This document summarizes a study that assessed the correlation between levels of the cytokine TNF-alpha and hemoglobin in patients infected with Plasmodium falciparum (Pf malaria) compared to healthy controls. The study found significantly higher levels of TNF-alpha and lower hemoglobin levels in Pf malaria patients compared to controls. A strong negative correlation was observed between TNF-alpha and hemoglobin levels in Pf malaria patients, indicating that higher TNF-alpha is associated with more severe anemia. The results suggest TNF-alpha may play an important role in the pathogenesis of anemia caused by Pf malaria.

1. Apollo Medicine 2012 December
Original Article
Volume 9, Number 4; pp. 292e296
Serum cytokine TNF-alpha and hemoglobin levels in Plasmodium
falciparum malaria e A correlative study in coastal districts of Odisha
Anshuman Sarangia,*, P.C. Mohapatrab, R.K. Dalaic
ABSTRACT
Background and objectives: This study was conducted to assess the correlation between TNF alpha levels and
Hemoglobin in patients affected by Plasmodium falciparum along with sex and age matched healthy controls.
Methods: This study included 32 patients of P. falciparum malaria, and 30 age and sex matched healthy controls.
Blood samples were drawn from malaria patients admitted to medicine ward along with healthy controls and trans-
ferred to the Biochemistry department for the routine hematological parameters and ELISA for TNF-alpha, IL-12 and
IL-4.
Results: The levels of TNF-alpha were significantly elevated in falciparum malaria compared to healthy controls.
Further Hb levels in Pf malaria showed significant decline compared to healthy controls. A strong negative correlation
was observed between TNF-alpha and Hb levels in Pf malaria.
Conclusion: The results indicate that in falciparum are characterized by up regulation of TNF-alpha and down
regulation of Hb levels. Present study shows close association between severe anemia and high TNF-alpha levels.
Copyright © 2012, Indraprastha Medical Corporation Ltd. All rights reserved.
Keywords: Pf malaria, Hb, Cytokine, TNF-alpha, Anemia
INTRODUCTION increased red cell loss through hemolysis and underproduc-
tion of red cells. Such a combination will lead to acceler-
Malaria is the most important parasitic infection affecting ated development of anemia.
an estimated 350e500 million people world-wide and Severe malarial anemia and cerebral malaria are the
resulting in between 0.5 and 2 million deaths annually.1 main complications of Plasmodium falciparum infection.
The pathogenesis of the anemia of malaria is complex, They are responsible for most of the estimated one to three
and this has been recognized from the earliest days of million malaria-related deaths every year in the world,
malariology. The anemia of malaria can certainly not be mainly among children below 5 years of age in sub-Saharan
explained by the hemolysis of parasitized red cells alone: Africa.2
it is frequently disproportional to the degree of parasitemia, Severe malarial anemia is reported to be the earliest
is often worst at a time when the parasitemia is waning, and complication, usually affecting children below 2 years of
may persist or deteriorate during periods of low-grade para- age Although severe anemia is a major concern in malaria
sitemia. The balance between red cell production and pathology due to its high mortality rates, milder forms of
destruction normally determines hemoglobin levels, and anemia also are important, since this manifestation is
in malaria, anemia may arise from the combination of responsible for considerable morbidity and is one of the
a
Department of Biochemistry, bProfessor & H.O.D., Department of Biochemistry, cAssociate Professor, Department of Medicine, S.C.B. Medical
College, Cuttack, Odisha, India.
*
Corresponding author. Tel.: þ91 9861232063, email: anshumanbbsr@rediffmail.com
Received: 23.3.2012; Accepted: 4.8.2012; Available online 23.8.2012
Copyright Ó 2012, Indraprastha Medical Corporation Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.apme.2012.08.003

2. Serum cytokine TNF-alpha and hemoglobin levels in Plasmodium falciparum malaria Original Article 293
major factors for the high disability-adjusted life years affected by P. falciparum along with sex and age matched
attributed to malaria.3 The mechanisms of severe malarial healthy controls.
anemia are the subject of intense study.4,5 Many factors
have been reported to influence its pathogenesis, but the
mechanisms themselves remain controversial.6
MATERIALS AND METHODS
The increased destruction and phagocytosis of infected
and uninfected erythrocytes, the suppression of erythropoi-
esis by relatively impaired erythropoietin production, the Study area and patients
autoimmune lysis of both parasitized and normal erythro-
The study was carried out in S.C.B. Medical, College,
cytes, and reticuloendothelial hyperfunction seem to be
Hospital, Cuttack, Odisha, Department of Medicine and
important causative factors, but they do not adequately
Department of Biochemistry. This hospital is the largest
explain the severity and extent of anemia. Furthermore,
government institution catering to the entire coastal belt
anemia can persist for weeks after effective antimalarial treat-
of Odisha. Therefore patients from interior rural areas visit
ment.7 Although the pathological basis for the development
the hospital for treatment.
of malarial anemia is not yet well understood, the participa-
Subjects were chosen from amongst malaria patients
tion of cytokines8 and of autoantibodies has been considered.
admitted to medicine indoor ward of S.C.B. Medical
Some works have suggested that severe anemia is associ-
College Hospital. P. falciparum malaria was defined
ated with predominant T-helper 1 (Th1) responses, character-
according to modified World Health Organization criteria.
ized by high levels of tumor necrosis factor alpha (TNF-a) in
Graph showing correlation between TNF-alpha and
relation to interleukin-10 (IL-10) levels, and conversely,
hemoglobin in pf malaria patients.
protection from this complication was associated with an
inverse relationship, i.e., with a balance toward a high
IL-10/TNF-a ratio. Other cytokines and chemokines, partic-
ularly those involved in macrophage migration and activity,
such as migration inhibitory factor (MIF) and monocyte
chemotactic protein-1 (MCP-1), also can be involved. Since
high TNF-a production by macrophages can be influenced
by phospholipids such as the Plasmodium-derived gly-
cosylphosphatidylinositol,9,10 anti-phospholipid antibodies,
which are common in malaria infections could play a role
in this system. Antibodies directed to erythrocyte membrane
antigens, which also are present in malaria infections,
could act by destroying parasitized and non-parasitized
erythrocytes.
Optimal immune response to malaria infection is charac-
terized by early intense pro-inflammatory cytokine-mediated
effectors mechanisms that kill or clear parasite-infected cells
and which are then equally rapidly suppressed by anti-inflam-
matory effectors once parasite replication has been brought
under control. The outcome of infection depends on a dedi-
cated balance between appropriate and inappropriate induc-
tion of these mediators. Early pro-inflammatory cytokine As a control group for cytokine determination 30 healthy
responses seem to mediate protective, whereas late response volunteers of same range of age and sex were also included
contributes to pathology. This suggests that a crucial balance in the study. All volunteers enrolled as control group were
might exist during the inflammatory response to malaria negative at the thick smear examination for P. falciparum
infection. without febrile episodes during the last 6 months and
The first characterized parasite-induced cytokine was without sign of anemia (Hb > 10 g/dl).
TNF alpha, induced in macrophages by erythrocytes Inclusion and classification of each case were based
infected by Plasmodium malarial pigment and certain on the symptoms, physical signs and laboratory finding
glycolipids such as GPI moiety. of malaria at the onset of the disease. On the basis of
This study was conducted to assess the correlation hematological parameters and evidence of neurological
between TNF alpha levels and Hemoglobin in patients involvement severity of malaria was established. The

3. 294 Apollo Medicine 2012 December; Vol. 9, No. 4 Sarangi et al.
malaria patients admitted to Medicine ward from correlation between hemoglobin and TNF-alpha
November 2009 to October 2010 were included in the (r ¼ À0.96) was found indicating an important association
study. If as per the inclusion criteria the patient was correlating TNF-alpha levels with anemia.
found fit to be included in the study then the consent
form was obtained duly signed and the patients were
included in the study. Blood hemoglobin was measured DISCUSSION
by cyanmethemoglobin method. Patient informed consent
form and Volunteer’s informed consent form were obtained In human malaria altered immune reactivity appears late in
from the participants in the study and the ethical clearance the acute phase of the diseases and can last a long time after
certificate from the Institutional Ethics Committee of the clearance of parasites from the circulation. An explana-
S.C.B Medical College, Cuttack is attached. tion for the poor acquisition of malaria unit in naturally
exposed population is that the parasite actively modulates
of specific immune response.11 The inflammatory response
Sample collection
that is needed to remove parasites lead to considerable
Blood samples were drawn from malaria patients admitted tissue damage and activation of phagocytes to kill intracel-
to medicine ward and transferred to Department for Hb esti- lular or extracellular parasites requires the production of
mation and ELISA for TNF alpha. Blood samples were inflammatory cytokines which can cause systemic effects
collected for immunological assessment in sterile tubes. such as severe anemia and cerebral malaria.12 The immune
All the samples were centrifuged and serum was refriger- response to malaria likely is regulated by the balance of
ated at À70 C in the Department of Biochemistry for pro- and anti-inflammatory cytokines that culminate in
determination of TNF-alpha. either immunoprotection or pathogenesis. In our study the
Blood samples were drawn from healthy volunteers not TNF alpha was elevated in all patients of malaria as expres-
infected with malaria and transferred to Department of sion of immune activation in response to the presence of
Biochemistry for Hb estimation and ELISA for TNF-alpha. parasites are determinants of malaria severity and
outcome13 and can represent potential target for therapeutic
interventions if their effect will be highlighted.
Cytokine determination Cytokines TNF-alpha induced in macrophages by eryth-
rocytes infected by Plasmodium malaria pigment and
Cytokine analysis was performed by Ray Biotech ELISA certain glycolipids such as GPI moiety. It has been shown
assay kits (Ray Biotech Inc 3607 Parkway Lane, Suite that GPI moiety induces NOS in macrophages and activates
200, Norcross GA 30092) for TNF-alpha as specified by endothelial cells by tyrosineekinase-mediated signal trans-
the manufacturer. Each plate included a standard curve of duction. The amount of TNF-alpha produced by malaria
recombinant human cytokine on logelog graph paper. All parasites seems to vary between people in the same
specimens were measured in duplicate and the mean of endemic area exposed to similar parasites and inoculation
the two values was taken. rates .TNF alpha has a role in the regulation of macrophage,
interleukin 12 induced production and it has been shown
Statistical analysis that TNF alpha is an important co-factor for interleukin
production of interferon gamma by NK cells. Luty et al14
Serum cytokine concentration was determined in dupli- showed a close association between the presence of severe
cate and expressed as mean Æ SE of the mean. Compari- anemia, high TNF alpha concentrations and large numbers
sons between groups were made using Z test with of circulating haemozoin-containing monocytes, suggesting
statistical significance set at SE value of 1% level of that haemozoin-induced TNF alpha reduction plays a part
significance. in either initiation or exacerbation of anemia as a clinical
outcome of chronic, uncontrolled parasitemia.
TNF alpha levels in falciparum malaria in the present
RESULTS study showed significant elevation (272.48 Æ 335.05 pg/
ml) compared to healthy controls (42.90 Æ 13.5 pg/ml).
In our study TNF alpha in falciparum malaria was signifi- TNF alpha levels were higher in falciparum malaria signi-
cantly elevated (631.25 Æ 382.26 pg/ml) compared to fying that TNF alpha may be an important component in
healthy controls (42.90 Æ 13.5 pg/ml) whereas Hb levels the pathogenesis of severe falciparum malaria and in partic-
were significantly reduced (8.2 Æ 0.54 g/dl) compared to ular in the cerebral syndrome and the hypoglycemia which
healthy controls (13.21 Æ 0.85). A strong negative can complicate this disease.

4. Serum cytokine TNF-alpha and hemoglobin levels in Plasmodium falciparum malaria Original Article 295
The anemia of malaria is due to a combination of prema- fall was soon realized to be out of proportion to the number
ture red cell destruction and inadequate red cell production. of red cells parasitized, so other factors were realized to
The two mechanisms often overlap and each may be the contribute. Phagocytosis of unparasitized red cells was
result of different pathways. Red cell destruction, for also recorded decades ago in monkey and human malaria,
example, involves both infected and uninfected cells whose and for many years was regarded as sufficient explanation
lifespan may be shortened by nonimmune and immune for this discrepancy.
mechanisms. Inadequate production may be due to the Others had been investigating dyserythropoiesis in the
suppression of erythropoiesis, or dysfunctional erythropoi- bone marrow of patients with falciparum malaria and
esis (dyserythropoiesis), where the bone marrow erythroid stressed its contribution to malarial anemia. A group in
cells are increased but mature red cell output is inadequate. Oxford seeking an explanation for this dyserythropoiesis
As recently reviewed,15 critical illness associated with an through an electron microscopy study of bone marrow,
inflammatory response invariably causes multifactorial observed sequestration of parasitized red cells and argued
anemia. It has often been noted that anemia could that this caused the bone marrow dysfunction in falciparum
contribute to poor oxygenation of tissues in malaria and malaria by restricting blood flow and thus inducing hypoxic
there is general acceptance that it can be severe enough changes.
to reduce the supply of oxygen to mitochondria to danger- Twenty-five years ago our group proposed that TNF
ously low levels. Thus it can be a major component of might cause the bone marrow depression seen in malaria.
malarial pathology. Subsequently an undefined product in macrophage superna-
Erythrocytes have a limited life, determined by how long tants, later identified as TNF, was found to inhibit the
they can remain flexible enough to squeeze through fenes- growth and differentiation of erythroid progenitor cells.
trations in specialized vessels in the red pulp of the spleen. When rTNF became available (but before it had become
A red cell that cannot pass this test is phagocytosed by adja- technically possible to assay for this cytokine in human
cent macrophages, and lost. In health this loss is balanced serum) the dyserythropoiesis and erythrophagocytosis
by erythropoiesis, and hematocrit remains normal. Should seen in terminal Plasmodium vincke infected mice were
red cells develop a premature poor deformability they are reproduced when a single injection of rTNF was given early
removed from the circulation correspondingly earlier. in the course of the infection.
Like other cells, erythrocytes stay intact by constantly Phagocytosis of erythroblasts in bone marrow,
extruding Naþ in exchange for Kþ through an energy a phenomenon also reported by Wickramasinghe in human
dependant “pump” in their cell membrane that was defined malaria, was commonly observed. Decreased erythropoiesis
by the ability of certain digitalis gylcosides to block it. This was subsequently reported in mice receiving continuous
Naþ/Kþ pump fails, and intracellular Naþ accumulates TNF infusions via implanted osmotic pumps, and increased
in (non-parasitized as well as parasitized) red cells erythropoiesis in malarial mice after injecting neutralizing
during human or monkey malaria. Since inhibition of the antibody directed against murine TNF. TNF-induced dyser-
Naþ/Kþ pump in vitro correlates with a reduced red cell ythropoiesis has since been confirmed in rats, and mice
deformability plus a parallel decrease in red cell filter- expressing high levels of human TNF become markedly
ability, any influence, such as NO, that inhibits this pump anemic during malaria infections, even though parasite
could potentially cause poor red cell deformability. Cyto- numbers, and therefore red cell loss post-schizogony, are
kine-induced iNOS provides a demonstrable16 way for considerably reduced.
these changes to occur in severe malaria. There is good During the intraerythrocytic phase of its life cycle, the
evidence that, when measured on admission, a severe malaria parasite matures within a cell in which hemo-
reduction in red cell deformability is a strong predictor of globin is the single major cytosolic protein. While in the
malarial mortality, but whether this is cause and effect, or trophozoite stage, the parasite avidly ingests and degrades
the two phenomena are simply inevitable co-travelers in host erythrocyte hemoglobin by means of a specialized
a strong pro-inflammatory milieu, is unclear. It seems clear structure called a cytostome, which spans the double
that poor red cell deformability (which affects parasitized membrane between erythrocyte and parasite cytoplasm.
and unparasitized red cells equally) and dyserythropoiesis Hemoglobin-containing vesicles are pinched off from the
can lead to severe anemia in various diseases, particularly cytostome and travel to the digestive vacuole where the
in chronic infections such as malaria. Because the parasite hemoglobin is broken down. The process of hemoglobin
inhabits erythrocytes, which must burst if the parasite is degradation releases heme, which accumulates in crystal-
to propagate, the obvious initial conclusion was that this line particles within the digestive vacuoles .The formation
source of red cell loss was central to the fall in hematocrit of these pigmented crystals (called hemozoin) is poorly
seen in this disease. As reviewed nearly 60 years ago this understood.

5. 296 Apollo Medicine 2012 December; Vol. 9, No. 4 Sarangi et al.
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