This review explores and positions the value of serology testing to support current immunization policies and the broader policy response to the coronavirus disease 2019 (COVID-19) crisis in Europe. We applied an exploratory approach to analysing existing evidence, international recommendations, and national policies using desk research from secondary sources, document analysis, and expert information. Regional and country-level resources from five focus countries were included: France, Germany, Italy, Spain, and the United Kingdom. Seven experts in the fields of COVID-19 immunization, serology testing, seroepidemiology, and vaccine safety and effectiveness studies contributed to the review and convened in two online panel sessions. The paper includes an overview of (1) the impact of the pandemic to date, (2) testing strategies, (3) COVID-19 vaccination policies, (4) lessons on using serology testing to support immunization, (5) current policies and recommendations on the use of a serology testing strategy, and (6) implementation barriers and challenges. Finally, this paper also provides a set of knowledge-based recommendations to advance the effective and timely inclusion of serology testing and resolve impeding knowledge gaps. The recommendations herein are intended to support timely decision-making, raise awareness, guide advocacy initiatives, and inspire future studies.

Under covid-19-pandemin har avloppsvattenbaserad epidemiologi (WBE) använts i stor utsträckning som ett komplement till kliniska tester över många delar av världen. Detta projekt syftade till att detektera och kvantifiera belastningen av Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) i avloppsvattenprover med hjälp av Revers transkriptas kvantitativ polymeraskedjereaktion (RT-qPCR). De analyserade proverna kom från fyra olika avloppsreningsverk i Sverige, under perioden november 2022 till maj 2023. Studien omfattade en översikt över olika provtagnings- och analytiska tekniker och normaliseringsmetoder som används i WBE-studier, vilket betonade vikten av metodval. SARS-CoV-2-RNA upptäcktes i alla analyserade prover och infektionstrender kunde identifieras effektivt, inklusive COVID-19-vågen som observerades under semesterperioden. De dominerande varianterna som upptäcktes under denna övervakningsperiod var omikron variantens undergrupper, BA.2. och BA.2.75. Den veckovisa kvantifierade SARS-CoV-2-belastningen i avloppsvattenproverna visade en signifikant positiv korrelation till de kliniska fall som rapporterats i motsvarande avrinningsområden. Denna associering förstärktes ytterligare genom att normalisera SARS-CoV-2-innehållet med fekal biomarkör peppar milt fläckvirus (PMMoV). Dessutom har två metoder för tidig varning, nämligen medelvärdet plus två standardavvikelser (MSD) och positiv procentuell förändring (PPC), implementerats på avloppsvattendata, vilket pekar på vikten av att tillämpa sådana varningsmetoder för att ge förståeliga och tolkbara resultat. Denna studie ger värdefulla insikter om övervakning och analys av SARS-CoV-2 i avloppsvatten, vilket bidrar till utvecklingen av robusta system för tidig varning och folkhälsostrategier. / During the COVID-19 pandemic, wastewater-based epidemiology (WBE) has been applied extensively as a complementary tool to clinical testing across many parts of the globe. This project aimed to detect and measure the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) load in wastewater samples using Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). The analyzed samples were from four different wastewater treatment plants (WWTPs) in Sweden, covering the period from November 2022 through May 2023. The study encompassed an overview of various sampling and analytical techniques and normalization approaches employed in WBE studies, highlighting the importance of method selection. SARS-CoV-2 RNA was detected in all the samples analyzed, and infection trends could be identified effectively, including the COVID-19 peak observed during the holiday season. The dominant variants detected during this monitoring period were the omicron variants; omicron BA.2. and omicron BA.2.75. The weekly quantified SARS-CoV-2 load in the wastewater samples showed a significant positive correlation to the clinical cases reported in the corresponding catchment areas. This association was further enhanced by normalizing SARS-CoV-2 content with the fecal biomarker pepper mild mottle virus (PMMoV). Furthermore, two early warning methods, namely the mean plus two standard deviations (MSD) and positive percentage change (PPC), were implemented on the wastewater data pinpointing the importance of applying such warning methods to provide understandable and interpretable results. This study provides valuable insights into the monitoring and analysis of SARS-CoV-2 in wastewater, contributing to the development of robust early warning systems and public health strategies.

Viruset SARS-CoV-2 binder till det membranbundna enzymet angiotensinkonvertas 2 (ACE2). ACE2 är även en del av Renin-Angiotensin-Aldosteron-systemet (RAAS) och Kallikrein-Kinin-systemet (KKS). Den enzymatiska delen av ACE2 klyvs från cellmembranet vid inbindningen av viruset eller genom enzymet ADAM17 och bildar då cirkulerande ACE2 (sACE2). Syftet med den här litteraturstudien var att undersöka om sACE2 kan kopplas till allvarlighetsgraden av covid-19. Studierna visade blandade resultat. Tre artiklar visade ökande halter sACE2 vid svår covid-19. Två studier visade sjunkande halter sACE2 vid svår covid-19. Två artiklar visade ingen koppling mellan sACE2 och allvarlighetsgraden av covid-19. Då forskningsområdet omkring SARS-CoV-2 är nytt finns det stora variationer i studiernas upplägg, vilket resulterar i att resultaten är osäkra och svåra att jämföra med varandra. Eventuellt kan det finnas en koppling mellan mängden sACE2 och allvarlighetsgraden av covid-19. Mer forskning behövs för att säkerställa om sACE2 i kombination med andra tester kan användas för att upptäcka patienter i risk för allvarlig covid-19. / The virus SARS-CoV-2 binds to the enzyme Angiotensin Converting Enzyme 2 (ACE2) which causes shedding of the extracellular part of the enzyme and produces soluble ACE2 (sACE2). ACE2 is also a part of Renin-Angiotensin-Aldosterone-system (RAAS) and Kallikrein-Kinin-system (KKS). The enzyme ADAM17 also causes ACE2 to shed during inflammation. Since inflammation is an important part of the Covid-19 pathogenesis, the sACE2 levels may affect the pathogenesis of Covid-19. The purpose of this literature study was to investigate a possible connection between sACE levels and Covid-19 severity. Three articles showed high levels of sACE2 in severe Covid-19. Two articles showed low levels of sACE2 in severe Covid-19. Two articles showed no connection between severity of Covid-19 and sACE2 levels.  Due to the novelty of the research area, there are variations in the study set-up. Although inconclusive study results, sACE2 may be used as a secondary biomarker to detect indications of severe Covid-19, but more research is needed.

Surveillance of the evolving SARS-CoV-2 genome combined with epidemiological monitoring and emerging vaccination became paramount tasks to control the pandemic which is rapidly changing in time and space. Genomic surveillance must combine generation and sharing sequence data with appropriate bioinformatics monitoring and analysis methods. We applied molecular portrayal using self-organizing maps machine learning (SOM portrayal) to characterize the diversity of the virus genomes, their mutual relatedness and development since the beginning of the pandemic. The genetic landscape obtained visualizes the relevant mutations in a lineage-specific fashion and provides developmental paths in genetic state space from early lineages towards the variants of concern alpha, beta, gamma and delta. The different genes of the virus have specific footprints in the landscape reflecting their biological impact. SOM portrayal provides a novel option for ‘bioinformatics surveillance’ of the pandemic, with strong odds regarding visualization, intuitive perception and ‘personalization’ of the mutational patterns of the virus genomes.

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Les études de séroprévalence portant sur les infections au SRAS-CoV-2 doivent souvent composer avec des échantillons non-aléatoires et non-représentatifs, limitant ainsi parfois la validité externe de leurs résultats lorsque ceux-ci sont appliqués à la population générale. Dans le cadre de ce mémoire, il s’agit d’investiguer la représentativité d’une cohorte pédiatrique d’une étude longitudinale de séroprévalence (Enfants et COVID-19 : Étude de séroprévalence) et d’évaluer dans quelle mesure ses estimations de séroprévalence peuvent s’appliquer à la population pédiatrique montréalaise en général. 1 632 enfants ont fourni au point de départ un échantillon sanguin afin de déterminer leur séropositivité aux anticorps contre le SRAS-CoV-2. À l’aide d’une modélisation par régression logistique et d’un procédé de « standardisation marginale », une pondération post-stratification calculée à partir des données du recensement canadien de 2016 a été appliquée à la population d’étude. Les variations dans les estimations de séroprévalence ont finalement été évaluées. D’importantes différences dans la distribution de certaines caractéristiques sociodémographiques peuvent être observées lorsqu’on compare la population d’étude et la population générale en se basant sur les données du recensement canadien de 2016. En comparaison des estimations non-pondérées, les estimations de séroprévalence générées à partir du procédé de « standardisation marginale » montrent une variation de plusieurs points de pourcentage, allant de -0,4% à +3,2%. La pondération n’a pas induit de changement dans l’estimation de mesures relatives comme les ratios de séroprévalence. Lorsque la population d’étude est non-représentative de la population-cible, il est nécessaire de pondérer les caractéristiques sociodémographiques associées à l’issue si l’on veut appliquer les résultats plus généralement. / Prevalence studies on SARS-CoV-2 infections have often based on study populations with non-random and non-representative samples, which limits the external validity of their results when applied to the general population. The aim of this thesis was to investigate the representativeness of a pediatric cohort of a longitudinal seroprevalence study (Children and COVID-19: Seroprevalence study) and to assess to what extent its baseline estimates of seroprevalence can be applied to the Montreal pediatric population. There were 1 632 children participants who provided a blood sample at baseline, which was used to determine their seropositivity to SARS-CoV-2 antibodies. Using logistic regression modeling and a "marginal standardization" method, post-stratification weights calculated from 2016 Canadian census data were applied to the study population. Variations in seroprevalence estimates were then assessed. Significant differences in the distribution of certain sociodemographic characteristics were observed when comparing the study population and the target population based on 2016 Canadian census data. Seroprevalence estimates were generated from the “marginal standardization” approach which differed to that of the non-standardized estimates, and the differences ranges from -0,4% to +3,2%. Weighting did not change relative measures estimates, such as seroprevalence ratios. When the study population is not representative of the target population, it is necessary to weight the sociodemographic characteristics associated with the prevalence estimates, if the results will be applied more broadly.

The rapid development, approval, and production of vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than 1 year after the first reports of a new infectious disease was a real game changer, providing 80%–90% efficacy in preventing severe etiopathologies of the coronavirus disease 2019 (COVID-19). These vaccines induce an immune response against the SARS-CoV-2 spike (S) protein located on the surface of the virus particle. Antibodies (Abs) recognizing the S-protein can inhibit binding of the virus via the S-protein to the angiotensin-converting enzyme-2 (ACE-2) receptor expressed on different human cells, especially when these Abs bind to the interaction site, the so-called receptor-binding domain (RBD). We have expressed the RBDs of wild-type SARS-CoV-2 and five variants of concern (VOCs) to test the immune response in people before vaccination with mRNA vaccines BNT162b2 and mRNA-1273 and after up to three vaccinations using in-house ELISA and inhibition assays. The methods of both assays are provided. Both vaccines initiated similarly high IgG titers after two vaccinations against the wild-type and even two VOC-RBDs (alpha and delta) and strongly inhibited the corresponding RBD-ACE-2 binding. The IgG titers and inhibition of ACE-2 binding were lower for beta and gamma RBDs and much lower for omicron RBD. The third vaccination after 6 months strongly increased both the IgG titers and the neutralizing effect against all variants, especially for omicron, leading to 63% ± 13% neutralization potential. Importantly, neutralization linearly increased with the IgG titers.

An unprecedented use of high-throughput sequencing for routine monitoring of SARS-CoV-2 viruses in patient samples has created a dataset of over 6 million SARS-CoV-2 genomes. To monitor genomes, deposited in the GISAID database, and to track the continuous sequence evolution of molecular assay oligonucleotide target sequences. A simple pipeline tool for non-experts was developed to mine this database for nucleotide changes in oligonucleotides and tested with the long oligonucleotides of a Recombinase polymerase amplification (RPA) assay targeting the RNA-dependent RNA polymerase (RdRP) gene of the SARS-CoV-2. Results indicate the emergence of a single nucleotide change in the reverse oligonucleotide from 0.03 to 26.23% (January to May 2021) in Alpha variant genomes, which however reduced to 17.64% by September after which the Alpha variant was completely displaced by the Delta variant. For all other variants, no relevant nucleotide changes were observed. The oligonucleotide screening pipeline allows efficient screening of nucleotide changes in oligonucleotides of all sizes in minutes.

There is an ongoing need for high-precision serological assays for the quantitation of anti-SARS-CoV-2 antibodies. Here, a trimeric SARS-CoV-2 spike (S) protein was used to develop an ELISA to quantify specific IgG antibodies present in serum, plasma, and dried blood spots (DBS) collected from infected patients or vaccine recipients. The quantitative S-ELISA was calibrated with international anti-SARS-CoV-2 immunoglobulin standards to provide test results in binding antibody units per mL (BAU/mL). The assay showed excellent linearity, precision, and accuracy. A sensitivity of 100% was shown for samples collected from 54 patients with confirmed SARS-CoV-2 infection more than 14 days after symptom onset or disease confirmation by RT-PCR and 58 vaccine recipients more than 14 days after vaccination. The assay specificity was 98.3%. Furthermore, antibody responses were measured in follow-up samples from vaccine recipients and infected patients. Most mRNA vaccine recipients had a similar response, with antibody generation starting 2–3 weeks after the first vaccination and maintaining positive for at least six months after a second vaccination. For most infected patients, the antibody titers increased during the second week after PCR confirmation. This S-ELISA can be used to quantify the seroprevalence of SARS-CoV-2 in the population exposed to the virus or vaccinated.

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