Coronavirus infection and neutralizing antibody responses among liver transplant recipients: single-center study

Background Liver transplant (LT) recipients are classified as a high-risk group and should receive regular surveillance for COVID-19 and are expected to have higher viral load and prolonged viral shedding. Virus-specific neutralizing antibodies (NAb), induced by infection, can prevent viral infection by deactivating viral access to host receptors. Aim To estimate the incidence of SARS-CoV-2 infections and ascertain the levels of NAb among LT recipients. Method This cross-sectional study included LT recipients. The survey included 14 parameters about demography, date since operation, and immunosuppressant medications and 11 parameters about COVID-19 infection. NAb was done by electrochemiluminescence immunoassay. Results Only 39 LT recipients responded to this survey. Their median age was 57, and 74.4% of them were men. Comorbidities were present in 64% of cases, and DM was the most common comorbidity. Immunosuppressants used were Tacrolimus in 56.4% and Tacrolimus with mycophenolate mofetil in 15.4%. Fifteen cases (38.5%) had suspected infection, and 2 (5.1%) had confirmed infection. Three patients (17.6%) were hospitalized with no mortality. NAb was done in 34 cases, and it was positive in 5 cases (14.7%) with a median level (range) of 0.075 (0.04–27). Three out of 5 cases with a positive NAb test did not give a history of either suspected or confirmed COVID-19 infection. Conclusion COVID-19 surveillance in LT recipients is important and suggests a relatively favorable clinical course despite the presumed challenges of immunosuppression. COVID-19 was associated with low hospitalization in LT recipients. NAb indicates a potential immune response even in asymptomatic LT recipients.


Introduction
Severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2) was responsible for the coronavirus infectious disease 2019 (COVID-19) [1].It soon spread to almost every country around the world, and the World Health Organization declared that COVID-19 was a public health emergency on January 30, 2020.
Liver transplant recipients are classified as a risk group and should receive regular surveillance for COVID-19.A great viral load and prolonged shedding in transplant recipients resulting in more infectivity and spread to others were anticipated [2].
Liu et al. [3] stated that liver transplant (LT) patients could be susceptible to SARS-CoV-2, with a worse prognosis compared to the normal population.
The LT society of India (LTSI) stated that SARS-CoV-2 can be transmitted to LT recipients and they can behave as asymptomatic carriers and sources of viral spread [4].
Virus-specific neutralizing antibodies (NAb), which are induced by infection or vaccination, can help to block viral infection later or during subsequent encounters with the pathogen [5,6].In general, Nabs help confer immunity by deactivating viral access to receptors used to enter host cells and/or binding to viral capsids so as to block the key step of uncoating the viral genome [7].Neutralizing antibodies (NAbs) to SARS-CoV-2 can be found in most infected persons 10-15 days after the onset of presenting symptoms [8].The titers of NAbs were variable in different patients [9].

Rational
It is essential to screen for the occurrence of SARS-CoV-2 infections among liver transplant recipients.To our understanding, no study to date has been undertaken in our country to determine levels of NAb in liver transplant recipients.

Aim of the study
The study was designed to estimate the occurrence of SARS-CoV-2 infections among liver transplant recipients.Also, to ascertain the levels of Nab in liver transplant recipients.

Type of the study
Prospective cross-sectional study.Study setting: Al-Rajhi Liver Hospital.

Study subjects Inclusion criteria
All liver transplant recipients in Al-Rajhi Liver Hospital who accept to participate.

Exclusion criteria
Those who refuse to participate in the study.

Ethical approval
The study protocol was reviewed and approved by the Committee of Medical Ethics of the Faculty of Medicine, Assiut University (IRB local approval number: 17300475).All participants gave their written consent to participate.Data collection was done in October, and November 2020, after obtaining the Ethical Approval.

Study tools
1-Survey included 21 parameters (14 parameters cover the demographic data, date since the operation, and immunosuppressant medications, and 11 parameters related to coronavirus infection).The authors developed and validated the survey used in this study.Internal validity was established in a pilot study of 10% of participants.Cronbach-α was found to be 0.72.SARS-CoV-2 infection was defined according to the Ministry of Health and population definitions of suspected and confirmed cases [10].2-Serological test searching for Neutralizing Ab against SARS-Corona virus 2 in liver transplant recipients (whether who give symptoms or are asymptomatic) was done.It was detected by the Electrochemiluminescence immunoassay (ECLIA) method.For the measures of the presence of NAB in the serum samples, a Cobas 411 immunoassay analyzer that employed an electrochemiluminescence immunoassay (ECLIA) was used.Specifically, an Elecsys Anti-SARS-CoV-2 immunoassay kit was used for the in vitro qualitative detection of antibodies (including IgG) to SARS-CoV-2 in human serum and plasma.The assay used a recombinant protein representing the nucleocapsid (N) antigen in a double-antigen sandwich assay format.The test aids in the determination of the immune reaction to SARS-CoV-2.Elecsys Anti-SARS-CoV-2 detects antibody titers, which have been shown to positively correlate with neutralizing antibodies in neutralization assays.Results were considered non-reactive when kit values were < 1 and reactive when kit values were ≥ 1.The titers measured in reactive samples were recorded.

Statistical analysis
Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS version 17, SPSS Inc., Chicago, IL, USA).Continuous data were all presented as the median and interquartile range, while categorical data were presented as frequencies and percentages.

Results
Fifty-one cases were subjected to living donor liver transplantation in our center from November 2014 to May 2019.Only 39 liver transplant recipients responded to this survey as 2 cases were non-Egyptian, and 10 cases refused to participate.
The median age of the respondents was 57 and 74.4% were men.61.5% were living in rural areas.HCV-related LC was the most common cause of transplant in Egypt, followed by HCC in 35.9%.Comorbidity was present in 64% of cases and DM was the most common.Duration since transplant was (1 -3 years) in 53.8% and more than 3 years in 30.8%.The most common immunosuppressant is Tacrolimus only in 56.4% and Tacrolimus with mycophenolate mofetil in 15.4%.About 69.2% of the respondents did routine follow-up laboratory investigations and imaging in a regular manner despite the occurrence of the pandemic (Table 1).
Answers to parameters regarding COVID-19 infection among liver transplant recipients during the first wave of the pandemic are shown in Table 2.
Out of 17 cases, only 3 were managed in the isolation hospital (17.6%).Reduction of the Tacrolimus dose was done in 4 cases (23.5%).Reduction of MMF or Everolimus doses was done in 9 cases (52.9%).
Neutralizing Ab test was done in 34 cases.Neutralizing Ab positivity was reported in only 5 cases out of 34 (14.7%) with a median (range) of 0.075 (0.04-27).Three out of 5 cases with positive neutralizing Ab test did not give a history of COVID-19 infection.
Complications occurred in 2 cases (ischemic hepatitis in 1 case and segmental and subsegmental pulmonary embolism in 1 case) and both improved and were discharged from the hospital.Three months later, the case with ischemic hepatitis died because of progressive liver failure.

Discussion
Neutralizing antibody (NAb) evaluations are useful for the determination of individual or herd immunity against SARS-CoV-2, vaccine efficacy, and long-term humoral protective response [9].Serological assays represent the test of choice to determine prior exposure to SARS-CoV-2.However, whether the detected antibodies can neutralize the virus and provide protection on subsequent exposure remains a point of debate.Antibodies against RBD of the S protein have been shown to be the primary source of neutralizing antibodies against the virus [11,12].This single-center study was designed to estimate the occurrence of SARS-CoV-2 infections among liver transplant recipients and to ascertain the levels of Nab in liver transplant recipients.
Despite the use of immunosuppressive drugs, neutralizing Ab positivity was reported in only 5 cases out of 34 (14.7%) with a median (range) of 0.075 (0.04-27).[14].However, in the current study, reduction of the tacrolimus dose was done in 4 cases (23.5%) and reduction of Mycophenolate mofetil or Everolimus doses was done in 9 cases (52.9%).
The national Egyptian protocol for living donor liver transplantation (LDLT) recommended reduction/ hold of calcineurin inhibitors (CNI) dose, according to disease severity and radiological findings in CT chest.It is essential to consider reducing azathioprine or mycophenolate dosages [15].Kulkarni et al. (2021), in their systematic review and metaanalysis, reported that immunosuppression was modified in 55.9% of patients after COVID-19 infection [16].
Only 17.6% of our cases need hospital admission; however, none of them need ICU admission.High numbers of infected cases in Egypt pass officially undetected and homely recovered [17].The incidence rate of hospitalization for COVID-19 was 14.4% in the general population in Italy [18].While in the study of Anwar et al. (2021), 42.6% of LT cases need hospitalization and only one of them needs ICU admission [14].Their cases were recruited from March 2020 to August 2020, while our cases were recruited in October and November 2020.LT and non-LT patients with COVID-19 infection have a similar risk of adverse outcomes in a systematic review and meta-analysis [16].
Chronic immunosuppression is considered a doubleedged sword in COVID-19.
While it may facilitate viral replication in the early phase of the infection, it could also, ameliorate the aberrant immune response which is responsible for the most severe forms of the disease [19].Liver transplant recipients show a lower prevalence of anti-SARS-CoV-2 antibodies and more pronounced antibody levels decline [20].

Limitations of the study
First, this was a single-center study.Second, the absence of a control group comprising immunocompetent individuals makes it challenging to compare the outcomes of liver transplant recipients with the general population and assess the impact of immunosuppression on COVID-19 severity.Third, although the study assesses neutralizing antibodies, it does not provide comprehensive data on the overall immune response, such as T cell responses, which could contribute to a more thorough understanding of immune protection.

Conclusion
COVID-19 infection surveillance in LT recipients is important and suggests a relatively favorable clinical course despite the presumed challenges of immunosuppression.COVID-19 infection was associated with low hospitalization.NAb indicates a potential immune response even in asymptomatic LT recipients.

Table 1
Demographic data, cause of liver transplant, comorbidity, and immunosuppressant regimens of liver transplant recipients