Purpose: SARS-CoV-2 has spread in Japan since January 2020 and has mutated repeatedly, resulting in shifts in the dominant circulating strains. To clarify the usefulness of antigen testing for mutant strains, we compared qualitative and quantitative antigen tests using different SARS-CoV-2 isolates. Methods: Culture supernatants of Delta (AY.54) and Omicron (BA.2, BA.4, and BA.5.2) variants isolated in Vero E6/TMPRSS2 cells were used as samples. Dilution series were prepared from the samples using PBS (−) and performed according to the package insert of each test kit and reagent. Results: The quantitative test tended to have better detection sensitivity than the qualitative tests, although some qualitative tests had comparable detection sensitivity to the quantitative test. The detection sensitivities of the tests ranged 105–106 copies/40 μL. The detection sensitivities of the test did not differ among the variant strains. Conclusion: Although differences in detection sensitivity were observed among the tests, all kits could detect high viral loads. There was little difference in detection sensitivity between the different mutant strains, indicating that the antigen test method is also valid for Omicron variants.
目的：2020年1月から日本でも感染が拡大したSARS-CoV-2は次々と変異し，主流株が移り変わってきた。今回，変異株に対する抗原検査の有用性について明らかにするために，SARS-CoV-2の分離株を用いて，抗原定性検査キットと抗原定量検査の比較検討を行った。方法：Vero E6/TMPRSS2細胞で分離したデルタ株（AY.54），オミクロン株（BA.2, BA.4, BA.5.2）の培養上清を試料とした。試料をPBS（−）を用いて希釈系列を作製し，各抗原定性検査キット及び抗原定量検査試薬の添付文書に従い実施した。結果：4種類の変異株を用いた検討の結果，抗原定性検査キットよりも抗原定量検査の方が検出感度がよい傾向にあったが，一部の抗原定性検査キットで抗原定量検査と同程度の検出感度が認められた。抗原定性検査キットの製品間の比較では，105～106 copies/40 μL程度で製品間に検出感度の差が認められた。変異株の違いによる検出感度に顕著な違いはなかった。結論：抗原定性検査キットの製品間で検出感度に差を認めたが，ウイルス量の多い時期にはいずれの検査キットでも検出可能であった。また，変異株の違いによる検出感度の差は少なく，オミクロン株でも抗原検査法が有効であることが示された。
Coronavirus disease 2019 (COVID-19) is an acute respiratory illness caused by SARS-CoV-2 that was first reported in China in December 2019.1) In Japan, the first case was reported in January 2020, and COVID-19 subsequently spread across the country. Regarding SARS-CoV-2 variants, the Delta variant was replaced by the Omicron variant, BA.1, and then BA.2 across Japan around February 2022, followed by BA.5 and its subvariant by the end of July 2022.2) Subsequently, several variants have emerged, and as of June 2023, the XBB variant is the predominant variant.3)
Nucleic acid (e.g., real-time RT-PCR) and antigen tests (qualitative and quantitative) are usually performed to detect COVID-19.4) Nucleic acid tests are considered the most sensitive COVID-19 tests.5) However, they require special testing equipment and advanced testing techniques. Conversely, among antigen tests, qualitative tests based on immunochromatography can be performed in any location with simple procedures, and such tests were distributed free of charge to symptomatic persons during the period of infectious spread.6) In addition, the quantitative antigen test based on chemiluminescent enzyme immunoassay requires specialized testing equipment.4) Currently, 51 types of qualitative antigen test kits and 9 types of quantitative test reagents have been approved through the efforts of test reagent manufacturers.7)
Although antigen tests are widely used, comparative study data on the detection sensitivity for SARS-CoV-2 variants among qualitative and quantitative tests are sparse. In this study, we compared the detection sensitivity of five qualitative test kits currently in use with that of a quantitative test, using Delta and Omicron variants isolated from clinical specimens in Tokyo, Japan.
Four variants strains isolated using Vero E6/TMPRSS2 cells at a BSL3 laboratory, namely hCoV-19/Japan/TKYK01734/2021 (Delta variant: B.1.617.2: AY.54), hCoV-19/Japan/TKYS15536/2022 (Omicron variant: B.1.1.529: BA.2), hCoV-19/Japan/TKYS16295/2022 (Omicron variant: B.1.1.529: BA.4), and hCoV-19/Japan/TKYS16224/2022 (Omicron variant: B.1.1.529: BA.5.2), were used in this study (Table 1). PBS (−) was prepared to dilute the culture supernatant by dissolving 9.6 g of Dulbecco’s PBS (−) powder (Nissui Pharmaceutical Co., Ltd.) in purified water to a total volume of 1,000 mL and autoclaving the mixture at 121°C for 15 min. The culture supernatant was diluted 10-, 50-, 100-, 250-, 500-, and 1,000-fold with this solution, aliquoted, and stored at −80°C. Each dilution was thawed at the time of use, and all dilutions were frozen once.
| Virus name | Collection date | WHO Label | Lineage | Amino acid mutations in the N protein region |
Number of amino acid mutations in the S protein region |
|---|---|---|---|---|---|
| hCoV-19/Japan/TKYK01734/2021 | unknown | Delta | AY.54 | D63G D377Y R203M | 7 |
| hCoV-19/Japan/TKYS15536/2022 | May 25, 2022 | Omicron | BA.2 | G204R P13L R203K S413R | 29 |
| hCoV-19/Japan/TKYS16295/2022 | June 20, 2022 | Omicron | BA.4 | G204R P13L P151S R203K S413R | 30 |
| hCoV-19/Japan/TKYS16224/2022 | June 14, 2022 | Omicron | BA.5.2 | G204R P13L R203K S413R | 29 |
Using the SARS-CoV-2 Direct Detection RT-qPCR Kit (Takara Bio), a calibration curve was prepared by real-time PCR, and the virus copy number per microliter of each 100-fold dilution of variant strains was calculated. This value was also calculated for each dilution based on the viral load of the 100-fold dilution. The RNA-positive control provided with the SARS-CoV-2 Direct Detection RT-qPCR Kit was used to generate the calibration curve, and an eight-step dilution series from 1.0 × 100 copies/μL to 1.0 × 107 copies/μL was generated using the provided Easy Dilution. The calibration curves prepared were confirmed using AcroMetrixTM Coronavirus 2019 (COVID-19) Low Positive Control 500 copies/μL and Ultra Low Positive Control 100 copies/μL (both from Thermo Fisher Scientific). The QIAamp Viral RNA Mini Kit (QIAGEN) was used for nucleic acid extraction, and 60 μL of RNA extract were purified from 140 μL of each 100-fold dilution. A QuantStudio 12K Flex Real-time PCR System (Thermo Fisher Scientific) was used as the real-time PCR device. The PCR protocol consisted of reverse transcription at 52°C for 5 min and 95°C for 10 s, followed by 45 cycles of 95°C for 5 s and 60°C for 30 s.
3. Testing process for qualitative antigen test kitsFive commercially available test kits were used according to the instructions provided (Table 2).
| Qualitative antigen test kit name | Name of manufacturer and distributor | Sample Type | Judgment time | Reaction temperature | Approval Date | LOT No. | |
|---|---|---|---|---|---|---|---|
| 1 | ESPLINE® SARS-CoV-2 | FUJIREBIO Inc | nasopharynx nasal cavity | 10–30 min | 20–37°C | May 13, 2020 | K4BCD003 |
| 2 | QuickNaviTM-COVID19 Ag | Denka Company Limited | nasopharynx nasal cavity | 8 min | 15–30°C | August 11, 2020 | 1882031 |
| 3 | ImunoAce SARS-CoV-2 II | TAUNS Laboratories, Inc. | nasopharynx nasal cavity | 15 min | room temperature | October 13, 2020 | 201228003 |
| 4 | PanbioTM COVID-19 Antigen rapid test | Abbott Diagnostics Medical Co., Ltd. | nasopharynx | 15 min | 15–30°C | January 22, 2021 | 41ADH252A |
| 5 | COVID-19 AND INFLUENZA A + B ANTIGEN COMBO RAPID TEST | NICHIREI BIOSCIENCES Inc | nasopharynx nasal cavity | 15 min | 15–30°C | April 14, 2021 | AT22060 |
Forty microliters of each dilution were added to the sample treatment solution and mixed well to create a sample solution. After allowing the sample solution to stand for 5 min, two drops were dropped into the reaction cassette at room temperature, and the determination was made after 30 min.
2) Test kit 2: QuickNaviTM-COVID19 AgForty microliters of each dilution were added to the sample treatment solution and mixed well to create a sample solution. Three drops of the sample solution were dropped onto the test device at room temperature, and the determination was made after 15 min.
3) Test kit 3: ImunoAce SARS-CoV-2 IIForty microliters of each dilution were added to the sample treatment solution and mixed well to create a sample solution. Three drops of the sample were dropped onto the test plate at room temperature, and the determination was made after 15 min.
4) Test kit 4: PanbioTM COVID-19 Antigen rapid testForty microliters of each dilution were added to the sample treatment solution and mixed well to create a sample solution. Five drops of the sample solution were dropped onto the test device at room temperature, and the determination was made after 15 min.
5) Test kit 5: COVID-19 AND INFLUENZA A + B ANTIGEN COMBO RAPID TESTForty microliters of each dilution were added to the sample treatment solution and mixed well to create a sample solution. Four drops of the sample solution were dropped into the test cassette at room temperature, and the determination was made after 15 min.
4. Testing process for the quantitative antigen testThe antigen quantification test LUMIPULSE SARS-CoV-2 Ag was performed on the LUMIPULSE G600II analyzer (Table 3). A sample solution was prepared by adding 40 μL of each dilution prepared in the sample processing solution. After allowing the sample solution to stand for 5 min, the entire sample solution was dropped into a sample cup for testing.
| Quantitative antigen test name | Name of manufacturer and distributor | Sample Type | Reaction time | Approval Date | LOT No. |
|---|---|---|---|---|---|
| Lumipulse® SARS-CoV-2 Ag | FUJIREBIO Inc | nasopharynx nasal cavity saliva | Approx. 30 min | June 19, 2020 | L2B3045 |
For the qualitative antigen test kits, the presence of a test line was visually judged by several persons after the appearance of a control line was confirmed for each test kit. The criteria for judgment were as follows: clear appearance of test lines, (+); slight appears of test lines, (±); and no visible test lines, (−).
For the quantitative antigen test, values of 10.00 pg/mL or more, 1.00 pg/mL to less than 10.00 pg/mL, and less than 1.00 pg/mL were judged as (+), (±), and (−), respectively, according to the package insert.
6. Ethical considerationsThe Ethics Committee of the Tokyo Metropolitan Institute of Public Health and Safety determined that there were no ethical issues with this study.
Using real-time PCR, the viral copy number of SARS-CoV-2 in 1 μL of a 100-fold dilution in each variant strain was measured (N = 3), and the average value was calculated. The amount of virus in 1 μL of the 100-fold dilution was approximately 104 copies for all four strains (Table 4).
| Virus name | Pango Lineage (WHO Label) | Lineage | 1st | 2nd | 3rd | average |
|---|---|---|---|---|---|---|
| hCoV-19/Japan/TKYK01734/2021 | B.1.617.2 (Delta) | AY.54 | 4.7 × 104 | 5.2 × 104 | 5.9 × 104 | 5.3 × 104 |
| hCoV-19/Japan/TKYS15536/2022 | B.1.1.529 (Omicron) | BA.2 | 1.8 × 104 | 3.5 × 104 | 2.7 × 104 | 2.7 × 104 |
| hCoV-19/Japan/TKYS16295/2022 | B.1.1.529 (Omicron) | BA.4 | 4.7 × 104 | 3.6 × 104 | 3.5 × 104 | 4.0 × 104 |
| hCoV-19/Japan/TKYS16224/2022 | B.1.1.529 (Omicron) | BA.5.2 | 2.0 × 104 | 1.5 × 104 | 1.7 × 104 | 1.8 × 104 |
The results using five qualitative test kits and one quantitative test using the culture supernatants of the four mutant strains diluted at six levels (10-, 50-, 100-, 250-, 500-, and 1,000-fold) illustrated that the proportion of positive samples did not significantly differ among the strains, and there was no significant difference in detection sensitivity among the strains (Table 5). In the 10-fold dilution (107 copies/40 μL), all test kits obtained positive results. Although differences in the detection sensitivity was noted among the kits for the 50–500-fold dilutions (105–106 copies/40 μL), the quantitative antigen tests successfully detected all variants. In the quantitative test, only the Delta variant was detected in the 1,000-fold dilution. The level of the Delta variant was 12.41 pg/mL, which is close to the range of reservation of judgment (±). The detection sensitivity of qualitative test kit A was comparable to that of the quantitative antigen test.
| Virus name | hCoV-19/Japan/TKYK01734/2021 | hCoV-19/Japan/TKYS15536/2022 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Pango Lineage | B.1.617.2 | B.1.1.529 | ||||||||||
| WHO Label | Delta | Omicron | ||||||||||
| Lineage | AY.54 | BA.2 | ||||||||||
| Dilution ratio | 10 | 50 | 100 | 250 | 500 | 1,000 | 10 | 50 | 100 | 250 | 500 | 1,000 |
| Qualitative antigen test kit A | + | + | + | + | + | − | + | + | + | + | + | − |
| Qualitative antigen test kit B | + | + | ± | − | n.t. | − | + | + | − | n.t. | n.t. | − |
| Qualitative antigen test kit C | + | − | − | n.t. | n.t. | − | + | − | − | n.t. | n.t. | − |
| Qualitative antigen test kit D | + | + | ± | ± | − | − | + | + | + | − | n.t. | − |
| Qualitative antigen test kit E | + | + | + | + | ± | ± | + | + | + | + | ± | ± |
| Quantitative antigen test | + | + | + | + | + | + | + | + | + | + | + | ± |
| Virus name | hCoV-19/Japan/TKYS16295/2022 | hCoV-19/Japan/TKYS16224/2022 | ||||||||||
| Pango Lineage | B.1.1.529 | B.1.1.529 | ||||||||||
| WHO Label | Omicron | Omicron | ||||||||||
| Lineage | BA.4 | BA.5.2 | ||||||||||
| Dilution ratio | 10 | 50 | 100 | 250 | 500 | 1,000 | 10 | 50 | 100 | 250 | 500 | 1,000 |
| Qualitative antigen test kit A | + | + | + | + | + | − | + | + | + | + | + | − |
| Qualitative antigen test kit B | + | − | − | n.t. | n.t. | − | + | + | ± | − | n.t. | − |
| Qualitative antigen test kit C | + | ± | − | n.t. | n.t. | − | + | − | − | n.t. | n.t. | − |
| Qualitative antigen test kit D | + | + | + | ± | − | − | + | + | ± | ± | − | − |
| Qualitative antigen test kit E | + | + | + | + | ± | ± | + | + | + | + | ± | ± |
| Quantitative antigen test | + | + | + | + | + | ± | + | + | + | + | + | ± |
n.t.: not tested
The outer portion of SARS-CoV-2 consists of the envelope (E), and spike (S), and membrane (M) proteins.8) The structure of the virion features a nucleocapsid composed of genomic RNA, and the nucleocapsid (N) protein is located inside the viral particle. The five qualitative test kits and the quantitative test used in this study all detect the N protein. Comparing each mutant strain used in this study with the Wuhan strain (hCoV-19/Wuhan/Hu-1/2019), the S protein was mutated in 7, 29, 30, and 29 locations in hCoV-19/Japan/TKYK01734/2021 (GISAID AccessionID: EPI_ISL_2080609), hCoV-19/Japan/TKYS15536/2022 (EPI_ISL_13337215), hCoV 19/Japan/TKYS16295/2022 (EPI_ISL_13502129), and hCoV-19/Japan/TKYS16224/2022 (EPI_ISL_13711216), respectively. By contrast, N protein mutations were detected at three, four, five, and four sites in hCoV-19/Japan/TKYK01734/2021, hCoV-19/Japan/TKYS15536/2022, hCoV-19/Japan/TKYS16295/2022, and hCoV-19/Japan/TKYS16224/2022, respectively. No significant difference in detection sensitivity was observed among the variants for both the qualitative and quantitative tests, which was attributed to the fact that the N protein is less likely to be mutated than the S protein and the small differences in mutations among the strains (Table 1, 5). This result is consistent with that reported by Yamazaki using Alpha and Delta strains.9) These results indicate that the antigen tests should remain effective even in an outbreak of Omicron variants, the current dominant strain, because the detection sensitivity was similar for Omicron and Delta strains.
This study observed differences in detection sensitivity among various antigen qualitative test kits. All strains were positive in the 10-fold dilutions, but in the 50–500-fold dilutions, differences in detection sensitivity were observed among the tests. Qualitative test kit B gave positive results for dilutions up to 50-fold for many mutant strains, but one mutant strain was only detected in the 10-fold dilution. For qualitative test kit C, all mutant strains were detected only in the 10-fold dilution. Qualitative test kit A, which tended to have the best detection sensitivity, could detect the variants in dilutions up to 500-fold, indicating a 50-fold difference in the detection sensitivity among the products. The study suggests that variations in detection sensitivity among the antigen qualitative test kits might be caused by the use of different anti-SARS-CoV-2 monoclonal antibodies across manufacturers and variations in the color tone of the judgement line. The quantitative value of the 100-fold dilution by real-time PCR was 1.8 × 104–5.3 × 104 copies/μL (Table 4), and because a volume of 40 μL was used in the study, the limit of detection for all qualitative test kits used in this study ranged 7.2 × 106–2.1 × 107 copies. The number of viral copies that could be detected using qualitative test kit A in the 500-fold dilutions corresponded to 1.4 × 105–4.2 × 105 copies. This result was similar to the virus concentration reported in previous studies reported by Sakai et al.10) and Yamazaki et al.9) The limit of detection for the quantitative test ranged 1.4 × 105–3.2 × 105 copies. Katsumi et al.11) reported virus isolation rates of 100% for virus copy numbers of 1 × 106–107 copies/μL and 94.4% for virus copy numbers of 1 × 105–106 copies/μL. Virus separation indicates the presence of infectious viruses. This suggests that both the qualitative and quantitative tests can detect infected individuals with high viral loads who are capable of transmitting the virus to the surrounding environment, although differences in detection sensitivity were noted among the products. However, the results suggest that both types of tests might be unable to detect infected individuals with viral copy numbers of 1 × 104–105 copies/μL or lower even if they are capable of spreading the virus.
According to the current guidelines4) for SARS-CoV-2 testing in Japan, quantitative antigen tests are indicated for the same specimens as the nucleic acid detection test for both symptomatic and asymptomatic subjects. Conversely, qualitative antigen tests, in principle, are indicated for symptomatic patients within the first 9 days of onset of illness. In a report by Nishimura et al.,12) who used the Alpha variant in their study, more than 90% of specimens that tested positive for isolation were positive for antigen regardless of whether the patient was symptomatic or asymptomatic. In addition, as previously mentioned, Yamazaki et al.,9) who conducted a study including the Alpha variant, found no marked difference in the detection sensitivity of qualitative antigen test kits attributable to differences in mutant strains. This suggests that even with the Omicron variant, which is the current dominant strain, qualitative antigen tests could be capable of detecting infected persons with the ability to transmit infection among both symptomatic and asymptomatic individuals.
It is expected that new variant strains will continue to emerge and replace current dominant strains. In addition, there is concern about a concurrent epidemic with influenza. We will continue to study the efficacy of antigen testing methods for variant strains and the use of simultaneous testing kits for SARS-CoV-2 and influenza virus.
Although differences in detection sensitivity were observed among the qualitative antigen test kits, all of the kits could detect high viral loads. There was no significant difference in detection sensitivity among the different strains, indicating that antigen tests are effective for the Omicron variant, which is the current dominant strain. The rapid detection of positive cases utilizing the advantages of each antigen test will facilitate infection control.
There is no potential conflict of interest to disclose.
