A Study of Analytical and Clinical Sensitivity of Aptima SARS-CoV-2 Assay (Hologic) and Proposals of Complementary Tests for SARS-CoV-2 Detection in Low Viral Load Specimens

Background: Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. Materials and Methods: This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally-collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. Results: Study conduct was between November 2nd 2020 and January 21st 2021. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI: 75.2%-87.5%). Specificity was 99.3% (CI: 98.3-99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI: 86.2%-97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2-56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. Conclusion: The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling.

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Evaluation of accuracy, exclusivity, limit-of-detection and ease-of-use of LumiraDx™: An antigen-detecting point-of-care device for SARS-CoV-2

Infection ◽

10.1007/s15010-021-01681-y ◽

2021 ◽

Author(s):

Lisa J. Krüger ◽

Julian A. F. Klein ◽

Frank Tobian ◽

Mary Gaeddert ◽

Federica Lainati ◽

...

Keyword(s):

Viral Load ◽

Limit Of Detection ◽

Point Of Care ◽

Scale Up ◽

Cross Reactivity ◽

Ease Of Use ◽

Analytical Sensitivity ◽

Rt Pcr ◽

Clinical Sensitivity ◽

Lateral Flow Assays

Abstract Purpose Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤ 85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. Methods This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. Results The study was conducted between November 2nd 2020 and 4th of December 2020. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI 75.2–87.5%). Specificity was 99.3% (CI 98.3–99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI 86.2–97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2–56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. Conclusion The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling. Trial registration number and registration date DRKS00021220 and 01.04.2020

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Analytical sensitivity and effectiveness of different SARS-CoV-2 testing options

10.1101/2021.11.26.21265946 ◽

2021 ◽

Author(s):

Nico Lelie ◽

Marco Koppelman ◽

Harry Van Drimmelen ◽

Sylvia Bruisten

Keyword(s):

Viral Load ◽

Probit Analysis ◽

Nucleic Acid Amplification ◽

Analytical Sensitivity ◽

Working Standard ◽

Infectious Dose ◽

Clinical Sensitivity ◽

Nucleic Acid Amplification Technology ◽

Sensitivity Data ◽

Antigen Tests

We prepared severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) working standards and reference panels from a pool of swab fluid samples before and after inactivation by beta-propiolactone and quantified viral load in nucleic acid amplification technology (NAT) detectable RNA copies/mL using limiting dilution analysis. The following 50% lower limits of detection (LOD) were estimated by probit analysis as compared to detection limits of rapid antigen tests on 1.5 fold dilutions of the native material: Roche cobas PCR 1.8 (1.0-3.3), Hologic Aptima TMA 6.6 (4.4-9.9), DRW SAMBA 15 (7-30), Molgen LAMP 23 (13-42), Fluorecare antigen 50,000, Abbott Panbio antigen 75,000 and Roche antigen 100,000 copies/mL. One 50% Tissue Culture Infectious Dose (TCID50)/mL of culture fluid was estimated to be equivalent to approximately 1000 RNA copies/mL (2700- 4300 International Units) in our working standard. When assuming this level as start of contagiousness in a log-linear ramp up viremia model with 10-fold rise of viral load per day for the B.1 (Wuhan) type we estimated relative time points of first detectability of early infection by the different SARS-CoV-2 assays from the LODs mentioned above. The four NAT assays would be able to detect early viremia 40-66 hours earlier than the 1000 copies/mL infectivity threshold, whereas the three antigen tests would become positive 41-48 hours later. Our modeling of analytical sensitivity data was found to be compatible with clinical sensitivity data of rapid antigen tests and confirms that NAT assays are more reliable than antigen assays for identifying early infected asymptomatic individuals who are potentially infectious.

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SARS-CoV-2 viral load monitoring by extraction-free testing of saliva

10.1101/2021.08.02.21261502 ◽

2021 ◽

Author(s):

Yue Qiu ◽

Ling Lu ◽

Dexiang Gao ◽

Patrick McGrath ◽

Chann Han ◽

...

Keyword(s):

Viral Load ◽

Clinical Sensitivity ◽

Viral Loads ◽

Clinical Assay ◽

Performance Variability ◽

Assay Performance ◽

Viral Load Monitoring ◽

Load Monitoring ◽

Droplet Pcr ◽

Polymerase Chain

Real-time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) remains the foundation of SARS-CoV-2 testing due to its accessibility, scalability, and superior assay performance. Variability in specimens and methods prevent standardization of RT-qPCR assays and reliable quantitative reporting to assess viral load. We developed an extraction-free RT-qPCR assay for detection of SARS-CoV-2 in saliva and monitored viral load until convalescence in COVID-19 patients. Comparison of 231 matched anterior nares swab and saliva specimens demonstrated that extraction-free testing of saliva has equivalent analytical and clinical assay performance compared to testing of RNA extracts from either anterior nares or saliva specimens. Analysis of specimen pairs revealed higher viral loads in the nasal cavity compared to the oral cavity, although this difference did not impact clinical sensitivity for COVID-19. Extraction-free testing of a combination specimen consisting of both nasal swab and saliva is also demonstrated. Assessment of viral load by RT-qPCR and parallel digital droplet PCR (ddPCR) revealed that cycle threshold (Ct) values less than approximately 30 correlated well with viral load, whereas Ct values greater than 30 correspond to low viral loads <10 copies/uL. Therefore, extraction-free saliva testing maximizes testing efficiency without compromising assay performance and approximates viral loads >10 copies/uL. This technology can facilitate high-throughput laboratory testing for SARS-CoV-2, monitor viral load in individual patients, and assess efficacy of therapies for COVID-19.

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