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Indian Journal of Medical Microbiology, Vol. 28, No. 4, October-December, 2010, pp. 354-357 Original Article IgG - Indirect fluorescent antibody technique to detect seroprevalence of Toxoplasma gondii in immunocompetent and immunodeficient patients in southern districts of Tamil Nadu G Sucilathangam1, N Palaniappan1, C Sreekumar2, T Anna2 1 Department of Microbiology , Tirunelveli Medical College, Tirunelveli - 627 011, Tamil Nadu, India Date of Submission: 21-Dec-2009 Code Number: mb10104 PMID: 20966568 Abstract Purpose: The purpose of the study was to determine the prevalence of Toxoplasma antibodies in selected immunocompetent and immunodeficient patients in and around Tirunelveli District of Tamil Nadu. Keywords: Seroprevalence, Toxoplasma gondii, indirect fluorescent antibody test, Tamil Nadu Introduction Toxoplasmosis is caused by a coccidian protozoan parasite Toxoplasma gondii. Humans and other warm-blooded animals are its intermediate hosts. The infection has a worldwide distribution. Approximately one-third of all humanity has been exposed to this parasite. Although usually asymptomatic in immunocompetent adults, it can cause severe disease manifestations and even death in immunocompromised patients. If acquired during pregnancy, it can cause various congenital anomalies in the child. Several serological assays may be used to demonstrate Toxoplasma antibody, including the Sabin-Feldman dye test, the complement fixation test, the indirect haemagglutination test, and the indirect fluorescent antibody (IFA) test. Excellent correlation exists between relative titres obtained in the IFA test and those obtained using the longer and more laborious procedures. In addition, the IFA test detects both IgG and IgM-class antibody. If the test is positive using anti-human IgG, a monospecific anti-human IgM fluorescein conjugate may be employed to distinguish the early antibody response characteristic of primary infection. The added advantages of IFAT are simplicity in routine use, indefinite storage of reagents, economy of mice, sharp end-point, avoidance of numerical counts and accessory factor, absence of prozones permitting screening procedures, and finally, ease of standardization. [1] The indirect fluorescent antibody test (IFAT) measures the IgG antibodies as the dye test. Titres were parallel to dye test titres. In India, the exact seroprevalence of this infection is not known. However, using various diagnostic tests, the prevalence has been reported to be as low as 1% and as high as 80% in adults. [2] However, the knowledge about this infection, diagnosis and interpretation of the test results is a major problem in the Indian context. Though, Toxoplasma infection does not cause repeated foetal losses, this is the most common indication for investigation of toxoplasmosis in India. There are several diagnostic test kits available in Indian markets, however, their qualities are not assessed by most of the laboratories before they are procured. [3] There is no baseline data on seroprevalence and antibody titres of toxoplasmosis in various subpopulations in different parts of our country. There is a lack of awareness and knowledge about this zoonotic infection. Further complicating the situation, there are several commercial organizations that are promoting their products without proper background knowledge and baseline data from India. Under this situation, it is highly imperative to assess the status of seroprevalence of toxoplasmosis in Tamil Nadu especially in pregnant women and immunodeficient patients, based on suitable IgG-based "in-house" IFAT using laboratory-prepared antigen and known positive serum. Perusal of available literature revealed no systematic studies on seroprevalence of toxoplasmosis in humans in southern parts of Tamil Nadu. The study was aimed to bring out status of toxoplasmosis in southern districts of Tamil Nadu for diagnosis of toxoplasmosis especially in pregnant women and immunodeficient patients. Materials and Methods Study population A total of 350 peripheral blood samples were collected from 175 immunodeficient patients (HIV and patients with malignancy) and 175 immunocompetent patients including pregnant women (135), ocular chorioretinitis cases (20), and patients with lymphadenopathy (20), in and around Tirunelveli district of Tamil Nadu. Inclusion criteria for immunodeficient group were of HIV-infected patients with CD 4 T-cell counts < 200/΅l and proved cancer patients. Exclusion criteria for immunodeficient group were of transplant recipients under immunosuppressive therapy or haemodialysis patients with chronic renal failure. Inclusion criteria for immunocompetent group were of asymptomatic pregnant women, pregnant women with bad obstetrics history (BOH), ocular chorioretinitis cases, and lymphadenopathy. Exclusion criteria for immunocompetent group were of myocarditis and pericarditis. Preparation of antigen As per the procedure outlined in USHDEW (U.S. Department of Health, Education and Welfare) manual, [4] the T. gondii (RH strain) tachyzoite antigen was prepared by in vivo mice propagation and in vitro cell culture system in MDCK (Madin Darby Canine Kidney) fibroblast cell line. The tachyzoite count was 1 Χ 10 7 and 1 Χ 10 9 per ml of antigen in vivo and in vitro methods, respectively. Formalin-killed T. gondii tachyzoite antigen was prepared for IFAT. Preparation of antigen slides Antigen slides were prepared as per the procedure outlined in USHDEW manual with minor modifications. [4] The formalin- killed T. gondii tachyzoite antigen harvested from the cell line culture was resuspended in an appropriate volume of PBS (so as to obtain 100−200 tachyzoites per high power field). The suspension was then drawn into a capillary tube and spotted onto grease-free Teflon-coated slides on which the circular wells were previously etched using a glass marking pencil. Once the spots had dried up, the slides were placed in cold acetone overnight. The fixed slides were air-dried, wrapped in aluminium foil and stored at -20 o C until further use. Reference sera The known positive and negative sera obtained from the reference laboratory (Dr. C. Sreekumar, Toxo laboratory, USDA, Maryland, USA) were aliquoted and stored at -20 o C until use. Conjugates Anti-human IgG-FITC conjugate raised in goat was obtained from Bangalore Genei, Peenya, Bangalore. IFA test procedure Indirect fluorescent antibody test was performed as described by Renterghem and Nimmen with minor modifications. [5] Positivity and negativity were determined as per the guidelines described in USHDEW manual. [4] Test sera samples that were positive at 1 : 16 dilution were further tested at two-fold serial dilutions until an endpoint was reached. Standardization of IFAT An IFAT was standardized using reference control positive and negative sera. The optimum dilution of conjugate was standardized. The conjugate dilutions of 1 : 25, 1 : 50, 1 : 100, and 1 : 200 were tested with known positive and negative reference sera. The optimum conjugate dilution, of 1 in 50, which gave the highest titre with positive sera and lowest negative sera titre, was selected. The positive sera from minimum dilution of 1 : 16 to maximum up to 1 : 512 showed good immunoreactivity with either bright yellow-green fluorescence over the entire tachyzoite or distinct yellow-green fluorescence around the entire periphery of the organism (which appeared reddish). With negative control serum (1 : 8), the organisms did not show any fluorescence, or sometimes polar fluorescence was also seen. Therefore the cut-off titre for positive was arrived at 1 : 16 and above. Results Evaluation of IgG IFAT The sensitivity, specificity, positive, and negative predictive value of IgG IFAT in detecting toxoplasmosis was 80, 90, 88.89, and 81.81%, respectively. Seroprevalence of toxoplasmosis by IgG IFAT Out of 350 patients tested by IgG IFAT, 41 patients (11.71%) had antibodies for T. gondii with mean IFAT titre of 43.32 ± 58.7 and the titre ranged from 1 : 16 to 1 : 256. Among the immunocompetent group of 175 patients, 19 patients (10.86%) had antibodies to T. gondii, whereas in immunodeficient group of 175 patients, 22 patients (12.57%) had antibodies for T. gondii. Out of 135 pregnant women tested, 13 (9.63%) were seropositive with mean IFAT titre of 51.69 ± 74.23 (titre ranged from 1 : 16 to 1 : 256). Among the 20 cases of lymphadenopathy, only 3 (15%) were seropositive with mean titre of 26.6 ± 9.23 (titre ranged from 1 : 16 to 1 : 32). Of the 20 ocular cases tested, only 3 (15%) were seropositive with mean titre of 58.67 ± 60.57 (titre ranged from 1 : 16 to 1 : 128). Among the 160 HIV-positive individuals tested by IFAT, 19 (11.88%) were seropositive with mean titre of 45.47 ± 61.33 (titre ranged from 1 : 16 to 1 : 256). Out of 15 cases of malignancy, only 3 (20%) were seropositive with mean titre of 26.6 ± 9.23 (titre ranged from 1 : 16 to 1 : 32) [Table - 1]. Discussion The IgG IFAT was standardized and the cut-off titre for positive was arrived at 1 : 16 and above. The sensitivity and specificity of IgG IFAT in the present study in detecting T. gondii was 80 and 90%, respectively. Most of the recent authors taken dilutions of 1 : 16 and above were positive for IFAT. [6],[7] The overall seroprevalence of T. gondii in and around Tirunelveli district of Tamil Nadu was 11.71% based on IgG IFAT. In India, the exact seroprevalence of T. gondii is not known. However, using various diagnostic tests, the prevalence has been reported to be as low as 1% and as high as 80% in adults. [2] Until recently, the prevalence of T. gondii in the general population of India was considered to be low when compared with Western countries. [8],[9] This may be partially true, however, the data are based on samples obtained from patients admitted to hospitals in big cities. Because most of the population of India lives in rural villages with little means to get to city hospitals, the data are not reflective of the population as a whole. The type of serological tests used and the titres taken as evidence of infection are also important factors. However, only studies during 1990 indicated that the prevalence may be a lot higher than previously considered. [10],[11] In earlier studies, only 12% seropositivity for T. gondii was reported from South India, [12] whereas, of late, a high prevalence (75−77%) of T. gondii was reported from North India. [10],[13] In this study, among the immunocompetent group of 175 patients, 19 patients (10.86%) had antibodies to T. gondii whereas, in immunodeficient group of 175 patients, 22 patients (12.57%) had antibodies for T. gondii. The overall seroprevalence from Bombay was 30.9% (51/165) in the immunocompetent adult and in immunodeficient patients it was 67.8%. [14] Out of 135 pregnant women tested, 13 (9.63%) were seropositive in the current study. The prevalence of toxoplasmosis in Indian pregnant women was variably reported. [2] The prevalence of toxoplasmosis in pregnant women from Calcutta was reported as 7.1% whereas; it was 45% in pregnant women from New Delhi by using IgG Avidity ELISA test. [2],[15],[16] However, the knowledge about this infection, diagnosis and interpretation of the test results in pregnant women was a major problem in the Indian context. [2] Though, Toxoplasma infection does not cause repeated foetal losses, this is the most common indication for investigation of toxoplasmosis in India. In this present study also, only 3 out of 19 (15.79%) cases with bad obstetric history (BOH) were seropositive for toxoplasmosis. Therefore, BOH alone was not taken as criteria for routine screening of toxoplasmosis. In the present study, among the 20 cases of lymphadenopathy, only 3 (15%) were seropositive. Of the 20 ocular cases tested, only 3 (15%) were seropositive. An outbreak of ocular toxoplasmosis occurred in Coimbatore, Tamil Nadu, in which 71.5% of particular area had high titres of both IgM and IgG antibodies, indicating that it was an acute infection, acquired through municipal water contamination. [17] Present study revealed that out of 15 cases of malignancy, only 3 (20%) was seropositive and 11.88% seropositive among the 160 HIV-positive individuals tested by IgG IFAT whereas, a high seroprevalence rate of T. gondii of 67.8% in HIV patients was reported from Bombay.[14] The study has highlighted an overall 11.71% of seroprevalence of T. gondii, which constitutes 12.57% in immunocompromised and 10.86% in immunocompetent patients in and around Tirunelveli district of Tamil Nadu. The study underlines the importance of screening of this parasite especially in the immunocompromised patients. The seroprevalence of T. gondii in pregnant women was about 9.63%, which necessitates initial screening for IgG antibodies and then, a paired sera sample after three weeks should be tested for rise in titer. Thereafter, the IgM antibodies should be tested to exclude the recent infection. All seropositive women who seroconvert during pregnancy should be monitored and their amniotic fluid/foetus should be screened by PCR and ultrasound, respectively, to assess the infection. References
Copyright 2010 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb10104t1.jpg] |
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