key: cord-0764425-8bk9xpdk
authors: Martin, M. C.; Gonzalez, M. I.; Holgado, N.; Jimenez, A.; Ortega, N.; Page, I.; Parrado, A.; Perez, M.; Blanco-Peris, L.
title: Sustained seroprevalence of anti SARS-CoV-2 total immunoglobulins in asymptomatic blood donors
date: 2021-05-01
journal: nan
DOI: 10.1101/2021.04.28.21256242
sha: df4f4c7b3834bb9a27128a167343ab8b1d1a204d
doc_id: 764425
cord_uid: 8bk9xpdk

Background: Seroprevalence analysis of SARS-CoV-2 is one of the keys to accurately monitor pandemics and help the authorities make health decisions and adjust the current social interventions. The aim of this study was to determine retrospective seroprevalence evolution among blood donors before pandemic, and along the first wave in Spain. A secondary objective was to determine whether age, blood group or haematological parameters are related to recent past infection. Material and Methods: A total of 12719 donations SARS-CoV-2 from July 2019 to October 2020 were analysed. Donors were 60.9% males and their average age was 46+/-13. An automated chemiluminiscence double-antigen sandwich immunoassay for the in vitro semi quantitative detection of total antibodies to SARS-CoV-2 in human serum and plasma was performed. Results: Seropositive donation rate grew up from week 11 to week 21, reaching plateau by near 8% donations, sustained until week 43 when 2nd wave arose in our country. 6.7% individuals were positive by the end of 1st wave. No differences by sex age or blood group were found regarding antibodies. Lymphocyte were significantly higher in positive woman as compared to negative ones and haemoglobin were lower in positive men as compared to negative ones. Discussion: Seroprevalence due to asymptomatic cases would be equivalent to that of general population. Sex and age would not affect COVID-19 susceptibility but its severity. Gender differences are present even in asymptomatic individuals: females are possibly protected by their relative lymphocytosis and neutropenia whereas males are would be weaker as seropositive men show a decrease of haematocrit and haemoglobin. Further studies are needed to confirm these gender differences not only in severe but as well in asymptomatic cases as they can help better understand COVID-19 pathogenesis and prognosis.

SARS-CoV-2 infection curses asymptomatic or with just mild symptoms in a 31 number of cases. This make it hard to calculate accurate infection or fatality rates 32 and prevalence. These calculations are important for evaluating risks of COVID-33 19 disease, predicting the spread of the virus and managing health resources 1 . 34

Most cases develop an effective immune response during infection, leading to 35 viral eradication and the production of specific T cell responses and antibodies 36

against SARS-CoV-2 that are usually detectable 10-21 days after infection. 37

Antibody assays are quite different: they either detect antibodies against different 38 viral proteins (S1, S1/S2, RBD or NC) or different immunoglobulin classes: IgG, 39

IgM, IgA or their combinations. To make it even messier, many factors can 40

influence test performance, including cross-reactivity with other coronaviruses or 41 platform (laboratory-based vs point-of-care, lateral flow). Chemiluminiscence 42 assays have suitable performances regarding both sensibility and sensitivity, 43 even though those values have been estimated as testing populations with 44 compatible symptoms and would be presumably lower when performing 45 serosurveillance analysis on apparently healthy population. Chemiluminiscent 46 immunoassays exhibit a significantly higher specificity score but a lower 47 sensitivity, as compared to ELISA immunoassays. Moreover, immunoassays 48

detecting IgG antibodies against SARS-CoV-2 N protein instead of S protein 49

alone are more reliable, considering both specificity and sensitivity scores 2 . 50 SARS-CoV-2 disease burden is suspected to be much larger than reported 51

COVID-19 cases due to low detection rates, especially at the beginning of 52

pandemics. Seroprevalence surveys are needed to monitor the pandemic, both 53 before and after vaccination strategies. 3 Reported COVID-19 cases do not 54

represent the full SARS-CoV-2 disease burden. Case reports are dependent on 55 patients seeking health care, massive local screenings or regional tracking 56

activities. Analysis of data from seroprevalence and serosurveillance is a 57 common strategy for estimating underreporting and real disease burden. There 58

are several features that should be taken into account, including time between 59 infection and antibody development or antibody waning, which must be 60 considered to understand seroprevalence surveys avoiding biases as. Congo fever. The use of blood donor samples means we're sampling mainly 72 asymptomatic and recovered cases of COVID-19 (normal blood donation is 73 allowed after 28 days following COVID-19 symptoms' resolution). 74

Heterogeneity of susceptibility and transmission is hard to evaluate but does 75 exist 4 . A portion of the population is not susceptible to infection from the first 76 pathogen contact. Some may have pre-existing immunity via cross-reactivity or 77 particular host factors such as mucosal immunity or trained innate immunity 78 protection (as it has been reported to be conferred by DTP or BCG vaccination 5 ).

There is as well a proportion of seronegative individuals that will develop 80 immunity by T cell mediated responses but without exhibiting an antibody 81 response 6 . 82 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 preprint this version posted May 1, 2021.

B.0000264 and holds an ISO 9001: 2015 certification endorsing our granting of 92 safety and traceability of any human biological sample we distribute, always 93

behaving Spanish and European rules on human samples and data protection 94 management. 95

The starting hypothesis is the existence of a certain number of asymptomatic 96

carriers of the SARS-CoV-2 virus that would develop antibodies against the new 97 coronavirus 6 (and subsequently contribute to herd immunity). There are recently 98 described early cases in France 7 , arising the question of whether the pathogen 99 could have been circulating even before the official recording of the first cases in 100

Spain by January 31 st , 2020. 101

The main aim of this study is to detect serum antibodies to determine what 102

percentage of the population has had contact with the virus at different times and 103

has therefore developed antibodies against it. A secondary goal is to establish 104 whether sex, age, blood group or haematological abnormalities are related to 105 recent past infection. This epidemiological features would make it possible to 106 optimize hospital management of COVID19 cases, make a good forecast of 107 resources for possible future outbreaks, and would facilitate decisions in the 108 social sphere by reliably knowing the percentage of immunized patients. 109

Aim, design and setting of the study 111

The aim of this study was to obtain, a reliable knowledge of asymptomatic 112 COVID-19 cases and their immunological and haematological characteristics, 113

seroprevalence, rate of positive donations and its temporal evolution along the 114 first wave. So, a retrospective observational analysis was performed. 115

The study population comprised randomised samples from blood, plasma 117 (excluding convalescent) and platelet donations from July 2019 to October 2020.

A total of 12718 samples of 11444 donors over 18 years old were included. 119

Data collection 120 121

All haematological and demographical data were extracted from electronic 122 medical records. The collection form included age, sex, blood group, and 123 laboratory data.

Laboratory data an analyses 126 127

Major laboratory markers were extracted from medical records. Routine blood 128 examinations included leukocyte (WBC), neutrophil, lymphocyte, platelet, 129 monocyte, eosinophil and basophil counts (cells*10^3/µL) and their percentages. 130

Serum biochemical tests recorded were immunoglobulins IgG, IgA and IgM 131

(mg/dL). Haemoglobin (Hb), haematocrit (HCT) and corpuscular mean volume 132

were analysed as well. An automated chemiluminiscence double-antigen 133 sandwich immunoassay for the in vitro semi quantitative detection of total 134

antibodies to SARS-CoV-2 in human serum and plasma was performed. Target  135 antigen of the immunoassay is a recombinant nucleocapsid (N) protein. Elecsys® 136

Anti-SARS-CoV-2 detects antibodies correlating with virus-neutralizing ones and 137

is therefore useful to help characterize the immune reaction to SARS-CoV-2 9,10 . 138

Immunoassay was validated by testing of 6 pairs of samples (plasma EDTA and 139 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 1, 2021. serum) from diagnosed PCR-positive, symptomatic cases infected by mid-April, 140

that were previously reported positive by the CMV, and checked to be as well 141

positive for IgG (Chemiluminiscence, N protein, Abbott Alinity S) and IgA (ELISA, 142 S protein, Euroimmunn) antiSARS-CoV-2 and another set of ten prepandemic 143 samples therefore supposed to be negative. A 100% concordance was yielded by 144 these validation assays. The cut-off was that recommended by manufacturer 145

(OD>1 to report reactivity of the first declared case. As can be seen in Figure 1 , a pronounced slope can be 199 appreciated from then to plateau at week 22. October) but none of them were positive when testing separately IgG, IgA or IgM. 202

Their OD were 1.05, 1.08 and 5.46 respectively. 1142 donations from year 2019 203 were analysed. 204

No differences in anti SARS-CoV2 reactivity due to sex, age or blood group of 205 donors were found ( The first case in Spain was reported on 31/01/2020, but 3 positive donations 232

were found as testing samples collected along 2019. That could be meaningful in 233 two opposite ways: one possible explanation would be cross reactivity to 234 seasonal cold coronaviruses and the other would be that SARS-COV-2 would 235

have been circulating at least since 2019 summer. 236 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 1, 2021. ; https://doi.org/10.1101/2021.04.28.21256242 doi: medRxiv preprint Although the possibility of cross reactivity has been widely commented in mass 237 media, little scientific literature can be found to date. It has been checked that 238 long-term cross-reactive both T-cell and antibodies can be a correlate of 239 protections against COVID-19 8 . It will be therefore important to determine the 240 magnitude and prevalence of this correlates to accurately determine the 241 immunization status of populations, the so named herd immunity. 242

There are as well reports about SARS-CoV-2 circulating in Europe along 2019. 243

Italy's Ministry of Health reported high levels of "unusual" strains of flu and 244 pneumonia concentrated in the area around Milan and appearing in 17 of Italy's 245 20 regions 12 . Our three donors would be even earlier cases. 246

Conversely to clinical forms of COVID-19 13 neither age nor sex have an 247

influence on the probability to develop an asymptomatic infection. Another 248

seroprevalence reports from european countries support this feature 14-18 . It may 249 be therefore ascertained that age and sex do not have a role in SARSCoV-2 250

infection susceptibility, but only in their progression to severe forms. 251

Blood group has been reported elsewhere 6, 19, 20 to confere susceptibility or 252

determine severity (most studies do not split these two concepts) to SARS-CoV2 253

infection. Relationship with susceptibility is not supported by our data. We 254 excluded convalescent plasma donors, opposite to other studies, that focused 255 into these donors 20 .Perhaps blood group might be related only to clinical cases 256 and therefore linked to severity but not related to asymptomatic infection. Another 257 surface antigens such as HLA or KIR might be as well involved and should be 258 studied. 259

CoV2 infection, notably lymphopenia and neutrophilia 21 . Conversely, our data 261 reveal that asymptomatic positive women had a significant neutropenia and 262

lymphocytosis as compared to negative ones. Perhaps this feature is a correlate 263 of protection against clinical severity. Mortality in COVID-19 is associated to low 264 haemoglobin 22 . In our series, haemoglobin and haematocrit were present just in 265 positive males as compared to negative ones. A hampering in O2 transport due 266

to hem-group 23 destruction has been reported to be responsible for that bad 267

prognosis. It seems as if males were more labile to this damage after infection, 268

being this effect visible even when no symptoms are present. 269

We can conclude that seroprevalence due to asymptomatic cases is equivalent to 271 that of general population. Sex and age would not affect COVID-19 susceptibility 272 but its severity. Gender differences are present even in asymptomatic individuals: 273 females are possibly protected by relative lymphocytosis and neutropenia 274

whereas males are would be weaker as seropositive men show a decrease of 275 haematocrit and haemoglobin. Further studies are needed to confirm these 276 gender differences not only in severe but as well in asymptomatic cases as they 277

can help better understand COVID19 pathogenesis and prognosis. 278 279

The authors thank to all blood donors making this work possible by allowing 281 research use of their samples by the Biobanco del Centro de Hemoterapia y 282 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 1, 2021. ; https://doi.org/10.1101/2021.04.28.21256242 doi: medRxiv preprint Hemodonación de Castilla y León. They also thank the staff in charge of blood 283 donation, and lab technicians for their efforts and Roche for its support. 284

This work has been carried out provided free equipment and test reagents from 286

Roche Diagnostics International Ltd. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 1, 2021. . CC-BY 4.0 International license It 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) CC-BY 4.0 International license It 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 preprint this version posted May 1, 2021. ; https://doi.org/10.1101/2021.04.28.21256242 doi: medRxiv preprint

320 Lower prevalence of antibodies neutralizing SARS-CoV-2 in group O 321 French blood donors

Immune Correlates of COVID-19 Control

Brief clinical 325 evaluation of six high-throughput SARS-CoV-2 IgG antibody assays

Sensitivity of anti-SARS-CoV-2 328 serological assays in a high-prevalence setting

Infection fatality risk 331 for SARS-CoV-2 in community dwelling population of Spain: nationwide 332 seroepidemiological study

COVID-19: age, Interleukin-6, C-338 reactive protein, and lymphocytes as key clues from a multicentre retrospective 339 study

Enhanced surveillance of COVID-342 19 in Scotland: population-based seroprevalence surveillance for SARS-343

CoV-2 during the first wave of the epidemic

SARS-CoV-2 antibody prevalence in 346 Brazil: results from two successive nationwide serological household 347 surveys

Repeated leftover serosurvey 349 of SARS-CoV-2 IgG antibodies

Estimated Community 352 Seroprevalence of SARS-CoV-2 Antibodies -Two Georgia Counties

CoV-2 IgG antibodies in Geneva, Switzerland (SEROCoV-POP): a 356 population-based study

ABO Blood 358 Types and COVID-19: Spurious, Anecdotal, or Truly Important 359 Relationships? A Reasoned Review of Available Data