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  In this Article
 »  Abstract
 »  Introduction
 »  Material and methods
 »  Results
 »  Discussion
 »  Acknowledgement
 »  References

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Year : 2001  |  Volume : 49  |  Issue : 4  |  Page : 375-9

Evaluation of cysticercus fasciolaris antigen for the immunodiagnosis of neurocysticercosis.


Department of Pathology, King George's Medical College, Lucknow - 226003, India.

Correspondence Address:
Department of Pathology, King George's Medical College, Lucknow - 226003, India.
[email protected]

  »  Abstract

Cysticercus cellulosae antigen has been frequently used to detect antibodies for immunodiagnosis of neurocysticercosis. We have, for the first time, used membrane extract of cysticercus fasciolaris, the larval stage of Taenia taeniaeformis, in ELISA, with successful results. IgM and IgG antibodies against cysticercus were measured in serum from cases of neurocysticercosis (217), normal and diseased controls (89). 203 sera from cases of neurocysticercosis were positive for either or both IgG and IgM antibodies while 157/217 cases showed IgM and 158/217 showed IgG antibodies. Ten controls showed false postivity in IgG ELISA. Eight of these cases also had IgM antibodies. The test had an overall sensitivity of 93.54% and a specificity of 84.2% with a positive predictive value of 93.54% and a negative predictive value of 84.2%. Cysticercus fasciolaris can be conveniently produced in the experimental laboratory host, Rattus rattus, and would be of practical value in the immunodiagnosis of cysticercosis in humans.

How to cite this article:
Husain N, Jyotsna, Bagchi M, Husain M, Mishra M K, Gupta S. Evaluation of cysticercus fasciolaris antigen for the immunodiagnosis of neurocysticercosis. Neurol India 2001;49:375


How to cite this URL:
Husain N, Jyotsna, Bagchi M, Husain M, Mishra M K, Gupta S. Evaluation of cysticercus fasciolaris antigen for the immunodiagnosis of neurocysticercosis. Neurol India [serial online] 2001 [cited 2021 Jan 19];49:375. Available from: https://www.neurologyindia.com/text.asp?2001/49/4/375/1217




   »   Introduction Top

Neurocysticercosis (NCC) is the commonest parasitic disease of the nervous system. In a multicentric population survey conducted by Cokervann et al,[1] 3-13% sera from Indonesia, Burma, Vietnam and the Philippines were found to react to cysticercus antigen. They observed the highest proportion of positive sera in a village on the island of Bali, Indonesia, where 21% of the sera tested positive. In Indian children with acute and chronic meningitis, an incidence of
5.47 % has been observed.[2] Sero-epidemiological studies conducted in school children in South Africa revealed an overall positivity rate of 5.5%.[3] A number of immunological tests have been evaluated in the diagnosis of cysticercosis, such as complement fixation,[4] indirect hemeagglutination assay,[5] immun-oelectrophoresis,[6] ELISA[7],[8],[9],[10],[11] and enzyme linked immunoblot assay.[12],[13],[14] The antigen used for detection of antibodies was derived, in most studies, from Cysticercus cellulosae (source: pigs and humans). Taenia crassiceps (murine cysticercus)[15],[16],[17] and fluid of T. Hyadatigena have also been used as a source of antigen.[18] In the current study we have, for the first time, evaluated ELISA using crude membrane extract of Cysticercus fasciolaris, the larval stage of Taenia taeniaeformis, for the detection of IgM and IgG antibodies in sera of patients with neurocysticercosis.



   »   Material and methods Top

A case control study was carried out for evaluation of the experimental ELISA in the diagnosis of neurocysticercosis at King George's Medical College, Lucknow, India. Study samples were obtained from 217 cases of neurocysticercosis and 36 normal controls and 53 sera from diseased controls. All patients were assessed for: i) Single or multiple ring lesions in CT/MRI Scan with visualisation of scolex; ii) Positivity in commercial cysticercus ELISA for IgG antibodies against Taenia solium antigen (United Biotech Inc., USA).
The criteria for inclusion of a patient as a 'case' in the study was taken as any of the following (Brutto, 1997) : a) Parenchymal single or multiple ring lesion in CT/MRI Scan with visualisation of scolex; b) Single or Multiple ring lesions in CT Scan with a positive commercial cysticercus ELISA. Normal controls (36) had no parenchymal ring lesions in CT/MRI scan and no subcutaneous nodules on clinical evaluation. Stool samples were examined on three consecutive days to rule out presence of intestinal parasites. Disease controls included 46 cases of tuberculoma diagnosed by MRI spectroscopy, 4 cases of Hydatid disease and 3 cases of intestinal taeniasis.
Experimental Technique : Gravid segments of Taenia taeniaeformis were collected from the intestines of cats. The eggs were gently separated and suspended in normal saline at a concentration of 100 eggs/ml. Rattus rattus were orally infected with 0.2 ml of the egg suspension. After 60 days the rats were sacrificed, Cysticercus fasciolaris cysts were dissected from the liver on ice, and membranes separated from the scoleces were washed with cold phosphate buffered saline at pH 7.4 (PBS), homogenised in five volumes of PBS, sonicated three times at 30S and cold centrifuged for 30 min. at 20,000 g. The supernatant, at a protein concentration of 150 mg/dl, was used as the concentrated antigen.
ELISA was performed as a blinded assay with all samples (cases and controls). The optimal dilution of antigen and serum to be used was standardised by checker board titration. Polystyrene microtitre plates (Nunc, U S A) were coupled with antigen diluted to 1:2000 in carbonate buffer (pH 9.6) and left overnight at 37oC. The plates were washed ten times with wash buffer ( PBS containing 0.1% Tween 20, [Sigma], USA) and blocked with 2% bovine serum albumin (BSA) (J.Mitra and Co, India) in washing buffer for 2 hours. Serum diluted to 1:1000 in wash buffer with 1% BSA, was allowed to react in each well for one hour. After five washes the wells were incubated with horse radish peroxidase labelled antihuman IgM or IgG antibody raised in mouse (Dakopatts, Denmark) at a dilution of 1:1000, for half an hour at 37oC. After washing, the substrate tetra methyl benzidine (Bangalore Genie, India) at a dilution of 1:20 was added. Reaction was stopped after 10 min. with 5 N sulphuric acid and Optical Density (OD) was measured at 450 nm. The tests were performed in triplicate and average absorbance was taken as the final OD. The tests were repeated on two occasions to check for reproducibility of results.
Statistical Analysis : Cut off value for negative and positive results was taken as two times the standard deviation above the mean of the controls. The sensitivity, specificity, negative and positive predictive values of the test were assessed.



   »   Results Top

CT scan findings : Cases of neurocysticercosis showed parenchymal ring enhancing lesions in CT scan which were single (172 cases) or multiple (45 cases). In 73 cases, the scolex could be visualized as an eccentric nodule. Perifocal oedema, calcification and mass effect was also seen.
Commercial cysticercus ELISA : ELISA for cysticercosis was performed using kits from United Biotech Inc., USA. The antigen was derived from Taenia solium (Cysticercus cellulosae) and conjugate detected IgG antibodies. One hundred and fourteen of 217 cases tested positive for antibodies in serum. All controls tested negative in the commercial ELISA.
Experimental ELISA using membrane antigen extract of Cysticercus fasciolaris : IgM and IgG antibodies in sera of 217 cases and 89 controls were assayed. Blanks, high positive and low positive controls were included in each batch. Cut off value of 559 was obtained in IgM ELISA while IgG ELISA had a cut off value of 535. Optical densities obtained were higher for IgM antibodies and lower for IgG antibodies [Figure. 1] and [Figure. 2].One hundred and fifty [eight] cases had IgG and 157 cases had only IgM antibodies. When presence of either or both antibodies (combined assay) was taken as diagnostic criterion for immunodiagnosis, 203 out of 217 cases tested positive and 14 out of 89 controls gave a false positive result.
Statistical Analysis : The sensitivity of the combined assay was 93.54% and the specificity was 84.2%, with a positive predictive value of the test being 93.5% and a negative predictive value of 84.2%. IgM ELISA had sensitivity of 72.3% and specificity of 91.0% as compared to IgG ELISA which had a sensitivity of 72.8% and a specificity of 88.7%. The reproducibility of the test was found to be 100% on two different occasions using the same batch of antigen.


   »   Discussion Top

A practical problem in evaluating any diagnostic test for neurocysticercosis is that no single gold standard is available for diagnosis of all cases. Diagnostic criteria has been enumerated with degrees of certainty for diagnosis of NCC.[19] Magnetic resonance imaging and CT scans are diagnostic only when the scolex can be visualised within the multiple or single ring lesions in CT scan.[20] However in many cases the scolex is not seen and the differential diagnosis, in India and other regions endemic for tuberculosis, is frequently tuberculoma.[21] Small cystic gliomas[22] and parenchymal abscesses may also, sometimes, present as ring enhancing lesions. Hence only cases with ring lesions having a positive commercial cysticercus ELISA or scolex in MRI/CT scans were included in the study for evaluation of the new test.
This is the first report of ELISA using membrane extract of Cysticercus fasciolaris for the immunodiagnosis of neurocysticercosis in humans. Ito et al[23] have used antigen extracts from oncospheres of Taenia taeniaeformis for immunodiagnosis in rats. Previous studies have been directed at the detection of IgM and IgG type antibodies against Cysticercus cellulosae.[1],[7],[8],[9],[11] The crude[10] and purified membrane extracts,[1] Cyst fluid[25] have all been used in various tests including ELISA. The adult worm of bovine cysticercosis, Taenia saginata, has also been tested antigenically in ELISA and a lower sensitivity and specificity has been reported as compared to the cyst fluid of Cysticercus cellulosae.[25] Antibody based ELISA have also been designed for the detection of circulating antigens in CSF of patients with NCC.[26],[27] Murine cysticercus, Cysticercus crassiceps, has been tested in antigen based ELISA for the detection of antibodies in patients with cysticercosis.[15],[17]
The sensitivity and specificity of ELISA for detection of antibodies in serum has varied from poor[11] to 80%.[9] Better results have been reported for detection of IgM antibodies as compared to IgG antibodies.[8] We have observed a similar sensitivity in IgM and IgG ELISA. It is interesting to note that in the current study, either or both antibodies were present in 203/217 cases. We would therefore advocate the evaluation of both IgG and IgM antibodies in sera of patients to increase the sensitivity of ELISA. As with other parasitic infestations of the body, one would expect the IgM antibodies to be present in the early stages of infection, both IgG and IgM positivity in active stages of disease and IgG to persist in disease of long duration. However such a correlation is not possible in cases of neurocysticercosis since the duration of the infestation does not frequently correlate with the duration of symptoms which may appear very early or late in the stage of the disease. In the current study we did not find any significant difference in duration of symptoms between cases with only IgG antibodies as compared to cases with IgM antibodies.
A higher positivity has been observed in CSF samples as compared to serum samples.[8],[11] Diaz et al, in a comparison of enzyme immunotransfer blot assay (EITB) and ELISA have observed a sensitivity of 94% by EITB and 65 % by antibody ELISA.[28] The specificity of the tests was 100% in EITB and 63 % in Antibody ELISA. Ito et al have also reported 100% sensitivity in confirmed NCC samples by immunoblot assay.[24] It appears, therefore, that among the currently available tests EITB is the most sensitive and specific, while CSF samples are the better samples for diagnosis. Both the test and the sample lack practical applicability in clinical labs and field conditions. An ELISA with a high sensitivity and specificity in serum is still on the requirement list and therefore in the present study we have attempted to use a different species of cysticercus with successful results. Secondly, in any parasitologic research the availability of the parasite is of prime importance. Cysticercus fasciolaris can be easily produced in the laboratory animal, Rattus rattus within a limited time period of 60 days. The small animals are easy to maintain. The sensitivity and specificity of ELISA using membrane extract of Cysticercus fasciolaris for the detection of IgM and/or IgG antibodies in serum is higher than the best results obtained with cysticercus cellulosae.[9]


   »   Acknowledgement Top

The study has been supported by a grant from the Council of Science and Technology, U.P., India.
 

  »   References Top

1.Cokervann M, Brown P, Gajdusek DC : Serodiagnosis of human cysticercosis using a chromatofocussed antigenic preparation of Taenia solium cysticerci in enzyme linked immunosorbent assay (ELISA). Transactions of the Royal Society of Tropical Health and Hygiene 1984; 78 : 492-496.   Back to cited text no. 1    
2.Chandramukhi A, Nayak P : Sub acute and chronic meningitis in children - an immunological study of cerebrospinal fluid. Indian J Pediatr 1990; 57 : 685-691.   Back to cited text no. 2    
3.Shasha W, Pammenter WD : Seroepidemiological studies of cysticercosis in school children from two rural areas of Transkei, South Africa. Ann Trop Med Parasitol 1991; 85 : 349-355.   Back to cited text no. 3    
4.Neito D : Cysticercosis of the nervous system: diagnosis by means of the spinal fluid complement fixation test. Neurology (Minneap) 1956; 6 : 725-732.   Back to cited text no. 4    
5.Proctor EM, Purel SJ, Elsdon-Dew : The Serological diagnosis of cysticercosis. Ann Trop Med Parasitol 1966; 60 : 146-151.   Back to cited text no. 5    
6.Katti MK, Jagannath C, Gokul BN et al : Antigenic analysis of cysticercus cellulosae by crossed immunoelectrophoresis and its role in the immunodiagnosis of neurocysticercosis. Indian J Med Res 1990; 91 : 39-43.   Back to cited text no. 6    
7.Diwan A, Cokervann M, Brown P : Enzyme linked immunosorbent assay (ELISA) for the detection of antibody to cysticerci of Taenia solium. Am J Trop Med Hyg 1982; 31 : 364-369.   Back to cited text no. 7    
8.Rosas N, Soleto J, Neito D : ELISA in the diagnosis of neurocysticercosis. Arch of Neurol 1986; 43 : 353-356.   Back to cited text no. 8    
9.Pammenter MD, Rossouw EJ : The value of antigenic fraction of Cysticercus cellulosae in the serodiagnosis of cysticercosis. Ann Trop Med Parasitol 1987; 81 : 117-123.   Back to cited text no. 9    
10.Plancarte A, Espinoza B, Flisser A : Immunodiagnosis of human neurocysticercosis by enzyme -linked immunosorbent assay. Childs Nerv Syst 1987; 3 : 203.   Back to cited text no. 10    
11.Malla N, Kaur M, Kaur U et al : Evaluation of enzyme linked immunosorbent assay for the detection of anticysticercus antibodies in cerebrospinal fluid from patients with neurocysticercosis. Journal of Hygiene Epidemiology Microbiology and Immunology 1992; 36 : 181-190.   Back to cited text no. 11    
12.Grogl M, Estroda JJ, Macdonald G et al : Antigen-antibody analysis in neurocysticercosis. J Parasitol 1985; 71 : 433-442.   Back to cited text no. 12    
13.Wilson M, Bryan RT, Fried JA et al : Clinical evaluation of cysticercosis enzyme-linked immuno electophoresis blot in patients with neurocysticercosis. J Infect Dis 1991; 164 : 1007-1009.   Back to cited text no. 13    
14.Pathak KM, Allan JC, Frsfeld K et al : Western blot and ELISA assay for the diagnosis of Taenia solium infection in pigs. Vet Parasitol 1994; 53 : 209-217.   Back to cited text no. 14    
15.Larraldo C, Soleto J, Montoya RM et al : Immunodiagnosis of human cysticercosis in cerebrospinal fluid. Antigens from murine Taenia crassiceps effectively substitute those from porcine Taenia solium. Arch Pathol Lab Med 1990; 114 : 926 -928.   Back to cited text no. 15    
16.Garcia E, Ordonez G, Sotelo J : Antigens from Taenia crassiceps cysticerci used in comlement fixation, enzyme linked immunosorbent assay, and western blot (immunoblot) for diagnosis of neurocysticercosis. J Clin Microbiol 1995; 33 : 3324-3325.   Back to cited text no. 16    
17.Bueno EC, Vaz AJ, Machado LD et al : Specific Taenia crassiceps and Taenia solium antigenic peptides for neurocysticercosis immunodiagnosis using serum samples. J Clin Microbiol 2000; 38 : 146-151.   Back to cited text no. 17    
18.Tamburrini A, Gomez Morales M A, Pozio E : Development of an immunoenzyme test for the diagnosis of human cysticercosis using a heterologous antigen. Parassitologia 1995; 37 : 195-198.   Back to cited text no. 18    
19.Brutto Oscar H Del Neurocysticercosis.Curr Opin Neurol 1997; 10 : 268-272.   Back to cited text no. 19    
20.Rajsekhar V, Hasan RP, Prakash H : Differenciating solitary small cysticercus granulomas and tuberculomas in patients with epilepsy. J Neurosurg 1993; 78 : 402-407.   Back to cited text no. 20    
21.Chandy MJ, Rajsekhar V, Ghosh J : Single small ring enhancing lesions in Indian patients with epilepsy: clinical, radiological and pathological considerations. J Neurol Neurosurg Psychiatry 1991; 54 : 702-705.   Back to cited text no. 21    
22.Wankate N, Takeda R, Takeda N : Magnetic resonance imaging and histopathology of cerebral gliomas. Neuroradiology 1992; 35 : 463-469.   Back to cited text no. 22    
23.Ito A, Plancarte A, Ma L et al : Novel antigens for neurocysticercosis: simple method for preparation and evaluation for serodiagnosis. Am J Trop Med Hyg 1998; 59 : 291-294.   Back to cited text no. 23    
24.Ito A, Sakakibara Y, Ma L et al : Ultrasonographic and serologic studies of experimental cysticercosis in rats infected with Taenia taeniaeformis. Parasite Immunol1998; 20 : 105-110.   Back to cited text no. 24    
25.Morakote N, Nawacharoen W, Sukonthasun K et al : Comparison of cysticercus extract, cyst fluid and Taenia saginata extract for use in ELISA for serodiagnosis of neurocysticercosis. Southeast Asian J Trop Med Public Health1992; 23 : 77-81.   Back to cited text no. 25    
26.Wang CY, Zhang HH, GE L Y : A M Ab-based ELISA for detecting circulating antigen in CSF of patients with neurocysticercosis. Hybridoma 1992; 11 : 825 -827.   Back to cited text no. 26    
27.Brandt JR, Geerts S, De Deken R et al : A monoclonal antibody based ELISA for the detection of circulating excretory-secretory antigens in Taenia saginata cysticercosis. Int J Parasitol 1992; 22 : 471-477.   Back to cited text no. 27    
28.Diaz JF, Verastegui M, Gilman RH et al : Immunodiagnosis of human cysticercosis (Taenia solium): a field comparison of an antibody-enzyme-linked immunosorbent assay (ELISA) , an antigen-ELISA, and an enzyme-linked immunoelectrotransfer blot (EITB) assay in Peru. Am J Trop Med Hygi1992; 46 : 610-615.   Back to cited text no. 28    

 

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