Cytokine and Chemokine Profiles in Acute Severe Fever with Thrombocytopenia Syndrome and Scrub Typhus in South Korea.

In East Asia, severe fever with thrombocytopenia syndrome (SFTS) and scrub typhus, which are common endemic tick- and mite-mediated diseases sharing common clinical manifestations, are becoming public health concerns. However, there are limited data on the comparative immunopathogenesis between the two diseases. We compared the cytokine profiles of SFTS and scrub typhus to further elucidate immune responses that occur during the disease courses. We prospectively enrolled 44 patients with confirmed SFTS and 49 patients with scrub typhus from July 2015 to December 2020. In addition, 10 healthy volunteers were enrolled as healthy controls. A cytometric bead array was used to analyze plasma samples for 16 cytokines. A total of 68 plasma samples, including 31 (45.6%) from patients with SFTS and 37 (54.4%) from patients with scrub typhus, were available for cytokine measurement. There were three cytokine expression patterns: increased levels in both SFTS and scrub typhus (interleukin 6 [IL-6], IL-10, interferon gamma induced protein 10 [IP-10], and granulocyte-macrophage colony-stimulating factor [GM-CSF]), highest levels in SFTS (interferon alpha [IFN-α], IFN-γ, granulocyte-CSF [G-CSF], monocyte chemotactic protein 1 [MCP-1], macrophage inflammatory protein 1α [MIP-1α], and IL-8), and distinct levels in scrub typhus (IL-12p40, tumor necrosis factor alpha [TNFα], IL-1ß, regulated on activation and normally T-cell expressed and secreted [RANTES], IL-17A, and vascular endothelial growth factor [VEGF]). Although patients with acute SFTS and scrub typhus exhibited partly shared expression patterns of cytokines related to disease severity, the different profiles of cytokines and chemokines might contribute to higher mortality in SFTS than in scrub typhus. Discrete patterns of helper T cell-related cytokines and VEGF might reflect differences in CD4 T-cell responses and vascular damage between these diseases.

Diagnostic Usefulness of Varicella Zoster Virus-Specific Immunoglobulin (Ig) A and IgG in Patients With Herpes Zoster.

BACKGROUND: Whether varicella zoster virus (VZV) antibody titer could discriminate patients with herpes zoster (HZ) from healthy controls (HCs) is unclear. We evaluated the diagnostic usefulness of VZV-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies in patients with confirmed HZ. METHODS: Study subjects comprised patients with confirmed HZ by salivary VZV DNA positivity and control age- and sex-matched HCs. Saliva was collected and concurrent blood samples were obtained on the first visit day (acute phase) and after 4 weeks (convalescent phase) from 44 HZ patients. All 44 healthy volunteers provided blood and saliva samples once. RESULTS: The median VZV IgA titers in acute-phase (5.2 mIU/mL, P < 0.001) and convalescent-phase (15.8 mIU/mL, P < 0.001) serum samples from HZ patients were significantly higher than those in HCs (1.35 mIU/mL). VZV IgA positivity was detected in about 20% of acute phase serum and convalescent-phase serum of HZ patients. The median VZV IgG antibody titers of HZ patients during acute (1,471.0 mIU/mL, P < 0.001) and convalescent (4,934.7 mIU/mL, P < 0.001) phases were significantly higher than the median titer reported for HCs (591.6 mIU/mL). A four-fold or higher increase in VZV IgG antibody titer was observed in 36.4% of HZ patients. CONCLUSION: VZV IgA positivity or four-fold or higher increase in VZV IgG antibody titers were not detected in a satisfactory proportion of HZ-infected patients. However, the titer of VZV IgA or IgG antibody particularly in convalescent-phase sera may discriminate HZ patients from HCs.

Comparison of the rapidity of SARS-CoV-2 immune responses between primary and booster vaccination for COVID-19.

BACKGROUND/AIMS: The rapidity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific memory B or T cell response in vaccinated individuals is important for our understanding of immunopathogenesis of coronavirus disease 2019 (COVID-19). We therefore compared the timing of adequate immune responses between the first and booster doses of COVID-19 vaccines in infection-naïve healthcare workers. METHODS: We enrolled healthcare workers who received two doses of either the BNT162b2 vaccine or the ChAdOx1 vaccine, all of whom received the BNT162b2 vaccine as the booster (the third) dose. Spike 1 (S1)-immunoglobulin G (IgG) antibodies and interferon gamma producing T cell responses were measured at 0, 7, 14, and 21 days after the first dose, and at 0 and between 2 to 7 days after the booster dose. RESULTS: After the first-dose vaccination, the S1-IgG antibody responses were elicited within 14 days in the BNT162b2 group and within 21 days in the ChAdOx1 group. After the booster dose, the S1-IgG antibody responses were elicited within 5 days in both groups. The SARS-CoV-2-specific T cell responses appeared at 7 days after the first dose and at 4 days after the booster dose. CONCLUSION: SARS-CoV-2-specific immune responses by memory B cells and T cells may be expected to appear around 4 to 5 days after the booster dose.

Comparison of antibody responses after the 1st and 2nd doses of COVID-19 vaccine with those of patients with mild or severe COVID-19.

BACKGROUND/AIMS: Data comparing the antibody responses of different coronavirus disease 2019 (COVID-19) vaccine platforms according to dose with natural severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection-induced antibody responses are limited. METHODS: Blood samples from adult patients with mild and severe COVID-19 and healthcare workers who received ChAdOx1 nCoV-19 vaccine (2nd dose at 12-week intervals) and BNT162b2 vaccine (2nd dose at 3-week intervals) were collected and compared by immunoglobulin G immune responses to SARS-CoV-2 specific spike protein using an in-house-developed enzyme-linked immunosorbent assay. RESULTS: A total of 53 patients, including 12 and 41 with mild and severe COVID-19, respectively, were analyzed. In addition, a total of 73 healthcare workers, including 37 who received ChAdOx1 nCoV-19 and 36 who received BNT162b2, were enrolled. Antibody responses after the first and second doses of the ChAdOx1 nCoV-19 vaccine or the first dose of the BNT162b2 vaccine were similar to those in convalescent patients with mild COVID-19, but lower than those in convalescent patients with severe COVID-19, respectively. However, after the second dose of the BNT162b2 vaccine, the antibody response was comparable to that in convalescent patients with severe COVID-19. CONCLUSION: Our data suggest that the second dose of mRNA vaccination may be more beneficial in terms of long-term immunity and prevention of SARS-CoV-2 variant infection than a single dose of COVID-19 vaccination or homologous second challenge ChAdOx1 nCoV-19.

Immune Responses to the ChAdOx1 nCoV-19 and BNT162b2 Vaccines and to Natural Coronavirus Disease 2019 Infections Over a 3-Month Period.

BACKGROUND: There are limited data directly comparing immune responses to vaccines and to natural infections with coronavirus disease 2019 (COVID-19). This study assessed the immunogenicity of the BNT162b2 and ChAdOx1 nCoV-19 vaccines over a 3-month period and compared the immune responses with those to natural infections. METHOD: We enrolled healthcare workers who received BNT162b2 or ChAdOx1 nCoV-19 vaccines and patients with confirmed COVID-19 and then measured S1 immunoglobulin (Ig) G and neutralizing antibodies and T-cell responses. RESULTS: A total of 121 vaccinees and 26 patients with confirmed COVID-19 were analyzed. After the second dose, the BNT162b2 vaccine yielded S1 IgG antibody responses similar to those achieved with natural infections (mean IgG titer [standard deviation], 2241 [899] vs 2601 [5039]; P = .68) but significantly stronger than responses to the ChAdOx1 vaccine (174 [96]; P < .001). The neutralizing antibody titer generated by BNT162b2 was 6-fold higher than that generated by ChAdOx1 but lower than that by natural infection. T-cell responses persisted for 3 months with BNT162b2 and natural infection but decreased with ChAdOx1. CONCLUSIONS: Antibody responses after the second dose of BNT162b2 are higher than after the second dose of ChAdOx1 and like those occurring after natural infection. T-cell responses are maintained longer in BNT162b2 vaccinees than in ChAdOx1 vaccinees.

Diagnostic usefulness of subgenomic RNA detection of viable SARS-CoV-2 in patients with COVID-19.

OBJECTIVES: The development of a rapid diagnostic test for viable SARS-CoV-2 is important for infection control. Real-time RT-PCR assays detect non-viable virus, and cell culture differentiates viable virus but it takes several weeks and is labour-intensive. Subgenomic RNAs may reflect replication-competent virus. We therefore evaluated the usefulness of subgenomic RNAs for diagnosing viable SARS-CoV-2 in patients with COVID-19. METHODS: Patients with various severities of confirmed COVID-19 were enrolled at a tertiary hospital between February and December 2020. RT-PCR assay results for genomic and subgenomic RNA of SARS-CoV-2 from nasopharyngeal swab, sputum and saliva specimens were compared with cell culture results. RESULTS: A total 189 specimens from 20 COVID-19 patients were tested in genomic and subgenomic PCR assays and cultured on Vero cells. Of these 189 samples, 62 (33%) gave positive culture results, 93 (49%) negative results and the remaining 34 (18%) indeterminate results. Compared with cell culture results, the sensitivities of genomic RNA and subgenomic RNA of the N and S genes were comparable at 100%, but the specificity of subgenomic RNA (N, 65% and S, 68%) was higher than that of genomic RNA (N, 23% and S, 17%, p < 0.001). The mean durations of positive culture and subgenomic RNA were 11.39 ± 10.34 and 13.75 ± 11.22 days after symptom onset (p 0.437), respectively, while that of genomic RNA was 22.85 ± 11.83 days after symptom onset (p < 0.001). DISCUSSION: Our comparison of subgenomic RNA detection with symptom duration and SARS-CoV-2 culture positivity provides a significant advancement on the transmissibility-based approach beyond the detection of SARS-CoV-2 genomic RNA, and warrants further studies on the development of better diagnostic strategy.

Viral and Immunologic Factors Associated with Fatal Outcome of Patients with Severe Fever with Thrombocytopenia Syndrome in Korea.

Significant progress has been made on the molecular biology of the severe fever with thrombopenia virus (SFTSV); however, many parts of the pathophysiological mechanisms of mortality in SFTS remain unclear. In this study, we investigated virologic and immunologic factors for fatal outcomes of patients with SFTS. We prospectively enrolled SFTS patients admitted from July 2015 to October 2020. Plasma samples were subjected to SFTSV RNA RT-PCR, multiplex microbead immunoassay for 17 cytokines, and IFA assay. A total of 44 SFTS patients were enrolled, including 37 (84.1%) survivors and 7 (15.9%) non-survivors. Non-survivors had a 2.5 times higher plasma SFTSV load than survivors at admission (p < 0.001), and the viral load in non-survivors increased progressively during hospitalization. In addition, non-survivors did not develop adequate anti-SFTSV IgG, whereas survivors exhibited anti-SFTSV IgG during hospitalization. IFN-α, IL-10, IP-10, IFN-γ, IL-6, IL-8, MCP-1, MIP-1α, and G-CSF were significantly elevated in non-survivors compared to survivors and did not revert to normal ranges during hospitalization (p < 0.05). Severe signs of inflammation such as a high plasma concentration of IFN-α, IL-10, IP-10, IFN-γ, IL-6, IL-8, MCP-1, MIP-1α, and G-CSF, poor viral control, and inadequate antibody response during the disease course were associated with mortality in SFTS patients.

Dynamics of Viral Shedding and Symptoms in Patients with Asymptomatic or Mild COVID-19.

We conducted a prospective cohort study at a community facility designated for the isolation of individuals with asymptomatic or mild COVID-19 between 10 January and 22 February 2021 to investigate the relationship of viral shedding with symptom changes of COVID-19. In total, 89 COVID-19 adult patients (12 asymptomatic, 16 presymptomatic, 61 symptomatic) were enrolled. Symptom scores, the genomic RNA and subgenomic RNA of SARS-CoV-2 from saliva samples with a cell culture were measured. Asymptomatic COVID-19 patients had a similar viral load to symptomatic patients during the early course of the disease, but exhibited a rapid decrease in viral load with the loss of infectivity. Subgenomic RNA and viable virus by cell culture in asymptomatic patients were detected only until 3 days after diagnosis, and the positivity of the subgenomic RNA and cell culture in symptomatic patients gradually decreased in both from 40% in the early disease course to 13% at 10 days and 4% at 8 days after the symptom onset, respectively. In conclusion, symptomatic patients have a high infectivity with high symptom scores during the early disease course and gradually lose infectivity depending on the symptom. Conversely, asymptomatic patients exhibit a rapid decrease in viral load with the loss of infectivity, despite a similar viral load during the early disease course.

Rapid COVID-19 Molecular Diagnostic System Using Virus Enrichment Platform.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus (SARS-CoV)-2, is rapidly spreading and severely straining the capacities of public health communities and systems around the world. Therefore, accurate, rapid, and robust diagnostic tests for COVID-19 are crucial to prevent further spread of the infection, alleviate the burden on healthcare and diagnostic facilities, and ensure timely therapeutic intervention. To date, several detection methods based on nucleic acid amplification have been developed for the rapid and accurate detection of SARS-CoV-2. Despite the myriad of advancements in the detection methods for SARS-CoV-2, rapid sample preparation methods for RNA extraction from viruses have rarely been explored. Here, we report a rapid COVID-19 molecular diagnostic system that combines a self-powered sample preparation assay and loop-mediated isothermal amplification (LAMP) based naked-eye detection method for the rapid and sensitive detection of SARS-CoV-2. The self-powered sample preparation assay with a hydrophilic polyvinylidene fluoride filter and dimethyl pimelimidate can be operated by hand, without the use of any sophisticated instrumentation, similar to the reverse transcription (RT)-LAMP-based lateral flow assay for the naked-eye detection of SARS-CoV-2. The COVID-19 molecular diagnostic system enriches the virus population, extracts and amplifies the target RNA, and detects SARS-CoV-2 within 60 min. We validated the accuracy of the system by using 23 clinical nasopharyngeal specimens. We envision that this proposed system will enable simple, facile, efficient, and inexpensive diagnosis of COVID-19 at home and the clinic as a pre-screening platform to reduce the burden on the medical staff in this pandemic era.

Comparison of Antibody and T Cell Responses Induced by Single Doses of ChAdOx1 nCoV-19 and BNT162b2 Vaccines.

There are limited data directly comparing humoral and T cell responses to the ChAdOx1 nCoV-19 and BNT162b2 vaccines. We compared Ab and T cell responses after first doses of ChAdOx1 nCoV-19 vs. BNT162b2 vaccines. We enrolled healthcare workers who received ChAdOx1 nCoV-19 or BNT162b2 vaccine in Seoul, Korea. Anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S1 protein-specific IgG Abs (S1-IgG), neutralizing Abs (NT Abs), and SARS-CoV-2-specific T cell response were evaluated before vaccination and at 1-wk intervals for 3 wks after vaccination. A total of 76 persons, comprising 40 injected with the ChAdOx1 vaccine and 36 injected with the BNT162b2 vaccine, participated in this study. At 3 wks after vaccination, the mean levels (±SD) of S1-IgG and NT Abs in the BNT162b2 participants were significantly higher than in the ChAdOx1 participants (S1-IgG, 14.03±7.20 vs. 6.28±8.87, p<0.0001; NT Ab, 183.1±155.6 vs. 116.6±116.2, p=0.035), respectively. However, the mean values of the T cell responses in the 2 groups were comparable after 2 wks. The humoral immune response after the 1st dose of BNT162b2 developed faster and was stronger than after the 1st dose of ChAdOx1. However, the T cell responses to BNT162b2 and ChAdOx1 were similar.

SARS-CoV-2-Specific Antibody and T Cell Response Kinetics According to Symptom Severity.

Data on the longevity of humoral and cell-mediated immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients with coronavirus disease 2019 (COVID-19) are limited. We evaluated the detailed kinetics of antibody and T-cell responses at the acute, convalescent, and post-convalescent phases in COVID-19 patients with a wide range of severity. We enrolled patients with COVID-19 prospectively from four hospitals and one community treatment center between February 2020 and January 2021. symptom severity was classified as mild, moderate, or severe/critical. Patient blood samples were collected at 1 week (acute), 1 month (convalescent), and 2 months after symptom onset (post-convalescent). Human SARS-CoV-2 IgG and IgM antibodies were measured using in-house-developed ELISA. The SARS-CoV-2-specific T-cell responses against overlapping peptides of spike proteins and nucleoprotein were measured by interferon-γ enzyme-linked immunospot assays. Twenty-five COVID-19 patients were analyzed (mild, n = 5; moderate, n = 9; severe/critical, n = 11). IgM and IgG antibody responses peaked at 1 month after symptom onset and decreased at 2 months. IgG response levels were significantly greater in the severe/critical group compared with other groups. Interferon-γ-producing T-cell responses increased between 1 week and 1 month after symptom onset, and had a trend toward decreasing at 2 months, but did not show significant differences according to severity. Our data indicate that SARS-CoV-2-specific antibody responses were greater in those with severe symptoms and waned after reaching a peak around 1 month after symptom onset. However, SARS-CoV-2-specific T-cell responses were not significantly different according to symptom severity, and decreased slowly during the post-convalescent phase.

Varicella zoster virus (VZV)-specific immunity and subclinical VZV reactivation in patients with autoimmune diseases.

BACKGROUND/AIMS: The risk of herpes zoster (HZ) is increased in patients with autoimmune diseases (AID), probably due to immunosuppressive therapy. METHODS: This prospective cross-sectional study investigated varicella zoster virus (VZV)-specific immunity in relation to subclinical VZV reactivation in 48 AID patients and 48 healthy controls (HCs). We assessed humoral immunity (serum VZV immunoglobulin g [IgG], IgA, and IgM) and cell-mediated immunity (interferon-γ [IFNγ]-releasing assay) to VZV as well as salivary VZV DNA status. Subclinical VZV reactivation was confirmed by detecting VZV DNA in saliva or VZV IgM in serum in the absence of typical HZ symptoms. RESULTS: Median IgA levels were higher in the AID group than in the HC group, while VZV IgG and IgM levels were comparable between the groups. AID patients showed fewer IFNγ spot-forming cells (SFCs) upon VZV stimulation than HCs (58.2 vs. 122.0 SFCs/106 peripheral blood mononuclear cells [PBMCs], p < 0.0001). Subclinical VZV reactivation was more frequent in AID patients than in HCs (12.5% vs. 0%, p = 0.01). AID patients with VZV reactivation received prednisolone more frequently and at a higher dose than AID patients without reactivation. VZV-specific IFNγ SFCs were significantly lower in patients with VZV reactivation among AID patients (26.3 vs. 62.6 SFCs/106 PBMCs, p < 0.0001). CONCLUSION: Results suggest that poor cellular response against VZV might cause clinical and subclinical reactivation of VZV in AID patients.

Frequency of putative enteric zoster diagnosed using saliva samples in patients with abdominal pain: a prospective study.

BACKGROUND: Varicella-zoster virus (VZV) infects and establishes latency in neurons in the ganglia of the cranial nerve, dorsal root and enteric ganglia. VZV reactivation in enteric neurons (enteric zoster) can cause non-specific abdominal pain and/or serious gastrointestinal dysfunction without cutaneous manifestations. Detection of VZV DNA in saliva may be useful for identifying enteric zoster. We evaluated the frequency of putative enteric zoster based on the presence of salivary VZV DNA in patients with acute abdominal pain. METHODS: Adult patients who visited the emergency room due to moderate to severe acute abdominal pain were prospectively enrolled at a tertiary hospital between May 2019 and November 2019. Abdominopelvic computed tomography (APCT) was performed in all patients. We also evaluated the presence of salivary VZV DNA in patients with confirmed coronavirus disease-19 (COVID-19) who were under stressful conditions. Saliva samples were collected from all studied patients. Enteric zoster was suspected based on the presence of salivary VZV DNA, detected using real-time polymerase chain reaction (PCR). RESULTS: Fifty patients with moderate to severe abdominal pain were enrolled. Five of 50 patients exhibited positive VZV-DNA PCR results. APCT revealed that among these five patients, two had pancreatic head cancer, two had small bowel obstruction after intra-abdominal surgery, and one had no remarkable findings. However, all 14 patients with COVID-19 showed negative salivary VZV-DNA PCR results. CONCLUSIONS: Approximately 10% of patients with moderate to severe acute abdominal pain showed positivity for salivary VZV DNA. Further studies are warranted on whether antiviral therapy based on salivary VZV-DNA PCR results may relieve abdominal pain in the studied patient population. TRIAL REGISTRATION: clinicaltrial.gov, number NCT03862092.

Correlation between Reactogenicity and Immunogenicity after the ChAdOx1 nCoV-19 and BNT162b2 mRNA Vaccination.

Correlation between vaccine reactogenicity and immunogenicity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unclear. Thus, we investigated to determine whether the reactogenicity after coronavirus disease 2019 vaccination is associated with antibody (Ab) titers and T cell responses. This study was prospective cohort study done with 131 healthcare workers at tertiary center in Seoul, South Korea. The degrees of the local reactions after the 1st and 2nd doses of ChAdOx1 nCov-19 (ChAdOx1) vaccination were significantly associated with the S1-specific IgG Ab titers (p=0.003 and 0.01, respectively) and neutralizing Ab (p=0.04 and 0.10, respectively) in age- and sex-adjusted multivariate analysis, whereas those after the BNT162b2 vaccination did not show significant associations. T cell responses did not show significant associations with the degree of reactogenicity after the ChAdOx1 vaccination or the BNT162b2 vaccination. Thus, high degree of local reactogenicity after the ChAdOx1 vaccine may be used as an indicator of strong humoral immune responses against SARS-CoV-2.

Factors of Severity in Patients with COVID-19: Cytokine/Chemokine Concentrations, Viral Load, and Antibody Responses.

The severity of COVID-19 ranges from mild to critical diseases. However, limited data have been published on the detailed kinetics of viral load and host immune response throughout the disease course depending on disease severity. In this study, we comprehensively analyzed viral load, antibody responses to SARS-CoV-2, and cytokines/chemokines during the disease course, and identified the factors related to severity. Nasopharyngeal (NP) and plasma specimens were obtained from 31 patients with COVID-19 during hospitalization. Viral RNA in NP specimens was quantified by reverse transcription-PCR. Anti-SARS-CoV-2 antibodies and cytokines/chemokines in plasma specimens were analyzed by ELISA and cytometric bead array. The viral load in patients with COVID-19 peaked at the early stage of the disease and continuously decreased. Severe and critical cases showed higher viral load and prolonged viral shedding than asymptomatic and mild cases. Whereas plasma IgG was gradually increased and maintained during hospitalization, plasma IgM peaked at 3 weeks after symptom onset and dissipated. The antibody response in severe and critical cases was slightly delayed but stronger than those in others. High levels of interferon (IFN)-α, IFN-γ-induced protein-10, monokine induced by IFN-γ, and interleukin-6 at 5-10 days from symptom onset were associated with the severity of COVID-19. Our data indicate that high viral load in the respiratory tract and excessive production of cytokines and chemokines between 1 and 2 weeks from the symptom onset were significantly associated with the severity of COVID-19.

Immunophenotyping of COVID-19 and influenza highlights the role of type I interferons in development of severe COVID-19.

Although most SARS-CoV-2-infected individuals experience mild coronavirus disease 2019 (COVID-19), some patients suffer from severe COVID-19, which is accompanied by acute respiratory distress syndrome and systemic inflammation. To identify factors driving severe progression of COVID-19, we performed single-cell RNA-seq using peripheral blood mononuclear cells (PBMCs) obtained from healthy donors, patients with mild or severe COVID-19, and patients with severe influenza. Patients with COVID-19 exhibited hyper-inflammatory signatures across all types of cells among PBMCs, particularly up-regulation of the TNF/IL-1ß-driven inflammatory response as compared to severe influenza. In classical monocytes from patients with severe COVID-19, type I IFN response co-existed with the TNF/IL-1ß-driven inflammation, and this was not seen in patients with milder COVID-19. Interestingly, we documented type I IFN-driven inflammatory features in patients with severe influenza as well. Based on this, we propose that the type I IFN response plays a pivotal role in exacerbating inflammation in severe COVID-19.