|Year : 2015 | Volume
| Issue : 1 | Page : 40-44
|Comparison of immunofluorescence and enzyme-linked immunosorbent assay and immunoglobulin G avidity techniques for screening of anti: Toxoplasma antibodies among single serum sample pregnant women in Tabriz, Iran
Mehrangiz Rajaii1, Mohammad Reza Aliparasti2, Behrouz Nagilli3, Shohreh Almasi4, Najibeh Asle-Rahnamaie-Akbari5
1 Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz; Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
2 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
3 Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
4 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
5 Department of Parasitology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
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|Date of Web Publication||11-Feb-2015|
| Abstract|| |
Background: Congenital toxoplasmosis is that pregnant women acquire the infection during gestation; diagnosis of the acute infection during pregnancy is a complex subject of maternal toxoplasmosis. Thus, the presence of immunoglobulin G (IgG) and/or IgM Toxoplasma antibodies in a single serum sample drawn during gestation cannot be used to define whether the infection was recently acquired or chronic. Materials and Methods: At this cross-sectional descriptive study, sera of 391 pregnant women examined and compared. They were in an age range of 21-35 years, referred by gynecologists and infectious disease specialists, during March 2012-April 2013. They have referred, 215 (54.98%), 102 (26%), 74 (18.92%) in the first, second and third trimesters of gestation, respectively. For each of them, a questionnaire was completed and serum samples were prepared in an equal condition, examined according to the procedures of indirect immunofluorescence (IIF), enzyme-linked immunosorbent assay (ELISA) and IgG Avidity techniques. Results: We have found 111 (28.38%) seronegative and 280 (71.61%) seropositive cases by IIF and 124 (31.70%) seronegative, 267 (68.28%) seropositive cases by ELISA. The IgG avidity test confirmed 45 (69.23%) and 7 (10.76%) doubtful cases of IgM test in IIF and ELISA techniques. Conclusions: This study highlights how to manage pregnant women with toxoplasmosis, especially in a single serum sample condition.
Keywords: Enzyme-linked immunosorbent assay, immunoglobulin G avidity, Toxoplasmosis-Diagnostic tests-immunofluorescence
|How to cite this article:|
Rajaii M, Aliparasti MR, Nagilli B, Almasi S, Asle-Rahnamaie-Akbari N. Comparison of immunofluorescence and enzyme-linked immunosorbent assay and immunoglobulin G avidity techniques for screening of anti: Toxoplasma antibodies among single serum sample pregnant women in Tabriz, Iran. Indian J Pathol Microbiol 2015;58:40-4
|How to cite this URL:|
Rajaii M, Aliparasti MR, Nagilli B, Almasi S, Asle-Rahnamaie-Akbari N. Comparison of immunofluorescence and enzyme-linked immunosorbent assay and immunoglobulin G avidity techniques for screening of anti: Toxoplasma antibodies among single serum sample pregnant women in Tabriz, Iran. Indian J Pathol Microbiol [serial online] 2015 [cited 2018 Aug 22];58:40-4. Available from: http://www.ijpmonline.org/text.asp?2015/58/1/40/151183
| Introduction|| |
Toxoplasma gondii is a world wide zoonotic intracellular protozoon parasite and approximately one-third of the world's population is infected. , It can infect a variety of cell types, including a wide range of mammals, birds and humans. Domestic and free ranging felines are considered the key animal species in the transmission of T. gondii, because they are the only hosts who can produce and shed environmentally resistant oocysts through the feces. ,,,, Anti-Toxoplasma immunoglobulin G (IgG) production and antibody titers in adults are quite variable depending upon various factors such as age, geographical background and assay method. Toxoplasmosis is often asymptomatic in humans, or is associated with mild, nonspecific clinical symptoms in immunocompetent subjects, but in pregnant women seroconversion, reactivation of a chronic infection caused fetal damages as congenital toxoplasmosis. The significant fetal damage has been documented in offspring of women, who were entirely asymptomatic or who had no apparent exposure to the parasite during gestation. ,, Classically serodiagnosis includes titration of specific IgG, showing past exposure and screening for specific IgM, which is suggestive of recent exposure or ongoing active infection. Pregnant women should be systematically screened for T. gondi IgG and IgM antibodies during their first medical visit (ideally during their first trimester) for obstetrical care. A systematic approach to such program does not exist in Iran, but it is mandated by law in countries such as France and Austria. ,, In these countries; serial specimens facilitate the unequivocal diagnosis of a recently acquired infection by demonstration of seroconversion or a significant rise in IgG Toxoplasma antibodies accompanied by the presence of detectable IgM titers. The problem with serologic diagnosis is further complicated by the fact that antibodies to T. gondi may persist for years in healthy people. ,, Thus, positive IgG and IgM test results are not necessarily diagnostic of a recently acquired infection. Accurate diagnosis of recently acquired infection with T. gondii is important for the proper clinical management of pregnant women, since the parasite can be transmitted from a recently infected mother to her fetus. In Iran, the decision whether a woman was recently infected, thus placing her fetus at risk, is often made from serologic test results obtained with a single sample of serum.  Therefore, it is critical to determine as accurately as possible whether the infection was acquired prior to or during gestation. For this reason, a number of assays as Toxoplasma serologic profiles must be performed (enzyme-linked immunosorbent assay [ELISA], immunosorbent agglutination assay [ISAGA], IgG avidity, indirect immunofluorescence [IIF], Dye test). The objective of the present study was to examine and compare ELISA and IIF as an acceptable or gold standard methods and IgG avidity test as a confirmatory assay for distinguishing acute toxoplasmosis in pregnant women.
| Materials and Methods|| |
At this cross-sectional descriptive study sera of 391 pregnant women during March 2012 April 2013 examined and compared. They were in an age range of 21 to 35 years, referred by gynecologists and infectious disease specialists, and were tested by IIF, ELISA and IgG avidity methods. These patients were in the different stages of pregnancy. A questionnaire was prepared for recording the demographic, epidemiologic and pregnancy information. Serum samples were prepared in the same condition and examined simultaneously according to the procedures of IIF and ELISA and IgG avidity techniques. In this study, we have used of SAS.8.1 (SAS Institute Inc, Cary, NC) to analyze data. Statistic analysis included estimation of diagnostic values such as sensitivity, specificity and their 95% confidence intervals.
Ready for use previously coated slides with antigen, polyvalent antihuman immunoglobulin conjugated serum and serum controls were purchased from Baharafshan Company Tehran, Iran. Antihuman serum conjugated with fluorescin isothiocyanate dilution of 1:20 in phosphate buffered saline (PBS) in 0.1% Evans blue More Details was used. Diluted sera were placed on antigen spots, next incubated at the 37°C for 30 min. The slides were then rinsed in PBS (PH 7.6) 2 times for 5 min each and dried. A drop of fluorescein isothiocyanate labeled antihuman immunoglobulin diluted in PBS was added to each spot. Slides were incubated at 37°C for 30 min again, washed, dried and mounted with buffered glycerol. The slides were examined under a fluorescent microscope, equipped with an Osram HBO mercury lamp. Fluorescent isothiocyanate labeled antihuman immunoglobulin visible fluorescence of cytoplasm membrane contrasting with red dyed cytoplasm of parasite regardless as positive or negative by IIF based on bright and continuous fluorescence around the total periphery of Toxoplasma, where in a negative reaction, no florescence is observed. A borderline reaction occurs, when fluorescence is not continuous around the Toxoplasma edges and decrease in parallel to the increase of serum dilution. For screening of Toxo-specific IgG, based on other investigations in this area and laboratory experiences starting serum dilution of 1/100 prepared, then if necessary continued of high dilutions, 1/200, 1/400, 1/800, for screening of Toxo-specific IgM only 1/100 serum dilution prepared, but in suspected conditions such as weak positive or doubtful cases, which could be resulted of excess of antibody (Prozone phenomena) or low concentrations of antibodies (weak positive), prepared 1/50 and 1/200 dilutions respectively.
Enzyme-linked immunosorbent assay
Detection of immunoglobulin G Toxo-specific antibody
Toxo-specific - IgG test (RADIM - Italy) with the sensitivity and specificity has been estimated to be 97.1% and 99.1%, respectively. Accurately, all the assays (Toxo-IgG, IgM, IgG avidity) were done as the brochures of kits.
For IgG test, the optical density (OD) of each negative control and cut-off calibrator (15 IU/ml) must be considered. The presence or absence of anti-Toxo-IgG antibody is defined by comparing the sample absorbance with the absorbance of the cut-off calibrator. Samples with OD lower than the 15 IU/ml (cut-off) are to be considered negative for anti Toxo-IgG antibodies. Samples with OD higher than the cut-off calibrator are to be considered positive for anti-Toxo-IgG antibodies. Samples with absorbance values varying within ± 10% of the cut-off calibrators are to be considered questionable (gray zone). Measurement of anti-Toxo-IgG antibodies were done quantitatively based on calibrators of, 30, 60,120,240 IU/ml concentrations.
Detection of Toxo-IgM
The Toxo specific IgM antibody, which is a rheumatoid factor-captured ELISA (RADIM - Italy). The sensitivity and specificity of Toxo-IgM test has been estimated at 98.1% and 98.2%, respectively.
For IgM test, the OD of each negative, positive and cut-off must be considered. The presence or absence of anti-Toxo-IgM antibody is defined by comparing the sample absorbance with the absorbance of the cut-off control. Samples with OD lower than the cut-off control are to be considered negative for anti-Toxo-IgM antibodies. Samples with OD higher than the cut-off control are to be considered positive for anti-Toxo-IgM antibody. Samples with absorbance values ranging within ± 10% of the cut-off control are to be considered questionable, and should be retested for confirmation (gray zone).
Immunoglobulin G avidity test
Toxo-specific - IgG avidity EIA - test (RADIM - Italy) with the sensitivity and specificity has been estimated to 89% and 90%, respectively.
The percent avidity <20% considered as low avidity, >30% considered as high avidity, 20-30% considered as gray zone.
| Results|| |
391 cases were screened for Toxo-specific IgG and IgM antibodies by IIF and ELISA and compared. Ultimately, the IgG avidity test has revealed recent and doubtful IgM cases.
111 and 280 serum samples were IgG negative and IgG positive respectively (1/100 dilution cut-off). 280 seropositives were comprised of 215 (54.99%) G + M - and 65 (16.62%) of G + M + cases; most of them were in titers of 1/200, 94 (33.5%) and 1/400, 90 (32%), [Table 1], [Table 2], [Table 3]. Regarding IgM, between 65 cases of IgM positive, 20 of them were clearly IgM positive without any doubt, and 45 cases were weakly reaction or doubtful.
|Table 1: Summary of positive and negative test results for toxoplasmosis in IIF and ELISA tests|
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|Table 2: Prevalence of Toxo-specific IgG serum dilutions (IIF) and concentrations (ELISA) and IgM antibodies among 391 pregnant women measured by IIF and ELISA|
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|Table 3: Confirmatory role of IgG avidity test in discrimination of active or acute toxoplasmosis|
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With enzyme-linked immunosorbent assay
124 and 267 serum samples were IgG negative and positive respectively, (<30 IU/ml cut-off), 267 seropositive cases comprised of 202 (51.66%) G+ M- and 65 (16.63%) G+ M+ cases. Most of the IgG positive cases (158, 40.41%) were in the range of 60-120 IU/ml serum concentration of antibody [Table 1], [Table 2], [Table 3]. Out of 65 cases of IgM positives, 58 cases were clearly positive, but five cases were in the gray zone or doubtful, which confirmed by IgG avidity (≤20%) test, and for two cases of IgM positives, which were positive for >1 year, IgG avidity index (AI) was above 30%, which confirmed as a past infection or IgM false positive.
| Discussion|| |
Estimating the gestational age of the primary Toxoplasma infection precisely as possible for the proper clinical management of pregnant women in a single serum is in fact, impossible to determine whether the infection occurred during gestation. In the absence of systematic serological testing during gestation (e.g., every month or trimester as performed in France), Diagnosis of serious toxoplasmosis practically hopeless. , Because the infection is asymptomatic in 93-97% of pregnant women, a low IgG titer and a negative IgM test on a pregnant woman in the first 24 weeks of gestation essentially placed the acquisition of the infection prior to gestation. A positive IgM test result in a single serum sample may reflect a lately obtained infection, an infection acquired in the distant past, or a false-positive result.  It is a fact that a significant percentage of the Iranian pregnant women population has preexisting IgG antibodies to T. gondii, reflecting infection acquired in the obscure past rather than during the present pregnancy. ,,,, Thus, early diagnosis of acute infection in the pregnant woman is critical to determine if treatment of the mother is indicated to attempt to prevent transmission of T. gondii to the fetus.  If initial serologic testing suggests the possibility of a recently acquired infection, obtaining a second serum for confirmatory testing is recommended, but can delay treatment and expose the fetus to increased risk of infection. Therefore, what is needed is a serologic approach that has allowed the rapid diagnosis of recently acquired infection in a single sample of serum? Diagnostic tests for the detection of oocysts in cat feces are of little significance owing to short potency (15 days). Histological examinations of biological samples show a lack of reliability, when the animals are infected with few parasites. Serological tests are the most-used diagnostic methods, especially dye test and IIF that require intact tachyzoites are more sensitive and specific, because during the infection, the first significant increase in IgM and IgG antibodies were observed against uticular antigens. polymerase chain reaction to identify T. gondii DNA in canine and feline biological samples was developed.  However, the prevalence of high IgG antibody titers to T. gondii among normal individuals in most populations and the sustained persistence in some persons of specific IgM antibodies has complicated the interpretation of serological tests when acute toxoplasmosis is suspected. , We have found 111 (28.39%) seronegative and 280 (71.61%) seropositive case by IIF and 124 seronegative (31.71%) and 267 (68.29%) seropositive by ELISA procedures [Table 1], so IIF could be able detected more IgG positives than ELISA. IIF test has advantages as: safer, doesn't require living organisms, killed tachyzoit are used and prepared smears of killed tachyzoit can be stored frozen for several months is required. Nevertheless, disadvantage of this method is requiring a fluorescent microscope, which may not be available in small laboratories, and an experienced lab technician who could be able discriminate doubtful or weak positive cases (resulting of low concentration of serum samples) or zone phenomena (resulting in high concentration of serum samples). In this procedure (IIF), we have found two groups of feeble positive reactions; first group consisted of 45 cases with a serological pattern of G+ M+ which results from an excess of antibody or zone phenomena, which have resolved in elevated serum dilutions. The second group consisted of 42 cases, with a serologic pattern of G± M- which resultant of low concentrations of antibody and will be negative in high serum prepared dilutions [Table 3]. With ELISA procedure, we have found seven doubtful IgM cases which 5 out of them could be in gray zone or initial course of an active infection but reminder, or two cases, which were continued IgM positive for >1 year. Collectively, a positive IgM test result should undergo confirmatory testing. IgG avidity testing helps for distinguishing recently or false-positive serologic reactions. The strength of specific IgG binding to the multivalent Toxoplasma antigen depends to stage of infection or this binding strength is called IgG avidity. In this assay, the hydrogen-bond disrupting agent, urea, is used to elute IgG from the immobilized antigen. As a result, the low affinity antibodies produced at an early stage of infection are separated from those with a higher binding affinity that reflect past immunity. It has the advantage of distinguishing between acute and chronic acquired infections using a single serum specimen. The avidity result, generally stated as the AI, is expressed mainly as the percent ratio of antibody titers or OD values with and without the urea washing step. 45 doubtful or uncertain IgM detected by IIF with low AI ≤ 20% clarified them as an acute infection [Table 3].  As observed in this study, I have shown an important diagnostic effect for detecting and interpretation of such obscure and uncertain serologic conditions. Low avidity results may persist for >1 year, for these reasons a panel of serological tests must be performed (ELISA, ISAGA, IgG avidity, IIF, Day test) for IgM, IgA, IgE.  Of course, other studies in different parts of Iran, including of pregnant women, have explained some of these complexes. , Collectively, it can be concluded that IIF needs an experienced technician for working with fluorescent microscopy or interpretation of doubtful cases. On the other hand, because of the ELISA is easier technique and lower expense, preferred in order to screening toxoplasmosis infection. However, in some literature, IIF reminded a standard technique. Finally, educational plans regarding the risk of toxoplasmosis at reproductive ages are recommended to be implemented in the national maternity health care programs.
| Acknowledgments|| |
This study was financially supported by Tropical and Infectious Diseases Research Center (TIDRC), Tabriz, Iran. The authors would like to thank the staff and authorities in TIDRC for supporting the conduct of this research. We are grateful to research development and coordination center (RDCC) in Tabriz University of medical sciences.
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Immunology Research Center, Tabriz University of Medical Sciences, Golgasht St., Daneshgah St., Tabriz
Source of Support: This study was financially supported by Tropical and
Infectious Diseases Research Center (TIDRC), Tabriz, Iran. The authors
would like to thank the staff and authorities in TIDRC for supporting the
conduct of this research. We are grateful to research development and
coordination center (RDCC) in Tabriz University of medical sciences., Conflict of Interest: None
[Table 1], [Table 2], [Table 3]
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