Toxoplasma gondii is an obligate intracellular protozoan parasite and an important zoonotic pathogen that causes severe diseases in congenitally infected patients and in immunocompromised patients such as AIDS victims in addition to the parasite infected persons.1-3 T. gondii infects macrophages primarily and is capable of invading and replicating within a wide variety of nucleated host cells. Acute T. gondii infection at early pregnancy in women without a history of infection may lead to fetal death in the uterus or severe neurological damage. 4-6 Tran’s placental infection of the fetus occurs in 12% of cases in which the mothers acquire infection during the first trimester. The incidence of transmission increases thereafter to more than 90% when maternal infection occurs during the last weeks before delivery, which is more likely to be asymptomatic, yet may proceed to choreoretinitis later in childhood or in adolescence. One third of mothers who acquire a primary T. gondii infection during the pregnancy transmits the infection to their fetuses.7-9 Therefore, an antenatal diagnostic method should be employed to screen this fraction.10,11 Then, the ELISA avidity methods help to detect the time of infection in mothers with toxoplasma gondii. This would help the mothers to make an informed decision on either treatment or therapeutic abortion. In Iran, up to present, most of the decisions on infected fetuses were made based on serological findings in their mothers, which might have led to abortion of many uninfected fetuses.12-14 The low avidity of immunoglobulin G (IgG) has been reported to be a useful marker of recent infection with Toxoplasma. Nevertheless, discrepancy results in the maturation of avidity over time have been reported. Alpha fetoprotein (AFP) is a protein normally produced by the liver and yolk sac of a developing baby during pregnancy. AFP levels decrease soon after the birth. AFP probably has no normal function in adults. The normal values in males or no pregnant females are generally less than 40 micrograms/liter. The examples above are common measurements for results of these tests. Normal value ranges may vary slightly among different laboratories.15-17 Greater than normal levels of AFP may be due to: Birth defections, including: Anencephaly; Duodenal atresia; Gastroschisis; Omphalocele; Spina bifida; Tetralogy of Fallot; Turner syndrome Controlling the albumin and a-fetoprotein (AFP)’ synthesis in the mammalian liver provides a powerful model system for investigating the molecular mechanisms of gene regulation during normal development and oncogenesis. The plasma levels of these two proteins exhibit a reciprocal relationship during development.15-17 AFP is dominant in the plasma of the developing fetus where it is synthesized by the yolk sac and liver. Its concentration is drastically reduced after the birth in rodents to reach a basal level in adult animals. However, re-expression of this protein occurs in cases of liver neoplastic and germinal tumors.15-17 The aim of this study was to evaluate of serum concentration of AFP in toxoplasmosis pregnant women with high level of IgG and IgM toxoplasma antibody by ELISA assay.
This research was a cross sectional study. In this study, it was randomly collected 255 serum samples from toxoplasmosis pregnant women from Avecina Center, Tehran, Iran. They had high levels of serum IgG and IgM antibody against toxoplasma gondii parasite. This study was based on comparative study. The mothers that were chosen were patients who had one or more abortion in their life. It was followed up these mothers’ disease process to understand the cause of their children abortion. All of mothers had high levels of serum IgG and IgM antibody against toxoplasma gondii parasite and about 13% of these mothers had high serum levels of AFP marker and the child’ of these patients had suffered from encephalitis disorders with attention of their radiography experiments. All of 255 toxoplasmosis mothers were selected from Avesina laboratory center and were sent to radiography test and other experiments on NTD disorders. It was divided all of infected pregnant women to 13 groups that in groups 3 and 10 these mothers had high levels of AFP markers. The main procedure was
ELISA assay to detect AFP marker in toxoplasmosis pregnant women. Because of sampling in equal conditions at the same time and devoting aborted 20 mothers in each group we divided all of the infected pregnant women to 13 groups. Materials used in this study were: Two hundred and fifty five serum samples from toxoplasmosis pregnant women; ELISA kit to detect IgG and IgM antibody against toxoplasma gondii parasite (This method was gold standard test); ELISA kit to detect AFP marker in toxoplasmosis pregnant women; ELISA washer device to wash ELISA plates to discard unwanted proteins and antibodies from ELISA wells; ELISA reader devices with 450 nm wavelength to measure OD of each ELISA well. With measurement of OD from each ELISA well, it was evaluated the precise concentration of each antibody or marker such as IgM or IgG or AFP marker; SPSS software for analyzing data. ELISA kit contents: 1. Six standard of IgG, IgM or AFP marker to delineated curve of quantitative ELISA assay; 2. Assay buffer or diluents buffer to dilute 125 serum samples; 3. Wash buffer to wash ELISA wells; 4. Enzyme conjugates solution to trace IgG, IgM or AFP marker; 5. TMB solution as substrate of ELISA assay; 6. Stop solution to halt total ELISA reaction. Method to detect IgG and IgM toxoplasma antibody: 1. We added 100λ of serum or amniotic fluid to ELISA avidity wells; 2. Incubated what? For 30 minutes in 370C; 3. We washed plates for 3 times with PBS (ELISA Washer); 4. We added 100λ enzyme conjugate to these plates; 5. Incubated for 30 minutes in 370C; 6. We added 100λ substrate (TMB); 7. After 15 minutes we added stop solution and read it at 450nm ELISA reader. Method to detect AFP marker: 1. To select appropriate wells; 2. To pour 50 λ standards, control serum and samples into wells; 3. To pour 100 λ assay buffer into
wells; 4. To shake plate gently and incubate it in room temperature until 30 minutes; 5. To discard every content of plate and wash 5 times by washing buffer; 6. To add 100 λ enzyme conjugate to each wells and incubate 30 minutes in room temperature; 7. To discard every content of plate and wash 5 times by washing buffer; 8. To add 100 λ TMB into each wells; 9. To incubate plate 15 minutes in room temperature at dark condition; 10. To add 100 λ stop solution into each wells and read on 450nm wavelength by ELISA reader device. It was suggested to read this plate on 630 nm wavelength such as reference filter. Before starting this procedure, it was reached any sample and standards to room temperature and shakes them gently. Total experiment should be done without stopping. It was used disposable sampler tip to achieved good results. After adding stop solution, it should be read OD each well until 30 minutes. In each time of washing procedure it was added about 300 λ of washing buffer to achieve good results. The important factor to achieve proper results is incubation time.
It was divided total toxoplasmosis mothers into 13 groups (AFP1 to AFP 13). The statistical surveillances were done by SPSS software consisted of t-test, case number, comparative study, and Q-Q plot evaluations. In this table it was showed that in the group 3 and 10 the toxoplasmosis pregnant women had high level of AFP marker and in these mothers, their fetus had any kind of encephalitis in radiography experiments. The normal range of AFP in serum was 0.2 to 8.5 ng/ml. It was detected toxoplasma gondii IgG and IgM antibody by fresh designed ELISA method and it was detected acute toxoplasmosis by ELISA avidity IgG antibody assay.
Table 1: T-test analyses infected pregnant women for AFP serum levels
Groups of infected mothers
t Df Mean Difference
95% Confidence Interval of the Difference Lower Upper AFP1 9.153 19 2.69400 2.0780 3.3100 AFP2 16.448 19 2.70000 2.3564 3.0436 AFP3 25.192 19 101.60000 93.1588 110.0412 AFP4 9.159 19 2.99400 2.3098 3.6782 AFP5 13.199 19 3.37550 2.8402 3.9108 AFP6 10.443 19 3.49100 2.7913 4.1907 AFP7 11.453 19 3.25300 2.6585 3.8475 AFP8 12.203 19 2.46500 2.0422 2.8878 AFP9 12.260 19 2.74500 2.2764 3.2136 AFP10 20.642 14 86.37333 77.3988 95.3479 AFP11 11.917 19 3.03500 2.5020 3.5680 AFP12 14.498 19 3.40150 2.9104 3.8926 AFP13 13.552 19 2.90000 2.4521 3.3479
In the table 1, it was showed that the t-test analyzes the serum levels of AFP for toxoplasmosis pregnant women. In this table, it was showed that in the group 3 with 95% Confidence Interval of the difference the serum levels of AFP were between 93 to
110 ng/ml and in the group 10 with 95% Confidence Interval of the difference the serum levels of AFP were between 77 to 95 ng/ml and in these toxoplasmosis pregnant women the encephalitis symptoms in the fetus were detected by radiology tests.
Figure 1: Case number histogram to show serum concentration of AFP IN group 3 infected mothers
In the figure 1 (two lines that are above of other lines), it was showed that in the groups 3 and 10 (toxoplasmosis pregnant women), the serum levels of AFP were in a higher levels than in the other groups and in these groups the encephalitis symptoms in the fetus were detected by radiology tests.
Figure 2: Comparative study between 13 groups of toxoplasmosis pregnant women by AFP evaluation
In the figure 2, it was showed that (with 95% Confidence Interval) the groups that had high levels of AFP were groups 3 and
10 that in these groups of toxoplasmosis pregnant women the NTD symptoms in their fetus were detected by radiology tests.
Figure 3: Q-Q plot of AFP3
In the figure 3 it was showed that in AFP3 toxoplasmosis pregnant women the mean range of serum AFP was between 80 to 120 ng/ml.
The AFP test is done to: Check the developing baby (fetus) of a pregnant woman for brain or spinal problems (called neural tube defects). Such defects occur in about 2 out of every 1,000 pregnancies. The chance of a neural tube defect in a baby is not related to the mother's age. Most women whose babies have neural tube defects had no family history of these problems such as toxoplasmosis.18-20 AFP plus IgM and IgG detection in toxoplasmosis mothers are very important factors in cerebral toxoplasmosis. 21-23 Our objective was evaluation of serum concentration of AFP in toxoplasmosis pregnant women with high level of IgG and IgM toxoplasma antibody by ELISA assay. Toxoplasmosis is a very common disease in the world. Two types of acquired toxoplasmosis are detected; 1) In the chronic toxoplasmosis, the abnormality of liver function is little but, 2) In acute congenital toxoplasmosis liver function became suppressed and the assessment of liver transaminases in this stage was very important.24-26 Prevention of congenital toxoplasmosis in pregnant women has been mainly based on serological tests for anti-Toxoplasma antibodies. Many serological tests, including the haemagglutination test, latex agglutination test (LAT), ELISA, and indirect fluorescence antibody test, have been utilized in the detection of antibodies against T. gondii. There have been several reports regarding the screening of anti-T. gondii antibodies among Koreans. Ryu et al. (1996) demonstrated 4.3% and 0.94% of positive rates, using ELISA and LAT kits among pregnant women who visited medical institutes in Yangpyong-gun and Kwangju-gun of Kyonggi-do. Distinguishing of acute and chronic phases of toxoplasmosis has critical importance in pregnant women and immuno- compromised patients. Some assays have been used to measure Toxoplasma specific IgM antibodies as indicators of the acute phase infection. Toxoplasma IgM antibodies can be detected for 1 year or longer in some cases. So, in asymptomatic individuals with stable titers of Toxoplasma IgG antibodies, positive IgM results are not easy to interpret and in acute phase of toxoplasmosis NK cells also increased that is specific factor.27-28 Han M and et al. showed that under the situation of T. gondii-infected dNK cells co-cultured with trophoblast cells, the up-regulation of sHLA-G could induce dNK cells apoptosis which ultimately may contribute to the abnormal pregnancy outcomes with T. gondii infection. Fan CK and et al. showed that Parents' educational level and cats kept indoors seemed to be the high risk factors for PSC in acquisition of T. gondii infection. While, ocular manifestation and/or headache of PSC should be checked for the possibility of being T. gondii elicited. Measures such as improving environmental hygiene and intensive educational intervention to both PSC and their parents should be performed immediately so as to reduce T. gondii infection of DRSTP inhabitants including PSC and adults. Rojas Y and etc showed that using serial serum AFP levels as the preferred method of surveillance in children with AFP-positive hepatoblastoma, reserving imaging for the early postoperative period, for children at high risk of relapse, and for determination of the anatomical site of clinically suspected recurrence. Given the small size of this preliminary study, validation in a larger patient population is
warranted. Carr BI and et al. showed that Patients with low AFP were the majority in this cohort, and patients with elevated GGTP had worse prognosis than those with low GGTP. GGTP may be a useful tumor and prognosis marker in low-AFP patients. AFP- negative patients are important to identify due to their enhanced survival.27-30 AFP is a glycoprotein molecule with 70000 D molecular weight that resembles to human albumin that both of them code on chromosome 4. AFP is the major protein in blood circulation of fetus that synthesizes primarily by yolk sac and then by liver. AFP concentration reaches to maximum level at 9th weeks of pregnancy in blood circulation of fetus (3000000 ng per ml). Then, gradually it decreases and becomes 20000 ng per ml in final period of pregnancy. AFP transmits from pregnant woman to her fetus and its level becomes high in serum of pregnant woman at 3th months of pregnancy.29.30. AFP concentration reaches to 5 ng per ml in pregnant woman at 10th weeks of pregnancy and these concentration increases 15% for each week that its level becomes 180 ng per ml at 25th weeks of pregnancy and its level reaches to 2ng per ml after delivery. AFP penetrates to amniotic fluid and its concentration reaches to 15000 ng per ml at 16th weeks of pregnancy. AFP levels increase in many disorders such as hepatocellular carcinoma, testis tumors and in adults. Its levels increase in hepatoblastoma and nephroblastoma tumors in children. Its level increases in metastatic malignancies from intestine to liver. Nowadays the evaluation of AFP serum levels in pregnant women are used to detect NTD disorders such as congenital toxoplasmosis in fetus.29,30
CONFLICT OF INTEREST
The authors declare that they have no conflict of interests.
This study was financially supported by Tarbiat Modares University. The authors wish to thank Dr Marsusi from Medical University of Tehran.