Sero-epidemiological studies are helpful in estimating the true magnitude of the disease in a population by estimating the number of individuals with subclinical /asymptomatic infections. Further, they can provide insights into the immune protection present in the population. In this study, we found nearly 53% of healthy donors that visited our blood bank at the time of the second COVID-19 wave in Pakistan between December 2020 and February 2021, showed positivity for IgG against spike protein of SARS- CoV-2. This seroprevalence is significantly higher than the previously reported data for healthy blood donors from Europe such as; 0.9% in Italy, 1.9% in Denmark, 2.7% in France and 0.91% in Germany (17–19) (20). A study conducted in healthy blood donors in Lombardy, Italy (9), one of the first lock down region in Italy reported a frequency of 19.7% (378/1922) for SARS-CoV2 anti-S1 and anti-S2 IgG antibodies and 21.6% for neutralizing antibodies during March to June 2020 (21). A weighted SARS-CoV-2 seroprevalence of 4.0% was observed in 2857 blood donors from Brazil in April 2020 (22) while New York, United States observed a seroconversion of 1 in every 8 blood donors in June-July 2020 (23). A high level of exposure to the virus was observed in Saudi Arabia, where IgG to spike proteins was observed in 234/1212 blood donors (19.3%) during May to July 2020 (24). Moreover, an increase in seroprevalence was reported in blood donors in parallel with the rise of COVID-19 infection in Jordan (25). Previously, reports of healthy blood donors from Karachi tested in June 2020 (at the peak of the first COVID-19 wave) showed a seropositivity in 15/70 (21.4%) with an increase (to 37.7%) in July 2020 (12).
Seroprevalence of anti-SARS-CoV-2 antibodies are shown to persist for up to 6 months post-disease onset (16). In Portgual, it was shown that seroprevalence was 2.1% in September 2020 and 13.4% in March 2021 (9). In Pakistan, a multi-center study (during 15-30th July 2020) reported seropositivity ranging from 31.1 to 48.1% in 15,390 individuals from seven randomly selected cities from three most populous provinces of Pakistan (Khyber Pakhtunkhwa, Punjab, and Sindh) (13). Younas et al. reported seroprevalence of approximately 38% during peak time of July 2020 at Karachi, one of the largest cities of Pakistan (12). The current study performed December 2020 until February 2021 at the time of the second COVID-19 wave shows a further rise (to 53%) in seropositivity in Karachi. On January 1, 2021, 41,000 tests were conducted each day and approximately 2500 positive COVID-19 cases (6.1%) were confirmed within a 24 h period (7). Our data therefore alludes to much greater infection rates in Karachi than identified from PCR based testing alone. A higher seropositivity is suggestive of active disease transmission in the community and this may be related to over population, congested living conditions and low compliance with the SOPs like wearing mask in public and maintaining social distancing.
Our data showed an increasing frequency of seropositivity to spike and RBD in individuals who were between their second to fourth decades of life. Similar findings have been reported from Saudi Arabia and Pakistan (12, 24). Declining seropositivity in the fifth and later decades might be attributed to decreasing immunity with advancing age, but no conclusion can be made as the studied population comprised predominantly of young adults. Notably, we had very few individuals aged 50 years and over as it was younger individuals who comprised the blood donor cohort.
Though several population-based studies have demonstrated a high seroprevalence in males compared to females (19, 22, 26, 27), such a comparison could not be undertaken because of gender imbalance in the current study with predominantly more male donors. Another limitation of this study is that a detailed COVID-19 history was not taken prior to enrollment of study subjects and any individual who had no signs and symptoms of disease within the past four weeks was eligible for the study. The secondary questionnaire was administered later and only 64% of the study subjects could be followed up.
Previous studies have shown COVID-19 to be present more in individuals with blood group A (24). as compared to those having blood group O (28). However, here, we did not find any significant difference in seroprevalence among different ABO blood group types, corroborating an earlier report from Pakistan (12).
One or more symptoms that are associated with COVID-19 including fever, cough, and loss of smell/taste, fatigue, and malaise were observed in 71(37.4%) individuals. This suggests that almost 60% of the individuals who were infected by SARS-CoV-2 remained asymptomatic. This finding is congruent with a study in Quebec where half to two third seropositive donors were found to be asymptomatic (29). Almost 25% donors had national or international travelling and 15–18 % of them also had an exposure to a suspected or conformed patient with COVID-19. Another interesting finding is that 44/298 of the spike seropositive donors had also been previously tested by RT- PCR because of suggestive symptoms, but only 11 (25%) were positive. The reasons for this low PCR positivity are not known, but a low viral load at the time of PCR testing, inadequate sampling technique or variability in PCR-based diagnosis may be the possible explanations.
Interestingly, we observed a discrepancy between individuals who had positive IgG to spike and those who were positive to RBD. Overall, only 31.2% subjects having IgG to spike demonstrated IgG to RBD. This ratio of spike to RBD positivity was comparable across the age groups investigated. Previously, it has been shown that IgG to spike and RBD both demonstrate high specificity against SARS-CoV-2 (15, 30) and a good correlation with virus neutralization (VN) titers. However, Salazar et al. in 2020 reported anti IgG-RBD to have slightly better correlation with VN in his study on 68 patients with COVID-19 (31). In this data set, we were not able to directly test all samples here for virus neutralizing activity. However, we did test selected RBD antibody positive samples for neutralizing activity and found a correlation between IgG titers and SARS-CoV-2 neutralising effect (32). Therefore, we speculate that RBD antibodies found in our healthy blood donors reflected previous SARS-CoV-2 infection whilst a higher frequency of spike antibodies was secondary to cross-reactive antibodies. Cross-reactive antibodies against SARS-CoV-2 in pre-pandemic sera have been shown in studies from Africa (33, 34). Moreover, a high prevalence of cross-reactive antibodies has been identified in sub-Saharan Africa (35). While SARS-CoV-2 antibody positivity may protect against reinfection (36), the protective role of cross-reactive antibodies against SARS-CoV-2 needs further probing. No definitive conclusion can be drawn as we did not screen for antibodies against other coronaviruses in this study, due to lack of access to testing kits. Another limitation is that the study was conducted at a single institution and a relatively small sample size. Further, as with other seroprevalence studies, the rate of exposure may be under-estimated depending on the time of sampling after COVID-19 as antibody responses wane with time (37).
Factors such as, bacilli Calmette-Guerin vaccination (38), high temperature and humidity (39), and exposure to other viral/bacterial pathogens (40) have all been suggested in contributing to increasing protective immunity against SARS-CoV-2 in the population. High seroprevalence rates suggest active virus transmission in the community and highlights the importance of use of protective measures such as use of mask and social distancing.
Overall, this study provides important insights into SARS-CoV-2 seroprevalence rates in the local population just prior to the introduction of COVID-19 vaccination in February 2021. Further, we propose that cross- reactive antibodies could enhance protective immunity against SARS-CoV-2 in this population.