Cycle Threshold (Ct) Values of SARS-CoV-2 Detected with the GeneXpert® System and a Mutation Associated with Different Target Gene Failure.

SARS-CoV-2 nucleic acid detection tests enable rapid virus detection; however, it is challenging to identify genotypes to comprehend the local epidemiology and infection routes in real-time qRT-PCR. At the end of June 2022, our hospital experienced an in-hospital cluster of COVID-19. When examined using the GeneXpert® System, the cycle threshold (Ct) value of the N2 region of the nucleocapsid gene of SARS-CoV-2 was approximately 10 cycles higher than that of the envelope gene. Sanger sequencing revealed a G29179T mutation in the primer and probe binding sites. A review of past test results revealed differences in Ct values in 21 of 345 SARS-CoV-2-positive patients, of which 17 cases were cluster-related and 4 were not. Including these 21 cases, 36 cases in total were selected for whole-genome sequencing (WGS). The viral genomes in the cluster-related cases were identified as BA.2.10, and those in the non-cluster cases were closely related and classified as being downstream of BA.2.10 and other lineages. Although WGS can provide comprehensive information, its use is limited in various laboratory settings. A measurement platform reporting and comparing Ct values of different target genes can improve test accuracy, enhance our understanding of infection spread, and be applied to the quality control of reagents.

Ensitrelvir is effective against SARS-CoV-2 3CL protease mutants circulating globally.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a public health concern worldwide. Ensitrelvir (S-217622) has been evaluated as an antiviral treatment for COVID-19, targeting SARS-CoV-2 3C-like protease (3CLpro). Ensitrelvir has been reported to have comparable antiviral activity against some of the SARS-CoV-2 variants: alpha, beta, gamma, delta, and omicron (BA.1.18). In this paper, we describe that ensitrelvir is effective against newly emerging SARS-CoV-2 variants and globally prevalent 3CLpro mutations. Ensitrelvir exhibited comparable antiviral activity against SARS-CoV-2 variants, including recently emerging ones: omicron (BA1.1, BA.2, BA.2.75, BA.4, BA.5, BQ.1.1, XBB.1, and XE), mu, lambda, and theta. Genetic surveillance of SARS-CoV-2 3CLpro, the target of ensitrelvir, was conducted using a public database and identified 11 major 3CLpro mutations circulating globally (G15S, T21I, T24I, K88R, L89F, K90R, P108S, P132H, A193V, H246Y, and A255V). The 3CLpro mutation from proline to histidine at amino acid position 132 was especially identified in the omicron variant, with prevalence of 99.69%. Enzyme kinetic assay revealed that these 3CLpro mutants have enzymatic activity comparable to that of the wild type (WT). Next, we assessed the inhibitory effect of ensitrelvir against mutated 3CLpro, with it showing inhibitory effects similar to that against the WT. These in vitro data suggest that ensitrelvir will be effective against currently circulating SARS-CoV-2 variants, including omicron variants and those carrying 3CLpro mutations, which emerging novel SARS-CoV-2 variants could carry.

Differential Dynamics of Humoral and Cell-Mediated Immunity with Three Doses of BNT162b2 SARS-CoV-2 Vaccine in Healthcare Workers in Japan: A Prospective Cohort Study.

Vaccines against SARS-CoV-2 with good efficacy are now available worldwide. However, gained immunity diminishes over time. Here, we investigate the course of both humoral and cell-mediated immunity in response to three doses of the Pfizer mRNA BNT162b2 SARS-CoV-2 vaccine in healthcare workers in Japan. SARS-CoV-2 anti-receptor-binding domain (RBD) antibodies (total Ig, IgG), neutralizing antibodies (NAb), and ELISpot were measured in serum and whole blood samples collected after each vaccine dose. ELISpot numbers were higher than the cutoff values in most participants at all times. It was suggested that the difference in behavior between humoral immunity and cell-mediated immunity with age is complementary. Anti-RBD total Ig, IgG, and NAb indicated a high correlation at each time point after vaccine doses. Total Ig was retained long-term after the second dose and increased significantly faster by the booster dose than IgG. Nab levels of all subjects were ≤20% six months after the second dose, and the correlation coefficient was greatly reduced. These are due to the avidity of each antibody and differences among commercial kits, which may affect the evaluation of immunokinetics in previous COVID-19 studies. Therefore, it is necessary to harmonize reagents categorized by the same characteristics.