High SARS-CoV-2 seroprevalence in blood donors from Romania after the third COVID-19 pandemic wave

To the Editor,

Recently in this journal, a 15.5% seroprevalence to SARS-CoV-2 in Portugal was reported after the third epidemic wave, of which 13.5% was attributable to natural infection and 2.0% to vaccination [1]. The seroprevalence was 1.7 times higher than the cumulative incidence based on PCR- testing and 2.7 times higher in children. We here report seroprevalence data from Romania indicating an escalating spread of the virus within a year time span, regardless of age, gender, area of residence, or blood type. Our data provide evidence regarding the magnitude of virus transmission in the population.

In Romania, as of September 2020, the seroprevalence of antibodies against SARS-CoV-2 in healthy blood donors was 1.51% [2]. To evaluate the degree of community spread of the virus and to further understand the evolution of the pandemic, this study aimed to estimate the seroprevalence of SARS-CoV-2 infection in blood donors residing in the same geographic area (Timis County) as in our previous study [2], between July and September 2021. Sampling, serologic tests, and statistical methods were identical to those used in our previous study conducted in 2020 [2].

Serum samples of 2395 consecutive volunteer blood donors were collected between 2 July 2021 and 2 September 2021 from residents of Timis County (705,113 inhabitants), who donated blood at the Regional Blood Transfusion Centre in Timisoara, Western Romania. The software used in the Regional Blood Centre provided subjects’ demographic data (age, gender, area of residence) using a code, without their identification.

Blood donors complied with the donation eligibility criteria set by the Romanian Ministry of Health [3]. Individuals underwent a health screening procedure and had to certify that they had no symptoms of COVID-19. Subjects with fever on the day of donation (forehead temperature > 37.3 °C), flulike symptoms 14 days before donation, and persons with a history of travel abroad or those who had close contact with suspected or confirmed COVID-19 cases 14 days before donation were not accepted. Individuals with anaemia, chronic hepatitis, liver cirrhosis, HIV, cancer, schizophrenia, epilepsy, asthma, diabetes type I were excluded from the blood donation procedure [3].

Participants classified as eligible for donation and enrolled in the study were grouped according to their age (18–29 years, 30–49 years and 50–64 years).

Tests were performed at the Clinical Laboratory of the Municipal Clinical Emergency Teaching Hospital in Timisoara, a reference laboratory for COVID-19 testing in Romania. Samples were tested for SARS-CoV-2 antibodies using the Elecsys^®, a double-antigen sandwich electrochemiluminescence assay, designed for Cobas e analysers (Roche Diagnostics GmbH, Mannheim). This test uses a recombinant nucleocapsid protein for identifying specifically the presence of the total antibodies (IgM, IgA, IgG) against SARS-CoV-2 infections, which are not generated after vaccination [4]. Antibodies to the nucleocapsid protein detect natural SARS-CoV-2 infection because this antigen is not targeted by the currently available vaccines in Europe [5]. Testing, including the quality control, and interpretation of results were based on the manufacturer’s criteria as follows: samples with cut-off index <1.0 were considered nonreactive/negative and samples with cut-off index ≥1.0, reactive/positive for anti-SARS-CoV-2 antibodies. The Elecsys Anti-SARS-CoV-2 immunoassay has a specificity of 99.81% and a sensitivity of 100% for past infection in patients at ≥14 days after PCR confirmation.

Statistical analyses were performed using Epi Info Version 7.2 (CDC, Atlanta, GA, USA) and Stata 16.1 (StataCorp, College Station, Texas, USA). For comparison between SARS-CoV-2 positive and SARS-CoV-2 negative blood donors, we used Chi-squared tests, with a p-value of <.05 to represent statistical significance. For between-group comparisons of categorical variables, logistic regression was used. Crude odds ratios and their 95% confidence intervals (95% CIs) were calculated.

This study was approved by the Ethics Committee of the Municipal Clinical Emergency Teaching Hospital in Timisoara, Romania. Written informed consent was obtained from all study participants.

Of the 2395 healthy blood donors aged between 18 and 64 years (mean age = 36.76 ± 10.11 years), 1448 were males (60.46%), and 1660 (69.31%) were residents of urban areas.

The overall SARS-CoV-2 seroprevalence was 41.04% (983/2395). No association between SARS-CoV-2 seroprevalence and age groups, gender, area of residence, ABO blood types or Rhesus blood groups was found (Table 1).

Table 1. Seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid antibodies in Romanian blood donors by age, gender, area of residence, ABO blood types, and Rhesus blood groups, July–September 2021.

Variables	Nr. positive /Nr. tested (%)	OR (95% CI)	p value
Age groups (years)
18–29	262/644 (40.68)	–	–
30–49	581/1419 (40.94)	1.01 (0.84–1.22)	0.911
50–64	140/332 (42.17)	1.06 (0.81–1.39)	0.655
Gender
Male	579/1448 (39.99)	1.12 (0.95–1.32)	0.193
Female	404/947 (42.66)	–
Area of residence
Urban	663/1660 (39.94)	0.862 (0.72–1.03)	0.099
Rural	320/735 (43.54)	–
ABO blood type
O	340/874 (38.90)	–	–
A	399/955 (41.78)	1.13 (0.93–1.36)	0.210
B	157/374 (41.98)	1.14 (0.88–1.45)	0.309
AB	87/192 (45.31)	1.30 (0.95–1.78)	0.101
Rhesus blood group
Rh positive	813/1999 (40.67)	1.10 (0.88–1.36)	0.404
Rh negative	170/396 (42.93)
Total	983/2395 (41.04)	NA	NA

OR: odds ratio; CI: confidence interval; NA: not applicable.

Results of the present study indicate a significantly higher SARS-CoV-2 seroprevalence compared to the prevalence (1.51%) reported in our cohort of blood donors tested in 2020 (p < .001) (Table 2) [2]. The increase in seroprevalence was significant regardless of gender, area of residence, age groups, Rhesus blood groups or ABO blood types (p < .001).

Table 2. Seroprevalence of SARS-CoV-2 nucleocapsid antibodies in blood donors (tested in 2020 and 2021) and the rate of confirmed COVID-19 reported cases in Timis County, Romania.

Nr. positive/ Nr. tested (%) in 2020*	Nr. positive/ Nr. tested (%) in 2021**	Nr. reported cases/ Nr. inhabitants (%), 19 July 2021***
32/ 2,115 (1.51)	983/ 2,395 (41.04)	54,393/ 705,113 (7.71)

*Tests were performed between 8 July 2020 and 1 September 2020.

**Tests were performed between 2 July 2021 and 2 September 2021.

***Confirmed COVID-19 cases reported in Timis County on 19 July 2021, two weeks before the midpoint of sample collection.

The seroprevalence of 41.04% in our study group was 5.3 times higher than the 7.71% rate of the COVID-19 confirmed cases reported in Timis County as of 19 July 2021 (Table 2).

In this study, we provided up-to-date information regarding the seroprevalence of SARS-CoV-2 antibodies in blood donors from Romania. Results indicate a significantly higher SARS-CoV-2 seroprevalence (41.04%) compared to the prevalence (1.51%) reported in our cohort of blood donors tested in 2020 (p < .001) [2]. The increase in seroprevalence was significant regardless of age groups, gender, area of residence, ABO blood types, or Rhesus blood groups [2]. The 1.51% seroprevalence of SARS-CoV-2 antibodies observed in 2020 may be explained by the social restrictions applied in Romania, including strict lockdown until 15^th May 2020, aimed to prevent and control the virus from spreading. The present study was performed one year later, after the third COVID-19 pandemic wave in Romania. Data from this survey shows a marked increase in seroprevalence after the third pandemic wave in blood donors. Our results are similar to previous reports: the seroprevalence of SARS-CoV-2 antibodies rapidly increased up to over 40% in Malagasy blood donors in nine months [6] and from 4.3% to 48.5% in Kenyan blood donors over one year [7]. This confirms the rapid spread of the virus and the hidden magnitude of infection worldwide.

The estimated seroprevalence of 41.04% in our study group also indicates that the rate of SARS-CoV-2 infections was 5.3 times higher than the 7.71% rate of the confirmed cases in Timis County (54,393 cases of 705,113 inhabitants as of 19 July 2021, two weeks before the midpoint of sample collection). This suggests that a significant number of asymptomatic SARS-CoV-2 infections have been detected using serologic tests.

In line with our previous study [2], gender, Rhesus blood groups, and ABO blood types are not associated with SARS-CoV-2 infection and our results are consistent with those reported by Uyoga et al. [7].

This study has several limitations. Study participants are blood donors, apparently healthy individuals, and are not fully representative of the Romanian adult general population. However, screening blood donors could represent a valid tool to evaluate the true extent of SARS-CoV-2 infection within a population [8]. In addition, persons with acute or long-term COVID-19 symptoms have been excluded from donating and the antibody response against SARS-CoV-2 can be low or transient in some individuals [9]. Previous studies indicated that antibody titres may fall over 3–4 months after infection [10]. It is also possible that some convalescents from the first or second COVID-19 pandemic waves were not detected during this survey [9]. Therefore, the real prevalence and true extent of SARS-CoV-2 infection might be higher.

The present study revealed a significant rise in the spread of the virus within a year time span, regardless of age, gender, area of residence, or blood type. This information provides new insights into the degree of herd immunity and evidence regarding the magnitude of virus transmission in the population.

Acknowledgments

We gratefully thank Darius Barbatei, Niculina Raducan, Alina Casu and Codruta Ologu-Schintee for their technical assistance during the study.

Disclosure statement

All authors declare no conflicts of interest. The Elecsys Anti-SARS-CoV-2 immunoassay kits were donated by Roche Diagnostics Romania.