Indian Journal of Pathology and Microbiology

ORIGINAL ARTICLE
Year
: 2013  |  Volume : 56  |  Issue : 1  |  Page : 20--23

Anti-Toxoplasma gondii antibody detection in serum and urine samples by enzyme-linked immunosorbent assay in HIV-infected patients


Sayan Bhattacharyya1, Sumeeta Khurana2, Mohan Lal Dubey2,  
1 Department of Microbiology, KGMU, Lucknow, Uttar Pradesh, India
2 Department of Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Correspondence Address:
Sayan Bhattacharyya
481, P. Majumdar Road, Kolkata -700 078, West Bengal
India

Abstract

Background: Toxoplasmosis is a common parasitic infection of man, and reactivation of latent disease in HIV-infected patients can cause fatal encephalitis. Diagnosis depends on demonstration of parasite-specific antibodies in serum. In HIV-infected patients, IgM is often undetectable, whereas IgG remains detectable in the majority. Urine sample is very easily available and has not been evaluated for immunodiagnosis of toxoplasmosis. Aim: The study was an effort to find whether urine sample can be used in place of serum for immunodiagnosis of toxoplasmosis. Materials and Methods: Enzyme-linked immunosorbent assay (ELISA) was carried out in serum and urine samples collected from 100 HIV-infected patients to detect anti-toxoplasma IgG and IgM antibodies and whether positivity correlated with the CD4 T-cell counts of patients. Results: In this study, we observed that there was no significant difference in positivity of anti-toxoplasma IgM and IgG between serum and urine samples of HIV-infected patients by ELISA. There was a negative correlation between CD4 count and seropositivity. Conclusion: Urine sample can be satisfactorily used in place of serum for immunodiagnosis of toxoplasmosis.



How to cite this article:
Bhattacharyya S, Khurana S, Dubey ML. Anti-Toxoplasma gondii antibody detection in serum and urine samples by enzyme-linked immunosorbent assay in HIV-infected patients.Indian J Pathol Microbiol 2013;56:20-23


How to cite this URL:
Bhattacharyya S, Khurana S, Dubey ML. Anti-Toxoplasma gondii antibody detection in serum and urine samples by enzyme-linked immunosorbent assay in HIV-infected patients. Indian J Pathol Microbiol [serial online] 2013 [cited 2020 Aug 7 ];56:20-23
Available from: http://www.ijpmonline.org/text.asp?2013/56/1/20/116143


Full Text

 Introduction



Toxoplasmosis, caused by Toxoplasma gondii, is one of the most common parasitic infections of humans and other warm-blooded animals. [1] The agent requires two hosts for completing its life cycle. Cat is the usual definitive host, and mice and rats are intermediate hosts. [2] Infection is acquired by oral route from tissue cysts in infected meat and oocysts in cat feces, or by organ transplant and blood transfusion. Congenital infection also occurs by vertical route. Clinical features range from mild febrile illness and lymphadenopathy in the immunocompetent host to encephalitis in the immunosuppressed host like the HIV-infected patient. Congenital infection manifests commonly as chorioretinitis, hydrocephalus, and intracerebral calcification. [2]

Diagnosis rests on direct demonstration of the parasite, immunologic methods, and imaging. Whereas imaging modalities such as computed tomography (CT) and magnetic resonance imaging (MRI) are less specific, direct demonstration is hazardous to the worker, is labor intensive, needs several weeks for results, and is often not conclusive of acute infection. [3]

Therefore, serological tests are the mainstay of correct diagnosis. Among the serological tests, indirect hemagglutination (IHA) is less sensitive and more labor intensive. [4] Indirect fluorescent antibody (IFA) test gives false-positive results with rheumatoid factor, and Sabin-Feldman dye test titres do not correlate with the severity of the illness. [5] Enzyme-linked immunosorbent assay (ELISA) (both conventional and avidity) can be used for diagnosis and prognosis. ELISA has the sensitivity of about 88%, with a range of 83.3-98.6% and is 97% specific for clinching a diagnosis of toxoplasmosis. [6],[7]

Diagnosing toxoplasma encephalitis (TE) in HIV-infected patients is particularly difficult as the CT/MRI features closely mimic central nervous system (CNS) lymphoma, and ELISA for anti-toxoplasma IgM, IgG, and IgA antibodies has low sensitivity. [8] Sensitivity of Polymerase Chain Reaction (PCR) from peripheral blood for diagnosis of TE varies from 11% to 77%. [9] Direct brain biopsy has only 50-75% sensitivity for TE and sampling faces ethical issues. [3] Although IgM is often undetectable, anti-toxoplasma IgG levels remain elevated in more than 97% of patients with toxoplasmosis and AIDS. [8],[10]

Antigen or antibody detection in urine has been tried in several parasitic infections and appears to be useful in the rural and remote areas where venepuncture is not possible due to lack of trained staff. [11],[12] ELISA in urine has not been tried so far for diagnosis of toxoplasmosis. Keeping these points in view, this study was undertaken to compare IgM and IgG antibody detection with T. gondii by ELISA in serum and urine samples of HIV-infected patients.

 Materials and Methods



A total of 100 HIV-infected patients were randomly selected from the immunodeficiency clinic of a tertiary care center. The sample size was calculated by the formula 3.84 × square of standard deviation (SD) (from other studies)/square of error. Every 10 th patient in the clinic was selected for the study.

Fifty HIV-seronegative healthy subjects were included as healthy controls and 30 patients (10 each with cysticercosis, amebiasis, and malaria) were enrolled as controls having other diseases. From each patient, 5 ml of serum and 10 ml of urine were collected after obtaining informed written consent. The samples were stored at –20°C till tested. The study was approved by the ethics committee of the institute.

Antigen preparation

The toxoplasma antigen was prepared from RH strain of T. gondii tachyzoites harvested from the peritoneal cavity of five Swiss albino mice infected 3 days earlier by intraperitoneal route (1 × 10 4 tachyzoites in 0.5 ml isotonic saline). The harvested tachyzoites were suspended in phosphate buffer saline (PBS) and thereafter subjected to differential centrifugation for removing leukocytes. [13] Finally, the resulting pellet containing the purified tachyzoites was suspended in 300 μl of distilled water, kept overnight at 4΀C, and subjected to sonication in ice.

Subsequently, the sonicated material was subjected to cold centrifugation at 4°C to remove membranes. The protein concentration of the final supernatant was determined by a one-step method using nitration of tyrosine. [14] This antigen was used for serodiagnosis by ELISA.

Detection of anti-toxoplasma antibody

The antibody detection in serum and urine samples was carried out by ELISA. Urine was centrifuged at 400 g for 5 min and then the supernatant was used for ELISA, which was performed as per standard protocol. [15] Optimum concentrations of antigen, conjugate, serum, and urine samples were determined by checkerboard titration. Toxoplasma antigen (1 μg/well) was coated on microtiter plates and ELISA was performed by conventional technique, using 1 in 400 dilution of serum (1 in 800 for IgG), 1 in 2 dilution of urine samples, and appropriate dilutions of horse radish peroxidase (HRP)-labeled anti-IgM and anti-IgG conjugates. [15] Readings were taken at 492 nm wavelength in Emax spectrophotometer (Molecular Device Corp., Sunnyvale, CA, USA). All samples were tested in duplicate and mean absorbance (Optical Density) values of two tests were taken as value for that sample. The cut-off OD for positivity was calculated by adding 2 SD to the mean of the multiple negatives (minimum of 3).

CD4 lymphocyte count

CD4 T-cell counts of 72 HIV-infected patients, performed by flow cytometry, were available from the immunodeficiency clinic of the department of internal medicine for correlation with serological test results. [16]

Statistical evaluation

Appropriate statistical methodologies, i.e., Chi-square test and z-test, were employed for analyzing results.

 Results



Of the 100 HIV-positive patients, 38 (38%) were asymptomatic. The rest had various coexisting illnesses such as pulmonary tuberculosis, chronic or persistent diarrhea, and oral thrush. The mean age of the HIV-infected patients was 32 years (24-64 years). The mean age of the subjects in the other disease control group was 19 years ( 14-23 years) and in the healthy control group, it was 29 years (24-32 years). Among the HIV-infected patients, 68% were men and 32% were women. In the healthy control group, 70% were men and 30% were women, whereas in the other disease control group, 71% were male and 29% were female patients.

Anti-Toxoplasma antibodies in serum samples

IgM antibodies

The mean cut-off OD for IgM in serum samples tested in 1 in 400 dilution was 0.384. Anti-toxoplasma IgM antibody was detected in 32% patients, whereas 6.6% other disease controls and 4% healthy controls showed IgM positivity [Table 1].{Table 1}

IgG antibodies

The mean cut-off for IgG ELISA for sera tested in 1 in 800 dilution was 0.469. IgG antibodies were positive in 42% of HIV-positive patients, whereas 10% of other disease control subjects and 4% healthy subjects demonstrated IgG antibodies [Table 1].

Anti-Toxoplasma antibody in urine samples

IgM antibody

The mean cut-off OD for sample was 0.116. ELISA for IgM antibodies was positive in urine of 25% patients, whereas 3.3% other disease control subjects and 2% healthy control subjects showed IgM antibodies. Results have been shown in [Table 2].{Table 2}

IgG antibody

The mean cut-off OD value for undiluted sample was 0.165. ELISA for IgG antibodies was positive in urine of 42% patients, whereas 6.6% other disease controls and 6% healthy controls showed IgG antibodies.

Both IgM and IgG anti-toxoplasma antibodies in urine were significantly higher (P < 0.05) in HIV-positive patients than in both control groups.

Correlation of anti-toxoplasma antibody positivity and CD4 counts in serum samples

The mean CD4+ cell count was significantly lower (P < 0.05) in patients with seropositivity for IgM anti-toxoplasma antibody (125 cells/μl) than that in IgM seronegative patients (375 cells/μl). Similarly, the mean CD4+ cell count was significantly lower ( P value<0.05) in patients with seropositivity for IgG (204/μl) than that in IgG seronegative patients (275/μl).

Correlation of CD4 counts and anti-toxoplasma positivity in urine samples

Mean CD4 cell count in the patients positive for IgM antibodies in urine was lower (163/μl) than that in IgM seronegative patients (238/μl). However, statistically the counts were not different ( P > 0.05) in two groups as determined by unpaired t-test. Mean CD4 count in the patients positive for IgG antibodies in urine was lower (179/μl) than that in IgG-negative patients (243/μl). However, statistically the counts were not different ( P > 0.05) in two groups as determined by unpaired t-test.

The positivities in both serum and urine samples for both IgG and IgM antibodies were compared by applying Chi-square test. There was no significant difference in IgM positivity in serum and urine samples (χ2 value = 1.5; P > 0.05). Similarly, positivity of IgG antibodies was not statistically different (χ2 value = 0.2; P > 0.05) in serum and urine samples in HIV-infected patients.

 Discussion



Toxoplasmosis is one of the most common parasitic illnesses affecting all ages. It is self-limiting in immunocompetent hosts but manifests as severe encephalitis and chorioretinitis in immunocompromised states such as in HIV-infected patients. Detection of parasite-specific antibodies is one of the pillars for diagnosis, but in HIV-infected patients, IgM antibody is often not detectable due to profound immunosuppression. IgG antibody, however, remains detectable in about 97% of patients infected with T. gondii in the past. [8],[10] In our study, the percentage of HIV patients showing IgG and IgM positivity in serum was 42% and 32%, respectively. Compared to this, the prevalence of anti-toxoplasma antibody in sera of HIV-infected patients in East Africa was found to be 27% in Uganda and 4% in Zambia. [17] One Indian study has reported toxoplasma-specific IgG, IgM, and IgA antibody response and antigenemia to be detectable in 12%, 6%, 7%, and 14% of HIV-infected patients, respectively. [18] Several studies mention the positive correlation between high anti-toxoplasma IgG titer and development of TE in HIV-infected patients having toxoplasmosis; hence, seropositive patients need to be considered for prophylaxis for the disease. [19]

Urine sample has been reported to be useful for the diagnosis of parasitic illnesses such as hydatidosis, schistosomiasis, and visceral leishmaniasis, and has the greatest advantage of being easily obtainable and noninvasive, especially in peripheral settings where venepuncture may be difficult to carry out [11],[12],[20] The percentage of HIV patients showing IgG and IgM positivity in urine was 42% and 25% in urine in our study. Comparison of IgM and IgG antibody detection by ELISA in serum and urine samples of HIV-infected patients showed that there was no significant difference in positivity between the two samples.

Hence it follows that urine sample can be used as an alternative or adjunct to serum for immunodiagnosis of toxoplasmosis. Earlier studies have also reported saliva as a useful sample for immunodiagnosis of toxoplasmosis, with a sensitivity and specificity of 64% and 94.67%, respectively, for detection of IgG and 81.25% and 85.71%, respectively, for IgM. [21] This emphasizes the utility of samples collected by minimally invasive procedures for immunodiagnosis of toxoplasmosis. The mean CD4 counts of the patients who tested positive in serum for anti-toxoplasma antibodies were lower than those of the patients who tested negative, thus suggesting that those individuals with lower CD4 counts have a higher probability of developing CNS toxoplasma infection. However, this association was not found in urine samples. There are a few reports in the literature of association between seropositivity for toxoplasmosis and CD4 count in HIV-positive patients. [22] At least one study mentions that in their study population, there was a negative correlation between CD4 count and opportunistic infections such as toxoplasmosis in HIV-positive patients. [23]

Some studies have established a significant association between low CD4 count and development of TE. [24] In the light of these studies and our findings, we strongly recommend that anti-toxoplasma serology be carried out as a screening test in HIV-infected patients, as early serodiagnosis and treatment can prevent the development of toxoplasma encephalitis in these patients. In remote rural areas where collection of blood samples may not be possible, urine samples can be used for such screening.

References

1Dubey JP. Toxoplasmosis. In: Collier L, Balows A, Sussman M, editors. Topley and Wilson's Microbiology and Microbial Infections. 9 th ed. New York: Arnold and Hodder; 1998. p. 303-18.
2Montoya JG, Kovacs JA, Remington JS. Toxoplasma gondii. In: Mandell GL, Bennett JE, Dolin R, editors. Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases. 6 th ed. Philadelphia: Elsevier Churchill Livingstone; 2005. p. 3170-93.
3Kasper LH. Toxoplasma infection. In: Kasper DL, Braunwald E, Fauci AS, Hauser SL, Longo DL, Jameson JL, editors. Harrison's Principles of Internal Medicine. 16 th ed. New York: McGraw Hill; 2005. p. 1243-8.
4Malhotra VL, Bharadwaj Y, Lakshmy A, Kapur H, Prakash K. Comparison of enzyme linked immunosorbent assay and indirect haemagglutination test in serologic diagnosis of toxoplasmosis. J Commun Dis 1991;23:154-6.
5McCabe RE, Remington JS. The diagnosis and treatment of toxoplasmosis. Eur J Clin Microbiol 1983;2:95-104.
6Pietkiewicz H, Hiszczyǹska-Sawicka E, Kur J, Petersen E, Nielsen HV, Stankiewicz M, et al. Usefulness of Toxoplasma gondii-specific recombinant antigens in serodiagnosis of human toxoplasmosis. J Clin Microbiol 2004;42:1779-81.
7Gamble HR, Dubey JP, Lambillotte DN. Comparison of a commercial ELISA with the modified agglutination test for detection of Toxoplasma infection in the domestic pig. Vet Parasitol 2005;128:177-81.
8Wong SY, Hajdu MP, Ramirez R, Thulliez P, McLeod R, Remington JS. Role of specific immunoglobulin E in diagnosis of acute Toxoplasma infection and toxoplasmosis. J Clin Microbiol 1993;31:2952-9.
9Joseph P, Calderón MM, Gilman RH, Quispe ML, Cok J, Ticona E, et al. Optimization and evaluation of a PCR assay for detecting toxoplasmic encephalitis in patients with AIDS. J Clin Microbiol 2002;40:4499-503.
10Marra C. Distinguishing central nervous system lymphoma from Toxoplasma encephalitis. Ann Intern Med 1994;120:812-3.
11Ravinder PT, Parija SC, Rao KS. Urinary hydatid antigen detection by coagglutination, a cost-effective and rapid test for diagnosis of cystic echinococcosis in a rural or field setting. J Clin Microbiol 2000;38:2972-4.
12Itoh M, Ohta N, Kanazawa T, Nakajima Y, Sho M, Minai M, et al. Sensitive enzyme-linked immunosorbent assay with urine samples: A tool for surveillance of schistosomiasis japonica. Southeast Asian J Trop Med Public Health 2003;34:469-72.
13Hughes HP, Van Knapen F, Atkinson HJ, Balfour AH, Lee DL. A new soluble antigen preparation of Toxoplasma gondii and its use in serological diagnosis. Clin Exp Immunol 1982;49:239-46.
14Bible KC, Boerner SA, Kaufmann SH. A one-step method for protein estimation in biological samples: Nitration of tyrosine in nitric acid. Anal Biochem 1999;267:217-21.
15Voller A, De Savigny D. Diagnostic serology of tropical parasitic diseases. J Immunol Methods 1981;46:1-29.
16Barbesti S, Soldini L, Carcelain G, Guignet A, Colizzi V, Mantelli B, et al. A simplified flow cytometry method of CD4 and CD8 cell counting based on thermoresistant reagents: Implications for large scale monitoring of HIV-infected patients in resource-limited settings. Cytometry B Clin Cytom 2005;68:43-51.
17Zumla A, Savva D, Wheeler RB, Hira SK, Luo NP, Kaleebu P, et al. Toxoplasma serology in Zambian and Ugandan patients infected with the human immunodeficiency virus. Trans R Soc Trop Med Hyg 1991;85:227-9.
18Malla N, Sengupta C, Dubey ML, Sud A, Dutta U. Antigenaemia and antibody response to Toxoplasma gondii in human immunodeficiency virus-infected patients. Br J Biomed Sci 2005;62:19-23.
19Colombo FA, Vidal JE, Penalva de Oliveira AC, Hernandez AV, Bonasser-Filho F, Nogueira RS, et al. Diagnosis of cerebral toxoplasmosis in AIDS patients in Brazil: Importance of molecular and immunological methods using peripheral blood samples. J Clin Microbiol 2005;43:5044-7.
20Islam MZ, Itoh M, Shamsuzzaman SM, Mirza R, Matin F, Ahmed I, et al. Diagnosis of visceral leishmaniasis by enzyme-linked immunosorbent assay using urine samples. Clin Diagn Lab Immunol 2002;9:789-94.
21Singh MP, Dubey ML, Sud A, Malla N. Antibody response to Toxoplasma gondii in saliva samples from human immunodeficiency virus-infected patients. Br J Biomed Sci 2005;62:81-4.
22Nagalingeswaran K, Solomon S, Madhivanan P, Yepthomi T, Venkatesan C, Amalraj E, Flanigan T, Thyagarajan SP, Mayer K. Correlation between plasma viral load and CD4+T cell count to opportunistic infections in persons with HIV in South India.Int Conf AIDS 2000 Jul 9-14; 13:(abstract no. MoPeB2267).
23Zufferey J, Sugar A, Rudaz P, Bille J, Glauser MP, Chave JP. Prevalence of latent toxoplasmosis and serological diagnosis of active infection in HIV-positive patients. Eur J Clin Microbiol Infect Dis 1993;12:591-5.
24Nissapatorn V, Lee CK, Khairul AA. Seroprevalence of toxoplasmosis among AIDS patients in Hospital Kuala Lumpur, 2001. Singapore Med J 2003;44:194-6.