statistics & numerical data , Humans , Male , Middle Aged , Nasopharynx/virology , Patient Acceptance of Health Care , Sensitivity and Specificity , Tropical Climate
15.
Viruses ; 13(9)2021 08 28.
Article in English | MEDLINE | ID: covidwho-1374538

ABSTRACT

The SARS-CoV-2 pandemic has required the development of multiple testing systems to monitor and control the viral infection. Here, we developed a PCR test to screen COVID-19 infections that can process up to ~180 samples per day without the requirement of robotics. For this purpose, we implemented the use of multichannel pipettes and plate magnetics for the RNA extraction step and combined the reverse transcription with the qPCR within one step. We tested the performance of two RT-qPCR kits as well as different sampling buffers and showed that samples taken in NaCl or PBS are stable and compatible with different COVID-19 testing systems. Finally, we designed a new internal control based on the human RNase P gene that does not require a DNA digestion step. Our protocol is easy to handle and reaches the sensitivity and accuracy of the standardized diagnostic protocols used in the clinic to detect COVID-19 infections.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/virology , Polymerase Chain Reaction , SARS-CoV-2 , COVID-19 Nucleic Acid Testing/standards , Humans , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/standards , Reagent Kits, Diagnostic , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Reproducibility of Results , SARS-CoV-2/genetics , Sensitivity and Specificity , Viral Load
16.
J Med Virol ; 93(9): 5538-5543, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1363694

ABSTRACT

In the current coronavirus disease 2019 (COVID-19) pandemic there is a mass screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) happening around the world due to the extensive spread of the infections. There is a high demand for rapid diagnostic tests to expedite the identification of cases and to facilitate early isolation and control spread. Hence this study evaluates six different rapid nucleic acid detection assays that are commercially available for SARS-CoV-2 virus detection. Nasopharyngeal samples were collected from 4981 participants and were tested for the SARS-CoV-2 virus by the gold standard real-time reverse-transcription polymerase chain reaction (RT-PCR) method and with one of these six rapid methods of detection. Evaluation of the rapid nucleic acid detection assays was done by comparing the results of these rapid methods with the gold standard RT-qPCR results for SARS-COV-2 detection. AQ-TOP had the highest sensitivity (98%) and a strong kappa value of 0.943 followed by Genechecker and Abbot ID NOW. The POCKIT (ii RT-PCR) assay had the highest test accuracy of 99.29% followed by Genechecker and Cobas Liat. Atila iAMP showed the highest percentage of invalid reports (35.5%) followed by AQ-TOP with 6% and POCKIT with 3.7% of invalid reports. Genechecker system, Abbott ID NOW, and Cobas Liat were found to have the best performance and agreement when compared with the standard RT-PCR for COVID-19 detection. With further research, these rapid tests have the potential to be employed in large-scale screening of COVID-19.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/standards , Humans , Nasopharynx/virology , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity , United Arab Emirates
17.
J Med Virol ; 93(9): 5481-5486, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1363685

ABSTRACT

As severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections continue, there is a substantial need for cost-effective and large-scale testing that utilizes specimens that can be readily collected from both symptomatic and asymptomatic individuals in various community settings. Although multiple diagnostic methods utilize nasopharyngeal specimens, saliva specimens represent an attractive alternative as they can rapidly and safely be collected from different populations. While saliva has been described as an acceptable clinical matrix for the detection of SARS-CoV-2, evaluations of analytic performance across platforms for this specimen type are limited. Here, we used a novel sensitive RT-PCR/MALDI-TOF mass spectrometry-based assay (Agena MassARRAY®) to detect SARS-CoV-2 in saliva specimens. The platform demonstrated high diagnostic sensitivity and specificity when compared to matched patient upper respiratory specimens. We also evaluated the analytical sensitivity of the platform and determined the limit of detection of the assay to be 1562.5 copies/ml. Furthermore, across the five individual target components of this assay, there was a range in analytic sensitivities for each target with the N2 target being the most sensitive. Overall, this system also demonstrated comparable performance when compared to the detection of SARS-CoV-2 RNA in saliva by the cobas® 6800/8800 SARS-CoV-2 real-time RT-PCR Test (Roche). Together, we demonstrate that saliva represents an appropriate matrix for SARS-CoV-2 detection on the novel Agena system as well as on a conventional real-time RT-PCR assay. We conclude that the MassARRAY® system is a sensitive and reliable platform for SARS-CoV-2 detection in saliva, offering scalable throughput in a large variety of clinical laboratory settings.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , Diagnostic Tests, Routine/standards , RNA, Viral/genetics , SARS-CoV-2/genetics , Saliva/virology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/standards , Benchmarking , COVID-19/virology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/methods , Diagnostic Tests, Routine/instrumentation , Diagnostic Tests, Routine/methods , Humans , Limit of Detection , Nasopharynx/virology , Specimen Handling/standards , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/instrumentation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
18.
J Med Virol ; 93(9): 5333-5338, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1363672

ABSTRACT

The accurate laboratory detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a crucial element in the fight against coronavirus disease 2019 (COVID-19). Reverse transcription-polymerase chain reaction testing on combined oral and nasopharyngeal swab (ONPS) suffers from several limitations, including the need for qualified personnel, the discomfort caused by invasive nasopharyngeal sample collection, and the possibility of swab and transport media shortage. Testing on saliva would represent an advancement. The aim of this study was to compare the concordance between saliva samples and ONPS for the detection of SARS-CoV-2 on various commercial and laboratory-developed tests (LDT). Individuals were recruited from eight institutions in Quebec, Canada, if they had SARS-CoV-2 RNA detected on a recently collected ONPS, and accepted to provide another ONPS, paired with saliva. Assays available in the different laboratories (Abbott RealTime SARS-CoV-2, Cobas® SARS-CoV-2, Simplexa™ COVID-19 Direct, Allplex™ 2019-nCoV, RIDA®GENE SARS-CoV-2, and an LDT preceded by three different extraction methods) were used to determine the concordance between saliva and ONPS results. Overall, 320 tests were run from a total of 125 saliva and ONPS sample pairs. All assays yielded similar sensitivity when saliva was compared to ONPS, with the exception of one LDT (67% vs. 93%). The mean difference in cycle threshold (∆C t ) was generally (but not significantly) in favor of the ONPS for all nucleic acid amplification tests. The maximum mean ∆​​​​​C t was 2.0, while individual ∆C t varied importantly from -17.5 to 12.4. Saliva seems to be associated with sensitivity similar to ONPS for the detection of SARS-CoV-2 by various assays.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , Diagnostic Tests, Routine/standards , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/epidemiology , COVID-19/virology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/methods , Diagnostic Tests, Routine/instrumentation , Diagnostic Tests, Routine/methods , Humans , Mouth/virology , Nasopharynx/virology , Quebec/epidemiology , Saliva/virology , Sensitivity and Specificity , Specimen Handling/standards
19.
Sci Rep ; 11(1): 16193, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1351975

ABSTRACT

We have optimised a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from extracted RNA for clinical application. We improved the stability and reliability of the RT-LAMP assay by the addition of a temperature-dependent switch oligonucleotide to reduce self- or off-target amplification. We then developed freeze-dried master mix for single step RT-LAMP reaction, simplifying the operation for end users and improving long-term storage and transportation. The assay can detect as low as 13 copies of SARS-CoV2 RNA per reaction (25-µL). Cross reactivity with other human coronaviruses was not observed. We have applied the new RT-LAMP assay for testing clinical extracted RNA samples extracted from swabs of 72 patients in the UK and 126 samples from Greece and demonstrated the overall sensitivity of 90.2% (95% CI 83.8-94.7%) and specificity of 92.4% (95% CI 83.2-97.5%). Among 115 positive samples which Ct values were less than 34, the RT-LAMP assay was able to detect 110 of them with 95.6% sensitivity. The specificity was 100% when RNA elution used RNase-free water. The outcome of RT-LAMP can be reported by both colorimetric detection and quantifiable fluorescent reading. Objective measures with a digitized reading data flow would allow for the sharing of results for local or national surveillance.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , COVID-19 Nucleic Acid Testing/standards , Humans , Molecular Diagnostic Techniques/standards , Nucleic Acid Amplification Techniques/standards , Sensitivity and Specificity
20.
J Infect Dev Ctries ; 15(7): 913-917, 2021 07 31.
Article in English | MEDLINE | ID: covidwho-1339633

ABSTRACT

Early diagnosis is among the crucial measures to control the spread of SARS-CoV-2 infection. To date, reverse transcription polymerase chain reaction (RT-PCR) is the gold standard for COVID-19 testing, but various factors can affect its performance leading to false negative results. Hereby we present a patient with a high clinical suspicion for COVID-19 and had multiple negative RT-PCR results over 5 days. A 22-year-old woman presented with fever, dry cough, nausea, myalgia, headache, and mild dyspnea. Eleven days before, she was in close contact with her father who had tested positive for COVID-19. RT-PCR on nasopharyngeal and oropharyngeal swabs were performed on day 8, 9, and 12 of illness which all came back negative even after she started having a worsening dyspnea and showing an increased lung opacity from radiographic findings on day 11 of illness. Interestingly, her rapid antibody test (VivaDiag™ COVID-19 IgM/IgG rapid test by VivaChek Biotech (HangZhou,China) was positive for anti-SARS-CoV-2 Ig M and Ig G. Due to the worsening condition, she was referred to a tertiary hospital where her RT PCR result was positive on day 13 of illness. After 28 days from her first symptom, she was discharged from the hospital with improved symptoms and chest X-ray. As conclusions, in patients with high suspicion of COVID-19, repeat swab tests are mandatory if previous tests were negative. The diagnosis and treatment plan of COVID-19 should not solely be based on RT-PCR, but also consider the patient's history, symptoms, laboratory result, and radiographic findings.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/standards , SARS-CoV-2/genetics , Antibodies, Viral/blood , COVID-19/virology , False Negative Reactions , Female , Humans , Immunoglobulin M/blood , Nasopharynx/virology , Sensitivity and Specificity , Specimen Handling , Time Factors , Young Adult
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