Seroprevalence of SARS-CoV-2 anti-nucleocapsid total Ig, anti-RBD IgG antibodies, and infection in Thailand: a cross-sectional survey from October 2022 to January 2023

Seroprevalence studies on SARS-CoV-2 are essential for estimating actual prevalence rates of infection and vaccination in communities. This study evaluated infection rates based on total anti-nucleocapsid immunoglobulin (N) and/or infection history. We determined the seroprevalence of anti-receptor binding domain (RBD) antibodies across age groups. A cross-sectional study was conducted in Chonburi province, Thailand, between October 2022 and January 2023. Participants included newborns to adults aged up to 80 years. All serum samples were tested for anti-N total Ig and anti-RBD IgG. The interviewer-administered questionnaires queried information on infection history and vaccination records. Of 1459 participants enrolled from the Chonburi population, ~ 72.4% were infected. The number of infections was higher in children aged < 5 years, with evidence of SARS-CoV-2 infection decreasing significantly with increasing age. There were no significant differences based on sex or occupation. Overall, ~ 97.4% of participants had an immune response against SARS-CoV-2. The anti-RBD IgG seroprevalence rate was lower in younger vaccinated individuals and was slightly increased to 100% seropositivity at ages > 60 years. Our findings will help predict the exact number of infections and the seroprevalence of SARS-CoV-2 in the Thai population. Furthermore, this information is essential for public health decision-making and the development of vaccination strategies.

The prevalence of infections.The number of SARS-CoV-2 infections was estimated based on seropositivity of total anti-N Ig antibodies and/or self-reported history of previous SARS-CoV-2 infection.The number of infections stratified by age group is shown in Fig. 3.The total number of total anti-N Ig seropositive and/or COVID-19 infection histories was 73.7% (1076/1459); 74.4% (888/1193) in the general population; and 70.7% (188/266) among healthcare workers.Sex and occupation did not have a significant impact in the risk of developing SARS-CoV-2 infection (p-value = 0.552, and 0.128, respectively).The number of infections in each age group ranged from 60.0 to 83.3% and was significantly different between age groups (p-value < 0.001).In general, the highest percentage of infections was found in the young age group (83.3%, in children aged < 5 years) and decreased slightly in older age groups (60.0%, in individuals aged > 70 years) (Fig. 3a.).The trend in the number of infections observed in the general population (Fig. 3b) was comparable to that observed among healthcare workers (Fig. 3c).Notably, healthcare workers aged above 60 years old exhibited a significant proportion of individuals with natural infection but anti-N total Ig negative, despite the small sample size (n = 6) (Fig. 3c).

Seroprevalence of anti-RBD IgG antibodies.
To assess anti-RBD IgG seroprevalence, anti-RBD IgG was measured.The seroprevalence classified by anti-RBD IgG titer for each age group is shown in Fig. 4. Overall, 97.1% (1416/1459) of the participants were anti-RBD IgG seropositive.Of whom, there were 96.4% (1150/1193) of the general population and 100% (266/266) of healthcare workers seropositive.This result found that the relative risk of seropositivity was significantly higher in healthcare workers than in the general population (Relative risk [RR] = 1.23; 95% CI 1.20-1.26,p-value < 0.01).Seroprevalence was significantly different in each age group (p-value < 0.001) and ranged from 82.3 to 100%.The lowest age stratified seropositivity was observed in individuals under 5 years of age, and it increased slightly to 100% in individuals over 60 years of age.As classified by anti-RBD titer, 43.9% (640/1459) had a high anti-RBD IgG titer, 41.3% (603/1459) had a medium anti-RBD IgG titer, and 11.9% (173/1459) had a low anti-RBD IgG titer.The highest percentage of high anti-RBD IgG titers was found in participants aged 21-30 years (53.3%, 136/255).The anti-RBD IgG titer showed significant differences between the general population and the healthcare worker group (Chi-square = 73.19,p-value < 0.001).Many healthcare workers (63.2%, 168/266) showed a high level anti-RBD IgG titer, while most in the general population (42.5%, 507/1193) had a medium level anti-RBD IgG titer.

Estimated numbers of infections and overall seroprevalence in the Chonburi population.
In this study, the number of infections (anti-N positive and/or self-reported history of previous SARS-CoV-2 infection) and anti-RBD seroprevalence were used to predict the number of infections and the overall seroprevalence in the Chonburi population.

Discussion
Serosurveys play an important role in determining the prevalence and monitoring transmission trends of infectious diseases and their burden in the general population or in selected groups 31 .The reported morbidities and mortality of SARS-CoV-2 vary worldwide.As of January 2023, the cumulative incidence reported of SARS-CoV-2 infection cases in Thailand and Chonburi Province was 4.7 million and 23.7 thousand cases, respectively 32 .This    www.nature.com/scientificreports/respectively.These data strongly suggested that the number of infections reported by the Ministry of Public Health (MoPH) is underestimated.
A previous seroprevalence study performed in Thailand using data from the first three waves of the epidemic between May 2020 and May 2021, found seropositivity of 1.9, 1.5, and 7.5%, respectively 27 .As an extension of that report, this study included data from October 2022 to January 2023-which corresponded to the Omicron wave-and found that approximately 73.7% of the population had experienced SARS-CoV-2 infection.It is possible that almost the entire Thai population was infected with SARS-CoV-2 during the Omicron wave, especially between March and August 2022, as shown in Fig. 2.This is similar to the findings of other studies, which found that antibodies attributed to natural SARS-CoV-2 infection had a detectable high seropositive rate during the Omicron wave 23,[33][34][35] .This positivity rate did not show any significant differences by sex or occupation.The number of infections in the young age group (age < 5 years old) was significantly higher than that in older age groups.These results may be associated with a later implementation of the COVID-19 vaccine in children, especially in those younger than 5 years (as shown in Table 1) and difficulties of controlling social distancing in child communities such as schools.In a small group of healthcare workers aged above 60 years old (n = 6), there was a notable proportion of individuals with natural infection.However, it was observed that anti-N total Ig in some individuals had disappeared, likely due to the waning of anti-N total Ig over time 18 .A larger sample size of participants in this age group will be necessary to understand the dynamics of anti-N total Ig following natural infection and/or vaccination.
At the end of 2021, most of the world's population may have developed antibodies against SARS-CoV-2, either through infection, vaccination, or both 7,8 .For Thailand and Chonburi province, the full doses of vaccine coverage is 77.2% and 84.9%, respectively, and the respective first booster dose coverage is 48.3% and 58.0% 35.The seroprevalence of anti-RBD IgG in this study found that 97.1% of the participants had developed antibodies against SARS-CoV-2, consisting of 96.4% of the general population and 100% among healthcare workers.With ongoing efforts for vaccination against COVID-19 and pandemic waves, current levels of hybrid immunity are likely to be substantially higher.This estimated seroprevalence showed a high prevalence rate, similar to previous studies in other countries that reported a seroprevalence rate greater than 90%, such as Chile (98.7% in 2022) 36 , Kenya (90.2% in January-February 2022) 37 , Portugal (95.8% in April-June 2022) 38 , and Switzerland (93.8% in April-June 2022) 38 .Furthermore, the anti-RBD IgG titer showed that almost all participants in the vaccinated age group were seropositive and maintained a medium to high titer, especially among healthcare workers.Thus, the vaccine had the potential to induce an immune response.Almost all healthcare workers may have had the opportunity to receive booster doses of the COVID-19 vaccine, while the general population may face restricted accessibility or hesitancy towards opting for the booster vaccination.According to a prior survey conducted in Bangkok in 2021, 82.2% of the participants expressed agreement with the idea of administering a third vaccine dose to Thai people to combat SARS-CoV-2 with mutations.Meanwhile, 12.2% remained unsure, and 5.6% disagreed with the statement 39 .
Limitations of this study were due to Thailand's vaccination policy during the first epidemic wave, and the inactivated vaccine being launched for a selected group of individuals at the end of February 2021.As of February 2022, 71.6% of the population received full doses of the vaccination, and the targeting of children aged 5-15 had begun 27 .In March 2022, booster doses of the mRNA vaccine were recommended.In the current study, the participants' vaccination history was obtained solely through questionnaires, which could potentially lead to missing the actual vaccination doses and dates.Since none of the participants in this study reported to have received an inactivated COVID-19 vaccine within six months prior to enrolment, it's possible that all instances of total anti-N Ig positivity were acquired naturally through infection.Although these results cannot fully ensure the representation of the general population in actual numbers, they can significantly affect the comparison of the number of infections and seroprevalence over time.By exploring additional regions in Thailand, the accuracy of seroprevalence data could be improved, leading to a more comprehensive representation of the entire country.
Most participants included in this study achieved a strong immune response against SARS-CoV-2 and had obtained hybrid immunity.This study demonstrates the importance of conducting updated serosurveys.The number of accumulative infections substantially exceeded previous estimates, and overall immunological exposure generated substantial population-level protective immunity.These findings can contribute to guide the planning of public health prevention protocols and the re-evaluation of vaccination strategies in the future, particularly for unvaccinated children.

Study design and ethical considerations.
A population-based, age-stratified, cross-sectional investigation was conducted in a selected province using random sampling to estimate the overall burden of the circulation of SARS-CoV-2 infection in Thailand.The study was part of a national serosurvey for vaccine-preventable diseases.The protocol was reviewed and approved by the Institutional Review Board of the Faculty of Medicine of Chulalongkorn University (IRB numbers 0706/65) and was conducted in accordance with the Declaration of Helsinki and the principles of good clinical practice.All participants or their parents were informed of the objectives of the study and written consent was obtained before enrollment in this study.
Chonburi province, located in the eastern Gulf of Thailand approximately 90 km from Bangkok, was chosen as the representative city of Thailand for several reasons.First, its geographical location offers a mix of urban areas and tourist attractions, with Pattaya City serving as a major tourist attraction for beaches, nightlife, shopping centers and entertainment venues.Second, Chonburi province has several growing industrial estates, including the Eastern Seaboard Industrial Estate, while also preserving rural agricultural landscapes where farmers cultivate a variety of crops.Third, the diverse population of the province engages in various occupations, including tourism, industrial manufacturing, and agriculture.This multifaceted agricultural community not only cultivates

Figure 1 .
Figure 1.Map of Thailand showing the blood sampling sites in 11 districts, Chonburi province, Thailand.The number of blood samples collected for the individual district is indicated.

Figure 2 .
Figure 2. The timeline of infected cases in this study.The left Y-axis represents the number of SARS-CoV-2 infections in this study (bar graph).The right Y-axis represents the number of SARS-CoV-2 infections in Thailand reported by the Ministry of Public Health (area graph).The timeline of infected cases and the duration of the SARS-CoV-2 variant strain outbreaks have been reported in previous studies28,29 .

Figure 3 .
Figure 3.The number of infections measured by anti-N total Ig and/or history of infection (a) in all participants, (b) in the general population, and (c) in the healthcare worker group.
The percentage positivity rate in each age group was calculated based on the actual numbers reported for the Thai population in official government records for each age group.At the end of 2022, the actual census count in Chonburi province and Thailand reported 1,594,758, and 64,867,406 individuals, respectively.The population was classified according to age group in Table 2. Based on the calculations for each age group, the Chonburi population had approximately 1,154,984 (72.4%) infections and 1,553,969 (97.4%) immune responses to COVID-19.Compared to Thailand, the Thai population reported approximately 46,438,034 (71.6%) infections and 63,333,347 (97.6%) immune responses to COVID-19 (Table 2).

Table 1 .
Demographic data of the participants in this study.

Table 2 .
The approximate number of infections and immune responses for COVID-19 stratified by age in Chonburi and Thailand populations.§ Based on the actual census count in December 2022.Data available from https:// stat.bora.dopa.go.th/ new_ stat 30 .