Development of multiplex S-gene-targeted RT-PCR for rapid identification of SARS-CoV-2 variants by extended S-gene target failure.

BACKGROUND: Tracking SARS-CoV-2 variants of concern (VOC) by genomic sequencing is time-consuming. The rapid screening of VOCs is necessary for clinical laboratories. In this study, we developed a rapid screening method based on multiplex RT-PCR by extended S-gene target failure (eSGTF), a false negative result caused by S-gene mutations. METHODS: Three S-gene target (SGT) regions (SGT1, codons 65-72; SGT2, codons 152-159; and SGT3, codons 370-377) and an N-gene region (for internal control) were detected in single-tube. Four types of VOC (Alpha, Delta, Omicron BA.1, and Omicron BA.2) are classified by positive/negative patterns of 3 S-gene regions (eSGTF pattern). RESULTS: The eSGTF patterns of VOCs were as follows (SGT1, SGT2, SGT3; P, positive; N, negative): Alpha, NPP; Delta, PNP; Omicron BA.1, NPN pattern; and Omicron BA.2, PPN. As compared with the S-gene sequencing, eSGTF patterns were identical to the specific VOCs (concordance rate = 96.7%, N = 206/213). Seven samples with discordant results had a minor mutation in the probe binding region. The epidemics of VOCs estimated by eSGTF patterns were similar to those in Japan. CONCLUSIONS: Multiplex RT-PCR and eSGTF patterns enable high-throughput screening of VOCs. It will be useful for the rapid determination of VOCs in clinical laboratories.

Antibody responses and SARS-CoV-2 infection after BNT162b2 mRNA booster vaccination among healthcare workers in Japan.

INTRODUCTION: Vaccine effectiveness against SARS-CoV-2 infections decreases due to waning immunity, and booster vaccination was therefore introduced. We estimated the anti-spike antibody (AS-ab) recovery by booster vaccination and analyzed the risk factors for SARS-CoV-2 infections. METHODS: The subjects were health care workers (HCWs) in a Chiba University Hospital vaccination cohort. They had received two doses of vaccine (BNT162b2) and a booster vaccine (BNT162b2). We retrospectively analyzed AS-ab titers and watched out for SARS-CoV-2 infection for 90 days following booster vaccination. RESULTS: AS-ab titer eight months after two-dose vaccinations had decreased to as low as 587 U/mL (median, IQR (interquartile range) 360-896). AS-ab titer had then increased to 22471 U/mL (15761-32622) three weeks after booster vaccination. There were no significant differences among age groups. A total of 1708 HCWs were analyzed for SARS-CoV-2 infection, and 48 of them proved positive. SARS-CoV-2 infections in the booster-vaccinated and non-booster groups were 1.8% and 4.0%, respectively, and were not significant. However, when restricted to those 20-29 years old, SARS-CoV-2 infections in the booster-vaccinated and non-booster groups were 2.9% and 13.6%, respectively (p = 0.04). After multivariate logistic regression, COVID-19 wards (adjusted odds ratio (aOR):2.9, 95% confidence interval (CI) 1.5-5.6) and those aged 20-49 years (aOR:9.7, 95%CI 1.3-71.2) were risk factors for SARS-CoV-2 infection. CONCLUSIONS: Booster vaccination induced the recovery of AS-ab titers. Risk factors for SARS-CoV-2 infection were HCWs of COVID-19 wards and those aged 20-49 years. Increased vaccination coverage, together with implementing infection control, remains the primary means of preventing HCWs from SARS-CoV-2 infection.

Tracking SARS-CoV-2 variants by entire S-gene analysis using long-range RT-PCR and Sanger sequencing.

INTRODUCTION: Genomic surveillance of the SARS-CoV-2 virus is important to assess transmissibility, disease severity, and vaccine effectiveness. The SARS-CoV-2 genome consists of approximately 30 kb single-stranded RNA that is too large to analyze the whole genome by Sanger sequencing. Thus, in this study, we performed Sanger sequencing following long-range RT-PCR of the entire SARS-CoV-2 S-gene and analyzed the mutational dynamics. METHODS: The 4 kb region, including the S-gene, was amplified by two-step long-range RT-PCR. Then, the entire S-gene sequence was determined by Sanger sequencing. The amino acid mutations were identified as compared with the reference SARS-CoV-2 genome. RESULTS: The S:D614G mutation was found in all samples. The R.1 variants were detected after January 2021. Alpha variants started to emerge in April 2021. Delta variants replaced Alpha in July 2021. Then, Omicron variants were detected after December 2021. These mutational dynamics in samples collected in the Chiba University Hospital were similar to those in Japan. CONCLUSION: The emergence of variants of concern (VOC) has been reported by the entire S-gene analysis. As the VOCs have unique mutational patterns of the S-gene region, analysis of the entire S-gene will be useful for molecular surveillance of the SARS-CoV-2 in clinical laboratories.

Highly sensitive detection of SARS-CoV-2 RNA by multiplex rRT-PCR for molecular diagnosis of COVID-19 by clinical laboratories.

BACKGROUND: The detection of SARS-CoV-2 RNA by real-time reverse transcription-polymerase chain reaction (rRT-PCR) is used to confirm the clinical diagnosis of COVID-19 by molecular diagnostic laboratories. We developed a multiplex rRT-PCR methodology for the detection of SARS-CoV-2 RNA. METHODS: Three genes were used for multiplex rRT-PCR: the Sarbecovirus specific E gene, the SARS-CoV-2 specific N gene, and the human ABL1 gene as an internal control. RESULTS: Good correlation of Cq values was observed between the simplex and multiplex rRT-PCR methodologies. Low copies (<25 copies/reaction) of SARS-CoV-2 RNA were detected by the novel multiplex rRT-PCR method. CONCLUSION: The proposed multiplex rRT-PCR methodology will enable highly sensitive detection of SARS-CoV-2 RNA, reducing reagent use and cost, and time required by clinical laboratory technicians.