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Background: High titer COVID-19 convalescent plasma (CCP) reduces hospitalizations among immunocompetent outpatients. This study evaluated recipient post-transfusion S receptor binding domain (S-RBD) IgG antibody levels and the association of progressing to hospitalization among unvaccinated outpatients with COVID-19 treated with CCP or control plasma. Method(s): This analysis focused on participants from a multicenter doubleblind, randomized, controlled trial comparing treatment of outpatients with COVID-19 convalescent plasma (CCP) or control plasma without SARS-CoV-2 antibodies. Participants with confirmed SARS-CoV-2 infection were transfused within 9-days of symptom onset between June 2020 and October 2021 (n=110 vaccinated control;n=105 vaccinated CCP;n=464 unvaccinated control;n=472 unvaccinated CCP;total n=574 control and n=577 CCP recipients). All subjects had specimens collected the day prior to transfusion (D-1), within 30 minutes after transfusion (D0), 14 (D14), 28 (D28), and 90 (D90) days post-transfusion. Ancestral SARS-CoV-2 S-RBD was measured by an in-house validated ELISA. All 54 COVID-19-related hospitalizations occurred within 2 weeks of transfusion. Result(s): Post-transfusion anti-S-RBD IgG levels on D0 were significantly greater for CCP (median=4 titer,log3) compared to control (median=2 titer,log3;p< 0.001) recipients. Neither sex nor age impacted antibody levels following CCP treatment at D14, D28, and D90. Vaccinated recipients had greater titers than unvaccinated recipients prior to transfusion with little change in titers post-transfusion. Unvaccinated recipients had low antibody titers on D-1 with CCP recipients exhibiting a significant increase in titer from D-1 to D0 compared to controls (mean fold change=1.89;p< 0.001). Among unvaccinated recipients, those who received CCP transfusion late ( >5 days after symptom onset) and had low D0 antibody levels (< 4.24 titer, log3) had the greatest proportion of hospitalizations (5.5%). In contrast, those who received CCP transfusion early (< 5 days after symptom onset) with high D0 antibody levels ( >4.24 titer, log3) had no hospitalizations. Unvaccinated CCP recipient anti-S-RBD IgG antibody levels on D0 correlated with donor anti-S-RBD IgG antibody levels (r=0.30, p< 0.001). Conclusion(s): Among unvaccinated outpatients with COVID-19, CCP recipient antibody dilutional titers after transfusion over 540 titer correlated with protection against hospitalization when transfusion occurred within 5 days of symptom onset. (Figure Presented).
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Background: People who inject drugs (PWID) may be at a greater risk of SARS-CoV-2 infection and COVID-19 due to socio-structural inequities, high-risk behaviors and comorbidities;however, PWID have been underrepresented in case-based surveillance due to lower access to testing. We characterize temporal trends and correlates of SARS-CoV-2 seroprevalence among a community-based sample of current and former PWID. Method(s): A cross-sectional study was conducted among participants in the AIDS Linked to the IntraVenous Experience (ALIVE) study-a community-based cohort of adults with a history of injection drug use in Baltimore, Maryland. Participants' first serum sample collected at routine study visits between December 2020 and July 2022 was assayed for antibodies to the nucleocapsid (N) (past infection) and spike-1 (S) (past infection and/or vaccination) proteins using the MSD V-Plex Panel 2 IgG SARS-CoV-2 assay. For each correlate, we estimated adjusted prevalence ratios (PR) via separate Poisson regression models adjusted for calendar time, age, sex and race. Result(s): Of 561 participants, the median age was 59 years (range=28-77), 35% were female, 84% were Black, 36% were living with HIV (97% on ART), and 55% had received >=1 COVID-19 vaccine dose. Overall, anti-N and anti-S prevalence was 26% and 63%, respectively. Prevalence of anti-N increased from 23% to 40% between December 2020-May 2021 and December 2021-July 2022, with greater increases in the prevalence of anti-S from 34% to 86% over the same period (Figure). Being employed (PR=1.53 [95%CI=1.11-2.11]) and never being married (PR=1.40 [0.99-1.99]) were associated with a higher prevalence of anti-N, while female sex (PR=0.75 [0.55-1.02]) and a history of cancer (PR=0.40 [0.17-0.90]) were associated with a lower prevalence of anti-N. Younger age, female sex (PR=0.90 [0.80-1.02]), and homelessness (PR=0.78 [0.60-0.99]) were associated with a lower prevalence of anti-S. Although HIV infection was not associated with anti-N, it was associated with a higher prevalence of anti-S (PR=1.13 [1.02-1.27]). Substance use was not associated with anti-N or anti-S. Conclusion(s): Anti-N and anti-S levels increased over time, suggesting cumulative increases in SARS-CoV-2 incidence of infection and vaccination among PWID;however, disparities in seroprevalence remain. Younger and female PWID and those experiencing homelessness were less likely to be anti-S positive, suggesting programs should aim to improve vaccination coverage in such vulnerable populations.
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Background: Sero-studies of SARS-CoV-2 have used antibody (Ab) responses to spike (S) and nucleocapsid (N) antigens to differentiate mRNA vaccinated (S+/N-) from infected (S+/N+) individuals. We performed testing on wellcharacterized subjects to determine how repeated vaccination or infection, and time from those exposures, influence these Ab levels. Method(s): Samples from individuals with known infection status: prepandemic negative controls n=462;first-time infected n=237 (~45 days post);vaccinated after infection n= 34 (~40 days post-vaccination and ~180 days post-infection);fully vaccinated n=158 (~50 days post);boosted n=31 (~30 days post);breakthrough n=18 (~14 days post-infection);reinfected n=10 (varied). Longitudinal samples (n=51) from subjects with evidence of reinfection (symptoms and/or positive rapid antigen test), were tested to determine the impact of the order of infection and/or vaccination on the magnitude of the anti-S and anti-N IgG Ab detected in the blood. Testing was performed with MesoScale Diagnostics (Gaithersburg, MD) assay. Outcomes are presented in WHO International Binding Antibody Units (BAU/mL). The cutoff for a positive result was 18 BAU for S and 12 BAU for N. Result(s): The median amount of Ab (IQR) in BAU for each group (Figure A) was: pre-pandemic negative controls S 0.53(0.27,1.03), N 0.55(0.18,1.67);first-time infected S 114(51,328), N 70(29,229);vaccinated after infection S 4367(2479,4837), N 15(7,35);fully vaccinated S 998(586,1529), N 0.31(0.16,0.68);boosted S 2988(1768,3522), N 0.59(0.32,1.03);breakthrough S 2429(2032,3413), N 2.5(0.93,8.6);reinfected S 1533(486,4643), N 7.8(2.6,62). For the breakthrough and second infections 17% and 40% were seropositive to N, respectively. Longitudinal analysis (Figure B) of those with multiple infections showed that all those with a positive rapid antigen test for their second infection had an increase in N Ab. Conclusion(s): The prevalence of antibodies to nucleocapsid cannot be used to determine the proportion of individuals infected to SARS-CoV-2 in a vaccinated population. Booster, repeated, and breakthrough infections are associated with IgG Ab levels to S >400 BAU/mL. A majority of breakthrough infections did not elicit an Ab response to N. For those with repeated infection, a minority elicited antibody responses to N. This could be related to misdiagnosis or the burden of infection, as only those who were positive by rapid antigen assay (indicative of a high viral load) had an increase in N Ab.
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Background: COVID-19 in Africa was less severe with fewer reported cases, hospitalizations and deaths compared to other continents. However, the lack of adequate surveillance systems in Africa makes estimating the burden of infection challenging. Serosurveillance can aid in determining the frequency of infection within this population. This study is aimed to estimate SARS-CoV-2 seroprevalence, describe the SARS-CoV-2 antibody (Ab) levels, and examine associations of seroreactivity among Ugandan blood donors. Method(s): Samples were obtained from the Mirasol Evaluation of Reduction in Infections Trial (MERIT), a randomized, double blind, controlled clinical trial evaluating transfusion transmitted infections. MERIT blood donor samples (n=3,517) were collected from Kampala, Uganda between October 2019 to April 2022. Additional blood donor samples (n=1,876) were collected from around the country between November-December 2021. Samples were tested for Ab to SARS-CoV-2 nucleocapsid (N) and spike (S) using an electrochemiluminescence immunoassay assay (Meso Scale Diagnostics, Gaithersburg, MD) per manufacturer's protocol. Samples seroreactive to both N and S Ab were considered Ab positive to SARS-CoV-2. Seroprevalence among MERIT donors were estimated within each quarter. Factors associated with seroreactivity from November-December 2021 were assessed by chi-square test. Result(s): SARS-CoV-2 seroprevalence increased from < 2.0% in October 2019- June 2020 to 82.5% in January-April 2022. Three distinct peaks in seroreactivity were seen in October-November 2020, July-August 2021, and January-April 2022 (see Figure). Among seroreactive donors, median N Ab levels increased 9-fold and median S Ab 19-fold over the study period. In November-December 2021, SARS-CoV-2 seroprevalence was higher among donors from Kampala (58.8%) compared to more rural regions of Hoima (47.7%), Jinja (47.9%), and Masaka (54.4%;p=0.007);S seroprevalence was lower among HIV+ donors (58.8% vs. 84.9%;p=0.009). Conclusion(s): Blood donors in Uganda showed high prevalence of Ab to SARSCoV- 2 by March of 2022, indicating that the infection levels were similar to many other regions of the globe. Higher seroprevalence was observed in the capital compared to more rural areas in Uganda. Further, increasingly high antibody levels among seropositive donors may indicate repeat infections. The lower COVID-19 morbidity and mortality was not due to a lack of exposure of the virus, but other factors yet to be determined.
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Background: Historically, control of HIV infection in young men living with HIV (LWH) has been problematic. We examined the STI/HIV burden in young men with urethral discharge syndrome (UDS) in Kampala, Uganda. Methods: Between Oct 2019-Nov 2020, 250 men with UDS were enrolled at 6 urban sites. All HIV positive men (20%, 50/250) had plasma viral load testing (Abbott m2000 RealTime HIV-1);when VL>1000 copies/mL, resistance and recency testing (Asanté HIV-1 Rapid Recency Assay, Sedia Biosciences) were performed. Penile meatal swabs were retrospectively tested for gonorrhea, chlamydia, trichomoniasis, and Mycoplasma genitalium (Hologic Aptima CT/NG, TV, MG). Descriptive statistical analysis, logistic, and bivariable and multivariable regression were undertaken. Results: Among the men LWH, 92% (46/50) had VL<1000;4 were not suppressed, 1 of whom was previously undiagnosed. Among the viremic individuals, no major resistance mutations were found and none appeared recently infected. Men (median age 24[22;32]) reported sex partners/previous 2 months (median 2[1;2]), 61.6% engaged in transactional sex in the previous 6 months, and 48.4% reported alcohol use. 44.4% reported alcohol use before sex in the previous 6 months. Overall, 0.4% reported 'always' condom use, 21.8% continued condomless sex since onset of UDS symptoms. There was a high burden of active, undiagnosed STIs found in these men (see Table);of the 10% who had syphilis, 80% were previously undiagnosed. Agreement between HIV-and syphilis-POC and lab-based testing was 100% and 95% (19/20), respectively. By multivariable logistic regression, alcohol use (OR, 3.32 (95% CI:1.61, 7.11)), and condomless sexual activity since symptom onset (OR, 2.86 (95% CI:1.20, 6.84)) were significantly associated with HIV;92% had at least one other STI. Conclusion: Among men presenting with UDS, bacterial STIs were very common. 20% had HIV with a surprisingly high level of viral suppression and no evidence of resistance in those with detectable VL. Recency testing results were non-discriminatory;none appeared recently infected. Risk of future HIV acquisition is high in those not LWH. Given the high frequency of bacterial STI, alcohol use and unprotected high-risk sexual behavior in this population, men with UDS who test negative for HIV should be prioritized for PrEP. Future research, evaluating the effect of SARS-CoV-2 on the burden of STI and level of viral suppression in this population, is required.
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Background: The prevalence of vaccinated, previously infected, and individuals at risk of SARS-CoV-2 infection is important for epidemiologic studies and public health interventions. Asymptomatic infections and reluctance to disclose vaccination status hinder accurate assessments of the current state of the epidemic. Since COVID-19 vaccines generate immune responses to spike (S1), but not nucleocapsid (N), it is possible to differentiate between vaccinated, infected, and unexposed individuals by comparing antibody reactivity to each antigen. The MSD V-Plex SARS-CoV-2 IgG assay can potentially differentiate each state in one test by simultaneously evaluating IgG reactivity to the S1, receptor binding domain (RBD), and N proteins. Methods: The MSD assay was validated with three sample sets: known vaccination with no previous infection (n=158);known infected and not vaccinated (n=157);and samples collected prior to the COVID-19 pandemic in 2016 (n=144). Of the previously infected individuals, 15 (9.6%) were hospitalized;sample collection occurred a median of 48 days after a PCR-positive result. Using an algorithm, samples with positive results on both S1 and RBD but negative on N were classified as vaccinated. Samples with a positive result on all three proteins were considered to be infected with the possibility of subsequent vaccination. Any other result was classified as unexposed. Sensitivity and specificity for each state were calculated. Results: Reactivity to each antigen is shown in the figure. 100% (95% confidence interval [CI] 97.7-100), 92% (95% CI 86.3-95.5), and 0.7% (95% CI 0.02-3.8) of vaccinated, infected, and unexposed samples were positive for S1. 100% (95% CI 97.7-100.0%), 91% (95% CI 85.5-95.0%), and 0.7% (95% CI 0.02-3.8%) of vaccinated, infected and unexposed samples were positive for RBD. 0% (95% CI 0-2.3), 86% (95% CI 79.6-91.0), and 2.1% (95% CI 0.4-6.0) of vaccinated, infected and unexposed samples were positive for N. Algorithm sensitivity and specificity for classification of vaccinated samples were 100% (95% CI 97.7-100) and 96.7 (95% CI 94-98.4). For the classification of samples from previously infected individuals, sensitivity and specificity were 83.4% (95% CI 76.7-88.9) and 100% (95% CI 98.8-100). Conclusion: This study establishes the sensitivity and specificity for a high-throughput assay ideal for SARS-CoV-2 seroprevalence studies. Future research should focus on applying this assay in health care settings to guide practice and policy to mitigate the pandemic.
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Background: Seroprevalence studies of antibodies to SARS-CoV-2 are important for public health surveillance. Recent studies have shown that antibodies to SARS-CoV-2, both from natural infection and vaccination, decrease with time from exposure. Variation in the performance of antibody assays will impact the estimates of SARS-CoV-2 exposure and vaccination levels in a population. Using standardized serial dilutions, we evaluated 17 SARS-CoV-2 assays to establish an approximate limit of detection for each assay. Methods: The evaluated assays consisted of three chemiluminescent immunoassays (CLIAs), eight standard enzyme-linked immunosorbent assays (ELISAs), and six lateral flow assays (LFAs). All assays either evaluated IgG antibodies or total antibodies to SARS-CoV-2. The target antigen of 14 assays was the spike protein (S) or receptor binding domain (RBD);three assays evaluated antibodies to the nucleocapsid protein (N). A human SARS-CoV-2 serology standard with a WHO SARS-CoV-2 Serology International Standard binding antibody units (BAU) value of 764 BAU/mL to spike IgG and 681 BAU/mL to nucleocapsid IgG was obtained from the Frederick National Laboratory for Cancer Research. Half-logarithmic serial dilutions of the standard were then run in triplicate on each assay. Results: The MSD V-Plex chemiluminescent immunoassays (CLIAs) were the most sensitive by three logs, with positive results at a dilution greater than 1:106 (Figure). Standard ELISAs were less sensitive, with limits of detection ranging from dilutions of 1:20 (Euroimmun NeutraLISA) to 1:1620 (Euroimmun SARS-CoV-2 IgG and Euroimmun QuantiVac). Lateral flow assays (LFAs) were the least sensitive, with only one assay (Wondfo Colloidal Gold) having at least one positive result with a dilution greater than 1:180. Conclusion: As population seroprevalence to SARS-CoV-2 continues to rise, tests with a high limit of detection will be crucial for surveillance studies. As antibody levels decline after vaccination or infection, our data indicate that CLIAs like the MSD assay may provide the best opportunity to capture asymptomatic cases and individuals with low antibody titers.
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Background: Live virus micro-neutralization (MN) is the gold standard for quantifying the neutralizing titer (NT) of antibodies to SARS-CoV-2. However, performing MN is labor intensive and requires a biosafety level 3 laboratory. We assessed the performance of 8 immunoassays which measure SARS-CoV-2 NT and compared them to gold standard MN results. Methods: Samples from 269 individuals known to previously be SARS-CoV-2 PCR+ (i.e., convalescent individuals, <10% hospitalized) and 200 pre-pandemic individuals were evaluated on 3 lateral flow immunoassays (LFAs;Wondfo Colloidal Gold, Wondfo Colored Microsphere, Wondfo Finecare) and 5 enzyme-linked immunoassays (ELISAs;ImmunoRank, GenScript, Cusabio, Euroimmun NeutraLISA, Euroimmun QuantiVac). MN was performed on all samples from convalescent individuals;results were classified as undetectable vs any detection of MN NT (NT<20 vs. NT>20), as well as high and low MN NT (NT>80 vs. NT<80). Receiver operating curve analysis was used to assess accuracy for detecting levels of NT. The area under the curve (AUC) was calculated for the manufacturer's cut off and empirically to identify the best discriminatory cut off value. Cohen's kappa statistics were calculated to assess categorical agreement and Spearman's rank statistics were calculated to assess correlations. Results: Of the 269 convalescent plasma samples, 89 (33%) had MN NT values <20 (undetectable) and 117 (43%) >80 (high NT). Using the manufacturer's cutoffs, sensitivity for detection of samples with any NT ranged from 79% to 100%, and the false-positive rate (ie, classifying samples with undetectable NT as positive) was highest for LFAs (72% to 84%) and ranged from 14% to 69% for the ELISAs. For all assays except the ImmunoRank and NeutraLISA ELISAs, discrimination to identify samples with any NT was improved by raising the cut off values (Table). AUCs of ∼0.94 to discriminate high NT samples could be achieved for all quantifiable assays using an adjusted cut off value. Cohen's kappa statistic ranged from 0.20 to 0.69. Spearman's rank correlation between each assay and NT value ranged from 0.73 to 0.86. Using the manufacturer's cutoffs, specificity on pre-pandemic samples was ≥98% for all assays except for Cusabio which was 86%. Conclusion: The performance of immunoassays using manufacturer's cutoff to discriminate samples with any NT was accurate (AUC>0.83 for all assays), but could be improved by changing the cutoff. Identifying samples with high NT could be achieved using an alternative cutoff.
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Background. As the COVID-19 pandemic continues, growing attention has been placed on whether patients previously infected with SARS-CoV-2 have an increased risk of developing and/or exacerbating medical complications. Our study aimed to determine whether individuals with previous evidence of SARS-CoV-2 infection prior to their current emergency department (ED) visit were more likely to present with specific clinical sign/symptoms, laboratory markers, and/or clinical complications. Methods. A COVID-19 seroprevalence study was conducted at Johns Hopkins Hospital ED (JHH ED) from March 16 to May 31, 2020. Evidence of ever having SARSCoV-2 infection (PCR positive or IgG Ab positive) was found in 268 ED patients at this time (i.e. infected and/or previously infected). These patients were matched 1:2 to controls, by date, to other patients who attended the JHHED. Clinical signs/symptoms, laboratory markers, and/or clinical complications associated with ED visits and/ or hospitalizations at JHH within 6 months after their initial ED visit was ed through chart review for these 804 patients. Cox proportional hazards regression analyses were performed. Results. Among 804 ED patients analyzed, 50% were female, 56% Black race, and 15% Hispanic with a mean age of 47 years. 323 (40%) patients had at least 1 subsequent ED visit and additional 70 (9%) had been admitted to JHH. After controlling for race and ethnicity, patients with evidence of current or prior COVID-19 infection were more likely to require supplemental oxygen [hazards ratio (HR) =2.53;p=0.005] and have a cardiovascular complication [HR =2.13;p=0.008] during the subsequent ED visit than the non-infected patients. Conclusion. Our findings demonstrate that those previously infected with SARSCoV-2 have an increased frequency of cardiovascular complications and need for supplemental oxygen in ED visits in the months after their initial SARS-CoV-2 infection was detected. EDs could serve as a critical surveillance site for monitoring post-acute COVID-19 syndrome complications.
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Background: The performance of serological antibody tests to SARS-CoV-2 infection varies widely and little is known about their performance in Africa. We assessed the performance of CoronaCHEK Lateral Flow Point of Care Tests on samples from Rakai, Uganda and Baltimore, Maryland, USA. Methods: Samples from subjects known to be SARS-CoV-2 PCR+ (Uganda: 50 samples from 50 individuals, and Baltimore: 266 samples from 38 individuals) and samples from pre-pandemic individuals collected prior to 2019 (Uganda: 1077 samples, Baltimore: 580 samples) were analyzed with the CoronaCHEK assay per manufacturers protocol. Sensitivity by duration of infection and specificity among pre-pandemic samples were assessed for the IgM and IgG bands separately and for any reactivity. Poisson regression models were used to calculate prevalence ratios (PR) for factors associated with a false-positive test among pre-pandemic samples. Results: In Baltimore samples, sensitivity for any reactivity increased with duration of infection with 39% (95% CI 30, 49) during 0-7 days since first positive PCR, 86% (95% CI 79, 92) for 8-14 days, and 100% (95% CI 89,100) after 15 days (See Figure). In Uganda, sensitivity was 100% (95% CI 61,100) during 0-7 days, 75% (95%CI 53, 89) for 8-14 days, and 87% (95%CI 55, 97) after 14 days since first positive PCR. Specificity results among pre-pandemic samples from Uganda was 96.5% (95% CI 97.5, 95.2), significantly lower than the 99.3% (95% CI 98.2, 99.8) observed in samples from Baltimore (p<0.01). In Ugandan samples, individuals with a false positive result were more likely to have had a fever more than a month prior to sample acquisition (PR 2.9, 95% CI 1.1, 7.0). Conclusion: Sensitivity of the CoronaCHEK appeared to be significantly higher in Ugandan samples from individuals within their first week of infection compared to their Baltimorean counterparts. By the second week of infection the sensitivity appeared the same between geographic areas. The specificity was significantly lower in Ugandan samples than those from Baltimore. False positive results from pre-pandemic Uganda appear to be correlated with the convalescent disease state, potentially indicative of a highly cross-reactive immune response in these individuals from East Africa.