Efficient SARS-CoV-2 variant detection and monitoring with Spike Screen next-generation sequencing.

The emergence and rapid spread of SARS-CoV-2 prompted the global community to identify innovative approaches to diagnose infection and sequence the viral genome because at several points in the pandemic positive case numbers exceeded the laboratory capacity to characterize sufficient samples to adequately respond to the spread of emerging variants. From week 10, 2020, to week 13, 2023, Slovenian routine complete genome sequencing (CGS) surveillance network yielded 41 537 complete genomes and revealed a typical molecular epidemiology with early lineages gradually being replaced by Alpha, Delta, and finally Omicron. We developed a targeted next-generation sequencing based variant surveillance strategy dubbed Spike Screen through sample pooling and selective SARS-CoV-2 spike gene amplification in conjunction with CGS of individual cases to increase throughput and cost-effectiveness. Spike Screen identifies variant of concern (VOC) and variant of interest (VOI) signature mutations, analyses their frequencies in sample pools, and calculates the number of VOCs/VOIs at the population level. The strategy was successfully applied for detection of specific VOC/VOI mutations prior to their confirmation by CGS. Spike Screen complemented CGS efforts with an additional 22 897 samples sequenced in two time periods: between week 42, 2020, and week 24, 2021, and between week 37, 2021, and week 2, 2022. The results showed that Spike Screen can be applied to monitor VOC/VOI mutations among large volumes of samples in settings with limited sequencing capacity through reliable and rapid detection of novel variants at the population level and can serve as a basis for public health policy planning.

Influenza A, Influenza B, human respiratory syncytial virus and SARSCoV-2 molecular diagnostics and epidemiology in the post COVID-19 era.

BACKGROUND: The concurrent circulation of SARS-CoV-2 with other respiratory viruses is unstoppable and represents a new diagnostic reality for clinicians and clinical microbiology laboratories. Multiplexed molecular testing on automated platforms that focus on the simultaneous detection of multiple respiratory viruses in a single tube is a useful approach for current and future diagnosis of respiratory infections in the clinical setting. METHODS: Two time periods were included in the study: from February to April 2022, an early 2022 period, during the gradual lifting of COVID-19 prevention measures in the country, and from October 2022 to April 2023, the 2022/23 respiratory infections season. We analysed a total of 1,918 samples in the first period and 18,131 respiratory samples in the second period using a multiplex molecular assay for the simultaneous detection of Influenza A (Flu-A), Influenza B (Flu-B), Human Respiratory Syncytial Virus (HRSV) and SARS-CoV-2. RESULTS: The results from early 2022 showed a strong dominance of SARS-CoV-2 infections with 1,267/1,918 (66.1%) cases. Flu-A was detected in 30/1,918 (1.6%) samples, HRSV in 14/1,918 (0.7%) samples, and Flu-B in 2/1,918 (0.1%) samples. Flu-A/SARS-CoV-2 co-detections were observed in 11/1,267 (0.9%) samples, and HRSV/SARS-CoV-2 co-detection in 5/1,267 (0.4%) samples. During the 2022/23 winter respiratory season, SARS-CoV-2 was detected in 1,738/18,131 (9.6%), Flu-A in 628/18,131 (3.5%), Flu-B in 106/18,131 (0.6%), and HRSV in 505/18,131 (2.8%) samples. Interestingly, co-detections were present to a similar extent as in early 2022. CONCLUSION: The results show that the multiplex molecular approach is a valuable tool for the simultaneous laboratory diagnosis of SARS-CoV-2, Flu-A/B, and HRSV in hospitalized and outpatients. Infections with Flu-A/B, and HRSV occurred shortly after the COVID-19 control measures were lifted, so a strong reoccurrence of various respiratory infections and co-detections in the post COVID-19 period was to be expected.

Association of Persistent Symptoms after Lyme Neuroborreliosis and Increased Levels of Interferon-α in Blood.

Patients who have Lyme neuroborreliosis (LNB) might experience lingering symptoms that persist despite antibiotic drug therapy. We tested whether those symptoms are caused by maladaptive immune responses by measuring 20 immune mediators in serum and cerebrospinal fluid (CSF) in 79 LNB patients followed for 1 year. At study entry, most mediators were highly concentrated in CSF, the site of the infection. Those responses resolved with antibiotic therapy, and associations between CSF cytokines and signs and symptoms of LNB were no longer observed. In contrast, subjective symptoms that persisted after use of antibiotics were associated with increased levels of serum interferon-α (IFN-α), which were already observed at study entry, and remained increased at each subsequent timepoint. Highest IFN-α levels corresponded with severe disease. Although the infection serves as the initial trigger, sequelae after antibiotic therapy are associated with unremitting systemic IFN-α levels, consistent with the pathogenic role of this cytokine in interferonopathies in other conditions.

Intrahost Monkeypox Virus Genome Variation in Patient with Early Infection, Finland, 2022.

Monkeypox virus was imported into Finland during late May-early June 2022. Intrahost viral genome variation in a sample from 1 patient comprised a major variant with 3 lineage B.1.3-specific mutations and a minor variant with ancestral B.1 nucleotides. Results suggest either ongoing APOBEC3 enzyme-mediated evolution or co-infection.

In human astrocytes neurotropic flaviviruses increase autophagy, yet their replication is autophagy-independent.

Astrocytes, an abundant type of glial cells, are the key cells providing homeostasis in the central nervous system. Due to their susceptibility to infection, combined with high resilience to virus-induced cell death, astrocytes are now considered one of the principal types of cells, responsible for virus retention and dissemination within the brain. Autophagy plays an important role in elimination of intracellular components and in maintaining cellular homeostasis and is also intertwined with the life cycle of viruses. The physiological significance of autophagy in astrocytes, in connection with the life cycle and transmission of viruses, remains poorly investigated. In the present study, we investigated flavivirus-induced modulation of autophagy in human astrocytes by monitoring a tandem fluorescent-tagged LC3 probe (mRFP-EGFP-LC3) with confocal and super-resolution fluorescence microscopy. Astrocytes were infected with tick-borne encephalitis virus (TBEV) or West Nile virus (WNV), both pathogenic flaviviruses, and with mosquito-only flavivirus (MOF), which is considered non-pathogenic. The results revealed that human astrocytes are susceptible to infection with TBEV, WNV and to a much lower extent also to MOF. Infection and replication rates of TBEV and WNV are paralleled by increased rate of autophagy, whereas autophagosome maturation and the size of autophagic compartments are not affected. Modulation of autophagy by rapamycin and wortmannin does not influence TBEV and WNV replication rate, whereas bafilomycin A1 attenuates their replication and infectivity. In human astrocytes infected with MOF, the low infectivity and the lack of efficient replication of this flavivirus are mirrored by the absence of an autophagic response.

External quality assessment of orthohantavirus and lymphocytic choriomeningitis virus molecular detection and serology in Europe, 2021.

BackgroundRodent-borne viruses such as orthohantaviruses and arenaviruses cause considerable disease burden with regional and temporal differences in incidence and clinical awareness. Therefore, it is important to regularly evaluate laboratory diagnostic capabilities, e.g. by external quality assessments (EQA).AimWe wished to evaluate the performance and diagnostic capability of European expert laboratories to detect orthohantaviruses and lymphocytic choriomeningitis virus (LCMV) and human antibody response towards orthohantaviruses.MethodsWe conducted an EQA in 2021; molecular panels consisted of 12 samples, including different orthohantaviruses (Seoul, Dobrava-Belgrade (DOBV), Puumala (PUUV) and Hantaan orthohantavirus), LCMV and negative controls. Serological panels consisted of six human serum samples reactive to PUUV, DOBV or negative to orthohantaviruses. The EQA was sent to 25 laboratories in 20 countries.ResultsThe accuracy of molecular detection of orthohantaviruses varied (50‒67%, average 62%) among 16 participating laboratories, while LCMV samples were successfully detected in all 11 participating laboratories (91-100%, average 96%). The accuracy of serological diagnosis of acute and past orthohantavirus infections was on average 95% among 20 participating laboratories and 82% in 19 laboratories, respectively. A variety of methods was used, with predominance of in-house assays for molecular tests, and commercial assays for serological ones.ConclusionSerology, the most common tool to diagnose acute orthohantavirus infections, had a high accuracy in this EQA. The molecular detection of orthohantaviruses needs improvement while LCMV detection (performed in fewer laboratories) had 95% accuracy. Further EQAs are recommended to be performed periodically to monitor improvements and challenges in the diagnostics of rodent-borne diseases.

Clinical and Laboratory Characteristics and Outcome of Illness Caused by Tick-Borne Encephalitis Virus without Central Nervous System Involvement.

Information on febrile illness caused by tick-borne encephalitis virus (TBEV) without central nervous system involvement is limited. We characterized 98 patients who had TBEV RNA in their blood but no central nervous system involvement at the time of evaluation. Median duration of illness was 7 days; 37 (38%) patients were hospitalized. The most frequent findings were malaise or fatigue (98%), fever (97%), headache (86%), and myalgias (54%); common laboratory findings were leukopenia (88%), thrombocytopenia (59%), and abnormal liver test results (63%). During the illness, blood leukocyte counts tended to improve, whereas thrombocytopenia and liver enzymes tended to deteriorate. At the time of positive PCR findings, 0/98 patients had serum IgG TBEV and 7 serum IgM TBEV; all patients later seroconverted. Viral RNA load was higher in patients with more severe illness but did not differ substantially in relation to several other factors. Illness progressed to tick-borne encephalitis in 84% of patients within 18 days after defervescence.

Unique Clinical, Immune, and Genetic Signature in Patients with Borrelial Meningoradiculoneuritis1.

Lyme neuroborreliosis (LNB) in Europe may manifest with painful meningoradiculoneuritis (also known as Bannwarth syndrome) or lymphocytic meningitis with or without cranial neuritis (peripheral facial palsy). We assessed host immune responses and the prevalence of TLR1 (toll-like receptor 1)-1805GG polymorphism to gain insights into the pathophysiology of these conditions. Regardless of LNB manifestation, most mediators associated with innate and adaptive immune responses were concentrated in cerebrospinal fluid; serum levels were unremarkable. When stratified by specific clinical manifestation, patients with meningoradiculoneuritis had higher levels of B-cell chemoattractants CXC motif chemokine ligand (CXCL) 12 and CXCL13 and T-cell-associated mediators CXCL9, CXCL10, and interleukin 17, compared with those without radicular pain. Moreover, these patients had a higher frequency of TLR1-1805GG polymorphism and more constitutional symptoms. These findings demonstrate that meningoradiculoneuritis is a distinct clinical entity with unique immune and genetic pathophysiology, providing new considerations for the study of LNB and borrelial meningoradiculitis.

Unique human immune signature of Ebola virus disease in Guinea.

Despite the magnitude of the Ebola virus disease (EVD) outbreak in West Africa, there is still a fundamental lack of knowledge about the pathophysiology of EVD. In particular, very little is known about human immune responses to Ebola virus. Here we evaluate the physiology of the human T cell immune response in EVD patients at the time of admission to the Ebola Treatment Center in Guinea, and longitudinally until discharge or death. Through the use of multiparametric flow cytometry established by the European Mobile Laboratory in the field, we identify an immune signature that is unique in EVD fatalities. Fatal EVD was characterized by a high percentage of CD4(+) and CD8(+) T cells expressing the inhibitory molecules CTLA-4 and PD-1, which correlated with elevated inflammatory markers and high virus load. Conversely, surviving individuals showed significantly lower expression of CTLA-4 and PD-1 as well as lower inflammation, despite comparable overall T cell activation. Concomitant with virus clearance, survivors mounted a robust Ebola-virus-specific T cell response. Our findings suggest that dysregulation of the T cell response is a key component of EVD pathophysiology.

Transmission of SARS-CoV-2 from Human to Domestic Ferret.

We report a case of natural infection with severe acute respiratory syndrome coronavirus 2 transmitted from an owner to a pet ferret in the same household in Slovenia. The ferret had onset of gastroenteritis with severe dehydration. Whole-genome sequencing of the viruses isolated from the owner and ferret revealed a 2-nt difference.

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa.

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.

Case of Babesia crassa-Like Infection, Slovenia, 2014.

We report a case of Babesia crassa-like infection in an asplenic patient in Slovenia in 2014. We diagnosed the infection using microscopy, 18S rRNA sequencing, and serology and monitored parasitemia using digital PCR. With its increasing occurrence, babesiosis should be included in differential diagnoses for immunocompromised patients displaying fever.

Clinical Evaluation of the cobas SARS-CoV-2 Test and a Diagnostic Platform Switch during 48 Hours in the Midst of the COVID-19 Pandemic.

Laboratories are currently witnessing extraordinary demand globally for sampling devices, reagents, consumables, and diagnostic instruments needed for timely diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To meet diagnostic needs as the pandemic grows, the U.S. Food and Drug Administration (FDA) recently granted several commercial SARS-CoV-2 tests Emergency Use Authorization (EUA), but manufacturer-independent evaluation data are scarce. We performed the first manufacturer-independent evaluation of the fully automated sample-to-result two-target test cobas 6800 SARS-CoV-2 (cobas) (Roche Molecular Systems, Branchburg, NJ), which received U.S. FDA EUA on 12 March 2020. The comparator was a standardized 3-h SARS-CoV-2 protocol, consisting of RNA extraction using an automated portable instrument, followed by a two-target reverse transcription real-time PCR (RT-PCR), which our laboratory has routinely used since January 2020 [V. M. Corman, O. Landt, M. Kaiser, R. Molenkamp, et al., Euro Surveill 25(3):pii=2000045, 2020, https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045]. cobas and the comparator showed overall agreement of 98.1% and a kappa value of 0.95 on an in-house validation panel consisting of 217 well-characterized retrospective samples. Immediate prospective head-to-head comparative evaluation followed on 502 samples, and the diagnostic approaches showed overall agreement of 99.6% and a kappa value of 0.98. A good correlation (r2 = 0.96) between cycle threshold values for SARS-CoV-2-specific targets obtained by cobas and the comparator was observed. Our results showed that cobas is a reliable assay for qualitative detection of SARS-CoV-2 in nasopharyngeal swab samples collected in the Universal Transport Medium System (UTM-RT) (Copan, Brescia, Italy). Under the extraordinary circumstances that laboratories are facing worldwide, a safe diagnostic platform switch is feasible in only 48 h and in the midst of the COVID-19 pandemic if carefully planned and executed.

Zika Virus Associated with Microcephaly.

A widespread epidemic of Zika virus (ZIKV) infection was reported in 2015 in South and Central America and the Caribbean. A major concern associated with this infection is the apparent increased incidence of microcephaly in fetuses born to mothers infected with ZIKV. In this report, we describe the case of an expectant mother who had a febrile illness with rash at the end of the first trimester of pregnancy while she was living in Brazil. Ultrasonography performed at 29 weeks of gestation revealed microcephaly with calcifications in the fetal brain and placenta. After the mother requested termination of the pregnancy, a fetal autopsy was performed. Micrencephaly (an abnormally small brain) was observed, with almost complete agyria, hydrocephalus, and multifocal dystrophic calcifications in the cortex and subcortical white matter, with associated cortical displacement and mild focal inflammation. ZIKV was found in the fetal brain tissue on reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay, with consistent findings on electron microscopy. The complete genome of ZIKV was recovered from the fetal brain.

Delayed Interferon Type 1-Induced Antiviral State Is a Potential Factor for Hemorrhagic Fever With Renal Syndrome Severity.

Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia. Interferon (IFN) responses play an important role in HFRS pathogenesis and early IFN-ß response is delayed by pathogenic hantaviruses. The severity of HFRS caused by Dobrava virus (DOBV) and Puumala virus (PUUV) varies. Our aim was to determine whether differences in early activation of IFN type 1-induced antiviral state influence HFRS severity. Peripheral blood mononuclear cells (PBMCs) from healthy donors and HFRS patients were stimulated with DOBV or PUUV and expression of selected genes was measured. PUUV, but not DOBV, activated IFN type 1-induced antiviral state in stimulated PBMCs, and IFNß, STAT-1, and MxA were highly upregulated. Upregulation of MxA was earlier in acute-phase PBMCs and higher in convalescent-phase PBMCs from patients with mild compared with severe PUUV infection. Our study showed that delayed IFN type 1-induced antiviral state could contribute to HFRS severity, particularly in PUUV infection.

Kinetics of Soluble Mediators of the Host Response in Ebola Virus Disease.

Background: The pathophysiology of Ebola virus disease (EVD) is still poorly understood. This study aimed at identifying soluble biomarkers that inform on disease mechanisms. Methods: Fifty-four soluble mediators of the immune, coagulation, and endothelial system were measured in baseline and follow-up samples from hospitalized patients with EVD, using Luminex technology. Cross-sectional expression levels and changes over time were correlated with outcome. Results: Levels of circulating proinflammatory cytokines and chemokines, as well as markers of endothelial dysfunction and coagulopathy, were elevated on admission to hospital in patients who died from EVD as compared to survivors. These markers further increased in patients who died and/or decreased over time in survivors. In contrast, markers of gut integrity and T-cell response were higher in survivors and increased until discharge. Conclusions: Inflammatory response, endothelial integrity, gastric tissue protection, and T cell immunity play a role in EVD pathophysiology.

Discovery and genome characterization of three new Jeilongviruses, a lineage of paramyxoviruses characterized by their unique membrane proteins.

BACKGROUND: In the past decade, many new paramyxoviruses that do not belong to any of the seven established genera in the family Paramyxoviridae have been discovered. Amongst them are J-virus (JPV), Beilong virus (BeiPV) and Tailam virus (TlmPV), three paramyxovirus species found in rodents. Based on their similarities, it has been suggested that these viruses should compose a new genus, tentatively called 'Jeilongvirus'. RESULTS: Here we present the complete genomes of three newly discovered paramyxoviruses, one found in a bank vole (Myodes glareolus) from Slovenia and two in a single, co-infected Rungwe brush-furred rat (Lophuromys machangui) from Mozambique, that represent three new, separate species within the putative genus 'Jeilongvirus'. The genome organization of these viruses is similar to other paramyxoviruses, but like JPV, BeiPV and TlmPV, they possess an additional open reading frame, encoding a transmembrane protein, that is located between the F and G genes. As is the case for all Jeilongviruses, the G genes of the viruses described here are unusually large, and their encoded proteins are characterized by a remarkable amino acid composition pattern that is not seen in other paramyxoviruses, but resembles certain motifs found in Orthopneumovirus G proteins. CONCLUSIONS: The phylogenetic clustering of JPV, BeiPV and TlmPV with the viruses described here, as well as their shared features that set them apart from other paramyxoviruses, provide additional support for the recognition of the genus 'Jeilongvirus'.

Analysis of Diagnostic Findings From the European Mobile Laboratory in Guéckédou, Guinea, March 2014 Through March 2015.

BACKGROUND: A unit of the European Mobile Laboratory (EMLab) consortium was deployed to the Ebola virus disease (EVD) treatment unit in Guéckédou, Guinea, from March 2014 through March 2015. METHODS: The unit diagnosed EVD and malaria, using the RealStar Filovirus Screen reverse transcription-polymerase chain reaction (RT-PCR) kit and a malaria rapid diagnostic test, respectively. RESULTS: The cleaned EMLab database comprised 4719 samples from 2741 cases of suspected EVD from Guinea. EVD was diagnosed in 1231 of 2178 hospitalized patients (57%) and in 281 of 563 who died in the community (50%). Children aged <15 years had the highest proportion of Ebola virus-malaria parasite coinfections. The case-fatality ratio was high in patients aged <5 years (80%) and those aged >74 years (90%) and low in patients aged 10-19 years (40%). On admission, RT-PCR analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold (Ct) than analysis of blood specimens from survivors (18.1 vs 23.2). Individuals who died in the community had a median Ct of 21.5 for throat swabs. Multivariate logistic regression on 1047 data sets revealed that low Ct values, ages of <5 and ≥45 years, and, among children aged 5-14 years, malaria parasite coinfection were independent determinants of a poor EVD outcome. CONCLUSIONS: Virus load, age, and malaria parasite coinfection play a role in the outcome of EVD.

Detection, identification and genotyping of Borrellia spp. in rodents in Slovenia by PCR and culture.

BACKGROUND: Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, is mainly maintained in natural foci through the transmission cycles of competent tick vectors (Ixodes sp.) and a vertebrate reservoir. Specific rodents have been identified as the principal reservoir of Borrelia burgdorferi sensu lato in Europe. Borrelia miyamotoi is the only relapsing fever spirochete transmitted by the same tick. The aim of the present study was to perform an epidemiological survey to determine the presence of B. burgdorferi sensu lato in rodents occurring in Slovenia and to explore the presence of Borrelia miyamotoi. The study was performed in two parts, retrospective and prospective; a total of 297 rodents was analyzed. Detection and identification of borrelia was performed by molecular methods and additionally in the prospective study by isolation and genotyping (MluI-LRFP and MLST). RESULTS: During the prospective part of the study, borrelia was isolated from 2/46 (4.3 %) lung specimens and from 10/46 (21.7 %) heart specimens of rodents. All isolated strains were identified as B. afzelii subtype Mla1, and MLST analysis revealed 5 distinct sequence types. Borrelia DNA was successfully detected by one or other of the PCR methods in 18/46 (39.1 %) and 75/251 (29.9 %) samples in the prospective and retrospective studies, respectively. LightMix® was found to be more sensitive than the ''in-house" nested PCR (91/297 (30.6 %) vs 48/297 (16.1 %)). Borrelia miyamotoi DNA was detected in 1/251 (0.4 %) and in 1/46 (2.2 %) heart specimens, in the retrospective and prospective parts of the study, respectively. CONCLUSION: We determined the prevalence of B. afzelii in rodents and report for the first time the presence of B. miyamotoi in Slovenia.

Serological response after COVID-19 infection compared to vaccination against COVID-19 in children with autoimmune rheumatic diseases.

BACKGROUND: Paediatric patients with autoimmune rheumatic diseases (pARD) have a dysregulated immune system, so infections present a major threat to them. To prevent severe COVID-19 infections we aimed to vaccinate them as soon as possible. Studies have shown that the BNT162b2 vaccine is safe, effective, and immunogenic, however, in a short observation period, only. METHODS: The main objective was to compare the serological response between three groups of pARD: after SARS-CoV-2 infection, after vaccination against COVID-19 with two doses of the BNT162b2 vaccine, and after experiencing both events. Data on demographics, diagnosis, therapy, and serology (anti-SARS-CoV-2 IgG/IgA) were collected from March 2020 to April 2022. For statistical analysis ANOVA, Mann-Whitney U test, Chi-square test and Fisher's exact test were applied. To compare adverse events (AE) after vaccination we included a control group of healthy adolescents. RESULTS: We collected data from 115 pARD; from 92 after infection and 47 after vaccination. Twenty-four were included in both groups. Serological data were available for 47 pARD after infection, 25 after vaccination, and 21 after both events. Serological response was better after vaccination and after both events compared to after infection only. No effect of medication on the antibody levels was noted. The safety profile of the vaccine was good. Systemic AE after the first dose of the vaccine were more common in healthy adolescents compared to pARD. In the observation period of 41.3 weeks, 60% of vaccinated pARD did not experience a symptomatic COVID-19 infection. CONCLUSIONS: IgG and IgA anti-SARS-CoV-2 levels were higher after vaccination and after both events compared to after infection only. Six months after vaccination we observed an increase in antibody levels, suggesting that pARD had been exposed to SARS-CoV-2 but remained asymptomatic. TRIAL REGISTRATION: The study was approved by the Medical Ethics Committee of the Republic of Slovenia (document number: 0120-485/2021/6).