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RESEARCH ARTICLE
Reduced IFN-glevels along with changes in
hematologic and immunologic parameters are key to
COVID-19 severity in Bangladeshi patients
Mohammed Moinul Islam
a,b,
*, Shafiqul Islam
a,b,c,
*, Ridwan Ahmed
a,b
, Mohit Majumder
a,b
,
Bishu Sarkar
a,b
, Md. Ejajur Rahman Himu
a,b
, Md Kawser
a,b
, Alamgir Hossain
a,b
,
Mohammad Jewel Mia
a,b
, Rashed Rezwan Parag
a,b
, Md. Rakibul Hassan Bulbul
e
, Shakeel Ahmed
e
,
MA Sattar
f
, Rajdeep Biswas
g
, Moumita Das
g
, Md. Mizanur Rahman
b,d
, Rajib Kumar Shil
a,b
,
Ramendu Parial
a,b
, Srikanta Chowdhury
a,b
, Manisha Das
b
, Abu Shadat Mohammod Noman
a,b
, and
Muhammad Mosaraf Hossain
a,b
**
a
Department of Biochemistry & Molecular Biology, University of Chittagong, Chattogram, Bangladesh;
b
EuGEF Research Foundation, Chattogram, Bangladesh;
c
Stem Cell Genetics, Institute for Life and Medical
Sciences, Kyoto University, Kyoto, Japan;
d
Department of Biochemistry, Rangamati Medical College, Ranga-
mati, Bangladesh;
e
Bangladesh Institute of Tropical and Infectious Diseases, Chattogram, Bangladesh;
f
De-
partment of Medicine, Chittagong Medical College and Hospital, Chattogram, Bangladesh;
g
Anaesthesia & ICU department, General Hospital, Chattogram, Bangladesh
The manifestation of coronavirus disease 2019 (COVID-19) severity and mortality has been associated with dysre-
gulation of the immune response, often influenced by racial disparities and conferred by changes in hematologic
and immunologic parameters. These biological and hematologic parameters as well as cytokine profiles were
investigated in a cohort of 61 COVID-19-positive patients (categorized into mild, moderate, and severe groups)
from Bangladesh using standard analytical methods. The data reported that the interleukin (IL)-4 and IL-6 levels
were significantly increased, whereas the levels of interferon (IFN)-gwere significantly reduced in patients with
severe COVID-19 (p<0.05) compared with those in patients with mild and/or moderate COVID-19. The extent of
erythrocyte sedimentation rate (ESR); neutrophil count; and levels of ferritin, C-reactive protein (CRP), and D-dimer
(p<0.05) were found to be significantly increased, whereas the white blood cell (WBC), lymphocyte, eosinophil,
and platelet counts (p<0.05) were observed to be significantly reduced in patients with severe COVID-19 com-
pared with those in the patients in other 2 groups. Our study exhibited a significantly higher IL-6-to-lymphocyte
ratioinpatientswithsevereCOVID-19thaninthosewith mild and moderate COVID-19. The calculated neutro-
phil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR), and ferri-
tin-to-ESR ratio were significantly increased in patients with severe COVID-19. The increase in the IL-4 and IL-6
levels along with CRP and D-dimer levels may envisage a hyperinflammatory environment and immune dysregu-
lation, which contribute to prolonged viral persistence, leading to severe disease. However, the reduced level of
IFN-gcan be attributed to a less fatality toll in Bangladeshcomparedwiththatintherestoftheworld.©2022
ISEH –Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.
HIGHLIGHTS
Interleukin (IL)-4 and IL-6 levels along with erythrocyte sedimenta-
tion rate (ESR); neutrophil count; and the levels of ferritin, C-reac-
tive protein (CRP), and D-dimer were significantly increased
however, interferon (IFN)-glevels were significantly reduced in
patients with severe coronavirus disease 2019 (COVID-19) com-
pared with those in patients with mild and/or moderate COVID-19.
The white blood cell (WBC), lymphocyte, eosinophil, and platelet
counts are significantly reduced in patients with severe COVID-19
in comparison with those in patients with mild and moderate
COVID-19 symptoms.
The neutrophil-to-lymphocyte ratio (NLR), IL-6-to-lymphocyte
ratio, platelet-to-lymphocyte ratio (PLR), lymphocyte-to-mono-
cyte ratio (LMR), and ferritin-to-ESR ratio are also significantly
Offprint requests to: Muhammad Mosaraf Hossain, Department of Biochemistry &
Molecular Biology, University of Chittagong, Chattogram 4331, Bangladesh;
E-mail: mosarafbmb@cu.ac.bd.
MMH, MMI, RP, and ASMN designed the study. SI, RA, MM, MK, AH, MJM, and
MRHB collected samples and data of the patients. SI, RRP, BS, and MERH carried
out the experimental works in the laboratory. MMI, MMH, and RP validated the
data. MMI analyzed the data, prepared the figures, and drafted the manuscript.
MMH and ASMN contributed to finalizing the manuscript by reviewing and editing.
MMH, MMI, RP, SC, RKS, and MD were actively involved in supervision and
overall management of the project. SA, MAS, RB, MD, and MMR were the resour-
ces for technical and hospital support. MMH acquired the fund from the Bangla-
desh Medical Research Council and University of Chittagong. All authors read,
reviewed, and approved the manuscript for submission.
*
MMI and SI contributed equally to this article.
0301-472X/©2022 ISEH –Society for Hematology and Stem Cells. Published by
Elsevier Inc. All rights reserved.
https://doi.org/10.1016/j.exphem.2022.11.006
1
ARTICLE IN PRESS
higher in patients with severe COVID-19 than in those with mild
and/or moderate COVID-19.
The reduced level of IFN-gis attributed to a less fatality toll in
Bangladesh.
The coronavirus disease 2019 (COVID-19) pandemic, engendered by
severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2), has
been a global disaster since its outbreak in Wuhan, China, in December
2019 [1]. As of March 2022, there were over 507 million confirmed
COVID-19 cases and a death toll of more than 62 million, with the case
fatality rate ranging from 0.04% to 18.18% reported worldwide; how-
ever, Bangladesh experienced approximately 2 million cases and total
deaths of less than 30,000 [2]. The disparity in the mortality rates caused
by COVID-19 in different regions of the world has been a puzzling
conundrum but grossly alluded to demographic variables such as age,
sex, ethnicity, presence or absence of comorbidities, human leukocyte
antigen (HLA) genotype, genetically inherited immunity, acquired
immunity by exposure to a wide variety of pathogens, and a lifestyle
reinforcing immunity [3,4]. COVID-19 severity and mortality have been
associated with hyperinflammation caused by elevated levels of proin-
flammatory cytokines, such as interleukin (IL)-6, C-reactive protein
(CRP), and ferritin; decrease in T and B lymphocyte counts and their
activation; and lymphopenia [5−9]. However, even with mortality risk
factors such as dense population, elderly, having comorbidities, poor
maintenance of social distancing protocol, unsuccessful lockdown, and
failure to implement self-isolation and quarantine [10], Bangladesh has
seen a comparatively low mortality rate, which comprised a case-fatality
ratio of approximately 1.49% [2].
SARS-CoV-2 acts on the lower respiratory tract in humans and
exhibits a myriad of clinical characteristics ranging from no symptoms
to severe pneumonia, with acute respiratory distress syndrome and
death. The clinical spectrum of the disease has been manifested as
asymptomatic; mild, with fever, cough, sore throat, malaise, headache,
muscle pain, nausea, vomiting, diarrhea, loss of taste and smell but the
absence of shortness of breath, dyspnea; moderate, with evidence of
lower respiratory disease and an oxygen saturation (SpO
2
)of≥94%;
and severe, with an SpO
2
of <94%, an arterial oxygen partial pressure
(PaO
2
)-to-fractional inspired oxygen (FiO
2
) ratio (with PaO
2
in mm
Hg) of <300 mm Hg, a rate of respiration of >30 breaths/min, or
infiltrates in >50% of the lungs; and perilous, with respiratory failure,
septic shock, and/or multiple organ dysfunction, leading to death
[11]. However, because of limited resources and unavailability of data
from both patients and hospitals in Bangladesh, it was difficult to dif-
ferentiate between asymptomatic and mild illnesses and between
patients with severe and critical illness, which narrowed the category
to mild, moderate, and severe illness in our study.
Mild infection causes increased viral replication, which continues
to an inflammatory response in case of moderate illness, which
becomes predominant in patients with severe-to-critical illness [12].
The 3 phases of immunopathologic progression leading to COVID-
19 mortality, namely, initiation phase, amplification phase, and con-
summation phase, are distinguished based on cytokine and chemo-
kine profiles [13]. On that account, for the treatment of COVID-19,
it is very important to identify host or viral determinants that are criti-
cal for the development of mild or severe disease because inflamma-
tory cell influx and cytokine blockade cannot bring benefit until a
good time point or patient selection is not considered [14].
Because of our concern about the disproportionate burden of
COVID-19 outcomes, with significant immunopathologic disparities,
in our study, we aimed to elucidate the cytokine parameters in mildly,
moderately, and severely ill patients with COVID-19, which can help
distinguish the severity of the disease by determining the immune sig-
nature, explanation of immune regulation that may lead to determin-
ing better COVID-19 therapy. In addition, a study of the correlation
of biochemical and immunologic parameters with the severity of
COVID-19 in Bangladesh is yet to be performed. In line with that,
we analyzed biochemical and hematologic parameters for mildly,
moderately, and severely ill individuals with COVID-19 to determine
the association of cytokine profile and other biological parameters
with COVID-19 severity. Our data reported that the IL-4 and IL-6
levels were significantly increased, whereas the levels of interferon
(IFN)-gwere significantly reduced in patients with severe COVID-19
compared with those in patients with mild and/or moderate
COVID-19. The extent of erythrocyte sedimentation rate (ESR); neu-
trophil count; and levels of ferritin, CRP, and D-dimer were found to
be significantly increased, whereas the WBC, lymphocyte, eosinophil,
and platelet counts were observed to be significantly reduced in
patients with severe COVID-19 compared with those in patients in
the other 2 groups. The reduced level of I FN-gcan be attributed to a
less fatality toll in Bangladesh compared with that in the rest of the
world. This study will help us to explore the intensity of the severity
of the disease caused by SARS-CoV-2 infection and make a defined
plan for controlling and medication for such viral infections in the
future.
MATERIALS AND METHODS
Study Design
A total of 61 (n= 61) patients with COVID-19, who tested positive
for COVID-19 or were admitted with symptoms to Chittagong Med-
ical College Hospital (CMCH), the Bangladesh Institute of Topical
and Infectious Diseases (BITID) hospital, or Chittagong General Hos-
pital, were enrolled in this study. The presence of SARS-CoV-2 was
confirmed using the real-time reverse-transcription polymerase chain
reaction (qRT-PCR) test according to World Health Organization
(WHO) interim guidelines. Demographic and epidemiologic charac-
teristics as well as symptoms were recorded using a pretested ques-
tionnaire.
Ethics Statement
Blood samples were collected as per the guidelines of ethical permis-
sion (Memo No: CMC/PG/2021/183) granted by the Ethical
Review Committee of Chittagong Medical College, Chattogram,
Bangladesh.
Categorization of the Study Subjects Based on Clinical
Symptoms
The selected patients with COVID-19 were categorized as having
mild (n= 20), moderate (n= 21), or severe disease (n= 20) based on
their clinical symptoms. The patients with mild COVID-19 were not
called for hospitalization or had self-recovering disease courses,
whereas the patients with moderate COVID-19 were specified as
those hospitalized in a nonintensive care unit (non-ICU) setting. Fur-
thermore, severely ill patients experienced cardiac impairment, an
ARTICLE IN PRESS
2 M.M. Islam et al Experimental Hematology
&& 2022
exaggerated inflammatory response, and/or aggravation of underlying
comorbidities, organ failure, and rapid clinical deterioration, with
respiratory failure, necessitating mechanical ventilation and requiring
intensive therapy or ICU care.
Sample Collection and Preparation
A total of 5 mL of whole blood was collected from the study subjects
in Vacutainer (Invitrogen) blood collection tubes by an expert phle-
botomist (Technician) while maintaining proper safety procedures.
All the blood samples were collected 5−7 days after symptoms
appeared. After collecting blood, the tubes were kept at room tem-
perature for 30 minutes without any disturbance. Finally, serum was
collected by centrifuging the tubes at 2,000 gfor 10 minutes.
Investigation of Hematologic and Biochemical Parameters
The complete blood count (CBC) test for the estimation of red blood
cell (RBC), white blood cell (WBC), and platelet counts as well as a
differential count (DC) for the estimation of neutrophil, lymphocyte,
monocyte, eosinophil, and basophil counts were performed using
Hematology Analyzer Sysmex XT-2000i and checked manually. The
Cynmeth and Westergren methods were followed for determining
the hemoglobin levels and measuring ESR, respectively. Serum creati-
nine levels were estimated using the Jaffe alkaline picrate method,
and CRP levels were determined using a high-sensitivity assay based
on the principle of latex agglutination. In addition, the electrochemi-
luminescence immunoassay and fluorescence immunoassay were
used to perform D-dimer and ferritin tests, respectively.
Investigation of Selected Cytokines
The levels of IL-6, tumor necrosis factor (TNF)-a,IFN-gand IL-4
were measured in the serum samples using the enzyme-linked immu-
nosorbent assay (ELISA) as per the protocol and instruction of the
manufacturer sourced from Demeditec Diagnostics GmbH, Germany
(Cat No.: TNF-a[DE4641], IFN-g[DE4434], IL-6 [DE4640]) and
Drug Instruments GmbH, Germany (IL4 [EIA-5539]). In brief, the
following procedures were adopted to measure the concentration of
the 4 tested cytokines in the serum samples of the study subjects.
After proper labeling of each well (well for the samples, a control,
and a calibrator), a volume of 50 mL of buffer (incubation) was ali-
quoted into each selected well, followed by loading of 200 mL of the
samples, a control, and a calibrator in the respective wells. The wells
were allowed to incubate at 20°C on a flat shaker at 700 rpm for 120
minutes. After discarding the liquids, the wells were washed thrice
using 400 mL of washing buffer. A volume of 50 mL of horseradish
peroxidase (HRP)-conjugated anti-TNF-a, anti-IFN-g, anti-IL-6, or
anti-IL-4 was aliquoted into the wells of the respective plates. The
wells were allowed to incubate at 20°C on a flat shaker at 700 rpm
for 120 minutes. The washing steps were repeated. A total volume of
100 mL of a revelation solution was loaded in all the wells. The wells
were allowed to incubate at 20°C on a flat shaker at 700 rpm for 15
minutes. Finally, a total volume of 100 mL of a stop solution was ali-
quoted in all the wells and the absorbance measured at 450 and
490 nm. The ELI SA-AID software was used to calculate the concen-
tration of the tested cytokines.
Statistical Analysis
The SPSS 17.00 and Graph pad prism 9 statistical software were used
for performing all statistical analyses. Data were expressed as mean §
SD.
RESULTS
Basic and Clinical Characteristics of Patients’Cohorts
Our study included 20, 21, and 20 patients with mild, moderate, and
severe symptoms of SARS-CoV-2 infection, respectively. Among the
total of 61 patients (median age, 53 years; interquartile range [IQR],
35.50−60.00 years; range, 24−70 years), the mean ages of the 3
groups (39.3 §13.78, 48.67 §12.18, and 53.05 §11.16, respec-
tively) were significantly different (p= 0.0211), with the moderate
and severe groups having higher ages (Table 1 and Supplementary
Figure E1). Among all the patients, 31 (50.8%) were men, and the
proportions of men and women were almost equally distributed in
each group (men:women = 11:10 in the mild, 10:10 in the moderate,
and 10:10 in the severe groups). There were no significant differences
in the sex ratio of each group.
Among the mild, moderate, and severe groups of patients with
COVID-19, the most prominent symptoms were fever (95%,
94.74%, and 95%, respectively), followed by cough (65%, 93.75%,
and 100%, respectively), body pain (45%, 60%, and 62.50%, respec-
tively), and headache (60%, 50%, and 37.50%, respectively), accom-
panied by or without sore throat, vomiting, diarrhea, weakness, loss
of taste and smell when the most severe cases manifested fever,
cough, body pain, and dyspnea (shortness of breath) as well as an
oxygen saturation of less than 80% (Table 1). The oxygen saturation
level (%) was significantly lower (p<0.05) in the severe group
(79.44%) than in the mild group (96.2%) of COVID-19-positive
patients. A computed tomography (CT) scan and X-ray report of the
posteroanterior view of the chest showed normal trachea and dia-
phragm, free basal angles, normal heart in TD, and a normal lung field
in the patients with mild COVID-19, whereas in the patients with
moderate COVID-19, lung fibrosis, pulmonary inflammatory lesions,
and pneumonitis were seen in many cases. In most patients with
severe COVID-19, advanced-stage pneumonia was seen, with more
than 50% of lung infection, kidney failure, and heart attack in some
cases. Approximately 55% of the mildly ill patients, 76% of the mod-
erately ill patients, and 75% of the severely ill patients with COVID-
19 recruited in our study had at least 1 or more comorbidities such as
diabetes mellitus, hypertension, hypotension, asthma, chronic kidney
disease (CKD), sinusitis, allergy, cancer, lung disease, and liver disease.
There were no significant differences in comorbidities observed
among the patients with mild, moderate, and severe COVID-19 (p>
0.05) (Table 1). Among the patients included in our study, 15% of
those in the mild group, 61.9% of those in the moderate group, and
70% of those in the severe group had diabetes mellitus, whereas
approximately 40%, 42.9%, and 55% of the patients of the mild,
moderate, and severe groups, respectively, had hypertension. In all
the groups, approximately 5% of the patients had heart disease. In
addition, among the patients with mild COVID-19, approximately
5% and 10% had CKD and allergy, respectively, with no hypoten-
sion, asthma, sinusitis, cancer, lung disease, and liver disease. On the
other hand, among the patients with moderate COVID-19,
ARTICLE IN PRESS
Experimental Hematology
Volume 000 Number
M.M. Islam et al 3
approximately 4.8%, 4.8%, and 10% had asthma, CKD, and cancer,
respectively, with no hypotension, sinusitis, allergy, lung disease, and
liver disease. Approximately 15% and 10% of the patients severely
affected by COVID -19 had heart disease and asthma, respectively,
with no CKD, sinusitis, cancer, lung disease, or liver disease (Supple-
mentary Figure E1C).
Hematologic Parameters and Infection-related Biomarkers
Our study showed that the ESR was significantly elevated in the
moderate and severe groups compared with that in the mild group (p
<0.05 and p<0.0001, respectively) and severe-to-moderate group
(p<0.01) of patients with COVID-19. The moderate and severe
groups had an ESR of more than the normal range (1%−20%) [15]
(Figure 1A). The patients with mild and moderate COVID-19 had
normal WBC counts (45,000−10,000 cells/mm3) [16], and less-
than-normal levels were present in the patients with severe COVID-
19, which were significantly lower in comparison with those in the
patients with mild (p<0.001) and moderate COVID-19 (p<0.0001)
(Figure 1B). The patients with mild COVID-19 had a normal range of
lymphocyte count (20%−40%) [15], whereas those with moderate
and severe COVID-19 had slightly lower (17.32% §8.426%) and far
lower (9.714% §3.539%) lymphocyte counts than normal, respec-
tively. The moderate and severe groups showed significantly reduced
lymphocyte counts compared with the mild (p<0.0001) and moder-
ate groups (p<0.0001) (Figure 1C). On the other hand, the neutro-
phil counts were significantly increased in the moderate (p<0.01)
and severe groups (p<0.0001) in comparison with those in the mild
group and exceeded the normal limit (54%−62%) [15] (Figure 1D).
The moderate and severe cases had significantly reduced eosinophil
counts compared with the mild cases (p<0.001 and p<0.0001),
although all the groups manifested a normal range of eosinophil
count (0%−3%) [15] (Figure 1F). The patients with severe COVID-
19 had significantly lower platelet counts (p<0.05) than those with
moderate COVID-19 (Figure 1H). However, all 3 groups showed a
normal range of platelet count (1 £105−4.5 £105 cells/mm3) [15].
The patients with mild, moderate, and severe COVID-19 had 0%
−0.5% of basophils, which is below normal levels and was found to
be statistically insignificant among the groups. We observed the ferri-
tin level to be higher than the normal range (13−300 ng/mL) [16] in
both the moderate and severe groups and the D-dimer level to be
higher than the normal range (<0.4 mg/mL) [15] in the severe group.
The data showed that the severe group had significantly higher (p<
0.05) ferritin levels than the mild group and that the severe group
had significantly higher (p<0.05) D-dimer levels than the mild and
moderate groups of patients with COVID-19 (Figure 1J, M). There
were no significant differences in the monocyte and RBC counts and
in the hemoglobin and serum creatinine levels among the patients
with mild, moderate, and severe COVID-19 (Table 2; Figure 1E, G, I,
L), and all the groups manifested a normal range of monocyte count
(3%−7%), RBC count (men: 4.7 £10
6
−6.1 £10
6
cells/mL; women:
42 £10
6
−5.4 £10
6
million cells/mL), hemoglobin level (men: 13.5
−16.5 g/dL; women: 12.0−15.0 g/dL), and serum creatinine level
(0.5−1.4 mg/dL) in the patients with COVID-19 included in our
Table 1 Demographic characteristics and symptoms manifested in patients with mild, moderate, and severe coronavirus disease
2019
Characteristics Unit Mild (n= 20) Moderate (n= 21) Severe (n= 20) pvalue Statistical test
Age Mean §SD 39.3 §13.78 48.67 §12.18 53.05 §11.16 0.0211
a
ANOVA
Sex number (% of males) 11 (55) 10 (47.62) 10 (50) 0.9845 Chi-square
Symptoms
O2 saturation (mean §SD) 96.2 §4.207 89.81 §9.123 79.44 §14.67 0.0185
a
ANOVA
Fever (% present) 95.00 94.74 95.00 0.9991 Chi-square
Cough (% present) 65.00 93.75 100.00 0.0062
b
Chi-square
Runny nose (% present) 30.00 30.00 14.29 0.7010 Chi-square
Body pain (% present) 45.00 60.00 62.50 0.6073 Chi-square
Headache (% present) 60.00 50.00 37.50 0.5509 Chi-square
Sore throat (% present) 15.00 22.22 44.44 0.2241 Chi-square
Vomiting (% present) 30.00 36.36 25.00 0.641 Chi-square
Diarrhea (% present) 35.00 40.00 16.67 0.6142 Chi-square
Loss of taste and smell (% present) 10.00 20.00 16.67 0.7394 Chi-square
Dyspnea (% present) 35.00 72.73 61.54 0.0961 Chi-square
Weakness (% present) 5.00 30.00 12.50 0.1612 Chi-square
Comorbidities
c
(% present) 55.00 76.19 75.00 0.2633 Chi-square
ANOVA=Analysis of variance.
ap<0.05.
bp<0.01.
c One or more of the following diseases: diabetes mellitus (DM), hypertension (HTN), hypotension, heart disease, asthma, chronic kidney disease,
sinusitis, allergy, cancer, and lung disease.
ARTICLE IN PRESS
4 M.M. Islam et al Experimental Hematology
&& 2022
study [16]. The CRP level was more than the normal range (<0.3 mg/
L) [16] in all the groups of patients with COVID-19, and the severe
group had significantly higher CRP levels than the mild (p<0.001)
and moderate groups (p<0.05) (Figure 11K).
The neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte
ratio (PLR), lymphocyte-to-monocyte ratio (LMR), and ferritin-to-
ESR ratio (FER) were observed to increase with disease severity
(Figure 2A, C-E). The NLR was significantly higher in the patients
with moderate (p<0.01) and severe (p<0.0001) COVID-19 than in
the patients with mild COVID-19, and the patients with severe
COVID-19 (p<0.01) had a significantly higher ratio than those with
moderate COVID-19. The PLR and LMR were significantly higher in
the patients with severe COVID-19 than in the patients with mild
and severe COVID-19. Both the patients with moderate and severe
COVID-19 exhibited significantly higher FER than those with mild
COVID-19.
Table 2 and Figure 1 present a comparative analysis of hematologic
and biochemical parameters among patients with mild, moderate,
and severe COVID-19.
Immunologic Signature of Patients with COVID-19 (The
Cytokine Profile)
The levels of IL-4, IL-6, TNF-a, and IFN-gwere not significantly dif-
ferent among the age groups of the studied COVID-19 patients (Sup-
plementary Figure E2). The most important findings of our study
Figure 1 Hematologic and biochemical parameters of patients with mild, moderate, and severe coronavirus disease 2019 (COVID-19). The
level of (A) erythrocyte sedimentation rate (ESR); (B) white blood cell (WBC), (C) lymphocyte, (D) neutrophil, (E) monocyte, (F) eosinophil, (G)
red blood cell (RBC), and (H) platelet counts; levels of (I) hemoglobin (Hb), (J) ferritin, (K) C-reactive protein (CRP), (L) serum creatinine, and
(M) D-dimer among the mild, moderate, and severe groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Experimental Hematology
Volume 000 Number
M.M. Islam et al 5
ARTICLE IN PRESS
Figure 2 Calculated results of neutrophil-to-lymphocyte ratio (NLR): (A) NLR, (B) interleukin (IL)-6-to-lymphocyte ratio, (C) platelet-to-
lymphocyte ratio (PLR), (D) lymphocyte-to-monocyte ratio (LMR), and (E) ferritin-to-erythrocyte sedimentation rate (ESR) ratio in
patients with mild, moderate, and severe coronavirus disease 2019 (COVID-19). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Table 2 Comparisons of hematologic and biochemical parameters among patients with mild, moderate, and severe coronavirus
disease 2019 (mean §SD)
a
Parameters Unit Mild (n= 20) Moderate (n= 21) Severe (n= 20) pvalue Statistical tests
ESR (mm/h) Mean §SD 25 §16.91 46.93 §6.686 74.07 §26.64 <0.0001
b
Ordinary 1-way ANOVA
WBC (/mm
3
) Mean §SD 6,883 §2,229 9,807 §3,609 35 §21.64 <0.0001
b
Ordinary 1-way ANOVA
Neutrophil (%) Mean §SD 58.44 §11.82 77.26 §8.419 85.07 §3.362 <0.0001
b
Ordinary 1-way ANOVA
Lymphocytes (%) Mean §SD 35.56 §11.01 17.32 §8.426 9.714 §3.539 <0.0001
b
Ordinary 1-way ANOVA
Monocyte (%) Mean §SD 2.94 §1.056 3.778 §1.396 3 §1.519 0.1259 Ordinary 1-way ANOVA
Eosinophil (%) Mean §SD 3.056 §1.162 1.667 §0.767 1.357 §0.633 <0.0001
b
Ordinary 1-way ANOVA
RBC (million cells/mL) Mean §SD 5.624 §3.614 5.002 §2.538 5.046 §0.9602 0.7554 Ordinary 1-way ANOVA
Platelets (/mm
3
) Mean §SD 341,444 §142,308 292,706 §78,400 2,07,000 §80,472 0.0039
c
Ordinary 1-way ANOVA
Hb (g/dL) Mean §SD 12.56 §1.770 11.93 §1.702 12.75 §1.924 0.3803 Ordinary 1-way ANOVA
S creatinine (mg/dL) Mean §SD 1.025 §0.3845 1.362 §0.7571 0.8473 §0.2448 0.0828 Ordinary 1-way ANOVA
Ferritin (ng/mL) Mean §SD 245.6 §271.9 699.1 §485.7 982.3 §772.6 0.2022 Ordinary 1-way ANOVA
D-dimer (mg/mL) Mean §SD 0.267 §0.1021 0.9936 §1.610 1.651 §2.141 0.3851 Ordinary 1-way ANOVA
CRP (mg/L) Mean §SD 9.182 §5.43 59.83 §51.89 136.3 §74.65 <0.0001
b
Ordinary 1-way ANOVA
ANOVA=Analysis of variance; CRP=C-reactive protein; ESR=erythrocyte sedimentation rate; S=serum; WBC=white blood cell.
a GraphPadPrism 9 software was used for data analysis.
bp<0.0001.
cp<0.01.
6 M.M. Islam et al Experimental Hematology
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revealed an upward trend in the IL-4 and IL-6 levels and, interest-
ingly, a downward trend in the IFN-glevels from the mild to severe
groups of patients with COVID-19 (Table 3). The severe group mani-
fested significantly higher IL-4 levels (p<0.001) than its mild counter-
part, whereas the moderate and severe groups manifested
significantly higher IL-6 levels (p<0.05 and p<0.001, respectively)
than the mild group Figure 3A, B). The IFN-glevels were progres-
sively reduced by the severity of the disease. Our data showed that
the IFN-glevels were significantly lower (p<0.0001) in the patients
with moderate (p<0.0001) and severe (p<0.0001) COVID-19 than
in their counterparts with mild COVID-19 (Figure 3C). Although not
statistically significant, the TNF-alevels were higher in the moderate
and severe groups than in the mild group (Figure 3D). Our study also
exhibited significantly higher IL-6 levels in the patients with severe
COVID-19 than in those with mild (p<0.01) and moderate (p<
0.05) COVID-19 (Figure 3).
DISCUSSION
The roles of clinical hematology parameters and inflammatory mole-
cules are pivotal in orchestrating host defense against COVID-19
pathogenesis. Various acute phase and proinflammatory markers are
associated with disease progression-related mortality in different eth-
nic populations [17,18]. In this study, most of the patients with severe
COVID-19 in Bangladesh tended to have lymphopenia; lower plate-
let, lymphocyte, and eosinophil counts; higher NLR; and elevated lev-
els of infection-related biomarkers (i.e., ESR, serum ferritin or
hyperferritinemia, and CRP) in comparison with those with mild and
moderate COVID-19. Therefore, understanding the dysregulation in
the immune system and associated hyperinflammation is key to
undertaking the disease’s pathologic course of action.
Our study showed leukopenia (lower WBC count) in the patients
with severe COVID-19, although those with mild and moderate
COVID-19 had a normal WBC count (Figure 1B). Because leukopenia
increases the risk of infections when infected [19], higher age and
related comorbidities might have caused the lowering of the WBC
count, which, in turn, led to more severe COVID-19 pathogenesis.
This is also supported by the findings that asymptomatic patients typi-
cally have a higher WBC count, particularly lymphocytes, wherein high
leukocyte counts help SARS-CoV-2-infected individuals to fight against
the virus and prevent the severity of the disease by fast viral clearance
[20]. In alignment with this fact, our study also showed a higher lym-
phocyte count in the patients with mild COVID-19, which was signifi-
cantly reduced in those with moderate and severe COVID-19
(Figure 1C). The highly reduced lymphocyte counts in the moderate
and severe groups in our study compared with those in the mild (p<
0.0001) and moderate groups (p<0.0001), respectively, are in align-
ment with the study results published by Liao et al. [21].Besides,lym-
phopenia (lymphocyte count <18%) is associated with severe
COVID-19 manifestation and has been suggested as a prognostic
marker for the disease’s severity and mortality [18,22,23]. Lymphocytes
(B and T cells) produce antibodies, chemokines, cytokines, and other
immune molecules to fight against infections, and a lower lymphocyte
count can cause immune system compromise. The eosinophil counts,
although in the normal range, were reduced significantly in the patients
with moderate and severe COVID-19 compared with those in the
patients with mild COVID-19 (Figure 1F). A number of other studies
have found reduced WBC, lower lymphocyte (lymphopenia), and
lower eosinophil counts (eosinopenia) to be associated with severe ill-
ness and death in patients with COVID-19 [21,22,24−26]. In the
patients with moderate and severe COVID-19, neutrophilia (higher
neutrophil count) was observed, and the neutrophil count was signifi-
cantly higher than that in the patients with mild COVID-19
(Figure 1D). Although in inflammatory responses, neutrophils are con-
sidered the first recruitment to the infection site, conferring protection
against viral infection, prolonged and acute neutrophilia caused by viral
infection may deteriorate pneumonia and acute respiratory distress
syndrome [27]. Therefore, larger activation of neutrophils caused by
prolonged viral presence in patients with moderate and severe
COVID-19 may be correlated with increased severity of the disease.
Our study also exhibited a significant reduction in the platelet count in
the patients with severe COVID-19 in comparison with that in the
patients with mild COVID-19. This observation is also in alignment
with the result of the study by Fan et al. [23], which showed that
thrombocytopenia (reduced platelet count) in ICU patients with
COVID-19 is associated with hospital admission and death. The baso-
penia (0%−0.5% of basophil count) observed in our study might have
been caused by inflammatory migration of basophils to COVID-19
infection sites.
Furthermore, in this study, the moderate and severe cases had higher
NLR (Figure 2A), which indicates a dysregulated host innate immune
response. Several other studies have also manifested a strong association
between higher NLR and the severity and mortality of COVID-19
[18,28,29] and other infectious diseases, including acute coronary syn-
drome and pneumonia [30]. In an earlier report, neutrophilia and ele-
vated NLR were shown to be significantly increased in patients with
severe COVID-19 and patients requiring ICU care [31]. Additionally,
the calculated PLR and LMR, which are 2 important predictors of
inflammation and mortality for many diseases [32,33], were significantly
Table 3 Cytokines signatures among patients with mild, moderate, and severe coronavirus disease 2019
a
Cytokines Unit Mild Moderate Severe pvalue Statistical test
IL-4 (pg/mL) Mean §SD 0.1583 §0.1345 0.269 §0.3209 0.3758 §0.2734 0.0181
b
Ordinary 1-way ANOVA
IL-6 (pg/mL) Mean §SD 8.419 §15.25 20.5 §26.73 50.06 §77.29 <0.0001
c
Ordinary 1-way ANOVA
IFN-ɣ(IU/mL) Mean §SD 11.83 §6.768 4.28 §8.834 0.0595 §0.1111 <0.0001
c
Ordinary 1-way ANOVA
TNF-a(pg/mL) Mean §SD 0.037 §0.06681 1.834 §7.125 0.549 §1.277 0.3812 Ordinary 1-way ANOVA
ANOVA=Analysis of variance; IFN=interferon; IL=interleukin; TNF=tumor necrosis factor.
a GraphPad Prism 9 software was used for data analysis.
bp<0.05.
cp<0.0001.
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higher in the patients with severe COVID-19 than in those with mild
and moderate COVID-19 in our study (Figure 2C, D). More impor-
tantly, patients severely affected by COVID-19 have been shown to
have higher PLR and LMR in several previous studies [34,35]. On the
contrary, higher LMR has been shown to be associated with lower mor-
tality in other studies of patients with COVID-19 [36].Therefore,scru-
tiny of erythrocytes, lymphocyte subsets, NLR, PLR, and LMR is
predictive of the severity and mortality of COVID-19.
Inflammatory or infection markers such as ESR, CRP, and D-dimer
have been found to be positively associated with COVID-19 severity
[37]. Our study showed progressive and significant elevation of ESR
as well as CR P and D-dimer levels with increasing severity of the dis-
ease (Figure 1A, K, M). Elevated CRP levels are critically linked with
the overproduction of inflammatory cytokines and a hyperactive
immune system [38], which may damage respiratory tissues, causing
severe COVID-19. In addition, the serum creatinine level was signifi-
cantly decreased in the patients with severe COVID-19 (Table 1). In
our study, the monocyte and RBC counts as well as hemoglobin lev-
els were not significantly different in the mild, moderate, and severe
groups (Figure 1E,G, I). Layla et al. [39] did not find any differences
in the RBC counts among patients with mild, moderate, and severe
COVID-19 in Bangladesh.
We observed the ferritin level (Figure 1J) and FER (Figure 2E) to
be higher in the moderate group and significantly inflated in the
severe group compared with those in the mild group of patients
with COVID-19. A high level of ferritin exerts immune-suppres-
sive and proinflammatory effects, mediating immune dysregula-
tion, which is accompanied by a cytokine storm, and our data are
also supported by the findings of previous studies that have sug-
gested that hyperferritinemia (elevated ferritin levels) and FER are
Figure 3 Cytokine profiles of patients with mild, moderate, and severe coronavirus disease 2019 (COVID-19). The levels
of (A) interleukin (IL)-4, (B) IL-6, (C) interferon (IFN)-g, and (D) tumor necrosis factor (TNF)-aamong the mild, moderate, and severe
groups of patients with COVID-19. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
8 M.M. Islam et al Experimental Hematology
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associated with increased severity of pulmonary disorders and
mortality due to COVID-19 [40,41]. The regulation of proinflam-
matory and anti-inflammatory mediators is crucial in viral infec-
tion, wherein proinflammatory and anti-inflammatory cytokines as
well as ferritin can induce each other to orchestrate the immune
system.
The most intriguing findings of our study are the increment in the
IL-4 and IL-6 levels and, interestingly, a progressive reduction in the
IFN-glevels from the mild to severe groups of patients with COVID-
19 ( Figure 3A, B,C). Increases in the IL-4 and IL-6 levels, but not
reduction in the IFN-glevel, have also been reported by other studies
[42]. Previous studies also showed that IL-6 antagonists significantly
reduced the respiratory support requirements and the levels of
inflammatory markers in all patients with COVID-19 with cytokine
storms and implicated benefits for severely ill patients [43]. In addi-
tion, IL-6-mediated low expression of HLA-DR and lymphopenia
have been shown to be associated with prolonged cytokine produc-
tion and hyperinflammation in patients with severe COVID-19 [44].
Our study indicates a similar pattern, wherein lymphopenia
(Figure 1C) and elevated IL-6 levels (Figure 3B) can be associated
with the severity of COVID-19.
IL-6/LY reflects the imbalance of inflammatory response and the
disruption of immune function [45]. In this context, IL-6/LY
appeared to be crucial in predicting the immune-inflammatory com-
plex index in the prognosis of COVID-19. Our study exhibited signif-
icantly higher IL-6 to Lymphocyte ratio in the patients with severe
COVID-19 than in those with mild (p<0.01) and moderate (p<
0.05) COVID-19 (Figure 2B). Furthermore, a negative correlation
(Pearson r=2.51) between IL-6 levels and lymphocytes (Figure
4C) in our study indicates the association of higher cytokine levels
and lymphopenia with the severity of COVID-19. In other studies,
patients with severe disease were shown to have high levels of IL-6,
which are correlated with decreased lymphocyte counts [46]. Macro-
phages and dendritic cells also manifested high levels of IL-6 and IL-
1b, which might directly destroy human spleens and lymph nodes
and eventually lead to lymphopenia in vivo [47].
The cytokine environment determines the subsets of pathogenic T
helper (T
H
) cells by influencing their differentiation. In particular,
IFN-gis critical for determining T
H1
development and inhibiting T
H2
differentiation, whereas IL-4 is necessary for the development of T
H2
proliferation and downregulation of T
H1
growth, creating a T
H1
/T
H2
balance that determines the disease outcomes [48]. The T
H1
/T
H2
bal-
ance regulates cellular immune responses, which impacts the devel-
opment of inflammatory dysregulation and susceptibility to infectious
diseases [49]. It has also been reported that higher IL-4 and IL-6 lev-
els and lower IFN-glevels (lower T
H1
/T
H2
) mediate diminished cell-
mediated immunity and increased production of serum antibodies
[48]. The higher IL-4 and IL-6 levels and the lower IFN-glevels in
the patients with severe COVID-19 observed in our study may indi-
cate a preference for an antibody-mediated immune effector
response rather than a cell-mediated response. Therefore, a decrease
in the lymphocyte counts in our study may cause speculation of a
Figure 4 Correlation analysis. Correlation among hematologic and proinflammatory parameters (A) in patients with coronavirus dis-
ease 2019 (COVID-19). Correlations of the levels of (A) interleukin (IL)-4, (B) IL-6, (C) interferon (IFN)-g, and (D) tumor necrosis factor
(TNF)-awith lymphocyte count (%) in all patients with COVID-19 in the study. CRP=C-reactive protein; Hb=hemoglobin; WBC=white
blood count.
Figure 5 Survival curves among mild, moderate, and severe
coronavirus disease.
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lower T
H1
/T
H2
ratio and a decent increase in B-cell proliferation. IL-6
supports viral persistence by activating IL-4-producing CD4+ T cells
and suppressing IFN-g-producing CD4+ T cells [50]. In addition, IL-
4 has a role in the development of rhinitis, allergic response, and
acute inflammation, whereas IFN-g-mediated T
H1
plays a critical role
in antigen-specific defense mechanisms at the epithelial surface associ-
ated with multiple inflammatory airway diseases [51]. Other reports
have stated that IFN-gacts as a regulator of efficient antigen presenta-
tion [52], and lowering the IFN-glevel or deviation in the IFN-g-IFN-
gR system severely impedes host immune responses to infections
[53]. Surprisingly, this is the first report to show that the IFN-glevel is
extremely reduced in patients with severe COVID-19 within 5
−7 days of symptoms appearing. Gadotti et al. [54] showed a higher
IFN-glevel within 10 days and a decrease in the IFN-glevel after
10 days of symptoms appearing, wherein a sustained higher IFN-g
level was related to higher mortality. In addition, the patients with
severe COVID-19 did not have significantly higher TNF-alevels than
those with moderate and mild COVID-19 enrolled in our study.
Recent studies also showed IFN-gand TNF-alevels to be associated
with respiratory distress and high mortality in patients with COVID-
19 [55]. Other studies showed that the combined effect of IFN-g
and TNF-ainduces PANoptosis and a deadly cytokine storm, causing
acute lung damage and mortality among patients [56,57]. According
to these results, we speculated that in Bangladeshi patients, lower
levels of IFNg10 days after symptoms appear could result in worse
outcomes through the loss of antiviral and antiinflammatory mecha-
nisms and exacerbation of COVID-19 pathogenesis but with lower
mortality. Therefore, we can posit that increased IL-4 and IL-6 levels
with robustly reduced IFNglevels in Bangladeshi patients corrobo-
rates the enhanced severity of COVID-19, with lower mortality
(Figures 5 and 6). However, to prove it entirely, complete immune
signatures and genetic disparities are required to be examined in a
higher number of samples and wider types of ethnic people world-
wide.
Conflicts of Interest Disclosure
The authors do not have any conflicts of interest to declare in relation
to this work.
Acknowledgments
This work was supported by the Bangladesh Medical Research
Council (BMRC) (BMRC/HPNSP-Research Grant/2020-2021/52
(1-47) and Research and Publication Cell (216/GoBe/PoRi/ProKa/
Doptor/cu/2021) University of Chittagong (CU), Bangladesh.
Figure 6 Graphical abstract. COVID-19=Coronavirus disease 2019; CRP=C-reactive protein; IFN=interferon; IL=interleukin;
LMR=Low mortality rate.
10 M.M. Islam et al Experimental Hematology
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Supplementary Figure E1 (A) Age distribution, (B) oxygen saturation, and (C) comorbidities in patients with mild, moderate, and
severe coronavirus disease 2019 (COVID-19). *p<0.05, **p<0.01. CKD=Chronic kidney disease; DM=diabetes mellitus.
Supplementary Figure E2 Levels of (A) interleukin (IL)-4, (B) IL-6, (C) interferon (IFN)-g, and (D) tumor necrosis factor (TNF)-ain dif-
ferent age groups (≤35, 36−45, 46−55, >55 years) of patients with coronavirus disease 2019 (COVID-19).
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