Clinical and Experimental Medicine (2024) 24:1

https://doi.org/10.1007/s10238-023-01256-1

RESEARCH

The demographic, laboratory and genetic factors associated with long

Covid‑19 syndrome: a case–control study
Ensiye Torki1 · Fahimeh Hoseininasab1 · Marjan Moradi2 · Ramin Sami3 · Mark J. M. Sullman4,5 ·
Hamed Fouladseresht1

Received: 19 October 2023 / Accepted: 15 December 2023

© The Author(s) 2024

Abstract
Long Covid-19 syndrome (LCS) manifests with a wide range of clinical symptoms, yet the factors associated with LCS
remain poorly understood. The current study aimed to investigate the relationships that demographic characteristics, clinical
history, laboratory indicators, and the frequency of HLA-I alleles have with the likelihood of developing LCS. We extracted
the demographic characteristics and clinical histories from the medical records of 88 LCS cases ­(LCS+ group) and 96 indi-
viduals without LCS ­(LCS− group). Furthermore, we evaluated the clinical symptoms, serum levels of interleukin (IL)-6
and tumor necrosis factor-α, laboratory parameters, and the frequencies of HLA-I alleles. Following this we used multiple
logistic regression to investigate the association these variables had with LCS. Subjects in the ­LCS+ group were more likely
to have experienced severe Covid-19 symptoms and had higher body mass index (BMI), white blood cell, lymphocyte counts,
C-reactive protein (CRP), and IL-6 levels than those in the L ­ CS− group (for all: P < 0.05). Moreover, the frequencies of the
HLA-A*11, -B*14, -B*38, -B*50, and -C*07 alleles were higher in the ­LCS+ group (for all: P < 0.05). After adjusting for
the most important variables, the likelihood of suffering from LCS was significantly associated with BMI, CRP, IL-6, the
HLA-A*11, and -C*07 alleles, as well as a positive history of severe Covid-19 (for all: P < 0.05). Our study showed that a
history of severe Covid-19 during the acute phase of the disease, the HLA-A*11, and -C*07 alleles, higher BMI, as well as
elevated serum CRP and IL-6 levels, were all associated with an increased likelihood of LCS.

Keywords Long Covid-19 syndrome · Severity · Interleukin-6 · HLA class I

Introduction
In May 2020, the term “Long Covid-19 Syndrome (LCS)”
was introduced in response to reports of persistent initial
symptoms, the recurrence of previously resolved symptoms,
or the appearance of new symptoms in some individuals
who had recovered from the acute phase of severe acute
respiratory syndrome coronavirus (SARS-CoV)-2 infec-
tion [1, 2]. Subsequently, the World Health Organization
(WHO) established a clinical case definition to facilitate a
consensus description of LCS. According to this definition,
LCS is characterized by the continuation or emergence of

1
* Hamed Fouladseresht Department of Immunology, School of Medicine, Isfahan
fouladseresht@med.mui.ac.ir University of Medical Sciences, Isfahan, Iran
2
Ensiye Torki Department of Genetics, School of Science, Shahrekord
ensytor98@gmail.com University, Shahrekord, Iran
3
Fahimeh Hoseininasab Department of Internal Medicine, School of Medicine,
fahim60.h@gmail.com Isfahan University of Medical Sciences, Isfahan, Iran
4
Marjan Moradi Department of Life and Health Sciences, University
marjan.moradi800570@gmail.com of Nicosia, Nicosia, Cyprus
5
Ramin Sami Department of Social Sciences, University of Nicosia,
raminsami@yahoo.com Nicosia, Cyprus
Mark J. M. Sullman
Sullman.M@unic.ac.cy

Vol.:(0123456789)
 1 Page 2 of 13
new symptoms at least three months after the initial SARS-
CoV-2 infection. These symptoms persist for at least two
months and cannot be explained by any other diagnosis [3].
Global estimates indicate that 54% of hospitalized and 34%
of non-hospitalized Covid-19 patients experience long-term
symptoms following recovery from the acute phase of the
disease [4]. The diverse range of symptoms in LCS, includes
fatigue, dyspnea, cough, cognitive and mental disorders,
headache, chest and joint pains, loss of smell and taste, hair
loss, insomnia, as well as cardiovascular and gastrointestinal
issues, suggesting that LCS affects multiple organs [5, 6].
However, despite recent advances, the pathogenesis of LCS
remains poorly understood. Several hypotheses have been
suggested regarding the pathogenic mechanisms of LCS,
including persistent ineffective antiviral immune responses
[7, 8], which may result in long-term symptoms caused by
direct cytopathic effects, chronic inflammation, and autoim-
munity [2].
Given the substantial number of individuals affected, the
diverse array of symptoms and phenotypes, and the absence
of treatment and rehabilitation guidelines, LSC has emerged
as an important global health challenge. LCS not only
reduces an individual’s ability to resume their daily lives and
work, but also increases the healthcare burden and economic
losses caused by the long-term complications of Covid-19
[9, 10]. These issues highlight the need to identify diagnostic
and prognostic factors for LCS. Research has shown that sev-
eral factors, including gender, the SARS-CoV-2 variant, pre-
existing medical comorbidities, and Covid-19 vaccination,
can influence the likelihood of developing LCS. Specifically,
being female and having pre-existing medical comorbidities
may increase the risk of experiencing long-term Covid-19
symptoms [11]. On the other hand, individuals who are fully
vaccinated before SARS-CoV-2 infection and those infected
with the Omicron variant (compared to other variants) may
have a lower risk of developing LCS [12, 13]. The impact
of older age and post-infection vaccination on the risk of
LCS remains unclear [11, 12]. Moreover, potential targets
for predicting and diagnosing LCS may involve the immune
system’s cellular and molecular components, which could be
linked to SARS-CoV-2 infection progression and subsequent
chronic complications.
Due to their crucial role in stimulating adaptive immune
responses, human leukocyte antigens (HLAs) are likely
genetic indicators for predicting the prognosis of infectious
diseases [14–16]. These molecules consist of a group of
proteins expressed on the cell surface that stimulate cellu-
lar and humoral immune responses by presenting peptides
derived from non-self-antigens to appropriate T cells [17].
HLA class I (HLA-I) molecules specifically present endog-
enous antigen-derived peptides, including viral antigens, to
activate ­CD8+ T cells, while HLA class II (HLA-II) mole-
cules present exogenous antigen-derived peptides to activate
Clinical and Experimental Medicine (2024) 24:1
­ D4+ T cells [18]. The major HLA-coding genes are located
C
on the short arm of chromosome 6 (6q21) and are known
as the most polymorphic region of the human genome [17].
The high degree of polymorphism and heterozygosity in the
HLA genes enables the immune system to react dynamically
to a broad range of pathogens and microorganisms, while
also influencing the individuals’ susceptibility to infectious
diseases [19]. Previous research has found a strong relation-
ship between the specific HLA-I alleles carried by an indi-
vidual and the severity of viral infections, as well as their
long-term complications. For instance, individuals carrying
the HLA-A*02 allele exhibit heightened protection against
the human immunodeficiency virus (HIV), whereas those
with the HLA-B*13 allele may be more susceptible to the
progression and chronicity of HIV disease [20]. Further-
more, previous studies have found that individuals carrying
the HLA-A*33 or -A*44 alleles are more susceptible to the
progression of varicella-zoster virus (VZV) infection symp-
toms and to chronic and neurological symptoms. In contrast,
individuals with the HLA-A*02 allele demonstrate effective
control over the infection during its early stages [21, 22].
In the context of Covid-19, research has revealed potential
associations between specific HLA-I alleles and variations
in the severity and duration of the disease. Specifically, the
HLA-B*35 allele has been associated with milder and more
restricted Covid-19, while the HLA-A*11, -B*27, -B*51,
and -C*01 alleles have been linked with severe Covid-19
[23–25].
The inability to promptly control infectious agents dur-
ing the early stages of an infection, coupled with difficulties
in completely clearing them during the later stages, may
be the primary cause of severe and prolonged complica-
tions in individuals carrying certain HLA-I alleles [26, 27].
Repeated immune system activation due to the sustained
presence of viral antigens, iatrogenic damage, and oppor-
tunistic secondary infections, can skew immune responses
towards persistent and ineffective inflammation. This in turn
can result in different long-term complications depending on
the affected tissues and organs [28–30]. Recent studies have
shown that high levels of inflammatory mediators, such as
interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and
C-reactive protein (CRP) after the acute phase of Covid-19,
are associated with an increased incidence of neurological,
pulmonary, cytoskeleton, and fatigue-related complications
in LCS patients [31–33].
Therefore, investigating the interaction between the
HLA-I alleles and inflammatory mediators in relation to the
likelihood of LCS represent a highly practical approach to
identify potential predictors of chronic inflammatory condi-
tions and LCS following the acute phase of Covid-19. This
endeavor can significantly contribute to the prevention and
management of long-term complications, thereby reducing
the healthcare burden and economic losses attributable to
Clinical and Experimental Medicine (2024) 24:1
LCS. To achieve this objective, we conducted a compara-
tive analysis of demographic characteristics, clinical history,
serum levels of IL-6 and TNF-α, laboratory parameters, and
the frequency of the HLA-I alleles in both LCS patients and
their controls.
Materials and methods
Study design and participants
In this case–control study, all participants were selected
among Covid-19-recovered individuals, who were referred
to hospitals affiliated with the Isfahan University of Medical
Sciences in Iran between July and September 2021 (the peak
of the SARS-CoV-2 delta variant outbreak) due to acute
Covid-19 and whose medical records contained all required
details (i.e., details about the SARS-CoV-2 infection, Covid-
19 severity during hospitalization, the duration of hospital
and intensive care unit (ICU) stays, and any pre-existing
medical conditions).
Participants had to meet the following eligibility criteria:
Persian (Fars) ethnicity, aged between 30 and 65 years old,
confirmation of a SARS-CoV-2 infection through reverse
transcription-quantitative polymerase chain reaction (RT-
qPCR) test and computerized tomography (CT) scan, clas-
sification of disease severity according to WHO’s interim
guidance, and no pre-existing medical conditions (e.g., dia-
betes, hypertension, malignancy, immunodeficiency, auto-
immune, or chronic inflammatory diseases). Furthermore,
to minimize potential vaccine-related influences, we only
included participants who had received two doses of the Sin-
opharm Covid-19 vaccine after recovery, with the last dose
being at least a three months earlier. To prevent potential
perturbation of inflammatory parameters, individuals who
had experienced an infectious disease or received a vacci-
nation within the past three months were excluded from the
study. In addition, individuals with a history of smoking or
drug addiction were also excluded.
Eligible participants were contacted by phone and invited
for a clinical examination and face-to-face interview with a
specialist. During these interviews, a standardized question-
naire was used to investigate whether each participant had
any persistent symptoms, including fever, headache and diz-
ziness, tinnitus and earache, cough and shortness of breath,
sore throat, fatigue, loss of smell and taste, cardiac problems,
muscle and joint pain, insomnia, memory and concentration
problems, depression and anxiety, gastrointestinal problems,
hair loss, and vision problems. The symptoms were assessed
using guidelines provided by the Centers for Disease Con-
trol and Prevention (CDC) and the National Health Service
(NHS). Participants reporting one or more symptoms per-
sisting from the onset of Covid-19 to at least three months
Page 3 of 13 1
later were classified into the ­LCS+ (case) group, while those
without such symptoms were placed into the ­LCS− (control)
group. Following the receipt of written informed consent
and the interview, 10 mL of whole blood was collected from
each eligible participant for laboratory analysis. The flow
diagram of participant selection is illustrated in Fig. 1.
This study adhered to the Declaration of Helsinki for
research involving human participants and received ethical
approval from the Ethics Committee of the Isfahan Uni-
versity of Medical Sciences, with the ethics code: IR.MUI.
MED.REC.1400.795.
Laboratory assessments
For the laboratory tests, 7 mL of blood from each partici-
pant was immediately sent to a clinical laboratory. The tests
included complete blood count (CBC), assessment of pro-
thrombin time (PT) and partial thromboplastin time (PTT),
and measurement of various indicators, such as creatinine
(Cr), alanine transaminase (ALT), aspartate aminotrans-
ferase (AST), lactate dehydrogenase (LDH), CRP, eryth-
rocyte sedimentation rate (ESR), blood sugar (BS), and
ferritin.
Measurement of IL‑6 and TNF‑α
Serum was isolated from 3 mL of non-anticoagulated blood
within 2 h. The serum concentrations of IL-6 and TNF-α
were measured using a commercial Enzyme-Linked Immu-
nosorbent Assay (ELISA) kit (Biolegend, USA) in accord-
ance with the manufacturer’s instructions. The sensitivity
of the ELISA kits was 4 pg/mL for IL-6 and 2 pg/mL for
TNF-α.
DNA extraction and HLA typing
Genomic DNA was extracted from 200 μL of the remaining
blood samples using the proteinase K method and a DNA
extraction kit (AddBio, Korea), in accordance with the
manufacturer’s instructions. The quality and quantity of the
extracted DNA were determined using NanoDrop (Thermo
Scientific, UK). DNA samples with a concentration higher
than 10 ng/μL were stored at −20 C prior to HLA testing.
HLA-A, -B, and -C allele typing was performed using the
polymerase chain reaction with the sequence-specific prim-
ers (PCR-SSP) method, using the Olerup SSP® HLA-A-B-C
SSP Combi Tray kit (Olerup, Sweden) in accordance with
the manufacturer’s instructions. The PCR products were
electrophoresed on a 2% agarose gel stained with DNA-safe
stain, and specific bands were visualized under a UV transil-
luminator. HLA alleles were determined using both the soft-
ware and worksheet methods provided by the manufacturer.
 1 Page 4 of 13
Fig. 1  Flow diagram of participant selection. CT scan: computerized
tomography scan, LCS: long Covid-19 syndrome, RT-qPCR: reverse
transcription-quantitative polymerase chain reaction, SARS-CoV-2:
Statistical analyses
Chi-square (χ2) and Fisher exact tests were used to com-
pare the categorical variables between the ­L CS − and
­LCS+ groups, and the results were presented as counts and
Clinical and Experimental Medicine (2024) 24:1
severe acute respiratory syndrome coronavirus 2, WHO: world health
organization
percentages (%). For the quantitative variables, as the Kol-
mogorov–Smirnov test indicated the data was not normally
distributed, differences between the groups were assessed
using the Mann–Whitney U test. The results were reported
as medians with interquartile ranges (IQR). Furthermore,
Clinical and Experimental Medicine (2024) 24:1
to estimate the association between the variables and the
likelihood of LCS, we performed a multiple logistic regres-
sion using the “Enter” method. We selected variables with
a p-value ≤ 0.1 from the χ2 and Mann–Whitney U tests for
inclusion in the logistic regression model. Due to its high
correlation with white blood cell (WBC) and neutrophil-to-
lymphocyte ratio (NLR), the lymphocyte count variable was
excluded from the model. The odds ratios (OR) and corre-
sponding 95% confidence intervals (95%CI) were calculated
to measure the effect of the variables. All statistical analysis
was performed using SPSS (v26.0), with a significance level
of P < 0.05. GraphPad Prism (v9.0.0) software was used to
create all figures.
Results
Demographic characteristics, laboratory
parameters, and clinical history of the participants
We enrolled 88 subjects with LCS (43 males and 45 females)
in the ­LCS+ group and 96 individuals without LCS (55
males and 41 females) in the ­LCS− group. A comparison
of demographic characteristics, laboratory parameters, and
clinical histories of the two groups is presented in Table 1.
There were no significant differences between the ­LCS+ and
­LCS− groups for sex (P = 0.252) or age (P = 0.601). How-
ever, patients with LCS had a significantly higher body mass
index (BMI) than their controls (P = 0.002).
The ­LCS+ group had significantly higher WBC, lympho-
cyte count, CRP, and BS, when compared to the L ­ CS− group
(for all: P < 0.05). In contrast, the NLR and ferritin levels
were significantly lower in the ­LCS+ group (P = 0.023 and
P = 0.012; respectively). However, no significant differences
were observed between the two groups in any of the other
laboratory parameters (for all: P ≥ 0.05).
Moreover, when examining the clinical history of indi-
viduals during the acute phase of Covid-19, it became
evident that subjects in the ­LCS+ group had significantly
longer hospital stays, a higher rate of admission to ICU, and
a higher incidence of severe Covid-19, in comparison to the
­LCS− group (for all: P < 0.001).
Clinical characteristics of patients with LCS
Figure 2 presents the prevalence of persistent symptoms in
the ­LCS+ group. The most commonly reported persistent
symptoms were fatigue (88.6%), hair loss (86.4%), shortness
of breath (79.5%), memory/concentration problems (75.0%),
and muscle/joint pain (70.5%). Moreover, the L ­ CS+ par-
ticipants who experienced severe Covid-19 during the acute
phase exhibited a higher prevalence of enduring shortness of
breath and memory/concentration problems, when compared
Page 5 of 13 1
to those with a history of non-severe Covid-19 (P = 0.008
and P = 0.009, respectively). However, please note that this
data is not shown in the figure.
The serum concentration of IL‑6 and TNF‑α
We also measured the serum levels of IL-6 and TNF-α
in the two groups. This showed that the L ­ CS+ group had
higher serum levels of IL-6 than the L ­ CS− group (37.78
(4.60–87.33) vs. 2.62 (0.72–43.76)), and this difference
was statistically significant (P = 0.004; Fig. 3). Similarly,
the serum level of TNF-α was significantly higher in the
­LCS+ group (3.73 (1.55–15.09)) than in the ­LCS− group
(2.68 (0.87–4.36), P = 0.042; Fig. 3).
The frequency of HLA‑A, ‑B, and ‑C alleles
The frequencies of the HLA-A, -B, and -C alleles in the
­LCS+ and ­LCS− groups are presented in Table 2. The HLA-
A*11, -B*14, -B*38, -B*50, and -C*07 alleles were all more
frequently present in the ­LCS+ group than in the ­LCS− group
(for all: P < 0.05). In contrast, the HLA-A*32, -B*51, -C*08,
and -C*12 alleles were less frequently found in the L ­ CS+
group (for all: P < 0.05).
Furthermore, the frequency of the alleles differed in
accordance with the severity of Covid-19 during the acute
phase of the disease. Specifically, we found the HLA-A*11,
-B*08, -B*14, B*50, and -C*07 alleles were significantly
more common in patients who experienced severe symp-
toms during the acute phase of the disease (SC group), when
compared to those with a history of non-severe Covid-19
(NSC group) (for all, P < 0.05). In contrast, the HLA-A*32,
-B*35, -B*51, and -C*08 alleles were less frequently found
in the SC group than in the NSC group (for all, P < 0.05;
Supplementary Table 1).
Multiple logistic regression modeling for screening
LCS
The results from the multiple logistic regression model are
shown in Table 3. After adjusting for the most important
variables, including BMI, WBC, NLR, CRP, ferritin, BS,
IL-6, TNF-α, HLA-A*11,-A*32, -B*14, -B*38, -B*50,
-B*51, -C*07, -C*08, -C*12 and severe Covid-19, we
found that the likelihood of experiencing LCS was sig-
nificantly associated with BMI, CRP, ferritin, IL-6, the
HLA-A*11, -C*07, and -C*08 alleles, as well as a posi-
tive history of severe Covid-19. These results indicate that
there is a substantially higher likelihood of LCS in individ-
uals who experienced severe symptoms during the acute
phase of Covid-19, in comparison to those with a history
of non-severe Covid-19 [OR: 4.75, 95% CI: 1.56–14.44].
Similarly, both the HLA-A*11 and -C*07 alleles were
 1 Page 6 of 13 Clinical and Experimental Medicine (2024) 24:1

Table 1  Comparison of the Groups LCS+ (n = 88) LCS− (n = 96) P-value

demographic characteristics,
laboratory parameters, and Variable n (%) / Median (IQR1-IQR3) n (%) / Median (IQR1-IQR3)
clinical history of the two
Sex
groups
Male 43 (43.90) 55 (56.10) 0.252
Female 45 (52.30) 41 (47.70)
Age (years) 47.50 (39.00–55.25) 43.00 (37.25–58.00) 0.601
BMI (Kg/m2) 28.48 (26.49–30.81) 25.95 (24.14–28.29) 0.002*
WBC (× ­103µL) 6.99 (5.95–7.97) 6.36 (5.80–7.36) 0.016*
RBC (× ­106µL) 5.15 (4.84–5.50) 5.18 (4.88–5.63) 0.568
Hb (g/dL) 14.25 (13.50–15.70) 14.65 (13.42–15.67) 0.837
HCT (%) 43.55 (40.22–47.07) 44.85 (41.42–47.37) 0.171
Plt (× ­103µL) 319.00 (253.00–361.75) 290.50 (259.25–331.50) 0.129
Neu.C (× ­103µL) 4.00 (3.27–4.75) 3.72 (3.25–4.38) 0.208
3
Lym.C (× ­10 µL) 2.37 (1.95–3.02) 2.01 (1.79–2.48) < 0.001*
NLR 1.60 (1.25–2.11) 1.77 (1.44–2.12) 0.023*
ESR (mm/hr) 9.00 (4.00–13.00) 7.00 (4.00–11.00) 0.331
CRP (mg/L) 2.40 (1.32–5.25) 1.80 (1.10–3.17) 0.005*
LDH (U/L) 332.00 (291.00–425.00) 327.00 (291.00–387.00) 0.487
Ferritin (ng/mL) 41.95 (26.26–82.58) 77.49 (32.57–114.16) 0.012*
BS (mg/dL) 97.50 (89.00–111.75) 92.50 (85.00–102.00) 0.025*
Cr (mg/dL) 1.06 (0.91–1.19) 1.04 (0.90–1.17) 0.775
AST (U/L) 21.00 (19.00–24.00) 20.00 (18.00–24.00) 0.433
ALT (U/L) 20.50 (16.00–26.75) 22.00 (16.25–29.00) 0.285
PT (Sec) 13.00 (13.00–13.00) 13.00 (13.00–13.00) 0.706
PTT (Sec) 31.05 (29.22–33.00) 30.60 (29.37–32.77) 0.985
Duration of hospitaliza- 8.50 (6.00–13.00) 0.00 (0.00–6.00) < 0.001*
tion in the acute phase
ICU admission in the acute phase
Yes 21 (23.90) 4 (4.20) < 0.001*
No 67 (76.10) 92 (95.80)
Covid-19 Severity in the acute phase
Severe 49 (55.70) 11 (11.50) < 0.001*
Non-Severe 39 (44.30) 85 (88.50)

ALT: alanine transaminase, AST: aspartate transaminase, BMI: body mass index, BS: blood sugar, Cr: cre-
atinine, CRP: c-reactive protein, dL: deciliter, ESR: erythrocyte sedimentation rate, g: gram, Hb: hemo-
globin, HCT: hematocrit, hr: hour, ICU: intensive care unit, IQR: interquartile range, Kg: kilogram, L: liter,
LCS: long Covid-19 syndrome, LDH: lactate dehydrogenase, Lym.C: lymphocyte count, ­m2: square meter,
mg: milligram, mL: milliliter, mm: millimeter, n: number, Neu.C: neutrophil count, ng: nanogram, NLR:
neutrophil/lymphocyte ratio, Plt: platelet, PT: prothrombin time, PTT: partial thromboplastin time, RBC:
red blood cells, Sec: second, U: unit, WBC: white blood cells, µL: microliter. * indicates P-value < 0.05

linked to an increased likelihood of LCS [OR: 2.32, 95% Discussion

CI: 1.13–4.74, and OR: 2.16, 95% CI: 1.08–4.35, respec-
tively]. In contrast, the presence of the HLA-C*08 allele
had a significant protective effect against LCS [OR: 0.06,
95% CI: 0.01–0.60]. Moreover, the odds of LCS were sig-
nificantly increased with each one-unit rise in BMI (kg/m2)
[OR: 1.12, 95% CI: 1.00–1.25], CRP (mg/L) [OR: 1.21,
95% CI: 1.05–1.38], or serum IL-6 (pg/mL) [OR: 1.01,
95% CI: 1.00–1.01]. Conversely, each one-unit rise in fer-
ritin (ng/mL) was associated with a significant decrease in
the odds of LSC [(OR: 0.99, 95% CI: 0.98–1.00].
The present study investigated differences in the likelihood
of experiencing LCS using demographic characteristics,
laboratory indicators, clinical history, and the frequency of
the HLA-I alleles. The results showed that the odds of LCS
increase by 0.12 times per one-unit rise in BMI. Previous
findings have shown that individuals with a BMI greater
than 30 kg/m2 have a 10% higher relative risk of experienc-
ing long-term Covid-19 complications than those with a nor-
mal BMI [34]. Furthermore, obesity has been identified as
Clinical and Experimental Medicine (2024) 24:1 Page 7 of 13 1

Fig. 2  Symptom (lasting

for > 12 weeks from the onset Skin Complication 12.5
of Covid-19) prevalence in the
Hearing Loss 13.6
­LCS+ Group (%)
Vision problem 34.1
Hair Loss 86.4
Gastrointestinal problems 45.5
Depression/Anxiety 69.3
Memory/Concentration problems 75.0
Insomnia 40.9
Muscle/Joint pain 70.5
Cardiovasular problems 55.7
Smell & Taste Loss 45.5
Fatigue 88.6
Sore Throat 10.2
Shortness of breath 79.5
Tinnitus/Earache 20.5
Headache/Dizziness 25.0
Fever 3.4

0 20 40 60 80 100
Percent

400 1500
** *
300 1000
200
500
100
20
TNF- α (pg/mL)
IL-6 (pg/mL)

40 15

10

20
5

0
0
-5
LCS- LCS+ LCS- LCS+

­ CS+ and LCS.− groups. IL-6: interleukin-6, LCS: long Covid-19 syndrome, mL: milliliter, pg: pico-
Fig. 3  IL-6 and TNF-α serum levels in the L
gram, TNF-α, tumor necrosis factor-α. * indicates P-value = 0.042, ** indicates P-value = 0.004

an independent risk factor for an incomplete recovery, even to mitochondrial dysfunction and the production of reac-
one year after hospital discharge, among patients who have tive oxygen species caused by hyperglycemia in individu-
recovered from severe or critical Covid-19 [35]. The under- als with obesity. These processes lead to the production of
lying mechanism for this phenomenon has been attributed pro-inflammatory mediators like leptin (a pro-inflammatory
Table 2  The frequencies of the HLA-A, -B, and -C alleles in the L
­ CS+ and ­LCS− groups
1

Alleles LCS+ n (%) LCS− n (%) P-value OR (95%CI) Alleles LCS+ n (%) LCS− n (%) P-value OR (CI95%) Alleles LCS+ n (%) LCS− n (%) P-value OR (CI95%)

A*01 21 (11.9) 20 (10.4) 0.644 1.16 (0.60– B*07 10 (5.7) 8 (4.2) 0.501 1.38 (0.53– C*01 6 (3.4) 4 (2.1) 0.529 1.65 (0.46–5.97)
2.23) 3.59)
A*02 22 (12.5) 24 (12.5) 1.000 1.00 (0.53– B*08 4 (2.3) 3 (1.6) 0.714 1.46 (0.32– C*02 12 (6.8) 15 (7.8) 0.715 0.86 (0.39–1.89)
Page 8 of 13

1.85) 6.64)
A*03 18 (10.2) 30 (15.6) 0.125 0.61 (0.33– B*13 8 (4.5) 7 (3.6) 0.663 1.25 (0.44– C*03 4 (2.3) 4 (2.1) 1.000 1.09 (0.26–4.43)
1.14) 3.54)
A*11 29 (16.5) 17 (8.9) 0.027* 2.03 (1.07– B*14 12 (6.8) 1 (0.5) 0.001* 13.97 (1.79– C*04 26 (14.8) 26 (13.5) 0.735 1.10 (0.61–1.99)
3.84) 108.63)
A*23 4 (2.3) 7 (3.6) 0.440 0.61 (0.17– B*15 5 (2.8) 4 (9.7) 0.355 0.59 (0.19– C*05 22 (12.5) 17 (8.9) 0.256 1.47 (0.75–2.87)
2.13) 1.80)
A*24 37 (21.0) 44 (22.9) 0.661 0.89 (0.54– B*18 8 (4.5) 14 (7.3) 0.267 0.60 (0.24– C*06 23 (13.1) 18 (9.4) 0.261 1.45 (0.75–2.79)
1.46) 1.48)
A*25 NS 1 (0.5) 1.000 NA B*35 29 (16.5) 43 (22.4) 0.153 0.68 (0.40– C*07 31 (17.6) 17 (8.9) 0.013* 2.20 (1.17–4.13)
1.15)
A*26 12 (6.8) 10 (5.2) 0.515 1.33 (0.56– B*37 2 (1.1) 3 (1.6) 1.000 0.72 (0.12– C*08 7 (4.0) 32 (16.7) < 0.001* 0.20 (0.08–0.48)
3.16) 4.38)
A*29 4 (2.3) 2 (1.0) 0.432 2.20 (0.40– B*38 12 (6.8) 2 (1.0) 0.004* 6.95 (1.53– C*12 12 (6.8) 25 (13.0) 0.048* 0.48 (0.23–1.00)
12.21) 31.51)
A*30 7 (4.0) 8 (4.2) 0.927 0.95 (0.33– B*39 2 (1.1) 3 (1.6) 1.000 0.72 (0.12– C*14 9 (5.1) 7 (3.6) 0.490 1.42 (0.51–3.90)
2.68) 4.38)
A*31 2 (1.1) 1 (0.5) 0.608 2.19 (0.19– B*40 8 (4.5) 9 (4.7) 0.948 0.96 (0.36– C*15 3 (1.7) 6 (3.1) 0.506 0.53 (0.13–2.18)
24.42) 2.56)
A*32 4 (2.3) 14 (7.3) 0.026* 0.29 (0.09– B*41 4 (2.3) 6 (3.1) 0.753 0.72 (0.20– C*16 14 (8.0) 20 (10.4) 0.415 0.74 (0.36–1.52)
0.91) 2.59)
A*33 10 (5.7) 6 (3.1) 0.230 1.86 (0.66– B*44 12 (6.8) 12 (6.3) 0.825 1.09 (0.48– C*17 3 (1.7) NS 0.108 NA
5.24) 2.51)
A*66 NS 1 (0.5) 1.000 NA B*47 2 (1.1) NS 0.228 NA C*18 4 (2.3) 1 (0.5) 0.198 4.44 (0.49–40.12)
A*68 5 (2.8) 6 (3.1) 0.873 0.90 (0.27– B*48 1 (0.6) 2 (1.0) 1.000 0.54 (0.04–
3.02) 6.03)
A*69 1 (0.6) 1 (0.5) 1.000 1.09 (0.06– B*49 9 (5.1) 9 (4.7) 0.850 1.09 (0.42–
17.58) 2.82)
B*50 9 (5.1) 2 (1.0) 0.022* 5.12 (1.09–
Clinical and Experimental Medicine

24.03)
B*51 14 (8.0) 32 (16.7) 0.012* 0.43 (0.22–
0.84)
B*52 7 (4.0) 7 (3.6) 0.868 1.09 (0.37–
3.18)
(2024) 24:1

B*53 1 (0.6) 4 (2.1) 0.374 0.26 (0.03–

2.42)
 Clinical and Experimental Medicine (2024) 24:1 Page 9 of 13 1

adipokine), TNF-α, IL-1, IL-6, and IL-18 [36]. These medi-

OR (CI95%) ators can skew the immune response towards a low-grade
chronic local inflammatory state, resulting in tissue damage,
necrosis, and cellular dysfunction. Over time, both innate
and adaptive immune responses are systemically affected,
rendering obese individuals more vulnerable to viral persis-
tence and secondary infections [36, 37].
Alleles LCS+ n (%) LCS− n (%) P-value

Additionally, our study found a strong association

between the severity of Covid-19 during the acute phase of
the disease and the likelihood of LCS. Specifically, individu-
als who experienced severe symptoms during the acute stage
of Covid-19 had a 3.75 times higher likelihood of develop-
LCS: long Covid-19 syndrome, n: number, NA: not applicable, NS: not seen, OR: odds ratio, 95%CI: 95% confidence interval. * indicates P-value < 0.05 ing LCS, in comparison to those with a history of non-severe
Covid-19. Previous studies have reported that patients who
experienced severe Covid-19 and required invasive mechani-
cal ventilation, ICU admission, and prolonged hospital stays
were more likely to develop long-term tissue damage and
persistent symptoms [38–40]. This may be due to the sys-
temic inflammatory responses and cytokine storms induced
by severe Covid-19. The excessive production of pro-inflam-
Alleles LCS+ n (%) LCS− n (%) P-value OR (CI95%)

1.09 (0.34–

1.09 (0.06–

2.20 (0.40–

5.58 (0.64–

0.26 (0.03–

matory cytokines can exacerbate tissue damage by stimulat-

17.58)

12.21)

48.27)

ing the cytotoxic activity of the ­CD8+ T cells, promoting

3.45)

2.42)

apoptosis, and disrupting the control of leukocyte traffick-

ing [41, 42]. In particular, IL-6 fosters the differentiation of
naïve ­CD4+ T cells into Th17 cells and inhibits Treg differ-
0.878

1.000

0.432

0.108

0.374

entiation, resulting in immunological tolerance disruption

and the development of chronic inflammation [43]. TNF-α
further exacerbates this process by inducing IL-6 expres-
6 (3.1)

1 (0.5)

2 (1.0)

1 (0.5)

4 (2.1)

sion [44]. Moreover, some required medical procedures for

patients with severe Covid-19, such as intubation, some-
times cause chronic inflammation due to iatrogenic damage.
In addition, the overuse of corticosteroids in patients with
6 (3.4)

1 (0.6)

4 (2.3)

5 (2.8)

1 (0.6)

severe Covid-19 can result in the development of oppor-

tunistic secondary infections, triggering sustained inflam-
matory responses [45]. The sustained inflammatory condi-
B*55

B*56

B*57

B*58

B*73

tion observed in LCS may also be due to the persistence of

SARS-CoV-2 antigens in tissues, autoreactive reactions, and
the failure of the individual’s immune system to return to
Alleles LCS+ n (%) LCS− n (%) P-value OR (95%CI)

baseline homeostasis, which may underlie the development

of long-term Covid-19 symptoms [32].
Among the long-term symptoms, in our study we found
that fatigue, hair loss, shortness of breath, and concentration
and memory problems were the most commonly reported
by individuals with LCS, which is consistent with previous
findings [46–48]. Previous research has shown that the pro-
duction of pro-inflammatory cytokines such as IL-1β, IL-6,
IL-12, interferon (IFN)-γ, and TNF-α leads to the recruit-
ment and activation of Th1 and Th17 cells on microglia,
Table 2  (continued)

resulting in nerve fiber demyelination, which may explain

the chronic muscle fatigue and neurological disorders in
individuals with LCS [33, 49, 50]. In addition, persistent
inflammation, lung fibrosis, and damage to pulmonary vas-
culature caused by pro-inflammatory cytokines such as IL-6
 1 Page 10 of 13 Clinical and Experimental Medicine (2024) 24:1

Table 3  The results of the Variable B S.E Wald df P-value OR 95% C.I. for OR
multiple logistic regression
showing the associations Lower Upper
between the variables and the
odds of LCS BMI 0.114 0.057 3.994 1 0.047* 1.12 1.00 1.25
WBC 0.373 0.284 1.734 1 0.188 1.45 0.83 2.53
NLR -0.309 0.533 0.337 1 0.561 0.73 0.26 2.09
CRP 0.186 0.070 7.141 1 0.008* 1.21 1.05 1.38
Ferritin -0.013 0.006 3.991 1 0.046* 0.99 0.98 1.00
BS 0.035 0.019 3.287 1 0.070 1.04 0.99 1.08
IL-6 0.007 0.004 4.300 1 0.038* 1.01 1.00 1.01
TNF-α 0.003 0.003 1.104 1 0.293 1.00 0.99 1.01
A*11 0.840 0.365 5.299 1 0.021* 2.32 1.13 4.74
A*32 -1.137 1.069 1.131 1 0.288 0.32 0.04 2.61
B*14 1.618 1.322 1.498 1 0.221 5.04 0.38 67.32
B*38 0.945 1.347 0.492 1 0.483 2.57 0.18 36.03
B*50 -1.772 2.063 0.738 1 0.390 0.17 0.01 9.70
B*51 0.041 0.704 0.003 1 0.954 1.04 0.26 4.14
C*07 0.772 0.357 4.675 1 0.031* 2.16 1.08 4.35
C*08 -2.842 1.190 5.704 1 0.017* 0.06 0.01 0.60
C*12 -0.743 0.392 3.593 1 0.058 0.48 0.22 1.03
Severe Covid-19 1.557 0.568 7.525 1 0.006* 4.75 1.56 14.44

B: regression coefficient, BMI: body mass index, BS: blood sugar, CRP: c-reactive protein, df: degrees of
freedom, IL-6: interleukin-6, LCS: long Covid-19 syndrome, NLR: neutrophil/lymphocyte ratio, OR: odds
ratio, S.E.: standard error, TNF-α: tumor necrosis factor-α, WBC: white blood cell, 95%CI: 95% confi-
dence interval. * indicates P-value < 0.05

may potentially contribute to long-term respiratory symp-
toms [1, 51]. In line with these findings, our study showed
that higher levels of CRP and IL-6 were associated with
LCS. Specifically, we found that the odds of LCS increased
by 0.21 and 0.01-fold for each one-unit rise in CRP and IL-6,
respectively. In contrast, higher levels of ferritin were associ-
ated with lower odds of LCS (OR = 0.99). It is worth noting
that previous studies have reported contradictory findings
regarding ferritin, which could be attributed to the timing
of marker evaluations in relation to the acute phase of the
disease. Nevertheless, understanding this issue highlights
the need for further studies [32, 52–54].
The findings of a study have highlighted the vital role
of HLA molecules in the immune system, since they act
as crucial mediators in the defense against infections and
significantly impacting the development and persistence
of the clinical symptoms [55]. As the most polymorphic
molecules, HLA-I play a crucial role in mitigating clinical
complications by selectively presenting a wide array of viral
antigen-derived peptides to the C ­ D8+ T cells, which are cen-
tral cells of the acquired immune system’s defense against
viral infections [56]. In the context of the SARS-CoV-2
infection, previous studies have shown the HLA-I alleles to
be associated with variations in Covid-19 severity, as well
as the resulting outcomes. Furthermore, several studies
have found susceptibility to Covid-19 to be associated with
the HLA-B*08 [57], -B*35 [58], and -C*07 [59] alleles.
In addition, individuals carrying the HLA-A*11 and -B*51
alleles have a heightened susceptibility to severe Covid-19
and worse outcomes in comparison to those lacking these
alleles [60]. Moreover, patients who carry the HLA-A*11
allele have been observed to have higher mortality rates
from Covid-19 [61]. In contrast, the HLA-A*32 [24, 58]
and -B*14 [57] alleles have been found to have a protective
effect against Covid-19. However, to the best of our knowl-
edge, no previous research has investigated the relationship
the HLA-I alleles have with LCS.
The current research found significantly higher frequen-
cies of the HLA-A*11, -B*14, -B*38, -B*50, and -C*07
alleles in the LCS group than in the control group, while
lower frequencies of HLA-A*32, -B*51, and -C*08 alleles
were observed in the LCS group. After adjusting for the
important variables, the HLA-A*11 and -C*07 alleles
were still associated with an increased likelihood of LCS
(1.32 and 1.16-fold, respectively). In contrast, the HLA-
C*08 allele was found to have a protective effect against
LCS (OR = 0.06). Considering the higher frequencies of the
HLA-A*11 and -C*07 alleles in patients who experienced
severe symptoms during the acute phase of the disease,
compared to those with a history of non-severe Covid-19,
we posit a hypothetical mechanism to explain the occur-
rence of long Covid-19 syndrome. This mechanism involves
Clinical and Experimental Medicine (2024) 24:1
the incomplete clearance of SARS-CoV-2 during the acute
phase of the disease in individuals carrying these alleles,
ultimately leading to tissue damage due to persistent and
inefficient inflammatory responses over the long term [2].
It is reasonable to assume that the HLA-I alleles with
a stronger binding affinity for epitopes derived from the
SARS-CoV-2 antigens elicit more effective CTL responses.
Consequently, this prevents disease progression to severe
complications, facilitates complete virus clearance, and pro-
motes full recovery. Conversely, HLA-I alleles with a weaker
binding affinity are associated with sustained inflammation,
organ damage, and long-term complications. This is because
they trigger inefficient and dysregulated immune responses
that are unable to clear SARS-CoV-2 [55, 62]. In this con-
text, Meysman et al. previously reported that HLA-A*02:01-
restricted peptides triggered more robust immune responses
compared to HLA-A*24:02-restricted peptides, when
examining VZV IE-62-derived peptides [63]. For SARS-
CoV-2 infection, the HLA-A*01:01, -A*02:01, -A*24:02,
-B*15:01, and -B*44:02-restricted peptides were shown to
efficiently induce epitope-specific C­ D8+ T-cell responses
[64], while no specific spike-derived peptides were identified
for the HLA-B*08:01 and -B*27:05 alleles, suggesting the
absence of appropriate spike-specific C­ D8+ T cell responses
in individuals carrying these alleles [65].
Limitations and future directions
It is important to acknowledge the limitations of our study,
which should be considered in future research. One limita-
tion was the absence of well-defined diagnostic criteria for
LCS, potentially impacting the accuracy of classifying indi-
viduals into the LCS and non-LCS groups. In addition, the
small sample size posed challenges and may have influenced
the results. The scarcity of similar studies further restricts
our ability to make comparisons. Furthermore, our study
followed a case–control design and only assessed partici-
pants at a single point in time. Therefore, we recommend
conducting larger-scale longitudinal studies in diverse ethnic
populations to investigate changes in symptoms and levels of
inflammatory markers in recovered Covid-19 patients over
time.
Conclusion
Our study showed that an increased likelihood of LCS was
associated with higher BMI, a history of severe Covid-19
during the acute phase of the disease, the presence of the
HLA-A*11 and -C*07 alleles, as well as elevated levels of
serum CRP and IL-6. In contrast, the HLA-C*08 allele and
Page 11 of 13 1
high serum ferritin levels significantly decreased the odds
of LCS.
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 10238-0​ 23-0​ 1256-1.
Acknowledgements The authors would like to thank all individuals
who participated in the studies cited in this article.
Author Contributions ET reviewed the individuals’ medical records
and extracted their recorded data. RS evaluated the clinical symptoms
of the subjects and administered the questionnaire. ET, FH, and MM
performed the experiments and extracted the data. ET and HF ana-
lyzed the data. ET, HF, RS, FH, MM, and MJMS wrote and reviewed
the manuscript. HF supervised all stages of the research and article
production. All authors contributed to the article and have approved
the submitted version.
Funding The present study was a part of a M.Sc. thesis written by
Ensiye Torki, financially supported by a grant from the Isfahan Uni-
versity of Medical Sciences, Isfahan, Iran [Grant No. 3400839], and
technically supported by the Department of Immunology, School of
Medicine, Isfahan University of Medical Sciences.
Data availability Not applicable.
Declarations
Conflict of interest The authors declare that they have no conflict of
interest.
Ethics approval This project was approved by Isfahan University of
Medical Sciences Ethics Committee (IR.MUI.MED.REC.1400.795).
Consent to participate Not applicable.
Consent to publication Not applicable.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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