medRxiv preprint doi: https://doi.org/10.1101/2021.07.16.21260611; this version posted July 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

SARS-CoV-2 seroprevalence in Chattogram, Bangladesh before a National Lockdown, March-April 2021 Taufiqur Rahman Bhuiyan1*, Juan Dent Hulse2*, Sonia T. Hegde2, Marjahan Akhtar1, Md. Taufiqul Islam1, Zahid Hasan Khan1, Ishtiakul Khan3, Md Mamunur Rashid3, Rumana Rashid3, Emily S. Gurley2, Tahmina Shirin4, Ashraful Islam Khan1, Andrew S. Azman2,5¶, Firdausi Qadri1¶ 1Infectious Diseases Division, International Centre for Diarrhoeal Disease Research, Bangladesh 2Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA 3Bangladesh Institute of Tropical and Infectious Diseases, Chattogram, Bangladesh 4 Institute for Epidemiology, Disease Control and Research, Bangladesh Directorate General for Health, , Bangladesh 5Institute for Global Health, University of Geneva, Geneva, Switzerland

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2021.07.16.21260611; this version posted July 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

Abstract The absence of population-based seroprevalence estimates in Bangladesh have impeded efforts to understand the relatively low reported mortality and incidence rates of SARS-CoV-2 in this country. We report findings of a representative serosurvey of the subdistrict in the Chattogram division of Bangladesh before a nationwide lockdown in April 2021. After adjusting for age, sex, household clustering and test performance using a Bayesian modeling approach, we estimate the seroprevalence of SARS-CoV-2 to have been 63.1% (56.2–60.8%) in Sitakunda during this period. These results illustrate that going into the national lockdown in April 2021, the majority of this population had already been infected despite a relatively low incidence of medically attended COVID-19. medRxiv preprint doi: https://doi.org/10.1101/2021.07.16.21260611; this version posted July 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

After the first COVID-19 wave in Bangladesh in May 2020, more than 550,330 cases and 8,462 deaths had been reported nationally.1 Both reported mortality and incidence rates appeared to be substantially lower than what was observed in many other countries across the world. Without population-based seroprevalence estimates, it is difficult to know whether these differences are due to limited surveillance and a lack of healthcare seeking or true differences in incidence due to effective interventions or alternate biological responses to infection. In early March 2021, cases across Bangladesh began to rise at the same time the Delta variant was detected in neighboring India. We conducted a representative serosurvey of Sitakunda subdistrict in the Chattogram division of Bangladesh just before a nationwide lockdown was implemented in response to this wave (27-March to 13-April) to estimate the seroprevalence of SARS-CoV-2. We tested participant serum for total antibodies (IgG, IgM and IgA) against the S1-RBD of SARS-CoV-2 using the Wantai SARS-CoV-2 Ab ELISA kit (Beijing Wantai Biological Pharmacy Enterprise Co., China) following manufacturer instructions. We corrected seroprevalence estimates for imperfect test performance using a previously documented Bayesian modeling approach and post-stratified results to match the target population structure (supplement).2 We estimated test performance by testing 214 pre-pandemic healthy participant samples from a 2014 cholera vaccine study and 71 samples from symptomatic PCR-confirmed SARS-CoV-2 infections (all were non-hospitalized, 3 to 268 days post-symptom onset) from Dhaka, Bangladesh. We estimated a specificity (99.1%, n=212/214, 95% CI: 97.8–100%) and sensitivity (87.3%, n=62/71, 95% CI: 79.5––95.1%) for detecting historical infection and found little evidence of decreasing sensitivity with time since infection (supplement). We enrolled 169 households and 664 individuals in the Sitakunda serosurvey, a majority of whom were female (57%, n=376/664) and the median age was 28 (IQR: 16–42) years. Most participants reported working at home (40%), going to school (29%), or conducting business outside of their home (16%) as their main occupation in the last month from enrollment. There were 393 (59%) seropositive individuals, including 21 (of 24) individuals reporting to have been vaccinated with at least one dose of the Oxford-AstraZeneca vaccine. On average, 60% of participants in each household had antibodies against SARS-CoV-2 (intraclass correlation coefficient (ICC): 0.30; 95% CI: 0.21–0.37). We found significant evidence of a gradient in seropositivity by population density, with those living in areas of higher population density being significantly more likely to be seropositive; 73% of individuals living in the most population dense areas were seropositive compared with 44% of individuals living in the least population dense areas (p<0.001, Supplement Table 1). Adjusting for age, sex, household clustering, and test performance we estimate the seroprevalence of SARS-CoV-2 in Sitakunda to have been 63.1% (56.2–69.8%) among all individuals and 62.5% (55.5–69.5%) among unvaccinated individuals just before the national lockdown and Ramadan (Table 1, Supplemental Table 3). We found no significant differences between sex (RR: 1.04, 95% CI: 0.94–1.17) and lower seroprevalence in children less than 15 years (RR 0.71, 0.58–0.84, Table 1, Supplemental Table 4). In the catchment area of this serosurvey, only 865 cases and 19 deaths have been reported since the start of the pandemic, while crudely extrapolating our serologic estimates suggests 209,000 infections during the same period.3 This implies more than 240 infections per reported clinical medRxiv preprint doi: https://doi.org/10.1101/2021.07.16.21260611; this version posted July 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

case, much higher than what has been documented in most settings across the world.4 These results illustrate that going into the national lockdown in April 2021, the majority of the population had already been infected despite a relatively low incidence of medically attended COVID-19. In addition to providing important validation data on a widely used immunoassay, our results can help assess the impact of variant strains on immune evasion and help set expectations for SARS-CoV-2 control in the months to come in Bangladesh.

Conflict of Interest We declare no competing interests.

Acknowledgements This work was supported by Bill and Melinda Gates Foundation [grant number INV-021879]. The authors would like to thank the Ministry of Health and Family Welfare (MOHFW) of Bangladesh. The authors would also like to express their sincere thanks to the staff members of icddr,b for their dedicated work in the field and laboratory during this pandemic situation. icddr,b is thankful and supported by the Governments of Bangladesh, Canada, Sweden, and the UK.

Table 1. Estimated seroprevalence of SARS-CoV-2 in Sitakunda adjusted for sex, age, household clustering, and test performance among all individuals, both vaccinated and unvaccinated. Variable Category Obs Test Test Seroprevalence Relative risk (95% positive negative (95% CI) CI)

Age 0–4 22 10 (45.5%) 12 (54.5%) 53.9 (35.8-71.5) 0.83 (0.56-1.09) (years) 5–9 49 21 (42.9%) 28 (57.1%) 50.5 (36.1-65.3) 0.77 (0.56-0.99)

10–14 76 32 (42.1%) 44 (57.9%) 52.7 (41.0-64.1) 0.81 (0.63-0.99)

15–24 148 88 (59.5%) 60 (40.5%) 64.3 (54.8-73.2) 0.99 (0.85-1.16)

25–34 114 69 (60.5%) 45 (39.5%) 64.7 (55.9-72.8) --

35–44 96 68 (70.8%) 28 (29.2%) 76.0 (66.5-84.7) 1.18 (1.02-1.37)

45–54 71 46 (64.8%) 25 (35.2%) 69.3 (57.2-80.6) 1.07 (0.88-1.28)

55–64 54 36 (66.7%) 18 (33.3%) 69.7 (56.0-82.1) 1.08 (0.87-1.29)

65+ 34 23 (67.6%) 11 (32.4%) 74.7 (60.4-87.3) 1.16 (0.95-1.39)

Sex Male 288 172 (59.7%) 116 (40.3%) 64.5 (56.6-72.1) 1.04 (0.94-1.17)

Female 376 221 (58.8%) 155 (41.2%) 61.5 (54.1-68.7) --

Overall 664 393 (59.2%) 271 (40.8%) 63.1 (56.2-69.8) -- medRxiv preprint doi: https://doi.org/10.1101/2021.07.16.21260611; this version posted July 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .

References 1. Bangladesh: WHO Coronavirus Disease (COVID-19) Dashboard With Vaccination Data [Internet]. [cited 2021 Jun 14]. Available from: https://covid19.who.int 2. Stringhini S, Wisniak A, Piumatti G, et al. Seroprevalence of anti-SARS-CoV-2 IgG antibodies in Geneva, Switzerland (SEROCoV-POP): a population-based study. The Lancet. 2020 Aug 1;396(10247):313–319. 3. Sitakunda Upazila - Banglapedia [Internet]. [cited 2021 Jun 26]. Available from: https://en.banglapedia.org/index.php/Sitakunda_Upazila 4. Chen X, Chen Z, Azman AS, et al. Serological evidence of human infection with SARS- CoV-2: a systematic review and meta-analysis. The Lancet Global Health. Elsevier; 2021 May 1;9(5): e598–e609.