Clinical Performance and Analytical Sensitivity of Three SARS-CoV-2 Nucleic Acid Diagnostic Tests.
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ABSTRACT:
SUBMITTER: Freire-Paspuel B
PROVIDER: S-EPMC8045661 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in December 2019 and has quickly become a worldwide pandemic. In response, many diagnostic manufacturers have developed molecular assays for SARS-CoV-2 under the Food and Drug Administration (FDA) Emergency Use Authorization (EUA) pathway. This study compared three of these assays, the Hologic Panther Fusion SARS-CoV-2 assay (Fusion), the Hologic Aptima SARS-CoV-2 assay (Aptima), and the BioFire Defense COVID-19 test (BioFire), to determine analytical and clinical performance as well as workflow. All three assays showed similar limits of detection (LODs) using inactivated virus, with 100% detection, ranging from 500 to 1,000 genome equivalents/ml, whereas use of a quantified RNA transcript standard showed the same trend but had values ranging from 62.5 to 125 copies/ml, confirming variability in absolute quantification of reference standards. The clinical correlation found that the Fusion and BioFire assays had a positive percent agreement (PPA) of 98.7%, followed by the Aptima assay at 94.7%, compared to the consensus result. All three assays exhibited 100% negative percent agreement (NPA). Analysis of discordant results revealed that all four samples missed by the Aptima assay had cycle threshold (Ct ) values of >37 by the Fusion assay. In conclusion, while all three assays showed similar relative LODs, we showed differences in absolute LODs depending on which standard was employed. In addition, the Fusion and BioFire assays showed better clinical performance, while the Aptima assay showed a modest decrease in overall PPA. These findings should be kept in mind when making platform testing decisions.
| S-EPMC7448672 | biostudies-literature
Project description:PurposeTo cope with the SARS-CoV-2 epidemic, several rapid nucleic acid assays have been approved for use, but the analytical performance has not been well evaluated. In this report, two key performance parameters, analytical sensitivity (limit of detection) and reproducibility, of three approved rapid nucleic acid assays were assessed using heat-inactivated SARS-CoV-2 culture supernatants quantified by digital PCR.MethodsThe LOD (limit of detection) and reproducibility of three approved rapid nucleic acid assays using their own instruments were assessed, while the LOD and reproducibility of two assays on a 7500 Real-Time instrument were assessed at the same time.ResultsUsing their own instruments, 100% of samples with 1150 copies/mL viral RNA could be detected by the Da An and Coyote assays, while 90% of samples could be detected by the Ustar assay; yet, for 525 copies/mL and 287.5 copies/mL viral RNA, the detection rate of the Ustar assay was higher than that of either the Da An or Coyote assays. However, the three assays did not produce statistically significant results with the three different concentrations of viral RNA (P=0.46, 0.46 and 0.46). Using a 7500 Real-Time instrument, Da An and Coyote assays did not produce statistically significant results with the 1150, 525 and 287.5 copies/mL viral RNA (P>0.99, >0.99 and >0.99). The positive and negative detection rates of the three assays in the intra- and inter-assay stages were 100% on both their own instruments and the 7500 real-time PCR instrument.ConclusionPositive or strongly positive samples can be detected by the rapid nucleic acid assay, but the analytical performance should be optimized, and comprehensive evaluations are also required.
| S-EPMC8374845 | biostudies-literature
Project description:BackgroundCurrently, SARS-CoV-2 RNA detection using real-time reverse-transcription PCR (rRT-PCR) is the standard diagnostic test for COVID-19 infection. Various rRT-PCR assays are currently used worldwide, targeting different genes of the SARS-CoV-2. Here, we compared the analytical sensitivity and clinical performance (sensitivity and specificity) of Allplex SARS-CoV-2/FluA/FluB/RSV assay (Seegene), Standard M nCoV real-time detection kit (SD Biosensor), and U-TOP COVID-19 detection kit (Seasun Biomaterials) for SARS-CoV-2 detection.MethodsTwo hundred and forty-nine nasopharyngeal swab samples were evaluated to compare the clinical performance of the rRT-PCR assays. For the analytical performance evaluation, two RNA controls with known viral loads-SARS-CoV-2 RNA control and SARS-COV-2 B.1.351 RNA control-were used to investigate the potential impact of SARS-CoV-2 variants, particularly the B.1.351 lineage.ResultsLimits of detection ranged from 650 to 1300 copies/ml for rRT-PCR assays, and the mean differences in cycle threshold (Ct ) values of the two RNA controls were within 1.0 for each target in the rRT-PCR assays (0.05-0.73), without any prominent Ct value shift or dropouts in the SARS-COV-2 B.1.351 RNA control. Using the consensus criterion as the reference standard, 89 samples were positive, whereas 160 were negative. The overall clinical performance of rRT-PCR assays was comparable (sensitivity 98.88%-100%; specificity 99.38%-100%), whereas the sensitivities of each target gene were more variable.ConclusionsThe three rRT-PCR assays showed comparable analytical sensitivity and clinical performance. The analytical and clinical sensitivities of each target gene were influenced more by the primer and probe design than the target gene itself.
| S-EPMC8842162 | biostudies-literature
Project description:In December 2019, a novel coronavirus (SARS-CoV-2) was first isolated from Wuhan city, China and within three months, the global community was challenged with a devastating pandemic. The rapid spread of the virus challenged diagnostic laboratories to rapidly develop molecular diagnostic methods. As SARS CoV-2 assays became available for testing on existing molecular platforms, laboratories devoted unprecedented energy and resources into evaluating the analytical performance of the new tests and in some cases developed their own diagnostic assays under FDA-EUA guidance. This study compares the validation of three different molecular assays at the Johns Hopkins Molecular Virology laboratory: the RealStar® SARS-CoV-2 RT-PCR, ePlex® SARS-CoV-2, and the CDC COVID-19 RT-PCR tests. Overall, our studies indicate a comparable analytical performance of the three assays for the detection of SARS-CoV-2.
| S-EPMC7194987 | biostudies-literature
Project description:The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began as a cluster of pneumonia cases in Wuhan, China before spreading to over 200 countries and territories on six continents in less than six months. Despite rigorous global containment and quarantine efforts to limit the transmission of the virus, COVID-19 cases and deaths have continued to increase, leaving devastating impacts on the lives of many with far-reaching effects on the global society, economy and healthcare system. With over 43 million cases and 1.1 million deaths recorded worldwide, accurate and rapid diagnosis continues to be a cornerstone of pandemic control. In this review, we aim to present an objective overview of the latest nucleic acid-based diagnostic tests for the detection of SARS-CoV-2 that have been authorized by the Food and Drug Administration (FDA) under emergency use authorization (EUA) as of 31 October 2020. We systematically summarize and compare the principles, technologies, protocols and performance characteristics of amplification- and sequencing-based tests that have become alternatives to the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. We highlight the notable features of the tests including authorized settings, along with the advantages and disadvantages of the tests. We conclude with a brief discussion on the current challenges and future perspectives of COVID-19 diagnostics.
| S-EPMC7823986 | biostudies-literature
Project description:BACKGROUND:Tuberculous pleurisy (TBP) is the most common form of extrapulmonary tuberculosis (TB). However, rapid diagnostic methods with high accuracy for tuberculous pleurisy are urgently needed. In the present study, we evaluated the diagnostic accuracy of Xpert MTB/RIF, LAMP and SAT-TB assay with pleural fluids from culture-positive TBP patients. METHODS:We prospectively enrolled 300 patients with exudative pleural effusions used as the samples for Xpert MTB/RIF, LAMP and SAT-TB assay. Of these, 265 including 223 patients diagnosed with TBP and 42 non-TBP patients used as controls were analyzed. RESULTS:The sensitivities of Xpert MTB/RIF (27.4%), LAMP (26.5%) and SAT-TB assay (32.3%) were significantly higher than that of pleural effusion smear (14.3%, X2?=?20.65, P?<? 0.001), whereas they were much lower than expected for the analysis of pleural effusion samples. Both SAT-TB assay and Xpert MTB/RIF demonstrated high specificities (100%) and PPVs (100%), but the NPVs of all of the tests were?<?22%. The area under ROC curve of pleural effusion smear, LAMP, Xpert MTB/RIF and SAT-TB assays was 0.524 (95% CI 0.431-0.617), 0.632 (95% CI 0.553-0.71), 0.637 (95% CI 0.56-0.714) and 0.673 (95% CI 0.6-0.745). SAT-TB assays had the highest AUC. CONCLUSION:Nucleic acid amplification tests are not the first choice in the diagnosis of tuberculous pleurisy. In this type of test, SAT-TB is recommended because of its low cost, relatively more accurate compared with the other two tests. This prospective study was approved by The Ethics Committee of the Shanghai Pulmonary Hospital (approval number: K19-148). TRIAL REGISTRATION:ClinicalTrials.gov identifier: ChiCTR1900026234 (Retrospectively registered). The registration date is September 28, 2019.
| S-EPMC7092483 | biostudies-literature
Project description:BackgroundAn optimised standard experimental setup across different hospitals is urgently needed to ensure consistency in nucleic acid test results for SARS-CoV-2 detection. A standard comparison across different nucleic acid tests and their optimal experimental setups is not present. We assessed the performance of three common nucleic acid tests, namely digital PCR (dPCR), quantitative PCR (qPCR), and loop-mediated isothermal amplification (LAMP), to detect SARS-CoV-2 in clinical settings.MethodsIn this systematic review and meta-analysis we compared sensitivity and specificity of qPCR, dPCR, and LAMP and their performances when different experimental setups (namely specimen type used, use of RNA extraction, primer-probe sets, and RNA extraction methods) are applied. We searched PubMed, BioRxiv, MedRxiv, SciFinder, and ScienceDirect for studies and preprints published between Feb 29 and Dec 15, 2020. Included dPCR, qPCR, and LAMP studies using any type of human specimens should report the number of true-positive, true-negative, false-positive, and false-negative cases with Emergency Use Authorization (EUA)-approved PCR assays as the comparator. Studies with a sample size of less than ten, descriptive studies, case studies, reviews, and duplicated studies were excluded. Pooled sensitivity and specificity were computed from the true and false positive and negative cases using Reitsma's bivariate random-effects and bivariate latent class models. Test performance reported in area under the curve (AUC) of the three nucleic acid tests was further compared by pooling studies with similar experimental setups (eg, tests that used RNA extracted pharyngeal swabs but with either the open reading frame 1ab or the N primer). Heterogeneity was assessed and reported in I 2 and τ2.FindingsOur search identified 1277 studies of which we included 66 studies (11 dPCR, 32 qPCR, and 23 LAMP) with 15 017 clinical samples in total in our systematic review and 52 studies in our meta-analysis. dPCR had the highest pooled diagnostic sensitivity (94·1%, 95% CI 88·9-96·6, by Reitsma's model and 95·8%, 54·9-100·0, by latent class model), followed by qPCR (92·7%, 88·3-95·6, and 93·4%, 60·9-99·9) and LAMP (83·3%, 76·9-88·2, and 86·2%, 20·7-99·9), using EUA-approved PCR kits as the reference standard. LAMP was the most specific with a pooled estimate of 96·3% (93·8-97·8) by Reitsma's model and 94·3% (49·1-100·0) by latent class model, followed by qPCR (92·9%, 87·2-96·2, and 93·1%, 47·1-100·0) and dPCR (78·5%, 57·4-90·8, and 73·8%, 0·9-100·0). The overall heterogeneity was I 2 0·5% (τ2 2·79) for dPCR studies, 0% (4·60) for qPCR studies, and 0% (3·96) for LAMP studies. AUCs of the three nucleic acid tests were the highest and differed the least between tests (ie, AUC>0·98 for all tests) when performed with RNA extracted pharyngeal swabs using SARS-CoV-2 open reading frame 1ab primer.InterpretationAll three nucleic acid tests consistently perform better with pharyngeal swabs using SARS-CoV-2 open reading frame 1ab primer with RNA extraction. dPCR was shown to be the most sensitive, followed by qPCR and LAMP. However, their accuracy does not differ significantly. Instead, accuracy depends on specific experimental conditions, implying that more efforts should be directed to optimising the experimental setups for the nucleic acid tests. Hence, our results could be a reference for optimising and establishing a standard nucleic acid test protocol that is applicable in laboratories worldwide.FundingUniversity Grants Committee and The Chinese University of Hong Kong.
| S-EPMC8510644 | biostudies-literature
Project description:ObjectiveTo assess the diagnostic performance of lateral flow immunochromatographic assays (LFAs) of 4 different manufacturers to identify SARS-CoV-2 antibodies (IgM, IgG, or total), comparing them with the nucleic acid amplification test (NAAT) or the clinical defined test (definite or probable SARS-CoV-2 infection, respectively).MethodsOne hundred nineteen serum samples were randomly selected by convenience and distributed in the following groups: (1) group with SARS-CoV-2 infection (n = 82; RT-qPCR positive [definite, n = 70] and probable [n = 12]); (2) other diseases (n = 27; other viruses identified [n = 8] and SARS of other etiologies [n = 19]); and (3) healthy control group (n = 10). LFAs of 4 manufacturers were compared: MedTest Coronavirus (COVID-19) IgG/IgM (MedLevensohn, Brazil); COVID-19 IgG/IgM ECO Test (Ecodiagnóstica, Brazil); Camtech COVID-19 IgM/IgG Rapid Test Kit (Camtech Diagnostics Pte Ltd, Singapore); and 1-Step COVID-19 Test for total antibodies (Guangzhou Wondfo Biotech Co., China).ResultsThe 4 tests studied showed high diagnostic performance characteristics for the diagnoses of definite or probable SARS-CoV-2 infection. The best measures were for the Wondfo test: sensitivity (86.59%; 95% CI: 77.26-93.11%), specificity (100%; 90.51-100%), DOR (257; 60-1,008), LR+ (33.43; 4.82-231.85), LR- (0.13; 0.08-0.23), accuracy (90.76%; 84.06-95.29%), and Matthews correlation coefficient (MCC) 0.82. Although considering only the probable SARS-CoV-2 infection (PCR-) cases, all the kits studied showed limited values.ConclusionOur data demonstrate the excellent performance of LFA for the diagnoses of definite or probable SARS-CoV-2 infection. There was substantial heterogeneity in sensitivities of IgM and IgG antibodies among the different kits. LFA tests cannot replace molecular diagnostics but should be used as an additional screening tool.
| S-EPMC8339017 | biostudies-literature
Project description:BackgroundReliable testing for SARS-CoV-2 is key for the management of the COVID-19 pandemic.AimWe estimate diagnostic accuracy for nucleic acid and antibody tests 5 months into the COVID-19 pandemic, and compare with manufacturer-reported accuracy.MethodsWe reviewed the clinical performance of SARS-CoV-2 nucleic acid and antibody tests based on 93,757 test results from 151 published studies and 20,205 new test results from 12 countries in the European Union and European Economic Area (EU/EEA).ResultsPooling the results and considering only results with 95% confidence interval width ≤ 5%, we found four nucleic acid tests, including one point-of-care test and three antibody tests, with a clinical sensitivity ≥ 95% for at least one target population (hospitalised, mild or asymptomatic, or unknown). Nine nucleic acid tests and 25 antibody tests, 12 of them point-of-care tests, had a clinical specificity of ≥ 98%. Three antibody tests achieved both thresholds. Evidence for nucleic acid point-of-care tests remains scarce at present, and sensitivity varied substantially. Study heterogeneity was low for eight of 14 sensitivity and 68 of 84 specificity results with confidence interval width ≤ 5%, and lower for nucleic acid tests than antibody tests. Manufacturer-reported clinical performance was significantly higher than independently assessed in 11 of 32 and four of 34 cases, respectively, for sensitivity and specificity, indicating a need for improvement in this area.ConclusionContinuous monitoring of clinical performance within more clearly defined target populations is needed.
| S-EPMC8646979 | biostudies-literature
Project description:ObjectivesAntigen rapid diagnostic tests (Ag-RDT) have been developed as reliable tools to control the SARS-CoV-2 pandemic. The objective of our study was to evaluate the diagnostic performance of two Ag-RDTs.MethodsWe evaluated CerTest SARS-CoV-2 Ag One Step Card Test and Panbio COVID-19 Ag Rapid Test Device Ag-RDTs. We included 320 nasopharyngeal samples: 150 PCR negative samples to assess the specificity and 170 PCR positive samples to evaluate the sensitivity. We also evaluated their sensitivity according to cycle threshold (Ct) values and the time from the onset of symptoms. Tests were compared using the McNemar's test and agreement was evaluated using the kappa score (k).ResultsBoth Ag-RDTs showed a specificity of 100 %. Overall sensitivity was 53.5 % for CerTest and 60.0 % for Panbio. For samples with Ct≤ 25, sensitivity was 94.0 % for CerTest and 96.4 % for Panbio (p = 0.500). Regarding samples with Ct>25, sensitivity was 14.0 % for CerTest and 24.4 % for Panbio (p = 0.004). Sensitivity for samples within the first 5 days after the onset of symptoms were 84.8 % for CerTest and 91.3 % for Panbio (p = 0.250) and notably decreased for samples taken after the fifth day. Both Ag-RDTs showed an excellent agreement between them (agreement = 96.7 %, k = 0.920). Agreement with PCR was also excellent for high viral load samples (Ct<25) for CerTest (98.0 %, k = 0.954) and Panbio (98.8 %, k = 0.973).ConclusionsCerTest SARS-CoV-2 and Panbio COVID-19 Ag showed excellent performance and agreement results for samples with high viral loads (Ct ≤ 25) or samples taken within the first 5 days after the onset of symptoms.
| S-EPMC7897407 | biostudies-literature