ORIGINAL

Niger J Paed 2015; 42 (2): 142 –146

Okocha EC

Ibeh NC

Ukaejiofor EO

Ebenebe JC

Aneke JC

Serum Levels of Pro-inflammatory

Cytokines in relationship to

outcomes in Children with P.

falciparum malaria, in Nnewi-South

east Nigeria

Okonkwo KU

Onah C

DOI:http://dx.doi.org/10.4314/njp.v42i2.14

Accepted: 12th February 2015

Abstract: Background and Objec-

tive: In P. falciparum malaria (PFM)

infestation there are marked changes

in cytokine production as the body

mounts an immune response to it.

Hence we set out to study these

changes.

factor alpha (TNF-α) were seen in the

uncomplicated and severe forms of

PFM. It was observed that the elevated

cytokine values correlated with PD (in

uncomplicated PFM but not in the

severe forms). The difference between

PD/absolute monocyte count (AMC)

ratio was not significant (p=0.13);

while PD/platelet count (PC) and PC/

AMC ratios were significant (p=0.01,

and 0.03 respectively) when compared

between uncomplicated and severe

disease.

Conclusion: Our data seems to suggest

that subjects with an adequate immune

response to the parasite density, in

terms of pro-inflammatory cytokine

levels, presented with uncomplicated

disease; while those who have an in-

adequate response presented with se-

vere disease. The ratios of (PD/PC)

and (PC/AMC), in the positive and

negative directions respectively, may

be predictors of increased disease se-

verity. These observations may have

implications for predicting disease

outcome and PFM therapy.

(

)

Okocha EC

Aneke JC, Okonkwo KU

Department of Haematology,

Ibeh NC

Department of Medical Laboratory

Science,

Methods: A total of 158 cases of

PFM among children attending the

paediatric unit of our hospital and 56

healthy controls were studied. Chil-

dren with febrile illness were

screened for malaria using 10%

Giemsa stained blood smear. Patients

with positive smears were recruited;

co-infected patients – those infected

by another organism in addition to

plasmodium specie.- were excluded.

Whole blood was collected, some into

plain tubes for serum cytokine testing

and some into EDTA bottles for com-

plete blood count and parasite density

(PD) determination. Controls with

asymptomatic parasitaemia were ex-

cluded.

Ebenebe JC

Department of Paediatrics,

Onah C

Department of Chemical Pathology,

Nnamdi Azikiwe University Teaching

Hospital, PMB 5025,

Nnewi, Anambra State, Nigeria.

Email: onyichideokocha@yahoo.com

Ukaejiofor EO

Department of Medical Laboratory

Science, University of Nigeria, Enugu

Campus, Enugu State, Nigeria.

Results: Using the World Health

Organization criteria for defining

severe malaria; we identified 15 cases

of severe and 143 cases of uncompli-

cated PFM. Significantly elevated

levels of interleukin-1 (IL-1), inter-

leukin 6 (IL-6) and tumour necrosis

Key Words: plasmodium falciparum

malaria, pro-inflammatory cytokines,

Parasite density/Platelet count ratio,

Platelet count/Absolute monocyte

Introduction

IL1, IL-2, IL-12, and TNF-α has been confirmed in in-

fected individuals . Parasite factors such as the density

3

TNF-α has been implicated in the pathogenesis of ma-

laria fever and appears to play a central role; comple-

mented by the effects of other cytokines such as IL-1

and their ability to infect a high percentage of erythro-

cyte can correlate positively to the synthesis of inflam-

matory cytokines, and consequently contribute to the

1

,2

6

and IL-6 . Following infection by P.falciparum, the pro

severity of the disease . Other lines of evidence, how-

-

(

immune response . These mediators elicit anti-parasitic

activities resulting in inhibition of parasiteamia and

stimulation of phagocytosis to enhance parasite clear-

inflammatory cytokines such as TNF-α and interferon

ever seem to suggest that parasite density do not corre-

7

,8

IFN)-γ are produced during the early stages of the host

late with clinical symptoms and by extension severity .

Thus this present study involving aged matched Nige-

rian children with severe and uncomplicated malaria,

sought to examine cytokine production and to determine

whether differences in serum cytokine levels correlated

with varied disease severity; and if these varied manifes-

tation can be predicted by simple laboratory parameters

such as parasite density, platelet count and absolute

monocyte count

3

4

ance . At the later stages of infection, anti-inflammatory

cytokines such as IL-10 are produced and down regu-

lates the potentially pathogenic inflammatory resp₅onses

that are important for controlling parasiteamia. The

presence of increased type-1 cytokines, including IFN-γ,

1

43

Subjects, materials and methods

Study site and subject enrollment

manufactured by Sysmex Corporation 1-5 Wakinohama-

Kigandori, Chuo-ku, Kobe 651-0073, Japan. The 3ml

blood sample collected into the plain tubes was left to

0

Serum and sequestrated whole blood were obtained

from 210 Nigerian children (3 months to 144 months)

attending children outpatient clinic (CHOP), children

emergency room (CHER) and hospitalized patients of

our hospital in Nnewi, (population,4,177,828 - National

stand in a vertical position for 1hour at 25 C and al-

lowed to clot. This was then centrifuged at 1000 x g for

10 minutes. The separated serum was dispensed in four

0

aliquots and stored frozen at –70 C until cytokine testing

was performed in less than 3months of collection.

9

Bureau of Statistics, ) Anambra State, South- East Nige-

ria, between March and December, 2011. Nnewi has an

intense seasonal transmission -March to December of

Plasmodium falciparum malaria (PFM). Cases were

classified base₀d on criteria described by World Health

Measurement of circulating cytokines

The levels of TNF-α, IL-I, and IL-6 in the serum were

determined using Enzyme linked inmmunosorbent as-

say, (ELISA) Kit procured from Abcam, Cambridge,

United Kingdom, according to manufacturer’s recom-

mendations. The choice of this method was informed by

its high sensitivity and the fact that micro (100µl) quan-

tities of samples are required. The test kits are sensitive

enough to detect cytokines levels less than 0.8pg/ml and

cross reactivity has not been observed for any other pro-

teins tested. All quality controls measures as prescribed

by the manufacturer were observed.

1

Organization thus: 143 subjects had uncomplicated

malaria, 15 had complicated malaria and 52 subjects

without malaria served as the control group.

At enrollment, relevant clinical features including vital

signs were assessed and documented by the attending

paediatrican. Clinical information obtained was entered

into standardized forms. For the patients who were feb-

0

rile axillary temperature above 37.4 C) a drop of blood

through finger prick was obtained to make peripheral

blood smears for microscopy – both thick and thin- for

diagnosis and speciation respectively. The smear was

stained with 10% Giemsia stain for 10 minutes and ex-

amined under the microscope immediately (using x100

objective). The number of parasites per 200 white blood

cells and the number of parasites per microliter of blood

cells determined (parasite density), the presence of ma-

laria pigment (haemozoins) were checked for. A blood

slide was considered negative after scanning through

Statistical analysis

Statistical analysis was performed with SPSS version

10; (SPS Inc., Chicago IL). Descriptive statistics were

expressed as means and standard deviations while the

student’s t- test and Chi square analysis were used for

testing differences in cytokines levels between clinical

groups. Comparison of intra group differences was done

using a post-hoc Bonferoni multiple comparative analy-

sis while associations were tested using the Pearson’s

linear regression for bivariate correlation. The level of

statistical difference was set at p <0.05.

1

00 fields and no malaria parasite was encountered. Dif-

ferential WBC and morphological examination of cells

were done. The result was communicated immediately

to the attending doctor for management of the patient.

The patients that have positive smears were recruited as

subjects.

Results

Controls were recruited from healthy afebrile clients

attending the infant welfare clinic (Immunization Clinic)

of the hospital and from apparently healthy clients who

came in for routine checkup and medical examination.

The control subjects were of the same age brackets as

the patients (3 months to 144 months). Smears were also

made as in the study group and examined for possible

presence of asymptomatic parasiteamia; those with

negative smears for P. falciparum were enrolled as con-

trols.

A total number of five hundred and fifty-three (553)

febrile children presenting at peadiatrics unit of our hos-

pital were screened for possible presence of malaria

parasiteamia according to the study protocol between the

months of March and December 2011. One hundred and

fifty-eight (158) patients tested positive for P. falcipa-

rum by microscopic examination of Giemsa stained

blood slide, giving a prevalence of 35% (158/553). Five

(5) patients had mixed infection of P. falciparum and P.

malariae species (5/553) placing a prevalence of 3% for

P. malariae species. No other species were seen. Using

Study protocols were reviewed and approved by

Nnamdi Azikiwe University Teaching Hospital Ethics

Committee. Subjects and controls were recruited follow-

ing due ethical consent from their parents/guardians.

Further laboratory procedures

8

the criteria for severe malaria, (anemia with Hb level

5g/dL, acute respiratory distress, renal failure, prostra-

tion, shock, abnormal bleeding and/or disseminated in-

travascular coagulation, (parasitaemia >2,000/µl of

blood, repeated generalized seizures. 15 cases were

identified as severe malaria – 5 with anemia (as de-

scribed above), 2 with acute respiratory distress, 3 with

generalized seizures and 5 with high parasitaemia (as

described above). The mean duration of symptom before

presentation was 3 days in this category and 2.2 days in

the uncomplicated group. The mean ages for the various

For all recruited subjects who have met the clinical and

parasitological criteria, and also for the control subjects,

5

ml of venous blood sample was withdrawn; 2ml into

tubes containing EDTA and 3ml into sterile plain tubes.

The sequestrated blood sample was used for full blood

count determination which was carried out using Sys-

mex KX-21N automated haematology analyzer machine

1

44

groups – control, uncomplicated and complicated ma-

laria – were 29.5 ± 39.28, 33.9 ± 34.37 and 40.9 ±

and complicated malaria groups respectively; but did

not indicate any significant differences when percent-

ages of neutrophil values were compared between

groups.

Table 2 shows a statistically significant relationship in

the means of serum pro-inflammatory cytokines in con-

trol, uncomplicated and complicated malaria groups of

subjects.

2

5.77; the difference between them were not statistically

significant (p= 0.51). Table 1 shows the hematological

parameters of the subjects studied according to their

categories. A post-hoc multiple comparative analyses

revealed that the control group had significantly (P<0.01

or ) lower WBC but higher haemoglobin concentration

(

Hb) and haematocrit compared to the uncomplicated

Table 1: Haematological parameters of children with complicated and uncomplicated malaria compared

with healthy control group.

Variables

Control Group

(N = 52)

Uncomplicated

Malaria Group

Complicated

Malaria Group

(N = 15)

F-Stat

P

Value

(N = 143)

Age (months)

WBC (x109 /L)

RBC (x1012 /L)

Haemoglobin(g/dl)

Haematocrit (l/l)

Platelet (x109 /L)

PCT (%)

MCV (fl)

MCH (pg)

MCHC (g/l)

RDW

MPV

29.5 ± 39.28

4.8 ± 1.29

4.8 ± 0.52

33.9 ± 34.37

9.2 ± 5.02

4.8 ± 4.20

40.9 ± 25.77

13.2 ± 12.22

3.0 ± 1.12

0.67

20.03

1.78

51.71

59.52

9.92

27.13

17.43

0.85

0.51

0.000*

0.17

0.000*

0.43

11.8 ± 1.23

35.4 ± 3.65

267.9±84.55

21.0 ± 10.07

74.1 ± 4.80

24.6 ± 1.71

33.1 ± 1.53

16.6 ± 5.99

8.5 ± 0.79

10.4 ± 1.92

32.6 ± 5.36

278.4 ± 142.9

14.3 ± 14.54

72.5 ± 10.03

24.6 ± 16.34

32.2 ± 5.10

31.7 ± 23.67

9.3 ± 1.23

12.3 ± 3.75

42.9 ± 16.51

11.2 ± 4.69

45.7 ± 19.80

4096.4±169.54

5.9 ± 3.63

18.2 ± 9.58

125.7±54.01

3.6 ± 5.17

58.2 ± 14.53

19.8 ± 5.99

33.6 ± 2.75

19.8 ± 5.99

8.3 ± 0.94

11.2 ± 2.40

48.1 ± 15.29

7.1 ± 5.06

36.8 ± 22.69

55008.2±941.12 -14.42

1.38

0.25

11.87

10.48

1.89

7.91

8.57

0.000*

0.15

0.000*

0.029*

0.000*

PDW

11.3 ± 1.79

54.9 ± 24.61

9.0 ± 3.10

%

Lymphocytes

Monocytes

Neutrophils

38.9 ± 10.72

3.59

Parasite density (cells/ul)

Data is expressed as mean ± standard deviation. * Significant difference (P<0.05 or P<0.001). Abbreviations: WBC = White blood cell;

RBC = Red blood cell; PCT = Plateletcrit; MCV = Mean cell volume; MCH = Mean corpuscular haemoglobin; MCHC = Mean corpus-

cular haemoglobin concentration; RDW = Red cell distribution width; MPV = Mean platelet volume; PDW = Platelet distribution

width.

Table 2: Mean serum levels of the pro-inflammatory cytokines

in control, uncomplicated malaria and complicated malaria

groups of subjects.

data and the uncomplicated malaria group as shown in

Table 4. In the complicated malaria group, no significant

correlations were observed between parasite density and

all the cytokines (P>0.05). The difference between PD/

absolute monocyte count (AMC) ratio, was not signifi-

cant (p=0.13); while PD/platelet count (PC) and PC/

AMC ratios were significant (p=0.01, and 0.03 respec-

tively) when compared between uncomplicated and se-

vere disease.

Vari-

ables

Pg/Ml)

Control

group

(N = 52)

Uncomplicated

malaria group

(N = 143)

Complicated

malaria group

(N =15)

P

Value

(

IL-1

IL-6

TNF-α

45.6±37.04

48.0±35.27

48.9±58.98

177.9 ± 316.31

492.3 ± 596.84

132.2 ± 229.42

315.8± 233.71

1275.3±605.37

369.0 ± 453.45

0.001*

0.000*

Data is expressed as mean ± standard deviation. * Significant differ-

ence (P<0.001). Abbreviations: IL-1 = Interleukin - 1; IL-6 = Inter-

leukin - 6; TNF-α = Tumor Necrosis Factor - Alpha.

Table 3: Bonferonni Post-Hoc multiple comparison test

Variables

Control

Uncomplicated

verses

Uncomplicated

P - value

0.008

0.000

0.071*

complicated

P-value

0.002

0.000

Complicated

P-value

0.183

0.000

Bonferroni comparison test showed statistically lower

levels of IL-6 in controls compared to subjects with

complicated and uncomplicated malaria, as shown in

Table 3. The levels of TNF-α were statistically lower in

controls compared to subjects with complicated malaria

and similarly lower for subjects with uncomplicated

malaria compared to those with complicated malaria, as

shown in Table 3. The levels of IL-1 were statistically

lower in controls compared to subjects with complicated

malaria, but not in the other subjects as shown in Table

IL1

IL6

TNF-α

Abbreviations: IL-1 = Interleukin - 1; IL-6 = Interleukin - 6; TNF-α =

Tumor Necrosis Factor - Alpha.* Non significant difference (P> 0.05)

3

. Pearson’s linear regression test indicated significant

positive correlations between parasite density level and

all cytokines studied in the combined ‘all’ (test subjects)

1

45

Table 4: Bivariate correlation between parasite density level

and the pro-inflammatory cytokines in study subjects

causal relationship between onset of blood stage infec-

tion, initiation of the immune response and subsequent

1

5

15

Variables

All test subjects

Uncomplicated

malaria Group

Complicated ma-

laria Group

parasite growth . However, Walther and colleagues ,

also noted that expression of these pro-inflammatory

cytokines that brings about parasite control on the para-

site proliferation comes at the cost of developing clinical

symptoms, suggesting that the initial innate response

may have far reaching consequences on disease out

come. This highlights the question of balance proposed

Coeffi-

cient

P

Coeffi-

cient

P

Coeffi-

cient

P

Parasite

Density

vs IL-1

Parasite

Density

vs IL-6

Parasite

Density

vs. TNF-α

0.161

0.433

0.170

0.043

0.169

0.308

0.268

0.04

3

0.077

0.441

-0.345

0.784

0.000

0.033

0.00

0

0.100

0.207

1

6

by a number of scholars; Kresmer et al. , Perkins et

17

18

al. , Tiago et. al . Although this study did not deter-

mine the levels of type 2 cytokines, it is evident that the

pathological alterations and outcome of the infection

depend on the reciprocal regulation of type 1 and type 2.

Type 1 cytokines predominantly mediate cellular im-

mune response as a result play important roles in de-

layed-type hypersensitivity and in infections by intracel-

lullar pathogens (such as malaria). These include inter-

leukin-1 (IL-I), gamma interferon, interleukin-12 (IL-

0.00

1

Discussion

P. falciparium malaria is characterized by marked

19

I2), and tumor necrosis factor-β (TNF-β) . Type 2 cy-

changes in cytokine production arising from immune

1

,2

tokines are important in humoral immune response and

infections that involve the development of hyper-

gammaglobulinaemia, increased immunoglobulin E and

eosinophilia, they include interleukin-4 (IL-4), inter-

leukin-5 (IL-5), interleukin-6 (IL-6), interleukin-10 (IL-

responses to infection . Malaria outcomes vary from

1

mild to severe disease . This study documented ele-

vated serum levels of pro-inflammatory cytokines in

malaria infection. This study also found distinct differ-

ences in cytokine production correlating with disease

severity. Our results seem to suggest that individuals

who had an adequate immune response to the parasite

density they were infested with, in terms of proportion-

ate cytokine response, presented with uncomplicated

disease; while those who has an inadequate response

presented with severe disease. Thus the clinical category

of severity of malaria may be an indication of the degree

of cytokine response to the infection. This observation

shows that indeed the pathology observed in malaria is

not directly as a result of the activities of the invading

19

I0), interleukin-13 (IL-I3) . Both of these cytokine

types are produced by a number of cell types including

CD4+ (T helper-1 and 2), CD8+ , Natural killer (NK), T

20

and B cells . Malaria infection has been reported to tilt

the balance towards the production of more type 1 cyto-

20

kines . More so, marked imbalance between these two

kinds of cytokines has been variously₁₄_,a₁₅ssociated with

differences in severity of the infection . Therefore in

severe, acute infections such as malaria, the ability to

mount an effective innate response may have implica-

tion on survival as our data set demonstrated that the

cytokine response of those with severe outcome com-

pared to their parasite density seem to have been

blunted. The significant difference between PD/PC and

PC/AMC ratios in uncomplicated and severe disease

seems to suggest that these ratios used in combination

with other clinical methods, may be useful in predicting

disease outcome. More work, however needs to be done,

using larger data sets, to establish this.

1

2

organism but the response of the individual to it . Ex-

cess production of TNF-α may be responsible for the

clinical and pathology (such as fever) seen in malaria.

Previous studies had shown IL-6 as an important pro-

inflammatory cytokine that is unregulated by TNF-α and

acts in concert with₃ other inflammatory mediators to

1

control parasitaemia .

This study found that both IL-6 and IL-1 appear to cor-

relate with disease severity since elevated levels were

noted in the severe malaria patients compared to the

matched uncomplicated cases, as well as healthy con-

1

1,14

.

trols. This observation agrees with previous studies

Conclusion

We also observed that elevation in TNF-α level is asso-

ciated with high density of P. falciparium infection, sug-

gesting a correlation with level of parasiteamia. This

finding could be responsible for the statistical difference

observed in the mean TNF-α between the severe and

control groups. Chotivanich and co-workers had in their

previous study demonstrated that parasite factors such as

the parasite density and their ability to infect high per-

centage of erythrocytes correlate positively with the rate

of synthesis of inflammatory cytokines and disease

Subjects with an adequate immune response to the para-

site density, in terms of proportionate increase in pro-

inflammatory cytokine levels, presented with uncompli-

cated disease; while those who have an inadequate re-

sponse - in terms of disproportionate increase in pro-

inflammatory cytokine levels, presented with severe

disease. This observation makes immunotherapy an op-

tion for the treatment of malaria. More so, disease sever-

ity, may be predicted by ratios such as PD/PC and PC/

AMC in combination with other clinical methods. More

work, which should include anti-inflammatory cyto-

kines, need to be done to corroborate this finding.

6

11,12,14

.

severity and consequently result in fatal outcome

Other lines of evidence have demonstrated that increas-

ing cytokine concentrations were coincident with rise in

asexual parasiteamia, suggesting also that there is a

1

46

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