|Year : 2001 | Volume
| Issue : 3 | Page : 145-148
A new approach to differentiate recent vs chronic toxoplasma infection: Avidity elisa in toxoplasma serology
P Yasodhara, BA Ramalakshmi, M KJ Sarma
National Institute of Nutrition, Indian Council of Medical Research, Jamai Osmania PO, Hyderabad - 500 007, India
National Institute of Nutrition, Indian Council of Medical Research, Jamai Osmania PO, Hyderabad - 500 007, India
The diagnosis of toxoplamosis during pregnancy is based on maternal serology, due to the asymptomatic nature of the disease. Detection of specific IgM although is the method used all over the world to detect acute infection, persistence of IgM for long periods poses problems in distinguishing acute from chronic infection, which is of crucial importance in pregnancy. Avidity ELISA is a method recently developed to distinguish IgG antibodies developed at an early stage of infection from those that reflect past immunity. The avidity assay uses protein-denaturing agents and is a modification of an ELISA. The usefulness of this technique was tested on sera of 113 pregnant women screened for Toxoplasma specific IgG/IgM antibodies. Nine of the sixteen sera positive for IgM/IgG antibodies and three sera positive for IgG alone were subjected to avidity ELISA. Only three sera were positive for low avidity IgG indicative of recent infection. All the three sera positive for IgG alone showed high avidity.
|How to cite this article:|
Yasodhara P, Ramalakshmi B A, Sarma M K. A new approach to differentiate recent vs chronic toxoplasma infection: Avidity elisa in toxoplasma serology. Indian J Med Microbiol 2001;19:145-8
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Yasodhara P, Ramalakshmi B A, Sarma M K. A new approach to differentiate recent vs chronic toxoplasma infection: Avidity elisa in toxoplasma serology. Indian J Med Microbiol [serial online] 2001 [cited 2014 Apr 19];19:145-8. Available from: http://www.ijmm.org/text.asp?2001/19/3/145/8149
Primary Toxoplasma infection during pregnancy may lead to transmission to the fetus and cause damage. Detection of infection and antiparasitic treatment during pregnancy may prevent fetal infection and damage.,
The disease being often subclinical, diagnosis is by demonstration of specific antibodies. Detection of seroconversion from serial sampling, presence of specific IgM/IgA antibodies are the methods being presently used. However persistence of specific IgM/IgA for long periods and also high dye test values (formerly considered gold standard) pose problems in the diagnosis of primary infection which is so crucial during pregnancy. Assessment of the exact time of primary infection is important, especially in a pregnant woman.
A new uprising principle in clinical microbiology is the measurement of binding of specific IgG (called IgG avidity) to multivalent Toxoplasma antigen. Recently it has been shown that specific IgG response after primary infection consists of low avidity antibodies with maturation of avidity in few months., Earlier low avidity antibody denaturation techniques have been applied effectively for distinguishing recent primary infection from preexisting rubella immunity. The method was later developed for the diagnosis of primary Toxoplasma infection. Subsequently, several investigators have supported the usefulness of this technique in the diagnosis of primary infections for several infectious diseases including Herpes Simplex Virus (HSV), Varicella Zoster Virus (VZV), Cytomegalovirus (CMV) and Epstein-Barr virus(EBV).,,,, In the present paper, we report the usefulness of this technique tested in pregnant women.
| ~ Material and Methods|| |
Sera of 113 pregnant women belonging to low socio-economic group (LSE) attending a local maternity hospital during February, 1997 - May 1997, were screened for Toxoplasma specific IgG/IgM using commercial diagnostic kits (Diamedix, USA). Nine Sera positive for IgG and IgM, and three sera positive for IgG alone were further subjected to avidity ELISA using Avidity ELISA kits, Labsystems, Finland. Acute, borderline and chronic Toxoplasmosis sera (based on avidity percentages) and the avidity ELISA kit were supplied by Dr.Hedman, through Labsystems, Finland.
Toxoplama specific IgG - Avidity test:
The assay was performed as described by Hedman et al using IgG avidity kits supplied by Labsystems, Finland.
Principle of the test:
The differentiation between recent and preexisting infections is one of the greatest challenges encountered in microbiology. To date, the most common means of diagnosis has been the determination of specific IgM antibodies, which in general appear during the initial phase but whose determination is often unreliable and problematic.
The first reaction of the immune system following an infection is the formation of low avidity antibodies and avidity is defined as the strength of binding of a multivalent antibody to its multivalent antigen. In an ELISA, there are multiple wash steps and the antigen concentration is limiting. High avidity antibodies preferentially bind to the antigen whereas low avidity antibodies are less firmly bound.
Avidity ELISA identifies the low avidity antibodies by measuring the relative degree of dissociation of specific antibodies from the test antigen fixed on a solid phase. The test is conducted in the presence and absence of a hydrogen bond dissociating agent urea in the first buffer. Urea promotes the dissociation of the antigen antibody complexes. Low avidity antibodies are easily eluted and washed off with the buffer. OD of the sample with and without urea wash is measured. Avidity percent are calculated as a avidity of <15% - low avidity; 15-30% boderline avidity, and >30= high avidity.
Each serum sample was analysed in two four fold titration rows, with one row starting at a dilution of 1:50 (row A) and other starting at 1:200 (row B). After incubation at 37° C, row A was washed 3 times with avidity washing solution (6 M urea in PBS with 0.05% Tween 20) in order to remove the low avidity IgG antibodies and row B with 150 mL of PBS Tween washing solution. The rest of the steps comprising incubation with the conjugate, washing, incubation with the substrate and addition of stop solution were performed according to the manufacturer's instructions. OD of each well was read in an ELISA reader at 405 nm. Controls (acute, borderline, chronic) supplied by Dr.Hedman were also run with the assay.
Two titration curves were obtained for each control and test serum. The curves were plotted on a millimeter graph paper with each doubling serum dilution corresponding to 20 mm on X-axis and absorbencies on the Y-axis. A cut off of 0.200 absorption units was used and titration curves with and without urea were drawn on the graph paper. The distance in mm between straight lines connecting the points on titration curves was measured. Corresponding avidity percentages were calculated from a table given in the instruction manual and interpreted as <15% low avidity; 15-30% borderline and >30% as high avidity.
[Figure - 1] and [Figure:2] show serum samples with low and high toxoplasma specific IgG avidity respectively.
Titration curves showing low and high IgG avidity. The distance between straight lines joining points on the titration curves are measured. Avidity percent is calculated from a table given in the instruction manual.
Low avidity < 15%
High avidity > 30%
| ~ Results and Discussion|| |
Of the 113 sera screened, 49 (43.3%) tested positive for IgG of which 16 (32%) were positive for IgM. Nine of these sera positive for both IgG/IgM and three sera positive for IgG alone were subjected to avidity ELISA. Of the 9 sera with possible acute infection, only three samples (30%) showed low avidity percentage. All the three sera positive for IgG alone showed high avidity percentage.
The detection of Toxoplasmosis has to be based on the maternal serology on account of diversity or absence of symptoms., The distinction between primary and secondary infection becomes crucial during pregnancy, because if infection is detected, antiparasitic treatment during pregnancy may prevent fetal infection and damage. Toxoplasma infection can be detected by demonstration of specific antibodies. Seroconversions using serial samples, presence of specific IgA/IgM antibodies in serum, a high dye titre value (>300 IU/ml) are the tests indicative of recent infection. However, all these tests are unreliable because in some patients IgA antibodies persist for months while IgM may persist for years following primary infection. Sabin Feldman dye test titres also remain high for several months following seroconversion, and therefore might be present before pregnancy and consequently the fetus is not at risk.
Avidity ELISA test was developed by Hedman et al , to overcome these problems. Subsequently other investigators supported the usefulness of this test in the diagnosis of recent T.gondi infection,, and other infections.,,,,
In most countries, interpretation of IgM results for the diagnosis of primary infection is based on results obtained in a single serum sample. Misintepretation of the results can occur due to persistence of IgM in the chronic stage of infection or due to low specificity of test. IgM antibodies are most often determined in a single serum sample using commercial kits, which are reported to give false positive results. Several investigators have found avidity ELISA test as a useful complementary test to confirm acute infection.,,,, This test can be carried out in a single serum sample and save the mother of unnecessary anxiety and treatment.
To date, different investigators have used different antigen preparations and different methods of calculating avidity. ,,, Therefore there is a need for an agreement on how to perform and calculate the avidity. Nevertheless avidity ELISA test seems to be a promising complementary test. In the present study too, although our sample size was small, the observations are in agreement with the previous studies., Lappalainen et al also report that only 11% of their IgG/IgM positive sera had low avidity. Nine women would have been identified as having acute infection, based on the conventional criterion of IgM positivity. Using avidity test, it was found that only three women had acute infection. This observation is significant. Since our sample size is small, studies on a larger sample would confirm the findings.
The detection of Toxospecific IgM in pregnant women is an old but often embarrassing problem. Most often the microbiologist or the clinician faces the problem of a first serum sample with positive IgG and IgM or IgM alone in a pregnant women without a previous result. Avidity IgG test, if performed on a blood sample collected early in pregnancy can aid as a complementary test.
| ~ Acknowledgements|| |
The Authors thank Dr. Kamala Krishnaswamy, Director, and National Institute of Nutrition for her keen interest in the study. The authors gratefully acknowledge Dr. Hedman, Lab. Systems, Finland for kindly giving us the Avidity kits as gratis. The secretarial help rendered by Mr. T. Satyanarayana and the technical help by Mr. K. Lingarkar is acknowledged.
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