Seroprevalence and Genotype Distribution of Toxoplasma gondii Among Pregnant Women in Northwest Ethiopia: A Cross‐Sectional Study

ABSTRACT

Background

Toxoplasma gondii (T. gondii) infection poses significant risks during pregnancy, yet data on seroprevalence and genotype diversity in Ethiopia are scarce.

Objective

This study aimed to determine the seroprevalence, associated risk factors, and genotype distribution of T. gondii among pregnant women in Northwest Ethiopia.

Methods

A cross‐sectional study was conducted among 554 pregnant women attending antenatal care in public hospitals between January 2022 and April 2024. Systematic random sampling was used. ELISA detected T. gondii‐specific IgG and IgM antibodies, and PCR‐RFLP targeting B1 and SAG2 loci were used for genotyping.

Results

Overall seroprevalence was 54.3%, with 6.1% testing IgG‐positive only, 9.4% IgM‐positive only, and 38.8% positive for both. Cat ownership (AOR = 2.2; 95% CI: 1.4–3.5) and dog ownership (AOR = 4.9; 95% CI: 2.9–8.1) were significantly associated with infection. Among 28 IgM‐positive samples, Type II strains predominated (50%), followed by Types I and III (25% each).

Conclusion

These findings support targeted screening and pet‐handling education to reduce the risk of congenital toxoplasmosis. Further molecular surveillance is recommended to inform public health strategies.

Abbreviations

AIDS

acquired immune deficiency syndrome

ANC

antenatal care

AOR

adjusted odd ratio

CNS

central nervous system

COR

crude odd ratio

CT

congenital toxoplasmosis

ELISA

enzyme‐linked immunosorbent assay

FAT

immuno fluorescence antibody test

IgA

immunoglobulin A

IgE

immunoglobulin E

IgG

immunoglobulin G

IgM

immunoglobulin M

LAT

latex agglutination test

RDT

rapid diagnostic test

1. Introduction

Toxoplasma gondii (T. gondii) is a globally prevalent protozoan parasite estimated to infect nearly one‐third of the human population [1]. It causes toxoplasmosis, an illness resulting from an obligate intracellular organism of the Apicomplexa phylum, specifically within the coccidian subgroup [2, 3]. The parasite targets warm‐blooded species, with cats serving as the definitive hosts for sexual reproduction, while humans and other mammals act as intermediate hosts where asexual replication occurs [4, 5]. In humans, the infection typically progresses through an acute phase, often asymptomatic or presenting with mild flu‐like symptoms, and a latent phase, which can persist in tissues such as the brain, heart, and muscles [6, 7, 8]. While immunocompetent individuals usually control the infection, immunocompromised patients are at risk of severe complications. During pregnancy, primary infection can result in transplacental transmission, leading to miscarriage, stillbirth, or congenital anomalies such as hydrocephalus and retinochoroiditis [4, 9, 10]. Diagnosis relies on serological testing, particularly enzyme‐linked immunosorbent assay (ELISA), which detects T. gondii‐specific IgG and IgM antibodies. Molecular techniques such as polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) enhance detection and enable genotyping of infecting strains [11, 12, 13, 14, 15]. Global seroprevalence varies widely (2%–90%) due to differences in climate, hygiene, dietary habits, and animal exposure. In Ethiopia, reported rates range from 35.6% in Adwa to 81.8% in Hawassa, yet molecular data remain scarce [16, 17, 18]. Known risk factors include pet ownership, especially cats and dogs, and consumption of undercooked meat or raw vegetables, which facilitate transmission via oocysts and tissue cysts [4, 19]. Genotype distribution influences virulence and congenital transmission risk. Historically, three clonal lineages, Types I, II, and III, were recognized, with Type II predominating in human infections [20, 21]. However, recent studies in Africa and South America have identified greater genetic diversity, including atypical and recombinant strains [22, 23]. Despite the public health importance of congenital toxoplasmosis, few studies in Ethiopia have concurrently assessed seroprevalence, risk factors, and genotype distribution in pregnant women. Therefore, this study aimed to determine the seroprevalence of T. gondii antibodies, identify associated risk factors, and characterize infecting genotypes in this population.

2. Materials and Methods

2.1. Study Design and Setting

An institution‐based cross‐sectional study was conducted between December 1, 2022 and August 31, 2024 at public hospitals in Northwest Ethiopia. The study aimed to assess the seroprevalence and genotype distribution of T. gondii among pregnant women attending antenatal care (ANC) services.

2.2. Study Participants

Eligible participants were pregnant women aged 18–49 years who attended ANC services and provided written informed consent. Women unable to communicate due to medical conditions were excluded.

2.3. Sample Size Determination and Sampling Technique

The sample size (n = 554) was calculated using a single population proportion formula, assuming a prevalence of 70.8%, 95% confidence level, 4% margin of error, and 10% nonresponse rate. Systematic random sampling was applied using ANC registries, with a sampling interval (k) based on expected daily attendance.

2.4. Data Collection Procedure

Sociodemographic, clinical, and lifestyle data were collected using a pretested semi‐structured questionnaire in Amharic. The questionnaire's reliability was confirmed with a Cronbach's α of 0.82 for risk factor items. Medical records were reviewed to validate clinical history. Eleven trained data collectors conducted interviews and sample collection.

2.5. Sample Collection and Storage

In total, 10 mL of venous blood was drawn pre‐ANC consultation using a 21 G needle from the antecubital vein. Samples were centrifuged at 3000 rpm for 5 min. Serum was stored at 4°C for up to 48 h or at −70°C for long‐term preservation.

2.6. Serological Testing (ELISA)

Commercial ELISA kits (BioCheck Inc.; Catalog #BC‐1035 for IgG, #BC‐1036 for IgM) were used to detect T. gondii‐specific antibodies. Results were interpreted per manufacturer guidelines. Samples within ±10% of cutoff values were retested to confirm borderline results.

2.7. DNA Extraction

DNA was extracted from both serum and whole blood using the QIAamp DNA Blood Mini Kit (Qiagen). Serum was prioritized for IgM‐positive samples due to the higher likelihood of circulating parasite DNA. DNA concentration and purity were assessed using a NanoDrop spectrophotometer. Extracted DNA was stored at −20°C until analysis.

2.8. PCR Amplification

PCR targeted the B1 gene of T. gondii. The reaction mix included B1‐specific primers (B1F1, B1R1), dNTPs, Taq polymerase, MgCl₂, and buffer. Thermal cycling (Bio‐Rad T100 thermo‐cycler) involved initial denaturation at 95°C for 15 min, followed by 40 cycles of denaturation (95°C), annealing (62°C), and extension (72°C) for 30 s each, with a final extension at 72°C for 10 min. Amplified products (196 bp) were visualized on 2% agarose gels stained with 0.5 µg/mL ethidium bromide at 100 V for 45 min using a GelDoc XR+ system. Nested PCR was performed using internal primers (B1F2, B1R2) and an annealing temperature of 60°C.

2.9. Genotyping via PCR‐RFLP

Genotyping was performed on nested PCR‐positive samples by amplifying the SAG2 gene at both 5′ and 3′ ends. First‐round primers (SAG2F4, SAG2R4 and SAG2F3, SAG2R3) were followed by nested primers (SAG2F1, SAG2R2 and SAG2F2, SAG2R1). Digestion was performed with Sau3AI (5′ end) and HhaI (3′ end). Products were resolved on 2% agarose gels. Reference strains (Types I, II, III) were included as controls.

2.10. Quality Assurance

The questionnaire was developed in English, translated into Amharic, and back‐translated for consistency. A pilot test was conducted on 5% of participants. SOPs were followed for all procedures. The principal investigator supervised data collection and laboratory analysis.

2.11. Data Processing and Analysis

Data were entered using Epi Info v7.1.5.2 and analyzed in SPSS v21. Descriptive statistics summarized participant characteristics. Bivariate and multivariable logistic regression identified risk factors. Variables with p < 0.2 in bivariable analysis were included in the multivariable model. Model fit was assessed using the Hosmer–Lemeshow test (p = 0.45), and discrimination was evaluated via ROC curve (AUC = 0.78). PCR‐RFLP data were analyzed for genotype significance using 95% CI and p < 0.05.

3. Results

3.1. Participant Characteristics

A total of 554 pregnant women receiving ANC at public hospitals in Northwest Ethiopia were enrolled. The mean age was 28.0 ± 5.2 years (range: 18–43 years). Most participants were Orthodox Christians (91.2%) and resided in urban areas (78.9%). Regarding employment, 31.9% worked in government positions, and 40.6% had attained education at the certificate level or higher. Exposure‐related factors included cat ownership (30.1%), dog ownership (28.2%), consumption of undercooked meat (30.7%), and raw fruits or vegetables (93.5%). Additionally, 18.6% reported farm dust exposure, and 10.8% had a history of blood transfusion (Table 1).

Table 1.

Study participants' characteristics of seroprevalence and genotype distribution of Toxoplasma gondii among pregnant women in Northwest Ethiopia.

Variables	Frequency	Percentage (%)
Age
16–20	37	6.7
21–25	125	22.6
26–30	233	42.1
31–35	102	18.4
≥ 36	57	10.3
Resident
Urban	437	78.9
Rural	117	21.1
Job
Student	40	7.2
Government	177	31.9
Daily labor	34	6.1
Farmer	68	12.3
Merchant	39	7
Other	196	35.6
Religion
Orthodox	505	91.2
Protestant	10	1.8
Muslim	34	1.8
Other	5	6.1
Level of education
Illiterate	120	21.7
Elementary	66	11.9
Secondary	143	25.8
Certificate and above	225	40.6
Cat present
Yes	167	30.1
No	387	69.9
Dog present
Yes	156	28.2
No	398	71.8
Eating uncooked meat
Yes	170	30.7
No	384	69.3
Eating uncooked fruits or vegetables
Yes	518	93.5
No	36	6.5
Contact with dust from farming
Yes	103	18.6
No	451	81.4
Blood transfusion
Yes	60	10.8
No	494	89.2

3.2. Seroprevalence of Toxoplasma gondii

The overall seroprevalence of T. gondii antibodies was 54.3% (95% CI: 50.2%–58.3%). Among seropositive individuals, 6.1% (95% CI: 4.2%–8.3%) tested positive for IgG only, 9.4% (95% CI: 7.2%–12.1%) for IgM only, and 38.8% (95% CI: 34.7%–43.0%) for both IgG and IgM.

3.2.1. Associated Risk Factors

Multivariable logistic regression identified cat and dog ownership as statistically significant risk factors for T. gondii infection. Cat ownership was associated with 2.2 times higher odds of infection (AOR = 2.2; 95% CI: 1.4–3.5), while dog ownership showed nearly five times higher odds (AOR = 4.9; 95% CI: 2.9–8.1). Other variables including age, residence, occupation, education, dietary habits, farming exposure, blood transfusion history, and gestational stage were not significantly associated with seropositivity in the adjusted model (Table 2). Model diagnostics indicated good fit (Hosmer–Lemeshow p = 0.45) and acceptable discrimination (AUC = 0.78).

Table 2.

Bivariate and multivariate logistic regression analyses of the seroprevalence of T. gondii IgG and IgM antibodies about selected variables of seroprevalence and genotype distribution of Toxoplasma gondii among pregnant women in Northwest Ethiopia.

Variable		Seronegative (%)	Seropositive (%)	COR (95% CI)	AOR (95% CI)
Age	18–20	12 (32.4)	25 (67.5)	1.4 (0.5–3.3) 0.7 (0.3–1.3) 0.7 (0.4–1.4) 0.7 (0.3–1.4)	—
	21–25	61 (48.8)	64 (52.1)
	26–30	108 (46.3)	125 (53.6)
	31–35	49 (48.1)	53 (51.9)
	36–43	23 (40.3)	34 (59.6)
Resident	Urban	219 (50.1)	218 (49.9)	2.4 (1.5, 3.8)	1.4 (0.8, 2.6)
	Rural	34 (29)	83 (70.9)
Job	Student	15 (37.5)	25 (62.5)	1.6 (0.8, 3.2) 0.9 (0.6, 1.4) 1.2 (0.5, 2.5) 2.5 (1.3, 4.6) 1.0 (0.5, 2.0)	0.6 (0.3, 1.5) 0.8 (0.5, 1.5) 0.6 (0.2, 1.4) 1.1 (0.4, 2.9) 1.2 (0.5, 2.6)
	Government	88 (49.7)	89 (51.3)
	Daily labor	15 (44.1)	19 (55.9)
	Farmer	19 (27.9)	49 (72.1)
	Merchant	19 (48.7)	20 (51.3)
	Others	97 (49.5)	99 (50.5)
Level of education	Illiterate	40 (33.3)	80 (66.7)	1.8 (1.1, 2.9) 1.3 (0.7, 2.3) 0.8 (0.5, 1.2)	0.8 (0.3, 1.8) 0.7 (0.3, 1.5) 1.1 (0.6, 1.9)
	Elementary	27 (40.9)	39 (59.1)
	Secondary	77 (53.8)	66 (46.2)
	Certificate and above	109 (48.4)	116 (51.6)
Cat owner	Yes	39 (23.3)	128 (76.7)	4 (2.6, 6)	2.2 (1.4, 3.5)
	No	214 (55.2)	173 (44.8)
Dog owner	Yes	25 (16)	131 (84)	7 (4.3, 11)	4.9 (2.9, 8.1)
	No	228 (57.2)	170 (42.8)
Raw meat consumption	Yes	79 (46.4)	91 (53.7)	0.9 (0.6, 1.3)	—
	No	174 (45.3)	210 (54.7)
Raw vegetable consumption	Yes	241 (46.5)	277 (53.5)	0.5 (0.2, 1.1)	1.8 (0.8, 4.0)
	No	12 (33.3)	24 (66.7)
Farming	Yes	34 (33)	69 (67)	1.9 (1.2, 3)	0.9 (0.5, 1.6)
	No	219 (48.5)	232 (51.5)
Hand‐washing habit	Yes	252 (45.8)	298 (54.2)	0.3 (0.4, 3.8)	—
	No	1 (25)	3 (75)
History of blood transfusion	Yes	26 (43.3)	34 (56.7)	1.1 (0.6, 1.9)
	No	227 (45.9)	267 (54.1)
Pregnancy stage (trimester)	1st	30 (37.9)	49 (62.4)	1.2 (0.7, 2.1) 0.7 (0.5, 1)	—

3.3. Sensitivity Analysis

Excluding IgM‐positive/PCR‐negative discordant samples slightly reduced the overall prevalence estimate but did not materially alter the associations with pet ownership. These findings suggest that while some IgM results may represent false positives or persistent antibody responses, the overall epidemiological trends remain robust.

3.4. Genotyping of T. gondii Strains

Of the 301 ELISA‐positive samples, 28 IgM‐positive specimens were selected for molecular analysis. PCR amplification targeting the B1 and SAG2 gene regions achieved 100% success. Genotyping revealed that 50% (14 samples) were Type II, while Types I and III each accounted for 25% (7 samples each), indicating Type II as the predominant genotype among acutely infected women.

3.5. PCR and Nested PCR for the B1 Gene

Initial PCR targeting the B1 gene produced the expected 196 bp fragment. Nested PCR yielded a more specific 97 bp fragment, confirming enhanced sensitivity and specificity (Figures 1 and 2, 3).

Figure 1.

Amplification of the T. gondii B1 gene of seroprevalence and genotype distribution of Toxoplasma gondii among pregnant women in Northwest Ethiopia. Lane 1: positive control. Lanes 2–20: positive samples: molecular weight marker 100 bp.

Figure 2.

Nested PCR of the B1 gene of seroprevalence and genotype distribution of Toxoplasma gondii among pregnant women in Northwest Ethiopia. Lane 1: positive control; Lanes 2–19: positive samples; Lane 20: negative control. Molecular weight marker 50 bp.

Figure 3.

First amplification of the SAG2 locus at the 5′ end of seroprevalence and genotype distribution of Toxoplasma gondii among pregnant women in Northwest Ethiopia. Lane 1: positive control. Lanes 2–13: positive samples; Lane 14: negative control. Molecular weight marker 50 bp.

3.5.1. SAG2 Gene Amplification

First‐round PCR of the SAG2 gene produced a 341 bp fragment. Nested PCR refined this with 241 and 221 bp amplicons from the 5′ and 3′ ends, respectively, consistent with known T. gondii profiles (Figures 2, 3, 4, 5, 6).

Figure 4.

First amplification of the 3′ ends of the SAG2 locus of seroprevalence and genotype distribution of Toxoplasma gondii among pregnant women in Northwest Ethiopia. Lane 1: positive control; Lanes 2–18: positive samples; Lane 20: negative control. Molecular weight marker 100 bp.

Figure 5.

Second amplification of the 5′ end of the SAG2 locus of seroprevalence and genotype distribution of Toxoplasma gondii among pregnant women in Northwest Ethiopia. Lane 1: positive control; Lanes 2–12: positive samples. Molecular weight marker 50 bp.

Figure 6.

Second amplification of the 3′ ends of the SAG2 locus of seroprevalence and genotype distribution of Toxoplasma gondii among pregnant women in Northwest Ethiopia. Lane 1: positive control; Lanes 2–19: positive samples. Molecular weight marker 100 bp.

3.6. PCR‐RFLP Analysis

RFLP analysis differentiated genotypes using restriction enzymes. The 3′ end of SAG2 was digested with HhaI to identify Type II strains (Figure 7), while Sau3AI digestion of the 5′ end distinguished Types I and III (Figure 8). Fragment patterns validated the reliability of nested PCR and RFLP methods.

Figure 7.

HhaI restriction digestion of the 3′ end amplification products of seroprevalence and genotype distribution of Toxoplasma gondii among pregnant women in Northwest Ethiopia. Lanes 1, 8, 9, 13, 17, and 21: Type I strains; Lanes 2, 4–7, 10, 11, 14–16, 18–20, 22, and 23: Type II strains; Lanes 3 and 12: Type III strains.

Figure 8.

Sau3AI restriction digestions of the 5′ amplification products of seroprevalence and genotype distribution of Toxoplasma gondii among pregnant women in Northwest Ethiopia. Lanes 1, 6, 9, and 10: Type I strains. Lanes 2, 3, 7, 8, 14–16, 18, 20, 22, and 23: Type II strains. Lanes 4, 5, 11–13, 17, 19, and 21: Type III strains.

4. Discussion

This study identified a notably high seroprevalence of T. gondii infection (54.3%) among pregnant women in Northwest Ethiopia. This prevalence is comparable to findings from Ghana (57.3%) [24] and Northwest Cameroon (54.5%) [25], suggesting a similarly high burden of exposure in sub‐Saharan Africa. However, it remains lower than the exceptionally high rates reported in Hawassa and Yiregalem (both 81.8%) [26] and Arbaminch (79.3%) [27], which may reflect localized differences in environmental sanitation, dietary customs, and animal contact. These regional variations underscore the influence of cultural practices, climate, and socioeconomic conditions on the transmission dynamics of T. gondii.

In contrast, the prevalence observed in this study exceeds that reported in several countries outside the region, including Turkey (47%) [28], Morocco (43%) [29], Northeast Iran (34.4%) [30], Nigeria (29.4%) [31], and Adwa, Ethiopia (35.6%) [32]. Such disparities highlight the complex interplay between geographic location, food safety practices, water quality, and public health infrastructure in shaping infection risk. The relatively high seroprevalence in our setting may also reflect limited awareness of toxoplasmosis transmission routes and inadequate preventive measures among pregnant women.

The predominance of IgG antibodies among seropositive participants indicates widespread prior exposure to T. gondii, consistent with findings from Cameroon [25] and Jimma [33]. The detection of IgM antibodies in 9.4% of participants suggests a smaller proportion of recent or ongoing infections. Although this rate is higher than those reported in Turkey (0.4%) [34] and Jimma (2.5%) [33], it remains within the expected range for endemic settings. The elevated IgM prevalence may reflect true acute infections or persistent IgM responses, which are known to linger for months postinfection and may occasionally yield false positives depending on assay specificity.

Multivariable analysis revealed significant associations between T. gondii seropositivity and pet ownership, particularly of cats and dogs. Cats are the only known definitive hosts capable of shedding environmentally resistant oocysts, which can contaminate soil, water, and food sources [35, 36]. Although dogs do not support the parasite's sexual cycle, they may serve as mechanical vectors by transporting oocysts on their fur or paws, thereby facilitating indirect human exposure [37]. These findings are consistent with studies from northeastern Iraq and other endemic regions, reinforcing the role of domestic animals in the epidemiology of toxoplasmosis [38, 39, 40].

Molecular characterization of 28 IgM‐positive samples using nested PCR and PCR‐RFLP revealed that Type II strains were predominant (50%), followed by Types I and III (25% each). This genotype distribution aligns with patterns observed in Europe and the Americas, where Type II is frequently implicated in both asymptomatic and symptomatic infections, including congenital toxoplasmosis [41]. The use of nested PCR targeting the B1 and SAG2 genes provided high sensitivity and specificity, and the observed polymorphisms in SAG2 enabled effective differentiation among the three clonal lineages [42]. While this distribution aligns with global patterns, the limited sample size and reliance on SAG2 genotyping restrict the generalizability of these findings. SAG2 provides useful lineage differentiation but lacks the resolution of multilocus or whole‐genome approaches, which are better suited to detect atypical or recombinant strains known to circulate in Africa and South America [43]. Future studies should employ higher‐resolution methods to capture the full genetic diversity of T. gondii in Ethiopia.

The 100% PCR positivity rate among IgM‐positive samples is consistent with acute infection, though methodological factors such as DNA extraction efficiency and assay sensitivity may also contribute. Sensitivity analysis excluding discordant IgM‐positive/PCR‐negative cases confirmed the robustness of prevalence estimates [44, 45].

5. Conclusion

This study contributes important epidemiological and molecular data on T. gondii infection among pregnant women in Northwest Ethiopia. The findings highlight a substantial burden of exposure, with pet ownership emerging as a key risk factor. The predominance of Type II strains underscores the need for continued surveillance and molecular monitoring. These insights are critical for informing targeted public health interventions, including antenatal screening, health education on zoonotic risks, and future studies employing advanced genotyping techniques to map strain diversity and transmission pathways.

6. Limitations

This study has limitations that affect its conclusions. The cross‐sectional design prevents causal inferences, necessitating longitudinal research for clearer temporal associations. Sampling was limited to pregnant women in public hospitals, hindering generalizability to broader populations. Environmental factors impacting exposure risk were not considered, and the high IgM prevalence could be influenced by various factors, including assay inaccuracies. The small sample size for genotype distribution leads to wide confidence intervals and reduced statistical power. Additionally, the use of SAG2 genotyping offers limited resolution compared to more comprehensive methods, potentially overlooking atypical strains in Ethiopia.

Author Contributions

Eden Woldegerima and Mequanente Dagnaw were responsible for conceptualizing the research, preparing the proposal, managing data collection and curation, conducting the analysis, validating the results, and drafting the manuscript. Eden Woldegerima, Mequanente Dagnaw, Nega Berhane, Mastewal Birhan, Mequanint Melesse, Destaw Fetene Teshome, Getnet Fetene, Marye Alemu Eshetu, Tewodros Eshetie, and Mulualem Lemma Kebede contributed to the study design, methodology, supervision, and data interpretation. Asif Jan contributed to investigation, writing – original draft, methodology, validation, software, formal analysis, and supervision. All authors contributed to the review and final approval of the manuscript.

Funding

The authors received no specific funding for this work.

Disclosure

The lead author, Mequanente Dagnaw, affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.

Ethics Statement

Ethical clearance was obtained from the University of Gondar Ethical Review Committee (Ref No/VP/RTT/05/280/2022).

Consent

Written informed consent was obtained from all participants. No minors were enrolled, and no compensation was provided. Confidentiality was maintained throughout.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data generated and analyzed during this study are available from the corresponding author on reasonable request, subject to ethical considerations.