In-house real-time PCR to detection Toxoplasma gondii in pregnant women candidates for amniocentesis and comparison with nested PCR and serologic methods in several clinical samples

doi:10.21203/rs.3.rs-3339699/v1

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Research Article

In-house real-time PCR to detection Toxoplasma gondii in pregnant women candidates for amniocentesis and comparison with nested PCR and serologic methods in several clinical samples

https://doi.org/10.21203/rs.3.rs-3339699/v1

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Background: Toxoplasmosis is a common parasitic infection caused by Toxoplasma gondii (T. gondii), an obligate and intracellular parasite, that affects both humans and animals globally. It can cause severe damage to neonates during pregnancy and immunocompromised patients. Therefore, finding a new method with high sensitivity for the diagnosis of toxoplasmosis that involves low-risk sampling is crucial for patient management. This study aims to diagnose toxoplasmosis in pregnant women from plasma, amniotic fluid, buffy coat, and urine specimens using serological, nested PCR, and real-time PCR tests.

Methods: We collected amniotic fluid, blood, and urine samples from 100 pregnant women referred to a gynecologist in Mazandaran province, northern Iran, who were candidates for amniocentesis. We performed anti-T. gondii IgM, IgG, and IgG avidity tests using enzyme-linked immunosorbent assay (chemiluminescence, ELISA), as well as nested PCR, real-time PCR, and multilocus PCR genotyping using RE and GRA6 target genes, respectively.

Results: Based on the serologic test, 52% of subjects were seropositive (51 samples were positive for IgG, and 1 was positive for both IgG and IgM antibodies), and all cases showed high avidity. Among 100 amniotic fluid samples, 2 and 3 cases tested positive using nested and real-time PCR, respectively, while all samples from buffy coat and urine were negative. All positive DNA samples were genotyped as type I.

Conclusions Our study results suggest that diagnosing congenital toxoplasmosis is achievable using a combination of serological and molecular tests. We also found that the real-time PCR method is more sensitive than the nested PCR.

Amniocentesis

IgG avidity

nested PCR

pregnant women

real-time PCR

Toxoplasma gondii

Toxoplasmosis is one of the most common parasitic infections in humans and warm-blooded vertebrates, with a global distribution [1] and caused by an obligate intracellular protozoan parasite Toxoplasma gondii (T. gondii) [2, 3]. Although this disease is usually asymptomatic in healthy individuals, it can be severe and dangerous in women during pregnancy and in immunodeficient individuals [4]. When transmitted through the placenta, T. gondii can cause severe congenital infections, including neurological and ocular damage in the newborn child, or even fetal death, while primary contamination can occur throughout pregnancy [5, 6].

T. gondii is estimated to have infected over one-third of the world's population, but the prevalence varies in different geographical areas due to differences in climate, nutrition, and hygiene habits[7, 8]. In various countries and regions, this rate ranges from 10–80%. Several published studies have shown that 12.4 to 24.6% of individuals are IgG seropositive for T. gondii, and it is estimated that between 0.01 to 0.1% of children worldwide are congenitally infected[7, 8]. Based on regional data in the USA, there are approximately 750 deaths and 400-4,000 cases of congenital toxoplasmosis reported annually [7].

Diagnosing toxoplasmosis infection is crucial as soon as possible due to the risk of transmission to the fetus. Therefore, anti-Toxoplasma IgG, IgM, and IgG avidity detection are commonly used in clinical practice for laboratory diagnosis on blood samples [9]. However, the diagnosis is confirmed in the fetus by detecting T. gondii DNA using polymerase chain reaction (PCR) in the amniotic fluid [10, 11].

Molecular tests on amniotic fluid have a positive predictive value and specificity estimated at 100%, and the negative predictive value and sensitivity calculated as 88% and 64%, respectively[12]. Although molecular methods have shown acceptable results, it is important to note that amniocentesis is one of the most invasive sampling methods during pregnancy and has been reported to have a miscarriage rate of 1.4%[13]. Therefore, a safe, lower-risk, and sensitive sampling method could replace the invasive amniocentesis method. Previous studies have shown that toxoplasmosis can be diagnosed using semi-invasive and risk-free samples such as blood, urine, and tear specimens, which have produced acceptable results in some cases [14–16].

Various methods, including serological and molecular methods, are used to diagnose toxoplasmosis infection. Rapid detection of infection with high sensitivity and specificity in individuals undergoing seroconversion is one of the most crucial steps in managing toxoplasmosis [17]. Measuring antibodies to detect active toxoplasmosis has always been challenging. IgG is valuable when the initial test is negative but positive on a subsequent test. On the other hand, IgM may remain in the blood for months, which can delay antibody responses or lead to non-specific responses against different factors. IgG avidity, developed by Hedman in 1989, is based on the tendency of immunoglobulins to polyvalent antigens of T. gondii. Serological tests and molecular methods are commonly used to diagnose T. gondii in pregnant women and are considered the best diagnostic approaches [18].

Molecular diagnostic methods used to identify the T. gondii parasite include PCR, RFLP PCR, RAPD, Microsatellite PCR, LAMP, Nested PCR, and the newer real-Time PCR method [19–21]. In this study, we conducted an experimental investigation on pregnant women who were candidates for amniocentesis to determine toxoplasmosis. We used both serological methods (Chemiluminescence, Avidity IgG) and molecular methods including nested-PCR and real-time PCR (qPCR, quantitative PCR) to analyze various clinical samples, including invasive (amniotic fluid) and non-invasive (plasma and urine) ones.

2.1. Ethics

All analyses and sampling were conducted as part of routine clinical work at teaching hospitals and polyclinics in Mazandaran, northern Iran. Informed consent was obtained from all participants, and their demographic data were recorded via a questionnaire. The questionnaire had been approved by the Ethics Committee of Mazandaran University of Medical Sciences (IR.MAZUMS.REC.1400.11546), and all procedures adhered to ethical rules and principles set forth in the Declaration of Helsinki.

2.2. Subjects and clinical sample collections

From August 5, 2021 to April 9, 2022, we conducted an experimental study on 100 pregnant women who were candidates for amniocentesis in their 15th to 18th week of pregnancy. These women were admitted to teaching hospitals and polyclinics and underwent amniocentesis performed by a gynecologist using a ten cc syringe. The obtained amniotic fluid (approximately 10 mL) was spun at 500×g for 10 minutes and then stored at -20°C until further use. In addition, blood samples with EDTA anticoagulant and urine were collected from all cases and stored at + 4°C until use. The blood samples were centrifuged for 10 minutes at 500×g to separate plasma and buffy coat for serological and molecular tests, respectively. Negative serology samples were considered negative controls, while tachyzoites T. gondii (RH strain) were utilized as a positive control.

2.3. Immunodiagnostic tests

For the serological method, Chemiluminescense IgM, IgG (bioMérieux, Marcy-l’Etoile, France) and IgG Avidity ELISA (Platelia Toxo IgM; Bio-Rad, Marnes-la-Coquette, France) were used according to the manufacturer’s instructions. The IgG Avidity ELISA test is used to differentiate between acute and chronic infections; however, it cannot be used at the beginning stages of an infection because the level of IgG antibodies must be sufficient for the denaturing agents to take effect. The final result is expressed as a percentage under the relative avidity index (RIA) [15, 22, 23].

2.4. Molecular assay

2.4.1. DNA extraction and Nested-PCR

Four ml of blood samples into a vial containing EDTA anticoagulant and 10 ml of urine and amniotic fluid were taken from the patient, and five approved negative cases for toxoplasmosis. The samples were centrifuged, the supernatant was discarded, and their precipitate was used for DNA extraction using the ROCHE commercial kit according to the manufacturer’s instructions. The genomic DNA was extracted from the T. gondii tachyzoites RH standard strain as the positive control, and DNA was used for amplification. A nano-spectrophotometer was used to determine the concentration and quality of the DNA. Nested-PCR technique was performed based on the 164 bp gene (RE). The product of PCR1 was diluted with distilled water at a ratio of 1 to 25 and was used as a template in PCR2. The reaction was carried out like the PCR1 in a volume of 25 µl. Primers include of Outer primer (sense strand) F1: 5′-TGACTCGGGCCCAGCTGCGT-3′, Outer primer (nonsense strand) R1: 5′-CTCCTCCCTTCGTCCAAGCCTCC-3′, Inner primer (sense strand) F2: 5′-AGGGACAGAAGTCGAAGGGG-3′ and Inner primer (nonsense strand) R2: 5′-GCAGCCAAGCCGGAAACATC-3′and was carried out as previously described [14, 24].The electrophoresis method on 1.5% agarose gel was used to evaluate the amplification of the desired fragment.

2.4.2. In-house real-time PCR (qPCR)

The primer-probe was designed in this study, and then the real-time PCR (qPCR) test was optimized to detect T. gondii. First, the primer-probe design was done using Beacon Designer software, Metabion, Germany; with the sequences of forward primer 5′-CGTCTGTCGGGATGAGAC-3′, reverse primer 5′-TCCCCTTCGACTTCTGTCC-3′ and Primer Probe 5′-TTCTGTGGCTCCAAGCACAGGC-3′. Next, real-time PCR was done with the reagents of; DNA (5.0 ng) 10µl, Primer Probe (5 µM) 0.5 µl, mastermixn (1X) 12.5 µl and D. W 2µl with the final Volume of 25µl. After preparing 12.5 microliters of master mix (PCR BIO SYSTEM Company) with 10µL of template DNA, 0.5 microliters of the primer-probe and two microliters of distilled water were mixed until the final volume reached 25 µL. The temperature program of the device is as follows: 10 min hot-start at 95 ̊C; 10 S denaturation stage at 95 ̊C (denaturation); 40 S primer connection at 58 ̊C temperature (annealing) and 10 S amplification stage at 72 ̊C temperature [25].

2.4.3. Sensitivity and specificity measuring

To assess the analytical sensitivity of the nested PCR and real-time PCR techniques for detecting T. gondii (RH strain) DNA, ten-fold serial dilutions of T. gondii DNA with concentrations ranging from 1 pg to 0.01 fg were prepared and utilized in both assays that targeted RE-164 [1, 26]. To assess the analytical specificity of the nested PCR and real-time PCR techniques, extracted DNAs from other microorganisms, including Acanthamoeba and Leishmania spp., were used as templates using specific primers of RE-164. A positive result was observed only with Toxoplasma gondii tachyzoites, and DNA extracted from Acanthamoeba and Leishmania spp., had a negative impact. The construction is performed with various concentrations of DNA. Next, the cycle threshold (CT) values were plotted as mean (triplicate) against the standard curve values to determine the detection limit of both primer sets. Parasite concentrations are determined after the calculation of the linear regression equation (y = ax + b), where y = CT; a = curve slope (slope); x = parasite number; and b = where the curve intersects y-axis (y-intercept).

2.5. Statistical analysis

Data were analyzed using SPSS Software v.21 (SPSS Inc. Chicago, IL, USA). Agreements between the serological and molecular assays were demonstrated using Cohen’s kappa coefficient [33]. The curves were statistically analyzed using an unequal-variance t-test based on a critical value of p ≤ 0.05.

3.1. Distribution of participants

Out of a total sample size of 100 women, 1% were aged 15–20, 2% were aged 21–25, 16% were aged 26–30, 39% were aged 31–35, 31% were aged 36–40, and 11% were aged 41–45. Among the 52 positive serology samples, the majority (57.69%) were found in women who were in their 15th week of pregnancy (as shown in Table 1).

Table 1

The results of anti-*Toxoplasma* IgG antibodies tests in100 patient’s candidates for amniocentesis during the 15 to 19 weeks of pregnancy.
Age category	Number of positive cases	Percentage (%)
15th week	30	57.69
16th week	8	15.38
17th week	10	19.23
18th week	3	5.76
19th week	1	1.92
Total	52	100

3.2. Results serology, nested PCR, and real-time PCR methods

Out of the 100 pregnant women included in the study, 51% tested positive only for IgG anti-toxoplasma antibodies, 1% were positive for both IgM and IgG anti-Toxoplasma antibodies, and 48% tested negative on serological tests. Of the eight IgG-positive cases, one was positive for both IgG and IgM antibodies, while two were negative for IgG. These cases were selected for evaluation of IgG avidity. The results showed that all eight IgG-positive cases had high avidity, while the two IgG-negative cases did not have detectable IgG avidity. In terms of sensitivity, the Real Time PCR method using RE genes had a sensitivity of 0.1 fg, while the Nested PCR method using RE genes had a sensitivity of 10 pg (Table 2).

Table 2

Serological and molecular results in several clinical samples of pregnant women candidates for amniocentesis
Clinical Samples	No. of examined	No. of seropositive	No. of nested positive	No. of qPCR positive
Plasma	100	52	-	-
Amniotic fluid	100	-	2	3
Buffy Coat	100	-	0	0
Urine	100	-	0	0
Total	100	52	2	3

3.3. The agreement coefficient of the tests

The Kappa test results indicated a strong correlation between the Nested PCR and Real Time PCR tests, with a coefficient of agreement of 0.8 and a statistically significant p-value of < 0.001. However, there was no statistically significant agreement between the IgG and IgM tests, with a coefficient of agreement of 0.018 and a p-value of 0.334. The coefficients of agreement between IgG and nested PCR, IgG and Real-time PCR, IgM and nested PCR, and IgM and real-time PCR were also not significant (p > 0.05). In all the IgG positive samples, the avidity titer was high, indicating agreement between these two tests.

Out of the 100 participants, two cases tested positive with nested PCR, and three cases tested positive with qPCR using RE-164 only in amniotic fluid samples (Figs. 1 and 2). None of the participants tested positive for both nested PCR and qPCR in buffy coat or urine samples. One of the three positive cases with the PCR method was IgG and IgG avidity positive, while the other two were negative on serological tests. Among the two positive cases with nested PCR, one case was negative on the serology method. All two cases that tested positive with nested PCR were also positive with qPCR. The CT value of the samples was approximately 35, compared to the CT value of approximately 25 in the positive control.

3.4. PCR Sequencing products

Three positive and five negative samples by PCR; were genotyped using specific primers for the GRA6 marker gene. After determining the sequence and analysis of the samples by Chromas and Bioedit software, they were blasted into the NCBI database. The results indicated that all the positive cases were related to type I of the parasite gene, and all five negative samples were not amplified.

Toxoplasmosis is a parasitic disease caused by T. gondii, which can have serious consequences during pregnancy. Therefore, it is essential to develop fast, accurate and sensitive diagnostic methods for its diagnosis in pregnant women. Early detection of toxoplasmosis can prevent acute symptoms and even abortion [2]. The most common and requested tests used in diagnosing toxoplasmosis are anti-toxoplasma IgG and IgM. However, these tests may not be sensitive or specific in many cases. For instance, anti-toxoplasma IgM can only be used in the acute phases of the disease and measured in peripheral blood for a short period [18]. In contrast, the IgG avidity test is applicable for a long time after the onset of the disease. In this study, both serological and molecular tests were conducted on three different sample types: blood, amniotic fluid, and urine samples to diagnose toxoplasmosis and find a sensitive, specific diagnostic method. Testing multiple samples increases the likelihood of detecting the presence of the parasite, as different samples may contain varying amounts of the parasite or its genetic material[15]. The aim of this research is to develop a sensitive, specific, and efficient diagnostic method for the diagnosis of toxoplasmosis in pregnant women. By combining both serological and molecular tests and testing multiple sample types, this study will contribute to the development of an early and accurate diagnosis of toxoplasmosis, ultimately preventing severe health outcomes.

Our study results indicate that 51% of the participants tested positive only for IgG anti-toxoplasma antibodies, while 1% were positive for both anti-toxoplasma IgM and IgG antibodies. Furthermore, 48% tested negative in serological tests. All cases tested positive for high-avidity grade on the IgG avidity test. The coefficient of agreement (Kappa value) between IgM-IgG, IgM-IgG avidity, and IgG-IgG avidity revealed that the avidity test alone cannot serve as a reliable method for diagnosing the acute form of congenital toxoplasmosis.

Interestingly, two cases with negative serology yielded positive results in Nested PCR and Real-time PCR. While low IgG avidity and positive IgM results indicate active infection, it is important to note that low avidity cannot be considered a 100% sign of active infection. Nonetheless, high avidity can rule out primary infection. Overall, these findings suggest that a combination of serological and molecular methods may be necessary to achieve a more accurate diagnosis of toxoplasmosis [15].

In a 2020 study by Sharifi et al., it was found that IgG avidity had higher diagnostic priority than anti-toxoplasma IgM in the early stages of toxoplasmosis. The results also indicated that the IgG avidity test is a valuable tool in distinguishing between acute and chronic stages of the disease in pregnant women [9]. Similarly, Rahimi Esboei et al., applied anti-Toxoplasma IgG and IgM tests along with IgG avidity to diagnose ocular toxoplasmosis using blood samples. The results were compared to LAMP, Nested, and qPCR techniques based on REP-529 and B1 gene targets. The findings suggested that serological tests are not precise enough for the diagnosis of ocular toxoplasmosis, and a more reliable test, such as molecular experiments, is needed [1, 14, 15]. Another study by Masoumeh Amiri et al., investigated the seromolecular prevalence of Toxoplasma infection in diabetic pregnant women in southwestern Iran. The results showed a strong correlation between positive IgM, low IgG avidity, and positive molecular test results. Additionally, among 185 individuals who tested negative in serology, five tested positive through nested-PCR [16]. These findings suggest that molecular and serological tests do not always overlap, and a combination of methods may be necessary to diagnose toxoplasmosis infection accurately.

In a non-matched study by Nimri et al., 248 pregnant women were assessed for toxoplasmosis diagnosis using anti-toxoplasma IgG, IgM antibodies, and conventional PCR. The findings showed that 96 (38.70%) patients were positive for IgG, while four people tested positive for IgM antibodies, and 20 tested positive through conventional PCR. These results differ from our findings, where all individuals who tested positive through molecular techniques were seropositive [17].

In this study, molecular examinations were used to validate the results of serology tests. The findings indicated that two and three cases tested positive through nested and qPCR, respectively. Among the serologically positive samples, only one case was positive through nested and qPCR, while other positive samples by molecular tests were serologically negative. These findings highlight the superior characteristics of molecular methods, particularly the qPCR method.

The CT value for approximately 35 samples, compared to a CT of approximately 25 in the positive control, suggests a low amount of parasite gene load in these samples. This observation could indicate the presence of cell-free DNA (cf DNA) and the rejection of congenital toxoplasmosis. Furthermore, some samples that tested positive through Real-time PCR were negative in serology and nested PCR. This discrepancy may be due to the high sensitivity of Real-time PCR, which can detect low amounts of parasite genes in the samples. Molecular examinations on amniotic fluid are routinely performed to identify congenital toxoplasmosis infection in cases where serological results are inconclusive. Moreover, PCR-based methods can detect the presence of parasites in the amniotic fluid [18]. Thus, one of the main objectives of the recent study was to diagnose toxoplasmosis infection without performing amniocentesis and instead using blood and urine samples as semi-invasive and non-invasive alternatives. However, this hypothesis was rejected when molecular results were negative for all 100 blood and urine samples tested. The real-time PCR method using the RE-164 target gene had a detection limit of 0.1 fg of T. gondii genomic DNA, while the detection limit for conventional PCR was estimated at ten pg [1, 27]. In a study by Nagy et al., four molecular methods were employed to detect T. gondii in amniotic fluid. The researchers found that real-time PCR was more sensitive, simpler, and more accessible than other molecular methods [18]. Therefore, it was reasonable to expect that the number of positive cases detected through real-time PCR would be higher than those detected through nested PCR in our study.

Gene targets play a significant role in the sensitivity and specificity of molecular methods, which are crucial for the diagnostic power of a molecular approach. Several gene targets have been employed to detect T. gondii, including SAGs, ROPs, B1, RE-529bp, GRAs, GPI, and ITS. Many published studies indicate that RE-529bp is the most sensitive and specific gene target. The results of these studies demonstrate that RE-529bp gene targets exhibit significantly better sensitivity and specificity than B1 gene targets in diagnosing ocular toxoplasmosis [1]. Similar to our study, Teixeira et al. and Reischl et al., reported that the Real-Time PCR method is faster and more straightforward, and the RE marker gene is more suitable for diagnosing toxoplasmosis in pregnant women using amniotic fluid [28, 29]. In a study conducted by Shojaei et al., the PCR method was applied to diagnose toxoplasmosis on serum and urine samples in experimentally infected mice. Contrary to our study, both serum and urine samples were suitable for detecting T. gondii after three and five days of infection, respectively [30]. Based on the results of several published studies, T. gondii has been isolated from various samples, such as amniotic fluid, vitreous fluid, serum, urine, saliva, and even tears [31]. However, in our recent study, a urine sample was not considered sensitive enough for diagnosing toxoplasmosis infection.

Our study has three limitations. Firstly, due to time constraints and non-participation of some pregnant women, the number of samples collected was low. Secondly, we were unable to follow up with the pregnant women after delivery because of cultural limitations and local beliefs. Thirdly, we faced financial constraints that prevented us from using more valuable methods, such as gene targets and obtaining additional samples.

The findings of the recent study suggest that congenital toxoplasmosis can be diagnosed using a combination of serological and molecular testing methods. Serological tests, particularly IgG avidity, have a strong correlation with each other and may be used to differentiate recent infections from older ones. It was also observed that real-time PCR (qPCR) was more sensitive than nested PCR. The results of measuring IgG and IgM through the chemiluminescence method and IgG avidity test were found to be well-correlated. These tests can be used to distinguish between active and primary infections and inactive infections. However, it is important to note that negative serological results do not rule out toxoplasmosis. Blood sampling is considered a minimal-invasive and low-risk diagnostic method, while urine sampling is a non-invasive and safe alternative to invasive procedures such as amniotic fluid sampling. These methods are helpful in diagnosing toxoplasmosis, and better results can be obtained by using molecular techniques such as qPCR, which has high specificity and sensitivity compared to conventional PCR and nested PCR.

Author contributions

MF, OS and AM contributed to the study concept and design. RT, MN and MS collected the data. MM drafted the manuscript. All authors read and approved the final manuscript.

Conflict of Interest The authors declare no confict of interests.

Ethics approval and consent to participate

This study was ethically approved by the Ethics Committee of Mazandaran University of Medical Sciences (IR.MAZUMS.REC.1400.11546), and all procedures adhered to ethical rules and principles set forth in the Declaration of Helsinki. Also, Informed consent was obtained from all the participants.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this manuscript and Tables.

Competing interests

The authors declare no competing interest.

Funding

The authors declare that they have no funding source.

Authors' contributions

MF, BRE and HZH contributed to the study concept and design. RA, SK, HSR, ZR, BRE, MM collected the data. RA, BRE, HZH drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We would like to appreciate the assistance offered by the colleagues at departments of Obstetrics and Gynecology of Mazandaran province and Iranian National Registry Center for Lophomoniasis (INRCL), Mazandaran University of Medical Sciences, Sari, Iran.

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No competing interests reported.

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In-house real-time PCR to detection Toxoplasma gondii in pregnant women candidates for amniocentesis and comparison with nested PCR and serologic methods in several clinical samples

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Ethics

2.2. Subjects and clinical sample collections

2.3. Immunodiagnostic tests

2.4. Molecular assay

2.4.1. DNA extraction and Nested-PCR

2.4.2. In-house real-time PCR (qPCR)

2.4.3. Sensitivity and specificity measuring

2.5. Statistical analysis

3. Results

3.1. Distribution of participants

3.2. Results serology, nested PCR, and real-time PCR methods

3.3. The agreement coefficient of the tests

3.4. PCR Sequencing products

4. Discussion

Conclusion

Declarations

References

Additional Declarations

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