Individual level analysis of digital proximity tracing for COVID-19 in Belgium highlights major bottlenecks (preprint)

To complement labour-intensive conventional contact tracing, digital proximity tracing was implemented widely during the COVID-19 pandemic. However, the privacy-centred design of the dominant Google-Apple exposure notification framework has hindered assessment of its effectiveness. Between October 2021 and January 2022, we systematically collected app use and notification receipt data within a test and trace programme for university students in Leuven, Belgium. Due to low success rates in each studied step of the digital notification cascade, only 4.3% of exposed contacts (CI: 2.8-6.1%) received such notifications, resulting in 10 times more cases detected through conventional contact tracing. Moreover, the infection risk of digitally traced contacts (5.0%; CI: 3.0-7.7%) was lower than that of conventionally traced non-app users (9.8%; CI: 8.8-10.7%; p=0.002). Contrary to common perception as near instantaneous, there was a 1.2-day delay (CI: 0.6-2.2) between case PCR result and digital contact notifications. These results highlight major limitations of the dominant digital proximity tracing framework.

Unravelling the effect of winter holiday celebrations on SARS-CoV-2 transmission (preprint)

Public holidays have been associated with SARS-CoV-2 incidence surges, although a firm causal link remains to be established. This association is sometimes attributed to events where transmissions occur at a disproportionately high rate, known as superspreading events. Here, we describe a sudden surge in new cases with the Omicron BA.1 strain amongst higher education students in Belgium. Contact tracers classed most of these cases as likely or possibly infected on New Year's Eve, indicating a direct trigger by New Year celebrations. Using a combination of contact tracing and phylogenetic data, we show the limited role of superspreading events in this surge. Finally, the numerous simultaneous transmissions allowed a unique opportunity to determine the distribution of incubation periods of the Omicron strain. Overall, our results indicate that, even under social restrictions, a surge in transmissibility of SARS-CoV-2 can occur when holiday celebrations result in small social gatherings attended simultaneously and communitywide.

Resistance of Omicron subvariants BA.2.75.2, BA.4.6 and BQ.1.1 to neutralizing antibodies (preprint)

Convergent evolution of SARS-CoV-2 Omicron BA.2, BA.4 and BA.5 lineages has led to the emergence of several new subvariants, including BA.2.75.2, BA.4.6. and BQ.1.1. The subvariants BA.2.75.2 and BQ.1.1 are expected to become predominant in many countries in November 2022. They carry an additional and often redundant set of mutations in the spike, likely responsible for increased transmissibility and immune evasion. Here, we established a viral amplification procedure to easily isolate Omicron strains. We examined their sensitivity to 6 therapeutic monoclonal antibodies (mAbs) and to 72 sera from Pfizer BNT162b2-vaccinated individuals, with or without BA.1/BA.2 or BA.5 breakthrough infection. Ronapreve (Casirivimab and Imdevimab) and Evusheld (Cilgavimab and Tixagevimab) lost any antiviral efficacy against BA.2.75.2 and BQ.1.1, whereas Xevudy (Sotrovimab) remained weakly active. BQ.1.1 was also resistant to Bebtelovimab. Neutralizing titers in triply vaccinated individuals were low to undetectable against BQ.1.1 and BA.2.75.2, 4 months after boosting. A BA.1/BA.2 breakthrough infection increased these titers, which remained about 18-fold lower against BA.2.75.2 and BQ.1.1, than against BA.1. Reciprocally, a BA.5 breakthrough infection increased more efficiently neutralization against BA.5 and BQ.1.1 than against BA.2.75.2. Thus, the evolution trajectory of novel Omicron subvariants facilitated their spread in immunized populations and raises concerns about the efficacy of most currently available mAbs.

Natural ventilation, low CO2 and air filtration are associated with reduced indoor air respiratory pathogens (preprint)

Currently, the real-life impact of indoor climate, human behavior, ventilation and air filtration on respiratory pathogen detection and concentration are poorly understood. This hinders the interpretability of bioaerosol quantification in indoor air to surveil respiratory pathogens and transmission risk. We tested 341 indoor air samples from 21 community settings for 29 respiratory pathogens using qPCR. On average, 3.9 pathogens were positive per sample and 85.3% of samples tested positive for at least one. The number of detected pathogens and their respective concentrations varied significantly by pathogen, month, and age group in generalized linear (mixed) models and generalized estimating equations. High CO2 and low natural ventilation were independent risk factors for detection. CO2 concentration and air filtration were independently associated with their concentration. Occupancy, sampling time, mask wearing, vocalization, temperature, humidity and mechanical ventilation were not significant. Our results support the importance of ventilation and air filtration to reduce transmission.

Longitudinal analysis of serum neutralization of SARS-CoV-2 Omicron BA.2, BA.4 and BA.5 in patients receiving monoclonal antibodies (preprint)

The emergence of novel Omicron lineages, such as BA.5, may impact the therapeutic efficacy of anti-SARS-CoV-2 neutralizing monoclonal antibodies (mAbs). Here, we evaluated the neutralization and ADCC activity of 6 therapeutic mAbs against Delta, BA.2, BA.4 and BA.5 isolates. The Omicron sub-variants escaped most of the antibodies but remained sensitive to Bebtelovimab and Cilgavimab. Consistent with their shared spike sequence, BA.4 and BA.5 displayed identical neutralization profiles. Sotrovimab was the most efficient at eliciting ADCC. We also analyzed 121 sera from 40 immunocompromised individuals up to 6 months after infusion of 1200 mg of Ronapreve (Imdevimab + Casirivimab), and 300 or 600 mg of Evusheld (Cilgavimab + Tixagevimab). Sera from Ronapreve-treated individuals did not neutralize Omicron subvariants. Evusheld-treated individuals neutralized BA.2 and BA.5, but titers were reduced by 41- and 130-fold, respectively, compared to Delta. A longitudinal evaluation of sera from Evusheld-treated patients revealed a slow decay of mAb levels and neutralization. The decline was more rapid against BA.5. Our data shed light on the antiviral activities of therapeutic mAbs and the duration of effectiveness of Evusheld pre-exposure prophylaxis.

Environmental circulation of adenovirus 40/41 and SARS-CoV-2 in the context of the emergence of acute hepatitis of unknown origin (preprint)

The recent surge of hepatitis of unknown origin in children is hypothesized to be caused by adenovirus 41 and/or SARS-CoV-2 infections. A relatively high proportion of patients testing positive for these viruses concomitantly with the development of acute hepatitis supports this hypothesis. To formally incriminate these viral infections as causative agents of hepatitis, both a plausible physiopathological pathway and supporting epidemiological dynamics in the community need demonstration. In this study, we measured the level of circulation of adenovirus 40/41 and SARS-CoV-2 in the general population of the city of Leuven in Belgium using wastewater monitoring between December 2020 and May 2022 and indoor air sampling in day care centers between November 2021 and May 2022. We also retrospectively analyzed medical records of 12.672 children attending a tertiary hospital draining the same region between January 2019 and April 2022. Our results demonstrate a recent but modest increase in hepatitis of unknown origin concomitant with a surge of circulating adenovirus 41 and SARS-CoV-2 in the general population, including in children under 5.

Early SARS-CoV-2 reinfections within 60 days highlight the need to consider antigenic variations together with duration of immunity in defining retesting policies (preprint)

The emergence of the SARS-CoV-2 Omicron variant, characterized by a significant antigenic diversity compared to the previous Delta variant, had led to a decrease in antibody efficacy in both convalescent and vaccinees sera resulting in high number of reinfections and breakthrough cases worldwide. However, to date, reinfections are defined by the ECDC as two positive tests >/= 60 days apart, influencing retesting policies after an initial positive test in several European countries. In our manuscript, we illustrate by a clinical case supplemental by epidemiological data that early reinfections do occur within 60 days especially in young, unvaccinated individuals. In older patient groups, unvaccinated and patients with a basic vaccination scheme are more vulnerable to reinfections compared to patients who received a first booster vaccine. For this reason, we consider that the duration of protection offered by a previous infection should be reconsidered, in particular when a shift between consecutive SARS-CoV-2 variants occurs.

Risk factors for SARS-CoV-2 transmission in student residences: a case-ascertained study in Leuven, Belgium from October 2020 until May 2021 (preprint)

Background Student residences are at high risk for rapid COVID-transmission due to crowding and frequent close contact. Aim We aimed to investigate the overall secondary attack rates (SAR) in student residences and to discern risk factors for higher transmission in order to improve the evidence base for screening efforts and preventive measures. Methods In this retrospective case-ascertained study, we analysed data from student residences screened in Leuven, Belgium between October 2020 and May 2021, following detection of a COVID-19 case in the residence. We investigated the impact on the SAR in the living units screened of delay-time until follow-up, shared use of kitchen or sanitary facilities, presence of an external infection source and occurrence of social gatherings attended by the index case. Results We included 200 residence units, representing 2326 screened residents, of which 68 units showed secondary transmission. The overall SAR was estimated at 0.0813 (95%CI 0.0705-0.0936). Both sharing sanitary facilities (p=0.04) and social gatherings attended by the index case (p=0.033) significantly impacted SAR, which increased from 3% to 13% when both risk factors were present compared to absent. Conclusions We identify risk factors which should be considered when selecting students for screening during an outbreak of COVID-19 in student residences to improve comprehensiveness and proportionality of testing. The identified risk factors improve the evidence base for preventive measures aimed at limiting social gatherings and improving ventilation of shared spaces in outbreak-prone settings. Lastly, they should be considered when designing student accommodation and other shared households.

Comprehensive immunovirological and environmental screening reveals risk factors for fatal COVID-19 during post-vaccination nursing home outbreaks (preprint)

COVID-19 vaccination has resulted in excellent protection against fatal disease, including in the elderly. However, risk factors for post-vaccination fatal COVID-19 are largely unknown. We comprehensively studied three large nursing home outbreaks (20-35% fatal cases) by combining SARS-CoV-2 aerosol monitoring, whole-genome phylogenetic analysis, and immunovirological profiling by digital nCounter transcriptomics. Phylogenetic investigations indicated each outbreak stemmed from a single introduction event, though with different variants (Delta, Gamma, and Mu). SARS-CoV-2 was detected in aerosol samples up to 52 days after the initial infection. Combining demographic, immune and viral parameters, the best predictive models for mortality comprised IFNB1 or age, viral ORF7a and ACE2 receptor transcripts. Comparison with published pre-vaccine fatal COVID-19 signatures and reanalysis of single-cell RNAseq data highlights the unique immune signature in post-vaccine fatal COVID-19 outbreaks. A multi-layered strategy including environmental sampling, immunomonitoring, and early antiviral therapy should be considered to prevent post-vaccination COVID-19 mortality in nursing homes.

Seroneutralization of Omicron BA.1 and BA.2 in patients receiving anti-SARS-CoV-2 monoclonal antibodies. (preprint)

The SARS-CoV-2 Omicron BA.1 variant has been supplanted in many countries by the BA.2 sub-lineage. BA.2 differs from BA.1 by about 21 mutations in its spike. Human anti-spike monoclonalantibodies(mAbs)areusedforpreventionortreatmentofCOVID-19. However, the capacity of therapeutic mAbs to neutralize BA.1 and BA.2 remains poorly characterized. Here, we first compared the sensitivity of BA.1 and BA.2 to neutralization by 9 therapeutic mAbs. In contrast to BA.1, BA.2 was sensitive to Cilgavimab, partly inhibited by Imdevimab and resistant to Adintrevimab and Sotrovimab. Two combinations of mAbs, Ronapreve (Casirivimab + Imdevimab) and Evusheld (Cilgavimab + Tixagevimab), are indicated as a pre-exposure prophylaxis in immunocompromised persons at risk of severe disease. We analyzed sera from 29 such individuals, up to one month after administration of Ronapreve and/or Evusheld. After treatment, all individuals displayed elevated antibody levels in their sera and neutralized Delta with high titers. Ronapreve recipients did not neutralize BA.1 and weakly impaired BA.2. With Evusheld, neutralization of BA.1 and BA.2 was detected in 19 and 29 out of 29 patients, respectively. As compared to Delta, titers were more severely decreased against BA.1 (344-fold) than BA.2 (9-fold). We further report 4 breakthrough Omicron infections among the 29 participants. Therefore, BA.1 and BA.2 exhibit noticeable differences in their sensitivity to therapeutic mAbs. Anti-Omicron activity of Ronapreve, and to a lesser extent that of Evusheld, is reduced in patients sera, a phenomenon associated with decreased clinical efficacy.

Observed serial intervals of SARS-CoV-2 for the Omicron and Delta varaints in Belgium based on contact tracing data, 19 November to 31 December 2021 (preprint)

The SARS-CoV-2 Omicron BA.1 variant is rapidly spreading worldwide, possibly outcompeting the Delta strain. We investigated the empirical serial interval for both variants using contact tracing data. Overall, we observed a shorter serial interval for Omicron compared to Delta, suggesting faster transmission. Furthermore, results indicate a relation between the empirical serial interval and the vaccination status for both the Omicron and the Delta variant. Consequently, with the progression of the vaccination campaign, the reasons for and extent of dominance of Omicron over Delta may need further assessment.

Remdesivir, Molnupiravir and Nirmatrelvir remain active against SARS-CoV-2 Omicron and other variants of concern. (preprint)

The in vitro effect of GS-441524, remdesivir, EIDD-1931, molnupiravir and nirmatrelvir against the various SARS-CoV-2 VOCs, including Omicron, was determined. VeroE6-GFP cells were pre-treated overnight with serial dilutions of the compounds before infection. The number of fluorescent pixels of GFP signal, determined by high-content imaging on day 4 post-infection, was used as read-out, and the EC50 of each compound on a viral isolate of each VOC was calculated. These experiments were performed in the presence of the Pgp-inhibitor CP-100356 in order to limit compound efflux. A SARS-CoV-2 strain grown from the first Belgian patient sample was used as ancestral strain. All the other isolates were obtained from patients in Belgium as well. Our results indicate that GS-441524, remdesivir, EIDD-1931, molnupiravir and nirmatrelvir retain their activity against the VOCs Alpha, Beta, Gamma, Delta and Omicron. This is in accordance with the observation that the target proteins of these antivirals are highly conserved.

The omicron (B.1.1.529) SARS-CoV-2 variant of concern does not readily infect Syrian hamsters (preprint)

The emergence of SARS-CoV-2 variants of concern (VoCs) has exacerbated the COVID-19 pandemic. End of November 2021, a new SARS-CoV-2 variant namely the omicron (B.1.1.529) emerged. Since this omicron variant is heavily mutated in the spike protein, WHO classified this variant as the 5th variant of concern (VoC). We previously demonstrated that the other SARS-CoV-2 VoCs replicate efficiently in Syrian hamsters, alike also the ancestral strains. We here wanted to explore the infectivity of the omicron variant in comparison to the ancestral D614G strain. Strikingly, in hamsters that had been infected with the omicron variant, a 3 log10 lower viral RNA load was detected in the lungs as compared to animals infected with D614G and no infectious virus was detectable in this organ. Moreover, histopathological examination of the lungs from omicron-infecetd hamsters revealed no signs of peri-bronchial inflammation or bronchopneumonia. Further experiments are needed to determine whether the omicron VoC replicates possibly more efficiently in the upper respiratory tract of hamsters than in their lungs.

The role of airborne transmission in a large single source outbreak of SARS-CoV-2 in a Belgian nursing home in 2020 (preprint)

Abstract Objectives: To better understand the conditions which have led to one of the largest COVID-19 outbreaks in Belgian nursing homes in 2020. Setting: A nursing home in Flanders, Belgium, which experienced a massive outbreak of COVID-19 after a cultural event. An external volunteer who dressed as a legendary figure visited consecutively the 4 living units and tested positive for SARS-CoV-2 the next day. Within days, residents started to display symptoms and the outbreak spread rapidly within the nursing home. Methods: We interviewed key informants and collected standardized data from all residents retrospectively. A batch of 115 positive samples with a Ct value of <37 by qRT-PCR were analysed using whole-genome sequencing. Six months after the outbreak, ventilation assessment of gathering rooms in the nursing home was done using a tracer gas test with calibrated CO2 sensors. Results: Timeline of diagnoses and symptom onsets clearly pointed to the cultural event as the start of the outbreak, with the volunteer as index case. The genotyping of positive samples depicted the presence of one large cluster, suggesting a single source outbreak. The global attack rate among residents was 77% with a significant association between infection and presence at the event. Known risk factors such as short distance to or physical contact with the volunteer, and wearing of a mask during the event were not associated with early infection. The ventilation assessment showed a high background average CO2 level in four main rooms varying from 657 ppm to 846 ppm. Conclusions: Our investigation shows a rapid and widespread single source outbreak of SARS-CoV-2 in a nursing home, in which airborne transmission was the most plausible explanation for the massive intra-facility spread. Our results underscore the importance of ventilation and air quality for the prevention of future outbreaks in closed facilities.

Molecular Detection of SARS-Cov-2 in Exhaled Breath Using a Portable Sampler (preprint)

The SARS-CoV-2 pandemic has highlighted the need for improved technologies to help control the spread of contagious pathogens. While rapid point-of-need testing plays a key role in strategies to rapidly identify and isolate infectious patients, a cornerstone for any disease-control strategy, current test approaches have significant shortcomings related to assay limitations and sample type. Direct quantification of viral shedding in exhaled particles may offer a better rapid testing approach, since SARS-CoV-2 is believed to spread mainly by aerosols. It potentially measures contagiousness directly, the sample is easy to obtain, its production can be standardized between patients, and the limited sample volume lends itself to a fast and sensitive analysis. In view of these benefits, we developed and tested an approach where exhaled particles are efficiently sampled using inertial impaction in a micromachined silicon chip, followed by an in-situ RT-qPCR molecular assay to detect SARS-CoV-2 shedding. We demonstrate that sampling subjects using a one-minute breathing protocol, yields sufficient viral RNA to detect infections with a sensitivity comparable to standard sampling methods. A longitudinal study revealed clear differences in the temporal dynamics of viral load for nasopharyngeal swab, saliva, breath, and antigen tests. Overall, after an infection, the breath-based test is the first to consistently report a negative result, putatively signaling the end of contagiousness and further emphasizing the potential of this tool to help manage the spread of airborne respiratory infections.

Considerable escape of SARS-CoV-2 variant Omicron to antibody neutralization (preprint)

The SARS-CoV-2 Omicron variant was first identified in November 2021 in Botswana and South Africa 1,2 . It has in the meantime spread to many countries and is expected to rapidly become dominant worldwide. The lineage is characterized by the presence of about 32 mutations in the Spike, located mostly in the N-terminal domain (NTD) and the receptor binding domain (RBD), which may enhance viral fitness and allow antibody evasion. Here, we isolated an infectious Omicron virus in Belgium, from a traveller returning from Egypt. We examined its sensitivity to 9 monoclonal antibodies (mAbs) clinically approved or in development 3 , and to antibodies present in 90 sera from COVID-19 vaccine recipients or convalescent individuals. Omicron was totally or partially resistant to neutralization by all mAbs tested. Sera from Pfizer or AstraZeneca vaccine recipients, sampled 5 months after complete vaccination, barely inhibited Omicron. Sera from COVID-19 convalescent patients collected 6 or 12 months post symptoms displayed low or no neutralizing activity against Omicron. Administration of a booster Pfizer dose as well as vaccination of previously infected individuals generated an anti-Omicron neutralizing response, with titers 5 to 31 fold lower against Omicron than against Delta. Thus, Omicron escapes most therapeutic monoclonal antibodies and to a large extent vaccine-elicited antibodies.

Integrated PCR testing and extended window contact tracing system for COVID-19 to improve comprehensiveness and speed (preprint)

Testing and contact tracing are standard tools for controlling the spread of COVID-19 1 . Their effectiveness hinges on a sequence of processes encompassing testing coverage and timeliness, testing quality and speed of reporting, contact tracing speed and comprehensiveness and compliance with advice given 2–6 . We optimized this sequence of processes in the context of a public health program targeting around 33,000 higher education students through a combination of low barrier PCR testing with rapid turn-around-time, close integration of testing and tracing teams and IT infrastructure, community engagement and the implementation of bidirectional contact tracing by extending the contact tracing window from 2 to 7 days before symptom onset or test of the index case. We anticipate this combined intervention to help improve epidemic control.

Empirical evidence on the efficiency of backward contact tracing in COVID-19 (preprint)

Standard contact tracing practice for COVID-19 is to identify persons exposed to an infected person during the contagious period, assumed to start two days before symptom onset or diagnosis. In the first large cohort study on backward contact tracing for COVID-19, we extended the contact tracing window by 5 days, aiming to identify the source of the infection and persons infected by the same source. The risk of infection amongst these additional contacts was similar to contacts exposed during the standard tracing window and significantly higher than symptomatic individuals in a control group, leading to 42% more cases identified through contact tracing. Compared to standard practice, backward traced contacts required fewer tests and shorter quarantine, but if infected they were identified later in their infectious cycle. Our results support implementing backward contact tracing when rigorous suppression of viral transmission is warranted.

Genetic diversity and evolution of SARS-CoV-2 in Belgium during the first wave outbreak (preprint)

SARS-CoV-2, the causative agent of COVID-19 was first detected in Belgium on 3rd February 2020, albeit the first epidemiological wave started in March and ended in June 2020. One year after the first epidemiological wave hit the country data analyses reveled the temporal and variant distribution of SARS-CoV-2 and its implication with Belgian epidemiological measures. In this study, 766 complete SARS-CoV-2 genomes of samples originating from the first epidemiological were sequenced to characterize the temporal and geographic distribution of the COVID-19 pandemic in Belgium through phylogenetic and variant analysis. Our analysis reveals the presence of the major circulating SARS-CoV-2 clades (G, GH and GR) and lineages circulating in Belgium at that time. Moreover, it contextualizes the density of SARS-CoV-2 cases over time with non-intervention measures taken to prevent the spread of SARS-CoV-2 in Belgium, specific international case imports and the functional implications of the most representative non-synonymous mutations present in Belgium between February to June 2020.