Correlation between Parasite Density with Plasma Levels of TNF-α

and IL-10 in Malaria Mix Infection in East Sumba District, East Nusa

Tenggara Province

Meka Faizal Farabi

, Heny Arwati

Yoes Prijatna Dachlan

Sekolah Pascasarjana, Universitas Airlangga, Surabaya, Indonesia

Department of Parasitology, Universitas Airlangga, Surabaya, Indonesia

Keywords: Mix Malaria, Parasite Density, TNF-α, IL-10

Abstract: East Nusa Tenggara Province is a malaria endemic area located in Eastern part of Indonesia with Annual

Parasite Incidence of 7.04% including falciparum malaria, vivax malaria and mix malaria. The information

on parasite density and plasma levels of TNF- α and IL-10 in mix malaria infection is rarely found. This

research is conducted to collect the information mentioned above. Methods: Diagnosis of malaria infection in

subjects was done by Rapid Diagnostic Test (RDT) as well as microscopy examination. Parasite density was

calculated based on number of parasite per 200 leukocytes on the Giemsa-stained thick blood films. Levels

of TNF-α and IL-10 in plasma were measured using Enzyme-Linked Immunosorbent Assay (ELISA).

Results: Five patiensts were diagnosed as mix malaria infection, with average of parasite density was 4341

paracite/µL. Average level of TNF-α and IL-10 was 207.31 pg/mL and = 15.91 pg/mL, respectively. Ratio of

TNF-α : IL-10 was 13 : 1. This research concludes, increased levels of TNF-α will decrease the parasite

density based on the time of infection, while the increase in parasite density is directly proportional to elevate

levels of IL-10 (p= 0,032).

1 INTRODUCTION

Malaria is a long-standing disease that still threatens

the lives of children, pregnant women and many

others in East Nusa Tenggara (ENT). Indonesian

malaria reports show that the population of ENT,

which accounts for 2% of Indonesia's population,

contributes to 25% of the total incidence of malaria in

Indonesia. On the island of Sumba itself the Annual

Parasite Incidence is high (Pusdatin, 2016).

Indonesia is located in a tropical which is the

endemic area of P. falciparum and P. vivax and high

transmission rate of malaria through mosquito bites

makes it possible for repeating inoculation by both

Plasmodium and causing mixed infection (Imwong,

2011).

Clinical conditions of people infected with mixed

malaria differ from those infected with single malaria,

this is due to the interaction between Plasmodium in

human hosts. The P. falciparum parasite has a faster

life cycle than P. vivax. The temperature of fever with

mixed infection is higher than single infection

(McKenzie, 2006).

The aim of this research is to determine the

relationship between parasite density with plasma

levels of TNF-α and IL-10 in patients with P. vivax

and P. falciparum mixed infections in East Sumba

Distric, East Nusa Tenggara.

2 IMMUNE RESPONSE DURING

MALARIA INFECTION

2.1 Innate Immune Response

Innate immune system is the first step in the immune

response to pathogens. The system includes a variety

of nonspecific responses such as recruitment of

immune cells to the site of infection, complement

cascade activation, destruction of foreign objects by

specific leukocytes and activation of the adaptive

immune system through antigen presentation (Clark,

2010). The main function of innate immunity is to

limit the parasitic density of the initial infection and

to modulate the specific immune response necessary

to eliminate parasites (Stevenson, 2004).

418

Farabi, M., Arwati, H. and Dachlan, Y.

Correlation between Parasite Density with Plasma Levels of TNF-Î

s and IL-10 in Malaria Mix Infection in East Sumba District, East Nusa Tenggara Province.

DOI: 10.5220/0007544104180421

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 418-421

ISBN: 978-989-758-348-3

Non-specific immune responses are represented

by multiple cells and intercellular components. One

of the innate immune systems that will respond

during a malaria infection is the mononuclear

phagocyte system (MPS), which includes monocytes,

macrophages, and dendritic cells (Mac-Daniel, 2015).

2.2 Adaptive Immune Response

2.2.1 Cellular

In the cellular immune response, T lymphocytes are a

major component of the immune system that serves

to recognize and destroy antigens through cytotoxic

activity and cytotoxic activation and activation of

various cell types and cytokine production (Abbas,

2012).

2.2.2 Humoral

The main characteristic of the Plasmodium parasite

that infects the erythrocytes is increasing the

production of Tumor Necrosis Factor alpha (TNF-α)

cytokines from macrophages, malaria pigments and

glycolipids such as Glycosil Phosphatidyl Inositol

(GPI). TNF is a major cytokine in acute inflammatory

responses. Severe infections can trigger the

production of TNF in large quantities that lead to

systemic reactions. TNF-α is produced by

neutrophils, activated lymphocytes, macrophages,

Natural Killer (NK) cells and some non-lymphoid

cells such as astrocytes, endothelial cells and smooth

muscle cells (Dietrick, 2008).

TNF-α plays a role in regulating Interleukin 12

(IL-12) production by macrophages and shows that

TNF-α is important as a co-factor for IL-12 in

increasing Interferon (IFN) production by NK cells.

TNF-α concentrations in plasma are associated with

changes in fever and parasite clearance

(Malaguarnera, 2002).

IL-10 that produced by monocytes is found in

plasma of patients with acute malaria. Th2 cells and

B cells, inhibiting cytokine production in Th1 and

CD8+ cells. IL-10 increases cell proliferation and

immunoglobulin production necessary for the

development and maturation of anti-malarial

antibodies. IL-10 also serves as a down regulator in

macrophages, reduces antigen presentation, plays an

important role in neutralizing the pathology of

macrophages in cerebral malaria by inhibiting IFN-γ

and TNF-α secretions (Malaguarnera, 2002).

3 METHOD

3.1 Location

This research was done in East Sumba district of the

Province of ENT. This province is located in Eastern

part of Indonesia that contain 7 islands. East Sumba

distric also contains several small islands. Easter

Sumba district bordered by the Sumba strait on the

north, Hindia ocean to the south, Sabu sea to the east

and Central Sumba district to the west. This district

that located in tropical region has rainy during

January-April and the rest was dry season, causing

this region classified as dry area (BPS Sumba Timur,

2016).

3.2 Blood Samples Collection

Blood sample colletcion was done by active and

passive malaria case detections. Active case was

performed by visitsing malaria suspected patient in

high transmission areas. Passive case detection was

carried out by collecting the blood samples of patients

who visiting Public Health Centers (PHC) and

Lindimara Christian Hospital. Prior to blood colletion

the characteristics of patients were recorded including

name, gender, age and address. All individuals aged

from four to seventy years old of both sexes included

in the study. Three millilitres of blood were collected

by vena punctured after the patient signed the

informed consent to prepare thick and thin blood film

and plasma collection. Inclusion critera were people

who diagnosed positive mixed malaria infection

containing P. falciparum and P. vivax by Rapid

Diagnostic Test (RDT) and microscopy examination.

3.3 Microscopy Examination and

Parasite Density Count

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 malaria parasites.

Detection by RDT was conducted by the staff of

PHC. Parasite density were counted per 500

leucocyte based on the following formula:

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

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

(1)

Correlation between Parasite Density with Plasma Levels of TNF-Î

s and IL-10 in Malaria Mix Infection in East Sumba District, East Nusa

Tenggara Province

419

3.4 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

andnd analysed by a single operator, and standard

curves were derived from cytokine standards.

3.5 Data Analysis

A total of 110 individuals enrolled in this study, 64

were diagnosed negative malaria, 22 diagnosed as

positive P. falciparum, 19 diagnosed as positive P.

vivax and 5 diagnosed as positive mixed malaria.

4 RESULT AND DISCUSSION

4.1 Thick Blood Smear Examination

Subject diagnosed as positive mixed malaria have 2

types of Plasmodium that is P. falciparum and P.

vivax can be seen under microscope. The result of

thick blood film examination as follow:

Figure 1. Microfotography of mixed malaria thick blood

smear

Seen in Figure 1. the difference between the two

species of the ring and the trofoocytic nucleus P.

vivax is thicker and larger (blue arrow) than the ring

and nucleus of the thin and small P. falciparum

trophosoit (red arrow)

4.2 Parasite Density

Five samples were examied with ELISA and found

the Parasite Density as follow:

Figure 2. Parasite density of subject with mixed malaria

The highest parasitic density was 6,656

parasites/μL while the lowest parasitic density was

3,888 parasites/μL. From Figure 2. the mean ratio

between P. falciparum and P. vivax is 1.04: 1

indicating no dominant Plasmodium in mixed

infections found.

4.3 Plasma Cytokine Level

Five samples were examined with ELISA and found

the level of of TNF-α and IL-10 as follow:

Table 1. Plasma cytokine level of mixed malaria

No.

TNF- α

IL-10

10,523

94,861

35,559

101,085

0,807

425,983

11,369

Subjects 1 and 3 have very low levels of TNF-α

and can not be detected with ELISA. Subjects with

low TNF-α levels were subjects 2 and 4 of 94.861

pg/mL and 101.085 pg/mL respectively, whereas

highest TNF-α levels were in sample 5 of 425.983

pg/mL. Subjects 1 and 5 had relatively low IL-10

levels of 10.523 pg/mL and 11.369 pg/mL while the

highest IL-10 levels were in sample 2 of 35.559

pg/mL. Subject 4 had a very low IL-10 level of 0.807

pg/mL and subject 3 was very low and undetectable

at ELISA. From Figure 2. can be determined the ratio

of TNF-α and IL-10 for each subject. Subject 1 has

ratio TNF-α : IL-10 of 0, subjet 2 ratio 2,67:1, subject

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5

Parasite Density

Total Parasite Falciparum Vivax

ICPS 2018 - 2nd International Conference Postgraduate School

420

3 ratio 0, subject ratio 125,15:1 and subject 5 ratio

37,47:1.

4.4 Statistical Analysis

Statistical analysis were conducted to data from 5

subjects. Kolgorov-Smirnov test were used to

describe the distribution of samples. The results of

these tests show the distribution of the sample is

normal with p > 0,05.

With normal distribution sample, next analysis is

using Pearson corellation test to determine the

correlation between parasite density, TNF-α, IL-10

and the ratio of both cytokines. Correlation is

significant at the p < 0,05. The result shows that no

significant correlation between parasite density and

TNF-α with p= 0,213 also no significant correlation

between parasite density and cytokine ratio with p=

0,130. But there is a significant correlation between

parasite density and IL-10 with p= 0,032.

4.5 Discussion

Increased levels of IL-10 correlate with increased

parasitemia. Exemplified by the highest parasite

density in subjects No. 1 compared to 4 other

subjects. The increase in IL-10 accompanied by very

low levels of TNF-α shows IL-10 anti-inflammatory

cytokines suppress the performance of TNF-α as a

pro-inflammatory cytokine in eliminating parasites

(Othoro, 1999). Subjects No. 2 showed an imbalance

of pro and anti-inflammatory cytokines responses

because with parasite densities that were still 4,512

parasites / μL the levels of IL-10 had increased to

reduce the TNF-α: IL-10 ratio of 2.67: 1, if IL-10

levels were continues to increase whilst TNF-α level

were decreased, the correlation was parasite density

in subject No.2 would not decrease. Plasma level of

IL-10 subjects No.4 need to be re-examined to see the

direction of change, if IL-10 remains in a low state

then TNF-α levels will be uncontrolled and at risk of

causing malaria complications. If IL-10 levels

increase optimally, there will be a regulatory balance

between pro-inflammatory and anti-inflammatory

cytokines. Subjects No.3 has no detectable plasma

cytokine level, this correlates with the parasite

density that still considerably high because no

cytokines response from the subject. Subject No.5 has

a clinical manifestation of high body temperature, this

indicates that level of TNF-α were increased thus

inhibiting the growth of parasite. This correlation can

be seen by the Parasite Density of Subject No.5 was

the lowest among other subjects.

5 CONCLUSIONS

Increased levels of TNF-α will decrease the parasite

density based on the time of infection but an

excessive increase will lead to complications of

malaria. While the increase in parasite density is

directly proportional to elevate levels of IL-10 is

evidenced by statistically significant correlation

between parasite density and IL-10 (p = 0,032).

ACKNOWLEDGEMENTS

The author would like to thank all community health

centers’ staff in East Sumba that help us to collect

samples especially Nikson and Luluk which provide

us a lot of information.

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Correlation between Parasite Density with Plasma Levels of TNF-Î

s and IL-10 in Malaria Mix Infection in East Sumba District, East Nusa

Tenggara Province

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