|Year : 2010 | Volume
| Issue : 2 | Page : 143-146
Serological screening for antenatal toxoplasma infection in India
S Khurana, R Bagga, A Aggarwal, V Lyngdoh, Shivapriya, K Diddi, N Malla
Department of Parasitology, Research Block -A, PGIMER, Chandigarh - 160 012, India
|Date of Submission||03-Jul-2009|
|Date of Acceptance||14-Dec-2009|
|Date of Web Publication||16-Apr-2010|
Department of Parasitology, Research Block -A, PGIMER, Chandigarh - 160 012
Purpose: Detection of infection caused by Toxoplasma gondii during pregnancy to prevent congenital infection. Materials and Methods: This study was carried out from January 2005 to 2006 in 300 pregnant women. Antitoxoplasma IgG, IgM, IgA antibody and IgG avidity were assessed using ELISA. Atleast two samples were taken atleast 3 weeks apart preferably one in each trimester. Result: Of these 300 pregnant women, anti toxoplasma IgG antibodies were detected in 46 (15.33%) cases, while 9 (3%) had positive anti toxoplasma IgM with IgA and /low IgG avidity antibodies suggestive of acute infection during or just before pregnancy. Conclusion: The results indicate that about 85% of female population of Chandigarh is susceptible to toxoplasma infection and thus should be specifically educated about prevention of this infection during pregnancy
Keywords: Toxoplasma, avidity, serology, pregnancy, women
|How to cite this article:|
Khurana S, Bagga R, Aggarwal A, Lyngdoh V, S, Diddi K, Malla N. Serological screening for antenatal toxoplasma infection in India. Indian J Med Microbiol 2010;28:143-6
|How to cite this URL:|
Khurana S, Bagga R, Aggarwal A, Lyngdoh V, S, Diddi K, Malla N. Serological screening for antenatal toxoplasma infection in India. Indian J Med Microbiol [serial online] 2010 [cited 2013 May 22];28:143-6. Available from: http://www.ijmm.org/text.asp?2010/28/2/143/62492
| ~ Introduction|| |
Toxoplasma gondii, the causative agent of toxoplasmosis can cause significant morbidity and mortality in the developing foetus if the mother acquires acute infection during pregnancy.  Transmission rate to the foetus increases from 15-65% with increasing gestational age.  However, the severity of congenital disease decreases with increasing gestational age. The spectrum of outcome of congenital toxoplasmosis ranges from death in utero, chorioretinitis and/or hydrocephalus with mental impairment, or a healthy infant without clinical signs of toxoplasmosis. The symptoms may be present at birth or develop later in life, leading to blindness, psychomotor retardation, and hearing difficulties. ,, The risk of toxoplasma seroconversion in pregnant mothers has been reported to be 2.2 times higher than in women not pregnant, in the same age group and risk increased to 7.7 in adolescents (12 to 20 years old). 
On account of the diversity or absence of symptoms, the detection of toxoplasma infection during pregnancy is usually made by various serological procedures.  The antibody result from a single serum sample gives no clear indication about the time of occurrence of the infection. A four-fold rise in titre of IgG between two or more samples in the same test run; presence of low avidity IgG; presence of IgM and/ or IgA antibody are required to support the diagnosis of acute toxoplasmosis. However, a high percentage of false positive reactions by the use of IgM and IgA ELISA kits have been reported.  Avidity of IgG antibodies against T. gondii thus, is a useful marker to differentiate between recent and remote infection.  Seroprevalence average of T. gondii infection in India has been reported to be 24.3%, lowest being in the northern parts of India, and highest in the south and seroconversion rate of 1.5% has been reported.  The seroprevalence of toxoplasma in Chandigarh area has been reported to be about 5% with more women showing seropositivity than males.  Moreover, since only the acute infection during pregnancy is associated with congenital infections, it is important to know the incidence of acute infection during pregnancy. Therefore, strategies for early recognition of maternal infection and the institution of adequate treatment during pregnancy could have a substantial impact on the incidence and neonatal mortality that are associated with congenital infection.
There are only a handful of studies available from India that have estimated the incidence of acute infection during pregnancy , This study aims to determine prevalence and incidence of T. gondii infection among pregnant women and assess the association between the seroprevalence and selected variables of interest.
| ~ Materials and Methods|| |
Study population: A total of 300 pregnant women were enrolled in the study; 200 of them were attending the Antenatal clinic of Department of Obstetrics and Gynaecology, and 100 resided in rural areas, which were followed up by house-to-house visits. The subjects were enrolled at random after taking their due consent. Detailed relevant information regarding the age, eating habits like vegetarian/ non-vegetarian, contact with cats and socioeconomic status were recorded in a preplanned proforma. The obstetric details of the period of gestation and expected date of delivery were included. The outcome of delivery was recorded, wherever available. The patients were educated about the Toxoplasma infections during pregnancy, its implications and importance of testing through oral and written material. The patients were given appropriate therapy whenever acute infection was established.
Sample collection: Two ml of blood was collected by venepuncture from each subject at first time of enrolment (during first trimester) and subsequently second sample was collected, either after three weeks of the first sample or in II and/or III trimester of pregnancy, depending on feasibility. Sera were stored at -20°C till use. Each sample was tested for IgG and IgM antibodies and those found positive in any test were further tested for IgA and IgG avidity.
Enzyme Linked Immunosorbent Assay (ELISA)
Preparation of Antigen: Crude, sonicated soluble antigen was prepared from the T. gondii (RH strain) being maintained in our department. The T. gondii tachyzoites were harvested from the peritoneal cavity of six Swiss albino mice which were infected three days earlier by intraperitoneal route (1 x 10 4 tachyzoites in 0.5 ml). After purification of tachyzoites, the antigen was prepared by sonication at about 20 kilocycles per second in pulses of 20 seconds each for total time of 10 minutes. Then the sonicated material was subjected to cold centrifugation at 4 0 C at about 800xg for about 20 minutes to remove the cellular debris.  The final supernatant was taken as crude soluble Toxoplasma antigen, and its protein concentration was estimated by the Biuret method.
Antibody detection by ELISA
IgG/IgM antibodies: An in-house ELISA for anti-Toxoplasma IgG and IgM antibody detection was carried out as described earlier.  Optimum dilutions of the antigen, patients and control sera and the conjugates were determined by checker board titrations. The optimum concentration of the Toxoplasma antigen for coating of plates for both IgG and IgM ELISA was 1 ìg per well. The optimum dilutions of the anti-IgG and IgM enzyme-labelled conjugates (Sigma Aldrich Chemicals, USA) were 1 in 30,000 and 1 in 10000 respectively. The optimum dilution of serum for IgM and IgG ELISA was found to be 1:800. The optical density values were recorded in an ELISA reader. The cut off O.D value was calculated as mean + 2 S.D of five negative control samples from age and sex matched subjects and samples with OD value ≥ 2 S.D were considered positive. The samples which had detectable IgG/IgM antibodies or both were further subjected to IgA and IgG avidity testing.
IgA Antibodies : A commercial ELISA kit (Meddens diagnostics, Groningen, Netherlands) was used for antitoxoplasma IgA antibody detection as per manufacturer's instructions.
IgG Avidity ELISA: IgG avidity ELISA, performed as an in-house test and is standardized in our lab. This test is based on the principle of avidity of IgG antibodies formed during recent acute infections; it is low and is tested by calculating the difference between the absorbance values due to antibody binding in the absence and presence of a urea solution. The optical density (O.D.) at 405 nm was measured with an automatic microplate spectrophotometer (Bio-Tek Instruments Inc., Vermont, USA). The IgG avidity index (AI) was calculated as the ratio between the O.D. of the two wells [for the sample washed with the dissociating reagent (6 Molar urea solution in a buffer) and the O.D. for the sample washed with a standard washing solution (PBS-Tween 20 buffer)], and then it was expressed as percent avidity. An AI of >30% was considered an indicator of IgG anti-Toxoplasma with high avidity, an AI of < 20% indicated low avidity, and an AI between 21 and 30% suggested borderline avidity (grey zone), respectively and was considered inconclusive. 
The descriptive data was given as mean ± standard deviation. The Chi-square test was used for the analytical assessment. The differences were considered to be statistically significant when the P value obtained was less than 0.05.
Ethical consideration: The project was approved by the Institutional Ethical Committee.
| ~ Results|| |
The results of our study are shown in [Table 1]. In our study, 46 (15.33%) out of 300 pregnant women, were positive for antitoxoplasma IgG antibodies and 15 were positive for IgM antibodies only. However, only nine i.e. 3% (or 19.57% out of these 46 patients) were IgM positive along with positive IgA /low IgG avidity, showing evidence of acute infection during pregnancy. The results are presented in [Table 1]. Acute infection during pregnancy was diagnosed if IgM positivity was associated with either low avidity IgG or IgA positivity or both. The samples that showed only IgM positivity in absence of either IgA or low avidity IgG antibodies were not diagnosed with acute infection. The mean age of the infected patients was 24.13 years. To determine the influence of age, the patients were divided into four age groups. Most of the women belonged to age group 25-29 years, but acute infection was present in 80% of <20 years of age women. Seropositivity was also analyzed with respect to parity. Acute infection was most commonly seen in primigravida (31.25%). History of animal contacts (P = 1.0), residence (P = 0.7) and diet pattern (P = 0.7) did not have any significant bearing on seroprevalence and acute infection.
There was a significant difference in the seropositivity in urban vs. rural areas [24 (12%) vs. 22 (22%); P < 0.05]. However, no difference was observed in the incidence of acute infections during pregnancy [5 (2.5%) in urban vs. 4 (4%) in rural; P > 0.05]
The outcome of infection was not known in all the cases since many of the women went to their maternal house for delivery. Eight out of 300 seronegative women enrolled in the study spontaneously aborted mostly in first or second trimester. However, out of nine acutely infected women, two aborted spontaneously in the late first or early second trimester and Medical Termination of Pregnancy (MTP) was done on one woman at 20 weeks of gestation due to major congenital abnormalities in multiple organs including hydrocephaly, ascites and lesions in brain of the foetus.
| ~ Discussion|| |
Congenital, intra uterine infections are often the cause of congenital abnormalities, intra uterine growth deficiencies and foetal death, resulting in both economic and social concerns. In North India, the overall seropositivity for T. gondii antibodies has been reported to vary from 5 to 46.7%. [7,,,, and even higher in Kumaon region. 
In the present study, a seroprevalence of 15.33%was reported in pregnant women which is lower than the rates previously reported from other parts of India. ,,,, This may be because the women in this region are more educated, sanitation and hygiene are better and women are not commonly involved in farming unlike others parts of North India from where very high seroprevalence rates of 77% were reported.  An acute infection during pregnancy was documented in 3% of pregnant women. A high prevalence of toxoplasma infection has typically been associated with warm and humid environments, contaminated water supplies, poor cooking habits, lack of hygiene, and contact with cats. , Results of our study did not show any significant difference in seropositivity with eating habits or contact with pets. Systematic screening programs in countries such as France and Austria, where serological surveillance of seronegative pregnant women for seroconversion is compulsory, have indicated that 1 to 1.5 per, 1,000 newborns suffer from congenital toxoplasmosis in France, while in USA, the incidence of acute Toxoplasma infection during pregnancy has been estimated at about 0.2 to 1% and incidence of congenital infection ranging from 1 in 1000 to 1 in 8000 live births. , In some European countries, where educational measures have been incorporated into routine obstetric care, reductions in incidence of infection by 50% have been reported. Increased awareness and the education at patient and government level is required, with respect to eating adequately cooked meat, keeping pets, hygienic vegetables preparation and hand washing and good quality water supply may lead to a decrease in the infection of toxoplasmosis.  Moreover, early diagnosis during pregnancy is important as transmission to the foetus in utero can be prevented by appropriate treatment. So screening of pregnant women for anti toxoplasma antibody and to educate them about source of transmission and preventive measures for disease is very important.
In a study conducted by Carter et al. in Ottawa, a 10-minute audiovisual educational program, together with a three-page handout focusing on our current knowledge of congenital Toxoplasma infection, effect of foetal infection, and prevention of infection - was effective in modifying the behaviour of pregnant women with regards to cats, food preparation, and personal hygiene.  Maria et al., 2004, in an epidemiological survey over 22 years, reported that an educational program on primary prevention reduced the rate of T. gondii seroconversion among pregnant women by 60%.  In our study too women were educated about the toxoplasmosis and importance for its testing especially in IgG.
Therefore, based on the results of our study, it is recommended that the pregnant women should be screened and educated concerning the risk factors that contribute to toxoplasma infections, and the importance of taking preventive measures.
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