Sero-surveillance for IgG to SARS-CoV-2 at antenatal care clinics in three Kenyan referral hospitals: repeated cross-sectional surveys 2020-21

1 1 Sero-surveillance for IgG to SARS-CoV-2 at antenatal care clinics 2 in three Kenyan referral hospitals: repeated cross-sectional 3 surveys 2020-21 4 R. Lucinde 1,*, D. Mugo1, C. Bottomley2, A. Karani1, E. Gardiner1, R Aziza3, J. Gitonga1, H. Karanja1, 5 J. Nyagwange 1, J. Tuju1, P. Wanjiku1, E. Nzomo4, E. Kamuri5, K. Thuranira5, S. Agunda5, G. Nyutu1, 6 A. Etyang 1, I. M. O. Adetifa1,2, E. Kagucia1, S. Uyoga1, M. Otiende1, E. Otieno1, L. Ndwiga1 , C. N. 7 Agoti 1, R. A. Aman6, M. Mwangangi6, P. Amoth6, K. Kasera6, A. Nyaguara1 , W. Ng’ang’a7, L. B. 8 Ochola 9, E. Namdala10 , O Gaunya10, R Okuku10, E. Barasa1,8, P. Bejon1,8, B. Tsofa1, L. I. Ochola- 9 Oyier 1, G. M. Warimwe1,8+, A. Agweyu1+, J. A. G. Scott1,2,8+, K. E. Gallagher1,2+. 10 * Corresponding author: Ruth Lucinde, Epidemiology & Demography Department, KEMRI- 11 Wellcome Trust Research Programme CGMR-C, PO Box 230-80108, Kilifi, Kenya. Email: 12 [email protected] 13 Alternate corresponding author: Katherine Gallagher, Department of Infectious Disease 14 Epidemiology, Faculty of Epidemiology and Population Health, London School of Hygiene and 15 Tropical Medicine, Keppel Street, London, WC1E 7HT, United Kingdom. Email: 16 [email protected] 17 +Contributed equally 18 1 KEMRI-Wellcome Trust Research Programme, Kilifi, Kenya. 19 2 Department of Infectious Diseases Epidemiology, London School of Hygiene and Tropical 20 Medicine, Keppel Street, London, UK. 21 3 School of Life Sciences and the Zeeman Institute for Systems Biology & Infectious Disease 22 Epidemiology Research (SBIDER), University of Warwick, Coventry, United Kingdom . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 2 23 4 Kilifi County Hospital, Ministry of Health, Government of Kenya 24 5 Kenyatta National Hospital, Ministry of Health, Government of Kenya 25 6 Ministry of Health, Government of Kenya, Nairobi, Kenya. 26 7 Presidential Policy and Strategy Unit, The Presidency, Government of Kenya, Nairobi, Kenya 27 8 Nuffield Department of Medicine, Oxford University, Oxford, UK 28 9 Institute of Primate Research, Nairobi, Kenya 29 10 Busia Country Teaching & Referral Hospital, Busia, Kenya 30 31 Short title: SARS-CoV-2 seroprevalence at antenatal care clinics in three Kenyan referral 32 hospitals 33 Abstract word count: 250/300 34 Article: 2997/5000 35 Keywords: SARS-CoV-2, serology, sero-surveillance, ante-natal care . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 3 36 Abstract 37 Introduction 38 The high proportion of SARS-CoV-2 infections that have remained undetected presents a challenge to 39 tracking the progress of the pandemic and estimating the extent of population immunity. 40 Methods 41 We used residual blood samples from women attending antenatal care services at three hospitals in 42 Kenya between August 2020 and October 2021and a validated IgG ELISA for SARS-Cov-2 spike 43 protein and adjusted the results for assay sensitivity and specificity. We fitted a two-component 44 mixture model as an alternative to the threshold analysis to estimate of the proportion of individuals 45 with past SARS-CoV-2 infection. 46 Results 47 We estimated seroprevalence in 2,981 women; 706 in Nairobi, 567 in Busia and 1,708 in Kilifi. By 48 October 2021, 13% of participants were vaccinated (at least one dose) in Nairobi, 2% in Busia. 49 Adjusted seroprevalence rose in all sites; from 50% (95%CI 42-58) in August 2020, to 85% (95%CI 50 78-92) in October 2021 in Nairobi; from 31% (95%CI 25-37) in May 2021 to 71% (95%CI 64-77) in 51 October 2021 in Busia; and from 1% (95% CI 0-3) in September 2020 to 63% (95% CI 56-69) in 52 October 2021 in Kilifi. Mixture modelling, suggests adjusted cross-sectional prevalence estimates are 53 underestimates; seroprevalence in October 2021 could be 74% in Busia and 72% in Kilifi. 54 Conclusions 55 There has been substantial, unobserved transmission of SARS-CoV-2 in Nairobi, Busia and Kilifi 56 Counties. Due to the length of time since the beginning of the pandemic, repeated cross-sectional 57 surveys are now difficult to interpret without the use of models to account for antibody waning. 58 59 60 61 62 . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 4 63 Introduction 64 Globally, as countries are confronted with new waves of SARS-CoV-2 infections and new variants, 65 WHO recommendations have focused on enhancing population immunity with the available COVID- 66 19 vaccines(1). In Kenya, as with many lower-middle income countries, COVID-19 vaccine supplies 67 have been limited(2). Vaccination started in March 2021 and by 31 st October 2021, 3.7 million people 68 had received their first dose (13.5% of the adult population), 1.6 million had received their second 69 dose (6% of the adult population), with some geographic heterogeneity. In Nairobi 34% were partially 70 vaccinated, 18% were fully vaccinated, compared to Busia where 8% were partially vaccinated, 3% 71 fully vaccinated, and Kilifi where 5% were partially vaccinated, 2% fully vaccinated(3). 72 As vaccine coverage and vaccine-induced immunity is still considered to be low in Kenya, it remains 73 important to track the potential protection conferred by natural infection. By the 31 st October 2021, 74 Kenya had experienced four waves of infections, and reported a total of 253,310 confirmed cases and 75 5281 deaths. However, with just 3.9% of the population over 65 years of age(4), the proportion of 76 infections that have been asymptomatic is likely to be very high(5). Additionally, limited access to 77 tests and low uptake of testing makes it likely that a substantial proportion of cases have remained 78 undetected. Measuring the prevalence of antibodies to SARS-CoV-2 is an alternative way to estimate 79 the cumulative incidence of infection. A number of serological assays have been developed and 80 perform well with high sensitivity and specificity(6-9). We have shown that 5.2% of blood donors in 81 Kenya had SARS-CoV-2 antibodies in June 2020 and this had risen to 9.1% in September 2020 and 82 48.5% by March 2021(10-12). However, it is unclear whether blood donors are representative of the 83 population. 84 In the context of a pandemic, sentinel public health surveillance using residual aliquots of routinely 85 collected blood samples has the potential to overcome participation bias. For example, sero- 86 surveillance for HIV among women attending antenatal care was used to track the progress of the 87 HIV pandemic and showed prevalence estimates that were similar to population samples from the . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 5 88 same areas(13, 14). It remains unclear whether pregnancy alters susceptibility to SARS-CoV-2 89 infection(15); however, a large systematic review has found no difference in risk of becoming 90 symptomatic when comparing pregnant women with confirmed SARS-CoV-2 infection in women of 91 the same age(16, 17). 92 In Kenya, in 2014, 50% of women had had at least one pregnancy or were pregnant by 20 years of age 93 and the coverage of at least one antenatal care visit was 96%(18). Residual blood samples from 94 mothers visiting antenatal care for the first time may therefore represent a relatively unbiased sample 95 of young women, and an alternative sentinel surveillance population to blood donors. Testing an 96 aliquot of blood for antibodies to SARS-CoV-2 is feasible as a venous blood sample (5ml) is already 97 taken to screen mothers for malaria, HIV and syphilis at their first ANC visit. We aimed to determine 98 the prevalence of antibodies against SARS-CoV-2 in mothers attending ANC at three referral 99 hospitals in Kenya. 100 Methods 101 Setting 102 In a collaboration between the Kenyan Ministry of Health (MOH) and KEMRI-Wellcome Trust 103 Research Programme (KWTRP), three referral hospitals were engaged. Kenyatta National Hospital 104 (KNH) is the national referral tertiary hospital located in Nairobi, the country’s capital city, 105 approximately 3km from the central business district. The population of Nairobi city was 4,397,073 in 106 2019(4). Busia Country Teaching & Referral Hospital (BCTRH) serves Busia County, an area of 107 1628 km 2, with a population of 893,681 (548/ km2). Kilifi County Hospital (KCH) is the county 108 referral hospital in Kilifi Town. Kilifi county covers an area of 12,000 km 2, with a predominantly 109 rural population of 1.4 million (116/km 2)(4). . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 6 110 Study population 111 All women attending ANC for the first time, who provided a routine blood sample at their clinic visit, 112 were included in the study. Women who did not provide a sample at their first antenatal care visit, or 113 women attending their second or subsequent ANC visit, were excluded. 114 Sample collection and processing 115 In Kenya, a 5ml blood sample is routinely collected at the first ANC visit. After testing for malaria, 116 syphilis and HIV in the hospital laboratory, the residual volume is usually discarded. In this study, all 117 residual samples were set aside and collected daily for SARS-CoV-2 sero-surveillance. Where 118 possible, the following data were collected from hospital records and linked to the residual sample 119 identity number: date of sample, age, sub-county of residence, trimester of pregnancy, presence or 120 absence of COVID19-like symptoms in the last month and COVID-19 vaccination status ascertained 121 via verbal report confirmed via SMS or certificate. No personal identifiers were collected. All samples 122 were tested at the KWTRP laboratories for IgG to SARS-CoV-2 whole spike protein using an 123 adaptation of the Krammer Enzyme Linked Immunosorbent Assay (ELISA)(6). Validation of this 124 assay is described in detail elsewhere(10). Results were expressed as the ratio of test OD to the OD of 125 the plate negative control; samples with OD ratios greater than two were considered positive for 126 SARS-CoV-2 IgG. Sensitivity, estimated in 174 PCR positive Kenyan adults and a panel of 5 sera 127 from the National Institute of Biological Standards in the UK was 92.7% (95% CI 87.9-96.1%); 128 specificity, estimated in 910 serum samples from Kilifi drawn in 2018 was 99.0% (95% CI 98.1- 129 99.5%)(10). 130 Analysis 131 We estimated the proportion of samples seropositive for IgG to SARS-CoV-2. Sampling at least 135 132 women per month from each hospital would provide estimates of seroprevalence in the range 3-25% 133 with a precision of 3-7%. Bayesian modelling was used to adjust seroprevalence estimates for the 134 sensitivity and specificity of the assay. Non-informative priors were used for each parameter . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 7 135 (sensitivity, specificity and proportion true positive) and the models were fitted using the RStan 136 software package(19) (see supplementary files for code). Sub-county population densities were 137 extracted from the Kenya National Bureau of Statistics’ database(4). 138 To account for the effects of waning IgG in repeated cross-sectional samples, we fitted a two- 139 component mixture model to the log 2 OD ratios in unvaccinated individuals. In this model, we 140 assumed that antibody levels follow a normal distribution in previously uninfected individuals and a 141 skew-normal in previously infected individuals. To fit the model, we fixed the standard deviation of 142 the negative component at the value observed in pre-COVID 19 samples. The remaining parameters 143 were estimated using RStan. Details of the priors used in the estimation have been described 144 elsewhere(20). 145 Patient and Public Involvement 146 The study was conducted as anonymous public health surveillance at the request of the Kenyan MOH, 147 in response to the COVID-19 pandemic. The study directly addressed the needs of the MOH by 148 providing some information on the extent of the spread of SARS-CoV-2 pandemic within Kenya. The 149 public were not involved in the conceptualisation or implementation of this study. The need for 150 individual informed consent from the women whose samples were studied was waived, the protocol 151 was approved by the Scientific and Ethics Review Unit (SERU) of the Kenya Medical Research 152 Institute (Protocol SSC 4085), the Kenyatta National Hospital – University of Nairobi Ethics 153 Review Committee (Protocol P327/06/2020) and the Busia & Kilifi County health management 154 teams. 155 Results 156 Crude and adjusted seroprevalence across time and location 157 In Nairobi, samples were collected in three rounds: round 1, median date 11 th August 2020, round 2, 158 median date 22 February 2021, and round 3, median date 30 September 2021. In these time periods, . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 8 159 706 women (93%) provided a sample (Supplementary Figure 1). Women were aged between 17 and 160 45 years (mean 31 years); 275 (40%) attended their first antenatal care visit during their third 161 trimester of pregnancy, although this differed significantly between the rounds: 62% in August 2020, 162 28% in February 2021 and 35% in September 2021 (p<0.001; Supplementary Table 1). A total of 632 163 (90%) reported residence in 16 different sub-counties of Nairobi, 267 (42%) of mothers were resident 164 in Embakasi North, East or West sub-counties, and 110 (17%) were resident in Dagoretti North or 165 South sub-counties. The proportion of participants living in high vs. low population density sub- 166 counties did not differ by round (supplementary Table 1). Among women who had data on symptoms 167 during the preceding month, 7% reported symptoms in the first two rounds, this significantly differed 168 from the third round where 43% reported symptoms, coinciding with the end of the cold season (June- 169 September). Symptoms were not associated with seropositivity, controlling for age (data not shown). 170 In Nairobi, seroprevalence, adjusted for the sensitivity and specificity of the ELISA, was 50% in 171 August 2020, 32% in February 2021 and 85% in September 2021 (Table 1). In October 2021, 12.7% 172 of women were vaccinated with at least one dose of COVID-19 vaccine, seroprevalence among the 173 unvaccinated was 82%. 174 In Busia, samples were collected in 2 rounds: round 1, median date 3 rd May, and round 2, median date 175 5 th October. In this time period a total of 567 first ANC visits were conducted; 567 (100%) provided a 176 sample (Supplementary Figure 1). Women were aged between 14 and 44 (mean age 27 years). Most 177 women (66%) attended their first ANC in their second trimester, although this differed by round (73% 178 in May and 60% in October (p=0.007; Supplementary table 1). In May, 40% of women reported 179 symptoms in the last month, which differed significantly from October 2021 where 56% reported 180 symptoms. Symptoms were not associated with seropositivity, controlling for age (data not shown). 181 Adjusted seroprevalence in Busia increased from 31% in May 2021 to 71% in October 2021 (Table 182 2). Just 6 (2%) of women were vaccinated in October 2021, and seroprevalence remained 71% among 183 the unvaccinated. . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 9 184 Table 1. Seroprevalence of IgG to SARS-CoV-2 among mothers attending antenatal care in Kenyatta National Hospital (KNH), Nairobi 30th July – 25th August 2020 27th Jan- 11th March 2021 7th September-19th October 2021 KNH Seroprevalence Adjusted seroprevalence Seroprevalence Adjusted seroprevalence Seroprevalence Adjusted seroprevalence Nairobi n / N % % 95% CI n / N % % 95% CI n / N % % 95% CI All 91 / 196 46.4 49.9 42.1-58.2 80 / 265 30.2 32.1 26.2-38.4 193 / 245 78.8 84.9 78.3-91.5 Age 17-29 years 39 / 93 41.9 44.9 33.8-56.9 28 / 101 27.7 29.6 20.7-39.2 82 / 101 81.2 87.2 77.9-95.7 30-45 years 44 / 90 48.9 52.5 41.1-63.7 47 / 141 33.3 35.7 27.3-44.3 107 / 139 77.0 83.0 74.0-91.7 Trimester First 7 / 17 41.2 44.9 21.2-70.1 27 / 83 32.5 35.0 24.1-46.4 48 / 60 80.0 85.5 73.0-96.1 Second 21 / 53 39.6 42.9 29.8-57.4 34 / 106 32.1 34.5 25.1-44.3 77 / 96 80.2 86.1 76.0-95.5 Third 58 / 114 50.9 54.7 44.6-64.6 18 / 75 24.0 25.8 16.4-37.0 64 / 84 76.2 81.6 71.2-91.7 Any symptoms in last month* Yes 7 / 12 58.3 61.2 33.3-86.1 3 / 18 16.7 20.8 5.5-42.2 80 / 106 75.5 81.2 71.3-90.5 No 78 / 172 45.3 48.7 40.2-57.5 77 / 247 31.2 33.3 26.9-40.0 113 / 139 81.3 87.5 79.3-95.1 Population density of sub-county of residence <20000/km2 44 / 97 45.4 48.8 38.1-59.4 29 / 104 27.9 29.8 20.6-39.8 86 / 102 84.3 90.6 82.2-97.9 20-81000/km2 39 / 79 49.4 53.1 41.0-65.7 40 / 124 32.3 34.6 25.9-44.1 85 / 115 73.9 79.5 70.3-88.5 COVID-19 vaccine status† Vaccinated - - - - - - - - 30 / 31 96.8 96.1 86.2-99.9 Unvaccinated - - - - - - - - 163 / 214 76.2 82.1 75.1-89.2 185 186 * Women were asked about the full list of COVID-19 symptoms as per the MOH COVID-19 screening form i.e. fever/ chills, general weakness, cough, sore throat, runny 187 nose, shortness of breath, diarrhoea, nausea/ vomiting, headache, irritability/ confusion, pain (muscular/ chest/ abdominal/ joint). 188 † Vaccination status (at least one dose) was not available for the first two rounds of data collection, vaccination began in Kenya in March 2021 and at first targeted specific 189 groups only, we assume that the vaccine coverage among women attended ANC between 27th Jan-11th March was 0%. 190 Variations in seroprevalence by any of the explanatory variables were not statistically significant in any time period when tested with chi2 test. . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 10 191 Table 2. Seroprevalence of IgG to SARS-CoV-2 among mothers attending antenatal care in Busia County Teaching & Referral Hospital (BCTRH), 192 Busia 193 15th April - 21 May 2021 20 September – 22 October 2021 Seroprevalence Adjusted seroprevalence Seroprevalence Adjusted seroprevalence BCTRH, Busia n / N % % 95% CI n / N % % 95% CI All 78 / 270 28.9 30.6 24.7-37.1 195 / 297 65.7 70.7 64.1-77.4 Age 17-29 years 3 / 14 21.4 26.0 6.5-51.4 128 / 203 63.1 67.8 59.8-76.0 30-45 years 2 / 7 28.6 35.3 7.4-71.3 58 / 84 69.0 74.2 62.7-85.4 Trimester First 18 / 51 35.3 38.2 25.1-52.6 50 / 78 64.1 68.7 56.6-80.1 Second 50 / 192 26.0 27.7 21.3-34.7 114 / 175 65.1 70.1 61.8-78.4 Third 7 / 22 31.8 35.3 17.3-55.8 26 / 39 66.7 71.2 54.3-86.4 Any symptoms in last month* Yes 37 / 109 33.9 36.4 26.6-46.2 107 / 164 65.2 70.1 61.6-78.8 No 41 / 161 25.5 27.0 19.6-35.1 84 / 129 65.1 70.0 60.4-79.3 COVID-19 vaccination status† Vaccinated - - - - 4 / 6 66.7 67.0 29.8-96.1 Unvaccinated - - - - 188 / 288 65.3 70.5 63.8-77.3 194 1DOB was only available for 22 women in the first round of data collection 195 * Women were asked about the full list of COVID-19 symptoms as per the MOH COVID-19 screening form i.e. fever/ chills, general weakness, cough, sore throat, runny 196 nose, shortness of breath, diarrhoea, nausea/ vomiting, headache, irritability/ confusion, pain (muscular/ chest/ abdominal/ joint). 197 † Vaccination status (at least one dose) was not available for the first two rounds of data collection, vaccination began in Kenya in March 2021 and at first targeted specific 198 groups only, we assume that the vaccine coverage among women attended ANC between 27th Jan-11th March was 0%. 199 Variations in seroprevalence by any of the explanatory variables were not statistically significant in any time period when tested with chi2 test. . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 11 200 In Kilifi, 1707 samples were collected between the 18 th September 2020 and 22nd October 2021, 201 collection was continuous apart from during the healthcare worker strike (December 2020 to February 202 2021). No data were available on age, trimester, location, symptoms or COVID-19 vaccination status. 203 Adjusted seroprevalence increased over the period of sample collection from 1% in September 2020 204 to 63% in October 2021 (p=0.0001, Chi sq test for trend; Table 3). 205 Table 3: Seroprevalence of IgG to SARS-CoV-2 among mothers attending antenatal care in 206 Kilifi County Hospital (KCH), over time KCH, Kilifi1 Seroprevalence Adjusted seroprevalence n / N % % 95% CI Month Sept-Oct 2020 3 / 265 1.1 0.9 0.0-2.7 Nov-Dec 2020 32 / 236 13.6 14.0 9.4-19.5 Mar-Apr 2021 55 / 260 21.2 22.2 16.7-28.1 May-Jun 2021 104 / 382 27.2 28.9 23.9-34.4 Jul-Aug 2021 148 / 260 56.9 61.2 54.4-68.4 Sept-Oct 2021 178 / 305 58.4 62.7 56.2-69.1 207 1 No age, trimester or symptom data were available from the ANC records at KCH. Months were combined into 208 2-month batches due to low numbers 209 210 Mixture model results 211 When two distinct distributions were fitted to the data—corresponding to antibody levels in 212 previously infected and previously uninfected individuals—there were substantial overlaps in the 213 distributions, especially at low seroprevalences (Figure 1). As seroprevalence estimates increased, the 214 distributions became more distinct. The mixture model produced estimates of cumulative incidence 215 that were consistently higher than those of the threshold analysis except for the final round in Nairobi, 216 where the results of both analyses were the same (85%) and the distributions hardly overlapped (85%; 217 Figure 2). Median OD ratios among the unvaccinated, seropositive individuals increase over time in 218 all three areas, potentially indicating natural boosting through re-infections (Figure 1, Supplementary 219 Table 2). . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 12 220 Figure 1. Mixture distributions fitted to anti-spike IgG antibody data collected in Kilifi (KCH), 221 Busia and Nairobi (KNH) . The red distribution represents predicted responses in individuals 222 previously infected with SARS-CoV-2 and the blue distribution represents predicted responses in 223 previously uninfected individuals. 224 Figure 2. Adjusted and modelled estimates of the cumulative incidence of SARS-CoV-2 225 infection. Estimates are shown with 95% credible intervals. 226 Discussion 227 Surveillance for IgG antibodies to SARS-CoV-2 among mothers attending ANC services in three 228 county referral hospitals in Kenya has revealed evidence of a substantial amount of prior infection by 229 October 2021. Seroprevalence is currently highest in Nairobi, then Busia, then Kilifi, correlating with 230 the counties’ population densities. 231 In Nairobi, in August 2020, just after the peak of the first wave of SARS-CoV-2 infections, mixture 232 modelling, which attempts to account for the wide range of OD ratios among those exposed to the 233 virus better than the simple threshold analysis(20), indicates a cumulative incidence of 75%, just 4 234 months after the start of the pandemic. At the same timepoint, 6,727 PCR-confirmed infections had 235 been registered across the city (<1% of the County’s population; Supplementary Figure 2). In March 236 2021, a year after the pandemic began, seroprevalence was lower, 32%. This second group of women 237 reported residing in the same sub-counties and were on average the same age (supplementary table 1). 238 The high levels of transmission of the virus in these locations early in the pandemic may have meant 239 some of these women had been infected at some point in the last year, but had since seroreverted. 240 Data on the rate of seroreversion differs with the assay used(21) and the severity of the initial 241 infection(22); approximately 9-12% with mild symptoms may sero-revert 4-6 months post- 242 infection(23, 24). Additionally, such high seroprevalence earlier in the year may have reduced the 243 number susceptible and dampened transmission within the same communities by March 2021. 244 Modelling indicates the first wave could have predominantly affected communities of low-income . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 13 245 earners, more likely to use public hospitals, such as KNH, with the later waves affecting a group 246 including higher-income, private healthcare users(25). In October 2021, seroprevalence in a third 247 group of women was 85%, 76% among those unvaccinated. These women were very similar in age, 248 residence location and trimester to the second group. The survey was conducted just after the fourth 249 wave of cases in Kenya, natural boosting could have occurred in these communities if residents 250 encountered the virus repeatedly(26). This is supported by the higher median OD ratios among 251 unvaccinated, seropositive individuals in the third round compared to both of the previous rounds. 252 The proportion vaccinated with at least one dose (13%) is lower than the average for Nairobi adults at 253 this time of 34%(3), although nationally only 13.5% of the population were vaccinated with one dose 254 at this time point. 255 In Busia, seroprevalence in May 2021 was 31% using the threshold analysis, and 51% using the 256 mixture model analysis. The two distributions of OD ratios had a substantial amount of overlap 257 leading to uncertainty in the estimate from mixture modelling at this time point. Threshold 258 seroprevalence increased to 71% in October 2021. By 21 st October 2021 only 2% of our study 259 population were vaccinated with at least one dose, lower than the nationally reported coverage of 8% 260 in Busia(3). Western Kenya was affected in the fourth wave of the pandemic in July-September 2021 261 and the seroprevalence represents a substantial amount of natural infection. 262 Seroprevalence steadily increased in Kilifi during the sampling timeframe to 63% (using the threshold 263 analysis) or 72% (using the mixture model analysis) in October 2021. The slower increase in 264 seroprevalence in Kilifi compared to Nairobi is consistent with modelling suggesting that the initial 265 wave of the COVID-19 pandemic was concentrated in urban centres, with subsequent spread 266 increasingly affecting rural areas(25); Kilifi County reported a marked increase in the number of 267 infections in December 2020 (Supplementary Figure 2). . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 14 268 A strength of this analysis is the use of a rigorously validated serological assay, using locally relevant 269 control populations and reference panels from the National Institute for Biological Standards and 270 Control (NIBSC) in the UK(27). The threshold used to define seropositivity was chosen to prioritise 271 specificity over sensitivity, i.e. to minimize the number of false positives. The very low crude 272 seroprevalence (0/82; 0%) in the first month of samples from Kilifi adds confirmation that the 273 specificity of this assay is very high. 274 Although sero-surveillance among pregnant women has been used as a proxy for population-based 275 SARS-CoV-2 surveillance in high income countries(28-34), the representativeness of the sample in 276 Kenya is unknown. A national survey in 2014 indicated 18% of the population utilised public 277 hospitals at their last visit to outpatient services (a further 40% utilized public health centres or 278 dispensaries). Utilisation of public health services was correlated with lower education levels(35). 279 The seroprevalence estimates from women attending ANC differ from the seroprevalence estimates 280 available from blood donor samples. The Nairobi seroprevalence of 50% in August is substantially 281 higher than the 10% seroprevalence reported among blood donors in the same county in June-August 282 2020(10), however in March 2021, 32% seroprevalence in ANC was lower than the estimated 62% 283 seroprevalence in blood donors(12). The majority of expectant mothers attending ANC in KNH 284 consistently came from 5 sub-counties close to the hospital, which are densely populated with low- 285 income earners(4). Blood transfusion donors are likely to be more heterogenous and widely 286 distributed across Nairobi including areas of lower population density and greater affluence. The 287 estimates from ANC in Kilifi in September 2020 (1%) and April 2021 (22%) were lower than the 288 14.1% seroprevalence reported among blood donors from Coastal Counties in September 2020 and 289 the 43% seroprevalence among blood donors in Jan-March 2021(10-12). Blood donations come from 290 across the county including urban centres such as Malindi, whereas women attending ANC in Kilifi 291 represent a less heterogenous semi-urban group. It is clear that viral transmission has been 292 heterogenous in terms of geography and socioeconomic status, seroprevalence estimates from . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 15 293 multiple different sentinel populations provide more reliable indicators of the development of the 294 pandemic than any one estimate alone. 295 The impact of pregnancy on susceptibility to SARS-CoV-2 infection is unclear(15); however, 296 comparisons of infected pregnant women with non-pregnant women of the same age suggests that a 297 similar proportion of infections become symptomatic(16). This would suggest a similar proportion of 298 pregnant and non-pregnant women mount a protective antibody response and seroprevalence 299 estimates are generalisable to non-pregnant women of the same age. In a comparison of samples from 300 blood donors and ANC in Australia, the two sample sets estimated seroprevalence within 0.1% of 301 each other, although overall prevalence was very low(36). Additionally, seroprevalence in blood 302 donors in Kenya did not differ by sex(10), suggesting that these results from pregnant women may be 303 generalisable to men between 17-45 years of age, residing in the same areas. 304 Our analysis is constrained by the nature of the anonymised surveillance data available. Data on age, 305 trimester and location for the women in Kilifi would have allowed more valid comparisons with data 306 from other sources. It is difficult to assess how comparable the different rounds from the same 307 location are, without more data. As discussed, we lack local data on the rate of antibody waning, 308 which is important to estimate cumulative incidence of infection from snapshot seroprevalence 309 estimates(37, 38). This is especially important in populations, like those reported here, where ongoing 310 transmission may cause ‘natural boosting’(37, 39, 40). 311 Conclusions 312 This seroprevalence study of women attending ANC clinics suggests there has been substantial, 313 unobserved transmission of SARS-CoV-2 within communities in Nairobi, Busia and Kilifi Counties. 314 However, it is becoming difficult to interpret the results of cross-sectional seroprevalence studies due 315 to the length of the pandemic(41). To attempt to account, to some extent, for antibody waning, we 316 have used mixture modelling, this suggests that 85% of the population using a public hospital in . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 16 317 Nairobi have been previously infected with SARS-COV-2. At least in the short-term, asymptomatic 318 infection is protective(26) and these seroprevalence estimates should be taken into account when 319 estimating population level immunity. Increases in antibody concentration over time implies an 320 increasing level of population protection that may be attributable to reinfections. 321 Acknowledgements 322 We thank the Kenyatta National Hospital, Busia Country Teaching & Referral Hospital and Kilifi 323 County Hospital employees who collected the samples during routine ANC visits and the women 324 themselves for providing samples for routine health screening. We thank Rebeccah Ayako, Evalyne 325 Akinyi and Cedrick Shikoli at the Institute of Primate Research for processing the ANC samples from 326 KNH. We thank F. Krammer for providing the plasmids used to generate the spike protein used in this 327 work. Development of SARS-CoV-2 reagents was partially supported by the NIAID Centres of 328 Excellence for Influenza Research and Surveillance (CEIRS) contract HHSN272201400008C. The 329 COVID-19 convalescent plasma panel (NIBSC 20/118) and research reagent for SARS-CoV-2 Ab 330 (NIBSC 20/130) were obtained from the NIBSC, UK. We also thank the WHO SOLIDARITY II 331 network for sharing of protocols and for facilitating the development and distribution of control 332 reagents. This paper has been published with the permission of the director, Kenya Medical Research 333 Institute. 334 For the purpose of Open Access, the author has applied a CC-BY public copyright licence to any 335 author accepted manuscript version arising from this submission. 336 Funding 337 This project was funded by the Wellcome Trust (grants 220991/Z/20/Z and 203077/Z/16/Z), the Bill 338 and Melinda Gates Foundation (INV-017547), and the Foreign Commonwealth and Development 339 Office (FCDO) through the East Africa Research Fund (EARF/ITT/039) and is part of an integrated . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 17 340 programme of SARS-CoV-2 sero-surveillance in Kenya led by KEMRI Wellcome Trust Research 341 Programme. 342 A.A. is funded by a DFID/MRC/NIHR/Wellcome Trust Joint Global Health Trials Award 343 (MR/R006083/1), J.A.G.S. is funded by a Wellcome Trust Senior Research Fellowship (214320) and 344 the NIHR Health Protection Research Unit in Immunisation, I.M.O.A. is funded by the United 345 Kingdom’s Medical Research Council and Department For International Development through an 346 African Research Leader Fellowship (MR/S005293/1) and by the NIHR-MPRU at UCL (grant 347 2268427 LSHTM). G.M.W. is supported by a fellowship from the Oak Foundation. C.N.A. is funded 348 by the DELTAS Africa Initiative [DEL-15-003], and the Foreign, Commonwealth and Development 349 Office and Wellcome (220985/Z/20/Z). S.U. is funded by DELTAS Africa Initiative [DEL-15-003], 350 L.I.O.-O. is funded by a Wellcome Trust Intermediate Fellowship (107568/Z/15/Z). R.A is funded by 351 National Institute for Health Research (NIHR) (project reference 17/63/82) using UK aid from the UK 352 Government to support global health research. 353 The views expressed in this publication are those of the authors and not necessarily those of the 354 funding agencies 355 Conflict of Interest 356 All authors: No reported conflicts. 357 Author contributions 358 Conceptualisation: A. Agweyu, J. A. G. Scott, G. M. Warimwe, K. E. Gallagher 359 Data curation: G. Nyutu 360 Formal statistical analysis: K Gallagher, Christian Bottomley 361 Funding acquisition: A Agweyu J. A. G. Scott, G. M. Warimwe 362 Investigation (Data collection and Lab): R. Lucinde, D. Mugo, A. Karani, E. Gardiner, J. Gitonga, H. 363 Karanja, J. Nyagwange, J. Tuju, P. Wanjiku, E. Nzomo, E. Kamuri, K. Thuranira, S. Agunda, L. B. 364 Ochola, E. Namdala, O Gaunya, R Okuku . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint 18 365 Methodology: K. Gallagher, C Bottomley 366 Project Administration: R Lucinde, 367 Supervision: .A Agweyu, J. A. G. Scott, G. M. Warimwe, K. E. Gallagher 368 Validation: K Gallagher, G Warimwe 369 Vizualisation: K Gallagher, R Aziza, C Bottomley 370 Original draft preparation: R Lucinde, K Gallagher 371 Review and Editing: R. Lucinde, D. Mugo, C. Bottomley, A. Karani, E. Gardiner, R Aziza, J. 372 Gitonga, H. Karanja, J. Nyagwange, J. Tuju, P. Wanjiku, E. Nzomo, E. Kamuri, K. Thuranira, S. 373 Agunda, G. Nyutu, A. Etyang, I. M. O. Adetifa, E. Kagucia, S. Uyoga, M. Otiende, E. Otieno, L. 374 Ndwiga, C. N. Agoti, R. A. 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CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprintthis version posted March 6, 2022. ; https://doi.org/10.1101/2022.03.03.22271860doi: medRxiv preprint . CC-BY 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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