International Journal of Medicine and
Pharmaceutical Science (IJMPS)
ISSN(P): 2250-0049; ISSN(E): 2321-0095
Vol. 5, Issue 6, Dec 2015, 65-70
© TJPRC Pvt. Ltd.
SUSCEPTIBILITY OF HUMAN LEUKOCYTES ANTIGENS HLA-DRB1 TO
TOXOPLASMA GONDII INFECTION WITH PREGNANCY
BATOOL MUTAR MAHDI1, ISRAA MOHAMMAD ABD AL-KHALIQ2 & ALI HUSSEIN ALWAN3
1
Professor (M.B. Ch. B., M.Sc. FICMS. Clinical Immunology) Director of
HLA Research Unit Al-Kindi College of Medicine Baghdad, Iraq
2
M.Sc, Research Scholar, Parasitology, Department of Microbiology, Al, kindy College of
Medicine Baghdad University, AL, Nahda Square Baghdad, Iraq
3
Assistant Professor Department of Biology/College of Science Al Mustansiriyah UniversityBaghdad, Iraq
ABSTRACT
Background
Toxoplasma gondii is an intracellular parasite which infects a widespread in humans and mammalian hosts
and can cause severe disease in immune-compromised persons due to reactivation of a previous latent infection, and can
be life-threatening disease.
To investigate whether an immunogenetic (HLA-DRB1) predisposition exists in mothers infected with
Toxoplasma gondii parasite.
Patients and Methods
Thirty unrelated women with first trimester miscarriage, their age ranged from 18-35 years, referred to Private
Clinics at Baghdad were enrolled in this study from January, 2014 through September, 2015.Thirty normal non
Original Article
Aim of the Study
pregnant women with negative obstetric history of abortion ethnically matched with patients group selected as control
group. 18-34 years. DNA was extracted , amplification and hybridization was performed using a panel of sequencespecific oligonucleotide probes (SSOP) using HLA-DRB1amplification and hybridization kits (SSO HLA type DRB1
plus and master mix for HLA type DRB1 amp. plus kits -Innogenetics-Belgium) using automated method by Auto Lipa –
48Innogenetics-Belgum. The results were interpreted using LiRas version-5.0 software-Innogenetics-Belgium.
Results
There was an increased frequency of HLA-DRB1*03:01:01 in patients with Toxoplasma compared with
healthy controls ( P< 0.024, odd ratio=4.33, 95% CI:1.20 –15.60).The other alleles HLA-DRB1* 04:03:01 and HLADRB1*11:01:01 were also increased in Toxoplasma patients and not detected in control group.
Conclusions
There is a significant association between Toxoplasma infection and HLA-DRB1*03:01:01 in Iraqi Arab
Muslims patients.This study was conducted with a small sample. We suggest further studies with larger sample.
KEYWORDS: Toxoplasma, Genetic, HLA
Received: Nov 24, 2015; Accepted: Dec 09, 2015; Published: Dec 12, 2015; Paper Id.: IJMPSDEC20159
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66
Batool Mutar Mahdi , Israa Mohammad Abd Al-Khaliq & Ali Hussein Alwan
INTRODUCTION
Toxoplasma gondii is an intracellular parasite which infects a widespread in humans and mammalian hosts and
can cause severe disease in immune-compromised persons due to reactivation of a previous latent infection, and can be
life-threatening disease (1,2). The infection can cause severe ocular, neurologic, and systemic disease in immune deficient
patients (3).There are several factors affected the course of Toxoplasma infection like the inoculum size, virulence of the
organism, sex, and immunological status of the host. Genetic factor has been reported to play an important role and risk
factor in increased susceptibility to T. gondii in humans; for example HLA-DQ3 has been found to be a genetic marker
associated with susceptibility to developing Toxoplasma dependent encephalitis (4,5). In contrast, other study showed that
the frequency of HLA-DQ1 was lower in Toxoplasma encephalitis patients than in healthy controls (6). In Brazilian AIDS
patients, the HLA alleles (HLA-A11-Cw4-B35-DR1-DQ1) were also associated with toxoplasmic retino choroiditis, in
particular the HLA-B35antigen (7). The role of HLA in Toxoplasma explained by the ability of CD8(+) T cells to act as
cytolytic effectors and produce interferon-γ (IFN-γ) by the recognition of peptides restricted by major histocompatibility
complex (MHC) class I molecules. T.gondii-specific HLA-B*0702-restricted T cell epitopes, GRA7(20-28)
(LPQFATAAT) and GRA3(27-35) (VPFVVFLVA), displayed high-affinity binding to HLA-B*0702 and elicited IFN-γ
from peripheral blood mononuclear cells of seropositive HLA-B*07 persons (8).Therefore, the genes of HLA molecules
can modify the outcome of Toxoplasma gondii infection (5). The ideal benefit of HLA is to identify HLA-A02 super type
epitopes from Toxoplasma gondii protein selicit a protective T helper cell type 1 immune response, and generate long-lived
IFN-γ-producing CD8+ T cells.Thus, immunization of HLA-A*0201 transgenic mice with these pooled peptides that used
as vaccine leads to protection against Toxoplasma infection (9).
The aim of this study was to investigate whether an immunogenetic (HLA) predisposition exists in mothers
infected with Toxoplasma .
Patients and Methods
Thirty unrelated women with first trimester miscarriage, their age ranged from 18-35 years, referred to Private
Clinics at Baghdad were enrolled in this study from January, 2014 through September, 2015. The inclusion criteria were
women with spontaneous miscarriage at 2-8 weeks gestations. Gestational age was determined by the last menstrual period
and was confirmed by ultrasound scan. Demographic data, including maternal age, parity, occupation, duration of marriage
and educational level were recorded. Obstetrical history (total number of pregnancy,stillbirths, abortions and live births).
All of them were positive serological tests of Toxoplasma gondii antibodies (IgM and IgG) by ELISA and Immuno
chromatographic tests.
Thirty normal non pregnant women with negative obstetric history of abortion selected as control group,
ethnically matched with patients group (18-34 years).
The Scientific and Ethical Committee of Al-kindy medical college had approved the study. Written informed
consents were obtained from the patients with asthma and control normal blood donors.
HLA Class II Typing (DRB1)
Five ml of venous blood were collected by venipuncture from study and control population in EDTA containers
for DNA extraction by blood kit (QIAmp DNA blood Mini Kit, QIAGEN INC- Germany). DNA product was verified by
Impact Factor (JCC): 5.4638
NAAS Rating: 3.54
Susceptibility of Human Leukocytes Antigens
Hla-Drb1 to Toxoplasma Gondii Infection with Pregnancy
67
electrophoresis in a 2% agarose gel containing ethidium bromide and was visualized under UV light. Locus- and allelespecific amplificationof genomic DNA was performed for DRB1. Amplification and hybridization was performed using a
panel of sequence-specific oligonucleotide probes (SSOP) using HLA-DRB1amplification and hybridization kits (SSO
HLA type DRB1 plus and master mix for HLA type DRB1 Amp plus kits -Innogenetics-Belgium) using automated method
by AutoLipa – 48Innogenetics-Belgum. The results were interpreted using LiRas version-5.0 software-InnogeneticsBelgium.
Statistical Analysis
HLA frequencies were determined by direct counting.The frequency of each allele was compared between
asthmatics and normal control group using chi-square test fisher exact test using MiniTab version. 3.0 software. In each
comparison, the Odds ratio(OR) along with the 95% confidence interval (95% CI) was used. P-value less than 0.05 were
considered statistically significant.
RESULTS
In this study, the DRB1 locus was examined in a sample of 30 patients with Toxoplasma infection as compared to
age matched control sample using a DNA-based methodology PCR-SSOP method. Table -1- summarizes the results of the
HLA-DRB1 allele frequencies obtained in patients with Toxoplasma infection and controls. There was an increased
frequency of HLA-DRB1*03:01:01 in patients with Toxoplasma compared with healthy controls (
P< 0.024, odd
ratio=4.33, 95% CI:1.20 –15.60). The other alleles HLA-DRB1* 04:03:01 and HLADRB1*11:01:01 were also increased
in Toxoplasma patients and not detected in control group.
Table 1: Human Leukocytes Antigens (Hla-Drb1) Alleles Frequencies in
Patients with Toxoplasma Gondii and Healthy Control Groups
02:0301
Patients with
AA Group
No.=30
No.
%
0
0
Healthy Control
Group
No.=30
No.
%
2
6.66
03:0101
12
40
4
13.33
0
20
0
0
40
0
0
0
40
0
0
0
36.66
3.33
0
0
0
20
2
0
1
4
0
7
2
2
7
4
4
2
0
2
7
2
8
0
6.66
0
3.33
13.33
0
23.33
6.66
6.66
23.33
13.33
13.33
6.66
0
6.66
23.33
6.66
26.66
0
HLADRB1*
alleles
03:0102
0
03:02:01
6
03:1101
0
03:1701
0
04:03:01
12
07:0101
0
08:0101
0
08:0201
0
11:0101
12
11:0301
0
11:6701
0
12:0901
0
13:01:01
11
13:0501
1
13;1801
0
14:0101
0
14:0201
0
14:04:01
6
Na=not applicable
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Odd Ratio
(95%Confidenc
e Interval)
na
4.33
1.20-15.60
na
na
na
na
na
na
na
na
2.190.71-6.69
na
na
na
na
0.48. 0.04-5.62
na
na
na
na
P- value
na
0.024
na
na
na
na
na
na
na
na
0.169
na
na
na
na
0.561
na
na
na
na
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68
Batool Mutar Mahdi , Israa Mohammad Abd Al-Khaliq & Ali Hussein Alwan
DISCUSSIONS
T. gondii, an obligatory parasite with invasive capabilities, can only survive intracellularly where it gets nutrients
and escapes from the host’s immune defense mechanisms (10). Genetic factor had an important role in infection. In this
study founded that increased frequency of HLA-DRB1*03:01:01 in patients with Toxoplasma compared with healthy
controls ( P< 0.024, odd ratio=4.33, 95% CI:1.20 –15.60). Other study by Nussenblatt et al 1989 (11) demonstrated that
HIA typing in ocular toxoplasmosis patients reported an increased frequencies of the HLA-A29, Cw7, DR3, DRw8,
DRw52, DQwl, and DQw2 antigens. Other study showed an increased frequency of the HLA-Bw62antigen, a subtype of
the HLA-B15 antigen,was found in patients with severe congenital toxoplasmosis and in patients with severe bilateral
ocular involvement with macular involvement (12). Suzuki et al 1996 (13) showed that HLA-DQ3 to be a genetic marker
associated with susceptibility to developing Toxoplasma dependent encephalitis (14).The effect of Human Leukocytes
Antigens (HLA) that located on chromosome 6p21 are among the most polymorphic genes, which is associated with
susceptibility to certain diseases due to variations of the peptide binding cleft influencing the bound antigens and presented
to T lymphocytes (15). In general, HLA class I molecules present foreign antigens that binds the cleft to T CD8+ cytotoxic
T lymphocytes and HLA Class II molecules present foreign antigenic peptides to CD4+ T helper cells (16). HLA plays a
critical role in human immunological diseases and host defense against infections with Toxoplasma (17).
These differences in the results of above studies done among populations are due to race, religion and ethnicity
factors. A comparison of several studies with our study using samples of diverse ethnic backgrounds reveals differences in
the important allele as well as varying allele frequencies between populations. In addition to that, this study was conducted
with a small sample and different method of study. Moreover,it was difficult to collect some relatives of the patients,
especially the second degree, which refused to participate in the study.These factors were found limitations in this study.
Since no specific gene variant has yet been identified as a cause of Toxoplasma infection despite a positive
associations in each study, there is a need to identify associated alleles in each particular population.
CONCLUSIONS
There is a significant association between Toxoplasma infection and HLADRB1*03:01:01 in Iraqi Arab Muslims
patients. This study was conducted with a small sample. We suggest further studies with larger sample.
Conflict of Interest: There is no conflict of interest.
ACKNOWLEDGMENTS
We would like to thank Iraqi Research and development department- Ministry of higher education and scientific
research for their support. Our thanks extended to the Baghdad University and Al-Kindy College of Medicine who
facilitated this study.
Competing Interests: The authors have declared that no competing interest exists.
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