Technical validation protocol for SAMBA II SARS-CoV-2/RSV Test
Published 31 March 2022
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1. Assay description and intended purpose
1.1
The SAMBA II SARS-CoV-2/RSV Test is a nucleic-acid based amplification test for the qualitative detection of nucleic acids from SARS-CoV-2 and/or Respiratory Syncytial Virus (RSV) in human respiratory specimens (combined nasal and throat/nasal/nasopharyngeal swabs).
SAMBA II SARS-CoV-2/RSV test kit (List No. 8800-12):
- 8800A, 12 each, SCoV/RSV Cartridge 1
- 8800B and 8800D, 12 each, SCoV/RSV Cartridge 2 and SCoV/RSV Cartridge 4
- 8800C, 12 each, SCoV/RSV Cartridge 3
Other materials required and provided:
- 8800E SCoV/RSV Buffer, 2ml
Each tube contains 2ml of SAMBA Lysis Buffer which includes a detergent that inactivates the virus.
- C12-0002 Fixed volume pipet, 300 μl (+ Pipet tips, filtered) OR
- C20-0007 Transfer pipets, 300 μl
- C19-0015-12 Sample Collection Tube, red cap
- C19-0093 Sample Card, SAMBA II SARS-CoV-2/RSV
Other materials required:
- Sterile flocked, rayon or Dacron swabs with plastic shaft for sample collection
Equipment required but ordered separately:
- 19-0006-AM SAMBA II Assay Module
- 19-0006-TM SAMBA II Tablet Module
- SAMBA II printer
1.2
The SAMBA II SARS-CoV-2/RSV Test is a nucleic-acid based amplification test for the qualitative detection of nucleic acids from SARS-CoV-2 and/or Respiratory Syncytial Virus (RSV) in human respiratory specimens (combined nasal and throat/nasal/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.
1.3
The SAMBA II SARS-CoV-2/RSV 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 assay is based on the chemistry used for the SAMBA II SARS-CoV-2 test with the addition of RSV. The SAMBA II SARS-CoV-2/RSV Test detects 2 gene targets for SARS-CoV2 (ORF 1ab and nucleocapsid protein – N) and one target for RSV (non structural protein 1 - NSP1). The test requires the use of 4 cartridges that contain reagents for extraction of viral nucleic acid from the specimen, amplification of the nucleic acid target and the detection of the amplification products.
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/RSV Test were determined using various reference materials including prepared panels and clinical specimens:
- heat inactivated 2019 Novel Coronavirus, Isolate USA-WA1/2020 (VR-1986HK, ATCC), lot 70035039 (3.75x108 copies/ml)
- human respiratory syncytial virus A, strain Long (VR-26PQ, ATCC), lot 70024412 (1.6x107 TCID50/ml or 1.7 x 1010 copies/ml)
- human respiratory syncytial virus B, strain B/WV/1461/85 (VR-1400, ATCC), lot 70013461 (2.8 x 105 TCID50/ml)
- World Health Organization First International Standard for SARS CoV-2 RNA, NIBSC Code: 20/146
- clinical specimens (only nasal specimens were used for clinical evaluation for data presented in the IFU)
- 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/RSV Test was determined for SARS-CoV-2 using 2 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/RSV buffer. The initial LoD was determined by testing target concentrations of 1000, 500, 350, 300 250 copies/ml.
Each panel member was initially tested in at least 2 replicates.
The final LoD was confirmed as 250 copies/ml (100% in a total of 20 replicates). The results are summarised in Table 1.
For analytical specificity and cross-reactivity
For SARS CoV-2 target in silico analysis for possible cross-reactions with all the organisms in Table 2 was conducted by carrying out a BLASTn search for of each of the SAMBA primers and probes against NCBI databases and retrieving all sequences with homologies > 80%. Only one probe (N region) had greater than 80% homology (81%) to one of the high priority organisms (Pneumocystis jirovecii [PJP]). This marginal homology would not impact the performance of the test because the other primers have no homology to P. jirovecii and hence it would not amplify.
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/RSV 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 2-37℃. No inhibitory 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 ℃ for up to 2 weeks or at -80℃ 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/RSV Test was determined for SARS-CoV-2 using 2 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 target concentrations of 1,000, 500, 350, 300 250 copies/ml. Each panel member was initially tested in at least 2 replicates. The final LoD was confirmed as 250 copies/ml (100% in a total of 20 replicates).
The results are summarised in Table 1.
Table 1. Final LoD determination
| Target | Virus strain | Confirmed LoD | % positive |
|---|---|---|---|
| SARS-CoV-2 | USA-WA1/2020 (VR-1986HK) | 250 cp/ml | 100% (20/20) |
| RSV-A | Long (VR-26PQ) | 0.5 TCID50/ml (513 cp/ml) | 95% (21/22) |
| RSV-B | B/WV/1461/85 (VR-1400) | 1.5 TCID50/ml | 96% (24/25) |
5. Precision and robustness
Intra-assay precision
The reproducibility of the SAMBA SARS-CoV-2/RSV Test was assessed using low-level positive (1050 cps/ml), medium-level (3500 cps/ml) positive and negative samples, each run-in replicates of 12 by 2 operators.
All SARS-CoV-2 positive samples were SARS-CoV-2 positive/RSV negative, and all RSV A samples were SARS-CoV-2 negative/RSV positive.
Specimen types
A matrix equivalency study was performed on nasal, combined nasal/throat and nasopharyngeal swab specimens collected into SAMBA SCoV/RSV Buffer. Results indicated that all 3 swab specimens spiked with low (1,050 copies/mL for SARS-CoV-2, 1.5 TCID50/ml for RSV A) and high (2,100 copies/mL for SARS-CoV-2, 3 TCID50/ml for RSV A) viral load showed equivalent performance when tested with the SAMBA II SARS CoV-2/RSV Test.
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/RSV Test 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/RSV Test was observed.
Table 2. Percent homology of the SAMBA II SARS-CoV-2/RSV test primers, capture probes and detector probes with high priority organisms likely in the circulating area
| High priority organisms likely in the circulating area | Primer RSV R | Probe 1 RSV | Probe 2 RSV | Primer RSV F | Primer Orf F | Primer Orf R | Probe Orf 1 | Probe Orf 2 | Primer N F 2 | Primer N R | Probe N 1 | Probe N 2 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Adenovirus (for example C1 Ad. 71) | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Human Metapneumovirus (hMPV) | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Parainfluenza virus 1-4 | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Influenza A | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Influenza B | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Enterovirus (for example EV68) | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Respiratory syncytial virus | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | ||||
| Rhinovirus | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Chlamydia pneumoniae | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Haemophilus influenzae | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Legionella pneumophila | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Mycobacterium tuberculosis | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Streptococcus pneumoniae | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Streptococcus pyogenes | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Bordetella pertussis | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Mycoplasma pneumoniae | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Pneumocystis jirovecii (PJP) | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | 81% | <80% |
| Candida albicans | <80% | 80% Up to 16/20 | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Pseudomonas aeruginosa | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Staphylococcus epidermis | <80% | 90% Up to 18/20 | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Streptococcus salivarius | 80% Up to 16/20 | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| SARS-CoV-1 | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| SARS-CoV-2 | <80% | <80% | <80% | <80% | ||||||||
| Human coronavirus 229E | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Human coronavirus OC43 | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Human coronavirus HKNI63 | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| SARS-CoV HKU339849 | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| MERS-coronavirus | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Corynebacterium diptheriae | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Legionella non-pneumophila | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Bacillus anthracis (anthrax) | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Moraxella catarrhalis | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Neisseria elongata | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Neisseria meningitidis | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Morbillivirus (measles) | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
| Varicella zoster (chicken pox) | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% | <80% |
Wet cross reactivity testing was carried out on a subset of organisms as listed in Table 3, which lists representative strains from Table 2. Each organism was spiked individually at ~105TCID50/ml for viruses and ~106 cells/ml for bacteria and fungi into combined nasal and throat / SAMBA SCoV Buffer samples and tested unspiked or spiked with SARS CoV-2 (1,050 GE copies/ml) and RSV A (1.5 TCID50 /ml). This was intended to further verify the specificity of the assay against this microbial panel and at the same time assess if these microorganisms if present in high titer, would interfere with the assay to detect the target organisms (SARS CoV-2 and RSV). Results from wet cross reactivity testing indicated that the SAMBA II SARS CoV-2/RSV Test is not negatively impacted by these microorganisms.
Table 3. Cross-reactivity wet testing
| 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 |
Potential interfering substances
The effect of potentially interfering substances was evaluated on the following panel of elevated levels of endogenous substances.
Table 4. Interfering substances
| Substance | Active ingredient | Concentration tested | Inhibition (yes/no) |
|---|---|---|---|
| Control | None | N/A | No |
| Blood | Whole blood 5% v/v | 5% | 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 |
| Sudafed nasal spray | Xylometazoline hydrochloride 0.1% | 5% | No |
| Sinex nasal spray | Oxymetazoline hydrochloride 0.5mg/ml | 5% | No |
| Nurofen oral suspension | Ibuprofen 100mg/5ml | 5% | No |
| Tamiflu powder suspension | Oseltamivir 6mg/ml | 5% | No |
| Ventolin syrup | 1% w/v Salbutamol sulfate 2mg/5ml | 5% | No |
| Calpol suspension | Paracetamol 250mg/5ml | 5% | Yes |
| Calpol suspension | Paracetamol 250mg/5ml | 1% | No |
| Snufflebabe | Sodium chloride 0.9% w/v | 5% | No |
| Children’s allergy relief | Loratadine 5mg/5ml | 5% | No |
Calpol paracetamol suspension (250 mg/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. 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)
Diagnostic sensitivity
Overall sensitivity was calculated using 150 positive specimens – 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 280 respiratory samples. As shown in Table 5 the specificity of the assay is 100% (CL 98.6-100).
Table 5. Sensitivity and specificity
Sensitivity (95% CI): 100% (97.5-100)
Specificity (95% CI): 100% (98.6-100)
Limits of detection: 250cps/ml
| CT | Number of samples | Sensitivity | 95% CI |
|---|---|---|---|
| <25 | 68 (45.3%) | 100% | 94.7-100 |
| 25-30 | 44 (29.3%) | 100% | 92.0-100 |
| 30-35 | 36 (24.0%) | 100% | 90.4-100 |
| >35 | 2 (1.3%) | 100% | 34.2-100 |
Summary
The SAMBA II SARS-CoV-2/RSV test assay meets the diagnostic performance criteria for both sensitivity and specificity.
The sensitivity is achieved with a greater proportion of specimens with a ct<25 included in the application. However, if the number of specimens with a comparator ct<25 is reduced to bring the total proportion of specimen in that banding to 40% the lcl remains above 94%, above the requirement to reach category-1 status 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 nasal and throat swabs, nasal 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.