Electrochemiluminesence Epidemiologic Detection of Toxoplasma gondii Infection in Pregnant Women With Direct and Indirect Diagnostic Techniques ( ELISA Avidity Plus Biochemical Assay )

Background and aims: Toxoplasmosis is a very common disease in the world. Two types of acquired toxoplasmosis have been detected. In the chronic toxoplasmosis, the abnormality of tissue function is little but, in acute toxoplasmosis, function of RES system becomes interrupted. The assessment of toxoplasma antibody with tissue enzymes in this stage is very important. Furthermore, serum ferritin in some conditions became high in acute phase of infectious disease. Methods: This study was based on comparative abundance study. Materials consisted of 980 serum and amniotic fluid samples collected from human blood with high level of IgG antibody against Toxoplasma gondii in Rajaie center, Tehran, Iran. The standard test was ELISA assay to detect these antibodies and the main test was measurement of liver transaminases (SGOT, SGPT) bilirubin and ferritin to evaluate acute toxoplasmosis. Data analysis was done by SPSS version 18.0. This study was done during March to June, 2017. Results: Results showed that in some patients with high level of IgG AVIDITY antibody against T. gondii the level of liver transaminases, serum bilirubin and ferritin became increased. For example in 120 patients with acute toxoplasmosis the mean serum levels of SGOT were 108 IU/L and in 80 patients the mean serum bilirubin were about 5 mg/dL. Conclusion: In acute congenital toxoplasmosis, the evaluation of IgG AVIDITY was first step and then the measurement of biochemical factors such as serum transaminases, serum Bilirubin and serum Ferritin were important.


Introduction
Toxoplasma gondii is an intracellular parasite of many kinds of tissue, including muscle, liver, and intestinal epithelium.In heavy acute infections, the organism can be found free in the blood and peritoneal exudates.It may inhabit in nucleus of host cell but usually lives in the cytoplasm.The life cycle includes intestinal-epithelial and extra intestinal stages in domestic cats and other felines but only extra intestinal stages in other hosts such as human.Seropositivity to toxoplasma was comparable among liver disease patients and those in control group.Raw shellfish consumption and domestic cat exposure are risk factors for acquiring acute T. gondii diseases.][3] In spite of the fact that antibody to toxoplasma is widely prevalent in hosts throughout the world, the clinical toxoplasmosis is less common.A general use of the IgG avidity toxoplasma test can diagnose IgG positive in congenital toxoplasmosis.It is clear that most infections are asymptomatic or mild.In 1976, the global prevalence of toxoplasmosis was estimated over 500 million.In acute phase, tachyzoites proliferate in many tissues and this rapid reproduction tends to kill host cells at a faster rate than what the normal turnover of such cells does.As infections become chronic, the zoites infecting brain, Bahadory et al heart, liver, and skeletal muscles are multiplied much more slowly than in the acute phase.5][6][7] In many immune suppressed hosts, T. gondii became acute.Acute toxoplasmosis may lead to congenital toxoplasmosis with fetal complications results during pregnancy.[10] Toxoplasma gondii is one of the infectious agents of congenital TORCH infections and causes severe clinical outcomes in fetuses and newborns.Infection with T. gondii during pregnancy can lead to severe fetal squeal.Moreover, ocular toxoplasmosis is a disease caused by the infection by T. gondii through congenital or acquired routes.The different laboratory methods used for diagnosing the congenital toxoplasmosis have variable sensitivity and specificity levels such as IgM Western blotting (WB), IgA enzyme immunoassay (EIA), and DNA amplification by real-time polymerase chain reaction (PCR).In acute stage of toxoplasmosis, the level of IgG avidity antibody is high and in these conditions the level of tissue enzymes such as transaminases (SGOT, SGPT), serum bilirubin, and ferritin became high.In addition, total malfunction of RES system was observed.[13][14][15]

Objective
The purpose of this survey was to evaluate the biochemical factors using electrochemiluminescence and ELISA AVIDITY test in patients with acute congenital toxoplasmosis (IgG avidity positive) in an Iranian population.

Methods
The study was based on comparative study.Samples were serum and amniotic fluid specimens collected from 980 patients with high level of IgG avidity antibody against T. gondii in Rajaie center, Tehran, Iran.The gold standard test was ELISA assay to detect IgG avidity toxoplasma antibody.These patients have positive ELISA titer for toxoplasma antibody measured by laboratory technicians in Rajaie clinical laboratory.The main test to detect serum liver transaminase and bilirubin was based on electrochemiluminescence assay and measurement of serum ferritin was based on ELISA assay.
ELISA Method to Detect Toxoplasma Antibody or Serum Ferritin 1. 100 λ of blood serum, amniotic fluid sample, or calibrator 2 was added to ELISA avidity wells.2. Samples incubated for 30 minutes at 37 º C.
After 15 minutes, the added solution stopped, then read at 450 nm ELISA reader.The formula of ELISA avidity (semi-quantitative): OD patients serum or amniotic samples OD calibrator 2 The normal range is <0.8 but the result >1.1 is positive for congenital toxoplasmosis.
Formula of SGPT measurement: The reagent 1 to measure SGPT consists of TRIS buffer, l alanine, and LDH.The reagent 2 to measure SGPT consists of 2-oxoglutarate and NADH.
The reagent 2 to measure SGOT consists of 2-oxoglutrate and NADH.
In both measurements, we mixed 100 λ human serum + 1000 λ reagent 1 and after 5 minutes, we added 250 λ reagent 2, then read OD of reaction tubes against blank tube at 340 λ wave-length.The normal range (activity) of SGOT and SGPT in this study was: <31 U/L.The measurement of serum bilirubin based on photometric study by auto analyzer Cobas e411 electrochemiluminescence instrument and measurement of serum ferritin were done by ELISA method.
This study was a comparative study based on studies during March to June 2017.The calculation of results performed by SPSS software version 9.0.

Results
Results showed that in some patients with high level of IgG AVIDITY antibody against T. gondii, the level of liver transaminases, serum bilirubin, and ferritin increased.For example, in 120 patients with acute toxoplasmosis, the mean serum level of SGOT was 108 IU/L and in 80 patients, the mean serum bilirubin was about 5 mg/dL.
Figure 1 indicates that in 980 toxoplasmosis pregnant women, the mean of ELISA AVIDITY titer was higher in serum sample than in amniotic fluid sample.Moreover, in each abortion, the mean of ELISA AVIDITY titer became high.For example in mothers with fourth abortion, the mean of ELISA AVIDITY titer was higher than in mothers with first abortion.
In Table 1, it is shown that in 980 patients with acute toxoplasmosis, the serum levels of bilirubin with 95% CI of the difference were between 1.1 until 1.7 mg/dL which are normal range.
Figure 2A shows that in 980 patients with acute toxoplasmosis, only 12% of patients had high level of serum SGOT.
In Figure 2B, it is indicated that in 980 patients with acute toxoplasmosis, only 14% of patients had high level of serum SGPT.
Figure 2C shows that in 980 patients with acute toxoplasmosis, only 8% of patients had high level of serum bilirubin.
Figure 2D illustrates that in 980 patients with acute toxoplasmosis, only 14% of patients had high level of serum ferritin.
In Table 2, it is clear that in 980 patients with acute congenital toxoplasmosis, the minimum level of serum  Ferritin, for example, was 12 mg/dL and the maximum level of serum Ferritin was 994 mg/dL.The biochemical assay was based on electrochemiluminescence assay.

Discussion
Toxoplasmosis, one of the TORCH's infections in pregnant women, is caused by Toxoplasma gondii.The seroprevalence of Toxoplasmosis among pregnant women was found to be comparatively high compared with previous reports from Saudi Arabia, for instance, or the prevalence of Toxoplasma IgG antibodies in pregnant women was low in Sicilian population.The clinical spectrum of T. gondii infection varies from an asymptomatic state to a severe illness.The preliminary diagnostic utility of 2 mixtures of T. gondii recombinant antigens in IgG ELISA and IgG avidity test has been evaluated.In addition, an epidemiological study has reported an association of T. gondii infection with liver cirrhosis.Therefore, the high positive results should be treated with some skepticism until additional precise diagnostic tools are developed.][18][19][20] In a recent study in the United States, elevated risk of recent T. gondii infection was associated with eating rare lamb.Even frozen lamb has been associated with acute T. gondii infection in Brazil.Interestingly, toxoplasmosis with liver involvement has been reported in deer hunters who had eaten undercooked venison.Under normal immune conditions, Toxoplasma infection is largely asymptomatic, but in those individuals who are immunecompromised, such as individuals with AIDS, malignant patient under chemotherapy, or organ transplant recipients, the parasite can become widely disseminated, causing severe toxoplasmosis and/or encephalitis.Also the prevalence of acute T. gondii infection is high among children with non-specific reactive hyperplasia of the cervical lymph nodes.][23][24][25] Sometimes, congenital toxoplasmosis received from undercooked meat.In spite of the fact that antibody to Toxoplasma is widely prevalent in human throughout the world, clinical toxoplasmosis is less common.The occurrence of congenital toxoplasmosis is still a problem.It is clear that most infections are asymptomatic or mild.Several factors influence this phenomenon: the virulence of the strain of Toxoplasma, the susceptibility of the individual host species, the age of the host, and the degree of the acquired immunity of the host.Occasionally, circumstances conspire to make a mild case important.For example, Elahain et al suggested a significant relationship between the age of the youngest child and the infection rate.Since there seems to be an age resistance, influence of adults or weaned juveniles are asymptomatic, although some exceptions occur.Asymptomatic infections can suddenly become fulminating if immunosuppressive drugs such as corticosteroids are employed for other conditions.The first extra intestinal sites to be infected in both cats and other hosts (humans) are the mesenteric lymph nodes and the parenchyma of the liver.These sites, experience rapid regeneration of cells too and perform an effective preliminary screening of the parasites.The most common symptom of acute toxoplasmosis is painful, swollen lymph glands in the cervical and abnormality in the RES system.In chronic phase, the cyst of Toxoplasma can remain intact for years and produce no obvious clinical effect.The larger number of acute toxoplasmosis (about 70%) is asymptomatic.Mumcuoglu et al, 24 suggested T. gondii IgM, IgG, and IgG avidity tests were performed by VIDAS automated analyzer using TOXO IgM, TOXO IgG II, and TOXO IgG avidity kits.In this study, we showed that in most asymptomatic acute toxoplasmosis patients with good immune system, the biochemical factors such as serum SGOT, serum SGPT, serum bilirubin, and serum Ferritin had different levels and required further study such as ELISA AVIDITY test.][28][29][30][31][32][33] In Iran there are no study about biochemical factors in discrimination of acute and chronic toxoplasmosis but in this study, we found many differences in biochemical factors of acute and chronic toxoplasmosis.

Table 1 .
One Sample T-test From Toxoplasmosis Patients

Table 2 .
Descriptive Statistics of Biochemical Index in Acute Toxoplasmosis Patients