Spirulina platensis and biosynthesized selenium nanoparticles improve performance, antioxidant status, humoral immunity and dietary and ileal microbial populations of heat-stressed broilers.

This study was conducted to assess the impact of dietary incorporation of Spirulina platensis and selenium nanoparticles (SeNPs) individually or in combinations on growth performance, antioxidant status, humoral immune response, and microbial populations in diet and ileum of heat-stressed broilers. Ross-308 one-day chicks (n = 450) were fed one of 9 experimental diets with five replicate cages in 2 phases for 35 d. The experimental diets were a control basal diet without supplementation or with 0.1 mg SeNPs, 0.2 mg SeNPs, 5 g Spirulina, 10 g Spirulina, 0.1 mg SeNPs + 5 g Spirulina, 0.1 mg SeNPs + 10 g Spirulina, 0.2 mg SeNPs + 5 g Spirulina and 0.2 mg SeNPs + 10 g Spirulina per kg diet. Dietary supplementation with Spirulina and SeNPs significantly (P < 0.05) increased body weight gain and European production efficiency factor. Serum GPx and SOD were significantly (P < 0.05) increased with dietary Spirulina and SeNPs supplementation, while, TBARS was decreased (P < 0.05). Circulating immunoglobulin IgM, IgA and IgG were increased in treated birds compared to the control ones, while the antibody titers to IBD, AIV, and NDV were not significantly altered. The results showed that SeNPs and Spirulina exhibited dose-dependent antimicrobial activities against ileal counts of total bacterial, total molds and yeast, coliform, E. coli, Salmonella spp. and Enterococcus spp. However, ileal populations of Lactic acid bacteria were increased with dietary Spirulina and SeNPs in a dose-dependent manner. The microbial load in broilers' diets was reduced by dietary incorporation of S. platensis and SeNPs. These results indicate that Spirulina and SeNPs can be potentially used as growth promoters and antioxidant, immunostimulant, and antimicrobial agents in heat-stressed broilers.

Clinical and antibody characteristics reveal diverse signatures of severe and non-severe SARS-CoV-2 patients.

BACKGROUND: COVID-19 pandemic continues, clarifying signatures in clinical characters and antibody responses between severe and non-severe COVID-19 cases would benefit the prognosis and treatment. METHODS: In this study, 119 serum samples from 37 severe or non-severe COVID-19 patients from the First People's Hospital of Yueyang were collected between January 25 and February 18 2020. The clinical features, antibody responses targeting SARS-CoV-2 spike protein (S) and its different domains, SARS-CoV-2-specific Ig isotypes, IgG subclasses, ACE2 competitive antibodies, binding titers with FcγIIa and FcγIIb receptors, and 14 cytokines were comprehensively investigated. The differences between severe and non-severe groups were analyzed using Mann-Whitney U test or Fisher's exact test. RESULTS: Severe group including 9 patients represented lower lymphocyte count, higher neutrophil count, higher level of LDH, total bile acid (TBA) (P < 1 × 10-4), r-glutaminase (P = 0.011), adenosine deaminase (P < 1 × 10-4), procalcitonin (P = 0.004), C-reactive protein (P < 1 × 10-4) and D-dimer (P = 0.049) compared to non-severe group (28 patients). Significantly, higher-level Igs targeting S, different S domains (RBD, RBM, NTD, and CTD), FcγRIIa and FcγRIIb binding capability were observed in a severe group than that of a non-severe group, of which IgG1 and IgG3 were the main IgG subclasses. RBD-IgG were strongly correlated with S-IgG both in severe and non-severe group. Additionally, CTD-IgG was strongly correlated with S-IgG in a non-severe group. Positive RBD-ACE2 binding inhibition was strongly associated with high titers of antibody (S-IgG1, S-IgG3, NTD-IgG, RBD-IgA, NTD-IgA, and CTD-IgA) especially RBD-IgG and CTD-IgG in the severe group, while in the non-severe group, S-IgG3, RBD-IgG, NTD-IgG, and NTD-IgM were correlated with ACE2 blocking rate. S-IgG1, NTD-IgM and S-IgM were negatively associated with illness day in a severe group, while S-IgG3, RBD-IgA, CTD-IgA in the severe group (r = 0.363, P = 0.011) and S-IgG1, NTD-IgA, CTD-IgA in the non-severe group were positively associated with illness day. Moreover, GRO-α, IL-6, IL-8, IP-10, MCP-1, MCP-3, MIG, and BAFF were also significantly elevated in the severe group. CONCLUSION: Antibody detection provides important clinical information in the COVID-19 process. The different signatures in Ig isotypes, IgG subclasses, antibody specificity between the COVID-19 severe and non-severe group will contribute to future therapeutic and preventive measures development.

Analysis of Serological Biomarkers of SARS-CoV-2 Infection in Convalescent Samples From Severe, Moderate and Mild COVID-19 Cases.

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study is to identify biomarkers of humoral immunity that could be used to differentiate severe from mild or asymptomatic SARS-CoV-2 infections. Some of these biomarkers could be used to define CoP in further serological studies using samples from vaccination breakthrough and/or re-infection cases. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (IU) for virus neutralisation assays or in Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG/IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and an electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD/S antibodies. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

Elevated cytokines and chemokines in peripheral blood of patients with SARS-CoV-2 pneumonia treated with high-titer convalescent plasma.

The global SARS-CoV-2 coronavirus pandemic continues to be devastating in many areas. Treatment options have been limited and convalescent donor plasma has been used by many centers to transfer passive neutralizing antibodies to patients with respiratory involvement. The results often vary by institution and are complicated by the nature and quality of the donor plasma itself, the timing of administration and the clinical aspects of the recipients. SARS-CoV-2 infection is known to be associated with an increase in the blood concentrations of several inflammatory cytokines/chemokines, as part of the overall immune response to the virus and consequential to mediated lung pathology. Some of these correlates contribute to the cytokine storm syndrome and acute respiratory distress syndrome, often resulting in fatality. A Phase IIa clinical trial at our institution using high neutralizing titer convalescent plasma transfer gave us the unique opportunity to study the elevations of correlates in the first 10 days after infusion. Plasma recipients were divided into hospitalized COVID-19 pneumonia patients who did not (Track 2) or did (Track 3) require mechanical ventilation. Several cytokines were elevated in the patients of each Track and some continued to rise through Day 10, while others initially increased and then subsided. Furthermore, elevations in MIP-1α, MIP-1ß and CRP correlated with disease progression of Track 2 recipients. Overall, our observations serve as a foundation for further study of these correlates and the identification of potential biomarkers to improve upon convalescent plasma therapy and to drive more successful patient outcomes.

Diagnostic Vaccination in Clinical Practice.

Testing the antibody response to vaccination (diagnostic vaccination) is crucial in the clinical evaluation of primary immunodeficiency diseases. Guidelines from the American Academy of Allergy, Asthma & Immunology (AAAAI) provide detailed recommendations for diagnostic vaccination with pure pneumococcal polysaccharide vaccines (PPV). However, the degree of compliance with these guidelines and the utility of the guidelines in actual practice are undescribed. To address this, we systematically evaluated diagnostic vaccination in adult patients with suspected primary immunodeficiency diseases in a single tertiary center from 2011 to 2016 (n = 229). We found that full compliance with the AAAAI guidelines was achieved for only 39 patients (17%), suggesting that the guidelines are not easy to follow. Worse, interpretation according to the guidelines was heavily influenced by which serotype-specific antibodies that were used for the evaluation. We found that the arbitrary choices of serotype-specific antibodies could change the fraction of patients deemed to have 'adequate immunity' by a factor of four, exposing an inherent flaw in the guidelines. The flaw relates to dichotomous principles for data interpretation under the AAAAI guidelines. We therefore propose a revised protocol for diagnostic vaccination limited to PPV vaccination, subsequent antibody measurements, and data interpretation using Z-scores. The Z-score compiles multiple individual antibody levels, adjusted for different weighting, into one single continuous variable for each patient. In contrast to interpretation according to the AAAAI guidelines, the Z-scores were robust to variations in the choice of serotype-specific antibodies used for interpretation. Moreover, Z-scores revealed reduced immunity after vaccination in the patients with recurrent pneumonia (a typical symptom of antibody deficiency) compared with control patients. Assessment according to the AAAAI guidelines failed to detect this difference. We conclude that our simplified protocol and interpretation with Z-scores provides more robust clinical results and may enhance the value of diagnostic vaccination.

SARS-CoV-2 Antibody Isotypes in Systemic Lupus Erythematosus Patients Prior to Vaccination: Associations With Disease Activity, Antinuclear Antibodies, and Immunomodulatory Drugs During the First Year of the Pandemic.

Objectives: Impact of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic on individuals with arthritis has been highlighted whereas data on other rheumatic diseases, e.g., systemic lupus erythematosus (SLE), are scarce. Similarly to SLE, severe SARS-CoV-2 infection includes risks for thromboembolism, an unbalanced type I interferon response, and complement activation. Herein, SARS-CoV-2 antibodies in longitudinal samples collected prior to vaccination were analyzed and compared with SLE progression and antinuclear antibody (ANA) levels. Methods: One hundred patients (83 women) with established SLE and a regular visit to the rheumatologist (March 2020 to January 2021) were included. All subjects donated blood and had done likewise prior to the pandemic. SARS-CoV-2 antibody isotypes (IgG, IgA, IgM) to the cell receptor-binding S1-spike outer envelope protein were detected by ELISA, and their neutralizing capacity was investigated. IgG-ANA were measured by multiplex technology. Results: During the pandemic, 4% had PCR-confirmed infection but 36% showed SARS-CoV-2 antibodies of ≥1 isotype; IgA was the most common (30%), followed by IgM (9%) and IgG (8%). The antibodies had low neutralizing capacity and were detected also in prepandemic samples. Plasma albumin (p = 0.04) and anti-dsDNA (p = 0.003) levels were lower in patients with SARS-CoV-2 antibodies. Blood group, BMI, smoking habits, complement proteins, daily glucocorticoid dose, use of hydroxychloroquine, or self-reported coronavirus disease 2019 (COVID-19) symptoms (except fever, >38.5°C) did not associate with SARS-CoV-2 antibodies. Conclusion: Our data from early 2021 indicate that a large proportion of Swedish SLE patients had serological signs of exposure to SARS-CoV-2 but apparently with a minor impact on the SLE course. Use of steroids and hydroxychloroquine showed no distinct effects, and self-reported COVID-19-related symptoms correlated poorly with all antibody isotypes.

Dynamic Characteristic Analysis of Antibodies in Patients With COVID-19: A 13-Month Study.

There is a worldwide pandemic of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; yet our understanding remains limited on the characteristic of antibodies, especially for dynamic long-term tracking. Sequential serum samples were collected up to 416 days post onset of symptoms (POS) from 102 patients who were hospitalized with coronavirus disease 2019 (COVID-19). Immunoglobulin (Ig)G, IgM, and IgA levels targeting SARS-CoV-2 spike 1 receptor-binding domain (S1-RBD), spike 2 extracellular domain (S2-ECD), and nucleocapsid protein (N) were quantified as well as neutralizing activity. We were pleasantly surprised to find that the antibody remained detective and effective for more than a year POS. We also found the varied reactions of different antibodies as time passed: N-IgA rose most rapidly in the early stage of infection, while S2-IgG was present at a high level in the long time of observation. This study described the long traceable antibody response of the COVID-19 and offered hints about targets to screen for postinfectious immunity and for vaccination development of SARS-CoV-2.

Longitudinal analysis to characterize classes and subclasses of antibody responses to recombinant receptor-binding protein (RBD) of SARS-CoV-2 in COVID-19 patients in Thailand.

Serological assays to detect antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might contribute to confirming the suspected coronavirus disease 2019 (COVID-19) in patients not detected with molecular assays. Human antibodies that target the host angiotensin-converting enzyme 2-binding domain of the viral spike protein are a target for serodiagnosis and therapeutics. This study aimed to characterize the classes and subclasses of antibody responses to a recombinant receptor-binding protein (RBD) of SARS-CoV-2 in COVID-19 patients and investigated the reactivity of these antibodies in patients with other tropical infections and healthy individuals in Thailand. ELISAs for IgM, IgA, IgG and IgG subclasses based on RBD antigen were developed and tested with time series of 27 serum samples from 15 patients with COVID-19 and 60 samples from pre-COVID-19 outbreaks including acute dengue fever, murine typhus, influenza, leptospirosis and healthy individuals. Both RBD-specific IgA and IgG were detected in only 21% of the COVID-19 patients in the acute phase. The median IgA and IgG levels were significantly higher in the convalescent serum sample compared to the acute serum sample (P < 0.05). We observed the highest correlation between levels of IgG and IgA (rho = 0. 92). IgG1 and IgG3 were the major IgG subclasses detected in SARS-CoV-2 infection. Only acute IgG3 level was negatively associated with viral detection based on RT-PCR of ORF1ab gene (rho = -0.57). The median IgA and IgG levels in convalescence sera of COVID-19 patients were significantly higher than healthy individuals and convalescent sera of other febrile infectious patients. The analyses of antibody classes and subclasses provide insights into human immune responses against SARS-CoV-2 during natural infection and interpretation of antibody assays.

Longitudinal detection of SARS-CoV-2-specific antibody responses with different serological methods.

Serological testing for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies is used to detect ongoing or past SARS-CoV-2 infections. To study the kinetics of anti-SARS-CoV-2 antibodies and to assess the diagnostic performances of eight serological assays, we used 129 serum samples collected on known days post symptom onset (dpso) from 42 patients with polymerase chain reaction-confirmed coronavirus disease 2019 (COVID-19) and 54 serum samples from healthy blood donors, and children infected with seasonal coronaviruses. The sera were analyzed for the presence of immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA) antibodies using indirect immunofluorescence testing (IIFT) based on SARS-CoV-2-infected cells. They were further tested for antibodies against the S1 domain of the SARS-CoV-2 spike protein (IgG, IgA) and against the viral nucleocapsid protein (IgG, IgM) using enzyme-linked immunosorbent assays. The assay specificities were 94.4%-100%. The sensitivities varied largely between assays, reflecting their respective purposes. The sensitivities of IgA and IgM assays were the highest between 11 and 20 dpso, whereas the sensitivities of IgG assays peaked between 20 and 60 dpso. IIFT showed the highest sensitivities due to the use of the whole SARS-CoV-2 as substrate and provided information on whether or not the individual has been infected with SARS-CoV-2. Enzyme-linked immunosorbent assays provided further information about both the prevalence and concentration of specific antibodies against selected antigens of SARS-CoV-2.

Comparison of seroprevalence of SARS-CoV-2 infections with cumulative and imputed COVID-19 cases: Systematic review.

BACKGROUND: Accurate seroprevalence estimates of SARS-CoV-2 in different populations could clarify the extent to which current testing strategies are identifying all active infection, and hence the true magnitude and spread of the infection. Our primary objective was to identify valid seroprevalence studies of SARS-CoV-2 infection and compare their estimates with the reported, and imputed, COVID-19 case rates within the same population at the same time point. METHODS: We searched PubMed, Embase, the Cochrane COVID-19 trials, and Europe-PMC for published studies and pre-prints that reported anti-SARS-CoV-2 IgG, IgM and/or IgA antibodies for serosurveys of the general community from 1 Jan to 12 Aug 2020. RESULTS: Of the 2199 studies identified, 170 were assessed for full text and 17 studies representing 15 regions and 118,297 subjects were includable. The seroprevalence proportions in 8 studies ranged between 1%-10%, with 5 studies under 1%, and 4 over 10%-from the notably hard-hit regions of Gangelt, Germany; Northwest Iran; Buenos Aires, Argentina; and Stockholm, Sweden. For seropositive cases who were not previously identified as COVID-19 cases, the majority had prior COVID-like symptoms. The estimated seroprevalences ranged from 0.56-717 times greater than the number of reported cumulative cases-half of the studies reported greater than 10 times more SARS-CoV-2 infections than the cumulative number of cases. CONCLUSIONS: The findings show SARS-CoV-2 seroprevalence is well below "herd immunity" in all countries studied. The estimated number of infections, however, were much greater than the number of reported cases and deaths in almost all locations. The majority of seropositive people reported prior COVID-like symptoms, suggesting that undertesting of symptomatic people may be causing a substantial under-ascertainment of SARS-CoV-2 infections.

Determinants of Serum Immunoglobulin Levels: A Systematic Review and Meta-Analysis.

Background: An up-to-date overview of determinants of serum immunoglobulins in adults is pivotal for clinical practice and research, but currently lacking. We therefore performed a systematic review and meta-analysis to identify determinants of serum immunoglobulin levels. Methods: Embase, Web of Science, Medline, Cochrane, and Google Scholar were searched from inception to July 11th, 2019 for articles reporting on determinants of serum immunoglobulin A, G or M (IgA, IgG or IgM) in adult humans. Random and fixed effect models were applied to obtain pooled mean differences (MDs) and 95% confidence intervals (CIs) for the association of age and sex with serum immunoglobulins. Results: We retrieved 117 articles reporting on determinants of serum immunoglobulins, of which 28 could be meta-analyzed. Older compared to younger individuals had higher IgA (MD: 0.38; CI: 0.18 - 0.58), but lower IgM levels (MD: -0.40; 95%: -0.66 - -0.14). Men had higher IgA (MD: 0.22; CI: 0.03 - 0.42), but lower IgM levels (MD: -0.21; CI: -0.32 - -0.10) than women. Age and sex did not influence IgG. Caucasian ethnicity was associated with lower IgA, IgG, and IgM. Smoking and corticosteroid use were associated with lower IgG. Positive associations were reported of probiotics with IgG, alcohol with IgA, hypertension with IgA and IgG, and acute psychological stress with IgA, IgG, and IgM. Conclusions: Older age and male sex are associated with higher IgA, but lower IgM, and urge investigation of age- and sex-specific reference ranges of immunoglobulins. Other identified determinants were ethnicity, diet, lifestyle and cardio-metabolic factors.

Characteristics of Anti-SARS-CoV-2 Antibodies in Recovered COVID-19 Subjects.

Coronavirus Disease 2019 (COVID-19) is a global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While detection of SARS-CoV-2 by polymerase chain reaction with reverse transcription (RT-PCR) is currently used to diagnose acute COVID-19 infection, serological assays are needed to study the humoral immune response to SARS-CoV-2. Anti-SARS-CoV-2 immunoglobulin (Ig)G/A/M antibodies against spike (S) protein and its receptor-binding domain (RBD) were characterized in recovered subjects who were RT-PCR-positive (n = 153) and RT-PCR-negative (n = 55) using an enzyme-linked immunosorbent assay (ELISA). These antibodies were also further assessed for their ability to neutralize live SARS-CoV-2 virus. Anti-SARS-CoV-2 antibodies were detected in 90.9% of resolved subjects up to 180 days post-symptom onset. Anti-S protein and anti-RBD IgG titers correlated (r = 0.5157 and r = 0.6010, respectively) with viral neutralization. Of the RT-PCR-positive subjects, 22 (14.3%) did not have anti-SARS-CoV-2 antibodies; and of those, 17 had RT-PCR cycle threshold (Ct) values > 27. These high Ct values raise the possibility that these indeterminate results are from individuals who were not infected or had mild infection that failed to elicit an antibody response. This study highlights the importance of serological surveys to determine population-level immunity based on infection numbers as determined by RT-PCR.

The utility of immunoglobulin A/complement 3 and immunoglobulin G/immunoglobulin M ratios in the assessment of disease activation in patients with Behçet disease.

OBJECTIVE: Pathogenesis of Behçet disease (BD) has not yet been clearly revealed and there is no ideal test for the estimation of disease activation at present. This study aimed to assess the efficiencies of IgG/IgM and IgA/C3 ratios in determining activation of BD. METHOD: This retrospective cohort study consisted of 140 patients with BD. Patients were divided into two groups: (1) active BD (n = 89) and (2) inactive BD (n = 51) and were compared in terms of demographic features, clinical characteristics and laboratory test results. IgA/C3 and IgG/IgM ratios were compared according to organ system involvement; receiver operating characteristic (ROC) curve analysis was performed in order to assess the performance of IgA/C3 and IgG/IgM ratios in determining patient disease status. RESULTS: Significantly higher levels of erythrocyte sedimentation rate, C-reactive protein, IgA, G, C4, IgA/C3, IgG/IgM ratios (p = .007 for IgA and p < .001 for others) and significantly lower levels of IgM and C3 were observed in patients with active BD (p < .001). The IgG/IgM ratio was significantly higher in patients with vascular involvement (p = .017) and the IgA/C3 ratio was significantly higher in patients with arthritis (p = .007). Cut-off values of 0.019 (70.8% sensitivity, 62% specificity) and 7.08 (84.3% sensitivity, 80% specificity) were determined for IgA/C3 and IgG/IgM ratios, respectively. CONCLUSION: IgA/C3 and IgG/IgM ratios may be used as additional parameters for the assessment of BD status.

An evaluation of 4 commercial assays for the detection of SARS-CoV-2 antibodies in a predominantly mildly symptomatic low prevalence Australian population.

A total of 1080 individual patient samples (158 positive serology samples from confirmed, predominantly mildly symptomatic COVID-19 patients and 922 serology negative including 496 collected pre-COVID) from four states in Australia were analysed on four commercial SARS-CoV-2 serological assays targeting antibodies to different antigens (Roche Elecsys and Abbott Architect: nucleocapsid; Diasorin Liaison and Euroimmun: spike). A subset was compared to immunofluorescent antibody (IFA) and micro-neutralisation. Sensitivity and specificity of the Roche (n = 1033), Abbott (n = 806), Diasorin (n = 1034) and Euroimmun (n = 175) were 93.7 %/99.5 %, 90.2 %/99.4 %, 88.6 %/98.6 % and 91.3 %/98.8 %, respectively. ROC analysis with specificity held at 99 % increased the sensitivity for the Roche and Abbott assays from 93.7% to 98.7% (cut-off 0.21) and 90.2 % to 94.0 % (cut-off 0.91), respectively. Overall seropositivity of samples increased from a maximum of 23 % for samples 0-7 days-post-onset of symptoms (dpos), to 61 % from samples 8-14dpos and 93 % from those >14dpos. IFA and microneutralisation values correlated best with assays targeting antibodies to spike protein with values >80 AU/mL on the Diasorin assay associated with neutralising antibody. Detectable antibody was present in 22/23 (96 %), 20/23 (87 %), 15/23 (65 %) and 9/22 (41 %) patients with samples >180dpos on the Roche, Diasorin, Abbott and microneutralisation assays respectively. Given the low prevalence in this community, two-step algorithms on initial positive results saw an increase in the positive predictive value (PPV) of positive samples (39 %-65 % to ≥98 %) for all combinations. Similarly accuracy increased from a range of 98.5 %-99.4 % to ≥99.8 % assuming a 1 % seroprevalence. Negative predictive value (NPV) was high (≥99.8 %) regardless of which assay was used initially.

Framework for Characterizing Longitudinal Antibody Response in Children After Plasmodium falciparum Infection.

Human Plasmodium infection produces a robust adaptive immune response. Time courses for 104 children followed for 42 days after initiation of Plasmodium falciparum chemotherapy were assayed for antibody levels to the five isotypes of human immunoglobulins (Ig) and 4 subclasses of IgG for 32 P. falciparum antigens encompassing all 4 parasite stages of human infection. IgD and IgE against these antigens were undetectable at 1:100 serum concentration, but other Ig isotypes and IgG subclasses were consistently observed against all antigens. Five quantitative parameters were developed to directly compare Ig response among isotypes and antigens: Cmax, maximum antibody level; ΔC, difference between Cmax and the antibody level at Day 0; tmax, time in days to reach Cmax; t1/2, Ig signal half-life in days; tneg, estimated number of days until complete loss of Ig signal. Classical Ig patterns for a bloodborne pathogen were seen with IgM showing early tmax and IgG production highest among Ig isotypes. However, some unexpected trends were observed such as IgA showing a biphasic pattern for many antigens. Variability among these dynamics of Ig acquisition and loss was noted for different P. falciparum antigens and able to be compared both quantitatively and statistically. This parametrization methodology allows direct comparison of Ig isotypes produced against various Plasmodium antigens following malaria infection, and the same methodology could be applied to other longitudinal serologic studies from P. falciparum or different pathogens. Specifically for P. falciparum seroepidemiological studies, reliable and quantitative estimates regarding the IgG dynamics in human populations can better optimize modeling efforts for serological outputs.

Dried blood spots for the quantitative evaluation of IgG isotypes and correlation with serum samples in HIV-exposed uninfected (HEU) infants.

BACKGROUND: The determination of IgG levels and their subclasses can provide clinically relevant information on the status of the immune system. Here we determined the sensitivity and reproducibility of the quantification of IgG subclasses from Dried Blood Spots (DBS) in Malawian uninfected infants exposed to HIV (HEU). METHODS: Sixty paired samples of serum and DBS from HEU infants were used. Samples were collected from 1, 6, and 24-month old infants. IgGs concentrations from both serum and DBS were analyzed by BN ProSpec Siemens assay, using a different setting for sample dilutions. The reproducibility of the DBS method was tested on 10 samples run twice, starting from the DBS extraction process. To assess the systematic, proportional, and random differences, we computed the Passing-Bablok regression, and the Bland-Altman analysis to estimate the total mean bias between the two tests. RESULTS: The IgG isotypes concentrations from serum and DBS showed significant differences in all the comparisons. Generally, the DBS method underestimated IgG subclasses' values showing a recovery range between 51.2% and 77.6%. Passing Bablok regression on age-based groups showed agreement for IgG, IgG1, and IgG2, but not for IgG3 and IgG4. The mean bias obtained with the Bland Altman test varied largely depending on IgG isotypes (-0.02-2.21 g/l) Coefficient of variation <7.0% was found in the repeated tests for IgG, IgG1, IgG3, and IgG4, while it was 12.4% for IgG2. CONCLUSIONS: Varying degrees of differences were seen in the IgGs measurement in the two different matrices. In IgGs analysis, the DBS method offers promise for population-based research, but the results should be carefully evaluated and considered as a relative value since they are not equivalent to the serum concentrations.

Distribution of anti-melanoma differentiation associated gene 5 (MDA5) IgG subclasses in MDA5+ dermatomyositis.

OBJECTIVES: The anti-melanoma differentiation-associated gene 5 (MDA5) antibody is the main predictor of interstitial lung disease (ILD) in DM and clinically amyopathic DM (CADM). Nevertheless, a subset of MDA5+ patients have a favourable prognosis. We aimed to determine the possibility of using anti-MDA5 antibody isotypes and IgG subclasses for evaluating ILD risk. METHODS: The isotypes (IgG, IgA and IgM) of anti-MDA5 were detected in serum samples of 36 anti-MDA5+ patients with DM/CADM using ELISA. IgG subclasses of anti-MDA5 antibodies were further investigated. Laboratory findings and cumulative survival were analysed based on the isotypes of anti-MDA5 and subclasses of anti-MDA5 IgG. RESULTS: Among the MDA5+ patients with DM/CADM, the positive rates of anti-MDA5 IgG, IgA and IgM were 100, 97 and 6%, respectively. The positive rates of anti-MDA5 IgG1, IgG2, IgG3 and IgG4 were 72, 25, 0 and 28%, respectively. The incidence of acute interstitial pneumonia, mortality rate and serum ferritin were significantly higher in anti-MDA5 IgG1+ patients than in anti-MDA5 IgG1- patients with DM/CADM (P = 0.0027, 0.015, 0.0011, respectively). The sensitivity and specificity of anti-MDA5 IgG1 for predicting mortality were 100 and 41.7%, respectively. A combination of anti-MDA5 IgG1 and IgG4 for predicting mortality yielded better specificity (87.5%). CONCLUSION: IgA and IgG are the primary anti-MDA5 antibody isotypes. Anti-MDA5 IgG1 is the primary component of MDA5 IgG subclasses and anti-MDA5 IgG1 and IgG4 might serve as useful biomarkers for predicting mortality in DM-ILD.

Previous viral symptoms and individual mothers influenced the leveled duration of human milk antibodies cross-reactive to S1 and S2 subunits from SARS-CoV-2, HCoV-229E, and HCoV-OC43.

OBJECTIVE: The influence of previous viral symptoms on the level and duration of human milk antibodies reactive to SARS-CoV-2, and common human coronaviruses (HCoVs) was investigated. STUDY DESIGN: Antibodies reactive to S1 and S2 subunits from SARS-CoV-2, HCoV-OC43, and HCoV-229E were measured via ELISA in human milk samples collected from March to June 2020 in mothers with and without viral symptoms. RESULTS: The presence of viral symptoms influenced the levels of SARS-CoV-2 S2-reactive SIgA/IgA and tended to influence SARS-CoV-2 S1 SIgA/IgA and S2-reactive SIgM/IgM in human milk but did not relate to IgG. HCoV-229E S1 + S2-reactive SIgA/IgA and SIgM/IgM, as well as HCoV-OC43 S1 + S2-reactive IgG were related to the symptoms. The duration of antibody levels in human milk in mothers with viral symptoms varied between 3 and 4 months post maternal report of viral symptoms. CONCLUSION: Previous viral symptoms and individual mothers may change the antibody cross-reactive levels to SARS-CoV-2 and HCoVs in human milk.