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.

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.

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.

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.