Alterations of gut bacteria Akkermansia muciniphila and Faecalibacterium prausnitzii in late post-transplant period after liver transplantation

IBEROAMERICAN JOURNAL OF MEDICINE 0)1 (2022) 45-51

Original article

IBEROAMERICAN
JOURNAL OF
MEDICINE

iberoamericanjm

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Alterations of gut bacteria Akkermansia muciniphila and
Faecalibacterium prausnitzii in late post-transplant period after
liver transplantation

Alexander Kukov %"\, Milena Peruhova °, Atanas Syarov‘™, Iskra Altankova ~4,
Nonka Yurukova Tio} Andrei Goncharov eD, Radoslava Vazharova c(), Anoaneta
Mihova *4\, Tsvetelina Velikova *4\, Yordanka Uzunova °\

“University Hospital “Lozenets”, Laboratory of Clinical Immunology, Sofia, Bulgaria
’ Faculty of Medicine, Sofia University “St. Kliment Ohridski”, University Hospital “Lozenets”, Clinic of Gastroenterology, Sofia,

Bulgaria

© Faculty of Medicine, Sofia University “St. Kliment Ohridski”, University Hospital “Lozenets”, Laboratory of Medical Genetics, Sofia,

Bulgaria

4 Faculty of Medicine, Sofia University “St. Kliment Ohridski”, Department of Clinical Immunology, Sofia, Bulgaria
° Faculty of Medicine, Sofia University “St. Kliment Ohridski”, University Hospital “Lozenets”, Clinic of Pediatrics, Sofia, Bulgaria

ARTICLE INFO

ABSTRACT

Article history:

Received 18 November 2021
Received in revised form 15
December 2021

Accepted 17 January 2022

Keywords:

Akkermansia muciniphila
Faecalibacterium prausnitzii
Gut microbiota

Liver transplantation

Immunosuppression

Introduction: Recent studies have shown that the intestinal microbiota can modulate certain
systemic metabolic and immune responses, including liver graft function and the development
of complications in patients after liver transplantation (LT). Akkermansia muciniphila (AKM) and
Faecalibacterium prausnitzii (FAEP) are two of the most abundant gut commensal bacteria, with
mucosa-protective and anti-inflammatory effects that are important for maintaining normal
intestinal homeostasis and gut barrier function. Our objective was to quantify levels of
Akkermansia muciniphila and Faecalibacterium prausnitzii in immunosuppressed patients with
LT.

Materials and methods; Fecal samples from 23 liver transplant patients (15 adults and 8
children) and 9 non-LT controls were examined. Bacterial DNA was isolated from the samples
using the stool DNA isolation kit and the obtained DNA was analyzed with commercially
available qPCR kit for AKM and FAEP.

Results: We found a statistically significant decrease in the amount of AKM and FAEP compared
to the control group. The median values were: for AKM 8.75 for patients and 10.25 for the control
group (p = 0.030), and for FAEP 9.72 and 10.47, p = 0.003, respectively. In children after LT, this
difference was also statistically significant: AKM (p = 0.051) and FAEP (p = 0.014). In contrast
no statistically significant differences were found between adult patients and controls, AKM (p
= 0.283) and FAEP (p = 0.056), although the amount of both bacteria showed tendency for
reduction.

Conclusions: In this pilot study, we found a reduction in the total amount of the two studied
bacteria in transplanted patients compared to the control healthy group.

© 2022 The Authors. Published by Iberoamerican Journal of Medicine. This is an open access article under the
CC BY license (http://creativecommons. org/licenses/by/4.0/).

* Corresponding author.

E-mail address: a_dimitroff@abv.bg

ISSN: 2695-5075 / © 2022 The Authors. Published by Iberoamerican Journal of Medicine. This is an open access article under the CC BY license
(http://creativecommons. org/licenses/by/4.0/).

https://doi.org/10.53986/ibjm.2022.0010

46

IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 45-51

Alteraciones de las bacterias intestinales Akkermansia muciniphila y
Faecalibacterium prausnitzii en el postrasplante tardio tras trasplante hepatico

INFO. ARTICULO

RESUMEN

Historia del articulo:
Recibido 18 Noviembre 2021
Recibido en forma revisada
15 Diciembre 2021
Aceptado 17 Enero 2022

Palabras clave:
Akkermansia muciniphila
Faecalibacterium prausnitzii
Microbiota intestinal
Trasplante de higado

Inmunosupresién

Introduccién: Estudios recientes han demostrado que la microbiota intestinal puede modular
determinadas respuestas metabdlicas e inmunitarias sistémicas, entre ellas la funcion del injerto
hepatico y el desarrollo de complicaciones en pacientes tras un trasplante hepatico (TH).
Akkermansia muciniphila (AKM) y Faecalibacterium prausnitzii (FAEP) son dos de las bacterias
comensales intestinales mas abundantes, con efectos protectores de la mucosa y
antiinflamatorios que son importantes para mantener la homeostasis intestinal normal y la
funcién de barrera intestinal. Nuestro objetivo fue cuantificar los niveles de Akkermansia
muciniphila y Faecalibacterium prausnitzii en pacientes inmunodeprimidos con TH.
Materiales y métodos: Se examinaron muestras fecales de 23 pacientes trasplantados de higado
(15 adultos y 8 nifios) y 9 controles sin TH. El ADN bacteriano se aisl6 de las muestras utilizando
el kit de aislamiento de ADN de heces y el ADN obtenido se analizo con el kit qPCR disponible
comercialmente para AKM y FAEP.
Resultados: Encontramos una disminuci6n estadisticamente significativa en la cantidad de AKM
y FAEP en comparacion con el grupo control. Los valores medianos fueron: para AKM 8,75 para
los pacientes y 10,25 para el grupo control (p = 0,030), y para FAEP 9,72 y 10,47, p = 0,003,
respectivamente. En nifios tras TH, esta diferencia también fue estadisticamente significativa:
AKM (p = 0,051) y FAEP (p = 0,014). Por el contrario, no se encontraron diferencias
estadisticamente significativas entre pacientes adultos y controles, AKM (p = 0,283) y FAEP (p
= 0,056), aunque la cantidad de ambas bacterias mostr6 tendencia a la reduccion.
Conclusiones: En este estudio piloto, encontramos una reduccion en la cantidad total de las dos
bacterias estudiadas en pacientes trasplantados en comparacion con el grupo control sano.

© 2022 Los Autores. Publicado por Iberoamerican Journal of Medicine. Este es un articulo en acceso abierto

bajo licencia CC BY (http://creativecommons. org/licenses/by/4.0/).

HOWTO CITE THIS ARTICLE: Kukov A, Peruhova M, Syarov A, Altankova I, Yurukova N, Goncharov A, Varzharova R, Mihova A,
Velikova T, Uzunova Y. Alterations of gut bacteria Akkermansia muciniphila and Faecalibacterium prausnitzii in late post-
transplant period after liver transplantation. Iberoam J Med. 2022;4(1):45-51. doi: 10.53986/ibjm.2022.0010.

some bacteria and their metabolites have a beneficial effect

1. INTRODUCTION

Patients with end-stage liver diseases (ESLD), such as
cirrhosis of different etiologies, autoimmune liver diseases,
genetically linked liver malformations and other have a poor
prognosis and liver transplantation (LT) is a life-saving
treatment for them. Scientific studies discuss the influence
of gut bacteria on normal and pathological liver function.
With the advent of advanced molecular biological
techniques to facilitate culture-independent characterization
of microbiota, it is possible to study in details different
bacteria in the human microbiome [1]. This allows us to
understand their role in the clinical setting during and after
LT. The connection between intestinal microbiome,
immunity and its dysregulation is well established, as well
as the changes that occur in the post-transplant period under
the influence of immunosuppressive therapy. A number of
studies have shown that changes in the microbiome may be
associated with acute graft rejection, as well as other post-
transplant complications. Gut microbiota modulate some
systemic and immune responses via multiple mechanisms
that may affect allograft function [2]. It has been found that

on innate and acquired immunity, reducing T-cell activation
and promoting induction of regulatory T cells (Tregs) [3].
This is essential for building and maintaining graft tolerance
[4].

The barrier functions of the intestinal mucosa are very
sensitive to dysbiosis (condition when the gut bacteria
become imbalanced) and are often disrupted which could
lead the bacterial metabolites to enter the circulation [1]. Gut
dysbiosis could alter the barrier functions of the mucosa;
followed by reduced production of mucin, which coincides
with the loss of the beneficial microorganism Akkermansia
muciniphila (AKM) [5].
(FAEP), which is a bacterium with potent anti-inflammatory
properties, via nuclear factor «B inhibition and induction of
Treg [6], is also of great interest for research. It has been
found that the decrease in the total amount of FAEP is
accompanied by overgrowth of some opportunistic species,
which increases the risk of infections in patients [7].

Many factors can affect the gut microbiota after LT, such as
antibiotics, pro/prebiotics, and immunosuppressive therapy
that can additionally modify the baseline gut bacterial

Faecalibacterium prausnitzii

dysbiosis present in ESLD, emphasizing the importance of

IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 45-51 47

understanding the impact of gut microbiota post LT [1, 8].
However, it should be noted that there are age related
changes in the gut microbiota with infant microbiota being
relatively volatile [9]. Calcineurin (CND,
including tacrolimus and cyclosporine A (CsA), are the
main immunosuppressive drugs used in the treatment of
patients after LT [10]. Optimal dosing is extremely
important for a better allograft outcome. It has been shown
that both high and low doses of CNI could suppress some
beneficial bacteria, such as Faecalibacterium prausnitzii
[11]. Mycophenolate (MMF) is __ potent
immunosuppressive agent used as adjunctive therapy in
prevention of allograft rejection. MMF has been shown to
be associated with frequent diarrhea and dysbiosis, as well
as a reduction in some beneficial bacteria including AKM
[10]. The mammalian target of rapamycin (mTOR)
inhibitors, such as sirolimus and everolimus, have a
favorable adverse event profile and are effective in
protecting kidney function in LT patients. Everolimus
appear to have little impact on intestinal microbiota [12].
We studied the distribution and quantities of AKM and
FAEP in Bulgarian patients after LT and subjected to
immunosuppressive therapy. The aim of this pilot study was
to investigate the prevalence and quantity of these two
bacteria in the late post-transplant period.

inhibitors

mofetil

2. MATERIALS AND METHODS

This study was performed between May 2021 and October
2021 at the University Hospital "Lozenets", Sofia, Bulgaria.
In total 32 individuals, children and adults, designed into
two groups: control group (9 persons) and patients after LT
(23 persons) were included in the study. Patients were
enrolled according to
transplantation at least 3 months after the procedure and on
immunosuppressive therapy; without systemic or gut
infections and antibiotic usage at least two months before
enrolment; no data for acute rejection; no autoimmune
diseases, diabetes and carcinoma. Exclusion criteria: active
hepatitis B hepatitis C
immunodeficiency virus infections or tuberculosis; alcohol
and cigarette abuse, acute diarrhea. All patients with LT
were in good general condition. Control groups (children
and adults) were healthy individuals without infections and
antibiotic treatment in the last 2 months.

inclusion criteria: with liver

virus, virus, human

The study was conducted following the ethical guidelines of
the Declaration of Helsinki and was approved by the local
ethics committee (protocol No:2/2021). All participants
have declared and signed their informed consent. Parents or
guardians signed the informed consent for children (< 18

years old). The liver transplantions were performed at the
University Hospital (Sofia), being also
performed the follow-up monitoring in the post-transplant
period.

"Lozenets"

2.1. STOOL SAMPLES COLLECTION

Fecal samples were obtained from children and adult
patients admitted to the hospital for routine examination.
Sterile stool collection tubes (Prima, 20 ml) were used; the
samples were immediately transported to the laboratory and
stored at - 20°C until later analysis.

2.2. ISOLATION OF BACTERIAL DNA FROM FECAL
SAMPLES

Bacterial DNA was isolated from the fecal samples using the
stool DNA isolation kit (QIAamp Fast DNA Stool Mini Kit,
ref.51604, Qiagen, Germany), according to the
manufacturer's guidelines, with a slight modification.
Briefly, 1 ml InhibitEX buffer and sand particles were added
to 200 mg feces; the samples were incubated at 95°C for 10
minutes, to promote better lysis of Gram-positive bacteria.
Additional homogenization and lysis of the samples was
done on Precellys 24 (Peqlab,Gmbh) for 15 sec. at 4500 rpm.
The resulting homogenate was centrifuged (3 min at 17000
g) and samples were processed for DNA isolation. The
concentration and purity of the DNA was determined
spectrophotometrically using a NanoDrop 2000
spectrophotometer (Thermo Scientific, USA). The mean
purity of the DNA was (mean + SD) 1.90 + 0.10, ratio
Abs260/280 nm. The DNA samples were stored at -20°C
until further testing was performed.

2.3. QUANTIFICATION OF AKKERMANSIA
MUCINIPHILA AND FAECALIBACTERIUM PRAUSNIT ZII
BY QPCR METHOD

After extraction, fecal bacterial DNA was quantified and
adjusted in order to obtain DNA 12.5 ng/ul. qPCRs included
4 ul template (50 ng of DNA per reaction) and 16 ul of
Reaction/Master mix. To quantify the amount of AKM and
FAEP DNA we used MutaPLEX® AKM/FAEP real time
PCR kit (Immundiagnostik AG, Germany), following the
manufacturer's instructions. Samples were analyzed using
LightCycler 480 II thermocycler (Roche). For quantification
of Akkermansia muciniphila and Faecalibacterium
prausnitzii positive DNA in samples, a standard curve using
standards was applied. The data obtained in copies per
reaction were then converted to cells/g, according to the
protocol and the results are presented in log io of bacteria

cells per g feces.

48 IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 45-51

2.4. STATISTICAL ANALYSIS

The Mann-Whitney Exact U test was used to compare the
Control and Patients groups. In terms of statistical
significance, p < 0.05 was significantly different.

3. RESULTS

The average post-transplant period was 7 years (min-max: 2
— 17y). The indications for transplantation in adults were
decompensated _ liver etiologies
(ethylism - 4, viral - 4, autoimmune - 2 and 6 with other

cirrhosis of various
etiology). In children, the cause for transplantation was:
autoimmune hepatitis - 1, biliary atresia - 3, liver cirrhosis
of unknown etiology - 4. Control groups included healthy
individuals without LT. Gender and age of the subjects
groups are presented in Table 1.

measured amounts in the transplanted group were
significantly lower than healthy controls (p = 0.014, Table
3). The analysis of AKM in children found that 3 out of 8
children (38%) had AKM in the fecal samples, eg. 62% of
children with LT do not have AKM expression in their feces.
The statistical comparison between the control and LT
patient groups revealed reduced amounts of AKM in
patients (p = 0.051, Table 3).

The results of the research in adults are presented in Table
4. The frequency of positive FAEP samples is 100% in the
control group, while in the transplanted patients it is 73%. In
27% of patients with LT, this bacterium was absent. AKM
expression was also reduced in patients with LT - 67%
versus 80% in the control group. Notably, in 33% of
transplanted patients AKM were not detected. The median
amounts for both bacteria in patients were reduced
compared to healthy controls, but the differences were not

Table 1: Demographic features of patients and control groups
Control group

Tested individuals

(n= 9)
Gender
(M/F)

Liver transplantation group

Children 4
Adults 5

(n = 23)
Gender
(M/F)
6 (4 - 12) 8 5/3 8 (2 - 17)
40 (26 - 49) 15 12/3 50 (19 - 70)

M: Male; F: Female. Age expressed as mean as well as minimum and maximum values.

We studied the presence and amounts of AKM and FAEP in
fecal samples of all enrolled persons. The results are shown
on Table 2. We found that in the control group, all
individuals had FAEP and AKM and only one lacked AKM.
In patients with LT group, however, we found that FAEP
was observed in 83% and AKM in only 52% of transplant
recipients. These differences were statistically significant
when the amounts of bacterial expression was compared for
FAEP and AKM, respectively p = 0.003 and p = 0.03. AKM
was not detected in 48% of the patients and FAEP in 17% of
the patients.

statistically significant (Table 4).

One possible reason for the decreased bacterial expression
in the gut of LT patients might be the immunosuppressive
therapy of these patients. Seven adult patients were treated
with tacrolimus and MMF, four - received tacrolimus alone,
one patient was on everolimus and cyclosporine, and one on
cyclosporine and MMF. Seven of the children with LT were
treated with tacrolimus and one child (autoimmune
hepatitis) was on cyclosporine, methylprednisolone and
MMF. Our results concerning the reduced prevalence of
both bacteria in LT patients, subjected to different

Table 2: Prevalence and amounts of FAEP and AKM in fecal samples of control and liver transplanted patients

Control group
(n= 9)

Bacteria te Median value
Positive (%) .
(min — max)
10.47
FAEP a COUR): ~~ oe 1th
10.25
AKM 8 (89%) (0.00 - 11.54)

Liver transplantation group

e273) = P value*
We Median value
Positive (%) F
(min — max)
9.72
19 (83%) (0.00 - 11.15) 0.003
8.75
122%) (000-1157) ae

*Mann-Whitney Exact U Test was used to compare the amounts of the two bacteria. p<0.05. FAEP: Faecalibacterium prausnitzii;
AKM: Akkermansia muciniphila.

The separate analysis in Children and Adults groups shows
that this trend is more pronounced in children than in adult
transplant subjects. In all studied children (controls and
patients with LT) we found the presence of FAEP, but the

immunosuppressive regimens, are shown on Table 5.
The following conclusions can be drawn from Table 5:
1. FAEP was detected in all children fecal samples. In
contrast, the prevalence of AKM in both patient

IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 45-51 49

groups was lower in comparison with controls.
Furthermore, two adult patients were found to be
double negative.

2. It should be noted that all regimens of used
immunosuppressive therapy most likely influence
AKM growth, as it was not detected in 10 out of 23
patients with LT (43%). We can assume that the
calcineurine inhibitor tacrolimus might suppress
both studied bacteria, but apparently its
combination with MMF enhances this effect.

3. The two double-negative patients (lacking both
FAEP and AKM) are on therapy with MMF and
tacrolimus or everolimus.

in several disease conditions [13]. Moreover, both species
are now shown to have a role in a well-functioning gut and
thus are considered as promising next generation probiotics
[14, 15].

In our study, we found a reduction in the prevalence and
amount of AKM and FAEP in patients in the late post-
transplant period compared to the control healthy group.
This was more markedly seen in children with LT, where
AKM was not detected in 62% of the studied samples. The
limitations of the current study include small number of
patients and control individuals, single testing of all
participants, single-center design, the COVID 19 pandemic,
which complicates the

further enrolment of

Table 3: Distribution and quantity of FAEP and AKM in children control and liver transplantation groups

Control group

Bacteria a4) 5
re, Median value
Positive (%)
(min — max)
FAEP 4 (100%) 10.47 (10.16 - 10.68)
AKM 4 (100%) 10.85 (9.77 - 11.54)

Liver transplantation group
(n= 8)

co
oe Median value EEG
Positive (%) 4
(min — max)
8 (100%) 9.63 (8.41 - 10.58) 0.014
3 (38%) 0.00 (0.00 - 11.20) 0.051

*Mann-Whitney Exact U Test was used to compare the amounts of the two bacteria. p<0.05. FAEP: Faecalibacterium prausnitzii;
AKM: Akkermansia muciniphila.

Table 4: Distribution and quantity of FAEP and AKM in adult control and liver transplantation groups

Control group

Bacteria ine) =
ye Median value
Positive (%) i
(min — max)
FAEP 5 (100%) 10.47 (10.31 - 11.12)
AKM 4 (80%) 9.71 (0.00 - 11.22)

Liver transplantation group
(n= 8)

Eo
Are Median value Evalue
Positive (%) 3
(min — max)
11 (73%) 9.88 (0.00 - 11.15) 0.056
10 (67%) 9.00 (0.00 - 11.57) 0.283

*Mann-Whitney Exact U Test was used to compare the amounts of the two bacteria. p<0.05. FAEP: Faecalibacterium prausnitzii;
AKM: Akkermansia muciniphila.

4. DISCUSSION

Akkermansia muciniphila and Faecalibacterium prausnitzii
are two commensal bacteria, symbiotic and numerically
abundant members of the gut microbiota. Recent studies
have demonstrated their possible association with dysbiosis

immunosuppressed patients.

Recently, it has been shown that after LT there is a decrease
in gut bacterial diversity and dysbiosis [1, 8]. It has been
found that changes in gut microbial composition can result
in disruption of the mucosal barrier, facilitating the
translocation of bacteria and microbial products pathogen
associated molecular patterns in the portal circulation
affecting the inflammatory cytokine milieu in the liver [16].

Table 5: Patients with liver transplantation with no detection of AKM or/and FAEP in context of immunosuppressive

regimens
FAEP negative AKM negative FAEP and AKM
Treatment n=4 n=10 double negative
(adults) (5 children and 5 adults) ri)

Tacrolimus 0 6" 0
Tacrolimus + MMF 3 2 1
Everolimus + CsA 0 1 0
Everolimus + MMF 1 1 1

*5 children + I adult patients. FAEP: Faecalibacterium prausnitzii; AKM: Akkermansia muciniphila; MMF: Mycophenolate
mofetil; CsA: Cyclosporine A.

50 IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 45-51

The pre- and post-LT comparative analysis observed a
decrease in gut microbial diversity in the early post-
transplant period (1 month), with improvement in diversity
after at least 6 months [17]. Furthermore, in a qPCR-based
study of 111 LT recipients it was found that the amount of
FAEP was significantly reduced in recipients, which
corresponds results [7].
prausnitzii 1s an anaerobe with a fecal-mucosal distribution,
one of the major producers of SCFAs (short-chain fatty
acids) of which butyrate is the main energy source for

with our Faecalibacterium

colonic epithelium and possesses potent anti-inflammatory
properties [18]. This bacterium is considered as an anti-
inflammatory with an essential role for the maintenance of
the colonic mucosa, the induction of regulatory T cells [18]
and the regulation of Treg/Th17 balance [19].

Our data shows a significant reduction of AKM load in LT
patients. In the literature, there are discrepant data.
Satapathy SK et al. found loss of AKM in patients after LT,
especially in patients with recurrent NAFLD. It has been
suggested that AKM may play a protective role in the
development of complications (such as de novo NAFLD)
after LT [20]. However, Sun et al., found an increase in
potentially beneficial bacteria, such as AKM, Blautia and
Clostridiales cluster XIVa, 3 months after LT. The authors
suggested that the transplanted liver could significantly
improve gut function, hence leading to an increase in
[21]. A.
muciniphila is an anaerobic, mucin-degrading bacterium
[15], which is considered to have a protective role in the
barrier function of the intestinal mucosa by strengthening

beneficial bacteria in the fecal microbiota

the contacts between intestinal epithelium cells (tight
junctions). Furthermore, it has been suggested that a
decrease in the total amount of AKM can be associated with
thinning of the mucin layer, which may contribute to liver
inflammation [5].

In addition, we try to analyze our data in the context of the
applied immunosuppressive therapy. The results showed
that AKM is mostly affected by tacrolimus and the
combination of tacrolimus with MMF. The available in
literature data on this topic is limited and there is
unequivocal opinion on how tacrolimus affects AKM. Most
studies have been performed on mouse models of LT and
immunosuppressive treatment. Same as us, some authors
indicate a reduction in the load of this bacterium after
tacrolimus treatment [22], in contrast others report that
tacrolimus increases the amount of AKM [10]. It has been
found in mice that the medium dose of tacrolimus increased
the amount of FAEP, while both low and high dose of
immunosuppressant reduced the bacteria [11]. Concerning
FAEP, we found a reduction in the bacterial load after

combined immunosuppression of tacrolimus/everolimus
and MMF, whereas patients in tacrolimus only do not
showed lack of F. prausnitzii. Intriguingly, it has been
observed that in patients who require higher drug doses to
reach optimal tacrolimus plasma concentrations, the amount
of FAEP is increased [23]. The authors found that some
Clostridiales including FAEP could transform tacrolimus
into a less potent metabolite. The aforementioned study
provides evidence for new pathways of tacrolimus
metabolism and the role of the gut microbiota in it.

5. CONCLUSIONS

Our pilot study shows that in patients with LT on
immunosuppressive therapy in the late post-transplant
period, the frequency and amount of beneficial bacteria
AKM and FAEP in gut microbiota are significantly reduced
in comparison with healthy individuals. This may be due to
a variety of possible factors, one of which is
immunosuppressive therapy. Studies in a larger number of
patients are needed for confirmation of our results and

further analysis.

6. ACKNOWLEDGEMENTS

This work was funded by the scientific project grant Ne80-
10-127/26.03.2021 of the Sofia University “St. Kliment
Ohridski’, Sofia, Bulgaria and was executed in University
Hospital "Lozenets", Sofia, Bulgaria. We appreciate the
generosity of the patients and the staff of all clinics for their
dedication and careful sample collection. We are thankful to
Dr. Georgi Vasilev, MD, PhD for statistical help.

7. CONFLICT OF INTERESTS

The authors declare no conflict of interest.

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