Antibody Response to the BA.5 Bivalent Vaccine Shot: a Two-Year Follow-Up Study following Initial COVID-19 mRNA Vaccination.

Although many studies have been conducted on the increase in spike antibody levels after vaccination, there is insufficient prospective and longitudinal information on the BA.5-adapted bivalent vaccine up to the fifth vaccination. In this study, we conducted a follow-up study of spike antibody levels and infection history in 46 health care workers who received up to 5 vaccinations. Monovalent vaccines were administered for the first to fourth vaccinations, and a bivalent vaccine was administered for the fifth vaccination. 11 serum samples were collected from each participant, and antibody levels were measured in a total of 506 serum samples. During the observation period, 43 of the 46 health care workers had no infection history, and 3 had a history of infection. Spike antibody levels peaked at 1 week after the second booster vaccination and gradually declined until the 27th week after the second vaccination. After 2 weeks following the fifth BA.5-adapted bivalent vaccine, the spike antibody levels significantly increased (median: 23,756 [IQR: 16,450 to 37,326]), compared to those measured before vaccination (median: 9,354 [IQR: 5,904 to 15,784]) (paired Wilcoxon signed-rank test, P = 5.7 × 10-14). These changes in antibody kinetics were observed regardless of age or sex. These results suggest that booster vaccination increased the spike antibody levels. Regular vaccination is effective in maintaining long-term antibody levels. IMPORTANCE A COVID-19 bivalent mRNA vaccine was developed and administered to health care workers. The COVID-19 mRNA vaccine induces a robust antibody response. However, little is known about the antibody response to vaccines in serially collected blood samples from the same individuals. Here, we provide two-year follow-up data on the humoral immune response to COVID-19 mRNA vaccines in health care workers who received up to five vaccinations, including the BA.5-adapted bivalent vaccine. The results suggest that regular vaccination is effective in maintaining long-term antibody levels and have implications for vaccine efficacy and booster dose strategies in health care settings.

Classification of Omicron BA.1, BA.1.1, and BA.2 sublineages by TaqMan assay consistent with whole genome analysis data.

OBJECTIVES: Recently, the Omicron strain of SARS-CoV-2 has spread and replaced the previously dominant Delta strain. Several Omicron sublineages (BA.1, BA.1.1, and BA.2) have been identified, with in vitro and preclinical reports showing that the pathogenicity and therapeutic efficacy differs between BA.1 and BA.2. We sought to develop a TaqMan assay to identify these subvariants. METHODS: A TaqMan assay was constructed for rapid identification and genotyping of Omicron sublineages with 171 samples. We analyzed three characteristic mutations of the spike gene, Δ69-70, G339D, and Q493R, by TaqMan assay. The accuracy of the TaqMan assay was examined by comparing its results with the results of whole genome sequencing (WGS) analysis. RESULTS: A total of 171 SARS-CoV-2 positive samples were analyzed by WGS and TaqMan assay. The 127 samples determined as BA.1/BA.1.1 by WGS were all positive for Δ69-70, G339D and Q493R by TaqMan assay. A total of 42 samples, determined as BA.2 by WGS, were negative for Δ69-70 but positive for G339D and Q493R by TaqMan. Two samples with G339N were determined to be inconclusive by the TaqMan method. Except for these two samples, the concordance rate between WGS and the TaqMan assay was 100% (169/169). CONCLUSION: TaqMan assays targeting characteristic mutations are useful for identification and discrimination of Omicron sublineages.

Direct comparison of Xpert Xpress, FilmArray Respiratory Panel, Lumipulse antigen test, and RT-qPCR in 165 nasopharyngeal swabs.

BACKGROUND: The nucleic acid amplification test (NAAT) and antigen test are approved diagnostic tests for COVID-19. In this study, we aimed to investigate the assay performance of two NAATs (Xpert Xpress SARS-CoV-2 and FilmArray Respiratory Panel) and a quantitative antigen test (Lumipulse). METHODS: One hundred and sixty-five nasopharyngeal swabs were subjected to Xpert, FilmArray, Lumipulse, and RT-qPCR assays. RESULTS: Of 165 samples, RT-qPCR showed 100 positives and 65 negatives. The Xpert had an overall agreement of 99.4% (95% confidence interval [CI]: 96.7-99.4%), sensitivity of 99% (95% CI: 96.8-99%), and specificity of 100% (95% CI: 96.6-100%). FilmArray had an overall agreement of 98.8% (95% CI: 95.9-98.8%), sensitivity of 98% (95% CI: 95.6-98%), and specificity of 100% (95% CI: 96.3-100%). Lumipulse had an overall agreement of 95.5% (95% CI: 91.8-95.5%), sensitivity of 92.3% (95% CI: 89.2-92.3%), and specificity of 100% (95% CI: 95.5-100%). The κ coefficient showed excellent agreement between each test and RT-qPCR. There was a high correlation between Xpert Ct values, RT-qPCR Ct values, viral loads and antigen level. CONCLUSIONS: Xpert Xpress and FilmArray Respiratory Panel exhibited an equivalent performance. The Lumipulse antigen test was slightly less sensitive than the NAATs, but showed high assay performance except for samples with low viral load. The Xpert Xpress, FilmArray Respiratory Panel and Lumipulse antigen tests offer rapid sample-to-answer data, allowing random access detection on automated devices.

Non-pharmaceutical interventions during the COVID-19 epidemic changed detection rates of other circulating respiratory pathogens in Japan.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has circulated worldwide and causes coronavirus disease 2019 (COVID-19). At the onset of the COVID-19 pandemic, infection control measures were taken, such as hand washing, mask wearing, and behavioral restrictions. However, it is not fully clear how the effects of these non-pharmaceutical interventions changed the prevalence of other pathogens associated with respiratory infections. In this study, we collected 3,508 nasopharyngeal swab samples from 3,249 patients who visited the Yamanashi Central Hospital in Japan from March 1, 2020 to February 28, 2021. We performed multiplex polymerase chain reaction (PCR) using the FilmArray Respiratory Panel and singleplex quantitative reverse transcription PCR targeting SARS-CoV-2 to detect respiratory disease-associated pathogens. At least one pathogen was detected in 246 (7.0%) of the 3,508 samples. Eleven types of pathogens were detected in the samples collected from March-May 2020, during which non-pharmaceutical interventions were not well implemented. In contrast, after non-pharmaceutical interventions were thoroughly implemented, only five types of pathogens were detected, and the majority were SARS-CoV-2, adenoviruses, or human rhinoviruses / enteroviruses. The 0-9 year age group had a higher prevalence of infection with adenoviruses and human rhinoviruses / enteroviruses compared with those 10 years and older, while those 10 years and older had a higher prevalence of infection with SARS-CoV-2 and other pathogens. These results indicated that non-pharmaceutical interventions likely reduced the diversity of circulating pathogens. Moreover, differences in the prevalence of pathogens were observed among the different age groups.

Lung Cancer Surgery With Persistent COVID-19 Infection.

A 71-year-old man with a history of drug-induced interstitial pneumonia was diagnosed with COVID-19 infection and simultaneously found to have a pulmonary mass, suggesting a coexisting lung cancer. Approximately 1 month after COVID-19 pneumonia resolved, the patient electively underwent right upper lobectomy. Postoperatively, acute exacerbation of interstitial pneumonia occurred and the patient died on the fifteenth postoperative day. By quantitative reverse transcription polymerase chain reaction, high levels of COVID-19-derived RNA were detected in the specimen of lung parenchyma. Despite resolved COVID-19 infection, it may persist locally in the lungs, with the risk of acute exacerbation of interstitial pneumonia due to secondary stressors including surgery.

Robust Antibody Responses to the BNT162b2 mRNA Vaccine Occur Within a Week After the First Dose in Previously Infected Individuals and After the Second Dose in Uninfected Individuals.

Background: Vaccines against severe acute respiratory syndrome coronavirus 2 can trigger acquired immunity in infection-naïve individuals and offer a path toward ending the coronavirus disease pandemic that began in 2019. However, the kinetics of early antibody responses in vaccinated individuals remain poorly understood. Method: We followed BNT162b2 mRNA-vaccinated health care workers (HCWs, N=108) including 103 infection-naïve and five previously infected individuals. A total of 763 blood samples were collected weekly or hourly basis before and after vaccination. Serological analysis of anti-spike and anti-nucleocapsid antibodies was performed. Results: Seroconversion occurred in all infection-naïve HCWs 3 weeks after the first dose (just before the second vaccination) and a marked boosting effect was observed at 4 weeks (1 week after the second dose). Among previously infected HCWs with pre-existing antibodies against the spike protein, a remarkable boosting effect was observed during the first week after vaccination, and a further increase in antibody titres was observed after the second dose. In one previously infected patient, daily blood sampling was conducted. Antibody titres began to increase 96 hours (4 days) after the first dose. Conclusion: The BNT162b2 mRNA vaccine remarkably enhanced antibody responses after the second dose in infection-naïve individuals and after the first dose in previously infected HCWs of all ages and genders. Antibody titres decreased slightly after the 5th week post-vaccination. The robust boosting effect of immunisation suggests that increased antibody titres following exposure to the virus may restrict viral replication, prolong the incubation period, or lessen the severity of disease.

Comparison of Roche and Lumipulse quantitative SARS-CoV-2 antigen test performance using automated systems for the diagnosis of COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide. Here, we evaluated the performance of two quantitative antigen (Ag) tests, the Roche and Lumipulse Ag tests, using automated platforms. METHODS: We collected 637 nasopharyngeal swab samples from 274 individuals. Samples were subjected to quantitative reverse transcription PCR (RT-qPCR), the Roche Ag test and Lumipulse Ag test. RESULTS: When RT-qPCR was used as a reference, the overall concordance rate of the Roche Ag test was 77.1% (491/637) with 70.0% (341/487) sensitivity and 100% specificity (150/150). When inconclusive results of the Lumipulse Ag test were excluded, the overall concordance rate of the Lumipulse Ag test was 88.3% (467/529) with 84.8% (330/389) sensitivity and 97.9% (137/140) specificity. The overall concordance rate between the Roche and Lumipulse Ag tests was 97.9% (518/529) with 96.7% (322/333) sensitivity and 100% (196/196) specificity. Quantitative Ag levels determined using the Roche and Lumipulse Ag tests were highly correlated (R2 = 0.922). The Roche and Lumipulse Ag tests showed high concordance up to nine days after symptom onset, with progressively lower concordance after that. CONCLUSIONS: The Roche and Lumipulse Ag tests showed equivalent assay performance and represent promising approaches for diagnosing coronavirus disease 2019.

Prospective study of 1308 nasopharyngeal swabs from 1033 patients using the LUMIPULSE SARS-CoV-2 antigen test: Comparison with RT-qPCR.

BACKGROUND: Reverse transcription polymerase chain reaction (RT-PCR) is the gold standard for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Previously, the accuracy of the quantitative LUMIPULSE SARS-CoV-2 antigen test was demonstrated using samples collected retrospectively. In this study, the LUMIPULSE antigen test was clinically validated using prospective samples. METHODS: In total, 1033 nasopharyngeal swab samples were collected from 1033 individuals, and an additional 275 follow-up samples were collected from 43 patients who subsequently tested positive for coronavirus disease 2019 (COVID-19). All 1308 samples were subjected to quantitative RT-PCR (RT-qPCR) and the antigen test. The antibody response was investigated for patients with discordant results to clarify if seroconversion had occurred. RESULTS: RT-qPCR identified 990 samples as negative and 43 as positive, while the antigen test identified 992 samples as negative, 37 as positive and four as inconclusive. The overall concordance rate was 99.7% (1026/1029). Sensitivity, specificity, positive predictive value and negative predictive value of the antigen test were 92.5% (37/40), 100% (989/989), 100% (37/37) and 99.7% (989/992), respectively, after exclusion of the four inconclusive results. The kappa coefficient was 0.960 (95% confidence interval 0.892-0.960), suggesting excellent agreement between the two tests. Seropositivity in five of seven patients with discordant results suggested that the discrepancy was caused by samples collected during the late phase of infection. Using follow-up samples, correlation was observed between the antigen level and the viral load or cycle threshold value. The concordance rate between these test results tended to be high among samples collected 0-9 days after symptom onset, but this decreased gradually in samples collected thereafter. CONCLUSIONS: This prospective study demonstrated that the LUMIPULSE antigen test is a highly accurate diagnostic test for SARS-CoV-2.

The dynamic change of antibody index against Covid-19 is a powerful diagnostic tool for the early phase of the infection and salvage PCR assay errors.

BACKGROUND: Currently, PCR assay is a golden standard for diagnosis of Covid-19. However, it needs nasopharyngeal swabs, expensive instruments and expertise. It even causes PCR errors. METHODS: We validated the antibody assay (Roche) in 36 followed patients and 1879 controls (medical staffs). RESULTS: Of 1879 medical staffs, only two (0.11%) were positive by Cut off Index (COI; 1.0) (mean ± SD, 0.094 ± 0.047). Thirty six patients were composed of three groups; Group A,4 from Diamond Princess cruise ship, Group B, 2 infected in Africa, and Group C, 30 infected in Japan. PCR assays were conducted at outside laboratories before and repeated in house after hospitalized. Of 36 at admission, positive antibody was seen in 4/4 from the ship, 0/2 from Africa, and 5/30 from Japan. Two from Africa showed the increase of COI and became positive on days 8 and 13. Thirty Japanese was divided in two groups, e.g., 23 showed dynamic increase of COI up to 84.4 within 3 days while active virus replication present (Group C). In remaining 7 (7/30, 23%) (Group C'), no rise of antibody nor positive in house PCR assays, indicative of false positive results of PCR at the beginning. CONCLUSION: This antibody testing has a wide dynamic ranges of COI and, thus, could be utilized in the early infection phase. This may also compliment and even help to avoid possible PCR errors. Therefore, this can serve as a powerful diagnostic tool, needed in the frontline of the clinic and hospitals.

Pooling RT-qPCR testing for SARS-CoV-2 in 1000 individuals of healthy and infection-suspected patients.

Severe acute respiratory coronavirus 2 (SARS-CoV-2) testing reagents are expected to become scarce worldwide. However, little is known regarding whether pooling of samples accurately detects SARS-CoV-2. To validate the feasibility of pooling samples, serial dilution analysis and spike-in experiments were conducted using synthetic DNA and nucleic acids extracted from SARS-CoV-2-positive and -negative patients. Furthermore, we studied 1000 individuals, 667 of whom were "healthy" individuals (195 healthcare workers and 472 hospitalized patients with disorders other than COVID-19 infection), and 333 infection-suspected patients with cough and fever. Serial dilution analysis showed a limit of detection of around 10-100 viral genome copies according to the protocol of the National Institute of Infectious Diseases, Japan. Spike-in experiments demonstrated that RT-qPCR detected positive signals in pooled samples with SARS-CoV-2-negative and -positive patients at 5-, 10-, 20-fold dilutions. By screening with this pooling strategy, by the end of April 2020 there were 12 SARS-CoV-2-positive patients in 333 infection-suspected patients (3.6%) and zero in 667 "healthy" controls. We obtained these results with a total of 538 runs using the pooling strategy, compared with 1000 standard runs. In a prospective study, we successfully detected SARS-CoV-2 using 10- to 20-fold diluted samples of nasopharyngeal swabs from eighteen COVID-19 patients with wide ranges of viral load. Pooling sample is feasible for conserving test reagents and detecting SARS-CoV-2 in clinical settings. This strategy will help us to research the prevalence infected individuals and provide infected-status information to prevent the spread of the virus and nosocomial transmission.

Analysis of a persistent viral shedding patient infected with SARS-CoV-2 by RT-qPCR, FilmArray Respiratory Panel v2.1, and antigen detection.

Various diagnostic tests utilizing different principles are currently under development for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, these tests can occasionally produce discrepant results, causing confusion in their interpretation. Here, we evaluated the performance and features of three diagnostic assays: quantitative reverse transcription polymerase chain reaction (RT-qPCR), FilmArray Respiratory Panel (RP) v2.1, and the LUMIPULSE antigen test. Twenty-seven serial nasopharyngeal swabs were collected from a prolonged viral shedding patient who had been hospitalized for 51 days. We examined the SARS-CoV-2 detection rates of the three tests. The overall agreement rate was 81% between RT-qPCR and FilmArray RP v2.1, 63% between the antigen test and FilmArray RP v2.1, and 59% between the antigen test and RT-qPCR. We obtained concordant results in samples with high viral loads (low threshold cycle values) by all three tests. RT-qPCR and FilmArray RP v2.1 accurately detected SARS-CoV-2 at the early to intermediate phases of infection, but the results varied at the late phase. The antigen test also produced a positive result at the early phase but varied at the intermediate phase and consistently produced negative results at late phase of infection. These results demonstrated FilmArray RP v2.1 could detect SARS-CoV-2 with accuracy comparable to RT-qPCR. Further, there were discrepant results using different types of diagnostic tests during the clinical course of prolonged viral shedding patient. We provided insights into how to utilize different types of kits to assess and manage SARS-CoV-2 infections.

Comparison of automated SARS-CoV-2 antigen test for COVID-19 infection with quantitative RT-PCR using 313 nasopharyngeal swabs, including from seven serially followed patients.

In routine clinical practice, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is determined by reverse-transcription PCR (RT-PCR). In the current pandemic, a more rapid and high-throughput method is in growing demand. Here, we validated the performance of a new antigen test (LUMIPULSE) based on chemiluminescence enzyme immunoassay. A total of 313 nasopharyngeal swabs (82 serial samples from 7 infected patients and 231 individual samples from 4 infected patients and 215 uninfected individuals) were analyzed for SARS-CoV-2 with quantitative RT-PCR (RT-qPCR) and then subjected to LUMIPULSE. We determined the cutoff value for antigen detection using receiver operating characteristic curve analysis and compared the performance of the antigen test with that of RT-qPCR. We also compared the viral loads and antigen levels in serial samples from seven infected patients. Using RT-qPCR as the reference, the antigen test exhibited 55.2% sensitivity and 99.6% specificity, with a 91.4% overall agreement rate (286/313). In specimens with > 100 viral copies and between 10 and 100 copies, the antigen test showed 100% and 85% concordance with RT-qPCR, respectively. This concordance declined with lower viral loads. In the serially followed patients, the antigen levels showed a steady decline, along with viral clearance. This gradual decline was in contrast with the abrupt positive-to-negative and negative-to-positive status changes observed with RT-qPCR, particularly in the late phase of infection. In summary, the LUMIPULSE antigen test can rapidly identify SARS-CoV-2-infected individuals with moderate to high viral loads and may be helpful for monitoring viral clearance in hospitalized patients.

Double-quencher probes improve detection sensitivity toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a reverse-transcription polymerase chain reaction (RT-PCR) assay.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in the city of Wuhan, Hubei Province, China, has spread worldwide and is threatening human life. The detection of SARS-CoV-2 is critical for preventing new outbreaks, curbing disease spread, and managing patients. Currently, a reverse-transcription polymerase chain reaction (RT-PCR) assay is used to detect the virus in clinical laboratories. However, although this assay is considered to have high specificity, its sensitivity is reportedly as low as 60-70 %. Improved sensitivity is, therefore, urgently required. METHODS: We used the primers and single-quencher probes recommended by the CDC (N1, N2 and N3) in the USA and the NIID (N1 and N2) in Japan. In addition, we designed double-quencher probes according to the virus sequence provided by the NIID to develop a further assay (termed the YCH assay [N1 and N2]). Using these assays, we conducted RT-PCR with serially diluted DNA positive controls to assess and compare the detection sensitivity of the three assays. Furthermore, 66 nasopharyngeal swabs were tested to determine the diagnostic performances. RESULTS: The threshold cycle (Ct) value of the RT-PCR was relatively low for the CDC and YCH assays compared with the NIID assay. Serial dilution assays showed that both the CDC and YCH assays could detect low copy numbers of the DNA positive control. The background fluorescence signal at the baseline was lower for the YCH assay compared with the NIID assay. We assessed the diagnostic performance between single- (NIID) and double-quencher (YCH) probes using 66 nasopharyngeal swabs. When the results of YCH-N2 assay were used as a reference, each assay detected SARS-CoV-2 with positive percent agreements of 56 % for NIID-N1, 61 % for YCH-N1, and 94 % for NIID-N2, and 100 % negative percent agreements for NIID-N1, YCH-N1 and NIID-N2. CONCLUSION: Double-quencher probes decreased the background fluorescence and improved the detection sensitivity of RT-PCR for SARS-CoV-2.

Analysis of Covid-19 and non-Covid-19 viruses, including influenza viruses, to determine the influence of intensive preventive measures in Japan.

BACKGROUND: Severe acute respiratory coronavirus 2 (SARS-CoV-2) has spread and caused death worldwide. Preventive measures and infection control are underway, and some areas show signs of convergence. Other viruses in addition to SARS-CoV-2 cause cold-like symptoms and spread in the winter. However, the extent to which SARS-CoV-2, influenza viruses and other causative viruses have prevailed since implementing preventive measures is unclear. OBJECTIVES: We aim to investigate the incidence of causative viruses and pathogens in patients. STUDY DESIGN: We collected 191 nasopharyngeal swabs from patients with cold-like symptoms in Japan. All samples were subjected to multiplex PCR with the FilmArray Respiratory Panel and reverse transcription PCR (RT-PCR) to detect SARS-CoV-2. RESULTS: FilmArray Respiratory Panel analysis detected at least one virus in 32 of 191 patients with cold-like symptoms (21 %). Of these, we frequently identified human rhinoviruses/enteroviruses (5.8 %, n=11), human metapneumovirus (3.7 %, n=7), coronavirus 229E (2.1 %, n=4) and coronavirus OC43 (1.6 %, n=3); while no influenza viruses were detected. RT-PCR analysis detected SARS-CoV-2 (4.2 %, n=8) in patients who were not infected with the aforementioned respiratory viruses. CONCLUSIONS: Co-infection with SARS-CoV-2 and other viruses was not observed. Causative viruses remain prevalent after implementing preventive measures. SARS-CoV-2 differs from influenza viruses in its infectivity.