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Background: This study aimed to compare the performance of Sanger-based SARS-CoV-2 spike gene sequencing and Next Generation Sequencing (NGS)-based full-genome sequencing for variant identification in saliva samples with low viral titer. Methods: Using 241 stocked saliva samples collected from confirmed COVID-19 patients between November 2020 and March 2022 in Hiroshima, SARS-CoV-2 spike gene sequencing (nt22735-nt23532) was performed by nested RT-PCR and Sanger platform using in-house primers. The same samples underwent full-genome sequencing by NGS using Illumina NextSeq2000. Results: Among 241 samples, 147 were amplified by both the Sanger and the Illumina NextSeq2000 NGS, 86 by Sanger only, and 8 were not amplified at all. The overall amplification rates of Illumina NextSeq2000 NGS and Sanger were 61% and 96.7%, respectively. At low viral titer (< 103 copies/mL), Illumina NextSeq2000 NGS provided 19.2% amplification, while Sanger was 89.7% (p < 0.0001). Both platforms identified 38 wild type, 54 Alpha variants, 84 Delta variants, and 57 Omicron variants. Conclusions: Our study provided evidence to expand the capacity of Sanger-based SARS-CoV-2 spike gene sequencing for variants identification over full-genome by Illumina NextSeq2000 NGS for mass screening. Therefore, the feasible and simple Sanger-based SARS-CoV-2 spike gene sequencing is practical for the initial variants screening, which might reduce the gap between the rapid evolution of SARS-CoV-2 and its molecular surveillance. [ABSTRACT FROM AUTHOR]

Perceived discrimination and work impairment are commonly observed in COVID-19 survivors, but their relationship has not been well understood. We aimed to evaluate the role of discrimination in the development of psychological distress and work impairment in COVID-19 survivors. From April 2020 to November 2021, 309 patients were recruited at two designated COVID-19 hospitals in Japan. Participants completed a standardized questionnaire including COVID-19 sequelae, psychological distress, impairments in work performance and perceived discrimination. The majority of participants (62.5%) experienced one or more COVID-19 sequelae. Psychological distress was observed in 36.9% and work impairment in 37.9%. In multivariate logistic regression analyses, COVID-19 sequelae and discrimination were associated with both psychological distress and work impairment. Mediation analysis demonstrated that the direct effect of sequelae on work impairment was non-significant after accounting for psychological distress, suggesting that the effect of sequelae on work impairment was mainly mediated through psychological distress. These findings were replicated in a subgroup analysis limited to patients with mild COVID-19. We conclude that discrimination plays an important role in the development of psychological distress and work impairment, and that both discrimination and psychological distress should be targets of intervention in COVID-19 survivors. [ABSTRACT FROM AUTHOR]

This cross-sectional study aimed to investigate the post-acute consequences of COVID-19. We conducted a self-administered questionnaire survey on sequelae, psychological distress (K6), impairments in work performance (WFun), and COVID-19–related experiences of stigma and discrimination in two designated COVID-19 hospitals in Hiroshima Prefecture, Japan, between August 2020 and March 2021. The prevalence of sequelae was calculated by age and COVID-19 severity. Factors independently associated with sequelae or psychological distress were identified using logistic regression analysis. Among 127 patients who had recovered from COVID-19, 52.0% had persistent symptoms at a median of 29 days [IQR 23–128] after COVID-19 onset. Among patients with mild COVID-19, 49.5% had sequelae. The most frequent symptoms were olfactory disorders (15.0%), taste disorders (14.2%), and cough (14.2%). Multivariate analysis showed that age was an independent risk factor for sequelae (adjusted odds ratios [AOR] for ≥ 60 years vs. < 40 years 3.63, p = 0.0165). Possible psychological distress was noted in 30.7% (17.9% of males and 45.0% of females). Female sex and the presence of sequelae were independent risk factors for psychological distress. Of all participants, 29.1% had possible impairments in work performance. Experiences of stigma and discrimination were reported by 43.3% of participants. This study revealed the significant impacts of Long COVID on health in local communities. A large-scale, long-term cohort study is desired. [ABSTRACT FROM AUTHOR]

Background: Symptoms after novel coronavirus disease 2019 (COVID-19) recovery by severe acute respiratory syndrome coronavirus 2 strains are unspecified., Methods: This self-administered questionnaire-based study was conducted to investigate symptoms after COVID-19 recovery at one of the main hospitals for COVID-19 treatment in Hiroshima, Japan, from September 2020 to March 2022 for patients who visited follow-up consultations after COVID-19. Study subjects were divided into four groups (Wild-type, Alpha, Delta, and Omicron periods) according to COVID-19 onset date. Hierarchical cluster analysis was performed to determine symptom clusters and investigate risk factors for each symptom cluster using multivariate analysis., Results: Among 385 patients who enrolled in this study, 249 patients had any persistent symptoms at a median of 23.5 (interquartile range, 20-31) days after COVID-19 onset. Among patients with any persistent symptoms, symptom clusters including olfactory or taste disorders, respiratory symptoms, and cardiac symptoms were found. Respiratory symptoms were more frequent among patients infected in the Omicron period compared to the Wild-type period (adjusted odds ratio [AOR] 3.13; 95% confidence interval [CI], 1.31-7.48). Compared to patients who recovered from mild COVID-19, patients who needed oxygen or ventilation support suffered fewer post-COVID-19 respiratory symptoms (AOR 0.46; 95% CI, 0.22-0.97) but more post-COVID-19 cardiac symptoms among them (AOR 2.67; 95% CI, 1.26-5.65). Olfactory or taste disorders were fewer among patients infected in the Omicron period compared to the Wild-type period (AOR 0.14; 95% CI, 0.04-0.46)., Conclusion: This study revealed that symptoms after COVID-19 may vary depending on the infected strain.

This study aimed to develop the feasible and effective universal screening strategy of the notable SARS-CoV-2 variants by Sanger Sequencing Strategy and then practically applied it for mass screening in Hiroshima, Japan. A total of 734 samples from COVID-19 confirmed cases in Hiroshima were screened for the notable SARS-CoV-2 variants (B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.1, C.37, B.1.1.529, etc.). The targeted spike region is amplified by nested RT-PCR using in-house designed primer set hCoV-Spike-A and standard amplification protocol. Additionally, randomly selected 96 samples were also amplified using primer sets hCoV-Spike-B and hCoV-Spike-C. The negative amplified samples were repeated for second attempt of amplification by volume-up protocol. Thereafter, the amplified products were assigned for Sanger sequencing using corresponding primers. The positive amplification rate of primer set hCoV-Spike-A, hCoV-Spike-B and hCoV-Spike-C were 87.3%, 83.3% and 93.8% respectively for standard protocol and increased to 99.6%, 95.8% and 96.9% after second attempt by volume-up protocol. The readiness of genome sequences was 96.9%, 100% and 100% respectively. Among 48 mutant isolates, 26 were B.1.1.7 (Alpha), 7 were E484K single mutation and the rest were other types of mutation. Moreover, 5 cluster cases with single mutation at N501S were firstly reported in Hiroshima. This study indicates the reliability and effectiveness of Sanger sequencing to screen large number of samples for the notable SARS-CoV-2 variants. Compared to the Next Generation Sequencing (NGS), our method introduces the feasible, universally applicable, and practically useful tool for identification of the emerging variants with less expensive and time consuming especially in those countries where the NGS is not practically available. Our method allows not only to identify the pre-existing variants but also to examine other rare type of mutation or newly emerged variants and is crucial for prevention and control of pandemic., (© 2022. The Author(s).)

Bleeding manifestations in primary immune thrombocytopenia (ITP) range from skin petechiae to life-threatening intracranial hemorrhage (ICH). However, the relation between these various bleeding manifestations and the platelet count in ITP remains poorly characterized. Using a nationwide database of patients with ITP during the years 2005 to 2014 (10 years) in Japan, we analyzed 19 415 adult patients newly diagnosed with ITP, including 222 with ICH. The frequency of skin purpura was 64.8%, and this increased linearly with thrombocytopenia without a specific platelet count threshold. In contrast, mucosal bleeding (epistaxis and gingival bleeding) and organ bleeding (melena, hematuria, and ICH) increased exponentially with thrombocytopenia at a platelet count threshold of 10 to 15 × 109/L. Age showed a much weaker correlation than platelet count with skin and mucosal bleeding. However, the incidence of organ bleeding increased exponentially above 60 years of age. Multivariate analysis showed that the presence of mucosal bleeding was a risk factor for occurrence of melena and hematuria but not for ICH. The frequency of ICH was 1.1% and risk factors for ICH were age ≥60 years (odds ratio [OR], 3.09; 95% confidence interval [CI], 2.13-4.47; P < .001), platelet count <10 × 109/L (OR, 2.96; 95% CI, 2.11-4.15; P < .001), and the presence of hematuria (OR, 1.56; 95% CI, 1.04-2.35; P = .033). The relation between ICH and platelet count varied with age. This large-scale analysis of risk factors for bleeding in ITP has revealed distinct characteristics of skin, mucosal, and organ bleeding in adult patients with newly diagnosed ITP, thus indicating those who are at a high risk of severe organ bleeding., (© 2020 by The American Society of Hematology.)

Objective: Although the rates of reported symptoms of Pandemic (H1N1) 2009 influenza virus infection are well studied, the course of progression of these symptoms is not clear. In this study, we carefully reviewed the progress of each patient after hospitalization and clarified the clinical course of the symptoms., Methods: We retrospectively examined the clinical data of 16 consecutive patients who had been hospitalized during the early stages of an influenza epidemic and observed the clinical progression of their symptoms., Results: Each symptom had a different time of onset and progression pattern. In roughly one-third of our patients, symptoms appeared before the onset of high fever. Acute respiratory symptoms tended to last longer than other symptoms; similarly, sore throat and cough lasted longer than rhinorrhea. The SpO(2) of the patients with influenza showed a declining trend. The point at which minimum SpO(2) levels were noted was approximately 1.5 days after onset of fever., Conclusion: In this H1N1 epidemic, patients typically tended to experience general fatigue, sore throat, and cough before the onset of fever, with sore throat and cough lasting longer than the other symptoms. Most patients showed decreased SpO(2) levels at -1.5 days after onset of fever.