key: cord-0757880-4h4phglo
authors: Meinus, C.; Singer, R.; Nandi, B.; Jagot, O.; Becker-Ziaja, B.; Karo, B.; Mvula, B.; Jansen, A.; Baumann, J.; Schultz, A.
title: SARS-CoV-2 prevalence and immunity: a hospital-based study from Malawi
date: 2021-12-17
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.12.336
sha: 5d56f6d05732e0fb4ccedd4562dba3cc48c898a8
doc_id: 757880
cord_uid: 4h4phglo

Background Viral transmission and disease dynamics of COVID-19 in sub-Saharan Africa are still not well understood. This study aims to provide further insight into the epidemiology of COVID-19 in Malawi by estimating the SARS-CoV-2 prevalence and immunity after SARS-CoV-2 infection in a hospital-based setting. Methods We conducted a hospital-based, convenience sampling, cross-sectional survey for SARS-CoV-2 in Lilongwe, Malawi. Participants answered a questionnaire and were tested for SARS-CoV-2 infection using ELISA and RT-PCR. To estimate immunity, a surrogate virus neutralization test was performed in all seropositive samples. Poisson regression was performed to assess the association of SARS-CoV-2 point prevalence and demographic and behavioral variables. Findings A total of 930 participants were included in the study. We found a combined point prevalence of 10.1% (95% CI 8.4-12.3). Separately analyzed, the RT-PCR positivity was 2.0 % (95% CI 1.2-3.4), and the seropositivity was 9.3% (CI 95% 7.6-11.4). Of the tested seropositive samples, 90.1% (95% CI 81.2%-95.1%) were also tested positive in the surrogate virus neutralization assay. We found a high rate of asymptomatic SARS-CoV-2 infection (45.7%). SARS-CoV-2 point prevalence was significantly associated with being health care worker (PR 1.7; 95% CI 1.1-2.5). Interpretation Our study suggests that official data underestimate the true magnitude of COVID-19 transmission. Performance of sVNT to estimate immunity in Malawi is feasible and revealed a considerable rate of post-infection immunity in our cohort. Subclinical infection and transmission are probably a game-changer with regards to surveillance, mitigation and vaccination strategies. Funding GIZ German Development and RKI Germany supported this study.

Background Viral transmission and disease dynamics of COVID-19 in sub-Saharan Africa are still not well understood. This study aims to provide further insight into the epidemiology of COVID-19 in Malawi by estimating the SARS-CoV-2 prevalence and immunity after SARS-CoV-2 infection in a hospital-based setting.

We conducted a hospital-based, convenience sampling, cross-sectional survey for SARS-CoV-2 in Lilongwe, Malawi. Participants answered a questionnaire and were tested for SARS-CoV-2 infection using ELISA and RT-PCR. To estimate immunity, a surrogate virus neutralization test was performed in all seropositive samples. Poisson regression was performed to assess the association of SARS-CoV-2 point prevalence and demographic and behavioral variables.

A total of 930 participants were included in the study. We found a combined point prevalence of 10.1% (95% CI 8.4-12.3). Separately analyzed, the RT-PCR positivity was 2.0 % (95% CI 1.2-3.4), and the seropositivity was 9.3% (CI 95% 7.6-11.4). Of the tested seropositive samples, 90.1% (95% CI 81.2%-95.1%) were also tested positive in the surrogate virus neutralization assay. We found a high rate of asymptomatic SARS-CoV-2 infection (45.7%). SARS-CoV-2 point prevalence was significantly associated with being health care worker (PR 1.7; 95% CI 1.1-2.5).

Our study suggests that official data underestimate the true magnitude of transmission. Performance of sVNT to estimate immunity in Malawi is feasible and revealed a considerable rate of post-infection immunity in our cohort. Subclinical

Sub-Saharan Africa (sSA) countries were expected to be highly vulnerable to COVID-19 due to fragile health care systems and potentially vulnerable populations (Gilbert et al., 2020) . However, according to official surveillance data, prevalence and mortality continue to be comparably low. Up to date, comparing Malawi to the USA, there is an about 25-fold lower number of registered cases and deaths, raising questions about the factual prevalence of the disease and possible preexisting immunity (https://covid19.who.int/). Serological surveillance has been implemented to estimate the true SARS-CoV-2 prevalence, however, predominantly in high-income countries (Bobrovitz et al., 2021) . Currently, there is an increasing but still small number of reported estimates of SARS-CoV-2 prevalence in African countries. In a recent meta-analysis from Chisale et al., 23 studies from 16 countries were included (Chisale et al., 2021) . In the published studies, there is a wide range of estimated prevalence (0%-63%) that reflects actual differences in regional prevalence and differences in selection criteria (Chisale et al., 2021) . In general, SARS-CoV-2 exposure is reported to be more extensive than indicated by case-based surveillance, suggesting that the total number of cases is likely largely underestimated.

Regarding further waves of the COVID-19 pandemic and the worldwide initiation of vaccination campaigns (Salyer et al., 2021) , not only disease prevalence but also acquired immunity after infection is of utmost interest for public health decisions.

Recently, a surrogate virus neutralization test has been established as an alternative to the gold standard conventional virus neutralization test to detect neutralizing antibodies without using live viruses (Tan et al., 2020) . It is a high-throughput serological test that mimics well virus-host interaction in an enzyme-linked immunoabsorbent assay (ELISA) plate, especially useful in settings with limited laboratory infrastructure (Marien et al., 2021) .

Malawi is a low-income country in south-eastern Africa with a population of approximately 17.6 million people. It is characterized by a predominantly rural (84%) and young (median age 17 years) population (http://www.nsomalawi.mw).

Multidimensional poverty, other pre-existing health-priorities such as malaria, malnutrition, tuberculosis and HIV, and deficiencies in the health infrastructure (Sonenthal et al., 2020) are considered key contextual contributors to COVID-19 severity and mortality. Given these circumstances, a revised estimate projected 16 million infections and 50,000 COVID-19 deaths over one year in an unmitigated scenario in Malawi (Green et al., 2020) .

On March 20 th , 2020, COVID-19 was declared a national disaster in Malawi, and a multisectoral approach to mitigate the spread of the disease in consideration of competing health priorities and the socio-economic context was initiated (Mzumara et al., 2021) . However, the current political mistrust and gatherings hampered mitigation strategies during anti-government demonstrations and mass political campaigns (Mzumara et al., 2021 , Nyasulu et al., 2021 . Community belief of unjustified national regulations and low-risk perception related to COVID-19 negatively affected adherence to prevention measures (Banda et al., 2021) . In addition, an already challenged health sector with limited financial and personal resources was further challenged by a strike of health providers demanding the provision of adequate personal protective equipment and staffing level (Nyasulu et al., 2021) . In 2020, the recorded incidence of SARS-CoV-2 infections peaked at the end of July and slowly descended until the beginning of September. As of August 24th, 2020, Malawi had cumulatively recorded 5,419 cases, including 169 deaths, much in contrast to the predicted or modulated numbers (https://www.malawipublichealth.org/). The study aimed to provide further insight into the epidemiology of COVID-19 in Malawi by estimating SARS-CoV-2 prevalence and immunity after SARS-CoV-2 exposure in a convenience sample from healthcare workers (HCWs) and non-HCWs in a hospitalbased setting.

Between August 24th and September 3rd, 2020, we conducted a hospital-based, cross-sectional survey for SARS-CoV-2 at Kamuzu Central Hospital in Lilongwe, Central Region, Malawi. Kamuzu Central Hospital is a tertiary level hospital that provides referral services for five district hospitals. The population in the direct community numbers 1.6 million but the hospital also serves the population across the wider region.

The study population included participants working or presenting at the facility.

HCWs were defined as health care professionals, allied health workers and administration; non-HCWs were defined as patients, guardians, and relatives.

Participants were selected through convenience sampling, aiming at an equal distribution of HCWs and non-HCWs. Participants under 18 years and those who presented with contraindications to venipuncture or unwilling or unable to consent were excluded. Written informed consent (in Chichewa or English) was obtained from all participants. Witnesses assisted participants who were not able to read in English or Chichewa.

The collection of serum samples and nasopharyngeal swabs was carried out following a standard protocol approved by the College of Medicine Research and Ethics Committee Malawi. Participants answered a questionnaire that included sociodemographic information, possible exposure with SARS-CoV-2, medical and travel history. Anonymized data entry was performed on a tablet using an electronic case report form. Serum samples were collected by venipuncture and were tested for SARS-CoV-2 antibodies by ELISA. Participants were also offered testing for acute SARS-CoV-2 infection by nucleic acid testing.

Serum samples were tested in a single replicate for the presence of SARS-CoV-2-IgG antibodies using a commercially available ELISA from Euroimmun (Lübeck, Germany). Assays were performed according to the manufacturer's guidelines. The microplate wells are coated with recombinant S1 structural protein, and the assay detects SARS-CoV2-IgG antibodies against the viral spike protein. Results were evaluated semi-quantitatively by calculating a ratio of the extinction of the control or patient sample over the extinction of the calibrator. According to the manufacturer's guidelines, the ratio was interpreted as following < 0.8 negative, ≥ 0.8 ≤ 1.1 borderline, > 1.1 positive. Borderline results were re-run in a single replicate and considered positive, negative, or borderline accordingly. Validation of the Euroimmun test kit in the Malawian setting was performed in 81 respective negative sera collected before July 2019.

In a subsequent step, a commercially available surrogate virus neutralization test (sVNT) from GenScript Biotech Corporation (New Jersey, USA) was performed in seropositive samples. Assays were performed according to the manufacturer's guidelines. This assay detects circulating neutralizing antibodies against SARS-CoV-2 that specifically inhibit the interaction between the receptor-binding domain of the viral spike glycoprotein and the angiotensin-converting enzyme 2 cell surface, without the use of live SARS-CoV-2. Results are evaluated semi-quantitatively by calculating the percent inhibition (1-Average optical density of sample/Average optical density of negative control) x 100%). According to the manufacturer's information, a sample with a percent inhibition ≥ 20% was considered positive and < 20% was considered negative. Validation of the sVNT test kit in the Malawian setting was performed in 44 respective negative sera collected before July 2019.

Nasopharyngeal swabs were collected whenever additional informed consent was given to determine SARS-CoV-2 prevalence by real-time reverse-transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted manually using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), and RT-PCR assays were 

All statistical analyses were performed using STATA (version17, Stata Corp, LP, TX, USA) software. A descriptive analysis was performed to investigate participants' characteristics. Prevalence and 95% CIs estimates were calculated for RT-PCR and ELISA separately. The point prevalence of SARS-CoV-2 and 95% CIs were estimated as a combined prevalence by dividing the number of individuals who had a positive result in the ELISA and/or a positive result in the RT-PCR over the number of all participants. To explore differences in SARS-CoV-2 point prevalence according to demographic and behavioral variables, we performed a univariable and multivariable Poisson regression model and calculated prevalence ratios (PR). We applied a multivariable regression model to estimate the adjusted PR, including all variables of interest associated with SARS-CoV-2 point prevalence at a p-value <0.20 or clinically appropriate (e.g., sex and age group) for the analysis. Also, we performed a multicollinearity test for independent variables in the multivariable model to assess any intercorrelations between them. A robust standard error for the parameter estimates was applied to control for mild violation of underlying assumptions (Tamhane et al., 2016) . All performed tests were two-tailed. Statistical significance was set at p-value < 0.05.

A total of 962 participants attended the study. Of those, 32 had to be excluded from the analysis because of incomplete data and/or withdrawal of consent. Of the 930 included participants, 914 provided a specimen for ELISA, and 734 provided a specimen for the RT-PCR. A total of 927 participants were with available results on ELISA or/and RT-PCR. For the subsequent sVNT assay, 4 seropositive samples had to be excluded because of insufficient remnant serum. Due to dissociative labeling, 75 laboratory specimens could not be assigned to a participant and were excluded (figure 1).

Of the 930 participants included for data analysis, 48.3% were HCWs, and 51.7%

were non-HCWs. Of the HCWs, 75.1% were direct-clinicians (such as doctors, nurses), 22.0% were direct-nonclinical staff (such as guards, housekeeping, and others), 0.9% were indirect-clinicians (such as staff working at the pharmacy or laboratory), and 2.0% were indirect-nonclinical staff (such as administration). Overall, 599 participants were female (64.4%). The majority of the participants were in the age groups from 18-29 years (33.3%) and from 30-39 years (33.2%). In addition, the majority of the participants resided in Lilongwe (89.1%) and were of Malawian nationality (99.0%). Further participants' demographics are summarized in table 1.

When combining the prevalence of SARS-CoV-2 seropositivity and the RT-PCR positive cases, there was a combined point prevalence of 10.1% (95% CI 8.4-12.3).

Separately analyzed, the RT-PCR positivity was 2.0% (95% CI 1.2-3.4) and the seropositivity was 9.3% (CI 95% 7.6-11.4) (table 2). Of those, tested positive for SARS-CoV-2 antibodies or/and RT-PCR, 45.7% did not report any symptoms in the last 6 months before data collection. None of the seropositive participants were hospitalized in the last six months. Of the 81 tested seropositive samples, 90.1% (95% CI 81.2%-95.1%) were tested positive in the surrogate virus neutralization assay.

In the univariable Poisson regression model, SARS-CoV-2 point prevalence was significantly associated with being a HCW (PR 1.7; 95% CI 1.1-2.5). Also, the SARS-CoV-2 point prevalence was 1.9 times greater in those who had contact with a COVID-19 case inside the hospital than those without contact with a COVID-19 case inside the hospital (PR 1.9; 95% CI 1.2-2.9). We found a significant association between higher education and SARS-CoV-2 point prevalence (PR 5.3; 95% CI 1.3-21.4) ( figure 2 and table 3) . However, this is because the majority of HCW have higher education than non-HCW (62.1% of HCW reported high education compared to 6.6% among non-HCW). In the multicollinearity test, both education attainment and COVID-19 contact inside the hospital showed strong intercorrelations with HCW status, and therefore they were not considered in the multivariable model. In the final model adjusted for sex, age group, and symptoms, HCW remained significantly associated with higher SARS-CoV-2-point prevalence (PR 1.6; 95% CI 1.1-2.4; pvalue 0.026) (table 4).

To our knowledge, this is the first large-scale SARS-CoV-2 serosurvey during the first wave of the coronavirus pandemic in sSA Africa (Malawi) that also included PCR testing to complement overall prevalence for recent SARS-CoV-2 infections plus an sVNT assay to investigate for immunity after SARS-CoV-2 infection. Our study adds to the growing evidence that official data might underestimate the true magnitude of COVID-19 transmission in sSA. We found a much higher than expected SARS-CoV-2 infection rate with a point prevalence of 10.1% (95% CI 8.4-12.3) that consists of a low rate of acute infections (positive RT-PCR (2.0%, 95% CI 1.2-3.4)) and high seropositivity of 9.3% (95% CI 7.6-11.4); compatible with the fact that our study was conducted after the peak of the first COVID-19 wave in Malawi (Mulenga et al., 2021) . With regards to a high SARS-CoV-2 prevalence and high rate of asymptomatic SARS-CoV-2 infections, we hypothesize that circulation of the virus in the population might have gone on silently and might have appeared much earlier and more than predicted, even before or despite the measures that were put in place.

The eventuality of high rates of subclinical infections and silent community transmission has implications when designing surveillance and mitigation strategies.

To understand and prevent widespread community transmission, asymptomatic surveillance would have to be incorporated. However, given the current constraints, even with upscaled testing capacities, and limited funding for other concurring health threats, asymptomatic surveillance does not seem feasible in a low-resource setting like Malawi. Seeing the costs and collateral damage of preventive measures in this crisis, such as widespread hunger, economic insecurity, disruption of essential medical services and access to education (Haider et al., 2020) , a targeted approach might be more resource-efficient in African countries.

It would be most helpful to now identify factors associated with SARS-CoV-2 infection, severity of infection and immunity in sSA. Furthermore, identifying and shielding the most vulnerable risk groups and adequate treatment options for the currently infected might allow a more targeted approach and be more effective than undirected lockdown and distancing measures to reduce socio-economic impact and maintain health likewise.

Our study found a higher SARS-CoV-2 prevalence in HCWs (12.8%) than non-HCWs (7.7%). Chibwana et al. found a similar rate (12.5%) of SARS-CoV-2 prevalence in 500 HCWs from Blantyre; interestingly, a finding detected at an earlier stage of the epidemic in Malawi (Chibwana et al., 2020) . HCWs are generally considered at risk for SARS-CoV-2 infection and transmission, especially in sSA, due to a shortage of personal protective equipment, training, and human resources (Bandyopadhyay et al., 2020 , Mukwege et al., 2021 , Olayanju et al., 2021 . Convenience sampling and the stigma related to coronavirus infection might have introduced a selection bias in our study, underestimating the true prevalence of SARS-CoV-2 infection in HCWs.

Limited caseloads in hospitals in Malawi due to concerns about the health system in the population, strikes and absenteeism of HCWs from work due to fear of infection might have limited the in-hospital transmission during the first wave of the pandemic.

SARS-CoV-2 infection was also attributable to COVID-19 exposure in the hospital, underlining the need for adequate personal protective equipment and training.

However, general (high) transmission in urban Lilongwe, where all HCWs resided, has to be considered.

Regarding the emergence of new virus variants and the desire to implement vaccination strategies, there is a pressing need to estimate immunity after SARS-CoV-2 infection. In our study, we used an sVNT as a surrogate assay test to measure neutralizing antibodies in individuals who tested positive for SARS-CoV-2

IgG. To our knowledge, this is the first study that included a surrogate assay for neutralizing antibodies in a serosurvey in sSA. We demonstrated that the performance of an sVNT was feasible, also with limited laboratory infrastructure. and a specificity of 98.2% (Marien et al., 2021) . With regards to potential crossreactivity of serological assays, we also validated the GenScript sVNT in a panel of 44 zero samples with no false-positive results observed. Of those that were tested positive for SARS-CoV-2 IgG, 90.1% displayed neutralizing antibodies. Although this is a snapshot and one might question generalizability, it is a considerable and higher than expected rate of seemingly protected individuals.

High rates of protection after infection and comparably low rates of severe infections do put in question the current public health approach also with regards to vaccination. Moreover, they would have an implication on the economics, logistics, and effectiveness of a country strategy, moving from a broad horizontal approach to a selective, targeted one, namely the vaccination of high-risk groups (e.g., the elderly, HCWs) only. In this light, the measurement of progress in African countries by vaccine coverage targets -as most recently posted by the WHO regional director for Africa (https://www.afro.who.int/news/eight-10-african-countries-miss-crucialcovid-19-vaccination-goal) -is not recognizing the immunological situation detected and presented here and is potentially misleading.

The emergence of new variants, possibly more transmissible, more lethal, and more easily escaping immunity, underlines the importance of continuous seroepidemiological surveillance of the COVID-19 pandemic. Serial population-based serosurveys, preferably in combination with excess mortality studies, need to be implemented into the national testing strategy to understand viral transmission and disease dynamics and tailor mitigation strategies to the local context.

In this study, we present African figures for African estimatescontext matters in predictions. Even revised epidemiological estimates proved to be wrong, predicting high morbidity and mortality, using European epidemiological data to calculate Malawi estimates. Conversely, we found a SARS-CoV-2 prevalence and postinfection immunity, much higher than expected from official numbers, while there was a high rate of presumably asymptomatic SARS-CoV-2 infections. A targeted approach that includes the identification and shielding of and adequate treatment options for the most vulnerable might be more effective than strict lockdown measures in a setting with high rates of subclinical infections to reduce health and socio-economic impact. Acceptable levels of immunity in sSA countries will most likely be reached by natural acquisition, enhanced by vaccination; diametrically opposed to European countries' vaccination enhanced by natural acquisition.

We need up-to-date epidemiological data to foster community-based decisions on COVID-19 mitigation and vaccination measures specific to the country. 

AS is staff member of GIZ German development who partly funded the study. RS, BB, BK and AJ are staff members of RKI Germany who gave technical and material support. All remaining authors declare no competing interests.

AS had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Deidentified participant data and study protocol with publication at Mendeley Data DOI (10.17632/hz5mfsmpk5.1) will be shared upon request and after review of a research proposal. All data requests should be directed to the corresponding author. 1.3 (0.9-2.0) 0.148 *All variables of interest associated with SARS-CoV-2 point prevalence with a pvalue <0.20 (Health care worker, sex, Education attainment, symptoms, contact to case inside the hospital) or clinically appropriate (sex, age group) for the analysis were considered for a multivariable model. Due to strong intercorrelations between HCW and education (60%) and mild intercorrelations between HCW and COVID-19 contact inside the hospital (30%), we removed the two variables from the model.

Malawi population and housing census main report

Public Health Institute of Malawi. COVID-19 daily situation report

Department of Disaster Management Affairs. Ministry of Health. National COVID-19 preparedness and response plan

COVID-19) Dashboard

Eight in 10 African countries to miss crucial COVID-19 vaccination goal

Knowledge, risk perceptions, and behaviors related to the COVID-19 pandemic in Malawi

Infection and mortality of healthcare workers worldwide from COVID-19: a systematic review

Global seroprevalence of SARS-CoV-2 antibodies: A systematic review and meta-analysis

CoV-2 seroprevalence in Health Care Workers but relatively low numbers of deaths in urban Malawi

Seroprevalence of anti-SARS-CoV-2 antibodies in Africa: A systematic review and meta-analysis

Limited specificity of commercially available SARS-CoV-2 IgG ELISAs in serum samples of African origin

Preparedness and vulnerability of African countries against importations of COVID-19: a modelling study

Estimating the Impact of COVID-19 Mitigation Strategies in Malawi

Lockdown measures in response to COVID-19 in nine sub-Saharan African countries

Clinical performance of different SARS-CoV-2 IgG antibody tests

Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections

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

High SARS-CoV-2 Seroprevalence in Healthcare Workers in Bukavu, Eastern Democratic Republic of Congo

Prevalence of SARS-CoV-2 in six districts in Zambia in July, 2020: a cross-sectional cluster sample survey

The health policy response to COVID-19 in Malawi

COVID-19 pandemic in Malawi: Did public sociopolitical events gatherings contribute to its first-wave local transmission?

SARS-CoV-2 Seropositivity in Asymptomatic Frontline Health Workers in Ibadan, Nigeria

The first and second waves of the COVID-19 pandemic in Africa: a cross-sectional study

COVID-19 preparedness in Malawi: a national facility-based critical care assessment

Prevalence odds ratio versus prevalence ratio: choice comes with consequences

A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike proteinprotein interaction

 18 We 

This study was funded by GIZ -German Development Cooperation by a research grant under the project number P.15.2090.9-001 and generously supported by Robert Koch Institute Germany in delivering diagnostics, material, and logistics.

Committee Malawi under the approval number P.07/20/3094. Written informed consent was obtained from all participants.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.