Correlation between Parasite Density with TNF-α and IL-10 in

Plasmodium Falciparum Infected Patients in East Sumba District,

East Nusa Tenggara Province

Akhmad Mubarok

, Heny Arwati

and Yoes Prijatna Dachlan

Department of ImmunologyPostgraduate School, Universitas Airlangga, Airlangga Street 4-6, Surabaya, Indonesia

Department of Parasitology Faculty of Medicine, UniversitasAirlangga, Surabaya, Indonesia

Keywords: Parasite density, Plasmodium falciparum, TNF-α, IL-10.

Abstract: Introduction: Malaria is caused by female mosquito Anopheles bite transmitting malaria parasite sporozoite

stage into the human body. According to Word Health Organization (WHO) due to malaria was 429.000

shows case causing malaria death, this need commitment of every country to overcome malaria. Indonesia is

still a malaria endemic area and includes a country with a high risk of malaria. Population migration from

malaria endemic areas to non-endemic areas of malaria is responsible for malaria transmission, especially in

Papua, West Papua, Maluku, North Maluku and East Nusa Tenggara. Malaria cases in East Nusa Tenggara

Province 7,05% per 1.000 opulation based on annual parasite incidence (API) and 36,039 positive. East

Sumba 12,84% per 1.000 population. Methods: A descriptive cross sectional study was conducted in East

Sumba district using the Logical Framework approach. Twenty two people were involved in this study with

ranging age 4 to 50 years old. Parasite density was examined by counting parasites on Giemsa-stained thick

smears under light microscope. Plasma level of TNF-α and Interleukin-10 were measured by using enzyme

linked immunosorbent assay (ELISA) Result: Significant values considered at p<0.05 The result show that

parasite density7.72 ±9.51/µl.TNF-α27.14 ± 50.22pg/ml IL-10 2.20± 3.23pg/ml Conclusion: The result

from this study conclude parasitedensity increases of TNF-αand IL-10 cytokine.

1 INTRODUCTION

Malaria one of deadly tropical parasite disease

caused by the genusPlasmodium, transmitted by the

femaleAnopheles bites they transmitted parasite into

the human (Mota et al, 2017).Data in 2015 people

death case cause malaria 429.000, need commitment

every country to against malaria (WHO,

2016).Indonesia one ofcountry with a high risk of

malaria endemic areaspecially in Papua, West

Papua, Maluku, North Maluku and East Nusa

Tenggara.East Sumba district part of East Nusa

Tenggara with high risk of malaria (Kemenkes,

2016).

Glycosylphosphatidylinositol (GPI) is malria

toxin that released along with merozoit and

hemozoin when P. falciparum¬ schizont-infected

erythrocytes rupture causing severe malaria

pathology through stimulation of pro-inflamatory

responses from NK cells and macrophages as

immune cells during innate immunity. Immune

response induced by GPI is mediated by pattern

recognition receptors such as TLR2 and TLR42

(Dunst et al, 2017). Intraerythrocytic malaria

parasite antigens trigger the early immune response

and cytokines production such as TNF-α, IL-1 and

IFN- from macrophages (Mana et al, 19910). The

interleukin-10 (IL-10) is an anti-inflamatory

cytokine produced by monocyte/lymphocyte which

has been shown to inhibit TNF-α (Burdin et al,

1994) protects against severe malaria anemia (SMA)

and cerebral malaria (Terazzaset al 2017).

IL-10 cytokines are found in plasma, produced

by monocytes, Th2 cells cyteand B cells, inhibiting

cytokine production in Th1 and CD8 + cells.IL-10

anti-inflammatory cytokine protects against severe

malaria anemia (SMA) withcerebral malaria

(Terazzaset al 2017).

During inflammation production of TNF-α is

increased. The dual of cytokines especially TNF-α

and IL-10 in the right level play role in protection

and healing (Irawati et al, 2008).

412

Mubarok, A., Arwati, H. and Dahlan, Y.

Correlation between Parasite Density with TNF-Î

s and IL-10 in Plasmodium Falciparum Infected Patients in East Sumba District, East Nusa Tenggara Province.

DOI: 10.5220/0007544004120417

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 412-417

ISBN: 978-989-758-348-3

In this study, parasite density, plasma level of

TNF-α and IL-10 of P. falciparum-infected patient

and residents in East Sumba District of East Nusa

Tenggara Province were measured to find out the

correlation between parasite density and both

cytokines to determine the immune status of the

patients.

2 MATERIAL AND METHODS

2.1 Location and Samples Collection

This research was done in East Sumba district of the

East Nusa Tenggara Province. This district that

located in tropical region has rainy season during

January-April and the rest was dry season, causing

this region classified as dry area (BPS Sumba Timur,

2016).

Blood were collected from P.falciparum-infected

patients who seeked medication in Lindimara

Hospital and Public Health Service (Puskesmas),

and from malaria-suspected residents who were

developing fever during samples collection. Only

P.falciparum-infected blood were used in this study.

Three milli liters (mL) of blood were collected from

median cubital vein and transffered to the heparin-

containing tube, furthermore, plasma were used to

measured the cytokines levels.

2.2 Microscopy Diagnosis and

Determination of Parasite Density

Microcopy examination was done on Giemsa-

stained thick and thin blood film using light

microscop under 1000x magnification with oil

immersion to detect and identify the species of

P.falciparum. Parasite density were counted per 500

leukocyte based on the following formula:

 = 

∑





(1)

Blood drops on the surface glass object, flattened

using the other glass object, thin blood smear attach

the glass objectwith a 45 degree in the blood, push

blood untill tip on ovale, thick and thin blood

fixation with metil alcohol dried and then staining

used giemsa.

2.3 Procedure of Giemsa Staining

The dried blood preparation fixation with methanol,

glass objects are placed on the stain rack, prepared

giemsa solution by mixing 3 cc giemsastock and 97

cc buffer solution, poured 3% giemsa solution to

cover the surface of the glass object, leave on for 30

– 45minute, poured clean water slowly on the glass

object until clean, dried and than staining used

giemsa.

2.4 Enzyme-Linked Immunosorbent

Assay (ELISA)

Plasma levels of TNF-α and IL-10 were measured

by using ELISA according to the manufacturer’s

protocol (Elabscience, USA), with all samples

running in a single assay. The ELISA was performed

and analysed by a single operator, and standard

curves were derived from cytokine standards.

Optical density (OD) value were measured at 450

nm immediately.

Add 100 µL sample of standard or plasma to

each well. Incubate for 90 minutes, remove the

liquid. Add 100µL of biotinylated detection antibodi

(Ab). Incubate for one hour at 37 degree, aspirate

and wash for three times, add 100 µL of HRP

conjugate. Incubate for 30 minutes at 37 degree,

aspirate and wash for 5 times, add 90 µL of substrate

reagent. Incubate for 15 minutes at 37 degree, add

50 µL of stop solution. Determine the optical density

(OD) value at 450 nm immdiately.

2.5 Statistical Analysis

All the statistical analyses were done using statistical

package for social science (SPSS). The normality

data was determine by Kolmogorov Smirnov test

with p > 0,05. Further analysis was using Pearson

corellation test to determine the correlation between

parasite density, TNF-α, IL-10 and the ratio of both

cytokines.

3 COPYRIGHT FORM

3.1 Microscopy Diagnosis of P.

falciparum Infection

Microcopy examination was done on Giemsa-

stained thick and thin blood film using light

microscop under 1000x magnification with oil

immersion to detect and identify the species of

P.falciparum. Parasite density were counted per 500

leucocyte based on the following formula:

 = 

∑





 (2)

Correlation between Parasite Density with TNF-Î

s and IL-10 in Plasmodium Falciparum Infected Patients in East Sumba District, East Nusa

Tenggara Province

413

Mycroscopy diagnosis of Giemsa-stained thin

films resulting in 22 out of 46 samples were

positive infected with P. falciparum,19 P. vivax and

5 mix of both species. Only P. falciparum positive

samples were used in this study.

Figure 1: Ringform (a) and gametocyte (b) stages of P.

falciparum on Giemsa-thin blood film

3.2 Characteristics of Subjects

The results of the characteristics subjects are

presented in Table 1. Mean age of malaria positive

group P.falciparum 16,64 years and negative group

24,10 years. Percentage of gender malaria positive

group 50% male, 50% female and malaria negative

group 30% male, 70% female.

Table 1: Characteristics of subjects

Total (%) Total (%)

Number of

the subject

Positive group

P.falciparum

(n = 22)

Negative group

P.falciparum

(n = 10)

Age (Year),

Mean ±SD

16,64 ±10,67 24,10 ± 5,74

Male 11(50,0) 3(30,0)

Female 11

(

50,0

)

(

70,0

)

3.3 Parasite Density

The calculation of parasite density P.falciparum are

presented in Figure 2 below.

Figure 2: Parasite density in subject infected with

P.falciparum

The highest parasite density was 38,768

parasites/μL while the lowest was 1,008

parasites/μL.

3.4 Plasma level of TNF-α

The results of measurements of TNF-α levels are

shown in Figure 3 below.

Figure 3: Plasma level of TNF-α in P. falciparum-infected

subjects

The highest levels of TNF-α were found in Pf6

1750.94 pg / mL and Pf20 1641.59 pg / mL samples.

The lowest TNF-α levels Pf3, Pf4, Pf5, Pf9, Pf10,

Pf12, Pf13, Pf18, Pf22 were below the standard

absorbance values of the ELISA measurement.

3.5 Plasma Level of IL-10

The plasma level of IL-10 by ELISA is shown in

Figure 4.

Figure 4: Plasma level of IL-10 levels in P. falciparum-

infected subjects

The highest levels of IL-10 were found in

samples of Pf6 with 112.04 pg / mL and Pf12 with

122.76 pg / mL. The lowest level of IL-10 were Pf2

with 2.52 pg / mL, Pf10 with 2.40 pg / mL and Pf15

with 1.05 pg / mL.

ICPS 2018 - 2nd International Conference Postgraduate School

414

The levels of parasite density, TNF-α and IL-10

vary widely, therefore, to explain the correlation

between parasite density and the levels of TNF-α

and IL-10, the discussion is grouped into 3 groups

and picked up several samples as follows:

1. Samples with high parasite density. 2. Samples

with high TNF-α level or high ratio of TNF-α: IL-

10, and 3. Samples with high IL-10 levels or low

TNF-α: IL-10 ratio.

Table 2: Grouping of representative samples with high

parasite densities, high TNF-α and high IL-10 levels

4 DISCUSSION

The higher levels of parasite density were observed

in uncomplicated P.falciparum infection compared

to the severe malaria infection. In the present study

high parasite density was followed by high TNF-α

and IL-10 levels. Activation of Th1 and Th2 balance

for parasite killing (Irawati, 2008). Several studies

have shown the involvement of pro-inflammatory

cytokine in patogenesis of severe falciparum malaria

where high plasma TNF-α and IL-10 show cerebral

malaria (CM) and severe malaria anemia (SMA)

(Parera, et al 2013)

Several studies indicated TNF-α is a critical

mediators of malarial fever P.falciparum. TNF-α

released in intermittent burst with the schizont

rupture. Not all proinflammatory cytokines are

equally relevant for the development of cerebral

malaria (Angulo, 2002). Cytokines play an

important role in human immune responses to

malarial disease. Balance between pro and anti

inflamatory cytokine are differ in each stage of

Plasmodium infection (Andrade, 2010).

4.1 Samples with High Parasite Density

There are 4 samples categorized as having high

parasite densities (> 10,000/µl blood) with different

levels of TNF-α and IL-10 as listed in the Table 2.

 Subject Pf21

This subject with high parasite density has

low TNF-α and IL-10 levels, make the ratio of

TNF-α : IL-10 also low (0). The low anti-

inflammatory response managed the host

immunity to suppress the levels of

proinflammatory cytokine (TNF-α) to the level

below standard (0). Possibility in defecting or

mutations in TNF-α encoding gene has occured

that can result in polymorphisms of the gene

(Qidwai and Khan, 2011).

 Subject Pf20

This subject has high parasite density with

high TNF-α levels but moderate IL-10 level, thus

the ratio of TNF-α : IL-10 is high. The blood

sample was collected at the Baing Health Center

when the subject was seeking medication due to

suffering from fever. Microscopy diagnosis has

confirmed the positive infection of P. falciparum.

The immune response of patient is indicated by

high levels of TNF-α. Fever is a clinical

manifestation of the TNF-α response. Fever and

TNF-α maintain parasitic density within safe

limits. Such immunity explains a host defense

mechanism that depends on parasite density

(Kwiatkowski 1989; 1991; 1994). TNF-α has

several other biological effects, such as the

deployment of neutrophils and monocytes to the

site of infection and activating these cells to

exclude pathogens, stimulating the expression of

vascular endothelial cell adhesion molecules for

leukocytes, stimulating macrophages to secrete

chemokines and inducing chemotactic and

leukocyte deployment, stimulating the

hypothalamus inducing fever (Bratawidjaja,

2014). High parasitic density induces TNF-α

production very strongly. The excessive level of

Correlation between Parasite Density with TNF-Î

s and IL-10 in Plasmodium Falciparum Infected Patients in East Sumba District, East Nusa

Tenggara Province

415

TNF-α indicate that parasites escape the immune

mechanism. The host fights infection by

producing anti-inflammatory cytokines, IL-10,

but is unable to kill parasites mimicking the

immunotolerance against pathogen. These

conditions can lead to splenomegaly and

hepatomegaly and maybe specific in East

Sumba Regency.

 Subject Pf19

The host responds to infection by producing

high level of IL-10 to suppress TNF-α

production, however, IL-10 produced by Th2

cells is unable to help B cells to produce anti-

parasitic antibodies that are strong enough to

eliminate parasites, so parasites continue to

increase. A research conducted by Shabani et al

(2017) described that density of P. falciparum

which >10,000 parasites µl of blood can cause

Cerebral Malaria (CM) and Severe Malaria

Anemia (SMA). In anemia, the parasite density

increases (Maina et al, 2010). P. falciparum

infects old erythrocytes. When erythrocytes

infected with schizont stage rupture release

merozoites to invade other erythrocytes, thus the

more erythrocytes infected by the schizont result

in reducing the number of erythrocytes. On the

other hand, the production of new erythrocytes is

not as fast as the invation of parasites. Parasitic

density is related to age, and clinical malaria

such as fever, chills, headache and splenomegaly

are associated with parasite density (Pryblyski et

al, 1999). Peripheral parasitic density is also

associated with plasma TNF-α level in pregnant

women (Ifeanyichukwu et al, 2017).

4.2 Samples with High TNF-α Level

 Subject Pf code 1

This subject has low parasite density, high

TNF-α levels and moderate IL-10 levels, thus the

ratio of TNF-α:IL-10 is moderate. High TNF-α

level plays a role in parasite killing mechanism,

because TNF-α levels can inhibit parasite growth

(Kwiatkowski, 1991), causing low parasitic

density. Moderate levels of IL-10 indicate the

host responds against infection to balance the

TNF-α. Subject Pf2 has similar immune status to

Pf 1.

 Subject Pf6

This subject has low parasite density, but

high level of TNF-α and IL-10 and moderate

ratio of both cytokines. This situation shows that

the host is fighting infection by producing IL-10

to compensate for TNF-α production. Parasite

killing mechanism has also occurred. The

presence of an anti-inflammatory response

indicates a tendency for patients to recover from

malaria infection.

4.3 Sample Group with High IL-10

Level

Subjects Pf5, Pf12 and Pf18 had defective TNF-α

coding genes (Qidwai and Khan, 2011), causing

very low TNF-α levels and unreadable by the system

on measurements with the ELISA method. High

respond of IL-10 indicates a strong fight against

infection followed by a decrease in parasite density,

thus indicates healing is very likely.

5 CONCLUSIONS

In summary, proinflammatory cytokine and anti-

inflammatory cytokine are both required for

adequate protection, Th-1 cytokine are important in

controlling early parasite malaria, although they

need to be counterbalanced later in the infection by a

Th-2 response which leads to antibody production.

REFERENCES

Angulo, I and Fresno, M. 2002. Cytokine in The

Pathogenesis of and Protection against Malaria.

Clinical and Diagnostic Laboratory Immunlogy.

American Society for Microbiology. Vol.9, No.6

Badan Pusat Statistik. 2017. Sumba Timur Dalam Angka.

Dachlan, Yoes Prijatna. 2013. Imunologi Malaria :Sistem

Imun Innate dan Adaptive pada Malaria. Rumah Sakit

Penyakit Tropik Infeksi. Univerersitas Airlangga.

Surabaya.

Harijanto, P.N. Nugroho, Agung. Gunawan, Carta A.

2012. Malaria dari Molekul ke Klinis. Edisi 2.

Penerbit Buku Kedokteran EGC. Jakarta.

Irawati, L. Acang, Nusirwan. Irawati, Nuzulia. 2008.

Ekspresi Tumor Necrosis Factor -Alfa (TNF-α) dan

Interleukin 10 (IL-10) pada Infeksi Falciprum.

Majalah Kedokteran Andalas No.1 Vol.32

Mota, M. Rodriguez, Ana. 2017. Malaria Immune

Response to Infection and Vaccination. Springer

International Publishing. Switzerland.

White, Nicholas J. Sasithon Pukritakamee, Tran Tinh

Hien, M Abul Faiz, Olugbenga A Mokuolu, Anjen M

Dondorp. 2014. Lancet Journal. Malaria. Vol.383

February 22.

ICPS 2018 - 2nd International Conference Postgraduate School

416

Terrazas, Cesar, James C Stock, Jennifer Kimble, Ellen

Morreti, Sanjay Varikuti and Abhay R. Satoskar.

2017. The Role of MIF in Parasitic Infections. USA.

World Health Organization (WHO). 2016. World Malaria

Report. Geneva. Switzerland

Parera M.K, Herath N.P, Pathirana L, Phone Kyaw M,

Alles H.K, Mendis K.N, Premawansa S,

HandunnettiS.M. 2013. Association of high plasma

TNF-alpha levels and TNF-alpha/IL-10 ratios with

TNF2 allele in severe P.falciparum malaria patients in

Sri Lanka. Phatogen and global health. Vol.107. No.1

Correlation between Parasite Density with TNF-Î

s and IL-10 in Plasmodium Falciparum Infected Patients in East Sumba District, East Nusa

Tenggara Province

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