RESUMEN
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.
RESUMEN
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.
RESUMEN
The gold standard test for identifying SARS-CoV-2, the causative agent of COVID-19, is polymerase chain reaction (PCR). Despite their limited sensitivity, SARS-CoV-2 antigen rapid diagnostic tests are vital tools in the fight against viral spread. Owing to its simplicity and low cost, the lateral flow assay (LFA) is the most extensively used point-of-care diagnostic test. Here, we report a newly designed LFA-NanoSuit method (LNSM) that works in conjunction with desktop scanning electron microscopy (SEM) to detect SARS-CoV-2. LNSM requires no standard SEM treatment, avoids cellulose and residual buffer deformation, and enables the capture of high-resolution images of antibody-labeled gold/platinum particles reacting with SARS-CoV-2 antigens. To assess its applicability, we compared clinical SARS-CoV-2 samples via visual detection of LFA, LSNM detection of LFA, and real-time reverse transcription-PCR (qRT-PCR). Compared to qRT-PCR, LNSM showed 86.7% sensitivity (26/30; 95% confidence interval (CI): 69.28-96.24%) and 93.3% specificity (14/15; 95% CI: 68.05-99.83%) for SARS-CoV-2. In samples with a relatively low SARS-CoV-2 RNA copy number (30 < Ct ≤ 40), the sensitivity of LNSM was greater (73.3%) than that of visual detection (0%). A simple, sensitive, and quantitative LNSM can be used to diagnose SARS-CoV-2.
RESUMEN
Owing to its simplicity and low cost, the lateral flow assay (LFA) is one of the most commonly used point-of-care diagnostic techniques, despite its low sensitivity and poor quantification. Here, we report a newly developed LFA-NanoSuit method (LNSM) combined with a desktop scanning electron microscope (SEM) for the direct observation of immunocomplexes labeled with a colloidal metal instead of signal enhancement strategies, such as using color, electrochemical signals, silver enhancement, magnetic properties, luminescent, and surface-enhanced Raman spectroscopy (SERS). The proposed LNSM suppresses cellulose deformity, thereby allowing the acquisition of high-resolution images of gold/platinum-labeled immunocomplexed pathogens such as influenza A, without conductive treatment as in conventional SEM. Electron microscopy-based diagnosis of influenza A exhibited 94 % clinical sensitivity (29/31; 95 % confidence interval [CI]: 79.3-98.2 %) and 100 % clinical specificity (95 % CI: 98.1-100 %), which was more sensitive (71.4 %) than visual detection (14.3 %), especially in the lower influenza A-RNA copy number group. The detection ability of our method was nearly comparable to that of real-time reverse transcription-PCR. This is the first report on the diagnosis of clinical diseases using LFA equipped with a desktop SEM. This simple and highly sensitive quantitative analysis method involving LFA can be used to diagnose various diseases in humans and livestock, including highly infectious diseases such as COVID-19.