|Year : 2016 | Volume
| Issue : 1 | Page : 24-30
Detection of T. gondii infection in blood donors in Alexandria, Egypt, using serological and molecular strategies
Omnya A El-Geddawi MSc 1, Mona H El-Sayad1, Nadia A Sadek2, Neveen A Hussien3, Mohamed A Ahmed4
1 Department of Applied and Molecular Parasitology, Parasitology, Medical Research Institute, Alexandria University, Alexandria, Egypt
2 Department of Hematology, Medical Research Institute, Alexandria University, Alexandria, Egypt
3 Department of Applied Medical Chemistry, Medical Research Institute, Alexandria University, Alexandria, Egypt
4 Department of Clinical Pathology, Military Medical Academy, Armed Forces Hospital, Alexandria, Egypt
|Date of Submission||26-Aug-2015|
|Date of Acceptance||16-Mar-2016|
|Date of Web Publication||25-Oct-2016|
Omnya A El-Geddawi
Department of Applied and Molecular Parasitology, Medical Research Institute, Alexandria University, Alexandria
Source of Support: None, Conflict of Interest: None
Toxoplasmosis is a widespread disease caused by the opportunistic parasite Toxoplasma gondii, with variable overall prevalence according to the different geographical areas. Blood donors pose as possible contributors for transfer of infection.
The aim of this study was to estimate the prevalence of exposure to Toxoplasma in blood donors using sensitive techniques in a cross-sectional study.
Materials and methods
An aggregate of 150 blood donors from the blood donation center of Alexandria University participated in this study. The blood samples were tested for the presence of T. gondii immunoglobulin (Ig) G antibody and target gene B1 using enzyme-linked immunosorbent assay and real-time PCR, respectively.
Of 150 participants, 65.3% tested were positive for anti-Toxoplasma IgG, and 10% showed parasitemia as B1 gene was successfully amplified in nine seropositive samples and in six seronegative samples.
The recorded IgG seropositivity in this selected group of individuals may be considered an indication of the general prevalence of toxoplasmosis in Alexandria. Detected parasitemia using real-time PCR draws attention to the possibility of transmission through blood transfusion even from seronegative donors and emphasizes the importance of specialized Toxoplasma DNA screening before donation of blood.
Keywords: B1 gene, blood donors, enzyme-linked immunosorbent assay, real-time PCR, Toxoplasma gondii
|How to cite this article:|
El-Geddawi OA, El-Sayad MH, Sadek NA, Hussien NA, Ahmed MA. Detection of T. gondii infection in blood donors in Alexandria, Egypt, using serological and molecular strategies. Parasitol United J 2016;9:24-30
|How to cite this URL:|
El-Geddawi OA, El-Sayad MH, Sadek NA, Hussien NA, Ahmed MA. Detection of T. gondii infection in blood donors in Alexandria, Egypt, using serological and molecular strategies. Parasitol United J [serial online] 2016 [cited 2019 Dec 9];9:24-30. Available from: http://www.new.puj.eg.net/text.asp?2016/9/1/24/192992
| Introduction|| |
Toxoplasma gondii is a parasitic protozoan of worldwide distribution in most warm-blooded creatures. Around 33% of the world’s human populace is reportedly infected with this parasite ,,. There is remarkable variation in the seroprevalence of toxoplasmosis between countries (10–80%) and even in a given country ,. Transmission of T. gondii to humans occurs by ingestion of cysts in improperly cooked infected meat, or feline mature oocysts contaminating food or water. Vertical transmission from mother’s placenta to fetus has drastic results. Organ transplantation and blood transfusion from infected donors are other important routes ,,.
Toxoplasmosis can bring about severe manifestations and complications in immunocompromised individuals and babies; however, it is typically understated and self-restricting in immunocompetent individuals ,. Diagnosis of toxoplasmosis cannot rely solely on clinical manifestations; confirmatory parasitological and serological assessments are required . Several serological tests recognizing specific immunoglobulins (IgG and IgM) against T. gondii in sera of patients have been used effectively . Among these assays, ELISA demonstrates a high sensitivity and specificity ; however, anti-Toxoplasma IgG or IgM may not be distinguished during the acute phase of infection, due to delayed production of these antibodies until after a few weeks of parasitemia. This is especially important in certain immunocompromised patients in whom the titers of specific anti-Toxoplasma antibodies may not be detectable .
PCR assays are extremely important for providing results of high sensitivity, especially when real-time PCR is used as it is useful for accurate and early diagnosis of toxoplasmosis. In real-time PCR, the 5′-nuclease movement of Taq DNA polymerase is used to slice a non-extendible, fluorescence-marked hybridization probe during the extension period of PCR. The fluorescence of the intact probe is replaced by 6-carboxy-tetramethyl-rhodamine (TAMRA) fluorescent dye. During PCR, the hybridization probe discharges an extinguishing impact of quenching bringing about an increase in fluorescence, corresponding to the measure of PCR product that can be observed by a sequence indicator . The regularly utilized focus for the location of T. gondii using PCR is the repetitive B1 gene , due to its presence in a repetitive manner as a 35-copy gene in the genome, and its conservation in each of the six T. gondii strains tested to date ,,,.
The variable predominance of T. gondii antibodies reported in past investigations of blood donors ,,,,,,,, portrays clear geographic and worldwide contrasts in transmission patterns between nations. In Egypt, information on the prevalence of T. gondii in the general population varied between 30 and 60% , with few reports focused on blood donors. An early study in 1986 on prevalence in blood donors to Ain Shams University Hospitals in Cairo revealed an IgG seropositivity of 19.5% . In another report from Mansoura governorate in 2009, 59.6% of cases were seropositive for Toxoplasma IgG antibodies using enzyme-linked immunosorbent assay (ELISA) . This information inspired us to conduct this cross-sectional study to distinguish between exposure to infection and T. gondii disease among blood donors in Alexandria, Egypt, using ELISA and real-time PCR.
| Materials and methods|| |
This cross-sectional study was performed during the period from March to July 2013 wherein 150 blood samples collected from donors attending the Blood Bank of Medical Research Institute, Alexandria University, were tested for anti-Toxoplasma IgG antibodies using ELISA and for circulating parasite DNA using quantitative real-time PCR. The study was approved by the ethics committee, Alexandria University. Donors were asked to participate in the study after clear explanation of the study objectives and informed verbal consent was obtained from all participants.
Blood sample collection
A volume of 10 ml of blood was collected from each donor and divided into two tubes: 4 ml was collected in a plain tube to allow coagulation of blood and separation of serum, and 6 ml was collected in a tube with EDTA for buffy coat separation. A total of 150 blood samples were enrolled in the study after excluding HIV, hepatitis B virus, hepatitis C virus, and Treponema pallidum infections.
Serum samples of blood donors were tested for anti-T. gondii IgG using commercially available ELISA (Biocheck Inc., Foster City, California, USA). The test was performed according to the manufacturer’s instructions; samples giving quantities of 32 or greater international units (IU/ml) were considered positive.
Buffy coat preparation
Anticoagulated blood of 3 ml was mixed with an equal volume of PBS, pH 7.4, and slowly layered on 3 ml Ficoll-Paque, and then centrifuged at 1900 rpm for 30 min at 25°C. The leukocyte-rich supernatant was collected, washed in PBS, and centrifuged at 1400 rpm for 10 min at 25°C. The pellets were kept frozen at −80°C to extract and subsequently amplify DNA ,.
Preparation of T. gondii tachyzoites
T. gondii tachyzoites, utilized as positive control and for standard curve preparation, were kindly provided by the Parasitology Department, Faculty of Medicine, Alexandria University. Tachyzoites were maintained in the laboratory by serial intraperitoneal inoculation of Swiss albino mice every 3 days. Parasites collected from mouse ascitic fluid were washed several times in PBS. The number of tachyzoites was determined using a hemocytometer (Neubauer chamber).
Total DNA was extracted from buffy coat and parasites using the genomic preparation blood DNA isolation kit (GeneJET Whole Blood Genomic DNA Purification Kit; Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) according to the manufacturer’s directions. Successful DNA extraction from the 150 samples was verified by running the extraction products on 1% agarose gel for 15 min at 100 V and stained with ethidium bromide ([Figure 1]). The extracted DNAs were kept frozen at −80°C until further testing using real-time PCR.
|Figure 1 Example of DNA extraction product (total DNA) from 24 stained samples visualized with ethidium bromide staining.|
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Quantitative detection of T. gondii B1 gene using real-time PCR
Real-time PCR was performed as described by Mei-Hui et al.  using the forward primer (TOXO-F) (5 μmol/l, 5′-TCCCCTCTGCTGGCGAAAAGT-3′), reverse primer (TOXO-R) (5 μmol/l, 5′-AGCGTTCGTGGTCAACTATCGATTG-3′), and Taqman probe (2 μmol/l, 6FAM-TCTGTGCAACTTTGGTGTATTCGCAG-TAMRA) to amplify the 98 bp segment of the 35-repititive B1 gene. The primers and the TaqMan probe were purchased from Life technologies, Applied Biosystems, Carlsbad, California, USA. The amplification reactions were performed in final volumes of 25 μl containing 5 μl of template DNA, 12.5 μl of 2× Taqman universal master mix, 2.5 μl of the forward primer (TOXO-F), 2.5 μl of the reverse primer (TOXO-R), and 2.5 μl of Taqman probe using a StepOne Real-Time PCR System (Applied Biosystems, CA, USA). After initial activation of AmpliTaqGold DNA polymerase at 95°C for 10 min, 40 PCR cycles of 95°C for 15 s (denaturation step) and 60°C for 1 min (annealing and extension steps) were performed. Ribonuclease P (RNase P) housekeeping gene was utilized as internal control to check the potential PCR inhibitors in the analyzed specimens.
The standard curve for the real-time PCR quantification was set by amplification of serial dilutions of T. gondii DNA corresponding to 10 000, 1000, 100, 10, 2, 0.2, 0.02, and 0.002 tachyzoites. The CT values resolved were plotted against log amount of equivalent tachyzoites ([Table 1] and [Figure 2]).
The SPSS software statistical program (version 20; SPSS Inc., Chicago, Illinois, USA) was utilized for both data presentation and statistical analysis of results.
| Results|| |
The age of the 150 participants ranged from 18 to 60 with a mean age of 32±8 years. Seropositivity of IgG T. gondii antibodies showed that, of the 150 blood donors, 98 (65.3%) were seropositive for toxoplasmosis ([Table 2]).
|Table 2 Seroprevalence of T. gondii IgG antibodies in 150 blood samples of donors|
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As demonstrated in [Table 3], the B1 gene was effectively amplified in only 15 blood tests (10%) ([Figure 3]), including nine samples with seropositive results and six samples with seronegative results. The measure of tachyzoites in every positive sample, as regards the CT values, is presented in [Table 4]. The values differed from 0.009 to 68.5 tachyzoite-equivalent.
|Figure 3 Amplification plot of B1-gene in positive samples represented by the different colors.|
Click here to view
|Table 4 Tachyzoite equivalent in real-time PCR positive samples (n=15) compared with ELISA|
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| Discussion|| |
Toxoplasmosis is prevalent everywhere throughout the world as an important protozoan parasite . Being an opportunistic pathogen, it affects organ transplant recipients and other immunocompromised patients, causing critical or life-debilitating complications as a consequence of either acute infection or reactivation of a latent dormant infection .
As reported previously, the predominance of toxoplasmosis among blood donors in Egypt varies according to locality and time . Accordingly, this study aimed to not only update the prevalence of toxoplasmosis in an important sector of the population in the second largest governorate in Egypt but also to evaluate the possibility of transmission of active infection through blood donation. To achieve this goal, assessment was carried out using serological (for prevalence) and molecular (for active infection) determinations. The outcome showed that the general prevalence of T. gondii IgG antibodies among the 150 blood donors was 65.3%. This rate varied, being slightly higher than that reported from Mansoura governorate, Egypt, in 2009, in which the prevalence of Toxoplasma -specific IgG in blood donors, as revealed using ELISA, was 59.6% ; both of these rates were higher than the 19.5% incidence reported much earlier in 1986 from Cairo .
When compared with different countries, our recorded seropositivity was much higher than that reported in blood donors in Mexico (7.4%) , Thailand (9%) , Chile (21.2%) , Malaysia (28.1%) , Czech Republic (33.1%) , Mali (41.2%) , and Saudi Arabia (52.1%) . Moreover, seropositivity was lower than that reported in blood donors in Brazil (79.0%)  and in Cuba (73.43%) . The variability in the prevalence levels of T. gondii infection among blood donor populaces may be attributed to dissimilarities in environmental pollutions as well as the individual habits and characteristics of the indigenous populations.
Diagnosis of toxoplasmosis is not conclusive without the monitoring of rise in antibody levels in an infected individual. Moreover, the serological diagnosis of active toxoplasmosis is restricted when reactivation of infection does not promote changes in the antibody levels, and when IgM antibodies persist during the chronic phase of infection, thus confusing interpretation of the serological results. Besides, serological techniques may not recognize below threshold levels of specific anti-Toxoplasma IgG or IgM produced during the active phase of disease in immunocompromised patients. In AIDS patients, these antibodies may be produced late following a few weeks of acquiring the contamination .
To improve diagnosis, the real-time PCR Taqman probe-based test was introduced to increase the sensitivity and specificity of the amplification reaction ,. Numerous studies utilized T. gondii B1 gene for toxoplasmosis detection using real-time PCR ,. Our results showed that the real-time PCR utilized for the identification of B1 gene proved to be sensitive, having recorded an equivalent of 0.002 tachyzoites, thus confirming its capacity to quantify the contamination load in a clinical example. It was successfully amplified in 15 (10%) blood donors’ samples (nine were IgG seropositive and six were IgG seronegative). This low detection rate cannot exclude recent infection and may be due to a small number of parasites circulating in the blood, a low amount of parasite DNA present in clinical samples compared with the total volume of blood in the human body, or short period of parasitemia duration ,,. The seronegative cases, whose B1 gene was decidedly identified as positive using real-time PCR, may be an indication of the presence of circulating Toxoplasma tachyzoites unassociated with antibodies. The proposed clarification is that it could be an extremely recent infection at the time of serological testing, prompting an inadequate distinguishable generation of antibodies by serology .
About 54% of blood donors were in the age group 26–35 years and 22.7% were in age group 36–45 years; those in the age group 18–25 years constituted 16% and those older than 45 years constituted 7.3%. Our study showed that the percentage of IgG seropositivity was nearly equal in two of the age groups, from 36–45 years and those over 45 years (73.5 and 72.7%, respectively); it was lower in the younger age groups. This finding is in agreement with many other studies reporting that the increased age-related rate of Toxoplasma infection is due to the greater likelihood of exposure to infection with increase in age ,,,.
As with other techniques, PCR may experience poor outcome due to variable execution. The absence of a consistent standard technique implies that sensitivity and specificity of molecular tests cannot generally be compared with those of other classical systems (e.g. serological testing, and cell cultures and animal inoculation). Proper correlation between protocols confirmed the high regard for molecular procedures, especially due to the shorter time needed to obtain results ,,. Accordingly, updating the diagnosis of toxoplasmosis using real-time PCR techniques will benefit the quick shut tube framework, and the reproducible quantitative results without the danger of pollution. This technique has great potential for use as a standardized technique applied at the clinical level . Furthermore, this strategy may be suitable for routine screening of T. gondii infection in the clinical research facility in conjunction with other diagnostic methods.
| Conclusion|| |
The revealed data represents the first report on the prevalence of toxoplasmosis in blood donors from Alexandria governorate, Egypt. The IgG seropositivity rate in this group of individuals may be considered as an indication of the general prevalence of exposure to infection in this governorate. The unexpected detection of circulating B1 gene in a number of seropositive as well as seronegative donors indicates the possibility of a high risk for transmission of infection to blood recipients. Thus, the development of a general screening system for toxoplasmosis in blood contributors in Alexandria is recommended, especially as transmission of the disease from seronegative individuals cannot be excluded.
All authors participated actively in the study and are in agreement with its contents which have not been published or considered for publication elsewhere. MH Sayad designed the study and was in charge of performing ELISA technique; NA Sadek provided the blood donors’ cases. NA Hussein, MA Ahmed, OA Geddawi were in charge of performing the molecular analysis (Real-Time PCR). OA Geddawi was in charge of collecting the samples, performing the ELISA and extracting the DNA for molecular analysis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]
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