Material and
Methods
Study
design
We performed a cross-sectional study to recruit renal transplant
recipients who were under regular follow-up in two university hospital’s
transplantation centers (CMF and KMF) and had RT-PCR confirmed COVID-19.
We also recruited consecutive renal transplant recipients who did not
have a history of COVID-19 and were attending transplantation outpatient
clinics in those institutions. We constructed two additional control
groups from a previously screened cohort of health care workers
[10]. None of the included patients were vaccinated for COVID-19
prior to antibody measurement.
We used descriptive comparative design to assess the outcomes. The study
protocol was approved by the local medical ethics committee (approval
no: 2021-2921) and the Ministry of Health’s Scientific Committee
(approval no: 2020-11-30T14_57_30). The study was conducted in
accordance with the 1975 Declaration of Helsinki, as revised in 2013.
Sampling
The study was conducted between December 7, 2020, and February 12, 2021.
The date of blood specimen collection for antibody measurement was
accepted as the enrollment date to the study. The date of RT-PCR testing
was accepted as the first day of infection.
Transplant
recipients
Based on previous studies we predicted seropositivity following COVID-19
as 60% for transplant recipients [9,11] and 90% for subjects from
the general population [12-14]. We performed a power analysis, and
we planned to recruit 42 participants to each group.
A total of 623 patients were attending outpatient transplantation
clinics during the year 2020. Target population was patients who had
COVID-19 following April, 2020. We did not formally screen all patients
under follow-up; however, all of our transplant patients who had an
RT-PCR confirmed COVID-19 history were eligible for the study. We
located 57 patients who had COVID-19, one of them died before the start
of the study, 46 of them accepted to participate in the study.
We recruited transplant patients who gave informed consent to the
COVID-19 negative group if they declared that they did not have a
diagnosis of COVID-19 as of the recruitment day. We also checked if they
had any RT-PCR test due to mandatory screening (before hospitalization
due to any cause, having a household member with COVID-19) and confirmed
that the RT-PCR test was negative.
Controls
We recruited control subjects from a cohort of health care workers that
we examined previously [10]. Details of recruitment and data
collection for those participants were previously described in detail
[10].
In that cohort, 116 subjects were RT-PCR positive. We excluded any
subjects with malignancy or using immunosuppressive drugs. We
transformed the duration between RT-PCR and antibody testing to binomial
data based on eight weeks’ cut-off value. RT-PCR positive control group
is formed by recruiting subjects using propensity score matching based
on age, sex, and transformed antibody testing duration data with a 1:1
ratio.
Among healthcare workers who do not have COVID-19 history, we selected
subjects designated as ”no risk” (health care workers who were not
attending the hospital because of administrative changes related to
pandemic) regarding COVID-19. We excluded any subjects with malignancy
or using immunosuppressive drugs; 106 subjects were eligible for
selection. We used age and sex-based propensity score matching to select
subjects from this cohort with a ratio of 1:1.
We used the same laboratory procedures to measure antibody levels in
those subjects and transplant recipients.
Data
Collection
We filled a standard form for every patient, and we used patient
interviews, medical records of the patients, the hospital’s electronic
database, and the national public health data management system to
collect data. Our form consisted of the following parts; demographics,
clinical data including transplantation history, drug use, laboratory
parameters, history, and clinical data related to COVID-19, and computed
tomography (CT) findings. We also used the COVID-19 severity index to
classify the patients into five mutually exclusive categories;
asymptomatic or presymptomatic, mild, moderate, severe, critical illness
[15].
PCR testing and Assessment of
Antibodies
We used the same methods for RT-PCR testing and SARS-CoV-2- antibody
measurement as described in detail previously [10,16]. For detection
of COVID-19 RNA, a commercial RT-PCR kit (Bio-Speedy COVID-19 RT-qPCR
kit; Bioeksen R & D Technologies Ltd., Istanbul, Turkey) was used. For
detection of SARS-CoV-2 IgG (anti-nucleocapsid protein antibodies),
chemiluminescent microparticle immunoassay (Abbott Laboratories, Cat no:
6R86, Lot no: 16253FN00) was carried out according to the manufacturer’s
instructions, and samples were run on the related instrument (ARCHITECT,
Abbott Laboratories, Abbott Park, IL, USA). Qualitative results were
reported by the instrument with the cut-off value of 1.40 S/C as
recommended.
Statistical
Analysis
Descriptive data were presented as mean and ± standard deviation (SD)
and median and interquartile range (IQR) for the continuous variables
and frequency and percentages (%) for the categorical variables.
Continuous variables were evaluated for normality distribution using
Shapiro-Wilk test. Kidney transplant recipients and control groups were
compared with an independent sample t test for normally distributed
variables and Mann-Whitney U test for non-normally distributed
variables. Categorical variables were compared by using Chi Square or
Fisher’s Exact test for proportion. Multivariate analysis was applied to
determine association between antibody level, group and post-infection
duration. All significance tests were 2-tailed, and values of p
<0.05 were considered statistically significant. All
statistical analyzes were performed by SPSS software version 21
(Chicago, IL).
We employed propensity score matching to balance in observed baseline
covariates and reduce the bias of treatment effect between kidney
transplant recipients and control groups. We assumed at ratio of 1:1 on
age and sex with nearest neighbor matching method. The propensity score
matching was performed using the RStudio (version 4.0.2 software).
According to previous studies, we accepted a seropositivity rate
following COVID-19 as 60% for transplant patients and 90% for the
general population. Therefore, considering the percentage of previous
studies, we performed power analysis (Gpower software, version 3.1,
Kiel, Germany) with 90% power and an error of 0.05 to determine the
minimum sample size for RT-PCR positive kidney transplant recipients and
control groups. A minimum sample size of 42 was estimated in each group.