Transfusion and Apheresis Science xxx (xxxx) xxx–xxx

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Transfusion and Apheresis Science

journal homepage: www.elsevier.com/locate/transci

Seroprevalence and molecular detection of Toxoplasma gondii in healthy

blood donors in southwest Iran
⁎
J. Sakia, M. Foroutanb, I. Khodkara, , A. Khodadadic, L. Nazarid
a
Alimentary Tract Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
b
Abadan School of Medical Sciences, Abadan, Iran
c
Department of Immunology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
d
Blood Transfusion Organization, Ahvaz, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: Background: Toxoplasmosis is a cosmopolitan parasitic disease caused by Toxoplasma gondii (T. gondii). Blood
Toxoplasma gondii transfusion is a probable route of T. gondii transmission. Due to lack of information about seroprevalence of T.
Blood donors gondii in healthy blood donors, this study was aimed to determine the chronic and acute infection using ser-
Seroprevalence ological and molecular methods.
PCR
Material and methods: In this cross-sectional investigation, 380 samples were collected from donated bloods.
Iran
Anti-Toxoplasma IgG and IgM antibodies were examined using enzyme-linked immunosorbent assay (ELISA).
Also, all IgG positive samples were tested by IgG avidity test. Eventually, to detection of active infection, DNA
was extracted from IgM positive and low IgG avidity samples and then tested using nested-polymerase chain
reaction (PCR).
Results: Among 380 blood donors, 131 (34.47%) were positive for only anti-T. gondii IgG, 2 (0.5%) were positive
for only anti-T. gondii IgM, and 11 (2.9%) were positive for both IgG and IgM antibodies. Then, 142 samples (131
IgG + and 11 IgG +IgM +) were evaluated using IgG avidity test. Of these, 115 (81%) had high avidity IgG
indicates past infection; 16 (11.26%) had low avidity IgG representing recent infection, and 11 (7.74%) were
equivocal. With nested PCR, 20 samples of 50 seropositive samples were diagnosed positive.
Conclusion: Detected active infection using nested-PCR draws attention to the possibility of T. gondii infection via
blood transfusion which emphasizes the importance of parasite DNA screening before donation of blood in high
risk groups such as: multi-transfused persons, immunosuppressed patient, and pregnant women.

1. Introduction
Toxoplasmosis is a ubiquitous zoonotic disease, caused by an ob-
ligate intracellular protozoan parasite known as Toxoplasma gondii (T.
gondii), which can influence various warm-blooded vertebrates such as
humans, livestock, birds, marine mammals, etc [1–4]. This protozoa
needs both intermediate and definitive hosts to complete its asexual and
sexual replication stages in the life cycle [1,5]. It is estimated at least
one-third of the world’s population are seropositive for the parasite and
carry it [6]. Toxoplasmosis in immunocompetent persons is mostly
without symptom; however, among immunocompromised individuals,
i.e. HIV+/AIDS patients, cancer patients, and organ transplant re-
cipients can cause serious consequences, even death [7–12]. According
to recently published systematic review in the Iranian im-
munocompromised patients, the pooled seroprevalence of T. gondii was
estimated 50.01% (95% CI, 43.85–56.17). Also, the rate of infection
among HIV/AIDS, transplant recipients and cancer patients in Iran were
reported 50.05%, 55.1% and 45.06%, respectively [8]. Unfortunately,
there is lack of a commercially licensed vaccine to prevent tox-
oplasmosis in humans [13–15].
It has been shown that T. gondii can be transmitted through whole
blood or leukocyte transfusion and could be alive in citrated blood at
5 °C for more than 50 days; accordingly, the refrigeration of blood packs
during storage can't delay and prevent from the transmission of infec-
tion [16]. Normal seropositive blood donors, particularly those in-
dividuals who are in the acute stage of infection, may have an im-
portant role in the transmission [17]. T. gondii in India country, after
malaria is the commonest protozoan infection, which transmitted by
blood transfusion [18]. In a survey conducted by Siegal et al., the
transmission of T. gondii in four leukemic patients by leukocyte trans-
fusion was reported [16]. Also, other transplantation recipients of
kidney, lung, heart and bone marrow are at risk to infection [19].

⁎
Corresponding author.
E-mail address: iman.khodkar@yahoo.com (I. Khodkar).

https://doi.org/10.1016/j.transci.2018.12.003
Received 23 June 2018; Received in revised form 15 November 2018; Accepted 5 December 2018
1473-0502/ © 2018 Elsevier Ltd. All rights reserved.

Please cite this article as: Saki, J., Transfusion and Apheresis Science, https://doi.org/10.1016/j.transci.2018.12.003
J. Saki et al.
Frequent blood transfusion from different donors regularly applied for
the individuals with sickle cell anemia, thalassemia and aplastic anemia
to survive, whereas pre-transfusion T. gondii screening has not been
considered yet [6,7].
In several investigations, seroprevalence of T. gondii was determined
in different population groups in Khuzestan province, southwest Iran
and indicated that this region has high prevalence [7,20–23]. Most
studies on T. gondii in the country and this region, have focused on high
risk categories like pregnant women and immunocompromised pa-
tients; hence, due to lack of information about seroprevalence of T.
gondii in healthy blood donors in Ahvaz city, southwest Iran, the current
study was aimed to determinate the T. gondii infection using enzyme-
linked immunosorbent assay (ELISA) and polymerase chain reaction
(PCR).
2. Materials and methods
2.1. Study area
Ahvaz city, capital of Khuzestan province, which located at south-
west Iran country (31°50ʹ N and 49°11ʹ E), is ranked as the 7th largest
city throughout the country and based on the latest census, its popu-
lation calculated about 1,395,184 in 352,128 families. Weather tem-
perature is highly variable throughout the year so that in summer
temperature exceeds than 50 °C whereas in winter fell down to −5 °C.
Also annual average rainfall is approximately 230 mm [7].
2.2. Study population
In this cross-sectional study that performed between April 2015 to
June 2015 in Ahvaz city, blood samples were taken from 380 healthy
volunteer blood donors who referred to central Blood Transfusion
Organization (BTO) of Khuzestan province located at Ahvaz city. The
collected samples were routinely examined for hepatitis B virus surface
antigen (HBsAg), hepatitis C virus (HCV), human immunodeficiency
virus (HIV), human T-lymphotropic virus 1 and 2 (HTLV1, 2) and
Treponema pallidum (T. pallidum) in BTO of Ahvaz city. Inclusion criteria
in our study were as follows: 1) individuals ≥ 18 years old; 2) persons
who tend to participate in the study (after obtaining a written informed
consent); 3) blood donors who tested negative for HbsAg, HCV, HIV,
HTLV 1, 2 and T. pallidum. The current study was approved by the
ethical committee of Ahvaz Jundishapur University of Medical Sciences
(Code no.93596).
2.3. Serology
From each subject who met the above-mentioned inclusion criteria
of the study, 5 mL blood was collected. All blood specimens im-
mediately after sampling transferred to the Department of Medical
Parasitology at Ahvaz Jundishapur University of Medical Sciences. At
first, the taken blood were centrifuged at 4000 rpm for 5 min. Then the
sera were separated in order to examination for specific anti-
Toxoplasma IgM and IgG antibodies and stored in −20 °C. Furthermore,
buffy coat separation was done and kept in −20 °C until DNA extrac-
tion for PCR. All samples were examined for IgM and IgG anti-
Toxoplasma antibodies using a commercial available ELISA kit
(NovaLisa T. gondii-IgG, Cat. No. TOXG0460; NovaLisa T. gondii-IgM,
Cat. No. TOXM0460) based on the manufacturer's instruction, as pre-
viously described by Saki et al., 2017 [22].
2.4. T. gondii IgG avidity test
The avidity determination is a diagnostic method which is used to
differentiate a recent (acute) and a more distant (past) infection with T.
gondii in patient sera. Avidity is the binding force of the antibody
(serum specimen) with the corresponding antigen. Low avid IgG
2
Transfusion and Apheresis Science xxx (xxxx) xxx–xxx
antibodies in the early stage of infection can be differentiated from high
avid antibodies associated with a past infection. The determination of
IgG antibody avidity is an additional analysis to the classic serology in
regard to the status of a T. gondii infection. All IgG positive samples
were tested by IgG avidity T. gondii commercial kit (NovaLisa T. gondii-
IgG avidity; NOVA TEC- GMBH) based on the manufacturer’s protocol
[22].
2.5. DNA extraction and molecular technique
Buffy coat separation was done for all samples and kept in −20 °C
until DNA extraction for nested-PCR. DNA from IgM positive and low
IgG avidity samples were extracted using SinaPure™ genomic DNA
isolation kit (Cat. No: EX6001) based on the manufacturer’s instructions
and kept in −20 °C till tested. Nested-PCR was employed for molecular
diagnosis. For this purpose, we used two specific set primers to detect
B1 gene as follows:
First stage primers
Forward (F1): 5′-TCAAGCAGCGTATTGTCGAG-3′
Reverse (R1): 5′−CCGCAGCGACTTCTATCTCT-3′
Second stage primers
Forward (F2): 5′-GGAACTGCATCCGTTCATGAG-3′
Reverse (R2): 5′-TCTTTAAAGCGTTCGTGGTC-3′
Ultimately, the PCR products were electrophoresed on 1.5% agarose
gel and visualized using Gel Documentation System (Uvidoc, Gel
Documentation System, Cambridge, UK).
2.6. Statistical analysis
Findings were analyzed by SPSS software (version 19) (SPSS Inc.,
Chicago, IL, USA).
3. Results
3.1. ELISA assay
Three hundred and eighty sera samples of donated bloods were
examined in this study. The overall prevalence of toxoplasmosis was
obtained 37.9% (144/380). Of these, 131 (34.47%) were seropositive
only for IgG, 2 (0.5%) were seropositive only for IgM, and 11 (2.9%)
were positive for both IgG and IgM.
3.2. IgG avidity ELISA
In order to differentiate between acute and chronic phases of T.
gondii infection, IgG avidity ELISA test was done on 131 samples with
IgG positive antibody and 11 samples with both IgG and IgM positive
antibodies. Among them, 115 (81%) indicated high avidity IgG, which
signifies past infection; 16 (11.26%) had low avidity IgG representing
recent infection, and 11 (7.74%) were equivocal.
3.3. Nested PCR
Nested-PCR was performed on 50 samples including, 21 samples
with high avidity IgG, 5 samples with low avidity IgG, 11 samples with
equivocal test, and 13 samples with IgM positive antibody. In total,
among 50 samples which tested by nested-PCR, 20 samples (40%) were
positive and 30 samples (60%) were negative. The results were as fol-
lows: 1 out of 21 persons with high avidity IgG, 5 persons with low
avidity IgG, 1 persons with equivocal test and all of 13 IgM positive
samples were positive by the nested-PCR method. Fig. 1 indicates the
results of nested-PCR test of some positive samples.
4. Discussion
Although the safety of the blood supply is always under strict
J. Saki et al.
Fig. 1. B1 nested-PCR analysis of selected seropositive (IgM, IgG) samples from blood donors. Lane 1, DNA size marker; Lane 2, Positive control; Lanes 3–5, IgM
positive samples; Lanes 6-13. Selected IgG positive samples from IgG avidity test.
control and surveillance, concerns remain regarding transfusion-trans-
mitted infections [6,24]. Blood transfusion is one of the probable
transmission routes for T. gondii infection [6].
The present study is the first survey to evaluate the seroprevalence
of T. gondii infection among apparently healthy blood donors in the
southwest region of Iran. In this study, 34.47% and 2.9% of blood do-
nors were found seropositive for only IgG and for both IgG & IgM an-
tibodies, respectively. Our results are in agreement with other studies
such as Tappeh et al. in Urmia city (37.8%) [25], Sadooghian et al. in
Khorasan Razavi province (37.5%) [26], Bahhaj et al. in Tabriz city
(38.6%) [27], and Zainodini et al. in Rafsanjan city (34.04%) [28],
while the lower seroprevalence of the infection was reported from Fars
province (12.3%) [29], Boyer-Ahmad county (16.3%) [30], and Za-
hedan city (25%) [31]. Also, the rate of T. gondii infection in blood
donors are varies in different zones worldwide. Based on a systematic
review and meta-analysis paper from a global perspective, the highest
and lowest seroprevalence of T. gondii infection among blood donors
were observed in Africa (46%; 95% CI, 14%–78%) and in Asia (29%;
95% CI, 23%–35%), respectively. Moreover, the seroprevalence in
America, Europe, and Oceania were 42% (95% CI, 21%–63%), 30%
(95% CI, 19%–40%), and 42% (95% CI, 36%–48%), respectively [6]. In
some regions such as: Egypt [32] and northeast Brazil [33], ser-
opositivity in healthy blood donors were reported more than 50%. Also
lower rate of infection have been reported from Thailand (9.6%) [34],
Taiwan (9.3%) [35], Namibia (0.96%) [36], and Mexico (7.4%) [37].
The reason for this discrepancy is due to type of sampling, sample size,
methodology, cultural habit of the region, etc.
The presence of T. gondii in blood during the period of infection,
especially in the acute stage of the infection may be lead to transmission
through blood transfusion [38]. In addition, the ability of the protozoa
to survive in the stored bloods, enhance the risk of transmission through
blood transmission. Also, it has been shown the presence of T. gondii
tachyzoites in stored bloods for several weeks. Therefore, T. gondii can
be transmitted via blood and leukocyte transfusions, particularly if
parasitized leukocytes are transfused in a high concentration [16]. It
has been reported that T. gondii seroprevalence in the Iranian general
population was 39.3%, while the rate of infection in patients with im-
munocompromised disorders was 50% [8,39]. Since these individuals,
particularly transplant recipients require whole blood or other blood
products; accordingly, the presence of T. gondii in blood products may
3
Transfusion and Apheresis Science xxx (xxxx) xxx–xxx
be lead to occurrence of irreparable consequences. Based on formal
statistics, more than 20,000 thalassemia patients are existing in Iran
that many of them live in the southwest Iran [40]. These individuals
and other patients with each hematopoietic disorders, needs multiple
transfusion for survival. It is estimated that approximately 25% of the
total national blood products are being used for thalassemia patients in
Iran [41].
In the current study, ELISA method was used. Multiple serological
techniques are routinely employed to detect specific IgG and IgM an-
tibodies against T. gondii. Among them, ELISA has high sensitivity and
specificity, which can differentiate between chronic and acute phase of
the infection [22,42,43]. Although there is evidence which suggests
despite the constant presence of anti-T. gondii IgM antibodies in serum,
any positive IgM-ELISA could not surely be considered as an acute in-
fection, thus the PCR technique to explore DNA of T. gondii is required
[20,43].
In conclusion, our results indicate that toxoplasmosis has high
prevalence in terms of chronic and active infection (IgM antibody and
circulating DNA using nested-PCR) among apparently healthy blood
donors in southwest Iran. Thus, it is strongly recommended to design an
appropriate screening program for preventing transfusion-transmitted
toxoplasmosis, especially among multi-transfused persons, im-
munosuppressed patient, and pregnant women to reduce the trans-
mission of T. gondii infection. This article provides a foundation for
further studies in the future.
5. Limitations
The limitations of the study are the lack of demographic information
such as age, sex, occupation, etc. These data were not provided by the
relevant organization.
Conflict of interest
None.
Author contributions
Conceptualization: Jasem Saki, Masoud Foroutan, Ali Khodadadi;
Data analysis: Jasem Saki, Iman Khodkar; Investigation: Jasem Saki,
J. Saki et al.
Masoud Foroutan, Iman Khodkar, Laleh Nazari; Methodology: Jasem
Saki, Masoud Foroutan, Iman Khodkar; Project administration: Jasem
Saki; Supervision: Jasem Saki; Writing - original draft: Masoud
Foroutan; Writing - review & editing: Jasem Saki, Masoud Foroutan.
Acknowledgments
The authors would like to kindly appreciate cooperation of per-
sonnel of Blood Transfusion Organization (BTO) of Ahvaz city for the
period of sampling. This study was conducted and financially supported
by Grant from Research Deputy of Ahvaz Jundishapur University of
Medical Sciences, Iran (No: RDC-9313).
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