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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
RdRP 1–10 34.73 100%
(95%CI:
93, 100)
100%
(95%CI:
96, 100)
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)
E 1–10 31.72 100%
(95%CI:
93, 100)
100%
(95%CI:
96, 100)
21. ThermoFisher Scientific TaqPath™ COVID-19
CE-IVD
RT-PCR Kit[f]
A48067 ORF1ab;
S protein;
N protein
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)
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
  *  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
N = 100 negative

E 97.7%
(95% CI:
88, 100)
100%
(95%CI:
96, 100)
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
N = 111 negative

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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