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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 28  |  Issue : 3  |  Page : 617-626

Seroprevalence and genotyping of Toxoplasma gondii in Menoufia governorate


1 Department of Parasitology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Zoonosis, National Research Centre, Cairo, Egypt

Date of Submission29-Aug-2014
Date of Acceptance14-Oct-2014
Date of Web Publication22-Oct-2015

Correspondence Address:
Salwa A Shams El-Din
Shams El-Din, Shebin El-kom, 32511 Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.165828

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  Abstract 

Objective
The aim of the study was to investigate the prevalence of Toxoplasma gondii in pregnant women in Menoufia governorate and identify the different genotypes of T. gondii in positive clinical samples.
Background
T. gondii is considered the most prevalent parasitic zoonotic disease worldwide, as at least one-third of the world's population is infected. T. gondii has a highly unusual population structure consisting of three clonal lineages known as types I, II, and III.
Patients and methods
The present study included 92 pregnant women who were divided into five groups: GI, consisting of 44 women with first trimester abortion; GII, consisting of 29 women with second trimester abortion; GIII, consisting of seven women with IUFD; GIV, consisting of six women with CFM; and GV, consisting of six women with normal pregnancy outcome. Serum samples were collected for detection of anti-Toxoplasma antibodies. Placental samples and products of conception were collected for PCR.
Results
A total of 48 (52.2%) women were seropositive for anti-Toxoplasma IgG and nine (9.78%) were seropositive for anti-Toxoplasma IgM antibodies. There was an association between IgG seroprevalence and age, gravidity, history of abortion, eating undercooked meat, and eating underwashed vegetables. Toxoplasma spp. DNA was detected in 26 (28.26%) women by means of PCR for the B1 gene. In nine of them amplification of the SAG2 gene was successful and typed as genotype I.
Conclusion
Seroprevalence of T. gondii among pregnant women in Menoufia governorate was 52.2%, which is considered high. Only genotype I was detected. The introduction of health educational programs should be considered.

Keywords: genotyping, PCR-RFLP, seroprevalence, Toxoplasma gondii


How to cite this article:
Nassef NE, Abd El-Ghaffar MM, El-Nahas NS, Hassanain MDA, Shams El-Din SA, Ammar AI. Seroprevalence and genotyping of Toxoplasma gondii in Menoufia governorate. Menoufia Med J 2015;28:617-26

How to cite this URL:
Nassef NE, Abd El-Ghaffar MM, El-Nahas NS, Hassanain MDA, Shams El-Din SA, Ammar AI. Seroprevalence and genotyping of Toxoplasma gondii in Menoufia governorate. Menoufia Med J [serial online] 2015 [cited 2020 Feb 24];28:617-26. Available from: http://www.mmj.eg.net/text.asp?2015/28/3/617/165828


  Introduction Top


Toxoplasma gondii is a coccidian obligate parasite of the phylum Apicomplexa. Toxoplasma spp. parasites are unusual in their capacity to parasitize a diverse array of cell types and infect any warm-blooded animal as well as in the diverse ways by which it can be transmitted [1].

Ingestion of tissue cysts in the intermediate hosts, ingestion of oocysts shed by the definitive host into the environment, and the transplacental crossing of tachyzoites from mother to fetus are the three main routes of transmission [2].

In humans, the disease is usually benign and asymptomatic in 60% of the cases. However, it can have serious consequences on the fetus, neonates, and in immunocompromised individuals [3].

The seroprevalence of T. gondii in pregnant women, in women of reproductive age, or in the general population varies greatly among countries (30-60%) and among geographical areas within the same country. The prevalence changes according to age, socioeconomic conditions, eating and hygiene habits, climate, and geographic location [4],[5].

T. gondii maintains a highly clonal population structure that consists of three lineages type I, type II, and type III which predominate in North America, Europe, and Africa [6],[7].

The presence of Toxoplasma spp. in biological samples can be diagnosed by molecular techniques such as different types of PCR and sequencers. The second step in the diagnosis of toxoplasmosis involves identification of the genetic group of T. gondii involved. This could allow the degree of pathogenicity and virulence of the parasite to be determined. Genotyping also has a key role in studies on the population biology and in epidemiological studies and in the identification of the infection source of T. gondii. The potential correlation between genotype and disease pattern may also be significant from the clinical viewpoint. Therefore, attention has been focused recently on the use of molecular biological tools in toxoplasmosis [8].

The aim of the present study was to evaluate the prevalence of T. gondii infection in pregnant women in Menoufia governorate and its relation with different risk factors. We also aimed to detect the most prevalent genotype of Toxoplasma spp. in these patients.


  Patients and methods Top


Study design

This cross-sectional descriptive study was carried out on 92 pregnant women attending Shebein El Kom Teaching Hospital and Menoufia General Hospital in Menoufia governorate.

The patients were divided into five groups: GI consisted of 44 patients who presented with first trimester abortion; GII consisted of 29 patients who presented with second trimester abortion; GIII consisted of seven patients who presented with a retained dead fetus after 28 weeks' gestation (IUFD); GIV consisted of six patients who presented with a fetus with congenital malformation (CFM); and GV consisted of six patients with normal pregnancy outcome (control group).

Blood samples were taken from each woman for serum separation for detecting anti-Toxoplasma IgG and IgM antibodies by means of enzyme-linked immunosorbent assay (ELISA). Placental samples or products of conception were taken from each woman for detection of the B1 gene of Toxoplasma spp. The B1 gene-positive samples were genotyped using nested PCR-restriction fragment length polymorphism (RFLP).

Some of the potential risk factors were investigated by using a short questionnaire, including eating undercooked meat, eating underwashed vegetables, and contact with a cat or with soil.

Clinical samples

Samples of blood were collected and centrifuged at 3000 rpm for 10 min and the separated serum was stored in labeled aliquots at -20°C. Tissue samples (products of conception in case of abortion or placental samples in cases of IUFD, CFM, or normal pregnancy outcome) weighing 50 g each, by evacuation and curettage or after deliveries, were collected from each woman.

The samples were washed three times with sterile PBS and each sample was well wrapped in a piece of foil, labeled, and stored at -20°C until being processed for PCR examination for Toxoplasma spp. DNA detection and characterization [9].

Detection of anti-Toxoplasma IgG and IgM antibodies by enzyme-linked immunosorbent assay

All serum samples were tested by ELISA to detect anti-Toxoplasma IgG and IgM antibodies using Prechek Bio Inc. (USA) product no. BC-1085 and product no. BC1087, respectively.

Isolation of DNA

DNA extraction from tissue samples (products of conception or placental samples) was performed using the Gene JET Genomic DNA Purification Kit (catalog no. K0721; Thermo Scientific, USA) in accordance with the manufacturer's instructions.

Detection of Toxoplasma spp. DNA by polymerase chain reaction

T. gondii infection was initially confirmed by PCR amplification of the repetitive and conserved B1 gene [10]. The primers used were as follows: forward 5′-GGA ACT GCA TCC GTT CAT GAG-3′ and reverse 5′-TCT TTA AAG CGT TCG TGG TC-3′. PCR reaction was set up in a final volume of 25 μl. Each PCR Eppendorf tube contained 12.5 μl of master mix [DreamTaq Green PCR Master Mix (2΄); catalog no. EP0712; Thermo Scientific], 1 μl of each primer, 5.5 μl of distilled water, and 5 μl of extracted DNA. The cycling conditions were denaturation at 94°C for 5 min, followed by 45 cycles at 94°C for 1 min, at 60°C for 1 min, and at 72°C for 2 min for each cycle. The final cycle was followed by an extension step at 72°C for 7 min. A 5 μl of each PCR product was subjected to gel electrophoresis in 2% agarose gel electrophoresis (product no. A9539; Sigma, USA). The reaction is expected to yield a product of 193 bp. RH strain of T. gondii was used as positive control, which was obtained as a gift from Zoonotic Diseases Department, National Research Centre (Cairo, Egypt).

Genotype analysis by nested-polymerase chain reaction-restriction fragment length polymorphism

The positive cases for B1 gene samples were subjected to strain typing by nested-PCR-RFLP of the amplified SAG2 gene of T. gondii. The nested PCRs separately amplified 5′ and 3′ ends of the gene, followed by restriction enzyme application according to Howe et al. [11] and Fuentes et al. [12].

The primers used are shown in [Table 1]. All the PCR reactions were performed in a thermocycler (MJ Research PTC-100; USA). The PCR products were analyzed by 2% agarose gel electrophoresis. The nested reactions were performed in a final volume of 25 μl as mentioned before. The 5′ end amplification was expected to yield a product of 241 bp, whereas the 3′ end amplification was expected to give a product of 221 bp in both cases for any strains of T. gondii.
Table 1: The nucleotide seque nces of the primers used for the nested-polymerase chain reaction assays targeting the SAG2 gene of Toxoplasma gondii

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The amplified products of the 5′ end were digested with Sau3AI enzyme (FastDigest Sau3AI, catalog no. FD0784; Thermo Scientific), whereas the 3′ end amplified products were digested with 3HhaI enzyme (FastDigest 3HhaI, catalog no. FD1854; Thermo Scientific), in order to determine the SAG2 type.

The RFLP reaction was performed in a total volume of 10 μl, consisting of 1 μl of nuclease-free water, 1 μl of FastDigest Green Buffer, 7 μl of PCR product, and 1 μl of restriction enzyme. The reaction was incubated at 37°C for 2 h, followed by 20 min at 65°C. The products were analyzed using 2% agarose gel electrophoresis.

Restriction digestion of 5′ end amplified products with Sau3AI distinguished the type III strain from type I and II strains and digestion of the 3′ end amplified fragments with HhaI differentiated type I and III strains. The restriction enzyme control genes were used for enzyme activity assessment [13].

Statistical analysis

The collected data were tabulated and analyzed by SPSS (version 12; SPSS Inc., Chicago, Illinois, USA). The χ2 -test and Fisher's exact test were used. P-values less than 0.05 were considered statistically significant.


  Results Top


This study included 92 women of a mean age of 27.44 ΁ 5.34 years: 68 (73.9%) women aged 30 years or younger; 23 (25%) women aged 31-40 years; and one (1.1%) woman older than 40 years. Seventy (76.1%) women were rural residents and 22 (23.9) were urban residents. Twenty-nine (31.5%) women were primigravida and the remaining, who constituted the majority, were multigravida (68.5%). Thirty-six (39.13%) patients had past experience of spontaneous abortion, four (4.3%) patients had past experience of IUFD, and only one patient had a past history of having a baby with congenital malformations.

Out of 92 women included in the present study, 52.2% were seropositive for anti-Toxoplasma IgG antibodies, 9.78% were seropositive for anti-Toxoplasma IgM antibodies, and 4.35% were positive for both IgG and IgM [Figure 1]. The rate of IgG seropositivity was highest in GIV and lowest in GV. The rate of IgM seropositivity was highest in GIV and GV and was lowest in GII; however, the difference between groups was statistically nonsignificant.

The mean age of IgG-seropositive women (28.83 ΁ 5.58 years) was significantly higher than that of seronegative women (25.93 ΁ 4.68 years).

There was a significant association between IgG seroprevalence and gravidity, history of abortion, eating underwashed vegetables, and contact with soil [Table 2]. There was no association between IgM seroprevalence and any of the studied variables [Table 3].
Figure 1: Percentage of women with To xoplasma spp. antibody titer.

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Table 2: Seroprevalence of anti-Toxoplasma IgG antibodies in relation to demographic data, relevant obstetric history, and relevant risk factors

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Table 3: Seroprevalence of anti-Toxoplasma IgM antibodies in relation to demographic data, relevant obstetric history, and risk factors

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Toxoplasma spp. DNA of the B1 gene was detected in 26 of 92 (28.26%) patients. Positive cases were higher in GIV. The difference between groups was statistically nonsignificant [Figure 2].
Figure 2: Agarose gel electrophoresis showing results of PCR amplification of the B1 gene (193 bp). Lane 1: DNA ladder; lane 2: negative control; lanes 3, 5, and 6: positive samples; lan es 4 and 7: negative samples.

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There was an association between positive B1 gene and eating undercooked meat and eating underwashed vegetables [Table 3] and [Table 4] and [Figure 2]; 69.23% of positive PCR women were IgG positive, 19.23% were IgM positive, and 11.54% were positive for both [Figure 3].
Figure 3: PCR results distributed according to anti-Toxoplasma spp. ant ibodies in the studied women.

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Table 4: Polymerase chain reaction results of B1 gene in relation to demographic data, relevant obstetric history, and risk factors

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Amplification of both ends of the SAG2 gene was successful in nine out of 26 (34.6%) positive samples for B1 gene and in the reference RH strain. In five samples out of 26 (19.2%), only the 5′ end of the SAG2 gene was amplified. In seven samples out of 26 (27%), only the 3′ end of the SAG2 gene was amplified. In the remaining five samples (19.2%), neither the 3′ end nor the 5′ end of the SAG2 gene was amplified. The nine fully studied samples were characterized as genotype I, as the 3′ and 5′ end amplified fragments of the SAG2 gene were undigested with the corresponding restriction enzymes. The seven samples that were partially characterized (3′ end only) resulted in type I or III (non-type II), as the 3′ end was undigested with the HhaI restriction enzyme. The five samples that were partially characterized (5′ end only) resulted in type I or II (non-type III), as the 5′ end was undigested with the Sau3AI restriction enzyme [Figure 4] and [Figure 5].
Figure 4: (a) Agarose gel electrophoresis showing results of amplifi cation of the 5¡ä end of the SAG2 gene (241 bp). Lane 1: DNA ladder; lane 2: negative control; lane 3: positive control; lane 8: positive sample; lanes 3, 5, 6, 7, 9, and 10: negative samples. (b) Agarose gel electrophoresis showing results of amplifi cation of the 3¡ä end of the SAG2 gene (221 bp). Lane 1: DNA ladder; lane 2: negative control; lane 3: positive control; lane 9: positive sample; lanes 4, 5, 6, 7 , 8, and 10: negative samples.

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Figure 5: Agarose gel electrophoresis showing digestion products of the 5¡ä and 3¡ä ends of the SAG2 gene. Lane 1: DNA ladder; lanes 2, 3, 5, and 6: undigested 5¡ä end (241 bp); lanes 7, 8, 9, and 10: undigested 3¡ä end (221 bp).

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There was significant association only between positive nested PCR and contact with soil [Table 5].
Table 5: Nested-polymerase chain reaction-restriction fragment length polymorphism results in relation to demographic data, relevant obstetric history, and risk factors

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  Discussion Top


Knowledge of the seroprevalence of T. gondii infection in pregnant women and risk factors of acquiring the infection would be valuable for planning appropriate preventive strategies [14].

In the present study, 52.2% of women were seropositive for anti-Toxoplasma IgG antibodies and 9.78% were seropositive for anti-Toxoplasma IgM antibodies.

A previous study in Menoufia reported that the seroprevalence of Toxoplasma spp. was 67.5% [15]. The seroprevalence of Toxoplasma spp. in pregnant women in Alexandria was 46.2% [16], that in El-Fayoum was 45.8% [17], that in Gharbia was 42.8% [18], and that in Dakhalia governorate was 44% [19]. However, seroprevalence of Toxoplasma spp. infection was 46.1% in women with early spontaneous miscarriage in Qena [20].

Comparable figures had been shown by El Mansouri et al. [21] in Morocco, Al-Mohammad et al. [22] in Saudi Arabia, and by Mousa et al. [23] in Libya who reported prevalences of 50.6, 51.4, and 44.8%, respectively.

However, a very low seroprevalence of toxoplasmosis of 6.4% was found in pregnant women in South Africa [24], and a very high seroprevalence of toxoplasmosis of 81.4% was found in women of child-bearing age in Central Ethiopia [25].

Toxoplasmosis seroprevalence is affected by complex environmental, socioeconomic, and health-related practices [26].

According to Cademartori et al. [27] and Dias et al. [28], the higher the age, higher the percentage of individuals seropositive for toxoplasmosis, which may be due to higher exposure rates. This is in agreement with the current results. Also the mean age of seropositive women (28.83 ΁ 5.58 years) was significantly higher than that of seronegative ones (25.93 ΁ 4.68 years; P < 0.01). A significant association between age and toxoplasmosis seroprevalence was also found in studies by El-Nahari and Al-Tamimi [29], Sartori et al. [30], and Moura et al. [31]. In contrast, Ayi et al. [32] and Babaie et al. [33] failed to find this association.

The seroprevalence of Toxoplasma-specific IgG antibodies was significantly higher in multigravida (61.9%) than in primigravida (31.03%; P < 0.01). This result was similar to that observed by Birgisdottir et al. [34]. This can be because of suppressed immune response or hormonal imbalance [35].

There was a significant association between IgG seropositivity and history of eating underwashed vegetables in the present study. This was in agreement with the results of several studies [36],[37],[38].

Contact with soil showed significant association with Toxoplasma spp. infection in this study. The studied population consisted mostly of rural residents. Soil is often contaminated with Toxoplasma oocytes shed by infected cats [39].

Regarding anti-Toxoplasma IgM antibodies in the present study, the prevalence rate of 9.78% was in consensus with that reported by El-Askari [9], who detected a similar rate of 7.8% among pregnant women attending Kasr El-Eini Maternity Hospital. Abd El-Ghany and Amin [40] revealed that anti-Toxoplasma IgM seropositivity was 10% in Sharkia governorate. A lower prevalence was reported by El-Deeb et al. [15].

Out of 92 women included in this study, 26 (28.26%) showed positive PCR results for Toxoplasma spp. All of them presented with abnormal pregnancy outcomes; none of the patients in the normal pregnancy outcome group (control group) were positive. PCR was positive in 34.1, 20.7, 28.6, and 50% of women in GI, GII, GIII, and GIV, respectively.

These results were closely related to the results of El-Fakahany et al. [41], who detected Toxoplasma spp. DNA in 20% of women who had undergone abortions and in 60% of women with babies with congenital anomalies. In another study by Abdel-Hameed and Hassanein [42], the positive percentage by PCR was 38% in women with miscarriage. Moreover, 29.9% of women with complicated obstetric history were positive for PCR according to El-Askari [9].

There was no statistically significant difference between positive and negative PCR with respect to age, residence, gravidity, parity, or history of bad pregnancy outcomes in the present study.

A higher percentage of women (66.7%) with a history of eating undercooked meat were positive for toxoplasmosis by PCR compared with women who did not (25.7%).

Women who ate underwashed vegetables and undercooked meat showed significantly higher rates of infection.

Similarly, Nimri et al. [43] found that the risk factor that was strongly associated with acute infection in PCR-positive women was eating raw meat.

The present study showed that Toxoplasma spp. DNA was found in 21 out of the total 48 IgG-seropositive women (18 women with positive IgG only and three with positive IgG and IgM). In contrast, Toxoplasma spp. DNA was detected in five women with seronegative IgG, which could be due to a recent infection.

In the present study, the sensitivity of PCR for detection of Toxoplasma spp. DNA in placental tissues was 50% and specificity was 100% when compared with ELISA. This low sensitivity was higher than that found by Fricker-Hidalgo et al. [44], who reported placental PCR sensitivity of 25% and specificity of 94.7%. Another study by Filisetti et al. [45] recorded a poor sensitivity of 25% and a good specificity of 99% for placental screening for Toxoplasma spp. infection by PCR.

In contrast, Robert-Gangneux et al. [46] obtained higher sensitivity (71%) and specificity (97%).

Indeed, various explanations could account for poor sensitivity, such as the amount of placental tissue analyzed, the homogenization technique used before PCR, and the PCR condition and technique itself (conventional or RT-PCR and gene target).

The main objective of the present study was to determine the genotypes of T. gondii strains obtained from pregnant women in Menoufia governorate using nested-PCR-RFLP at the polymorphic SAG2 locus. Amplification of both ends of the SAG2 gene was successful in nine out of 26 (34.6%) positive samples for the B1 gene. The nine fully studied samples were characterized as genotype I, as the 3′ and 5′ end amplified fragments of the SAG2 gene were undigested with the corresponding restriction enzymes.

These results were consistent with those of Tolba et al. [47], who investigated the genotypes of T. gondii in seropositive women in Alexandria governorate by using nested-PCR-RFLP for the GRA6 gene. The amplification was successful in 12 out of 100 seropositive samples. They found seven samples of genotype I (58.3%) and the remaining five samples (41.7%) were described as atypical genotypes.

In addition, the detection of type I in the present study is in agreement with the results from Tunisia, where, using the same marker, all Tunisian samples were classified as SAG2 type I. By using multilocus analysis, all Tunisian isolates examined, except one, harbored recombinant I/II and/or I/III strains [48]. This was also observed in studies reported in Ugandian HIV patients and other patients of African origin [49],[50],[51].

Predominance of type I is clearly in contrast with the findings reported by Abdel-Hameed and Hassanein [52] who detected type II in 87% of studied isolates and type I in only 13% of cases from the Obstetric and Gynecology Emergency Unit, Ain Shams University Hospitals, by using the same method of genotyping.

In reports from Europe and the USA, the SAG2 type II is the most prevalent in humans with toxoplasmosis [12],[53].

Different geographical locations and limited sample size may account for differences between studies.

In the present study, no amplification of the 5′ end in 7 samples (27%) or the 3′ end in 5 samples (19.2%) was reported. In another five (19.2%) samples, neither the 3′ end nor the 5′ end of the SAG2 gene was amplified. This phenomenon was similar to that reported in Spain by Fuentes et al. [12], with no amplification of the 5′ end in 18% and of the 3′ end in 6% of samples, and as in the Colombian study by Gallego et al. [54], in which no amplification of the 5′ end was detected in 9% of samples and of the 3′ end in 57% of samples. This could indicate the presence of polymorphisms in the primers binding site of these isolates [12].


  Conclusion Top


From the present study it was concluded that the seroprevalence of T. gondii among pregnant women in Menoufia governorate is high, with a significant proportion of women at risk for contracting T. gondii infections. Advanced age, increased gravidity, eating underwashed vegetables, and contact with soil were the independent risk factors associated with the presence of T. gondii infections. The determined genotype of Toxoplasma spp. isolates was type I.


  Acknowledgements Top


The authors are thankful to Dr Hassan Aly Elfadaly, Dr Raafat Shaaben, and Dr Khaled Abd El-Rahman for help and guidance in ELISA work and molecular techniques in the current study.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

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    Tables

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