A Dried Blood Spot protocol for high-throughput quantitative analysis of SARS-CoV-2 RBD serology based on the Roche Elecsys system.

SARS-CoV-2 spreads pandemically since 2020; in 2021, effective vaccinations became available and vaccination campaigns commenced. Still, it is hard to track the spread of the infection or to assess vaccination success in the broader population. Measuring specific anti-SARS-CoV-2 antibodies is the most effective tool to track the spread of the infection or successful vaccinations. The need for venous-blood sampling however poses a significant barrier for large studies. Dried-blood-spots on filter-cards (DBS) have been used for SARS-CoV-2 serology in our laboratory, but so far not to follow quantitative SARS-CoV-2 anti-spike reactivity in a longitudinal cohort. We developed a semi-automated protocol or quantitative SARS-CoV-2 anti-spike serology from self-sampled DBS, validating it in a cohort of matched DBS and venous-blood samples (n = 825). We investigated chromatographic effects, reproducibility, and carry-over effects and calculated a positivity threshold as well as a conversion formula to determine the quantitative binding units in the DBS with confidence intervals. Sensitivity and specificity reached 96.63% and 97.81%, respectively, compared to the same test performed in paired venous samples. Between a signal of 0.018 and 250 U/mL, we calculated a correction formula. Measuring longitudinal samples during vaccinations, we demonstrated relative changes in titers over time in several individuals and in a longitudinal cohort over four follow-ups. DBS sampling has proven itself for anti-nucleocapsid serosurveys in our laboratory. Similarly, anti-spike high-throughput DBS serology is feasible as a complementary assay. Quantitative measurements are accurate enough to follow titer dynamics in populations also after vaccination campaigns. This work was supported by the Bavarian State Ministry of Science and the Arts; LMU University Hospital, LMU Munich; Helmholtz Center Munich; University of Bonn; University of Bielefeld; German Ministry for Education and Research (proj. nr.: 01KI20271 and others) and the Medical Biodefense Research Program of the Bundeswehr Medical Service. Roche Diagnostics provided kits and machines for analyses at discounted rates. The project is funded also by the European-wide Consortium ORCHESTRA. The ORCHESTRA project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 101016167. The views expressed in this publication are the sole responsibility of the author, and the Commission is not responsible for any use that may be made of the information it contains.IMPORTANCESARS-CoV-2 has been spreading globally as a pandemic since 2020. To determine the prevalence of SARS-CoV-2 antibodies among populations, the most effective public health tool is measuring specific anti-SARS-CoV-2 antibodies induced by infection or vaccination. However, conducting large-scale studies that involve venous-blood sampling is challenging due to the associated feasibility and cost issues. A more cost-efficient and less invasive method for SARS-CoV-2 serological testing is using Dried-Blood-Spots on filter cards (DBS). In this paper, we have developed a semi-automated protocol for quantifying SARS-CoV-2 anti-spike antibodies from self-collected DBS. Our laboratory has previously successfully used DBS sampling for anti-nucleocapsid antibody surveys. Likewise, conducting high-throughput DBS serology for anti-spike antibodies is feasible as an additional test that can be performed using the same sample preparation as the anti-nucleocapsid analysis. The quantitative measurements obtained are accurate enough to track the dynamics of antibody levels in populations, even after vaccination campaigns.

Diagnostic accuracy of a three-gene Mycobacterium tuberculosis host response cartridge using fingerstick blood for childhood tuberculosis: a multicentre prospective study in low-income and middle-income countries.

BACKGROUND: Childhood tuberculosis remains a major cause of morbidity and mortality in part due to missed diagnosis. Diagnostic methods with enhanced sensitivity using easy-to-obtain specimens are needed. We aimed to assess the diagnostic accuracy of the Cepheid Mycobacterium tuberculosis Host Response prototype cartridge (MTB-HR), a candidate test measuring a three-gene transcriptomic signature from fingerstick blood, in children with presumptive tuberculosis disease. METHODS: RaPaed-TB was a prospective diagnostic accuracy study conducted at four sites in African countries (Malawi, Mozambique, South Africa, and Tanzania) and one site in India. Children younger than 15 years with presumptive pulmonary or extrapulmonary tuberculosis were enrolled between Jan 21, 2019, and June 30, 2021. MTB-HR was performed at baseline and at 1 month in all children and was repeated at 3 months and 6 months in children on tuberculosis treatment. Accuracy was compared with tuberculosis status based on standardised microbiological, radiological, and clinical data. FINDINGS: 5313 potentially eligible children were screened, of whom 975 were eligible. 784 children had MTB-HR test results, of whom 639 had a diagnostic classification and were included in the analysis. MTB-HR differentiated children with culture-confirmed tuberculosis from those with unlikely tuberculosis with a sensitivity of 59·8% (95% CI 50·8-68·4). Using any microbiological confirmation (culture, Xpert MTB/RIF Ultra, or both), sensitivity was 41·6% (34·7-48·7), and using a composite clinical reference standard, sensitivity was 29·6% (25·4-34·2). Specificity for all three reference standards was 90·3% (95% CI 85·5-94·0). Performance was similar in different age groups and by malnutrition status. Among children living with HIV, accuracy against the strict reference standard tended to be lower (sensitivity 50·0%, 15·7-84·3) compared with those without HIV (61·0%, 51·6-69·9), although the difference did not reach statistical significance. Combining baseline MTB-HR result with one Ultra result identified 71·2% of children with microbiologically confirmed tuberculosis. INTERPRETATION: MTB-HR showed promising diagnostic accuracy for culture-confirmed tuberculosis in this large, geographically diverse, paediatric cohort and hard-to-diagnose subgroups. FUNDING: European and Developing Countries Clinical Trials Partnership, UK Medical Research Council, Swedish International Development Cooperation Agency, Bundesministerium für Bildung und Forschung; German Center for Infection Research (DZIF).

Studying temporal titre evolution of commercial SARS-CoV-2 assays reveals significant shortcomings of using BAU standardization for comparison.

BACKGROUND: Measuring specific anti-SARS-CoV-2 antibodies has become one of the main epidemiological tools to survey the ongoing SARS-CoV-2 pandemic, but also vaccination response. The WHO made available a set of well-characterized samples derived from recovered individuals to allow normalization between different quantitative anti-Spike assays to defined Binding Antibody Units (BAU). METHODS: To assess sero-responses longitudinally, a cohort of ninety-nine SARS-CoV-2 RT-PCR positive subjects was followed up together with forty-five vaccinees without previous infection but with two vaccinations. Sero-responses were evaluated using a total of six different assays: four measuring anti-Spike proteins (converted to BAU), one measuring anti-Nucleocapsid proteins and one SARS-CoV-2 surrogate virus neutralization. Both cohorts were evaluated using the Euroimmun Anti-SARS-CoV-2-ELISA anti-S1 IgG and the Roche Elecsys Anti-SARS-CoV-2 anti-S1 assay. RESULTS: In SARS-CoV-2-convalesce subjects, the BAU-sero-responses of Euroimmun Anti-SARS-CoV-2-ELISA anti-S1 IgG and Roche Elecsys Anti-SARS-CoV-2 anti-S1 peaked both at 47 (43-51) days, the first assay followed by a slow decay thereafter (> 208 days), while the second assay not presenting any decay within one year. Both assay values in BAUs are only equivalent a few months after infection, elsewhere correction factors up to 10 are necessary. In contrast, in infection-naive vaccinees the assays perform similarly. CONCLUSION: The results of our study suggest that the establishment of a protective correlate or vaccination booster recommendation based on different assays, although BAU-standardised, is still challenging. At the moment the characteristics of the available assays used are not related, and the BAU-standardisation is unable to correct for that.

The representative COVID-19 cohort Munich (KoCo19): from the beginning of the pandemic to the Delta virus variant.

BACKGROUND: Population-based serological studies allow to estimate prevalence of SARS-CoV-2 infections despite a substantial number of mild or asymptomatic disease courses. This became even more relevant for decision making after vaccination started. The KoCo19 cohort tracks the pandemic progress in the Munich general population for over two years, setting it apart in Europe. METHODS: Recruitment occurred during the initial pandemic wave, including 5313 participants above 13 years from private households in Munich. Four follow-ups were held at crucial times of the pandemic, with response rates of at least 70%. Participants filled questionnaires on socio-demographics and potential risk factors of infection. From Follow-up 2, information on SARS-CoV-2 vaccination was added. SARS-CoV-2 antibody status was measured using the Roche Elecsys® Anti-SARS-CoV-2 anti-N assay (indicating previous infection) and the Roche Elecsys® Anti-SARS-CoV-2 anti-S assay (indicating previous infection and/or vaccination). This allowed us to distinguish between sources of acquired antibodies. RESULTS: The SARS-CoV-2 estimated cumulative sero-prevalence increased from 1.6% (1.1-2.1%) in May 2020 to 14.5% (12.7-16.2%) in November 2021. Underreporting with respect to official numbers fluctuated with testing policies and capacities, becoming a factor of more than two during the second half of 2021. Simultaneously, the vaccination campaign against the SARS-CoV-2 virus increased the percentage of the Munich population having antibodies, with 86.8% (85.5-87.9%) having developed anti-S and/or anti-N in November 2021. Incidence rates for infections after (BTI) and without previous vaccination (INS) differed (ratio INS/BTI of 2.1, 0.7-3.6). However, the prevalence of infections was higher in the non-vaccinated population than in the vaccinated one. Considering the whole follow-up time, being born outside Germany, working in a high-risk job and living area per inhabitant were identified as risk factors for infection, while other socio-demographic and health-related variables were not. Although we obtained significant within-household clustering of SARS-CoV-2 cases, no further geospatial clustering was found. CONCLUSIONS: Vaccination increased the coverage of the Munich population presenting SARS-CoV-2 antibodies, but breakthrough infections contribute to community spread. As underreporting stays relevant over time, infections can go undetected, so non-pharmaceutical measures are crucial, particularly for highly contagious strains like Omicron.

Nucleocapsid-specific T cell responses associate with control of SARS-CoV-2 in the upper airways before seroconversion.

Despite intensive research since the emergence of SARS-CoV-2, it has remained unclear precisely which components of the early immune response protect against the development of severe COVID-19. Here, we perform a comprehensive immunogenetic and virologic analysis of nasopharyngeal and peripheral blood samples obtained during the acute phase of infection with SARS-CoV-2. We find that soluble and transcriptional markers of systemic inflammation peak during the first week after symptom onset and correlate directly with upper airways viral loads (UA-VLs), whereas the contemporaneous frequencies of circulating viral nucleocapsid (NC)-specific CD4+ and CD8+ T cells correlate inversely with various inflammatory markers and UA-VLs. In addition, we show that high frequencies of activated CD4+ and CD8+ T cells are present in acutely infected nasopharyngeal tissue, many of which express genes encoding various effector molecules, such as cytotoxic proteins and IFN-γ. The presence of IFNG mRNA-expressing CD4+ and CD8+ T cells in the infected epithelium is further linked with common patterns of gene expression among virus-susceptible target cells and better local control of SARS-CoV-2. Collectively, these results identify an immune correlate of protection against SARS-CoV-2, which could inform the development of more effective vaccines to combat the acute and chronic illnesses attributable to COVID-19.

Rapid and Accurate Diagnosis of Pediatric Tuberculosis Disease: A Diagnostic Accuracy Study for Pediatric Tuberculosis.

INTRODUCTION: An estimated 1.2 million children develop tuberculosis (TB) every year with 240,000 dying because of missed diagnosis. Existing tools suffer from lack of accuracy and are often unavailable. Here, we describe the scientific and clinical methodology applied in RaPaed-TB, a diagnostic accuracy study. METHODS: This prospective diagnostic accuracy study evaluating several candidate tests for TB was set out to recruit 1000 children <15 years with presumptive TB in 5 countries (Malawi, Mozambique, South Africa, Tanzania, India). Assessments at baseline included documentation of TB signs and symptoms, TB history, radiography, tuberculin skin test, HIV testing and spirometry. Respiratory samples for reference standard testing (culture, Xpert Ultra) included sputum (induced/spontaneous) or gastric aspirate, and nasopharyngeal aspirate (if <5 years). For novel tests, blood, urine and stool were collected. All participants were followed up at months 1 and 3, and month 6 if on TB treatment or unwell. The primary endpoint followed NIH-consensus statements on categorization of TB disease status for each participant. The study was approved by the sponsor's and all relevant local ethics committees. DISCUSSION: As a diagnostic accuracy study for a disease with an imperfect reference standard, Rapid and Accurate Diagnosis of Pediatric Tuberculosis Disease (RaPaed-TB) was designed following a rigorous and complex methodology. This allows for the determination of diagnostic accuracy of novel assays and combination of testing strategies for optimal care for children, including high-risk groups (ie, very young, malnourished, children living with HIV). Being one of the largest of its kind, RaPaed-TB will inform the development of improved diagnostic approaches to increase case detection in pediatric TB.

Distinct and dynamic activation profiles of circulating dendritic cells and monocytes in mild COVID-19 and after yellow fever vaccination.

Dysregulation of the myeloid cell compartment is a feature of severe disease in hospitalized COVID-19 patients. Here, we investigated the response of circulating dendritic cell (DC) and monocyte subpopulations in SARS-CoV-2 infected outpatients with mild disease and compared it to the response of healthy individuals to yellow fever vaccine virus YF17D as a model of a well-coordinated response to viral infection. In SARS-CoV-2-infected outpatients circulating DCs were persistently reduced for several weeks whereas after YF17D vaccination DC numbers were decreased temporarily and rapidly replenished by increased proliferation until 14 days after vaccination. The majority of COVID-19 outpatients showed high expression of CD86 and PD-L1 in monocytes and DCs early on, resembling the dynamic after YF17D vaccination. In a subgroup of patients, low CD86 and high PD-L1 expression were detected in monocytes and DCs coinciding with symptoms, higher age, and lower lymphocyte counts. This phenotype was similar to that observed in severely ill COVID-19 patients, but less pronounced. Thus, prolonged reduction and dysregulated activation of blood DCs and monocytes were seen in a subgroup of symptomatic non-hospitalized COVID-19 patients while a transient coordinated activation was characteristic for the majority of patients with mild COVID-19 and the response to YF17D vaccination.

The diagnostic yield of nasopharyngeal aspirate for pediatric pulmonary tuberculosis: a systematic review and meta-analysis.

Background: Tuberculosis (TB) is a leading cause of death in children, but many cases are never diagnosed. Microbiological diagnosis of pulmonary TB is challenging in young children who cannot spontaneously expectorate sputum. Nasopharyngeal aspirates (NPA) may be more easily collected than gastric aspirate and induced sputum and can be obtained on demand, unlike stool. However, further information on its diagnostic yield is needed. Methods: We systematically reviewed and meta-analyzed the diagnostic yield of one NPA for testing by either culture or nucleic acid amplification testing (NAAT) to detect Mycobacterium tuberculosis from children. We searched three bibliographic databases and two trial registers up to 24th November 2022. Studies that reported the proportion of children diagnosed by NPA compared to a microbiological reference standard (MRS) were eligible. Culture and/or WHO-endorsed NAAT on at least one respiratory specimen served as the MRS. We also estimated the incremental yield of two NPA samples compared to one and summarized operational aspects of NPA collection and processing. Univariate random-effect meta-analyses were performed to calculate pooled diagnostic yield estimates. Results: From 1483 citations, 54 were selected for full-text review, and nine were included. Based on six studies including 256 children with microbiologically confirmed TB, the diagnostic yield of NAAT on one NPA ranged from 31 to 60% (summary estimate 44%, 95% CI 36-51%). From seven studies including 242 children with confirmed TB, the diagnostic yield of culture was 17-88% (summary estimate 58%, 95% CI 42-73%). Testing a second NPA increased the yield by 8-19% for NAAT and 4-35% for culture. NPA collection procedures varied between studies, although most children had NPA successfully obtained (96-100%), with a low rate of indeterminate results (< 5%). Data on NPA acceptability and specifically for children under 5 years were limited. Conclusions: NPA is a suitable and feasible specimen for diagnosing pediatric TB. The high rates of successful collection across different levels of healthcare improve access to microbiological testing, supporting its inclusion in diagnostic algorithms for TB, especially if sampling is repeated. Future research into the acceptability of NPA and how to standardize collection to optimize diagnostic yield is needed.

Enhanced Spike-specific, but attenuated Nucleocapsid-specific T cell responses upon SARS-CoV-2 breakthrough versus non-breakthrough infections.

SARS-CoV-2 vaccine breakthrough infections frequently occurred even before the emergence of Omicron variants. Yet, relatively little is known about the impact of vaccination on SARS-CoV-2-specific T cell and antibody response dynamics upon breakthrough infection. We have therefore studied the dynamics of CD4 and CD8 T cells targeting the vaccine-encoded Spike and the non-encoded Nucleocapsid antigens during breakthrough infections (BTI, n=24) and in unvaccinated control infections (non-BTI, n=30). Subjects with vaccine breakthrough infection had significantly higher CD4 and CD8 T cell responses targeting the vaccine-encoded Spike during the first and third/fourth week after PCR diagnosis compared to non-vaccinated controls, respectively. In contrast, CD4 T cells targeting the non-vaccine encoded Nucleocapsid antigen were of significantly lower magnitude in BTI as compared to non-BTI. Hence, previous vaccination was linked to enhanced T cell responses targeting the vaccine-encoded Spike antigen, while responses against the non-vaccine encoded Nucleocapsid antigen were significantly attenuated.

FujiLAM for the diagnosis of childhood tuberculosis: a systematic review.

BACKGROUND: Childhood tuberculosis (TB) remains underdiagnosed. The novel lateral flow FujiLAM assay detects lipoarabinomannan (LAM) in urine, but data on performance in children remain limited. METHODS: We conducted a systematic review assessing the diagnostic performance of FujiLAM for diagnosing paediatric TB. The last search was conducted in November 2021. RESULTS: We included three studies with data from 698 children for FujiLAM. For FujiLAM, sensitivity using a microbiological reference standard were 60% (95% CI 15 to 95), 42% (95% CI 31 to 53) and 63% (95% CI 50 to 75), respectively. Specificity was 93% (95% CI 85 to 98), 92% (95% CI 85 to 96) and 84% (95% CI 80 to 88). Using a composite reference standard, sensitivity was 11% (95% CI 4 to 22), 27% (95% CI 20 to 34) and 33% (95% CI 26 to 40), and specificity was 92% (95% CI 73 to 99), 97% (95% CI 87 to 100) and 85% (95% CI 79 to 89). Subgroup analyses for sensitivity of FujiLAM in children living with HIV (CLHIV) compared with those who were negative for HIV infection were inconsistent across studies. Among CLHIV, sensitivity appeared higher in those with greater immunosuppression, although wide CIs limit the interpretation of observed differences. Meta-analysis was not performed due to considerable study heterogeneity. CONCLUSION: The high specificity of FujiLAM demonstrates its potential as a point-of-care (POC) rule-in test for diagnosing paediatric TB. As an instrument-free POC test that uses an easy-to-obtain specimen, FujiLAM could significantly improve TB diagnosis in children in low-resource settings, however the small number of studies available highlight that further data are needed. Key priorities to be addressed in forthcoming paediatric evaluations include prospective head-to-head comparisons with AlereLAM using fresh specimens, specific subgroup analysis in CLHIV and extrapulmonary disease and studies in different geographical locations.CRD42021270761.

The interplay of viral loads, clinical presentation, and serological responses in SARS-CoV-2 - Results from a prospective cohort of outpatient COVID-19 cases.

Risk factors for disease progression and severity of SARS-CoV-2 infections require an understanding of acute and long-term virological and immunological dynamics. Fifty-one RT-PCR positive COVID-19 outpatients were recruited between May and December 2020 in Munich, Germany, and followed up at multiple defined timepoints for up to one year. RT-PCR and viral culture were performed and seroresponses measured. Participants were classified applying the WHO clinical progression scale. Short symptom to test time (median 5.0 days; p = 0.0016) and high viral loads (VL; median maximum VL: 3∙108 copies/mL; p = 0.0015) were indicative for viral culture positivity. Participants with WHO grade 3 at baseline had significantly higher VLs compared to those with WHO 1 and 2 (p = 0.01). VLs dropped fast within 1 week of symptom onset. Maximum VLs were positively correlated with the magnitude of Ro-N-Ig seroresponse (p = 0.022). Our results describe the dynamics of VLs and antibodies to SARS-CoV-2 in mild to moderate cases that can support public health measures during the ongoing global pandemic.

User perspectives and preferences on a novel TB LAM diagnostic (Fujifilm SILVAMP TB LAM)-a qualitative study in Malawi and Zambia.

Widely available tuberculosis (TB) diagnostics use sputum samples. However, many patients, particularly children and patients living with HIV (PLHIV), struggle to provide sputum. Urine diagnostics are a promising approach to circumvent this challenge while delivering reliable and timely diagnosis. This qualitative study in two high TB/HIV burden countries assesses values and preferences of end-users, along with potential barriers for the implementation of the novel Fujifilm SILVAMP TB-LAM (FujiLAM, Fujifilm, Japan) urine test. Between September 2020 and March 2021, we conducted 42 semi-structured interviews with patients, health care providers (HCPs) and decision makers (DMs) (e.g., in national TB programs) in Malawi and Zambia. Interviews were transcribed verbatim and analyzed using a framework approach supported by NVIVO. Findings aligned with the pre-existing Health Equity Implementation Framework, which guided the presentation of results. The ease and convenience of urine-based testing was described as empowering among patients and HCPs who lamented the difficulty of sputum collection, however HCPs expressed concerns that a shift in agency to the patient may affect clinic workflows (e.g., due to less control over collection). Implementation facilitators, such as shorter turnaround times, were welcomed by operators and patients alike. The decentralization of diagnostics was considered possible with FujiLAM by HCPs and DMs due to low infrastructure requirements. Finally, our findings support efforts for eliminating the CD4 count as an eligibility criterion for LAM testing, to facilitate implementation and benefit a wider range of patients. Our study identified barriers and facilitators relevant to scale-up of urine LAM tests in Malawi and Zambia. FujiLAM could positively impact health equity, as it would particularly benefit patient groups currently underserved by existing TB diagnostics. Participants view the approach as a viable, acceptable, and likely sustainable option in low- and middle-income countries, though adaptations may be required to current health care processes for deployment. Trial registration: German Clinical Trials Register, DRKS00021003. URL: https://www.drks.de/drks_web/setLocale_EN.do.

Understanding the interaction between cytomegalovirus and tuberculosis in children: The way forward.

Over 1 million children develop tuberculosis (TB) each year, with a quarter dying. Multiple factors impact the risk of a child being exposed to Mycobacterium tuberculosis (Mtb), the risk of progressing to TB disease, and the risk of dying. However, an emerging body of evidence suggests that coinfection with cytomegalovirus (CMV), a ubiquitous herpes virus, impacts the host response to Mtb, potentially influencing the probability of disease progression, type of TB disease, performance of TB diagnostics, and disease outcome. It is also likely that infection with Mtb impacts CMV pathogenesis. Our current understanding of the burden of these 2 diseases in children, their immunological interactions, and the clinical consequence of coinfection is incomplete. It is also unclear how potential interventions might affect disease progression and outcome for TB or CMV. This article reviews the epidemiological, clinical, and immunological literature on CMV and TB in children and explores how the 2 pathogens interact, while also considering the impact of HIV on this relationship. It outlines areas of research uncertainty and makes practical suggestions as to potential studies that might address these gaps. Current research is hampered by inconsistent definitions, study designs, and laboratory practices, and more consistency and collaboration between researchers would lead to greater clarity. The ambitious targets outlined in the World Health Organization End TB Strategy will only be met through a better understanding of all aspects of child TB, including the substantial impact of coinfections.

Host-Based Biomarkers in Saliva for the Diagnosis of Pulmonary Tuberculosis in Children: A Mini-Review.

The diagnosis of pulmonary tuberculosis (TB) in children remains a significant challenge due to its paucibacillary nature, non-specificity of symptoms and suboptimal sensitivity of available diagnostic methods. In young children particularly, it is difficult to obtain high-quality sputum specimens for testing, with this group the least likely to be diagnosed, while most at risk of severe disease. The World Health Organization (WHO) has prioritized research into rapid biomarker-based tests for TB using easily obtainable non-sputum samples, such as saliva. However, the role of biomarkers in saliva for diagnosing TB in children has not been fully explored. In this mini-review, we discuss the value of saliva as a diagnostic specimen in children given its ready availability and non-invasive nature of collection, and review the literature on the use of host-based biomarkers in saliva for diagnosing active pulmonary TB in adults and children. Based on available data from adult studies, we highlight that combinations of cytokines and other proteins show promise in reaching WHO-endorsed target product profiles for new TB triage tests. Given the lack of pediatric research on host biomarkers in saliva and the differing immune response to TB infection between children and adults, we recommend that pediatric studies are now performed to discover and validate salivary host biosignatures for diagnosing pulmonary TB in children. Future directions for pediatric saliva studies are discussed, with suggestions for technologies that can be applied for salivary biomarker discovery and point-of-care test development.

Head-to-head evaluation of seven different seroassays including direct viral neutralisation in a representative cohort for SARS-CoV-2.

A number of seroassays are available for SARS-CoV-2 testing; yet, head-to-head evaluations of different testing principles are limited, especially using raw values rather than categorical data. In addition, identifying correlates of protection is of utmost importance, and comparisons of available testing systems with functional assays, such as direct viral neutralisation, are needed.We analysed 6658 samples consisting of true-positives (n=193), true-negatives (n=1091), and specimens of unknown status (n=5374). For primary testing, we used Euroimmun-Anti-SARS-CoV-2-ELISA-IgA/IgG and Roche-Elecsys-Anti-SARS-CoV-2. Subsequently virus-neutralisation, GeneScriptcPass, VIRAMED-SARS-CoV-2-ViraChip, and Mikrogen-recomLine-SARS-CoV-2-IgG were applied for confirmatory testing. Statistical modelling generated optimised assay cut-off thresholds. Sensitivity of Euroimmun-anti-S1-IgA was 64.8%, specificity 93.3% (manufacturer's cut-off); for Euroimmun-anti-S1-IgG, sensitivity was 77.2/79.8% (manufacturer's/optimised cut-offs), specificity 98.0/97.8%; Roche-anti-N sensitivity was 85.5/88.6%, specificity 99.8/99.7%. In true-positives, mean and median Euroimmun-anti-S1-IgA and -IgG titres decreased 30/90 days after RT-PCR-positivity, Roche-anti-N titres decreased significantly later. Virus-neutralisation was 80.6% sensitive, 100.0% specific (≥1:5 dilution). Neutralisation surrogate tests (GeneScriptcPass, Mikrogen-recomLine-RBD) were >94.9% sensitive and >98.1% specific. Optimised cut-offs improved test performances of several tests. Confirmatory testing with virus-neutralisation might be complemented with GeneScriptcPassTM or recomLine-RBD for certain applications. Head-to-head comparisons given here aim to contribute to the refinement of testing strategies for individual and public health use.

From first to second wave: follow-up of the prospective COVID-19 cohort (KoCo19) in Munich (Germany).

BACKGROUND: In the 2nd year of the COVID-19 pandemic, knowledge about the dynamics of the infection in the general population is still limited. Such information is essential for health planners, as many of those infected show no or only mild symptoms and thus, escape the surveillance system. We therefore aimed to describe the course of the pandemic in the Munich general population living in private households from April 2020 to January 2021. METHODS: The KoCo19 baseline study took place from April to June 2020 including 5313 participants (age 14 years and above). From November 2020 to January 2021, we could again measure SARS-CoV-2 antibody status in 4433 of the baseline participants (response 83%). Participants were offered a self-sampling kit to take a capillary blood sample (dry blood spot; DBS). Blood was analysed using the Elecsys® Anti-SARS-CoV-2 assay (Roche). Questionnaire information on socio-demographics and potential risk factors assessed at baseline was available for all participants. In addition, follow-up information on health-risk taking behaviour and number of personal contacts outside the household (N = 2768) as well as leisure time activities (N = 1263) were collected in summer 2020. RESULTS: Weighted and adjusted (for specificity and sensitivity) SARS-CoV-2 sero-prevalence at follow-up was 3.6% (95% CI 2.9-4.3%) as compared to 1.8% (95% CI 1.3-3.4%) at baseline. 91% of those tested positive at baseline were also antibody-positive at follow-up. While sero-prevalence increased from early November 2020 to January 2021, no indication of geospatial clustering across the city of Munich was found, although cases clustered within households. Taking baseline result and time to follow-up into account, men and participants in the age group 20-34 years were at the highest risk of sero-positivity. In the sensitivity analyses, differences in health-risk taking behaviour, number of personal contacts and leisure time activities partly explained these differences. CONCLUSION: The number of citizens in Munich with SARS-CoV-2 antibodies was still below 5% during the 2nd wave of the pandemic. Antibodies remained present in the majority of SARS-CoV-2 sero-positive baseline participants. Besides age and sex, potentially confounded by differences in behaviour, no major risk factors could be identified. Non-pharmaceutical public health measures are thus still important.

In Search of the SARS-CoV-2 Protection Correlate: Head-to-Head Comparison of Two Quantitative S1 Assays in Pre-characterized Oligo-/Asymptomatic Patients.

BACKGROUND: Quantitative serological assays detecting response to SARS-CoV-2 are needed to quantify immunity. This study analyzed the performance and correlation of two quantitative anti-S1 assays in oligo-/asymptomatic individuals from a population-based cohort. METHODS: In total, 362 plasma samples (108 with reverse transcription-polymerase chain reaction [RT-PCR]-positive pharyngeal swabs, 111 negative controls, and 143 with positive serology without confirmation by RT-PCR) were tested with quantitative assays (Euroimmun Anti-SARS-CoV-2 QuantiVac enzyme-linked immunosorbent assay [EI-S1-IgG-quant]) and Roche Elecsys® Anti-SARS-CoV-2 S [Ro-RBD-Ig-quant]), which were compared with each other and confirmatory tests, including wild-type virus micro-neutralization (NT) and GenScript®cPass™. Square roots R of coefficients of determination were calculated for continuous variables and non-parametric tests were used for paired comparisons. RESULTS: Quantitative anti-S1 serology correlated well with each other (true positives, 96%; true negatives, 97%). Antibody titers decreased over time (< 30 to > 240 days after initial positive RT-PCR). Agreement with GenScript-cPass was 96%/99% for true positives and true negatives, respectively, for Ro-RBD-Ig-quant and 93%/97% for EI-S1-IgG-quant. Ro-RBD-Ig-quant allowed distinct separation between positives and negatives, and less non-specific reactivity versus EI-S1-IgG-quant. Raw values (95% CI) ≥ 28.7 U/mL (22.6-36.4) for Ro-RBD-Ig-quant and ≥ 49.8 U/mL (43.4-57.1) for EI-S1-IgG-quant predicted NT > 1:5 in 95% of cases. CONCLUSIONS: Our findings suggest both quantitative anti-S1 assays (EI-S1-IgG-quant and Ro-RBD-Ig-quant) may replace direct neutralization assays in quantitative measurement of immune protection against SARS-CoV-2 in certain circumstances. However, although the mean antibody titers for both assays tended to decrease over time, a higher proportion of Ro-RBD-Ig-quant values remained positive after 240 days. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40121-021-00475-x.

In Search of the SARS-CoV-2 Protection Correlate: Head-to-Head Comparison of Two Quantitative S1 Assays in Pre-characterized Oligo-/Asymptomatic Patients.

BACKGROUND: Quantitative serological assays detecting response to SARS-CoV-2 are needed to quantify immunity. This study analyzed the performance and correlation of two quantitative anti-S1 assays in oligo-/asymptomatic individuals from a population-based cohort. METHODS: In total, 362 plasma samples (108 with reverse transcription-polymerase chain reaction [RT-PCR]-positive pharyngeal swabs, 111 negative controls, and 143 with positive serology without confirmation by RT-PCR) were tested with quantitative assays (Euroimmun Anti-SARS-CoV-2 QuantiVac enzyme-linked immunosorbent assay [EI-S1-IgG-quant]) and Roche Elecsys® Anti-SARS-CoV-2 S [Ro-RBD-Ig-quant]), which were compared with each other and confirmatory tests, including wild-type virus micro-neutralization (NT) and GenScript®cPass™. Square roots R of coefficients of determination were calculated for continuous variables and non-parametric tests were used for paired comparisons. RESULTS: Quantitative anti-S1 serology correlated well with each other (true positives, 96%; true negatives, 97%). Antibody titers decreased over time (< 30 to > 240 days after initial positive RT-PCR). Agreement with GenScript-cPass was 96%/99% for true positives and true negatives, respectively, for Ro-RBD-Ig-quant and 93%/97% for EI-S1-IgG-quant. Ro-RBD-Ig-quant allowed distinct separation between positives and negatives, and less non-specific reactivity versus EI-S1-IgG-quant. Raw values (95% CI) ≥ 28.7 U/mL (22.6-36.4) for Ro-RBD-Ig-quant and ≥ 49.8 U/mL (43.4-57.1) for EI-S1-IgG-quant predicted NT > 1:5 in 95% of cases. CONCLUSIONS: Our findings suggest both quantitative anti-S1 assays (EI-S1-IgG-quant and Ro-RBD-Ig-quant) may replace direct neutralization assays in quantitative measurement of immune protection against SARS-CoV-2 in certain circumstances. However, although the mean antibody titers for both assays tended to decrease over time, a higher proportion of Ro-RBD-Ig-quant values remained positive after 240 days.

Prevalence and Risk Factors of Infection in the Representative COVID-19 Cohort Munich.

Given the large number of mild or asymptomatic SARS-CoV-2 cases, only population-based studies can provide reliable estimates of the magnitude of the pandemic. We therefore aimed to assess the sero-prevalence of SARS-CoV-2 in the Munich general population after the first wave of the pandemic. For this purpose, we drew a representative sample of 2994 private households and invited household members 14 years and older to complete questionnaires and to provide blood samples. SARS-CoV-2 seropositivity was defined as Roche N pan-Ig ≥ 0.4218. We adjusted the prevalence for the sampling design, sensitivity, and specificity. We investigated risk factors for SARS-CoV-2 seropositivity and geospatial transmission patterns by generalized linear mixed models and permutation tests. Seropositivity for SARS-CoV-2-specific antibodies was 1.82% (95% confidence interval (CI) 1.28-2.37%) as compared to 0.46% PCR-positive cases officially registered in Munich. Loss of the sense of smell or taste was associated with seropositivity (odds ratio (OR) 47.4; 95% CI 7.2-307.0) and infections clustered within households. By this first population-based study on SARS-CoV-2 prevalence in a large German municipality not affected by a superspreading event, we could show that at least one in four cases in private households was reported and known to the health authorities. These results will help authorities to estimate the true burden of disease in the population and to take evidence-based decisions on public health measures.

Publisher Correction to: Protocol of a population-based prospective COVID-19 cohort study Munich, Germany (KoCo19).

An amendment to this paper has been published and can be accessed via the original article.