FIND evaluation update: SARS-CoV-2 molecular diagnostics
In February 2020, FIND launched an expression of interest (EOI) for test developers of in vitro diagnostics (IVDs) that detect SARS-CoV-2 nucleic acid (molecular tests) to participate in independent evaluation studies. Over 200 submissions were received and applications were reviewed according to the following scoring criteria (Table 1).
Table 1: Scoring criteria | Max score |
Limit of detection | 3 |
Regulatory status | 2 |
Type of organization | 1 |
Quality management system | 1 |
Other products available in low- and middle-income countries | 3 |
TOTAL | 10 |
As of August 2020, FIND is no longer accepting applications to evaluate molecular tests.
FIND conducted independent evaluations at the University Hospitals of Geneva (HUG) to verify the limit of detection (LOD) – as reported by the manufacturers – and the clinical performance of 22 manual molecular test kits in comparison to an in-house PCR protocol that was optimized based on the Tib Molbiol assay. The LOD analysis was performed using cultured viral stocks from a clinical isolate from Switzerland that was quantified using an E gene standard. The clinical performance analysis was conducted on extracted samples from individuals suspected to have COVID-19, 50 of which were reference PCR positive and 100 of which were reference PCR negative. Data are summarized in Table 2.
Additionally, a limited clinical performance evaluation of the Cepheid Xpert Xpress SARS-CoV-2 assay was also performed at HUG. A second collaborating site, the Translational Health Science and Technology Institute (THSTI) conducted a similar limited clinical performance evaluation of the Molbio TrueNat SARS-CoV-2 assay. Both studies were performed using frozen, stored respiratory samples from COVID-19 suspects. Results on the performance of these automated near-POC assays are shown in Table 3.
molecular assay evaluation protocol summary
Table 2: Results for 22 manual (open) molecular tests evaluated at HUG | |||||||||||
Company | Product name | Product number | Gene target | Verified LOD (copies / reaction) | Avg Ct (lowest dilution 10/10) | Clinical sensitivity (50 positives) | Clinical specificity* (100 negatives) |
Lot No. | PCR platform** | Supplier recommended Ct cut-off | |
1. | altona Diagnostics | RealStar® SARS-CoV-2 RT-PCR Kit 1.0 |
821003/ 821005 |
E | 1–10 | 35.45 | 92% (95%CI: 81, 97) |
100% (95%CI: 96, 100) |
023567 | BioRad CFX96 deep well | None; any signal can be considered positive |
S | 1–10 | 35.99 | 92% (95%CI: 81, 97) |
100% (95%CI: 96, 100) |
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2. | Atila BioSystems Inc. | Atila iAMP COVID-19 Detection (isothermal detection) |
iAMP-COVID-100-RUO | ORF1ab | 50–100 | N/A | 100% (95%CI: 93, 100) |
99%* (95%CI: 95, 100) |
COVID20200320 | BioRad CFX96 deep well | Any signal is considered positive (isothermal) |
N | 1–10 | N/A | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
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3. | Beijing Wantai Biological Pharmacy Enterprise Co. Ltd | Wantai SARS-CoV-2 RT-PCR Kit |
WS-1248 | ORF1ab | 1–10 | 36.20 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
nCoVP20200305 | BioRad CFX96 deep well | ≤40 |
N | 1–10 | 37.12 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
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4. | BGI Health (HK) Co. Ltd | Real-time Fluorescent RT-PCR kit for detection 2019-nCOV (CE-IVD) |
MFG030010 | ORF1 | 1–10 | 32.43 | 100% (95%CI: 93, 100) |
99%* (95%CI: 95, 100) |
6220200305 | Roche LightCycler 480 | ≤38 |
5. | bioMérieux SA |
ARGENE® SARS-COV-2 R-GENE®[b] |
423720 (CE-IVD) 423717 (RUO) |
N | 10–50 | 36.44 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
1007989610 1007947520 |
BioRad CFX96 deep well | Any signal considered as positive |
RdRP | 10–50 | 32.44 | 96%[a] (95%CI: 87, 99) |
100% (95%CI: 96, 100) |
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6. | Bioneer Corporation |
AccuPower® SARS-CoV-2 Real-Time RT-PCR Kit |
SCV-2122 | E | 10–50 | 35.85 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
200931E | BioRad CFX96 deep well | <38 |
RdRP | 10–50 | 36.18 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
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7. | Boditech Med. Inc. | ExAmplar COVID-19 real-time PCR kit (L) |
UFPK-4 | E | 10–50 | 34.9 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
WLQCB02L | BioRad CFX96 deep well | ≤42 |
RdRP | 50–100 | 33.46 | 90% (95%CI: 79, 96) |
100% (95%CI: 96, 100) |
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8. | CerTest Biotec S.L. |
VIASURE SARS-CoV-2 Real Time PCR Detection Kit | VS-NC0112L VS-NC0212L |
ORF1ab | 10–50 | 35.16 | 98% (95%CI: 90, 100) |
100% (95%CI: 96, 100) |
NCO212L-023 | BioRad CFX96 deep well | <40 |
N | 1–10 | 35.46 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
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9. | DAAN Gene Co. Ltd of Sun Yat-Sen University | Detection Kit for 2019 Novel Coronavirus (2019-nCoV) RNA (PCR-Fluorescence Probing) | DA0930-DA0932 | ORF1 | 1–10 | 38.76 | 100% (95%CI: 93, 100) |
96%* (95%CI: 90, 98) |
2020007 | Roche LightCycler 480 | ≤40 |
N | 1–10 | 36.97 | 100% (95%CI: 93, 100) |
98%* (95%CI: 93, 99) |
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10. | EUROIMMUN AG | EURORealTime SARS-CoV-2[c] | MP 2606-0425 | ORF1ab/N | 1–10 | 37.88 | 100% (95%CI: 93, 100) |
98%* (95%CI: 93, 99) |
I200320AL | Light Cycler 480 II | Any signal considered positive |
11. | GeneFirst Ltd | The Novel Coronavirus (2019-nCoV) Nucleic Acid Test Kit | MPA-COVID19 | ORF1 | 1–10 | 35.45 | 100% (95%CI: 93, 100) |
99%* (95%CI: 95, 100) |
00072 | BioRad CFX96 deep well | ≤37.0 positive; 37-40 indeterminate; >40 negative |
N | 1–10 | 36.72 | 98% (95%CI: 90, 100) |
100% (95%CI: 96, 100) |
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12. | KH Medical Co. Ltd | RADI COVID-19 Detection Kit |
RV008 | S | 1–10 | 37.94 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
V008.200202 | BioRad CFX96 deep well | ≤40 |
RdRP | 10–50 | 36.74 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
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13. | PerkinElmer Inc. | PerkinElmer® SARS-CoV-2 Real-time RT-PCR Assay[c,d] |
SY580 | N | 1–10 | 39,43 | 100% (95%CI: 93, 100) |
99%* (95%CI: 95, 100) |
8220200303 | BioRad CFX96 deep well | ≤42 |
ORF1 | 1–10 | 38,99 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
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14. | Primerdesign Ltd |
Coronavirus COVID-19 genesig® Real-Time PCR assay[c] |
Z-Path-COVID-19-CE | RdRP | 1–10 | 36.7 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
JN-02780-0009 | LightCycler 480 | Any signal regarded as positive |
15. | QuantumDx | QuantuMDx SARS-CoV-2 RT-PCR Detection Assay | Q22003 | Orf1, N, S | 1–10 | 36.8 | 100% (95% CI: 92, 100) |
100% (95% CI: 96, 100 |
P01100 | BioRad CFX96 deep well | ≤40 |
16. | R-Biopharm AG | RIDA®GENE SARS-CoV-2 RUO | PG6815RUO | E | 1–10 | 37.99 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
21120N | BioRad CFX96 deep well | None; any signal can be considered positive |
17. | Sansure Biotech Inc. | Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing)[e] | S3102E | ORF1 | 10–50 | 35.16 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
2020007ZC | Thermofisher Quantstudio 5 | ≤40 |
N | 10–50 | 34.96 | 100% (95%CI: 93, 100) |
95%* (95%CI: 89–98) |
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18. | SD Biosensor Inc. | STANDARD M nCoV Real-Time Detection Kit |
M-NCOV-01 | E | 1–10 | 37.43 | 100% (95%CI: 93, 100) |
97%* (95%CI: 92, 99) |
MNC00120005 | Roche LightCycler 480 | ≤41 |
ORF1 | 1–10 | 36.99 | 100% (95%CI: 93, 100) |
99%* (95%CI: 95, 100) |
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19. | Seegene Inc. | Allplex™ 2019-nCoV Assay |
RP10244Y RP10243X |
E | 1–10 | 33.3 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
RP4520C24 | BioRad CFX96 | ≤40 |
N | 1–10 | 36.74 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
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RdRP | 1–10 | 34.73 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
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20. | Shanghai Kehua Bio-Engineering Co. Ltd |
KHB Diagnostic kit for SARS-CoV-2 Nucleic Acid (Real-time PCR) |
KH-G-M-574-48 | ORF1 | 1–10 | 30.39 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
20037410 | BioRad CFX96 deep well | More than two targets detected and curve is of S shape |
N | 1–10 | 32.95 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
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E | 1–10 | 31.72 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
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21. | ThermoFisher Scientific | TaqPath™ COVID-19 CE-IVD RT-PCR Kit[f] |
A48067 | ORF1ab; S N |
1–10 | NA | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
2225262 | Quantstudio 5 | Not Applicable (Automated software interpretation) |
22. | Vela Diagnostics | ViroKey™ SARS-CoV-2 RT-PCR Test[c] | 300682 | RdRP | 10–50 | 30.95 | 94% (95%CI: 84, 98) |
100% (95%CI: 96, 100) |
1000000597 | BioRad CFX96 deep well | ≤40 |
ORF1 | 1–10 | 35.57 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
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Tib Molbiol/Roche Diagnostics | ModularDx Kit SARS-CoV (COVID19) E-gene (Tib Molbiol) + LightCycler Multiplex RNA Virus Master (Roche) | 53-0776-96 6754155001 |
E | 1–10 | 33.34 | 100% (95%CI: 93, 100) |
100% (95%CI: 96, 100) |
48202019 48274100 |
Roche LightCycler 480 | Define the cut-off 2–4 cycles higher than observed Cp value for 10 copies |
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* Clinical specificity: Further investigation is needed to determine if apparent false positives are truly false positives or whether they are due to a false negative reference standard result ** PCR platform: All products were evaluated on a PCR platform recommended by the supplier, listed in this table. Each test can be performed on other PCR systems, detailed in the product’s instructions for use. [a] The two false negative samples tested positive with the second PCR (PCR 2) that targets E gene of SARS, SARS-COV-2 and/or SARS-like coronaviruses. [b] Samples for both analytical and clinical analyses were from already-extracted specimen, therefore the methods varied from those recommended by the supplier as the internal control was not included. [c] Samples for both analytical and clinical analyses were from already-extracted specimen, therefore the methods varied from those recommended by the supplier as the internal control was added to the master mix. [d] Evaluation procedure varied from recommended protocol. In order to achieve the recommended sample input volume, a 2.5 fold dilution of the samples was used. [e] Sansure claims a lower LOD of 6.4 cp/rxn, which has been independently verified. [f] Evaluation procedure varied from recommended protocol as source material was already-extracted RNA; extracted MS2 control was added directly to the master mix. |
Table 3: Results for evaluation of two near-POC automated tests | ||||||
Company | Product name | Product number | Gene target | Clinical sensitivity (50 positives) | Clinical specificity* (100 negatives) |
Comparator test |
Cepheid Inc. | Xpert® Xpress SARS-CoV-2 |
XPRSARS-COV2-10 | N2 | 100% (95%CI: 92,100) |
99% * (95%CI: 95, 100) |
Roche Cobas ® SARS-CoV-2
N = 44 positive |
E | 97.7% (95% CI: 88, 100) |
100% (95%CI: 96, 100) |
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Molbio Diagnostics Pvt Ltd | TrueNat SARS-CoV-2[1] |
601410020 | E+RdRP[2] | 98% (95% CI: 90.98) |
96% * (95% CI:90,98) |
altona Diagnostics (n=86) /LabGun™ (n=64) and/or Seegene, Inc. (n=12)
N = 51 positive |
601420050 | ||||||
* Clinical specificity: Further investigation is needed to determine if apparent false positives are truly false positives or whether they are due to a false negative reference standard result [1] Note: evaluation performed at THSTI [2] RdRP is only used as a reflex test; the results are for combined E+RdRP positives |
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For questions relating to the evaluation of molecular tests, please contact our Emerging Threats team.
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