Breakthrough COVID-19 infections – Analyzing our experience

Objective: There are many cases of post-vaccination COVID-19 globally. Also, literature on serum antibodies after vaccination is abundant. Our research focuses on breakthrough infections reported at our institution during the third wave of COVID-19. Methods: A total of 177 people recruited at the Indus Hospital Karachi between May to September 2021 with COVID-19 infection were divided into vaccinated, partially vaccinated, and unvaccinated cohorts. Furthermore, a subset of the vaccinated cohort was tested for anti-NP and anti-S antibodies. Results: There were 119 patients with breakthrough infection, however, 74% had mild symptoms. The antibodies against NP and S were found at a higher level in those who had a breakthrough infection in comparison to healthy vaccinated controls. Conclusion: Vaccination does not prevent disease but does confer some immunity causing less severe infection.


INTRODUCTION
The COVID-19 pandemic has negatively impacted global health and economy.Mass vaccination drives continue using vaccines of varying platforms and efficacy in attempts to decrease transmission and severity.2][3] These are defined as the detection of SARS-CoV-2 RNA in respiratory specimens of patients >14 days after vaccination. 4The CDC has noted 5800 breakthrough infections, mostly above 60 years, out of which one third were asymptomatic,470 severe and 74 fatal cases.65% were women. 5here are many studies that show robust antibody response post-COVID-19 vaccination, most data being for mRNA vaccines. 6One study found that low anti-Spike antibody levels post-vaccination pose a high risk for breakthrough infection and these antibodies may wane earlier in AstraZeneca as compared to Pfizer. 7nother study found maximum antibody response with Pfizer, followed by AstraZeneca, Sputnik, and Sinopharm showing that individuals were at a higher risk for breakthrough infections who got the latter three vaccines, especially Sinopharm. 8In Pakistan, Sinopharm was the primary vaccine administered to most populations initially, we are seeing breakthrough infections mandating a study on breakthrough COVID-19 and antibody levels.

METHODS
This study was conducted between 18 th May to 20 th September 2021 at the Indus Hospital Karachi under the Ethics committee approval number IHHN_ IRB_2021_06_017 on 13 th July 2021.All Health care workers (HCWs) who tested positive for COVID-19 by a nasopharyngeal swab RT-PCR were considered eligible after informed consent.All symptomatic HCWs and those with exposure to COVID-19 patients, reported to the Infection Control department (ICD) of the hospital for COVID-19 nasopharyngeal RT-PCR.We classified positive cases into three categories: 'breakthrough infection' (at least two weeks had passed after completion of vaccine), 'unvaccinated' (COVID within one week of first dose of vaccine) and 'partially vaccinated' (tested positive after one week of first dose and the second dose was missed).Demographic and relevant clinical details were taken.Hospitalized COVID-19 breakthrough infection patients were also recruited after consent.
A subset of HCWs with breakthrough infections were registered for serum anti-COVID-IgG testing, where first sample was taken within a week of testing positive for COVID-19 via nasopharyngeal swab RT-PCR, and the second sample after one month.A control group comprising of healthy vaccinated individuals who never had COVID-19, was recruited for comparison.Blood samples for the control were drawn at 4-8 weeks after vaccination.Anti-COVID-NP-IgG and anti-COVID-S-IgG were tested using enzyme linked immunosorbent assay (ELISA) kits provided by AESKU, Germany.The antibody titers of <8 IU/ml was taken as negative, between 8 and 12 IU/ml as indeterminate and >12 IU/ml was considered as positive according to manufacturer's instructions.
Data analysis was done using SPSS version 26.0.The normality of quantitative variables was assessed using Shapiro Wilk test.The normally distributed variables, the duration between symptoms and last day of vaccination was compared between the groups using students t-test.While for the non-normal variables such as age, duration of symptoms, total duration of illness and maximum ordinal score, Mann Whitney U test was applied.Chi- Needed high flow oxygen 4 Other categorical variables were compared using fisher exact test.P-value of less than 0.05 was considered significant.
A total of 206 participants were recruited; 177 were COVID-19 cases and 29 healthy controls.Among cases 119 were true breakthrough, 50 were partially vaccinated and eight were unvaccinated.Clinical characteristics of true breakthrough and partially vaccinated cases.No significant difference was found in the positivity rate of COVID-19 IgG antibodies between two groups, however, both anti-NP and anti-S antibodies coexisted in breakthrough patients at a higher rate while isolated anti-S antibodies was found in higher percentage of healthy controls.Median titers of both antiNP and antiS antibodies were significantly higher in breakthrough patients (Table-III).We then divided our patients and controls according to their post-vaccination period at which their samples were collected and found no difference in the positivity rates of both antibodies between groups except for anti-NPantibodies that were found higher in patients than controls when tested before four weeks post vaccination.However, median titers of these antibodies were significantly high in patients compared to controls at both time points.
Amongst the 27 patients, re-tested at four to eight weeks, most of them found initially positive for anti-NP antibodies remained positive (18; 95%) while one became indeterminate.Of six samples that were initially tested negative, five turned positive at this time (83%).While of two indeterminate samples, one became positive and one negative on repeat testing (P=0.097).For anti-S antibodies, 23 samples were initially positive, three were negative and one indeterminate had same results on repeat testing (Table-III).

It is a naive belief that previous COVID-infection or vaccination confers life-long immunity as SARS-
CoV-2 is a non-monotypic virus.Immune response to COVID-19 is heterogeneous and influenced by several host characteristics causing a clinical spectrum ranging from asymptomatic to fatal disease.Similarly, immune protection after vaccination is determined by a complex interplay between host factors like age, gender, genetics, ethnicity, comorbidity, the predominant viral strain, immune evasion, inoculum, community transmission, the type of vaccine and post-vaccination period. 9All vaccines in current use are efficacious at limiting the disease rather than blocking infection. 10][13] We observed similar findings in our study where breakthrough cases had predominantly mild symptoms, needed symptomatic care, and only 16.8% required hospitalization.
Unfortunately, 6.7% of breakthrough infections ultimately proved fatal, highlighting the fact that vaccination does not completely prevent death.Morbidity and mortality in patients hospitalized with severe disease were seen mainly in the unvaccinated cases.
The predominant circulating strain being reported globally and in Pakistan at the time of the study was the Delta variant, a highly transmissible variant causing severe disease with reported attenuated response from vaccination. 14The risk of severe breakthrough infection, however, remains low especially with mRNA vaccines like the BNT162b2 vaccine, while other vaccines being less effective. 14,15Breakthrough infection caused by Delta variant is comparable in transmission to that in unvaccinated individuals with similar viral burden in the upper respiratory tract. 16A decrement in vaccine efficacy has been reported after 12 weeks. 17The vaccine rollout in Pakistan has been mainly with the inactivated wholevirus vaccines Sinopharm, Sinovac and the Spike protein expressing adenovirus five vector based CanSino BIO, and robust literature on these vaccines is still unavailable.The overall reported efficacy from phase three trials for these three vaccines are 73%, 83.5% and 57.5% respectively, with CanSino BIO trial reporting 91.7% efficacy in prevention of severe disease. 18,19However, their performance against the Delta variant has not been described.The circulating Delta virus combined with types of vaccine used for the primary vaccination may be the prime reason for breakthrough infections among HCWs observed during the study period, who were at a high risk of exposure to the virus.
Ideally, a vigorous antibody response in any infection correlates with good outcome but till-date no antibody threshold has emerged to correlate the protection against COVID-19 breakthrough infections. 20eutralizing antibodies against the spike protein and its receptor-binding domain have been reported as primary immune predictors of protection against symptomatic infection and increasing antibody levels correlate with higher vaccine efficacy. 21,22In Pakistan, neutralizing antibody titers are not available, and only anti-S or anti-NPIgG antibody titers are available commercially.Some correlation between neutralizing antibody titers and anti-S antibodies has been reported but its application to clinical care is uncertain. 23n this study, significantly high antibody titers for both anti-NP and anti-S antibodies were seen in breakthrough patients confirming previous study findings. 24The reason can be explained that antigen challenge has stimulated preformed memory B-cells as a result of vaccination or previous COVID-19 infection.This is supported by the fact that in healthy controls, anti-S antibody titers were found low and anti-NP antibodies were mostly absent.Another reason of high positivity and high antibody titers in breakthrough patients could be an aberrant immune response leading to symptomatic COVID-19 on one hand and early, uneventful recovery due to the stimulation of already activated effector cells and molecules of immune system secondary to vaccination.Presence of anti-NP antibodies in some of our healthy controls can be due to prior asymptomatic COVID-19 infection.It has been reported that immunosuppression, comorbidity and old age result in a diminished immune response. 24In our cohort, the asthmatic patients on steroids confirm the pattern of more severe disease though the numbers are very low.Interestingly median age of patients was found low in breakthrough infections compared to controls. 24mitations: Our study has several limitations.Estimation of neutralizing antibodies and test for cell mediated immunity were not performed.Serum antibodies against COVID-19 were performed on only 63 patients, further splitted in cases and controls, a number too small to make an effective comparison.No immune correlates could be identified and repeat antibody testing could only be done in 27 patients; thus, no pattern of immunity overtime could be identified.Further, the variants/clades of SARS-CoV-2 were not determined.

CONCLUSION
Our study found that breakthrough infections are usually mild which points towards some protection provided by vaccines.The anti-NP and anti-S titers were higher in infected people in comparison to healthy controls which could be a booster response to viral exposure.Further studies are needed to understand human immunological response to COVID-19.
Grant Support & Financial Disclosures: None.
applied to compare categorical variables, gender, type of vaccination and history of COVID-19.

Table - I
: Characteristics of true breakthrough and partially vaccinated cases

Table -
II: Baseline characteristics of breakthrough cases vs Controls (in whom anti-COVID-19-IgG was tested).Although 80% of breakthrough cases were asymptomatic to mild symptomatic, moderate, severe, critical to fatal diseases were still observed in 5%, 5.8%, and 9.2% cases respectively.Eighteen point six percent of breakthrough cases were re-infections .Home isolation and symptomatic care were needed in 80.7% of breakthrough cases while 16.8% needed hospitalization (Table-I).COVID-19 IgG was estimated for 63 participants including 34 HCWs with breakthrough infections and 29 healthy vaccinated controls.Their baseline characteristics are described in Table-II.The cases were significantly younger by age than controls (P= 0.044).Gender, comorbidity, and type of vaccines administered among cases and controls were not statistically significant.Sinopharm was received by 79.4% of breakthrough infection cases and 82.7% of controls.

Table -
III: Comparison of anti-COVIDIgG between patients with breakthrough infection and vaccinated healthy controls.