Prevalence of Toxoplasma gondii in Women Population of Rafha City, Saudi Arabia

Akbar Ali1,*, Khalil Mohamed2 and Fawzia Toulah3

1Faculty of Pharmacy, Northern Border University, Rafha, Saudi Arabia

2Department of Epidemiology, Faculty of Public Health and Health Informatics, Umm Al-Qura University, Makkah, Saudi Arabia

3Department of Zoology, Faculty of Science, King Abdul Aziz University, Jeddah, Saudi Arabia

ABSTRACT

Toxoplasmosis, a disease caused by Toxoplasma gondii parasite, is one of the most worldwide parasitic infections of warm-blooded animals including man. In most adults it does not cause serious illness, but it can cause serious health problems in infants, pregnant women and immune compromised patients. Abortion is one of the most serious outcomes in pregnant women. In this cross-sectional study, a total of 162 women of child bearing age were included from Rafha city of Saudi Arabia. Demographic data was recorded. Specific Toxoplasma gondii IgG and IgM were detected in serum by ELISA method. Toxoplasma gondii DNA was detected using PCR. Most women were in the 20-29 years age group (48%), followed by 30-39 years (33%) and >40 years (14%). Overall prevalence of Toxoplasma gondii IgG was seen in 12%, with significantly more in women with age more than 30 years as compared to those with age less than 30 years (19% vs 8%; p=0.038). The percentage of IgG seroprevalence was higher in women who ever got pregnant as compared to those who were never pregnant (13% vs 5%). The percentage was also higher in women with direct contact to soil (14% vs 5%). Only one sample was PCR positive. The results show that overall prevalence of Toxoplasma gondii infection in young women of Rafha city is low as compared to other regions of Saudi Arabia.

Article Information

Received 13 January 2017

Revised 10 March 2017

Accepted 18 March 2017

Available online 15 May 2017

Authors’ Contribution

AA contributed in designing the study, sample collection, lab work, acquisition, analysis and interpretation of data and drafting the article. KM and FT contributed in conception and designing the study and drafting and revising the article.

Key words

Toxoplasma gondii, Seroprevalence, IgG, IgM, ELISA, Polymerase chain reaction.

DOI: http://dx.doi.org/10.17582/journal.pjz/2017.49.3.1039.1047

* Corresponding author: [email protected];

[email protected]

0030-9923/2017/0003-1039 $ 9.00/0

Introduction

Toxoplasma gondii is a single-celled, obligate intracellular protozoan parasite that causes the disease toxoplasmosis (Dardé et al., 2011). T. gondii can infect virtually all warm-blooded animals (Skariah et al., 2010), but domestic cats are the main hosts (CDC, 2016). Toxoplasmosis is one of the most common infections in developed countries; more than 60 million people in the United States may be infected with the Toxoplasma parasite (Cook et al., 2000; Jones et al., 2001). Among the infections causing food-borne deaths, Toxoplasmosis stands third after salmonellosis and listeriosis (Dubey and Jones, 2008). It has been estimated that 30–50% of the world population has been exposed to the parasite and a huge fraction may be chronically infected (Flegr et al., 2014; Pappas et al., 2009).

The lifecycle of T. gondii consists of two phases. The sexual phase takes place only within cats (felids, wild or domestic). The asexual phase can take place in all warm-blooded animals which are defined as intermediate hosts (Dubey and Jones, 2008; Hill and Dubey, 2002). In the epithelial cells of cat, the parasite sexual produces millions of thick-walled oocysts which are released in the feces (Dubey, 2009). When a warm-blooded animal ingests these oocytes, the sporozoites are released. These sporozoites differentiate into active and quickly proliferating tachyzoites (Dubey et al., 2011). Under the influence of the host’s immune system, the tachyzoites are converted to bradyzoites which form clusters. These clusters are called tissue cysts (Weiss and Kim, 2011). Tissue cysts are mostly observed in the brain, the eyes, and striated muscle (including the heart) approximately 7–10 days after initial infection (Dubey et al., 1998). The cysts can persist for the life time of the infected host (Weiss and Kim, 2011).

A Toxoplasma infection can occur by consuming undercooked, contaminated meat, food and water (Jones and Dubey, 2012). The parasite may also be transmitted from mother to child; receiving an infected organ transplant or infected blood via transfusion (Paquet and Yudin, 2013).

Among the infected people, very few have mild, flu-like symptoms; majority of infected people do not show any observable symptoms. However, in infants, pregnant women and immune compromised patients the infection can cause serious health problems (Paquet and Yudin, 2013). If pregnant mother is infected, the parasite can move to unborn fetus via the placenta resulting in congenital toxoplasmosis. This can result in with fetal death and abortion. In infants, congenital toxoplasmosis is associated with neurologic defects, neurocognitive defects, and chorioretinitis resulting in serious symptoms like blindness or mental disability (Torgerson and Mastroiacovo, 2013).

Materials and Methods

Ethics statement

Present study was approved by the Ethics Committee of Northern Border University, ArAr, Kingdom of Saudi Arabia (Approval No. 3-11-1436-5). The samples were collected strictly according to the recommendations of the committee. Informed consents were taken from each participant.

Collection of samples

The present study was carried out at faculty of pharmacy, Northern Border University Rafha branch. A total of 162 females of child bearing age (16-45) were included in this study. Samples were collected from Rafha Central Hospital. Blood (1 to 2 mL) and urine samples collected in appropriate containers. The demographic data was recorded on prescribed forms.

Serological detection of T. gondii infection

The serum IgM and IgG antibody (Ab) levels against T. gondii antigens were measured using the UDI (United Diagnostic Industries, KSA) Toxoplasma IgM ELISA kit for direct IgM antibody detection (REF EM127) and Toxoplasma IgG ELISA kit for direct IgG antibody detection (REF EG127). Both the kits are based on the classical ELISA technique. The manufacturer’s recommended protocols were followed. Serum/plasma samples were diluted to a ratio of 1:101 with sample diluent buffer. Blank and calibrators were also run along with samples. For both IgG and IgM ELISA assays, optical density (OD) was measured at 450 nm (reference filter 620 nm). The ratios between the OD value of unknown samples and that of calibrator were calculated. Any sample with the ratio equal or more than 1.100 was considered positive. On the other hand, if the ration was less than 0.900, the sample was supposed to be negative.

PCR detection of T. gondii infection

Isolation of DNA

The genomic DNA from blood samples were extracted using the solution based blood DNA preparation kit (Jena Biosciences, Germany, Cat# PP205S) according to the manufacturers’ instructions. The DNA pellet was dissolved in 70 µl of DNA Hydration Solution and stored at -20 0C.

For DNA extraction from urine, the urine samples were first centrifuged at 5000 rpm for 10 min to pellet down the parasite. Almost all the supernatant was discarded with only 300 µl of residual liquid, mixed vigorously with pipette and used for DNA extraction using above procedure.

PCR amplification

Two PCR approaches were used to amplify target; single PCR targeting B1 gene (GenBank accession number AF179871); Nested PCR targeting RE region (GenBank accession number AF146527). The oligonucleotide primers used for the amplification of the B1 and RE PCR are shown in Table I.

Positive control

ABI Toxoplasma gondii (RH strain) quantitated DNA (Catalog #: 08-948-250) was used as positive control. According to the manufacturers descriptions, this positive control contained 16000 copies of Toxoplasma gondii (RH strain) genomic DNA per microliter. A total of 8 fresh serial dilutions were made containing 16000 (provided), 1600, 160, 16, 8, 4, 2 and 1 copy per microliter in distilled water immediately before use.

Table I.- The oligonucleotide primers used for the PCR amplification.

Gene			Primer sequence	Product size
B1 gene
	B1F		5’–CTCAAGGAGGACTGGCAACC-3’	250 bp
	B1R		5’–GCCTCTCGACCGGAACTTTT-3’
RE region
First Round		REF	5’-GATGAAGGCGACGGTGAGGA–3’	267 bp
		RER	5’-CGCTGCAGACACAGTGCATCT–3’
Second Round		F3	5’-GAAGCGACGAGAGTCGGAGA – 3’	205bp
		B3	5’-GATTCCTCTCCTACGCCTCCT – 3’

For the detection of T. gondii DNA in this study, human blood samples were used for DNA extraction; the human genomic DNA is also extracted with the expected T. gondii DNA which obviously is the major part. This human DNA can affect the sensitivity and specificity of the PCR. In order to study this, about 30ng of human genomic DNA was also added to each PCR in separate PCR reactions when using the various dilutions of ABI Toxoplasma gondii (RH strain) quantitated DNA as positive control.

Amplification of the B1 gene

Single round amplification: In B1 PCR, a 250 base pair fragment covering B1 gene was amplified. The total 20μl PCR mixture contained 1X PCR buffer (Thermo Scientific) , 1.5mM MgCl2, 0.2mM of each dNTPs, 0.5μM each primer (B1F, B1R, Table I), 0.5 units Taq polymerase (Thermo Scientific) and 1μL of extracted DNA. Cycling conditions for PCR were: Initial denaturation at 95°C for 5 min and then 40 cycles each of 95°C for 10 seconds, 56°C for 20 seconds and 72°C for 30 seconds, followed by a final extension at 72°C for 5 min. A known negative control contained sterile water. The positive control is described above. Five microliters of each PCR product were run on a 2% agarose gel in 1X TBE buffer to confirm the presence of PCR product.

Amplification of the RE region

Nested PCR approach was used to amplify the RE gene as follows:

First round amplification: A 267bp selected region of the RE region of the parasite was amplified in the first round. Each 20μL PCR reaction contained 1X PCR buffer (Thermo Scientific), 1.5mM MgCl2, 0.2mM of each dNTPs, 0.5μM each primer (REF, RER, Table I), 0.5 units Taq polymerase (Thermo Scientific) and 1μL of extracted DNA. Cycling conditions for PCR were: Initial denaturation at 95°C for 5 min and then 35 cycles each of 95°C for 20 seconds, 56°C for 30 seconds and 72°C for 40 seconds, followed by a final extension at 72°C for 5 min. A known negative control containing sterile water and a positive sample containing T. gondii DNA was also amplified along with patient samples.

Second round amplification: One microliter of the first round PCR product was used as template in the second round. The 20μl PCR mixture contained 1X PCR buffer (Thermo Scientific), 1.5mM MgCl2, 0.2mM of each dNTPs, 0.5μM each primer (F3, B3, Table I), 0.5 units Taq polymerase (Thermo Scientific). Cycling conditions for PCR were: Initial denaturation at 95°C for 1 min and then 40 cycles each of 95°C for 10 seconds, 60°C for 15 seconds and 72°C for 20 seconds, followed by a final extension at 72°C for 5 min. Negative and positive control PCR from first-round amplified product were used in the second round negative and positive controls respectively. The size of the second round PCR product was 205bp. Finally, 5ul of each of positive and negative controls along with unknown samples were run on 2% agarose gel containing ethidium bromide and observed under UV light. A known molecular weight 50bp DNA marker was included in each run.

Table II.- The demographic data of the cases included.

	Total (162)	IgG Positive (n=19)	IgG Negative (n=143)	p Value
Nationality
Saudi (%)	124 (77)	15 (12)	109 (88)	0.79
Non-Saudi (%)	38 (23)	4 (11)	34 (89)	0.79
Age (years)
<30 (%)	103 (64)	8 (8)	95 (92)	0.038
>30 (%)	59 (34)	11 (19)	48 (81)	0.038
Pregnancy
Ever (%)	140 (86)	18 (13)	122 (87)	0.26
Never (%)	22 (14)	1 (5)	21 (95)	0.26
Abortion (No. of women)
Abortion (%)	30 (19)	6 (20)	24 (80)	0.12
Never (%)	132 (81)	13 (10)	119 (90)	0.12
Water source
Home filtered (%)	79 (49)	11 (14)	68 (86)	0.4
Bottled (%)	83 (51)	8 (10)	75 (90)
Drinking milk
Fresh (%)	29 (18)	1 (3)	28 (97)	0.13
Bottled (%)	133 (82)	18 (14)	115 (86)
Type of meat consumed
Camel (%)	53 (33)	4 (8)	49 (92)	0.38
Beef (%)	33 (20)	3 (9)	30 (91)
Mutton (%)	73 (45)	11 (15)	62 (85)
Non	3 (2)	1 (33)	2 (67)
Frequency of eating meet
Daily (%)	109 (67)	14 (13)	95 (87)	0.37
Weekly (%)	50 (31)	4 (8)	46 (92)	0.37
Never (%)	3 (2)	1 (33)	2 (67)
Meat preference
Partially cooked (%)	36 (22)	2 (6)	34 (94)	0.24
Completely cooked (%)	123 (76)	16 (13)	107 (87)
Vegetarian (%)	3 (2)	1 (33)	2 (67)
Contact with cats
Yes (%)	90 (56)	12 (13)	78 (87)	0.48
No (%)	72 (44)	7 (10)	65 (90)	0.48
Direct contact with soil
Yes (%)	88 (54)	14 (16)	74 (84)	0.08
No (%)	74 (46)	5 (7)	67 (93)	0.08
Dusting / Cleaning at home
Yes (%)	120 (74)	16 (13)	104 (87)	0.28
No (%)	42 (26)	3 (7)	39 (93)	0.28

Statistical analysis

Data were recorded, validated and analyzed for average and percentage prevalence between groups. Chi square test were used as tests of significance at 5 % level of significance. The P values were calculated online at following website http://www.quantpsy.org/chisq/chisq.htm

Results

Out of 162 women of child bearing age included in this study, 12% were IgG positive for Toxoplasma gondii. None of them was positive for Toxoplasma gondii IgM. Based upon IgG reactivity, two groups were made (Table II). Among the participants, 124 were Saudi national while 38 belonged to different nationalities including Pakistanis, Indians, and Sudanese. The prevalence of Toxoplasma gondii IgG among Saudi and non-Saudi women was almost the same (p=0.79). Two age groups were made and compared for the seroprevalence the IgG. It was found that the seroprevalence was significantly high in women with more than 30 years of age (p=0.038). Insignificantly higher incident was seen in women who ever got pregnant than those who were never pregnant (13% vs 5%, p=0.26). Although higher percentage of IgG seroprevalent cases was seen in women who ever had abortion than those who never experienced it, the correlation was not significant (20% vs 10v%, p=0.12). Other parameters also showed the similar correlation with seroprevalence of T. gondii IgG (Table II). Average number of pregnancies among seropositive women was slightly higher than the seronegative women (5.1 vs 4.25; p=0.5; data not shown in Table II).

Selection of the suitable PCR approach

To compare and select the better of the two, B1 and RE PCR were performed using serial dilutions of the positive control as described in methods. These dilutions were used as template to amplify a fragment of B1 gene (single round) and RE region (nested). The BI PCR was designed as a single round PCR as it is believed to be sensitive enough to detect a single copy of T. gondii DNA from human samples (Jones et al., 2000).

Sensitivity and specificity of the B1 gene PCR

The B1 PCR showed a single amplicon of 250bp on 2% agarose gel. Using different dilutions of the positive control, this reaction was capable of detecting 2 copies and more of T. gondii DNA (Fig. 1). It could not however, detect single copy of the target genome. It has been estimated that one copy of T. gondii contains ~50fg DNA which means that B1 PCR was able to detect 100 fg of Toxoplasma DNA. It was seen that the added human genomic DNA (30ng) decreased the sensitivity of the B1 PCR to 4 copies per microliter. It did not however, affect the specificity of the B1 PCR as a single amplicon of the same size was observed in this case too.

Sensitivity and specificity of the RE PCR

First round

The RE first round showed a 266bp amplified product on 2% agarose gel. Using different dilutions of the positive control, the sensitivity of the RE first round PCR was higher than that of B1 PCR as it detected single copy of the target DNA (Fig. 1). However, at higher concentrations of the target DNA (16000, 1600 and 160copies /ul) it showed nonspecific PCR bands on agarose gel along with the specific one. The nonspecific band(s) fainted at lower concentrations. No extra efforts were done to decrease the non-specificity as it could decrease the sensitivity of the assay too. The added human genomic DNA did not affect the specificity of the RE first round PCR but it decreased the sensitivity of the assay to 2 copies per microliter (Fig. 1). This time it could not detect single copy of the target DNA.

Second round

The second round PCR amplification was done using first round amplicons as a template with and without added human genomic DNA in the first round. It is to be noted that no human genomic DNA was added in the second round. The second round PCR showed extra bands and smear in reactions with high initial copy number of the target DNA added in the first round. In case of smaller initial copy number, little or no extra bands were observed (Fig. 2). Rather, a single sharp PCR band was seen in these cases. In this way this represent a semi quantitative PCR. So no efforts were done to get rid of the extra bands/smear. The sensitivity of this round was also higher than first round as the last dilution with single copy number (with added human genomic DNA) which was negative in the first round produced a good sharp single band in the second round.

Patient samples

The RE nested PCR approach was finally selected for the PCR amplification/detection of Toxoplasma gondii DNA in the samples. Known negative and positive controls were also run in each PCR batch. For positive control, the dilution with intermediate copy number (16 copies/μl) was used. Out of all 162 samples, only one was PCR positive in DNA sample extracted from blood (Fig. 3). It was however, negative in DNA extracted from urine.

Discussion

The copy number (repeats) of B1 gene within genome varies from 3 to 7 (Costa and Bretagne, 2012) depending on the type of strain while previously it was estimated to be around 35 (Wahab et al., 2010; Correia et al., 2010). The copy number of RE region varies from 30 to 60 while others have reported it to be 200 to 300 (da Silva et al., 2011; Costa and Bretagne, 2012).

Table III.- T. gondii seroprevalence studies done in Saudi Arabia.

Research Group	Total samples	IgG +ve (%)	IgM +ve (%)	PCR +ve (%)	Region
Alghamdi et al. (2016)	203	32.5	6.5	22.2	Riyadh
Almogren (2011)	2176	38	zero	--	Riyadh
Bin Dajem and Almushait (2012)	137	38.6	6.5	41	Aseer
Al-Mohammad et al. (2010)	554	51	9	--	Al Ahsa
Aqeely et al. (2014)	195	20	6.2	--	Jazan
Eida (2015)	226	27	13	3.5	Jazan
Almushait et al. (2014)	487	38.8	6.2	--	Southwest
El-Shahawy et al. (2014)	96	29.2	3.1	--	Najran
Imam et al. (2016)	150	21	0	--	Madina

For the detection of T. gondii, the most frequently used sequence is the B1 gene, first identified by Burg et al. (1989). Although Jones et al. (2000) reported that the BI PCR is sensitive enough to detect a single copy of T. gondii DNA; our protocol detected at least 2 copies of template DNA in a single round. With Human genomic DNA added to the B1 PCR, the detection limit further increased to 4 copies of the genome which is obvious. Yamada et al. (2011) reported the detection limit of B1 assay in a single PCR to be 100 copy numbers repeats of B1 gene (around 20 copies of the template DNA) which decreased to 10 copy number (around 2 copies of the template DNA) in the second round (nested PCR). In a study by Kong et al. (2012), the detection limit of the RE nested PCR was reported to be 600fg (around 12 copies of the genome) of the template DNA. We performed much better as we were able to detect single copy of the genome using RE nested PCR. Jones et al. (2000) reported that the addition of human lymphocyte DNA (10–150 ng) does not inhibit the B1 PCR amplification (193-bp PCR product) from 1 ng purified T. gondii genomic DNA. In the current study we found that the addition of human lymphocyte DNA (30 ng) decreases the sensitivity of B1 (250bp) and RE PCR amplification (266-bp) especially from lesser amount of purified T. gondii genomic DNA.

The prevalence of worldwide T. gondii infection in human varies from region to region ranging from less than 10% to over 90% (Bouratbine et al., 2001; Torgerson and Mastroiacovo, 2013). In Saudi Arabia, different studies have reported the seroprevalence of T. godii IgG ranging from 21 to 38.6% while that of IgM ranging from zero to 09% (Table III). In the current study, the overall seroprevalence of T. gondii IgG was low (12%). This shows that overall prevalence of T. gondii infection in this part of Saudi Arabia is low. All the samples were negative for T. gondii IgM. This means that there was no recent infection among the cases and all the IgG positive cases acquired the infection long ago. This is also evident from the PCR results as all the IgG positive cases were negative for T. gondii DNA. In Saudi Arabia, two studies have reported same results for IgM (M).

Higher seroprevalence of T. gondii IgG (> 50%) is seen in countries where people use raw or poorly cooked meat and in tropical regions with lots of cats and more favourable climate for the survival of T. gondii oocysts (Cook et al., 2000; Jones et al., 2001; Di Carlo et al., 2008). In the United States, 15% of women at child bearing age (15 to 44) have T. gondii, infection. The most common sources of acquiring infection are exposure to cats or soil (gardening without gloves), drinking contaminated water or consuming raw or undercooked meat (Skariah et al., 2010; Elmore et al., 2010). Transfusion or organ transplantation from an infected person can also be a source of infection (Dubey and Jones, 2008). The climate of Saudi Arabia is mostly hot and dry so it does not favour the survival of T. gondii oocysts. Traditionally most of the people do not like to keep pet cats. They like to eat thoroughly cooked meat. In the current study all the above mentioned factors and many more were correlated with the seroprevalence of the infection (Table II). Significantly high IgG seroprevalence was seen in the age group >30 years. This was in accordance with a study conducted in India in 2014 in which a higher incidence (41%) of IgG positivity was seen in age group >40years (Singh et al., 2014). They also reported higher prevalence in married women and in people living in mud houses. In the current study, higher percentage of IgG seroprevalence was seen in women who ever got pregnant than those who were never pregnant (13% vs 5%) but this was statistically insignificant (p=0.26). The percentage of IgG positive cases gradually increased with increasing number of pregnancies (Fig. 4). In Saudi Arabia, majority of people do not live in mud houses. So, we compared seroprevalence between women who are exposed to dust/soil and those who are not. Women with direct contact to soil had higher percentage of IgG seroprevalence than those who were not (14% vs. 5%). Similar trend was seen in women who do regular dusting/cleaning at home. As discussed earlier, cats are considered as one of the sources of T. gondii infection but this was not seen in the current study.

No significant association between seroprevalence and other factors was observed; the reason could be smaller number of samples. A future study with larger number of samples may reveal more interesting facts about the topic in this region.

Conclusion

Although antibodies against T. gondii (IgG and IgM) were seen in some study participants, the overall prevalence of T. gondii active infection in young women of Rafha city was very low as compared to other regions of Saudi Arabia and other parts of the world. The ministry of health is providing compulsory active immunization against many infectious diseases including toxoplasmosis. Considering the results of the current study, we hope that the disease will be eradicated from the region very soon.

Acknowledgements

The authors gratefully acknowledge the approval and the support of this research study by the grant no. 3-11-1436-5 from the Deanship of Scientific Research at Northern Border University, Arar, KSA. The authors would also like to thank Jawahir Alghamdi (Faculty of Sciences, King Saud University, Riyadh) and Elsamoual Ibrahim Ahmedani (Faculty of Pharmacy, Northern Border University, Rafha) for their technical help in this research work.

Statement of conflict of interest

Authors have declared no conflict of interest.

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