key: cord-0292579-k89s3264
authors: Mandolo, J.; Msefula, J.; Henrion, M. Y.; Brown, C.; Moyo, B.; Samon, A.; Moyo-Gwete, T.; Makhado, Z.; Ayres, F.; Motlou, T.; Mzindle, N.; Kalata, N.; Muula, A. S.; Kwatra, G.; Msamala, N.; Likaka, A.; Mfune, T.; Moore, P. L.; Mbaya, B.; French, N.; Heyderman, R. S.; Swarthout, T. D.; Jambo, K. C.
title: Dynamics of SARS-CoV-2 exposure in Malawian blood donors: a retrospective seroprevalence analysis between January 2020 and February 2021
date: 2021-08-22
journal: nan
DOI: 10.1101/2021.08.18.21262207
sha: b4f5d502546b532a12a213e8c611f03b72017e5c
doc_id: 292579
cord_uid: k89s3264

Background: As at end of July 2021, the COVID-19 pandemic has been less severe in sub-Saharan Africa than elsewhere. In Malawi, there have been two subsequent epidemic waves. We therefore aimed to describe the dynamics of SARS-CoV-2 exposure in Malawi. Methods: We measured the seroprevalence of anti-SARS-CoV-2 antibodies among randomly selected blood donor sera in Malawi from January 2020 to February 2021. In a subset, we also assesed in vitro neutralisation against the original variant (D614G WT) and the Beta variant. Findings: A total of 3586 samples were selected from the blood donor database, of which 2685 (74.9%) were male and 3132 (87.3%) were aged 20-49 years. Of the total, 469 (13.1%) were seropositive. Seropositivity was highest in October 2020 (15.7%) and February 2021 (49.7%) reflecting the two epidemic waves. Unlike the first wave, both urban and rural areas had high seropositivity by February 2021, Balaka (rural, 37.5%), Blantyre (urban, 54.8%), Lilongwe (urban, 54.5%) and Mzuzu (urban, 57.5%). First wave sera showed potent in vitro neutralisation activity against the original variant (78%[7/9]) but not the Beta variant (22% [2/9]). Second wave sera potently neutralised the Beta variant (73% [8/11]). Interpretation: The findings confirm extensive SARS-CoV-2 exposure in Malawi over two epidemic waves with likely poor cross-protection to reinfection from the first on the second wave. Since prior exposure augments COVID-19 vaccine immunity, prioritising administration of the first dose in high SARS-CoV-2 exposure settings could maximise the benefit of the limited available vaccines in Malawi and the region.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

[2/9]). Second wave sera potently neutralised the Beta variant (73% [8/11]) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Introduction 96

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 in 97

Hubei province, China as a cause of coronavirus disease 2019 (COVID-19) 1,2 . SARS-CoV-2 98 infection presents with a range of disease manifestations, from asymptomatic infection to 99 severe acute respiratory distress and death. SARS-CoV-2 has spread globally and the World 100

Health Organisation (WHO) declared COVID-19 a global pandemic on 11 th March 2020. As of 101 12th August 2021, more than 205 million people globally have been infected with more than 102 4.32 million deaths 3 . The pandemic has placed substantial pressures on health systems 103 delivering care for patients with COVID-19 and caused disruption of non-COVID-19 health-104 care provision, in addition to negative effects on the global economy 4 . 105

The potential risk from SARS-CoV-2 to Africa was identified early in the global pandemic 5 . 107

As the epicentre of transmission moved from East Asia to West Asia, Europe and then to 108

North America, there was speculation as to the likely impact of the pandemic on the African 109 continent with its younger populations, high rates of infectious diseases including HIV, 110 poverty and undernutrition, as well as a fragile health care infrastructure 5,6 . Evidence has 111

shown that the epidemiology of the COVID-19 pandemic in sub-Saharan Africa has been 112 different from the Americas, China and Europe 3 . Available seroprevalence data indicate 113 that SARS-CoV-2 has circulated more widely in African populations than can be deduced 114 from the number of reported confirmed cases, hospitalisations and deaths 7-9 . This has led 115 to speculations that SARS-CoV-2 could have circulated longer in sub-Saharan Africa than the 116 first confirmed cases, but current evidence is limited. Others have speculated that a high 117 prevalence of circulating seasonal coronaviruses could have induced some cross-reactive 118 protective immunity 10, 11 . 119 120 SARS-CoV-2 Spike and Nucleocapsid specific antibody responses in blood are detectable in 121 individuals between 5 to 21 days post symptom onset 12 . Subsequently, anti-SARS-CoV-2 122

IgM antibody levels drop sharply within the initial 14 days followed by a gradual decline in 123 anti-SARS-CoV-2 IgG antibodies within the next 6 months 13 , reaching undetectable levels in 124 some individuals 14 . Whether these antibodies are likely to be protective against subsequent 125 infections is becoming clearer. Anti-SARS-CoV-2 Spike antibodies generated following 126 exposure to the original viral variant (D614G) exhibit reduced neutralisation potency against 127 . CC-BY-NC 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 the alpha and beta variants of SARS-CoV-2 15-17 . Conversely, antibodies generated following 128 infection with the beta variant are able to potently neutralise the original variant 18-20 . 129 130 Though there were initial plans made by the Malawi government in April 2020 to implement 131 a national lockdown of social and commercial activities, these were never implemented 21 . 132

As such, unlike other countries in the region, including Kenya 22 , South Africa 23 and 133

Zimbabwe 24 , there were no systematic lockdowns and limited curfew restrictions. Schools 134 were officially closed (from 23 rd March 2020 to 7 th September 2020) and many social 135 gathering settings (including restaurants and places of worship) voluntarily closed or had 136 significantly reduced services 25, 26 . Malawi closed its borders and airports from April 2020 to 137

September 2020, with limited essential traffic allowed in and out of the country 27 . As of 12 th 138

August 2021, 56,952 people were confirmed to have been infected by SARS-CoV-2 in 139

Malawi, resulting in 1,895 deaths and 169,632 people had been fully vaccinated 3 . 140 141 Due to movement restrictions instituted to curb SARS-CoV-2 transmission, conducting large 142 national population-based seroprevalence studies has been a significant challenge. This has 143 led to the use of blood donor and healthcare worker serosurveys to estimate and monitor 144 the extent of the SARS-CoV-2 pandemic in several countries 7-9,28 . Here, we report results of 145 a national serosurvey using archived serum samples from blood transfusion donors across 146

Malawi from January 2020 through February 2021, generated using a WHO-recommended 147 anti-SARS-CoV-2 Receptor Binding Domain total antibody assay with high sensitivity and 148 specificity, supported by SARS-CoV-2 pseudovirus neutralisation assays to explore variant 149 specificity. 150 151

The Malawi Blood Transfusion Service (MBTS) maintains an archive of sera from all blood 154 donations received in their Malawi facilities (average of 70,000 annually before the COVID-155 19 pandemic). All blood donors undergo routine screening through a self-administered 156 questionnaire, one-on-one assessment and a mini-health screening by MBTS staff. Donors 157 meeting routine donor ineligibility criteria including age below 16 or above 65 years, 158 haemoglobin below 12.5g/dl, past medical history suggestive of HIV, hepatitis or syphilis, 159 . CC-BY-NC 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

We used the WHO-approved WANTAI SARS-CoV-2 Ab commercial Enzyme Linked-188

Immunosorbent Assay (ELISA) kit to detect total SARS-CoV-2 antibodies, following 189 manufacturer's instructions (Beijing Wantai Biological Pharmacy Enterprise co., Ltd, China; 190 WS-1096) as reported previously 31 . This ELISA was shown to have the best overall 191 . CC-BY-NC 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101/2021.08.18.21262207 doi: medRxiv preprint 7 characteristics to detect functional antibodies in different phases and severity of disease 31 . 192 It is a two-step incubation antigen "sandwich" enzyme immunoassay kit which uses 193 polystyrene microwell strips pre-coated with recombinant SARS-CoV-2 Receptor Binding 194 Domain (RBD) antigen. The optical density (OD) of each well was read at 450nm in a 195 microplate reader (BioTek ELx808, UK) within 10 minutes of adding Stop Solution. Cut-off 196 values were calculated following manufacturer's instructions. The ratio between a sample's 197 absorbance and the cut-off were calculated for each sample. Specimens giving a ratio of 198 <0.9 were reported as negative for this assay, a ratio of >1.1 were reported as positive, and 199 a ratio between 0.9-1.1 were reported as borderline. Samples with borderline results were 200 retested using a confirmatory assay described below. The sensitivity and specificity of the 201 

RUO commercial ELISA kit (Omega diagnostics, UK) kit uses 96-well microtiter plates pre-207 coated with purified SARS-COV-2 Spike (S2) and Nucleoprotein (N) antigens to detect SARS-208 COV-2 IgG. This was performed following manufacturer's instructions as reported 209 previously 7 . Results were interpreted as described above for the ELISA assay, with a ratio of 210 <0.9 reported as negative, a ratio of >1.1 reported as positive, and a ratio between 0.9-1.1 211 reported as borderline. 212

Samples were pre-screened using an in-house (NICD) spike ELISA 16 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Subsequently, 1x10 4 HEK 293T cells engineered to over-express ACE-2, kindly provided by Dr 224

Michael Farzan (Scripps Research), were added and incubated at 37°C, 5% CO 2 for 72 hours 225 upon which the luminescence of the luciferase gene was measured. 226 227

We performed statistical analyses and graphical presentation using R statistical package, 229 Results 240

A total of 3,586 blood donor serum samples were selected from the four regional blood 242 transfusion centres. Samples had been collected between January 2020 through February 243 2021. These included 823 from Mzuzu (Northern region), 946 from Lilongwe (Central 244 region), 830 from Balaka (Eastern region) and 987 from Blantyre (Southern region) ( Table 1) . 245 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint As part of quality control procedures, we randomly selected 76 samples that were positive 258 on the primary SARS-CoV-2 ELISA and retested them using the second ELISA targeting S2 259 and NP IgG. Seventy-one (71) The overall (all months aggregated) seroprevalence adjusted for the reported independent 267 sensitivity and specificity estimates was 11.2% [95% CI from 5.8% to 14.9%]. In a 268 multivariable logistic regression model, seroprevalence did not vary significantly by sex (p = 269 0.532) or age (p > 0.10 for all age groups using those 10-19 years old as reference) but did 270 vary geographically, from 5.5% in Balaka (reference) to 14.6% in Blantyre (p < 0.001), 9.8% in 271 Lilongwe (p < 0.001) and 14.6% in Mzuzu (p < 0.001) ( Table 2) . 272 273

The first PCR-confirmed case of COVID-19 identified by Malawi's national surveillance 275 system was on 2 nd April 2021, with the first peak of the national COVID-19 cases being July 276 2020 (2,813 cases) and subsequently a second peak in January 2021 (17,380 cases) ( Figure  277 1). In this blood donor SARS-CoV-2 serosurvey, the first seropositive samples were observed 278 in February 2020 in Balaka (1 sample) and March 2020 in Blantyre (2 samples) (Figure 2 , 279 Table 3 and Supplementary Table 1 ). Both unadjusted and adjusted seroprevalence 280 estimates increased with time, with two distinct waves that followed the same temporal 281 trend as the reported national COVID-19 cases (Figure 1 and Supplementary Table 1) . When 282 aggregating serum samples from all locations, the first wave among donors was observed 283 from July to November 2020 with the peak seroprevalence in October 2020 (15.7% adjusted 284 seroprevalence), while the second wave was observed between December 2020 to February 285 2021 with peak seroprevalence in February 2021 (49.4%) (Figure 1, Table 3 and 286 . CC-BY-NC 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101/2021.08.18.21262207 doi: medRxiv preprint Supplementary Table 1) . However, there were differences in the temporal trend in 287 seroprevalence according to location (Figure 2 , Table 3 and Supplementary Table 1) . Balaka 288 had a 0% adjusted seroprevalence from January to November 2020, subsequently increasing 289 from 1.9% to 37.5% between December 2020 and February 2021. The peak adjusted 290 seroprevalence in the first wave was July 2020 in the Blantyre (15.9%), August 2020 in 291

Lilongwe (13.1%) and October 2020 in Mzuzu (24.8%). Seroprevalence bubble plots at three 292 periods in time demonstrated widespread exposure amongst blood donors across the 293 country in February 2021 compared to the earlier snapshots (Figure 3) . reasoned that differential neutralisation potency by SARS-CoV-2 antibodies could be used to 301 estimate the predominance of specific variants driving seroprevalence. We therefore 302 measured neutralisation antibody responses against D614G WT and Beta variant in 303 randomly selected seropositive sera collected at the peak of the first wave (Wave 1) in June 304

to October 2020 compared to those collected in February 2021 (Wave 2). Wave 1 sera was 305 more potent against D614G WT than the Beta variant, while Wave 2 sera was relatively 306 more potent against the Beta variant than D614G WT (Figure 4a-b) . Only 22% (2/9) of the 307

Wave 1 sera was more potent against the Beta variant than D614G WT, while 47% 8/17 of 308 the Wave 2 sera was more potent against the Beta variant than D614G WT (Figure 4b-c) . In 309 contrast, 78% (7/9) of the Wave 1 sera was more potent against D614G WT than the Beta 310 variant, while 18% 3/17 of the Wave 2 sera was more potent against D614G WT than the 311 Beta variant (Figure 4c) . Furthermore, 35% (6/17) of the Wave 2 sera showed no 312 neutralisation against the Beta variant or D614G WT (Figure 4c) . Collectively, these results 313 support existing sequencing evidence that Malawi's second SARS-CoV-2 epidemic wave was 314 driven by the Beta variant. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101/2021.08.18.21262207 doi: medRxiv preprint

Our study provides important insights into the dynamics of the COVID-19 epidemic in both 320 urban and rural Malawi, with likely relevance to the southern Africa region. The 321 seroprevalence estimates from this blood donor serosurvey are very high compared to the 322 reported national surveillance figures for confirmed asymptomatic and symptomatic cases, 323 but they do reflect the emergence and magnitude of the first and second COVID-19 324 epidemic waves in Malawi. They also provide evidence against the speculation that SARS-325

CoV-2 had been circulating more widely in Malawi before the first detected case in April 326 2020 or that circulation of endemic coronaviruses 10,11 had generated cross-reactive 327 antibody responses. Data from pseudovirus neutralisation assays suggests that the second 328 wave was predominantly driven by the Beta variant, as sera from the first wave had poor 329 neutralising activity against the Beta variant of SARS-CoV-2. Collectively, our findings 330 provide further support to the emerging data that in terms of hospitalised disease and 331 death, the COVID-19 pandemic has so far been attenuated in sub-Saharan Africa, despite 332 high population exposure to SARS-CoV-2. 333

We report a high seroprevalence of 49.7% nationally in February 2021, mirroring the 335 upsurge in confirmed COVID-19 cases, hospitalisations and death in Malawi experienced 336 during this period 3,6 . This is consistent with a study in South African blood donors that 337 reported seroprevalence rates ranging from 31.8% to 62.5% in January 2021 37 . So far, the 338 epidemic trajectory in Malawi as determined by the national confirmed COVID-19 cases, 339 deaths and hospitalisations has paralleled the epidemic trajectory in South Africa 3 . This is 340 likely due to shared borders and the resulting frequent human migration between the two 341 countries, specifically in the last 12 months during which there has been an influx of groups 342 of returning residents from South Africa to Malawi. This movement of people coincided with 343 rapid surges in COVID-19 cases in the first and second epidemic waves in Malawi 38 . These 344 findings support the need for a consolidated and standardised regional public health effort 345 in effectively addressing the COVID-19 pandemic in southern Africa to reduce the risk of 346 further epidemic waves across the region. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 variant 36 . In agreement, our neutralisation data showed that sera collected from the second 351 wave (February 2021) was better at neutralising the Beta variant than that from the first 352

Beta variant rather than the original variant (D614G WT). This is consistent with previous 354 studies showing that homologous convalescent sera retain higher in vitro neutralisation 355 potency than heterogous convalescent sera against the infecting virus 15-20 . It is noteworthy 356 that 35% of the Wave two sera that were positive for antibodies to the SARS-CoV-2 RBD 357 showed no neutralisation against the Beta variant or D614G WT, highlighting a disconnect 358 between qualitative antibody detection and functional activity. This discordance could be 359 due to low anti-RBD antibody titers as they have been shown to be associated with poor in 360 vitro neutratisalion 19, 20 . 361

The temporal kinetics of the SARS-CoV-2 seroprevalence in this blood donor serosurvey 363

suggests that the first epidemic wave was largely confined to urban areas. This differs to the 364 second wave, which was not only more rapid in its growth but also in its geographic spread, 365 as shown by the high seroprevalence even in the rural areas. This is consistent with national 366 surveillance reports showing that confirmed COVID-19 cases in the first wave were primarily 367 from the three major cities, Blantyre, Lilongwe and Mzuzu, but in the second wave there 368 was an increase in reported cases in rural areas 39, 40 . Also noteworthy is the earlier 369 seroprevalence peak in Blantyre and Lilongwe, compared to the northern city of Mzuzu. This study has considerable strengths, including consistent monthly sampling, use of well-377 validated assays and national geographical representation. There are however some 378 important limitations. First, inherent in using a blood donor sampling frame is that the 379 convenience sampling was not representative of the general population, skewed towards 380 males and individuals aged 18 to 45 years old. In addition, we were unable to evaluate the 381 association between the behaviour of blood donors and risk of acquisition to SARS-CoV-2 382 . CC-BY-NC 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 infection, as this may bias the measured results away from the true population 383 seropositivity. Though random sampling from the larger population would have been ideal, 384 restrictions on movement and gathering make these study designs extremely challenging to 385 implement. Second, in some individuals, SARS-CoV-2 antibodies wane over time to 386 undetectable levels leading to false negatives, especially in those who had asymptomatic 387 COVID-19 12-14 . Therefore, the seroprevalence estimates are likely an underestimate of the 388 cumulative exposure within this sampled population. However, the selection bias and 389 waning of antibody levels is unlikely to substantially alter the temporal trends reported in 390 this study. 391

In conclusion, we report a dramatic rise in SARS-CoV-2 seroprevalence from 6.2% in July The authors thank all blood donors whose samples are used in this study, the staff of the 403

Transfusion Services (MBTS) for their support and co-operation during the study. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101/2021.08.18.21262207 doi: medRxiv preprint J a n is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101/2021.08.18.21262207 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 

Figure 1. Overall SARS-CoV-2 adjusted seroprevalence over time superimposed on Malawi 621 national PCR-confirmed COVID-19 cases. Black dots (together with 95% CI) are estimated 622 seroprevalence at each timepoint (month), adjusted for assay sensitivity and specificity. The 623 histograms represent confirmed national COVID-19 cases per month, including 624 asymptomatic and symptomatic cases. Case frequency reported above histograms

Vertical dotted line defines transition from 2020 to 2021. SARS-CoV-2 = Severe 627 acute respiratory syndrome coronavirus 2, COVID-19 = Coronavirus disease

Positive for detection of anti-SARS-CoV-2 receptor binding domain (RBD) antibody

Flexible seroprevalence models. The model fits provide a smoothed estimate of 654 the seroprevalence over time

The orange line is the line of best fit for the empirical data

using a smooth generalised additive model, along with light-orange shading indicating 95%

Black dots (together with 95% CI) are estimated seroprevalence at each timepoint 658 (month), adjusted for assay sensitivity and specificity. Grey dots (a top and bottom of 659 figures) show the individual-level data, which are either positive (1) or negative (0) for 660 detection of anti-SARS-CoV-2 receptor binding domain

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 2020-08-27-malawi-to-lift-covid-19-499 international-air-travel-restrictions-from-september/. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted August 22, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted August 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 24 704