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Serum Levels of Interferon-gamma in Patients with
Falciparum Malaria- Khartoum State- Sudan
Ahmed Abdalfatah Ahmed Galander
Department of Parasitology and Medical Entomology, College of Medical
Laboratory Science, Sudan University of Science and Technology, Khartoum, Sudan
Abstract:
A complex parasite such as human
Plasmodium is likely to generate
a variety of substances that injure the hosts directly or cause immunopathology. In
malaria, a blood concentration of anti-inflammatory cytokines, such as interferon-
gamma (IFN-γ) is increased. The present study was performed to analyze IFN-γ levels
inpatients with certain conditions of malaria with healthy controls and correlate
with malaria density infection as well as age groups. It is a cross-sectional study
was carried out in Khartoum State/ Sudan, a total of 49 febrile patients and 10
healthy volunteers as controls were included in this study. Blood samples were
collected from both patients and control candidates in (EDTA) containers for
parasitological and immunological tests. Microscopical examination of thick and thin
blood films rather than immunochromatography test (ICT) were used to detect the
positive samples for Plasmodia. Enzyme linked Immunosorbant Assay (ELISA) was
used for the determination of IFN-γ levels. The results showed that the prevalence
was occurred among all age groups and P.falciparum was the predominant species
with P.vivax mono or co-infections. Furthermore IFN-γ levels in malaria patients were
65.14and SD 64.56 while the levels in healthy participants were 12.33 and SD was
4.11. The difference was found to be statistically significant (P= 0.001). The levels of
cytokine mentioned above were observed to be raised in malaria individual compared
to healthy control candidates.
Keywords—
P.falciparum
;
P.vivax
; Giemsa; Immunochromatographic; IFN-
γ; Khartoum State
INTRODUCTION
Malaria is widespread in tropical and subtropical regions, there are
approximately 350-500 million cases, with 1 to 3 million morbidity, the majority
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among young children in sub-Saharan Africa [1], about 90% of them in Africa
and about half of the world‘s population (3.3 billion people) is at risk of malaria in
more than 100 countries. It’s spreading as a result of environmental changes and
drug resistance [2]. Malaria is commonly associated with poverty, but is also a cause
of poverty and a major hindrance to economic development [3]. Species of
medical importance are; the most serious forms are caused by
. Malaria caused by
and
causes milder disease
that is not generally fatal. A fifth species,
, causes malaria in
macaquesbut can also infect humans. [4,5]. The clinical manifestation ranging
from asymptomatic, uncomplicated to life-threading complications which
common found in sub-Saharan Africa [6]. Sudan is considered as a high burden of
malaria-related morbidity and mortality. However, with WHO’s support to the
national malaria control programme, morbidity and mortality has reduced the
cases from more than four million in 2000 to less than one million in 2010. Between
2001 and 2010, the number of deaths due to malaria reduced by 75%. WHO works
in close collaboration with the national malaria control programme to implement
appropriate and cost- effective malaria control interventions [7]. The pathogenesis
of malaria is due to massive hemolysis of infected cell rather than sensitized
uninfected cell induced by hemozoin and malarial pigment increasing the
severity of anaemia. Cytoadherence in falciparum is due knobes expressed on
parasitized red cell and specific receptor complex on the endothelial cells cause
the most severe complications [8]. The criteria and mechanism of severity in
P. vivax
may differ from falciparum malaria and not related to parasite biomass. There is a 4-
to 5-fold greater loss of uninfected red cells in
P.vivax
relative to
P. falciparum
infection at low parasite densities (<2%) that produce severe anaemia or other
complications [9]. Protective anti-malarial immunity reflects as antidiv production,
phagocytosis, cellular cytotoxicity and parasite inhibition exerted by lymphocytes,
neutrophils and mononuclear phagocytes. However, some of these cellular
activities may also cause tissue damage and the course of a malaria infection is
highly dependent on the balance between the cytokines secreted by the various cells
when activated [10]. In any event, proinflammatory cytokines such as IFN γ, IL- 1,
IL-6 and others may be protective by inducing parasite killing by
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monocytes/macrophages and neutrophils. IL-12, produced by mononuclear
phagocytes and other cells, contributes to protection against pre-erythrocytic and blood
infection by initiating a Th1 anti-malaria response in mice as well as in monkeys [11].
MATERIALS AND METHODS
Study design:
This is a cross-sectional study.
2.2 Study area and study period:
The study was conducted in Medical Diagnostic Center- Khartoum State-
Sudan during the period from December 2016 to December 2018.
Study population:
The study was carried out on patients that clinically diagnosed with
malaria in addition of ten volunteers as control.
Inclusion criteria:
Patients with history of fever proceeding in the past 24 hours with confirmed
malaria parasite positive slides and ICT were included study participant’s cases and
healthy volunteers were included as the study controls.
Exclusion criteria:
All patients who were negatively diagnosed for malaria parasites. Patients
with any known concurrent chronic illness and diseases such as malnutrition,
chronic infection as tuberculosis, pneumonia or urethritis in addition to known
cancer patients.
Sample size:
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The sample size was obtained according to the statistical method
for determining sample size as following equation as described by Mendenhall
etal
.
(1981) [12].
N = t2 *P
(
1-p)/M2
)
N= Sample size
t = The normal standard deviate (t = 1.96)
P = The frequency of occurrence of malaria (16%)
M= Degree of precision (0.05%)
According to the above equation, the study was conducted among
200 clinically suspected individuals. Only 49 patients whom were
diagnosed with positive malaria were included in the study. In addition to 10
volunteers were included as control.
METHODS
Sample collection and processing:
Fifty nine blood samples were collected from all participants. The blood was
drawn aseptically into EDTA Vacutainer® tubes (Becton Dickson and company,
Franklin Lakes, NJ, USA). From each specimen had been tested by the both
methods as dry films and Immune Chromatographic Test
(ICT), then vacutainers were centrifuged at 1,200 g for 10 minutes at room
temperature. The plasma was out and the samples were aliquoted and stored at -
20°C until assayed. All questionnaires were filled by participants [13,14].
Preparation of thick film:
After collection of blood on a clean and grease free glass slide, thick
film was made by spreading one drop of blood with a spreader evenly on an area
about 15×15 mm in diameter. Care was taken to avoid rouleaux formation. Then,
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the slide was labeled properly and allowed to air-dry by keeping the slide on
horizontal position. Precaution was taken during spreading and drying [13].
Preparation of thin film:
After collection of one drop of blood on a clean grease free slide, thin film
was made by spreading the blood using a smooth edged slide or spreader at an
angle of 45º from the horizontal plane. A well-prepared thin blood film was
judged by having a smooth tail end and free of vertical lines and holes. The
slide was then labeled properly and allowed to air-dry [13]. Absolute methanol or
ethanol was used to fix the thin film. Following steps were taken for fixing the
thin film as described by Cheeshbrough (1999) [13]:
- The slide was placed horizontally on a staining rack.
- A small drop of absolute methanol or ethanol was applied to the thin film.
- Then the slide was allowed to fix for 1-2 minutes. Staining of the films:
The slide was first placed on a staining rack. Then 10% Giemsa stain
having a pH of 7.2 was poured gently on the fixed thin film or de-hemoglobinized
thick film until the slide was totally covered. Then the slide was allowed to stain for
30-45 minutes out of the sunlight. Then the stain was washed with clean water.
Back of the slide was wiped and placed in a draining rack. The slide was then allowed
for air- dry [13].
Microscopic examination of the stained film Thick film:
The thick film was examined first by using the 40x objective, a well-
stained part of the thick film was selected which was well populated with
WBC. Then the selected portion of the film was examined with 100x oil immersion
objective by moving along the width of the slide. At least 100 fields were examined
before a slide was considered as negative for malaria parasite [13].
Thin film:
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The thin film was examined by the 40x objective first followed by 100 x
oil immersion objectives. After applying immersion oil the film was examined
by moving along the edge of the film. Then moving the slide inward by one
field, returning in a lateral movement and so on. At
least 100 fields were examined before a slide was considered as negative for
malaria parasite [13].
DISCUSSION
Malaria infection gives rise to host responses which are regulated by both
the innate and adaptive immune system as well as by environmental factors. Acquired
immunity is both species
-
and stage-specific that initiated by pro-inflammatory
cytokines such as IFN γ, IL- 1, IL-6 and other cytokines which may be protective
by inducing parasite killing by phagocytes and neutrophils [16]. However,
cytokines had crutial roles in both the clinical effects due to pathogenesis of malaria
in addition to the strong cellular immune response mainly due to the pro-
inflammatory cytokines IL-12 and IFN-γ [9].
This study aimed to determine the correlation between IFN-γ levels cytokine
and malaria infection. It is thought that many factors affect their levels.
The pro-inflammatory cytokines, IFN-γ levels were elevated in patients with
malaria compared to healthy controls; patients with low parasitemia had much
greater elevated levels of IFN-γ which initiated by early T helper type- 1 response
compared to the other patients with higher parasitemia [17], while in higher
parasitemia the regulatory role of T-helper type 2 responses activated the production
of anti-inflammatory cytokines were required to protect against excessive release of
IFN-γ to determine the beneficial effects on the host’s ability to cope with
infection and
combat disease [11].
Consistent with the present results, study carried by Artavanis
(2002), showed that rapid and intense IFN-γ response from malaria-naive PBMC
than do
P. falciparum
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schizont lysates correlating with rapid iRBC activation of the NK cell population
to produce IFN-γ [17]. In the same way this study showed that there was no
correlation between IFN-γ level and the age of infected patients because the study
area considered as non-endemic malaria transmission and this result was compatible
with Prakash
et al.
(2006) study that showed IFN-γ and other cytokines as IL-2, IL-
5, IL-6, and IL- 12 levels were increased during infection predominantly in
patients with mild malaria in comparison with severe malaria or complicated malaria
regardless the age [18].
Experimental malaria studies performed exclusively in adult hosts indicated
that both CD4+ and CD8+ T cells play an important role in the defense against
malaria. To address the role of these cells in an age-dependent model, where young
susceptible rats can be protected by the transfer of whole spleen cells from adult
protected rats, TCR cells were transferred to young infected rats. These
experiments indicated that 58% of young rats recovered from infection after T cell
transfer [16].
Furthermore, RDTs designed to detect
P.falciparum
and
P. vivax
antigens as
mixed infection, appear to be less accurate in malaria diagnosis compared to
microscopy, these tests fail to detect around 8.2% of the cases and this percent
was higher in compare of 4% failure as study done by Hamza
et al.
(2016) [19].
The results study showed that
P.falciparum
as the predominant
species which was consistent with Hamza
et al.
(2016) study [19], but
with presence of
P.vivax
infection rather than mixed
P.falciparum-
P.vivax
infections in the study area.
ACKNOWLEDGMENTS
We thank the director of
IJAHMR
for permission to publish, Dr. Fahad Sahih
Saber, Dr. Ahmed Bakeet Abdallah, Amna Abdallah Ali and Marwa M. Elhaj for
technical assistance.
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