Technical validation protocol for SAMBA II SARS-CoV-2/FluA/FluB Test
Published 31 March 2022
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1. Assay description and intended purpose
1.1
The SAMBA II SARS-CoV-2/FluA/FluB Test is a nucleic-acid based amplification test for the qualitative detection of nucleic acids from SARS-CoV-2, Influenza A and/or Influenza B in human respiratory specimens (combined nasal and throat, nasal or nasopharyngeal swabs). The assay is intended for use by professionals and trained operators on the SAMBA II instrument system in clinical and point-of-care settings.
Each kit includes (List No. 8600-12):
- enough cartridges to test 12 samples – all cartridges are for single-use only
- 8600A, 12 each, SCoV/Flu Cartridge 1
- 8600B and 8600D, 12 each, SCoV/Flu Cartridge 2 and SCoV/Flu Cartridge 4
- 8600C, 12 each, SCoV/Flu Cartridge 3
- 8600E SCoV/Flu Buffer, 2ml each tube contains 2ml of SAMBA Lysis Buffer which includes a detergent that inactivates the virus
- C12-0002 Fixed volume pipet, 300µl (+ Pipet tips, filtered) OR
- C20-0007 Transfer pipets, 300µl
- C19-0015-12 Sample Collection Tube, red cap
- C19-0092 Sample Card, SAMBA II SARS-CoV-2/FluA/FluB
Other materials required are sterile, flocked, rayon or Dacron swabs with plastic shaft for sample collection.
1.2
The SAMBA II SARS-CoV-2/FluA/FluB Test is a fully automated assay run on the CE-marked SAMBA II instrument system consisting of the SAMBA II Assay Module, and the SAMBA II Tablet Module control unit. The SAMBA II SARS-CoV-2/FluA/FluB Test requires the use of 4 cartridges that hold reagents for extraction of viral nucleic acid from the specimen, amplification of the nucleic acid target and the detection of the amplification products and consumables. The sample buffer is inactivating, and the instrument is intended for use in clinical and point-of-care settings.
2. Type of sample to be used in validation
The performance characteristics of the SAMBA II SARS-CoV-2/FluA/FluB Test were determined using various reference materials including prepared panels and clinical specimens. These were:
- heat inactivated SARS CoV-2 (Isolate USA-WA1/2020), ATCC VR-1986HK
- World Health Organization First International Standard for SARS CoV-2 RNA, NIBSC Code: 20/146
- Influenza A virus H1N1 (Isolate A/PR/8/34), ATCC VR-95PQ
- Influenza A virus H3N2 (Isolate A/PR/8/34), ATCC VR-544
- Influenza B, Victoria Lineage (Isolate B/Florida/78/201), ATCC VR-1931
- clinical specimens (nasal swabs, combined nose and throat swabs, nasopharyngeal swabs) – IFU states that data analysed within the IFU is for nasal swabs only; this report covers additional respiratory sample types
- microorganisms from the American Type Culture Collection and BEI Resources
This report only reviews the performance of the SARS CoV2 components of the combined assay.
For analytical sensitivity and linearity, the limit of detection (LoD) of the SAMBA II SARS-CoV-2/FluA/FluB Test was determined for SARS-CoV-2 using 3 lots of reagents. Serial dilutions of inactivated SARS-CoV-2 virus (ATCC VR-1986HK) were prepared in pooled negative combined nose and throat swab samples in SAMBA SCoV buffer. The initial LoD was determined by testing instructions for use C19-0094-EN V5 Approved: 17 January 2022, page 16 target concentrations of 200 and 150 cp/ml. Each panel member was initially tested in replicates of 3. The final LoD was confirmed by testing 200 cp/ml in replicates of 28. The results are summarised in Table 1.
For analytical specificity and cross-reactivity, for SARS CoV-2 target, one strain of SARS CoV-2 (ATCC 1986HK) was used in LoD determination and 2 closely related strains (human coronavirus NL93 and SARS, Table 2b) were tested. The assay only detected SARS CoV-2 and did not cross-react with the NL3 and SARS indicating that the primer set and probes for SARS CoV-2 are specific. In addition, monthly in silico analysis of a similar product (SAMBA II SARS CoV-2 Test, P/N 8500-12) indicates that the primer set and probes of the SARS CoV-2 target are able to detect circulating variants of concern (VOC) and variants under investigation (VUI).
Stipulate if the material is required to be extracted, that is volume received, volume extracted, volume eluted, elution buffer to be used in the assay.
The swab is soaked for 10 minutes in the SCoV/Flu Buffer to inactivate the sample and then 300ul transferred to the cartridge for extraction and amplification.
The assay has a shelf life of 6 months at 2-37oC. No inhibitory/interfering reagents have been identified. In addition, the company has stated that the performance of the test was assessed in both fresh and frozen clinical samples and showed equivalent performance. Samples may be frozen at -20 oC for up to 2 weeks or at -80 oC at least 3 months.
3. Equipment and reagents
Additional equipment required:
- sterile, flocked, rayon or Dacron swabs with plastic shaft for sample collection
- 19-0006-AM SAMBA II Assay Module
- 19-0006-TM SAMBA II Tablet Module
- SAMBA II printer
4. Performance characteristics
Analytical sensitivity and linearity of SARS COV-2 targets
Lowest limits of detection (LLoD)
SARS-CoV-2: the LoD of the SAMBA II SARS-CoV-2/FluA/FluB Test was determined for SARS-CoV-2 using 3 lots of reagents by the manufacturer. Serial dilutions of inactivated SARS-CoV-2 virus (ATCC VR-1986HK) were prepared in pooled negative combined nose and throat swab samples in SAMBA SCoV buffer. The initial LoD indicated an LoD of 150 to 200cps/ml. Each panel member was initially tested in replicates of 3. The final LoD was confirmed by testing 200 cp/ml in replicates of 28.
Table 1. Final LoD determination
| Target | Virus strain | Confirmed LoD | % positive |
|---|---|---|---|
| SARS-CoV-2 | USA-WA1/2020 (VR-1986HK) | 200 cp/ml | 96% (27/28) |
| FluA H1N1 | A/PR/8/34 (VR-95PQ) | 75 cp/ml | 95% (20/21) |
| FluA H3N2 | A/Hong Kong/8/68 (VR-544PQ) | 1500 cp/ml | 100% (23/23) |
| FluB | B/Florida/78/2015 (VR-1931) | 1.5 TCID50/ml | 97% (28/29) |
5. Precision and robustness
The reproducibility of the SAMBA SARS-CoV-2/FluA/FluB Test was assessed using low-level positive, medium-level positive and negative samples. Each run included replicates of 8 by 3 operators.
All SARS-CoV-2 positive samples (2,000 cp/ml-medium positive and 600 cp/ml-low positive) were SARS-CoV-2 positive/Flu A and Flu B negative; all Flu A samples (750 cp/ml-medium positive and 225 cp/ml-low positive) were Flu A positive/SARS-CoV-2 and Flu B negative and all Flu B samples (15 TCID50/ml medium positive and 4.5 TCID50/ml-low positive) were Flu B positive/SARS-CoV-2 and Flu A negative.
Specimen types
The manufacturer presented an equivalency study of matched swab samples collected from nose and throat, nasopharyngeal and nasal cavities of 5 healthy individuals were tested for suitability as specimen type(s) for the SAMBA II SARS CoV-2 Test. Each specimen type (except for nose and throat, which were combined) from the 5 individuals were individually spiked with the 3 inactivated viruses at 600 copies/mL for SARS-CoV-2, 225 cp/ml Flu A H1N1 and 4.5 TCID50/ml for Flu B. All 3 viruses were detected in all the specimen types while the unspiked samples all tested negative.
6. Analytical specificity (interferences and cross-reactions)
Cross-reactivity to non-target samples/organisms
A range of samples either direct clinical samples or spiked samples that are known positives for other diseases, both closely related (that is other coronaviruses), syndromic diseases (that is other respiratory viruses and bacteria) and common diseases (that is HIV, HBV, HCV, VZV, EBV, CMV) should be tested.
The analytical specificity (cross-reactivity) of the SAMBA II SARS-CoV-2/FluA/FluB assay targets were evaluated using both wet testing and in silico analysis. Results are shown in manufacturers IFU and reproduced in Tables 2 and 3.
The analytical specificity for this assay was confirmed by testing a panel of different microorganisms which represents the most common respiratory pathogens. No cross-reactivity of the SAMBA II SARS-CoV-2/FluA/FluB.
Table 2. Organisms tested for cross reactivity and concentration tested
| Microorganism | Source | Catalog/Accession No. | Concentration tested |
|---|---|---|---|
| Human coronavirus NI63 | BEI | NR-470 | 1 x 105pfu/ml |
| SARS Coronavirus, Gamma-irradiated and sucrose-purified | BEI | NR-9323 | 1 x 105pfu/ml |
| Adenovirus 21 | BEI | NR-51436 | 2.5 x 105 TCID50/ml |
| Human Parainfluenza Virus 4a, M-25 | BEI | NR-3237 | 1.0 x 103 TCID50/ml |
| Enterovirus 71, Tainan/4643/1998 | BEI | NR-471 | 1.6 x 105 TCID50/ml |
| Human Respiratory Syncytial Virus, A1998/3-2 | BEI | NR-28529 | 1.6 x 105 TCID50/ml |
| Human Respiratory Syncytial Virus B | BEI | NR-790 | 1 x 105 TCID50/ml |
| Rhinovirus 20, 15-CV19 | BEI | NR-51439 | 5 x 104 TCID50/ml |
| Chlamydophila pneumoniae Strain TW-183 | ATCC | VR-2282 | ~106cells/ml |
| Haemophilus influenzae | ATCC | 49766 | ~106cells/ml |
| Legionella pneumophila | ATCC | 33152 | ~106cells/ml |
| Mycobacterium tuberculosis | BEI | NR-49100 | ~106cells/ml |
| Bordetella pertussis | BEI | NR-42460 | ~106cells/ml |
| Mycoplasma pulmonis | BEI | NR-3858 | ~106cells/ml |
| Pneumocystis jirovecii (formerly P. carinii) | ATCC | PRA-159 | ~106cells/ml |
| Staphylococcus epidermidis | ATCC | 14990 | ~106cells/ml |
| Streptococcus salivarius | BEI | HM-121 | ~106cells/ml |
Table 3. Organisms used for in silico cross-reactivity analysis – only SARS CoV-2 data shown
| High priority organisms likely in the circulating area | Primer Orf F | Primer Orf R | Probe Orf 1 | Probe Orf 2 |
|---|---|---|---|---|
| Adenovirus (for example C1 Ad. 71) | <80% | <80% | <80% | <80% |
| Human Metapneumovirus (hMPV) | <80% | <80% | <80% | <80% |
| Parainfluenza virus 1-4 | <80% | <80% | <80% | <80% |
| Influenza A | <80% | <80% | <80% | <80% |
| Influenza B | <80% | <80% | <80% | <80% |
| Enterovirus (for example EV68) | <80% | <80% | <80% | <80% |
| Respiratory syncytial virus | <80% | <80% | <80% | <80% |
| Rhinovirus | <80% | <80% | <80% | <80% |
| Chlamydia pneumoniae | <80% | <80% | <80% | <80% |
| Haemophilus influenzae | <80% | <80% | <80% | <80% |
| Legionella pneumophila | <80% | <80% | <80% | <80% |
| Mycobacterium tuberculosis | <80% | <80% | <80% | <80% |
| Streptococcus pneumoniae | <80% | <80% | <80% | <80% |
| Streptococcus pyogenes | <80% | <80% | <80% | <80% |
| Bordetella pertussis | <80% | <80% | <80% | <80% |
| Mycoplasma pneumoniae | <80% | <80% | <80% | <80% |
| Pneumocystis jirovecii (PJP) | <80% | <80% | <80% | <80% |
| Candida albicans | <80% | <80% | <80% | <80% |
| Pseudomonas aeruginosa | <80% | <80% | <80% | <80% |
| Staphylococcus epidermis | <80% | <80% | <80% | <80% |
| Streptococcus salivarius | <80% | <80% | <80% | <80% |
| SARS-CoV-1 | <80% | <80% | <80% | <80% |
| SARS-CoV-2 | ||||
| Human coronavirus 229E | <80% | <80% | <80% | <80% |
| Human coronavirus OC43 | <80% | <80% | <80% | <80% |
| Human coronavirus HKNI63 | <80% | <80% | <80% | <80% |
| SARS-CoV HKU339849 | <80% | <80% | <80% | <80% |
| MERS-coronavius | <80% | <80% | <80% | <80% |
| Corynebacterium diptheriae | <80% | <80% | <80% | <80% |
| Legionella non-pneumophila | <80% | <80% | <80% | <80% |
| Bacillus anthracis (anthrax) | <80% | <80% | <80% | <80% |
| Moraxella catarrhalis | <80% | <80% | <80% | <80% |
| Neisseria elongata | <80% | <80% | <80% | <80% |
| Neisseria meningitidis | <80% | <80% | <80% | <80% |
| Morbillivirus (measles) | <80% | <80% | <80% | <80% |
| Varicella zoster (chicken pox) | <80% | <80% | <80% | <80% |
The in silico analysis indicates that significant amplification of non-target sequences that result in cross-reactivity or potentially interfere with detection of SARS-CoV-2 is not likely to occur. In silico analysis was taken from the IFU as no data was provided for independent analysis.
Potential interfering substances
The effect of potentially interfering substances was evaluated on the following panel of elevated levels of endogenous substances.
Table 4. Potentially Interfering substances
| Substance | Active ingredient | Concentration tested | Inhibition yes/no |
|---|---|---|---|
| Control | None | N/A | No |
| Blood | Whole blood | 5%/1% | Yes/no |
| MWE Sigma Virocult | Transport media | 1:1 | No |
| Copan UTM-RT | Transport media | 1:1 | No |
| Remel M4RT | Transport media | 1:1 | No |
| Benacort | Budenoside 64 µg/spray | 5% | Yes |
| Benacort | Budenoside 64 µg/spray | 1% | No |
| Nurofen oral suspension | Ibuprofen 100mg/5ml | 5% | No |
| Sudafed nasal spray | Xylometazoline hydrochloride 0.1% | 5% | No |
| Tamiflu powder suspension | Oseltamivir 6mg/ml | 5% | No |
| Sinex nasal spray | Oxymetazoline hydrochloride 0.5mg/ml | 5% | No |
| Ventolin syrup | 1% w/v Salbutamol sulfate 2mg/5ml | 5% | No |
| Calpol suspension | Paracetamol 250mg/5ml 33 μg/mL | 5% | Yes |
| Calpol suspension | Paracetamol 250mg/5ml μg/mL | 1% | No |
| Snufflebabe | Sodium chloride 0.9% w/v | 5% | No |
| Children’s allergy relief | Loratadine 5mg/5ml | 5% | No |
Calpol paracetamol suspension (250mg/5ml) and Benacort Budenoside nasal spray (64 µg/spray) inhibited at 5% v/v. However, neither showed any interference when tested at 1% v/v. Similarly, whole blood was not inhibitory at 1% but interfered with the Influenza A target but not Influenza B and Sars-CoV-2 at 5%. None of the other substances showed any interference in spiked or unspiked samples.
7. Diagnostic sensitivity and specificity (clinical validation with confirmed positives and negatives)
Overall sensitivity was calculated 151 positives specimens after removing duplicates. Three of these 156 samples were negative by SAMBA. Repeat testing of these specimens was not incorporated into the evaluation as a discriminator assay was not used and concordant results were not retested, 95% confidence limits were calculated. In addition, sensitivity was calculated across the dynamic range of the assay and the results shown in table 5. The comparator assay was the TaqPath Covid-19, FluA/B, RSV Combo kit.
For specificity the manufacturer provided data for 277 respiratory samples. Two samples were positive by SAMBA and negative by TaqPath. Repeat testing of these 2 results was not included in the analysis as a discriminator assay was not used and concordant results were not retested.
As shown in Table 5 the specificity of the assay is 99.3% (CL 97.4-99.8).
Table 5. Sensitivity and specificity
Sensitivity (95% CI): 98.0% (94.3-99.3)
Specificity (95% CI): 99.3% (97.4-99.8)
Limits of detection: 200cps/ml
| CT | Number of samples | Sensitivity | 95% CI |
|---|---|---|---|
| <25 | 69 (45.7%) | (68/69) 98.6% | 92.2-99.7 |
| 25-30 | 47 (31.1%) | (47/47) 100% | 92.4-100 |
| 30-35 | 34 (22.5%) | (33/34) 97.1% | 85.1-99.5 |
| >35 | 1 (0.6%) | (0/1) 0% | 0-79.3 |
Summary
The SAMBA II SARS-CoV-2/FluA/FluB assay meets the diagnostic performance criteria for both sensitivity and specificity.
However, the sensitivity is achieved with a greater proportion of specimens with a ct<25 included in the application. If the number of specimens with a comparator ct<25 is reduced to bring the total proportion of specimen in that banding to 40% then the sensitivity of the assay falls to 97.8 % and the lower 95% confidence limit to 93.7%, which is still above the lower limit for an extracted PCR assay under the Coronavirus Test Device Authority (CTDA) regulations. In addition, the number of specimens with a ct>35 does not reach the requirement of the CTDA guidance, however, the Technical Virology Group allows approval of assays where the combined numbers of specimens above ct30 is at least 20% of the total number of specimens.
The evaluation was performed using a mixture of upper respiratory sample types including combined nose and throat swabs, nasal swabs and nasopharyngeal swabs. For CTDA approval, sufficient numbers of specimens of each sample type, across the dynamic range of the assay, would need to be supplied.