Neutralization activity of IgG antibody in COVID­19­convalescent plasma against SARS-CoV-2 variants.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the anti-SARS-CoV-2 antibody levels, anti-spike (S)-immunoglobulin G (IgG) and anti-nucleocapsid (N)-IgG, and the neutralization activity of IgG antibody in COVID­19­convalescent plasma against variants of SARS-CoV-2, alpha, beta, gamma, delta, kappa, omicron and R.1 strains. The study included 30 patients with clinically diagnosed COVID-19. The anti-S-IgG and anti-N-IgG levels ranged from 30.0 to 555.1 and from 10.1 to 752.6, respectively. The neutralization activity (50% inhibition concentration: IC50) for the wild-type Wuhan strain ranged from < 6.3 to 81.5 µg/ml. IgG antibodies were > 100 µg/ml in 18 of 30 (60%) subjects infected with the beta variant. The IC50 values for wild-type and beta variants correlated inversely with anti-S-IgG levels (p < 0.05), but no such correlation was noted with anti-N-IgG. IgG antibodies prevented infectivity and cytopathic effects of six different variants of concern in the cell-based assays of wild-type, alpha, gamma, delta, kappa and R.1 strains, but not that of the beta and omicron strains. IgG is considered the main neutralizing activity in the blood, although other factors may be important in other body tissues.

Fatty liver on computed tomography scan on admission is a risk factor for severe coronavirus disease.

OBJECTIVES: To alleviate the overflow of coronavirus disease 2019 (COVID-19) patients in hospitals, less invasive and simple criteria are required to triage the patients. We evaluated the relationship between COVID-19 severity and fatty liver on plain computed tomography (CT) scan performed on admission. METHODS: In this retrospective cohort study, we considered all COVID-19 patients at a large tertiary care hospital between January 31 and August 31, 2020. COVID-19 severity was categorized into severe (moderate and severe) and non-severe (asymptomatic and mild) groups, based on the Japanese National COVID-19 guidelines. Fatty liver was detected on plain CT scan. Multivariate logistic regression analysis was performed to evaluate factors associated with severe COVID-19. RESULTS: Of 222 patients (median age: 52 years), 3.2%, 58.1%, 20.7%, and 18.0% presented with asymptomatic, mild, moderate, and severe COVID-19, respectively. Although 59.9% had no fatty liver on plain CT, mild, moderate, and severe fatty liver occurred in 13.1%, 18.9%, and 8.1%, respectively. Age and presence of fatty liver were significantly associated with severe COVID-19. CONCLUSION: Our study showed that fatty liver on plain CT scan on admission can become a risk factor for severe COVID-19. This finding may help clinicians to easily triage COVID-19 patients.

A Pilot Study on Viral Load in Stool Samples of Patients with COVID-19 Suffering from Diarrhea.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be detected in the stool samples of patients with coronavirus disease 2019 (COVID-19), and this virus can be transmitted via the oral-fecal route. However, there are only few reports on the viral load in the stool samples. In this pilot study, we aimed to evaluate the clinical characteristics and viral load of SARS-CoV-2 in the stool samples of 13 patients with confirmed COVID-19 using pepper mild mottle virus as a control, which has been proposed as a potential marker of human feces contamination in the environmental water bodies. SARS-CoV-2 RNA was detected in the stool samples of four patients (31%), and among them, three exhibited symptoms of diarrhea. One patient who suffered from long-term diarrhea (22 days) exhibited highest level of viral RNA in the stool sample (8.28 log10 copies/g). However, we could not harvest SARS-CoV-2 from the stool sample of any patient, even after culturing with VeroE6/TMPRESS2 cells for four weeks. Our results suggest that SARS-CoV-2 RNA can be detected in the stool samples of patients with COVID-19 suffering from diarrhea. However, further studies elucidating the relationship between SARS-CoV-2 viral load in the stool samples and symptoms of diarrhea in large cohorts and upon adjusting other causative factors and virus infectivity are still warranted.

Seroprevalence of SARS-CoV-2 antibodies among 925 staff members in an urban hospital accepting COVID-19 patients in Osaka prefecture, Japan: A cross-sectional study.

ABSTRACT: The subclinical severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection rate in hospitals during the pandemic remains unclear. To evaluate the effectiveness of our hospital's current nosocomial infection control measures, we conducted a serological survey of anti-SARS-CoV-2 antibodies (immunoglobulin [Ig] G) among the staff of our hospital, which is treating coronavirus disease 2019 (COVID-19) patients.The study design was cross-sectional. We measured anti-SARS-CoV-2 IgG in the participants using a laboratory-based quantitative test (Abbott immunoassay), which has a sensitivity and specificity of 100% and 99.6%, respectively. To investigate the factors associated with seropositivity, we also obtained some information from the participants with an anonymous questionnaire. We invited 1133 staff members in our hospital, and 925 (82%) participated. The mean age of the participants was 40.0 ±â€Š11.8 years, and most were women (80.0%). According to job title, there were 149 medical doctors or dentists (16.0%), 489 nurses (52.9%), 140 medical technologists (14.2%), 49 healthcare providers (5.3%), and 98 administrative staff (10.5%). The overall prevalence of seropositivity for anti-SARS-CoV-2 IgG was 0.43% (4/925), which was similar to the control seroprevalence of 0.54% (16/2970) in the general population in Osaka during the same period according to a government survey conducted with the same assay. Seropositive rates did not significantly differ according to job title, exposure to suspected or confirmed COVID-19 patients, or any other investigated factors.The subclinical SARS-CoV-2 infection rate in our hospital was not higher than that in the general population under our nosocomial infection control measures.

Time-course evaluation of the quantitative antigen test for severe acute respiratory syndrome coronavirus 2: The potential contribution to alleviating isolation of COVID-19 patients.

INTRODUCTION: The automated quantitative antigen test (QAT), which detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is suitable for mass screening. However, its diagnostic capability differentiated by time from onset and potential contribution to infectivity assessment have not been fully investigated. METHODS: A retrospective, observational study using nasopharyngeal swab specimens from coronavirus disease (COVID-19) inpatients was conducted using LumipulseⓇ SARS-CoV-2 antigen test. Diagnostic accuracy was examined for the early (up to 10 days after onset) and late (over 10 days after onset) stages. Time-course QAT changes and reverse-transcription quantitative polymerase chain reaction tests results were displayed as locally estimated scatterplot smoothing curve, and receiver operating characteristic curve (ROC) analysis was used to determine the appropriate cutoff value for differentiating the early and late stages. RESULTS: We obtained 100 specimens from 68 COVID-19 patients, including 51 early-stage and 49 late-stage specimens. QAT sensitivity and specificity were 0.82 (0.72-0.90) and 0.95 (0.75-0.99) for all periods, 0.93 (0.82-0.98) and 1.00 (0.39-1.00) for the early stage, and 0.66 (0.48-0.82) and 0.93 (0.69-0.99) for the late stage, respectively. The ROC analysis indicated an ideal cutoff value of 6.93 pg/mL for distinguishing early-from late-stage specimens. The sensitivity, specificity, positive predictive value, and negative predictive value for predicting the late stage were 0.76 (0.61-0.87), 0.76 (0.63-0.87), 0.76 (0.61-0.87), and 0.76 (0.63-0.87). CONCLUSIONS: QAT has favorable diagnostic accuracy in the early COVID-19 stages. In addition, an appropriate cutoff point can potentially facilitate rapid identification of noncontagious patients.

How we secured a COVID-19 convalescent plasma procurement scheme in Japan.

BACKGROUND: In order to tackle the COVID-19 pandemic, a COVID-19 convalescent plasma (CCP) procurement program was initiated in Japan in April 2020. The program was a collaboration between a government-managed national hospital, an infectious disease research institute, and a blood banking organization. Each party assumed different responsibilities: recruitment, SARS-CoV-2 antibody profiling, and plasmapheresis; conduction of screening tests; and SARS-CoV-2 blood testing, respectively. METHODS: We adopted a two-point screening approach before the collected CCP was labeled as a CCP product for investigational use, for which we mainly tested anti-SARS-CoV-2 antibody eligibility and blood product eligibility. Anti-SARS-CoV-2 spike protein titer was measured using enzyme-linked immunosorbent assay, and the IC50 value was denoted as the neutralizing activity. Blood donor eligibility was extended beyond the normal blood donation guidelines to include a broader range of participants. After both eligibility criteria were confirmed, participants were asked to revisit the hospital for blood donation, which is a unique aspect of the Japanese CCP program, as most donations are taking place in normal blood donation venues in other countries. Some donors were re-scheduled for repeat plasma donations. As public interest in anti-SARS-CoV-2 antibodies increased, test results were given to the participants. RESULTS: As of September 17, 2020, our collection of CCP products was sufficient to treat more than 100 patients. As a result, projects for administration and distribution are also being conducted. CONCLUSIONS: We successfully implemented a CCP procurement scheme with the goal to expand to other parts of the country to improve treatment options for COVID-19.

Does the SARS-CoV-2 rapid antigen test result correlate with the viral culture result?

Rapid antigen tests (RATs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have advantages over viral culture in terms of cost and rapidity of testing, but they have low sensitivity. In addition, RATs tend to be negative from approximately 11 days after symptom onset. To determine whether the antigen-negative state indicates a lack of infectiousness, we assessed the association between viral culture and RAT results. Viral culture, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and rapid antigen testing were performed on stored nasopharyngeal samples with threshold cycle values < 30, based on previous RT-qPCR testing. SARS-CoV-2 was isolated by viral culture from nine samples (45%) and one sample (17%) with positive and negative RAT results, respectively. The RAT and viral culture results were both associated with the viral load level and their cutoffs were similar, but the associations were not statistically significant. RAT might be a useful indicator of infectiousness, which can be helpful to control infection. However, further studies with larger sample size are warranted to confirm this observation.

HLA-A*11:01:01:01, HLA-C*12:02:02:01-HLA-B*52:01:02:02, Age and Sex Are Associated With Severity of Japanese COVID-19 With Respiratory Failure.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing coronavirus disease 2019 (COVID-19) was announced as an outbreak by the World Health Organization (WHO) in January 2020 and as a pandemic in March 2020. The majority of infected individuals have experienced no or only mild symptoms, ranging from fully asymptomatic cases to mild pneumonic disease. However, a minority of infected individuals develop severe respiratory symptoms. The objective of this study was to identify susceptible HLA alleles and clinical markers that can be used in risk prediction model for the early identification of severe COVID-19 among hospitalized COVID-19 patients. A total of 137 patients with mild COVID-19 (mCOVID-19) and 53 patients with severe COVID-19 (sCOVID-19) were recruited from the Center Hospital of the National Center for Global Health and Medicine (NCGM), Tokyo, Japan for the period of February-August 2020. High-resolution sequencing-based typing for eight HLA genes was performed using next-generation sequencing. In the HLA association studies, HLA-A*11:01:01:01 [Pc = 0.013, OR = 2.26 (1.27-3.91)] and HLA-C*12:02:02:01-HLA-B*52:01:01:02 [Pc = 0.020, OR = 2.25 (1.24-3.92)] were found to be significantly associated with the severity of COVID-19. After multivariate analysis controlling for other confounding factors and comorbidities, HLA-A*11:01:01:01 [P = 3.34E-03, OR = 3.41 (1.50-7.73)], age at diagnosis [P = 1.29E-02, OR = 1.04 (1.01-1.07)] and sex at birth [P = 8.88E-03, OR = 2.92 (1.31-6.54)] remained significant. The area under the curve of the risk prediction model utilizing HLA-A*11:01:01:01, age at diagnosis, and sex at birth was 0.772, with sensitivity of 0.715 and specificity of 0.717. To the best of our knowledge, this is the first article that describes associations of HLA alleles with COVID-19 at the 4-field (highest) resolution level. Early identification of potential sCOVID-19 could help clinicians prioritize medical utility and significantly decrease mortality from COVID-19.

Asymptomatic COVID-19 re-infection in a Japanese male by elevated half-maximal inhibitory concentration (IC50) of neutralizing antibodies.

INTRODUCTION: "Re-infection" with COVID-19 is a growing concern; re-infection cases have reported worldwide. However, the clinical characteristics of SARS-CoV-2 re-infection, including the levels and role of anti-SARS-CoV-2 Spike protein IgG antibodies and the half-maximal concentration (IC50) of neutralizing antibodies remain unknown. METHODS: Both the epidemiological and clinical information has been collected during two episodes of COVID-19 in a patient. Laboratory results, including RT-PCR, Ct values, anti-SARS-CoV-2 Spike protein IgG antibodies, and the IC50 of neutralizing antibodies levels were analyzed on the patient. RESULTS: The patient was a 58-year-old man who developed moderate COVID-19 pneumonia with oxygen demand (cannula 2 L/min) in the first episode. By day 30, he recuperated and was discharged after testing negative for SARS-CoV-2. After two and a half months, his three family members showed COVID-19 symptoms and tested positive for SARS-CoV-2. He tested positive for SARS-CoV-2 once again and was asymptomatic (the second episode). The IC50 of neutralizing antibodies against SARS-CoV-2 greatly increased from 50.0 µg/mL (after the first episode) to 14.8 µg/mL (after the second episode), and remained strongly reactive (20.1 µl/mL) after 47 days of the second episode. CONCLUSIONS: Epidemiological, clinical, and serological analyses confirmed that the patient had re-infection instead of persistent viral shedding from first infection. Our results suggest that SARS-CoV-2 re-infection may manifest as asymptomatic with increased neutralizing antibody levels. Further studies such as the virus characteristics, immunology, and epidemiology on SARS-CoV-2 re-infection are needed.

Association of HLA-DRB1*09:01 with severe COVID-19.

HLA-A, -C, -B, and -DRB1 genotypes were analyzed in 178 Japanese COVID-19 patients to investigate the association of HLA with severe COVID-19. Analysis of 32 common HLA alleles at four loci revealed a significant association between HLA-DRB1*09:01 and severe COVID-19 (odds ratio [OR], 3.62; 95% CI, 1.57-8.35; p = 0.00251 [permutation p value = 0.0418]) when age, sex, and other common HLA alleles at the DRB1 locus were adjusted. The DRB1*09:01 allele was more significantly associated with risk for severe COVID-19 compared to preexisting medical conditions such as hypertension, diabetes, and cardiovascular diseases. These results indicate a potential role for HLA in predisposition to severe COVID-19.

Neutralization of SARS-CoV-2 with IgG from COVID-19-convalescent plasma.

While there are various attempts to administer COVID-19-convalescent plasmas to SARS-CoV-2-infected patients, neither appropriate approach nor clinical utility has been established. We examined the presence and temporal changes of the neutralizing activity of IgG fractions from 43 COVID-19-convalescent plasmas using cell-based assays with multiple endpoints. IgG fractions from 27 cases (62.8%) had significant neutralizing activity and moderately to potently inhibited SARS-CoV-2 infection in cell-based assays; however, no detectable neutralizing activity was found in 16 cases (37.2%). Approximately half of the patients (~ 41%), who had significant neutralizing activity, lost the neutralization activity within ~ 1 month. Despite the rapid decline of neutralizing activity in plasmas, good amounts of SARS-CoV-2-S1-binding antibodies were persistently seen. The longer exposure of COVID-19 patients to greater amounts of SARS-CoV-2 elicits potent immune response to SARS-CoV-2, producing greater neutralization activity and SARS-CoV-2-S1-binding antibody amounts. The dilution of highly-neutralizing plasmas with poorly-neutralizing plasmas relatively readily reduced neutralizing activity. The presence of good amounts of SARS-CoV-2-S1-binding antibodies does not serve as a surrogate ensuring the presence of good neutralizing activity. In selecting good COVID-19-convalescent plasmas, quantification of neutralizing activity in each plasma sample before collection and use is required.

The Anticoagulant Nafamostat Potently Inhibits SARS-CoV-2 S Protein-Mediated Fusion in a Cell Fusion Assay System and Viral Infection In Vitro in a Cell-Type-Dependent Manner.

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.

Factors associated with anti-SARS-CoV-2 IgG antibody production in patients convalescing from COVID-19.

INTRODUCTION: Among patients with coronavirus disease 2019 (COVID-19), the factors that affect anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody production remain unclear. This study aimed to identify such factors among patients convalescing from COVID-19. METHODS: This study comprised patients who had been diagnosed with COVID-19 between January 1 and June 30, 2020 and gave consent for anti-SARS-CoV-2 spike protein antibody measurement using enzyme-linked immunosorbent assay during their acute and/or convalescent phases. Factors related to elevated antibody titers and the relationship between the days from disease onset and the development of antibody titers were assessed. RESULTS: A total of 84 participants enrolled in the study. Nineteen participants had antibody titers measured during the convalescent phase only, and 65 participants had antibody titers measured during the acute and convalescent phases. The antibody titers peaked in weeks 5 and 6. The stepwise multivariate log-normal analysis revealed that male sex (P = 0.04), diabetes mellitus (P = 0.03), and high C-reactive protein levels during the disease course (P < 0.001) were associated with elevated IgG antibodies. Glucocorticoid use was not associated with antibody titers. CONCLUSION: The study found that high values of maximum CRP levels during the acute phase, male sex, and diabetes mellitus were associated with elevated antibody titers. Antibody titers tended to be highest in the first 5 or 6 weeks after the onset of symptoms.

Myeloid cell dynamics correlating with clinical outcomes of severe COVID-19 in Japan.

An expanded myeloid cell compartment is a hallmark of severe coronavirus disease 2019 (COVID-19). However, data regarding myeloid cell expansion have been collected in Europe, where the mortality rate by COVID-19 is greater than those in other regions including Japan. Thus, characteristics of COVID-19-induced myeloid cell subsets remain largely unknown in the regions with low mortality rates. Here, we analyzed cellular dynamics of myeloid-derived suppressor cell (MDSC) subsets and examined whether any of them correlate with disease severity and prognosis, using blood samples from Japanese COVID-19 patients. We observed that polymorphonuclear (PMN)-MDSCs, but not other MDSC subsets, transiently expanded in severe cases but not in mild or moderate cases. Contrary to previous studies in Europe, this subset selectively expanded in survivors of severe cases and subsided before discharge, but such transient expansion was not observed in non-survivors in Japanese cohort. Analysis of plasma cytokine/chemokine levels revealed positive correlation of PMN-MDSC frequencies with IL-8 levels, indicating the involvement of IL-8 on recruitment of PMN-MDSCs to peripheral blood following the onset of severe COVID-19. Our data indicate that transient expansion of the PMN-MDSC subset results in improved clinical outcome. Thus, this myeloid cell subset may be a predictor of prognosis in cases of severe COVID-19 in Japan.

SARS-CoV-2 Leakage From the Gas Outlet Port During Extracorporeal Membrane Oxygenation for COVID-19.

Patients with the coronavirus disease 2019 (COVID-19) sometimes develop refractory respiratory failure and may require venovenous extracorporeal membrane oxygenation (VV-ECMO). It is known that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is sometimes present in the blood of COVID-19 patients. VV-ECMO is often used for several weeks, and plasma leaks can occur, albeit rarely. Hence, in terms of infection control, a concern is that SARS-CoV-2 may leak from the gas outlet port of the oxygenator during ECMO support of critically ill COVID-19 patients. The aim of this study was to clarify whether SARS-CoV-2 leaks from the oxygenator during ECMO support. Five patients with critical COVID-19 pneumonia were placed on VV-ECMO. Silicone-coated polypropylene membrane oxygenators were used in the ECMO circuits for these patients. SARS-CoV-2 ribonucleic acid (RNA) was measured by quantitative reverse transcription polymerase chain reaction in serum and at the gas outlet port of the ECMO circuit at the time of circuit replacement or liberation from ECMO. SARS-CoV-2 RNA was detected in the gas outlet port of the ECMO circuit for three of the five patients. None of the medical staff involved in the care of these five patients has been infected with COVID-19. In conclusion, SARS-CoV-2 could leak to the gas outlet port of the ECMO circuit through silicone-coated polypropylene membranes during ECMO support of critically ill COVID-19 patients.