Correlation of Parasite Density with Plasma Level of TNF-α and IL-

10 in Patients Infected by Plasmodium Vivax in East Sumba District,

East Nusa Tenggara Province

Frieti Vega Nela

, Heny Arwati

and Yoes Prijatna Dachlan

Department of Immunology Postgraduate School Universitas Airlangga,Surabaya, East Java, Indonesia

Department of Parasitology, Faculty of Medicine, Universitas Airlangga,Surabaya, East Java, Indonesia

Keywords: IL-10, Parasite Density, Plasmodium vivax, TNF-α.

Abstract: Introduction. Annual parasite incidence (API) in East Nusa Tenggara Province (NTT) 2015 per 1000

population is 7.04%. However, API in each Public Health Center (Puskesmas) Sumba Island remains high.

High levels of pro-inflammatory cytokines in malaria infection, such as TNF-α is associated with severe

pathology, whereas, anti-inflammatory cytokines such as IL-10 is associated with acute malaria. The objective

of the study was to analyze correlation between parasite densities and plasma level of both cytokines in P.

vivax-infected patients in East Sumba Regency East Nusa Tenggara Province. Methods. Parasite densities

were calculated per 500 leucocytes on Giemsa-stained thick blood smears. The levels of TNF-α and IL-10

were measured by Enzyme-Linked Immunosorbent Assay (ELISA) method. Statistical analyses were done

by Spearman test. Results. Correlation was observed significantly in parasite density and TNF-α p = 0.032

and parasite density and IL-10 p=0.000. This result indicated that the stage of immunity in patients was not

affected by the parasite density but clinical symptoms may have a greater role in increasing and decreasing

the plasma level of cytokines. Conclusion. There was correlation between parasite densities and plasma level

of TNF-α and IL-10 in P. vivax infected patients is the studied areas.

1 INTRODUCTION

Malaria incidence was still high in the eastern parts of

Indonesia including Papua Province, West Papua,

East Nusa Tenggara (NTT), Central Sulawesi and

Maluku (Kemenkes, 2013). During 2015 the Annual

parasite incidence (API) in East Nusa Tenggara

Province (NTT) per 1000 population was 7.04%, the

number of cases of positive malaria as high as 36,039

from 5,120,061 inhabitants. The API in each Public

Health Center (Puskesmas) in Sumba Island remains

high (Pusdatin, 2016).

Malaria has been known since 3,000 years ago

and is caused by protozoa of the genus Plasmodium

and transmitted by female Anopheles mosquitoes

(Gunawan, 2000). There are 5 species of parasite

causing malaria in humans, namely Plasmodium

falciparum, Plasmodium vivax, Plasmodium

malariae, Plasmodium ovale and Plasmodium

knowlesi (White et al., 2014).

Plasmodium vivax has a longer incubation time

(12 days to several months), has a erythrocyte cycle

42-48 hours and produces fewer merozoites per

schizon. It is generally known that P.vivax requires a

duffy antigen as a receptor needed to invade host

erythrocytes. In humans who do not have this antigen,

they will become resistant to the infection (Andrade

et al., 2010).

An immune response to malaria leads to parasite

elimination or persistent responses are mediated by

cytokines that cause immunopathology. In malarial

infection high levels of pro-inflammatory cytokines,

such as Tumor Necrosis Factor (TNF), Interferon

Gamma (IFN-γ) and Interleukin-6 (IL-6) are

associated with severe pathology whereas cytokines,

anti-inflammatory agents such as Transforming

Growth Factor Beta (TGF-ß) and IL-10, are

associated with acute malaria. IL-10 cytokines have

an important role as immuno-regulators from

infections caused by Plasmodium, by neutralizing

theeffects of cytokines produced by Th1 and CD8+

cells, which are responsible for immunopathology

associated with excess cytokine production (Medina

et al., 2011).

Nela, F., Arwati, H. and Dachlan, Y.

Correlation of Parasite Density with Plasma Level of TNF-Î

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

DOI: 10.5220/0007542203250328

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 325-328

ISBN: 978-989-758-348-3

325

Activated macrophages release pro-inflammatory

cytokines such as TNF-α, IL-1 and IL-6 (Tsokonas et

al., 2002). The release of TNF-α apart from activated

macrophages can also be directly induced by the

malaria parasite and its dissolved antigen such as

malaria pigment (haemozoin) and

Glycosylphosphatidylinositol (GPI). TNF-α

indirectly inhibits parasites by increasing phagocyte

activity of monocytes (Wipasa et al., 2002; Korbel et

al., 2004).

CD4+ T cells are classified into 2 major subsets

according to the cytokine production pattern. Th1

produces IL-12, IFN-γ, and TNF-α. While Th2

produces IL-4, IL-5, IL-6, IL-10. In general, Th1 cells

are responsible for cell-mediated immunity (CMI).

The cytokine activates macrophages and other cells to

produce mediators releasing inflammatory cytokines.

Th2 cells regulate humoral immune by helping B

cells to produce antibodies. Th2 cells promote the

production of immunoglobulins. Both Th1 and Th2

cells are involved in protective immunity against

malaria in the pre-eritrocytic stages, and the balance

of cytokine production by both Th is the determining

factor of the disease (Wipasa et al., 2002).

2 MATERIAL AND METHODS

2.1 Subject of Research

The blood samples were collected from East Sumba

residents by active case detection in the villages with

high API value. Passive case detection was done by

collecting blood samples from P.vivax-infected

patients who came to Puskesmas seeking medication.

Blood samples were taken from those who meet

inclusion criteria which is P.vivax positive by rapid

diagnostic test (RDT) and microscopic examination

followed by the signed informed consent.

2.2 Screening

A screening test is used to determine the inclusion

criteria described above using 2 methods: RDT and

microscopic tests. All samples were examined using

a microscopic. RDT is used when sampling is active

in the village because there is no microscope to

support microscopic examination. Microscopic

examination is still performed after the RDT results

show a positive P.vivax.

2.3 Enzyme-Linked Immunosorbent

Assay (ELISA)

Blood collection from vein cubitis patients with P.

vivax malaria three milliliters (ml), inserted blood

into the heparin tube, centrifuge for 15 minutes at a

speed of 3000 rpm, the plasma is taken and

transferred into ependorf tube using micropipette.

The measurements of TNF-α and IL10 levels use

the Enzyme-Linked Immunosorbent Assay (ELISA)

in accordance with manufacturer protocols, with all

samples running in a single assay. The ELISA was

performed and analyzed by a single operator, and

standard curves were derived from cytokine

standards.

3 RESULT AND DISCUSSION

3.1 Parasite Density

P.vivax positive samples that have been collected are

smeared in thick drops and examined using a 1000x

magnification microscope. The formula for

calculating parasite density is as follows:

Parasite density = ∑ Parasite x 8000

500

The following is a microphotography picture of

thick blood smear P.vivax:

Figure 1: Microphotography P.vivax thick blood smear

Parasite density of samples diagnosed positive

P.vivax as follows:

ICPS 2018 - 2nd International Conference Postgraduate School

326

Figure 2: Parasite density P.vivax

3.2 Category of TNF- α and IL-10

Measurement of TNF-α and IL10 levels using ELISA

can be categorized according to the following:

Table 1: Category of TNF-α levels

TNF-α

Category

Frequency %

(pg/ml)

–

Low

57,89

–

Intermediate

36,84

> 50

5,26

TNF-α level was categorized as 3 types that is

low, intermediate, high. Low IL-10 percentage is 0-

10 is 57,89%, intermediate 11-50 is 36,84% and

high> 50 is 5,26%.

3.3 Category of TNF- α and IL-10

Kolgorov-Smirnov Test was used to find out the

normality of data. When the data is evenly

distributed, then Pearson test was used to analyze the

correlation between parasite density with TNF-α and

IL-10. If the data is distributed unevenly, Spearman

test was used. The correlation is significant if p <0.05

is obtained. The results showed that significant

correlation was observed significantly parasite

density and TNF-α p = 0.032 and parasite density and

IL-10 p=0.000.

3.4 Discussion

Infections caused by P.vivax have long been regarded

as benign, especially when compared with infections

caused by P.falciparum, but vivax malaria causes

more severe disease than P.falciparum infection

(Borges et al., 2013).

TNF-α is a pro-inflammatory cytokine that is the

cause of fever (Hietbink et al., 2006). At high levels

TNF-α can cause severe tissue damage (Couper et al.,

2008). At the optimum level TNF-α can kill parasites

directly, provide protection and lead to malaria

recovery. Low levels of TNF-α can inhibit the growth

of parasites in the stadium in the blood by activating

the cellular immune system (Raza et al., 2013).

IL-10 is the main anti-inflammatory cytokine in

the natural immune response and adaptive

inflammatory response through the process of

inactivation of macrophages and T cells (Dodoo et al.,

2002). High level of IL-10 will prevent the

development of severe malaria anemia (Weatherall et

al., 2002). The occurrence of severe anemia is

associated with a decrease in the concentration of IL-

10 in the circulation and increases the ratio of TNF-α

and IL-10. This condition contributes to the reversible

suppression of bone marrow activity that occurs in

malaria patients (Malaguarnera, 2002).

4 CONCLUSIONS

There was correlation between parasite densities and

plasma level of TNF-α and IL-10 in P. vivax infected

patients is the studied areas. This result indicated that

the stage of immunity in patients was not affected by

the parasite density but clinical symptoms may have

more role in increasing and decreasing the plasma

level of cytokines.

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East Nusa Tenggara Province

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