Immune imprinting, breadth of variant recognition, and germinal center response in human SARS-CoV-2 infection and vaccination.

During the SARS-CoV-2 pandemic, novel and traditional vaccine strategies have been deployed globally. We investigated whether antibodies stimulated by mRNA vaccination (BNT162b2), including third-dose boosting, differ from those generated by infection or adenoviral (ChAdOx1-S and Gam-COVID-Vac) or inactivated viral (BBIBP-CorV) vaccines. We analyzed human lymph nodes after infection or mRNA vaccination for correlates of serological differences. Antibody breadth against viral variants is lower after infection compared with all vaccines evaluated but improves over several months. Viral variant infection elicits variant-specific antibodies, but prior mRNA vaccination imprints serological responses toward Wuhan-Hu-1 rather than variant antigens. In contrast to disrupted germinal centers (GCs) in lymph nodes during infection, mRNA vaccination stimulates robust GCs containing vaccine mRNA and spike antigen up to 8 weeks postvaccination in some cases. SARS-CoV-2 antibody specificity, breadth, and maturation are affected by imprinting from exposure history and distinct histological and antigenic contexts in infection compared with vaccination.

Diagnostic performance of host protein signatures as a triage test for active pulmonary TB.

The current four-symptom screen recommended by the World Health Organization (WHO) is widely used as screen to initiate diagnostic testing for active pulmonary tuberculosis (TB), yet the performance is poor especially when TB prevalence is low. In contrast, more sensitive molecular tests are less suitable for placement at primary care level in low-resource settings. In order to meet the WHO End TB targets, new diagnostic approaches are urgently needed to find the missing undiagnosed cases. Proteomics-derived blood host biomarkers have been explored because protein detection technologies are suitable for the point-of-care setting and could meet cost targets. This study aimed to find a biomarker signature that fulfills WHO's target product profile (TPP) for a TB screening. Twelve blood-based protein biomarkers from three sample populations (Vietnam, Peru, and South Africa) were analyzed individually and in combinations via advanced statistical methods and machine learning algorithms. The combination of I-309, SYWC and kallistatin showed the most promising results to discern active TB throughout the data sets meeting the TPP for a triage test in adults from two countries (Peru and South Africa). The top-performing individual markers identified at the global level (I-309 and SYWC) were also among the best-performing markers at country level in South Africa and Vietnam. This analysis clearly shows that a host protein biomarker assay is feasible in adults for certain geographical regions based on one or two biomarkers with a performance that meets minimal WHO TPP criteria.

Measurement of Severe Acute Respiratory Syndrome Coronavirus 2 Antigens in Plasma of Pediatric Patients With Acute Coronavirus Disease 2019 or Multisystem Inflammatory Syndrome in Children Using an Ultrasensitive and Quantitative Immunoassay.

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in blood has high sensitivity in adults with acute coronavirus disease 2019 (COVID-19), but sensitivity in pediatric patients is unclear. Recent data suggest that persistent SARS-CoV-2 spike antigenemia may contribute to multisystem inflammatory syndrome in children (MIS-C). We quantified SARS-CoV-2 nucleocapsid (N) and spike (S) antigens in blood of pediatric patients with either acute COVID-19 or MIS-C using ultrasensitive immunoassays (Meso Scale Discovery). METHODS: Plasma was collected from inpatients (<21 years) enrolled across 15 hospitals in 15 US states. Acute COVID-19 patients (n = 36) had a range of disease severity and positive nasopharyngeal SARS-CoV-2 RT-PCR within 24 hours of blood collection. Patients with MIS-C (n = 53) met CDC criteria and tested positive for SARS-CoV-2 (RT-PCR or serology). Controls were patients pre-COVID-19 (n = 67) or within 24 hours of negative RT-PCR (n = 43). RESULTS: Specificities of N and S assays were 95-97% and 100%, respectively. In acute COVID-19 patients, N/S plasma assays had 89%/64% sensitivity; sensitivities in patients with concurrent nasopharyngeal swab cycle threshold (Ct) ≤35 were 93%/63%. Antigen concentrations ranged from 1.28-3844 pg/mL (N) and 1.65-1071 pg/mL (S) and correlated with disease severity. In MIS-C, antigens were detected in 3/53 (5.7%) samples (3 N-positive: 1.7, 1.9, 121.1 pg/mL; 1 S-positive: 2.3 pg/mL); the patient with highest N had positive nasopharyngeal RT-PCR (Ct 22.3) concurrent with blood draw. CONCLUSIONS: Ultrasensitive blood SARS-CoV-2 antigen measurement has high diagnostic yield in children with acute COVID-19. Antigens were undetectable in most MIS-C patients, suggesting that persistent antigenemia is not a common contributor to MIS-C pathogenesis.

Patients with severe COVID-19 do not have elevated autoantibodies against common diagnostic autoantigens.

OBJECTIVES: Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative pathogen of coronavirus disease 2019 (COVID-19) presents occasionally with an aberrant autoinflammatory response, including the presence of elevated circulating autoantibodies in some individuals. Whether the development of autoantibodies against self-antigens affects COVID-19 outcomes remains unclear. To better understand the prognostic role of autoantibodies in COVID-19, we quantified autoantibodies against 23 markers that are used for diagnosis of autoimmune disease. To this end, we used serum samples from patients with severe [intensive care unit (ICU)] and moderate (ward) COVID-19, across two to six consecutive time points, and compared autoantibody levels to uninfected healthy and ICU controls. METHODS: Acute and post-acute serum (from 1 to 26 ICU days) was collected from 18 ICU COVID-19-positive patients at three to six time points; 18 ICU COVID-19-negative patients (sampled on ICU day 1 and 3); 21 ward COVID-19-positive patients (sampled on hospital day 1 and 3); and from 59 healthy uninfected controls deriving from two cohorts. Levels of IgG autoantibodies against 23 autoantigens, commonly used for autoimmune disease diagnosis, were measured in serum samples using MSD® U-PLEX electrochemiluminescence technology (MSD division Meso Scale Discovery®), and results were compared between groups. RESULTS: There were no significant elevations of autoantibodies for any of the markers tested in patients with severe COVID-19. CONCLUSIONS: Sample collections at longer time points should be considered in future studies, for assessing the possible development of autoantibody responses following infection with SARS-CoV-2.

Ultrasensitive assay for saliva-based SARS-CoV-2 antigen detection.

OBJECTIVES: Widespread SARS-CoV-2 testing is invaluable for identifying asymptomatic/pre-symptomatic individuals. There remains a technological gap for highly reliable, easy, and quick SARS-CoV-2 diagnostic tests suitable for frequent mass testing. Compared to nasopharyngeal (NP) swab-based tests, saliva-based methods are attractive due to easier and safer sampling. Current saliva-based SARS-CoV-2 rapid antigen tests (RATs) are hindered by limited analytical sensitivity. Here, we report one of the first ultrasensitive, saliva-based SARS-CoV-2 antigen assays with an analytical sensitivity of <0.32 pg/mL, corresponding to four viral RNA copies/µL, which is comparable to that of PCR-based tests. METHODS: Using the novel electrochemiluminescence (ECL)-based immunoassay, we measured the SARS-CoV-2 nucleocapsid (N) antigen concentration in 105 salivas, obtained from non-COVID-19 and COVID-19 patients. We then verified the results with a second, independent cohort of 689 patients (3.8% SARS-CoV-2 positivity rate). We also compared our method with a widely used point-of-care rapid test. RESULTS: In the first cohort, at 100% specificity, the sensitivity was 92%. Our assay correctly identified samples with viral loads up to 35 CT cycles by saliva-based PCR. Paired NP swab-based PCR results were obtained for 86 cases. Our assay showed high concordance with saliva-based and NP swab-based PCR in samples with negative (<0.32 pg/mL) and strongly positive (>2 pg/mL) N antigen concentrations. In the second cohort, at 100% specificity, sensitivity was also 92%. Our assay is about 700-fold more sensitive than the Abbott Panbio Rapid Test. CONCLUSIONS: We demonstrated the ultrasensitivity and specificity assay and its concordance with PCR. This novel assay is especially valuable when compliance to frequent swabbing may be problematic.

Correlation of SARS-CoV-2 Nucleocapsid Antigen and RNA Concentrations in Nasopharyngeal Samples from Children and Adults Using an Ultrasensitive and Quantitative Antigen Assay.

Diagnosis of COVID-19 by PCR offers high sensitivity, but the utility of detecting samples with high cycle threshold (CT ) values remains controversial. Currently available rapid diagnostic tests (RDTs) for SARS-CoV-2 nucleocapsid antigens (Ag) have sensitivity well below PCR. The correlation of Ag and RNA quantities in clinical nasopharyngeal (NP) samples is unknown. An ultrasensitive, quantitative electrochemiluminescence immunoassay for SARS-CoV-2 nucleocapsid (the MSD S-PLEX SARS-CoV-2 N assay) was used to measure Ag in clinical NP samples from adults and children previously tested by PCR. The S-PLEX Ag assay had a limit of detection (LOD) of 0.16 pg/ml and a cutoff of 0.32 pg/ml. Ag concentrations measured in clinical NP samples (collected in 3.0 ml of media) ranged from less than 160 fg/ml to 2.7 µg/ml. Log-transformed Ag concentrations correlated tightly with CT values. In 35 adult and 101 pediatric PCR-positive samples, the sensitivities were 91% (95% confidence interval, 77 to 98%) and 79% (70 to 87%), respectively. In samples with a CT of ≤35, the sensitivities were 100% (88 to 100%) and 96% (88 to 99%), respectively. In 50 adult and 40 pediatric PCR-negative specimens, the specificities were 100% (93 to 100%) and 98% (87 to 100%), respectively. Nucleocapsid concentrations in clinical NP samples span 8 orders of magnitude and correlate closely with RNA concentrations (CT values). The S-PLEX Ag assay showed 96 to 100% sensitivity in samples from children and adults with CT values of ≤35, and a specificity of 98 to 100%. These results clarify Ag concentration distributions in clinical samples, providing insight into the performance of Ag RDTs and offering a new approach to diagnosis of COVID-19.

A Novel Sensitive Immunoassay Targeting the 5-Methylthio-d-Xylofuranose-Lipoarabinomannan Epitope Meets the WHO's Performance Target for Tuberculosis Diagnosis.

The only currently commercialized point-of-care assay for tuberculosis (TB) that measures lipoarabinomannan (LAM) in urine (Alere LF-LAM) has insufficient sensitivity. We evaluated the potential of 100 novel monoclonal antibody pairs targeting a variety of LAM epitopes on a sensitive electrochemiluminescence platform to improve the diagnostic accuracy. In the screening, many antibody pairs showed high reactivity to purified LAM but performed poorly at detecting urinary LAM in clinical samples, suggesting differences in antigen structure and immunoreactivity of the different LAM sources. The 12 best antibody pairs from the screening were tested in a retrospective case-control study with urine samples from 75 adults with presumptive TB. The best antibody pair reached femtomolar analytical sensitivity for LAM detection and an overall clinical sensitivity of 93% (confidence interval [CI], 80% to 97%) and specificity of 97% (CI, 85% to 100%). Importantly, in HIV-negative subjects positive for TB by sputum smear microscopy, the test achieved a sensitivity of 80% (CI, 55% to 93%). This compares to an overall sensitivity of 33% (CI, 20% to 48%) of the Alere LF-LAM and a sensitivity of 13% (CI, 4% to 38%) in HIV-negative subjects in the same sample set. The capture antibody targets a unique 5-methylthio-d-xylofuranose (MTX)-dependent epitope in LAM that is specific to the Mycobacterium tuberculosis complex and shows no cross-reactivity with fast-growing mycobacteria or other bacteria. The present study provides evidence that improved assay methods and reagents lead to increased diagnostic accuracy. The results of this work have informed the development of a sensitive and specific novel LAM point-of-care assay with the aim to meet the WHO's performance target for TB diagnosis.

Exploratory factor analysis yields grouping of brain injury biomarkers significantly associated with outcomes in neonatal and pediatric ECMO.

In this two-center prospective cohort study of children on ECMO, we assessed a panel of plasma brain injury biomarkers using exploratory factor analysis (EFA) to evaluate their interplay and association with outcomes. Biomarker concentrations were measured daily for the first 3 days of ECMO support in 95 participants. Unfavorable composite outcome was defined as in-hospital mortality or discharge Pediatric Cerebral Performance Category > 2 with decline ≥ 1 point from baseline. EFA grouped 11 biomarkers into three factors. Factor 1 comprised markers of cellular brain injury (NSE, BDNF, GFAP, S100ß, MCP1, VILIP-1, neurogranin); Factor 2 comprised markers related to vascular processes (vWF, PDGFRß, NPTX1); and Factor 3 comprised the BDNF/MMP-9 cellular pathway. Multivariable logistic models demonstrated that higher Factor 1 and 2 scores were associated with higher odds of unfavorable outcome (adjusted OR 2.88 [1.61, 5.66] and 1.89 [1.12, 3.43], respectively). Conversely, higher Factor 3 scores were associated with lower odds of unfavorable outcome (adjusted OR 0.54 [0.31, 0.88]), which is biologically plausible given the role of BDNF in neuroplasticity. Application of EFA on plasma brain injury biomarkers in children on ECMO yielded grouping of biomarkers into three factors that were significantly associated with unfavorable outcome, suggesting future potential as prognostic instruments.

Higher levels of brain injury biomarker tau are associated with unfavorable outcomes in patients supported with ECMO following cardiac arrest.

Aim: We sought to determine if higher plasma levels of brain injury biomarkers neurofilament light (NfL), phosphorylated tau 181 (pT181), tau, and ubiquitin C-terminal hydrolase L1 (UCHL1) were associated with unfavorable outcomes in children supported on extracorporeal membrane oxygenation (ECMO) with and without preceding cardiac arrest. Methods: We conducted a secondary analysis of a two-center prospective observational study of ECMO patients 0-<18 years. Plasma concentrations of NfL, pT181, tau, and UCHL1 were measured on ECMO days 1, 2 and 3. Unfavorable outcome was defined as in-hospital mortality or discharge Pediatric Cerebral Performance Category (PCPC) >2 with decline from baseline PCPC among survivors. Results: Among 88 children on ECMO, mean tau levels were significantly higher on each of the first three ECMO days in children who underwent extracorporeal cardiopulmonary resuscitation (ECPR) compared to those with non-ECPR cardiac arrest or with no cardiac arrest preceding ECMO. Higher ECMO day 1 tau levels were significantly associated with increased hazard of unfavorable outcome in unadjusted (HR, 1.35, 95% CI 1.09-1.66) and adjusted (HR, 1.42; 95% CI 1.13-1.79) models. Higher levels of NfL or pT181 were not associated with increased hazard for unfavorable outcome in multivariable models. UCHL1 values were outside of detectable limits and thus deferred from analysis. Conclusions: Levels of tau were significantly associated with increased hazard of death or unfavorable neurologic outcome in unadjusted and adjusted models. Biomarkers of brain injury, particularly tau, may aid in detection of neurologic injury and neuroprognostication in patients on ECMO with and without preceding cardiac arrest.

Gestational SARS-CoV-2 Infection in a Ugandan Birth Cohort: High Incidence, Mild Maternal Disease, and Evidence of Association with Transient Infant Stunting.

Many questions remain about the prevalence and effects of SARS-CoV-2 infection in malaria-endemic African countries like Uganda, particularly in vulnerable groups such as pregnant women. We describe SARS-CoV-2 immunoglobulin (Ig)G and IgM antibody responses and clinical outcomes in mother-infant dyads enrolled in malaria chemoprevention trials in Uganda. From December 2020-February 2022, among 400 unvaccinated pregnant women enrolled at 12-20 weeks gestation and followed through delivery, 128 (32%) were seronegative for anti-SARS-CoV-2 IgG and IgM at enrollment and delivery, 80 (20%) were infected prior to or early in pregnancy, and 192 (48%) were infected or re-infected with SARS-CoV-2 during pregnancy. We observed preferential binding of plasma IgG to Wuhan-Hu-1-like antigens in individuals seroconverting up to early 2021, and to Delta variant antigens in a subset of individuals in mid-2021. Breadth of IgG binding to all variants improved over time, consistent with affinity maturation of the antibody response in the cohort. No women experienced severe respiratory illness during the study. SARS-CoV-2 infection in early pregnancy was associated with lower median length-for-age Z-score at age 3 months compared with no infection or late pregnancy infect (-1.54 versus -0.37 and -0.51, P = 0.009). These findings suggest that pregnant Ugandan women experienced high levels of SARS-CoV-2 infection without severe respiratory illness. Variant-specific serology testing demonstrated evidence of antibody affinity maturation at the population level. Early gestational SARS-CoV-2 infection was associated with transient shorter stature in early infancy. Further research should explore the significance of this finding and define targeted measures to prevent infection in pregnancy.

Evaluation of a multiplexed oligonucleotide ligation assay for SARS-CoV-2 variant identification.

BACKGROUND: SARS-CoV-2 variant surveillance informs vaccine composition and decisions to de-authorize antibody therapies. Though detailed genetic characterization requires whole-genome sequencing, targeted mutation analysis may complement pandemic surveillance efforts. METHODS: This study investigated the qualitative performance of a multiplex oligonucleotide ligation assay targeting 19 spike mutations using 192 whole genome sequenced upper respiratory samples representing SARS-CoV-2 variants of concern. RESULTS: Initial valid results were obtained from 95.8% [95% confidence interval (CI): 92.0 - 98.2; 184/192] of samples. All eight invalid samples were valid on repeat testing. When comparing SARS-CoV-2 oligonucleotide ligase assay SARS-CoV-2 variant calls with whole genome sequencing, overall positive percent agreement was 100% (95% CI: 98.1 - 100.0; 192/192), as was the positive and negative percent agreement for each of the tested variants; Gamma, Delta, Omicron BA.1, BA.2, and BA.4/BA.5. CONCLUSIONS: This multiplexed oligonucleotide ligation assays demonstrated accurate SARS-CoV-2 variant typing compared to whole genome sequencing. Such an approach has the potential to provide improved turnaround compared to sequencing and more detailed mutation coverage than RT-qPCR.

Elevation of neural injury markers in patients with neurologic sequelae after hospitalization for SARS-CoV-2 infection.

Patients with SARS-CoV-2 infection (COVID-19) risk developing long-term neurologic symptoms after infection. Here, we identify biomarkers associated with neurologic sequelae one year after hospitalization for SARS-CoV-2 infection. SARS-CoV-2-positive patients were followed using post-SARS-CoV-2 online questionnaires and virtual visits. Hospitalized adults from the pre-SARS-CoV-2 era served as historical controls. 40% of hospitalized patients develop neurological sequelae in the year after recovery from acute COVID-19 infection. Age, disease severity, and COVID-19 infection itself was associated with additional risk for neurological sequelae in our cohorts. Glial fibrillary astrocytic protein (GFAP) and neurofilament light chain (NF-L) were significantly elevated in SARS-CoV-2 infection. After adjusting for age, sex, and disease severity, GFAP and NF-L remained significantly associated with longer term neurological symptoms in patients with SARS-CoV-2 infection. GFAP and NF-L warrant exploration as biomarkers for long-term neurologic complications after SARS-CoV-2 infection.

Nutritional markers and proteome in patients undergoing treatment for pulmonary tuberculosis differ by geographic region.

INTRODUCTION: Contemporary phase 2 TB disease treatment clinical trials have found that microbiologic treatment responses differ between African versus non-African regions, the reasons for which remain unclear. Understanding host and disease phenotypes that may vary by region is important for optimizing curative treatments. METHODS: We characterized clinical features and the serum proteome of phase 2 TB clinical trial participants undergoing treatment for smear positive, culture-confirmed TB, comparing host serum protein expression in clinical trial participants enrolled in African and Non-African regions. Serum samples were collected from 289 participants enrolled in the Centers for Disease Control and Prevention TBTC Study 29 (NCT00694629) at time of enrollment and at the end of the intensive phase (after 40 doses of TB treatment). RESULTS: After a peptide level proteome analysis utilizing a unique liquid chromatography IM-MS platform (LC-IM-MS) and subsequent statistical analysis, a total of 183 core proteins demonstrated significant differences at both baseline and at week 8 timepoints between participants enrolled from African and non-African regions. The majority of the differentially expressed proteins were upregulated in participants from the African region, and included acute phase proteins, mediators of inflammation, as well as coagulation and complement pathways. Downregulated proteins in the African population were primarily linked to nutritional status and lipid metabolism pathways. CONCLUSIONS: We have identified differentially expressed nutrition and lipid pathway proteins by geographic region in TB patients undergoing treatment for pulmonary tuberculosis, which appear to be associated with differential treatment responses. Future TB clinical trials should collect expanded measures of nutritional status and further evaluate the relationship between nutrition and microbiologic treatment response.

mRNA vaccination compared to infection elicits an IgG-predominant response with greater SARS-CoV-2 specificity and similar decrease in variant spike recognition.

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, new vaccine strategies including lipid nanoparticle delivery of antigen encoding RNA have been deployed globally. The BioNTech/Pfizer mRNA vaccine BNT162b2 encoding SARS-CoV-2 spike protein shows 95% efficacy in preventing disease, but it is unclear how the antibody responses to vaccination differ from those generated by infection. Here we compare the magnitude and breadth of antibodies targeting SARS-CoV-2, SARS-CoV-2 variants of concern, and endemic coronaviruses, in vaccinees and infected patients. We find that vaccination differs from infection in the dominance of IgG over IgM and IgA responses, with IgG reaching levels similar to those of severely ill COVID-19 patients and shows decreased breadth of the antibody response targeting endemic coronaviruses. Viral variants of concern from B.1.1.7 to P.1 to B.1.351 form a remarkably consistent hierarchy of progressively decreasing antibody recognition by both vaccinees and infected patients exposed to Wuhan-Hu-1 antigens.

Measurement of SARS-CoV-2 antigens in plasma of pediatric patients with acute COVID-19 or MIS-C using an ultrasensitive and quantitative immunoassay.

BACKGROUND: Detection of SARS-CoV-2 antigens in blood has high sensitivity in adults with acute COVID-19, but sensitivity in pediatric patients is unclear. Recent data suggest that persistent SARS-CoV-2 spike antigenemia may contribute to multisystem inflammatory syndrome in children (MIS-C). We quantified SARS-CoV-2 nucleocapsid (N) and spike (S) antigens in blood of pediatric patients with either acute COVID-19 or MIS-C using ultrasensitive immunoassays (Meso Scale Discovery). METHODS: Plasma was collected from inpatients (<21 years) enrolled across 15 hospitals in 15 US states. Acute COVID-19 patients (n=36) had a range of disease severity and positive nasopharyngeal SARS-CoV-2 RT-PCR within 24 hours of blood collection. Patients with MIS-C (n=53) met CDC criteria and tested positive for SARS-CoV-2 (RT-PCR or serology). Controls were patients pre-COVID-19 (n=67) or within 24h of negative RT-PCR (n=43). RESULTS: Specificities of N and S assays were 95-97% and 100%, respectively. In acute COVID-19 patients, N/S plasma assays had 89%/64% sensitivity, respectively; sensitivity in patients with concurrent nasopharyngeal swab cycle threshold (Ct) ≤ 35 were 93%/63%. Antigen concentrations ranged from 1.28-3,844 pg/mL (N) and 1.65-1,071 pg/mL (S) and correlated with disease severity. In MIS-C, antigens were detected in 3/53 (5.7%) samples (3 N-positive: 1.7, 1.9, 121.1 pg/mL; 1 S-positive: 2.3 pg/mL); the patient with highest N had positive nasopharyngeal RT-PCR (Ct 22.3) concurrent with blood draw. CONCLUSIONS: Ultrasensitive blood SARS-CoV-2 antigen measurement has high diagnostic yield in children with acute COVID-19. Antigens were undetectable in most MIS-C patients, suggesting that persistent antigenemia is not a common contributor to MIS-C pathogenesis. KEY POINTS: In a U.S. pediatric cohort tested with ultrasensitive immunoassays, SARS-CoV-2 nucleocapsid antigens were detectable in most patients with acute COVID-19, and spike antigens were commonly detectable. Both antigens were undetectable in almost all MIS-C patients.

Direct comparison of antibody responses to four SARS-CoV-2 vaccines in Mongolia.

Different SARS-CoV-2 vaccines are approved in various countries, but few direct comparisons of the antibody responses they stimulate have been reported. We collected plasma specimens in July 2021 from 196 Mongolian participants fully vaccinated with one of four COVID-19 vaccines: Pfizer/BioNTech, AstraZeneca, Sputnik V, and Sinopharm. Functional antibody testing with a panel of nine SARS-CoV-2 viral variant receptor binding domain (RBD) proteins revealed marked differences in vaccine responses, with low antibody levels and RBD-ACE2 blocking activity stimulated by the Sinopharm and Sputnik V vaccines in comparison to the AstraZeneca or Pfizer/BioNTech vaccines. The Alpha variant caused 97% of infections in Mongolia in June and early July 2021. Individuals who recover from SARS-CoV-2 infection after vaccination achieve high antibody titers in most cases. These data suggest that public health interventions such as vaccine boosting, potentially with more potent vaccine types, may be needed to control COVID-19 in Mongolia and worldwide.

Diagnostic accuracy of 3 urine lipoarabinomannan tuberculosis assays in HIV-negative outpatients.

BACKGROUNDInadequate tuberculosis (TB) diagnostics are a major hurdle in the reduction of disease burden, and accurate point-of-care tests (POCTs) are urgently needed. We assessed the diagnostic accuracy of Fujifilm SILVAMP TB lipoarabinomannan (FujiLAM) POCT for TB diagnosis in HIV-negative outpatients and compared it with Alere Determine TB LAM Ag (AlereLAM) POCT and a laboratory-based ultrasensitive electrochemiluminescence LAM research assay (EclLAM).METHODSIn this multicenter diagnostic test accuracy study, we recruited HIV-negative adults with symptoms suggestive of pulmonary TB presenting to outpatient health care centers in Peru and South Africa. Urine samples were tested using FujiLAM, AlereLAM, and EclLAM, and the diagnostic accuracy was assessed against a microbiological reference standard (MRS) and a composite reference standard.RESULTSThree hundred seventy-two HIV-negative participants were included and the prevalence of microbiologically confirmed TB was 30%. Compared with the MRS, the sensitivities of AlereLAM, FujiLAM, and EclLAM were 10.8% (95% confidence interval [CI] 6.3%-18.0%), 53.2% (95% CI 43.9%-62.1%), and 66.7% (95% CI 57.5%-74.7%), respectively. The specificities of AlereLAM, FujiLAM, and EclLAM were 92.3% (95% CI 88.5%-95.0%), 98.9% (95% CI 96.7%-99.6%), and 98.1% (95% CI 95.6%-99.2%), respectively. Positive likelihood ratios of AlereLAM, FujiLAM, and EclLAM were 1.4, 46.2, and 34.8, respectively, and positive predictive values were 37.5%, 95.2%, and 93.7%, respectively.CONCLUSIONCompared with AlereLAM, FujiLAM detected 5 times more patients with TB in HIV-negative participants, had a high positive predictive value, and has the potential to improve rapid diagnosis of TB at the point-of-care. EclLAM demonstrated that additional sensitivity gains are possible, which highlights LAM's potential as a biomarker. Additional research is required to assess FujiLAM's performance in prospective cohorts, its cost-effectiveness, and its impact in real-world clinical settings.FUNDINGGlobal Health Innovative Technology Fund, the UK Department for International Development, the Dutch Ministry of Foreign Affairs, the Bill and Melinda Gates Foundation, the Australian Department of Foreign Affairs and Trade, the German Federal Ministry of Education and Research through Kreditanstalt für Wiederaufbau, and the NIH and National Institute of Allergy and Infectious Diseases.

Sensitive electrochemiluminescence (ECL) immunoassays for detecting lipoarabinomannan (LAM) and ESAT-6 in urine and serum from tuberculosis patients.

BACKGROUND: Tuberculosis (TB) infection was responsible for an estimated 1.3 million deaths in 2017. Better diagnostic tools are urgently needed. We sought to determine whether accurate TB antigen detection in blood or urine has the potential to meet the WHO target product profiles for detection of active TB. MATERIALS AND METHODS: We developed Electrochemiluminescence (ECL) immunoassays for Lipoarabinomannan (LAM) and ESAT-6 detection with detection limits in the pg/ml range and used them to compare the concentrations of the two antigens in the urine and serum of 81 HIV-negative and -positive individuals with presumptive TB enrolled across diverse geographic sites. RESULTS: LAM and ESAT-6 overall sensitivities in urine were 93% and 65% respectively. LAM and ESAT-6 overall sensitivities in serum were 55% and 46% respectively. Overall specificity was ≥97% in all assays. Sensitivities were higher in HIV-positive compared to HIV-negative patients for both antigens and both sample types, with signals roughly 10-fold higher on average in urine than in serum. The two antigens showed similar concentration ranges within the same sample type and correlated. CONCLUSIONS: LAM and ESAT-6 can be detected in the urine and serum of TB patients, regardless of the HIV status and further gains in clinical sensitivity may be achievable through assay and reagent optimization. Accuracy in urine was higher with current methods and has the potential to meet the WHO accuracy target if the findings can be transferred to a point-of-care TB test.

Application of multiplexed ion mobility spectrometry towards the identification of host protein signatures of treatment effect in pulmonary tuberculosis.

RATIONALE: The monitoring of TB treatments in clinical practice and clinical trials relies on traditional sputum-based culture status indicators at specific time points. Accurate, predictive, blood-based protein markers would provide a simpler and more informative view of patient health and response to treatment. OBJECTIVE: We utilized sensitive, high throughput multiplexed ion mobility-mass spectrometry (IM-MS) to characterize the serum proteome of TB patients at the start of and at 8 weeks of rifamycin-based treatment. We sought to identify treatment specific signatures within patients as well as correlate the proteome signatures to various clinical markers of treatment efficacy. METHODS: Serum samples were collected from 289 subjects enrolled in CDC TB Trials Consortium Study 29 at time of enrollment and at the end of the intensive phase (after 40 doses of TB treatment). Serum proteins were immunoaffinity-depleted of high abundant components, digested to peptides and analyzed for data acquisition utilizing a unique liquid chromatography IM-MS platform (LC-IM-MS). Linear mixed models were utilized to identify serum protein changes in the host response to antibiotic treatment as well as correlations with culture status end points. RESULTS: A total of 10,137 peptides corresponding to 872 proteins were identified, quantified, and used for statistical analysis across the longitudinal patient cohort. In response to TB treatment, 244 proteins were significantly altered. Pathway/network comparisons helped visualize the interconnected proteins, identifying up regulated (lipid transport, coagulation cascade, endopeptidase activity) and down regulated (acute phase) processes and pathways in addition to other cross regulated networks (inflammation, cell adhesion, extracellular matrix). Detection of possible lung injury serum proteins such as HPSE, significantly downregulated upon treatment. Analyses of microbiologic data over time identified a core set of serum proteins (TTHY, AFAM, CRP, RET4, SAA1, PGRP2) which change in response to treatment and also strongly correlate with culture status. A similar set of proteins at baseline were found to be predictive of week 6 and 8 culture status. CONCLUSION: A comprehensive host serum protein dataset reflective of TB treatment effect is defined. A repeating set of serum proteins (TTHY, AFAM, CRP, RET4, SAA1, PGRP2, among others) were found to change significantly in response to treatment, to strongly correlate with culture status, and at baseline to be predictive of future culture conversion. If validated in cohorts with long term follow-up to capture failure and relapse of TB, these protein markers could be developed for monitoring of treatment in clinical trials and in patient care.

Non-human Primate Total-body Irradiation Model with Limited and Full Medical Supportive Care Including Filgrastim for Biodosimetry and Injury Assessment.

An assessment of multiple biomarkers from radiation casualties undergoing limited- or full-supportive care including treatment with filgrastim is critical to develop rapid and effective diagnostic triage strategies. The efficacy of filgrastim with full-supportive care was compared with results with limited-supportive care by analyzing survival, necropsy, histopathology and serial blood samples for hematological, serum chemistry and protein profiles in a non-human primate (Macaca mulatta, male and female) model during 60-d post-monitoring period following sham- and total-body irradiation with 6.5 Gy 60Co gamma-rays at 0.6 Gy min-1 Filgrastim (10 µg kg-1) was administered beginning on Day 1 post-exposure and continued daily until neutrophil counts were ≥2,000 µL-1 for two consecutive days. Filgrastim and full-supportive care significantly decreased the pancytopenia duration and resulted in improved animal survival and recovery compared to animals with a limited-supportive care. These findings also identified and validated a multiparametric biomarker panel to support radiation diagnostic device development.