Variations in the persistence of 5'-end genomic and subgenomic SARS-CoV-2 RNAs in wastewater from aircraft, airports and wastewater treatment plants.

Wastewater sequencing has become a powerful supplement to clinical testing in monitoring SARS-CoV-2 infections in the post-COVID-19 pandemic era. While its applications in measuring the viral burden and main circulating lineages in the community have proved their efficacy, the variations in sequencing quality and coverage across the different regions of the SARS-CoV-2 genome are not well understood. Furthermore, it is unclear how different sample origins, viral extraction and concentration methods and environmental factors impact the reads sequenced from wastewater. Using high-coverage, amplicon-based, paired-end read sequencing of viral RNA extracted from wastewater collected directly from aircraft, pooled from different aircraft and airport buildings or from regular wastewater plants, we assessed the genome coverage across the sample groups with a focus on the 5'-end region covering the leader sequence and investigated whether it was possible to detect subgenomic RNA from viral material recovered from wastewater. We identified distinct patterns in the persistence of the different genomic regions across the different types of wastewaters and the existence of chimeric reads mapping to non-amplified regions. Our findings suggest that preservation of the 5'-end of the genome and the ability to detect subgenomic RNA reads, though highly susceptible to environment and sample processing conditions, may be indicative of the quality and amount of the viral RNA present in wastewater.

Results from the SARS-CoV-2 wastewater-based surveillance system in Denmark, July 2021 to June 2022.

The microbiological analysis of wastewater samples is increasingly used for the surveillance of SARS-CoV-2 globally. We described the setup process of the national SARS-CoV-2 wastewater-based surveillance system in Denmark, presented its main results during the first year of activities, from July 2021 to June 2022, and discussed their operational significance. The Danish SARS-CoV-2 wastewater-based surveillance system was designed to cover 85 % of the population in Denmark and it entailed taking three weekly samples from 230 sites. Samples were RT-qPCR tested for SARS-CoV-2 RNA, targeting the genetic markers N1, N2 and RdRp, and for two faecal indicators, Pepper Mild Mottle Virus and crAssphage. We calculated the weekly SARS-CoV-2 RNA concentration in the wastewater from each sampling site and monitored it in view of the results from individual testing, at the national and regional levels. We attempted to use wastewater results to identify potential local outbreaks, and we sequenced positive wastewater samples using Nanopore sequencing to monitor the circulation of viral variants in Denmark. The system reached its full implementation by October 2021 and covered up to 86.4 % of the Danish population. The system allowed for monitoring of the national and regional trends of SARS-CoV-2 infections in Denmark. However, the system contribution to the identification of potential local outbreaks was limited by the extensive information available from clinical testing. The sequencing of wastewater samples identified relevant variants of concern, in line with results from sequencing of human samples. Amidst the COVID-19 pandemic, Denmark implemented a nationwide SARS-CoV-2 wastewater-based surveillance system that integrated routine surveillance from individual testing. Today, while testing for COVID-19 at the community level has been discontinued, the system is on the frontline to monitor the occurrence and spread of SARS-CoV-2 in Denmark.

SARS-CoV-2 Variants BQ.1 and XBB.1.5 in Wastewater of Aircraft Flying from China to Denmark, 2023.

We analyzed wastewater samples from 14 aircraft arriving in Denmark directly from China during January 9-February 12, 2023. Wastewater from 11 aircraft was SARS-CoV-2-positive by PCR; 6 predominantly contained BQ.1 and XBB.1 subvariants. Wastewater-based surveillance can contribute to public health monitoring of SARS-CoV-2 and other emerging infectious agents.

Predicting COVID-19 Incidence Using Wastewater Surveillance Data, Denmark, October 2021-June 2022.

Analysis of wastewater is used in many settings for surveillance of SARS-CoV-2, but it remains unclear how well wastewater testing results reflect incidence. Denmark has had an extensive wastewater analysis system that conducts 3 weekly tests in ≈200 sites and has 85% population coverage; the country also offers free SARS-CoV-2 PCR tests to all residents. Using time series analysis for modeling, we found that wastewater data, combined with information on circulating variants and the number of human tests performed, closely fitted the incidence curve of persons testing positive. The results were consistent at a regional level and among a subpopulation of frequently tested healthcare personnel. We used wastewater analysis data to estimate incidence after testing was reduced to a minimum after March 2022. These results imply that data from a large-scale wastewater surveillance system can serve as a good proxy for COVID-19 incidence and for epidemic control.

Seroprevalence of SARS-CoV-2 Antibodies in Denmark: Results of Two Nationwide Population-Based Surveys, February and May 2021.

Background: Seroprevalence studies can be used to measure the progression of national COVID-19 epidemics. The Danish National Seroprevalence Survey of SARS-CoV-2 infections (DSS) was conducted as five separate surveys between May 2020 and May 2021. Here, we present results from the two last surveys conducted in February and May 2021. Methods: Persons aged 12 or older were randomly selected from the Danish Population Register and those having received COVID-19 vaccination subsequently excluded. Invitations to have blood drawn in local test centers were sent by mail. Samples were analyzed for whole Immunoglobulin by ELISA. Seroprevalence was estimated by sex, age and geography. Comparisons to vaccination uptake and RT-PCR test results were made. Results: In February 2021, we found detectable antibodies in 7.2% (95% CI: 6.3-7.9%) of the invited participants (participation rate 25%) and in May 2021 in 8.6% (95% CI: 7.6-9.5%) of the invited (participation rate: 14%). Seroprevalence did not differ by sex, but by age group, generally being higher among the <50 than 50+ year-olds. In May 2021, levels of seroprevalence varied from an estimated 13% (95% CI: 12-15%) in the capital to 5.2% (95% CI: 3.4-7.4%) in rural areas. Combining seroprevalence results with vaccine coverage, estimates of protection against infection in May 2021 varied from 95% among 65+ year-olds down to 10-20% among 12-40 year-olds. In March-May 2021, an estimated 80% of all community SARS-CoV-2 infections were diagnosed by RT-PCR and captured by surveillance. Conclusion: Seroprevalence estimates doubled during the 2020-21 winter wave of SARS-CoV-2 infections and then stabilized as vaccinations were rolled out. The epidemic affected large cities and younger people the most. Denmark saw comparatively low infections rates, but high test coverage; an estimated four out of five infections were detected by RT-PCR in March-May 2021.

Pre-vaccination vulnerability and suspected adverse events following HPV vaccination. A case-control study nested in the Danish national birth cohort.

Previous studies have indicated that girls experiencing suspected adverse events (AE) following HPV vaccination were more vulnerable prior to vaccination. However, no study has previously investigated differences in vulnerability using prospectively collected self-reported measures of vulnerability. The objective of this study therefore was to describe the distribution of biological and psychosocial indicators of vulnerability in girls referred to a hospital setting due to suspected adverse events and compare it with a sample of non-referred HPV vaccinated girls. The study was conducted as a case control study based within the Danish National Birth Cohort. Cases were defined as HPV vaccinated girls referred to a hospital setting between 2015 and 2017 due to suspected adverse events (n = 80), and 5 controls were randomly selected from the remaining source population, matched to cases on age at vaccination, region of residence and year of vaccination. The final study population consisted of 480 girls. Prior exposures were based on information gathered from an 11 year follow up of the DNBC and included information on self-rated health, frequent health complaints, medication use, bullying, stressful life events and physical activity. Conditional logistic regression analysis was used to estimate the association between each exposure and referral. The percentage of individuals in the exposed category of each exposure was generally higher for cases than controls. Particularly, the odds of being referred were higher for those with low self-rated health compared to high (OR [95%-CI] 2.43 [1.07-5.5]1), those being bullied (OR 3.19 [1.17-8.73]), and those who had taken medication (OR 2.22 [1.32-3.67]). Overall, these results indicated that girls experiencing suspected AE following HPV vaccination were more vulnerable prior to vaccination.

Infections in temporal proximity to HPV vaccination and adverse effects following vaccination in Denmark: A nationwide register-based cohort study and case-crossover analysis.

BACKGROUND: Public trust in the human papilloma virus (HPV) vaccination programme has been challenged by reports of potential severe adverse effects. The reported adverse symptoms were heterogeneous and overlapping with those characterised as chronic fatigue syndrome (CFS) and have been described as CFS-like symptoms. Evidence suggests that CFS is often precipitated by an infection. The aim of the study was to examine if an infection in temporal proximity to HPV vaccination is a risk factor for suspected adverse effects following HPV vaccination. METHODS AND FINDINGS: The study was a nationwide register-based cohort study and case-crossover analysis. The study population consisted of all HPV vaccinated females living in Denmark, born between 1974 and 2006, and vaccinated between January 1, 2006 and December 31, 2017. The exposure was any infection in the period ± 1 month around time of first HPV vaccination and was defined as (1) hospital-treated infection; (2) redemption of anti-infective medication; or (3) having a rapid streptococcal test done at the general practitioner. The outcome was referral to a specialised hospital setting (5 national HPV centres opened June 1, 2015) due to suspected adverse effects following HPV vaccination. Multivariable logistic regression was used to estimate the association between infection and later HPV centre referral. The participants were 600,400 HPV-vaccinated females aged 11 to 44 years. Of these, 48,361 (9.7%) females had a hospital-treated infection, redeemed anti-infective medication, or had a rapid streptococcal test ± 1 month around time of first HPV vaccination. A total of 1,755 (0.3%) females were referred to an HPV centre. Having a hospital-treated infection in temporal proximity to vaccination was associated with significantly elevated risk of later referral to an HPV centre (odds ratio (OR) 2.75, 95% confidence interval (CI) 1.72 to 4.40; P < 0.001). Increased risk was also observed among females who redeemed anti-infective medication (OR 1.56, 95% CI 1.33 to 1.83; P < 0.001) or had a rapid streptococcal test (OR 1.45, 95% CI 1.10 to 1.93; P = 0.010). Results from a case-crossover analysis, which was performed to adjust for potential unmeasured confounding, supported the findings. A key limitation of the study is that the HPV centres did not open until June 1, 2015, which may have led to an underestimation of the risk of suspected adverse effects, but stratified analyses by year of vaccination yielded similar results. CONCLUSIONS: Treated infection in temporal proximity to HPV vaccination is associated with increased risk for later referral with suspected adverse vaccine effects. Thus, the infection could potentially be a trigger of the CFS-like symptoms in a subset of the referred females. To our knowledge, the study is the first to investigate the role of infection in the development of suspected adverse effects after HPV vaccination and replication of these findings are needed in other studies.

General Practitioner Attendance in Proximity to HPV Vaccination: A Nationwide, Register-Based, Matched Case-Control Study.

AIM: This study aimed to describe and compare general practitioner (GP) attendance patterns in the years surrounding HPV-vaccination among cases suspected to experience adverse events and their matched controls in order to determine if a potential larger change in GP attendance among cases was observed in temporal relation to vaccination. METHODS: Register-based, matched case-control study. Cases were defined as women referred to specialized hospital settings (HPV-centers) for suspected adverse event between June 1st 2015 and December 31st 2015 (n=1458). Information on referral was obtained from the HPV-centers directly. Each case was matched with five controls on age at vaccination, region, time of first vaccine registration and total number of doses, resulting in a total study population of 8670 girls and women. Negative binomial regression analyses were used (i) to estimate mean yearly GP contacts among cases and controls, and (ii) to further investigate the relative difference in change in GP attendance following vaccination between cases and controls. RESULTS: Overall, cases displayed higher GP attendance than controls from five years before vaccination up until five years after. Compared to controls, cases increased their GP attendance more in the years following HPV vaccination, corresponding to a 40% increase in the incidence rate ratios (IRR) from before to after vaccination (ratio-IRR = 1.40 [1.36; 1.44]). The change occurred in close proximity to vaccination, and the pattern was the same independently of the year of vaccination. However, for the later vaccination years cases displayed an additional increase in their GP attendance around time of extensive media attention. CONCLUSION: Girls and women being referred for suspected adverse events after HPV-vaccination changed their GP attendance pattern in close proximity to their first HPV-vaccination and not solely in temporal linkage to the onset of public debate.

Are unexplained adverse health events following HPV vaccination associated with infectious mononucleosis? - A Danish nationwide matched case-control study.

BACKGROUND: In Denmark, the acceptance of the HPV vaccination program has been threatened by reports of suspected adverse events. Epstein Barr Virus (EBV) infection is associated with symptoms of long-lasting tiredness and may be misinterpreted as HPV vaccine adverse events. The main aim of this study was to examine if EBV infection around time of HPV vaccination was a risk factor for later suspected vaccine adverse events. METHODS: The study was a nationwide register-based matched case-control study. Cases were females vaccinated against HPV in the period 2011 throughout 2017 with suspected adverse events. For each case, five HPV vaccinated females without suspected adverse events were selected. Information about EBV infection was obtained from the Danish Microbiology Database and assessed for three time periods: (1) before first HPV vaccination, (2) around time of HPV vaccination, and (3) any time during the study period 2010-2017. Multiple logistic regression was used to estimate the association between EBV and suspected adverse events. RESULTS: We identified 1217 cases, matched to 6085 controls. A higher proportion of cases (38; 3.1%) than controls (31; 0.5%) were tested for EBV around time of HPV vaccination and cases had elevated odds for testing both EBV positive (OR 4.52, 95% CI 2.68-7.63) and EBV negative (OR 20.99, 95% CI 5.81-75.79). Only five females were classified with acute/recent EVB infection in this period. CONCLUSION: Misinterpretation of EBV infection late symptoms is not a leading explanation for Danish females experiencing suspected adverse events after HPV vaccination. Although EBV cannot be excluded as an explanatory factor for a very small proportion of suspected adverse events, the findings are more likely explained by protopathic bias, i.e. the fact that a larger proportion of females suspecting adverse events are tested for EBV.

Hospital contacts and diagnoses five years prior to HPV vaccination among females referred for suspected adverse vaccine effects: A Danish nationwide case-control study.

BACKGROUND: High health care utilization before Human Papilloma Virus (HPV) vaccination might be associated with increased risk of suspected adverse effects (AE's) after vaccination. We investigated the association between hospital contacts and diagnoses before HPV vaccination and risk of later referral to a specialized hospital setting (HPV center) for suspected AE's. METHODS: The study was a Danish register-based matched case-control study. Cases were females referred to an HPV center. Five controls per case were randomly selected in the source population of HPV vaccinated females. Information on hospital contacts and diagnoses was obtained from the Danish National Patient Registry. Conditional logistic regression analyses were used to investigate the association between having one or more diagnoses in each specific International Classification of Diseases 10th version (ICD-10) chapter five years before the HPV vaccination and subsequent referral to an HPV center. RESULTS: We identified 1496 cases and 7480 controls. In total, 80% of the cases versus 65% of the controls had at least one hospital contact prior to HPV vaccination (Prevalence Proportion Ratio - PPR: 1.24 (95% Confidence Interval - CI: 1.21-1.27)), with 24% vs 12% (PPR: 1.97 (95% CI: 1.76-2.19)) having six or more contacts. Cases were more likely to have had a diagnosis in 15 out of 19 ICD-10 chapters before the vaccination, with ORs larger than 1.8 for infectious diseases, psychiatric diseases, diseases of the nervous, circulatory, digestive and musculoskeletal system, unspecific symptoms and unspecific contacts. CONCLUSION: Pre-vaccination morbidity and health care utilization seem to play a role in the path leading to suspected AE's after HPV vaccination. Since many of the diagnoses that were particularly increased before vaccination in referred females are overlapping with the most frequent symptoms reported, we cannot exclude that for some of the females, the suspected AE's might have been existing already before the vaccination.

Psychiatric conditions and general practitioner attendance prior to HPV vaccination and the risk of referral to a specialized hospital setting because of suspected adverse events following HPV vaccination: a register-based, matched case-control study.

AIM: No association between human papilloma virus (HPV) vaccination and numerous diseases has been found. Still, a large number of Danish women are reporting suspected adverse events. Other factors may play a role, and the aim of this study is to examine the association between psychiatric conditions, general practitioner (GP) attendance and indicators of psychological symptoms prior to HPV vaccination and the risk of referral to an HPV center following vaccination. STUDY DESIGN AND SETTING: Register-based, matched case-control study. Cases were identified from five Danish, regional HPV centers, and health data for cases and controls were obtained from national registries. PARTICIPANTS: Cases were defined as women referred to an HPV center between January 1, 2015 and December 31, 2015 (n=1,496). Each case was matched with five controls on age, region and time of first vaccine registration. The total study population consisted of 8,976 women. RESULTS: Overall, women above 18 years who had been referred to an HPV center were more likely to have used psychiatric medication (odds ratio [OR]: 1.88 [95% CI 1.48-2.40]) or to have been hospitalized because of a psychiatric disorder within 5 years prior to the first vaccine registration (OR: 2.13 [95% CI 1.59-2.86]). Specifically, referred women were more likely to have used antipsychotics, antidepressants, attention deficit hyperactivity disorder (ADHD) medication or anxiolytics, and to have been hospitalized for affective disorders or anxiety, but not to have been hospitalized for schizoid, ADHD or eating disorders. In addition, they were more likely to have had talk therapy or psychometric test performed prior to vaccination (OR: 1.72 [95% CI 0.1.35-2.18] and OR: 1.67 [95% CI 1.30-2.13], respectively). Referred women of all ages had higher use of GP before vaccination. Population attributable fraction analyses indicated that psychiatric medication, hospitalization due to a psychiatric disorder and use of talk therapy, or psychometric test "explained" 13%, 10%, 12% and 11% of the referrals, respectively. Results did not change substantially when adjusted for potential confounders. CONCLUSION: Women referred to HPV centers because of suspected adverse events after vaccination more often had preexisting psychiatric conditions, psychological symptoms or frequent GP attendance prior to HPV vaccination.

Health care utilization in general practice after HPV vaccination-A Danish nationwide register-based cohort study.

OBJECTIVE: The Human Papillomavirus (HPV) vaccine has increasingly been suspected of adverse effects in Denmark since 2013. By using consultations with the general practitioner (GP) as an indicator for morbidity, this study aims to examine the association between HPV vaccination and morbidity in girls in the Danish childhood immunization program. METHODS: The study is a nationwide register-based cohort study. Both the HPV and the Measles, Mumps and Rubella (MMR) vaccines were offered to 12-year-old girls in Denmark in the study period (2008-2015). Therefore, both vaccines were included as exposures to allow differentiation between potential effects. This resulted in four exposure groups: HPV only vaccinated, HPV+MMR vaccinated, MMR only vaccinated, and Non-vaccinated girls. Outcomes were: daytime consultation rates and frequent GP attendance (> 7 annual GP consultations). We estimated consultation rates by negative binomial regressions analysis and frequent GP attendance by logistic regression analysis. Both analyses were stratified on the years 2008-2013 versus 2014. RESULTS: The study included 214,240 girls born in 1996-2002. All vaccinated groups consulted the GP more often than the non-vaccinated group, both before and after the vaccination. After the vaccination, an increase in consultations was observed for all three vaccinated groups; most distinct for girls vaccinated in 2014. For girls vaccinated before 2014, we found a slightly higher risk of frequent GP attendance after vaccination in the HPV only group compared to the non-vaccinated group, whereas in 2014, frequent GP attendance was seen for all three vaccinated groups; most substantial for the MMR only vaccinated group. CONCLUSION: In this study, no exclusive increase in health care utilization was detected after HPV vaccination. However, a general difference in the health care utilization pattern was found between vaccinated and non-vaccinated girls, which increased after the time of vaccination, primarily for girls vaccinated after 2013.