Mild and moderate COVID-19 during Alpha, Delta and Omikron pandemic waves in urban Maputo, Mozambique, December 2020-March 2022: a population-based surveillance study (preprint)

In sub-Saharan Africa, reported COVID-19 numbers have been lower than anticipated, even when considering populations younger age. The extent to which risk factors, established in industrialised countries, impact the risk of infection and of disease in populations in sub-Saharan Africa, remains unclear. We estimated the incidence of mild and moderate COVID-19 in urban Mozambique and analysed factors associated with infection and disease in a population-based surveillance study. During December 2020-March 2022, households of a population cohort in Polana Canico, Maputo, Mozambique, were contacted biweekly. Residents reporting any respiratory sign, anosmia, or ageusia, were asked to self-administer a nasal swab, for SARS-CoV-2 PCR testing. Of a subset of 1400 participants, dried blood spots were repeatedly collected three-monthly from finger pricks at home. Antibodies against SARS-CoV-2 spike glycoprotein and nucleocapsid protein were detected using an in-house developed multiplex antibody assay. We estimated the incidence of respiratory illness and COVID-19, and SARS-CoV-2 seroprevalence. We used Cox regression models, adjusting for age and sex, to identify factors associated with first symptomatic COVID-19 and with SARS-CoV-2 sero-conversion in the first six months. During 11925 household visits in 1561 households, covering 6049 participants (median 21 years, 54.8% female, 7.3% disclosed HIV positive), 1895.9 person-years were followed up. Per 1000 person-years, 364.5 (95%CI 352.8-376.1) respiratory illness episodes of which 72.2 (95%CI 60.6-83.9) COVID-19 confirmed, were reported. Of 1412 participants, 2185 blood samples were tested (median 30.6 years, 55.2% female). Sero-prevalence rose from 4.8% (95%CI 1.1-8.6%) in December 2020 to 34.7% (95%CI 20.2-49.3%) in June 2021, when 3.0% were vaccinated. Increasing age (strong gradient in hazard ratio, HR, up to 15.70 in [≥]70 year olds, 95%CI 3.74-65.97), leukaemia, chronic lung disease, hypertension, and overweight increased risk of COVID-19. We found no increased risk of COVID-19 in people with HIV or tuberculosis. Risk of COVID-19 was lower among residents in the lowest socio-economic quintile (HR 0.16, 95%CI 0.04-0.64), with no or limited handwashing facilities, and who shared bedrooms (HR 0.42, 95%CI 0.25-0.72). Older age also increased the risk of SARS-CoV-2 seroconversion (HR 1.57 in 60-69 year olds, 95%CI 1.03-2.39). We found no associations between SARS-CoV-2 infection risk and socio-economic, behavioural factors and comorbidities. Active surveillance in an urban population cohort confirmed frequent COVID-19 underreporting, yet indicated that the large majority of cases were mild and non-febrile. In contrast to industrialised countries, deprivation did not increase the risk of infection nor disease.

SARS-CoV-2 surveillance between 2020 and 2021 of all mammalian species in two Flemish zoos (Antwerp Zoo and Planckendael Zoo) (preprint)

The COVID-19 pandemic has led to millions of human infections and deaths worldwide. Several other mammal species are also susceptible to SARS-CoV-2, and multiple instances of transmission from humans to pets, farmed mink, wildlife and zoo animals have been recorded. We conducted a systematic surveillance of SARS-CoV-2 in all mammal species in two zoos in Belgium between September and December 2020 and July 2021 in four sessions, and a targeted surveillance of selected mammal enclosures following SARS-CoV-2 infection in hippos in December 2021. A total of 1523 faecal samples were tested for SARS-CoV-2 via real-time PCR. None of the samples tested positive for SARS-CoV-2. Additional surrogate virus neutralization tests conducted on 50 routinely collected serum samples during the same period were all negative. This study is a first to our knowledge to conduct active SARS-CoV-2 surveillance for several months in all mammal species of a zoo. We conclude that at the time of our investigation, none of the screened animals were excreting SARS-CoV-2.

Evaluation of a throat spray with lactobacilli in COVID-19 outpatients in a randomized, double-blind, placebo-controlled trial for symptom and viral load reduction (preprint)

Objectives: Primary care urgently needs treatments for COVID-19 patients because current options are limited, while these patients account for more than 90% of the people infected with SARS-CoV-2. Methods: We evaluated a throat spray containing three Lactobacillaceae strains with broad antiviral properties in a randomized double-blind placebo-controlled trial. Seventy-eight eligible COVID-19 patients were randomized to verum (n=41) and placebo (n=37) within 96 hours of positive PCR-based SARS-CoV-2 diagnosis and per-protocol analysis was performed. Symptoms and severity were reported daily via an online diary. Combined nose-throat swabs and dried blood spots were collected at regular time points in the study. Results: The daily reported symptoms were highly variable, with no added benefit for symptom resolution in the verum group. Specific monitoring of the applied lactobacilli strains showed that they were detectable via microbiome (27%) and qPCR analysis (82%) of the verum group. Their relative abundances were also negatively correlated with the acute symptom score. At the end of the trial, a trend towards lower SARS-CoV-2 viral loads was observed for the verum group (2/30, 6.7% positive) compared to the placebo group (7/27, 26% positive) (p = 0.07). Conclusions: Despite a trend towards lower SARS-CoV-2 viral loads at the end of the trial and a negative correlation between relative abundances of the applied lactobacilli in the microbiome and acute symptoms, we did not observe a significant effect on overall symptom score for the verum group. This suggests that studies with earlier application of the spray in larger study populations are needed to further assess application potential.

mRNA-1273 vaccine (Moderna): a better option than BNT162b2 (Pfizer) in kidney transplant recipients and dialysis patients? (preprint)

In this study of the humoral immune response after the first dose of SARS-CoV-2 mRNA vaccine, low seroconversion rates were noted in both kidney transplant recipients and dialysis patients. However, vaccination with the mRNA-1273 vaccine (Moderna) resulted in both higher seroconversion rates and mean antibody titers compared to BNT162b2 (Pfizer).

Prospective SARS-CoV-2 cohort study among general practitioners during the second COVID-19 wave in Flanders, Belgium (preprint)

Primary health care providers (PHCPs), especially general practitioners (GPs) are essential to organise health care efficiently. During the COVID-19 pandemic, they also keep the pressure off hospitals. PHCPs are assumed to be at high risk of a COVID-19 infection, as they are exposed to a large portion of the population (usually with less personal protective equipment than other frontline health care workers(HCWs)). Nevertheless, previous seroprevalence studies focussed on the general population or HCWs in hospital settings, rather than PHCPs. The aim of this study was to determine the seroprevalence of PHCPs after the first and during the second SARS-CoV-2 wave in Flanders (Belgium) and compare it to the seroprevalence in the general population (blood donors). A prospective cohort of PHCPs, mainly GPs (n=698) was screened for IgG antibodies against SARS-CoV-2 at five different time-points (June-December 2020). The dried blood spots they produced were analysed using a Luminex multiplex immunoassay. The seroprevalence of PHCPs remained stable between June and September 2020 (4.6-5.0%), but increased significantly from October to December (8.1-13.4%) 2020. The seroprevalence of PHCPs was not significantly higher than the seroprevalence of the blood donors at the end of December 2020. In conclusion, the sharp increase in seroprevalence during the second COVID-19 wave in Flanders shows that PHCPs were more at risk during the second wave compared to the first one. However, the increase was in line with the general population suggesting that PHCPs mainly got infected in their private settings.

SARS-CoV-2 surveillance in Norway rats (Rattus norvergicus) from Antwerp sewer system, Belgium (preprint)

Background: SARS-CoV-2 human-to-animal transmission can lead to the establishment of novel reservoirs and the evolution of new variants with the potential to start new outbreaks in humans. Aim: We tested Norway rats inhabiting the sewer system of Antwerp, Belgium, for the presence of SARS-CoV-2 following a local COVID-19 epidemic peak. In addition, we discuss the use and interpretation of SARS-CoV-2 serological tests on non-human samples. Methods: Between November and December 2020, Norway rat oral swabs, feces and tissues from the sewer system of Antwerp were collected to be tested by RT-qPCR for the presence of SARS-CoV-2. Serum samples were screened for the presence of anti-SARS-CoV-2 IgG antibodies using a Luminex microsphere immunoassay (MIA). Samples considered positive were then checked for neutralizing antibodies using a conventional viral neutralization test (cVNT). Results: The serum of 35 rats was tested by MIA showing 3 potentially positive sera that were later shown to be negative by cVNT. All tissue samples of 39 rats analyzed tested negative for SARS-CoV-2 RNA. Conclusion: This is the first study that evaluates SARS-CoV-2 infection in urban rats. We can conclude that the sample of 39 rats had never been infected with SARS-CoV-2. We show that diagnostic serology tests can give misleading results when applied on non-human samples. SARS-CoV-2 monitoring activities should continue due to the emergence of new variants prone to infect Muridae rodents.

Evaluation of a surrogate virus neutralization test for high-throughput serosurveillance of SARS-CoV-2 (preprint)

High-throughput serological tests that can detect neutralizing antibodies against SARS-CoV-2 are desirable for serosurveillance and vaccine efficacy evaluation. Although the conventional neutralization test (cVNT) remains the gold standard to confirm the presence of neutralizing antibodies in sera, the test is too labour-intensive for massive screening programs and less reproducible as live virus and cell culture is involved. Here, we performed an independent evaluation of a commercially available surrogate virus neutralization test (sVNT, GenScript cPass) that can be done without biosafety level 3 containment in less than 2 hours. When using the cVNT and a Luminex multiplex immunoassay (MIA) as reference, the sVNT obtained a sensitivity of 94% (CI 90-96%) on a panel of 317 immune sera that were obtained from hospitalized and mild COVID-19 cases from Belgium and a sensitivity of 89% (CI 81-93%) on a panel of 184 healthcare workers from the Democratic Republic of Congo. We also found strong antibody titer correlations (rs>0.8) among the different techniques used. In conclusion, our evaluation suggests that the sVNT could be a powerful tool to monitor/detect neutralising antibodies in cohort and population studies. The technique could be especially useful for vaccine evaluation studies in sub-Saharan Africa where the basic infrastructure to perform cVNTs is lacking.

Symptomatic SARS-CoV-2 re-infection of a health care worker in a Belgian nosocomial outbreak despite primary neutralizing antibody response. (preprint)

Background. It is currently unclear whether SARS-CoV-2 re-infection will remain a rare event, only occurring in individuals who fail to mount an effective immune response, or whether it will occur more frequently when humoral immunity wanes following primary infection. Methods. A case of re-infection was observed in a Belgian nosocomial outbreak involving 3 patients and 2 health care workers. To distinguish re-infection from persistent infection and detect potential transmission clusters, whole genome sequencing was performed on nasopharyngeal swabs of all individuals including the re-infection case's first episode. IgA, IgM, and IgG and neutralizing antibody responses were quantified in serum of all individuals, and viral infectiousness was measured in the swabs of the reinfection case. Results. Re-infection was confirmed in a young, immunocompetent health care worker as viral genomes derived from the first and second episode belonged to different SARS-CoV-2 clades. The symptomatic re-infection occurred after an interval of 185 days, despite the development of an effective humoral immune response following symptomatic primary infection. The second episode, however, was milder and characterized by a fast rise in serum IgG and neutralizing antibodies. Although contact tracing and virus culture remained inconclusive, the health care worker formed a transmission cluster with 3 patients and showed evidence of virus replication but not of neutralizing antibodies in her nasopharyngeal swabs. Conclusion. If this case is representative of most Covid-19 patients, long-lived protective immunity against SARS-CoV-2 might not be likely.

Evaluating SARS-CoV-2 spike and nucleocapsid proteins as targets for IgG antibody detection in severe and mild COVID-19 cases using a Luminex bead-based assay (preprint)

Large-scale serosurveillance of severe acute respiratory syndrome coronavirus type 2 (SARS- CoV-2) will only be possible if serological tests are sufficiently reliable, rapid and inexpensive. Current assays are either labour-intensive and require specialised facilities (e.g. virus neutralization assays), or expensive with suboptimal specificity (e.g. commercial ELISAs). Bead-based assays offer a cost-effective alternative and allow for multiplexing to test for antibodies of other pathogens. Here, we compare the performance of four antigens for the detection of SARS-CoV-2 specific IgG antibodies in a panel of sera that includes both severe (n=40) and mild (n=52) cases, using a neutralization and a Luminex bead-based assay. While we show that neutralising antibody levels are significantly lower in mild than in severe cases, we demonstrate that a combination of recombinant nucleocapsid protein (NP), receptor- binding domain (RBD) and the whole spike protein (S1S2) results in a highly sensitive (96%) and specific (99%) bead-based assay that can detect IgG antibodies in both groups. Although S1-specific IgG levels correlate most strongly with neutralizing antibody levels, they fall below the detection threshold in 10% of the cases in our Luminex assay. In conclusion, our data supports the use of RBD, NP and S1S2 for the development of SARS-CoV-2 serological bead- based assays. Finally, we argue that low antibody levels in mild/asymptomatic cases might complicate the epidemiological assessment of large-scale surveillance studies.